The Interactive Fly
Genes involved in tissue and organ development
Genes influencing glial differentiation
Glia constitute a support system for neurons. Neurons process and carry information from one neuron to the next, or between neurons and muscles. In their supportive role glia provide neurons with nutrition and growth factors; they maintain neural homeostasis by removing toxic substances, and provide neurons with electrical insulation, assuring proper neuron function. In addition, glia act as substrates for neural migration.
In the Drosophila embryonic CNS several subtypes of glial cells develop, which arrange themselves at characteristic positions and presumably fulfil specific functions. The mechanisms leading to the specification and differentiation of glial subtypes are largely unknown. By DiI labelling in glia-specific Gal4 lines the lineages of the lateral glia in the embryonic ventral nerve cord were clarified and each glial cell was linked to a specific stem cell. For the lineage of the longitudinal glioblast, it was shown to consist of 9 cells, which acquire at least four different identities. A large collection of molecular markers (many of them representing transcription factors and potential Gcm target genes) reveals that individual glial cells express specific combinations of markers. However, cluster analysis uncovers similar combinatorial codes for cells within, and significant differences between the categories of surface-associated, cortex-associated, and longitudinal glia. Glial cells derived from the same stem cell may be homogeneous (though not identical; stem cells NB1-1, NB5-6, NB6-4, LGB) or heterogeneous (NB7-4, NB1-3) with regard to gene expression. In addition to providing a powerful tool to analyse the fate of individual glial cells in different genetic backgrounds, each of these marker genes represents a candidate factor involved in glial specification or differentiation. This was demonstrated by the analysis of a castor loss of function mutation, which affects the number and migration of specific glial cells (Beckervordersandforth, 2008).
This report provides a comprehensive description of marker gene and enhancer trap expression in CNS glial cells of late Drosophila embryos. The markers include many transcription factors known to be involved in cell fate specification, as well as a number of still unknown factors. They were chosen for this analysis either because they were known to be expressed in subsets of glial cells or because they were known to be involved in cell fate determination in the nervous system. All together, more than 50 markers were tested, 39 of which showed expression in glial cells and hence were described in detail. Their specific expression patterns, though in many cases not restricted to glia, enable identification of groups of cells, as well as individual cells (Beckervordersandforth, 2008).
The lateral CNS glial cells have been assigned to three categories, according to their spatial distribution and morphology: the surface-, the cortex-, and the neuropile-associated glial cells. Categories were further divided into subgroups, as for example the surface-associated glial cells into subperineural glial cells and channel glia. Several of the molecular markers described in this study exhibit expression patterns, which correspond to the spatial/morphological definition of glial categories or subgroups. For example, moody or svp-lacZ are expressed in all surface-associated glial cells, whereas P101-lacZ is only expressed in the SPG-subgroup and engrailed only in the CG-subgroup. In addition, nearly each individual glial cell expresses a specific combination of markers indicating that they develop unique identities. Yet, nothing is known about how these identities are acquired. Comparing subtype affiliation or lineage ancestry of all glial cells with their respective marker gene expression patterns, it becomes obvious that glial cell specification is a process occurring on the level of individual cells. Cells might have a predisposition for a particular subtype laid down by lineage (e.g. NB5-6 derived cells to become subperineurial glia). In contrast, a temporal cascade within a lineage could determine individual cell identities (as it might be the case for the NB7-4 lineage) (Beckervordersandforth, 2008).
DiI labelling of the lineages of various progenitor cells in combination with cell-specific enhancer trap lines revealed that the composition of glial progeny within the lineages is invariant. Clonally related glia cells often express similar combinations of marker genes. The LGs, a prominent subgroup of the neuropile-associated glia and the only interface glia in the embryonic VNC, have been defined as the progeny of the LGB, which become aligned along the longitudinal connectives. However, there has been confusion about the size and composition of the LGB-lineage. By means of DiI labelling and marker gene expression, the size of this lineage was determined to be 9 cells. Although all cells of the LGB-lineage express a similar set of markers, a few markers are restricted to only parts of the lineage. Based on such markers, as well as positional criteria, the group of LGs was further subdivided. One of the cells, the LP-LG, is located slightly more lateral than the other LGs, and seems to be geared towards the ISN. Since it lies close to, and expresses a similar combination of markers as the M-ISNG, it would be justified to assign this cell to the group of nerve root glia; however, this was not done in order to avoid conflicts with the established nomenclature. Despite of their similarities, these two cells are of different origin: the LP-LG derives from the LGB, and the M-ISNG is generated by NB1-3 (Beckervordersandforth, 2008).
Most of the NBs that arise at corresponding positions and times in thoracic and abdominal segments (called serially homologous NBs) acquire the same fate, i.e. they generate the same lineages expressing corresponding sets of markers. However, some of these serially homologous lineages develop characteristic, tagma-specific differences with regard to cell number and/or cell types. Tagma-specific characteristics of these lineages have been shown to be under the control of Hox genes (Beckervordersandforth, 2008).
Although the total number of CNS glial cells is identical in thoracic and abdominal neuromeres, there are some differences in their origin and distribution of subtypes. This is due to tagma-specific differences among serially homologous lineages of NBs 1-1, 2-2, 5-6, and 6-4, which give rise to CBGs and SPGs. NB6-4A (A, abdominal) generates only two CBGs: MM-CBG and M-CBG, whereas the NB6-4T (T, thoracic) lineage comprises an additional MM-CBG2 and a neuronal sublineage. NB1-1T generates only neurons, whereas NB1-1A produces three SPGs (A-SP-G, B-SPG, and LV-SPG) in addition to neurons. In the thorax, the LV-SPG is presumably generated by NB5-6T, a cell at the position of A-SPG is produced by NB2-2T, and a cell at the B-SPG position is missing. Despite their different origin, the NB1-1A- and NB2-2T-derived SPGs specifically express hkb-lacZ and mirr-lacZ. Furthermore, the NB1-1A- and the NB2-2T-derived A-SPG appear to assume the same identity, as they express the same set of markers (including castor, which is not found in the abdominal B-SPG. Taken together, the differences between thorax and abdomen are restricted to only few glial cells, most of which acquire similar cell fates in thorax and abdomen (as judged by marker gene expression) irrespective of their progenitor (Beckervordersandforth, 2008).
The collection of marker genes and enhancer trap lines presented in this study provides powerful tools for the identification of specific glial cells in different genetic backgrounds. Each of markers also represents a candidate factor involved in glial subtype specification and/or differentiation. Many of these genes encode transcription factors known to be involved in cell fate specification, like fushi tarazu, mirror, and muscle segment homeobox. Other genes encode factors involved in cell signalling, e.g moody and CG11910, or enzymes like CG7433 and CG6218 (Beckervordersandforth, 2008).
moody is expressed in all cells belonging to the surface-associated glia. At the end of embryogenesis, surface-associated glial cells form a thin layer ensheathing the entire CNS, thereby establishing the blood-brain barrier. Moody is a G-protein coupled receptor, which acts in a complex pathway to regulate the cortical actin, thereby stabilizing the extended morphology of the surface-glia. This is necessary for the formation of septate junctions to achieve proper sealing of the nerve cord (Bainton, 2005; Daneman, 2005; Schwabe, 2005). Moody therefore represents a protein, which is essential for establishing and maintaining a specific function of surface glia (Beckervordersandforth, 2008).
Two of the markers analyzed represent metalloproteases: Neprilysin4 (Nep4) and Invadolysin. Invadolysin has been described to play a role in mitotic progression and in migration of germ cells (McHugh, 2004), but as for Nep4, its function in the nervous system is unknown. In vertebrates it has been shown that metalloproteases are involved in various processes in the CNS: they are associated with neurite outgrowth, migration of neurons and myelination of axons. For one matrix-metalloprotease, MMP-12, it has been shown that it is expressed in oligodendrocytes, where it functions in maturation and morphological differentiation of OL lineages (Larsen, 2004; Larsen, 2006). It has been postulated that LGs are analogous to vertebrate oligodendrocytes (Hidalgo, 2000), as both groups of cells enwrap axonal projections in the CNS, although to different degrees (no myelination in Drosophila). The two metalloproteases analysed are exclusively expressed in neuropile- (LGs) and cortex-associated glial cells (CBGs). Thus, both Nep4 and Invadolysin may possibly be involved in the differentiation of LGs. In invadolysin loss of function mutants, the specification of lateral glial cells does not seem to be affected, but the LGs show a very subtle phenotype in their positioning (data not shown). An explanation for the subtle phenotype may be redundant function of both enzymes. Indeed, in vertebrates it has been shown that metalloproteases have many overlapping substrates in vitro, and redundancy and compensation has been shown for matrix-metalloproteases (MMPs) in double mutants. Furthermore, it has been shown for members of the neprilysin family of metalloendopeptidases in Caenorhabditis elegans and Drosophila melanogaster, that many of the enzymatic properties have been conserved during evolution (Beckervordersandforth, 2008).
Making use of the molecular markers, this study characterized the phenotype of a cas loss of function mutation. Cas is a transcription factor, which acts in temporal cell fate specification. Together with Pdm, Cas is involved in the determination of late progeny cells in CNS lineages. In late embryonic stages, cas is specifically expressed in four glial cells per hemisegment, the V-CG and D-CG, the A-SPG and the LV-SPG, as well as in many neurons. The A- and LV-SPG, which are late progeny of the NB1-1A, are not affected in cas mutants, whereas the NB7-4-derived CGs seem mislocalized, with the medial migration of both CGs being impaired in cas mutants. This points to different functions of Cas in distinct NB lineages. As can be deduced from Repo stainings, general aspects of glial differentiation do not seem to be affected in cas mutants. Further analysis will have to clarify whether the role of Cas in NB7-4 derived glial cells is on the level of cell fate determination and/or whether it directly acts on specific aspects of differentiation (migration, motility). It also remains to be shown whether Cas acts cell-autonomously in this process (Beckervordersandforth, 2008).
Glia origins and diversity mirror that of the neurons they accompany. Longitudinal glial of the ventral nervous system (CNS) are derived from longitudinal gliobasts that have a neuroectodermal origin. Other CNS and brain glia are derived from the same neuroblasts that give rise to neurons. Midline glia have a separate mesectodermal origin, being derived from midline neuroblasts. There are at least two other classes of glia sharing precursors with neurons: those associated with peripheral axons and those associated with sensory organ neurons (Nelson, 1994 and Jones, 1995).
In the developing Drosophila visual system, glia migrate into stereotyped positions within the photoreceptor axon target fields and provide positional information for photoreceptor axon guidance. Conversely, glial migration depends on photoreceptor axons, as glia precursors stall in their progenitor zones when retinal innervation is eliminated. These results support the view that this requirement for retinal innervation reflects a role of photoreceptor axons in the establishment of an axonal scaffold that guides glial cell migration. Optic lobe cortical axons extend from dorsal and ventral positions toward incoming photoreceptor axons and establish at least four separate pathways that direct glia to proper destinations in the optic lobe neuropiles. Photoreceptor axons induce the outgrowth of these scaffold axons. Most glia do not migrate when the scaffold axons are missing. Moreover, glia follow the aberrant pathways of scaffold axons that project aberrantly, as occurs in the mutant dachsous. The local absence of glia is accompanied by extensive apoptosis of optic lobe cortical neurons. These observations reveal a mechanism for coordinating photoreceptor axon arrival in the brain with the distribution of glia to multiple target destinations, where they are required for axon guidance and neuronal survival (Dearborn, 2004).
The Drosophila optic ganglia are derived from a pair of
neuroectodermal proliferative centers known as the inner and outer anlage, located on the ventrolateral surface of each larval brain hemisphere. The onset of Wingless (Wg)
expression has been described in a few cells located at the presumptive dorsal and ventral margins of the disc-shaped outer anlagen in young larvae. These two `Wg domains' are positioned as adjacent wedges within the disc-shaped outer anlage. In mid-metamorphosis, the
disc opens at a fissure located between the two Wg domains; the disc unfurls linearly, such that the Wg domains move to the future dorsal and ventral poles of the ganglion. Interestingly, the Wg domains are roughly coincident with sites revealed by clonal analyses to be the origins of glia that migrate into the lamina neuropile. This relationship was investigated in greater detail (Dearborn, 2004).
A marker of wingless expression, the lacZ
reporter construct 17en40 (or wg-lacZ) was used to
examine wg expression with respect to glial cell development and
movement. In cells expressing wg-lacZ, cytoplasmic ß-galactosidase fills cellular processes and permits the visualization of
their detailed morphology. The paths of glial cell migration are observed to coincide with a stereotyped pattern of cytoplasmic extensions emanating from
wg-lacZ expressing cells. The extensions follow stereotyped routes
and terminate at specific destinations in the lamina, medulla and lobula.
Glia form chains along these extensions and accumulate in neuropile
destinations beyond the extension's termini. Marker analysis
indicates that the glia undergo differentiation as they progress along these paths. Lamina glia express the early glial differentiation factor glial cells missing (gcm) at their points of entry onto the extensions. Further along the path toward the neuropile, glia commence
expression of the homeodomain protein Repo, a marker
downstream of Gcm expression. Thus, in the case of lamina marginal glia,
differentiation can be assessed relative to glial progression along the
migratory pathway (Dearborn, 2004).
The wg-lacZ positive extensions are evidently axons. Their cell
membranes label by anti-horseradish peroxidase antibody, a neuronal
marker. Their cell bodies label positive for Elav, also a neuronal marker. The axons extending toward the same neuropile destination bundle together in a fascicle, as indicated by labeling of individual axons in mosaic animals. Four wg-lacZ labeled fascicles were resolved emerging from each of the dorsal and ventral Wg domains. One fascicle from each domain extends to the border of the lamina field, terminating at a position adjacent to layers of glia known as the lamina epithelial (Ep) and marginal (Ma) glia (established nomenclature is found at Flybrain). These layers of glia lie, respectively, above and below the layer of the axon termini of the R1-R6 photoreceptors. Glia can be observed migrating in a chain along the fascicles. Owing to the absence of specific markers, epithelial
and marginal glia could not be distinguished prior to their separation into distinct layers. It seems
likely, however, that both glial types migrate on the same pathway. One
fascicle from each Wg domain extends to the cortex, neuropile boundary of the medulla, and is associated with the chain-like migration of medulla neuropile (MNG) glia. One fascicle from each domain extends to the boundary between the medulla and lobula, and corresponds to a pathway for migration of inner chiasm (Xi) glia, which demarcate the border between medulla and lobula neuropiles. The final pair of fascicles extends into the lobula neuropile and forms a pathway associated with lobula neuropile glia (LoG). These four sets of putative migratory guides are referred to as 'scaffold axons'. Notably, the migration of
these four glia types depends on retinal innervation of the optic lobe, a
requirement that is explored in this study (Dearborn, 2004).
Earlier stage specimens were examined in order to determine the temporal relationship between glial migration and the outgrowth of scaffold axons. During the first two larval stages, most neuroectodermal cells of the outer anlagen express the cell adhesion protein Fasciclin 2 (Fas2), with the exception of those in the two Wingless domains; Wingless-expressing cells are Fas2 negative. As development proceeds to the third instar stage, the neuroectodermal populations mostly convert to blast cells that produce the neurons and glia of the lamina and medulla. The differentiation of wg-lacZ positive scaffold neurons and the extension of their axons follow this general temporal scheme, a small population of glia precedes the arrival of the first
photoreceptor axons in the target field of the retinal axon. A small population of centrally located glia have been described that precede the
arrival of photoreceptor axons in the lamina. At these early time points, when the Wg domains consist of fewer than a hundred cells; scaffold axon fascicles are not detected. Photoreceptor axons subsequently arrive in the temporal order that follows the posterior to anterior pattern of eye development. The elaboration of wg-lacZ positive scaffold axons is first detected as the first photoreceptor axons arrive in the target field, when only the first one or two columns of ommatidia have initiated differentiation in the eye disc. As retinal innervation continues, the number of glia in the neuropile regions increases
steadily. Thus, Wingless-positive cells, though present long before the arrival of photoreceptor axons in the brain, appear to first extend axons when retinal axons begin to arrive in the brain (Dearborn, 2004).
The wg-lacZ labeled-axons also were examined in pupal stage
animals, where mature axon projections into the optic lobe neuropiles can be resolved. wg-lacZ positive axons can still be detected extending from cell bodies located at dorsal and ventral cortical positions. The axons project, respectively, to dorsal and ventral targets in the medulla and lobula neuropiles. It is
evident that the wg-lacZ-positive neurons include intrinsic neurons of the proximal medulla (Pm neurons), which extend arbors tangentially within small regions of the proximal medulla. Projections into a specific tangential layer of the lobula
were also observed. Projections into the lamina neuropile were not observed;
at the third instar stage the extensions terminate at the border of the
developing lamina neuropile. These axons thus may not have become part of
lamina circuitry, or alternatively have ceased wg-lacZ expression at the pupal stages examined (Dearborn, 2004).
Attempts were made to determine the location of progenitors that give rise to the distinct types of migratory glia and the neurons that form their migratory pathways. The Wingless expressing cells of the dorsal and ventral domains are located in areas of complex gene expression controlled by Wingless (Wg) signaling activity. Adjacent
to the Wg domains are non-overlapping cell populations that express the
TGF-ß family member Decapentaplegic (Dpp). Both the Wg- and
Dpp-positive cell populations express the transcription factor Optomotor Blind (Omb). Dachsous (Ds), a Cadherin family member, is expressed in a graded fashion with respect to the Wg
domains. These three genes, though expressed in different patterns, are under the control of Wg activity (Dearborn, 2004 and references therein).
By performing clonal analysis with tissue labeled to provide positional landmarks, it was possible to localize glial progenitors and scaffold neurons to distinct sites of origin. The
FLP/FRT system was used to generate somatic clones that were positively
marked by membrane-bound GFP (UAS-CD8::GFP). Rare
recombination events were induced such that most specimens harbored only one or a few labeled cell clones in the developing optic ganglia, which were examined in late third instar larvae. Clones including lamina marginal and epithelial glia were found to label progenitors located at the dorsal and ventral margins of the outer anlagen. Clones that
included lamina epithelial glia, lamina marginal glia, medulla neuropile glia, inner chiasm glia or lobula neuropile glia were all found within the domain of cells that express Wg, Omb and Ds (Domain I). A majority of these clones contained both labeled scaffold neurons and glia (16/26); clones with only neurons or glia were less frequent. In the majority of specimens in which glia were labeled, the clone extended into multiple domains and included Domain I. With rare exception, these larger clones also contained neurons. Thus, at the time that somatic recombination was induced (mid-second instar, starting 75 hours after egg laying), most progenitor cells retained the potential to produce both neurons and glia. When the specimens were analyzed with respect to the glial types that were labeled, an interesting pattern emerged with regard to the position of labeled progenitor cells in the Domain I region. Labeled progenitors for each of the glial cell types appeared in distinct domains on the proximal-distal axis. For example, the lamina epithelial and marginal glial
progenitors were found in a more lateral position than medulla
neuropile glial progenitors. Inner chiasm glial progenitors were observed in an even more proximal location.
Hence, the progenitor domains for distinct glial types appear to be organized into a proximal distal stack within Domain I (Dearborn, 2004).
Labeled scaffold neurons were most often included in the glial clones, and were found in close proximity to glial progenitors that used the axons as migratory guides. In a minority of cases, small clones were recovered which included only scaffold neurons. These clones were contained within Domain I (Wg, Omb, Ds expression) in all but two of nineteen cases. These data do not resolve when the glial and neuronal lineages diverge. However, they do permit the conclusion that glia and the neurons that appear to establish their migratory pathways are generated in close proximity and with a lineage relationship (Dearborn, 2004).
The migration of glia from the prospective dorsal and ventral margins of the developing optic lobe depends on the arrival of photoreceptor axons in the
target field. When photoreceptor axons are absent, as occurs in mutants
that eliminate ommatidial development (sine oculis, eyes absent and eyeless) most glia remain stalled in their progenitor domains.
When photoreceptor axons innervate only part of the lamina field, glia migrate to the region that receives retinal innervation. It has thus been supposed that photoreceptor axons attract glia into the lamina target field. It was thus
thought it might be informative to examine the glial migratory scaffold under conditions where glial migration did not occur (Dearborn, 2004).
To this end, the axon scaffold was examined in sine
oculis1 (so) and EyelessD
(ey) animals. These mutants display variable penetrance, such that
photoreceptor neurons can be completely absent, or develop in variably sized clusters in a particular region of the developing retina. A lack of retinal innervation has compound effects on optic lobe development. Lamina neurons fail to develop because of the absence of axon-borne signals. The medulla is greatly reduced in cell number by extensive apoptosis. Employing mosaic analysis, Fischbach and Technau
(1984) showed that so1 acts in the eye to bring about these effects on the brain (Dearborn, 2004).
In the current analysis, the scaffold axons were labeled by the expression of wg-lacZ. When
photoreceptor axons were absent, the scaffold axons were likewise missing. However, wg-lacZ positive cells seemed to be present in normal number in the Wg domains, and expressed the neuronal HRP antigens.
In prior work, a number of markers expressed in the vicinity of the Wg domains were expressed normally in the absence of retinal innervation (e.g., Dpp and Omb). Therefore, it is thought unlikely that retinal innervation is required for the differentiation of these optic lobe neurons. When the scaffold axons were absent in so1 and eyD animals, glia accumulated at the edges of the Wg domains near the point where they would
have joined axon fascicles on paths toward neuropile destinations.
Most so1 and eyD animals develop part
of an eye, such that the corresponding optic lobe receives partial
innervation. In these cases, photoreceptor axons project to appropriate
retinotopic locations despite the absence of the usual array of neighboring axons. In such specimens, scaffold axons were found only in parts of the brain that received retinal innervation and not in regions that completely lacked innervation. A correlation between
retinal innervation, scaffold axon extension and glial migration was found in each of 32 animals with partial eye development
restricted to either dorsal or ventral regions. These observations thus argue strongly that scaffold axon outgrowth and glial migration depend on retinal innervation (Dearborn, 2004).
Retinal innervation might elicit scaffold axon outgrowth, thereby
establishing a necessary pathway for glial migration. Conversely, glial
migration, elicited by retinal innervation directly, might establish a
necessary pathway for scaffold axon outgrowth. Two approaches were undertaken in an attempt to resolve this issue. In the first, scaffold axons were eliminated by neuronal expression of activated Ras1 (Ras1N17) in
order to determine whether glial migration would occur in their absence. In the second, mutant animals with misdirected scaffold axons were examined in order to determine whether migratory glia follow the aberrant axon projections. Both approaches indicated that scaffold axons are necessary as glial migratory guides (Dearborn, 2004).
Prior work has revealed extensive apoptosis in the optic lobes of 'eyeless' mutants of Drosophila. In the mutant sine oculis (so), mosaic analysis has revealed that extensive cell death in the optic lobe is due to the lack of so function in the retina and not the brain. These and additional observations have led to the conclusion that the optic lobe phenotype of sine oculis is due to lack of retinal innervation. The 'trophic' function of photoreceptor axons could be direct, via provision of a survival factor, or indirect, e.g., by eliciting the migration of glia that provide a survival factor (Dearborn, 2004).
To address the issue, wild-type and so1 animals were
examined for the onset of apoptosis by their expression of activated caspase,
which can be monitored in Drosophila with an antibody against the
activated human caspase 3 protein. In third instar stage wild-type animals, few
cortical cells are labeled by the anti-Caspase labeling. By contrast,
so1 third instar larval optic lobes display an increased
number of caspase 3-positive cells throughout the medulla cortex. Putative apoptotic cells were concentrated in regions where glia were particularly few or absent. Caspase 3-positive cells were also particularly prevalent in cortical areas immediately adjacent to the neuropile. Areas particularly deficient in glia also displayed an irregular neuropile structure. These observations were subjected to a quantitative analysis. Areas of 50 µm2 in 1.3
µm confocal optical sections of the medulla cortex were counted for the number of caspase 3-positive cells. In wild-type animals, an average of 0.6 activated caspase-positive cells were found per 50
µm2 area. No regional differences in the density of apoptotic cells were observed in wild-type animals. so1 specimens displayed an average of 5.5 apoptotic cells in 50
µm2 areas adjacent to medulla neuropile regions that lacked medulla neuropile glia. Elevated apoptosis, an average of 6.5 activated caspase-positive cells, was also observed in distal
cortical cell populations whose axons would normally innervate glia-starved regions of neuropile. By contrast, cell populations adjacent to glia-rich regions of medulla in the same so1 animals showed only 2.4 apoptotic cells per 50 µm2, while in
the corresponding distal cortical cell populations that innervate these
glia-rich regions, only 2.5 apoptotic cells per 50 µm2 were found. Thus, although so1 animals displayed an overall increase in caspase-positive cells, the frequency was significantly greater in cell populations that innervated glia-starved regions of neuropile. The results of this analysis are statistically significant: for medulla neuropile proximal regions within so1 animals, a paired t-test analysis yielded a P-value of 1.3e-06 (comparing glia-poor and glia-rich regions), while the same statistical analysis comparing cortical regions yielded a P value of 4.8e-07. These observations suggest that both local and long-range trophic cues are provided by glia to cortical neurons. Additional studies show that the
absence of glia, rather than photoreceptor axons, appears the more likely
cause of extensive apoptosis and neural cell loss observed in eyeless strains of Drosophila (Dearborn, 2004).
It is concluded that Drosophila optic lobe glia use axon
fascicles as migratory guides and that the extension of these axon fascicles is induced by the ingrowth of photoreceptor axons from the developing retina. The migratory
scaffold axons emerge from optic lobe regions that are in close proximity to sites where glial cells originate; both arise in the dorsal and ventral domains where cells express the morphogen Wingless.
When the scaffold axons were eliminated by the autonomous expression of an activated Ras transgene, glia failed to migrate and stalled at the borders of their progenitor sites. Extensive cortical cell apoptosis ensued. When the scaffold axons projected aberrantly (in animals mutant for the cadherin Dachsous), glia followed the
aberrant routes to incorrect destinations. The longstanding observation that glial migration does not occur in eyeless mutant Drosophila might thus be explained by an indirect mechanism in which innervation controls the establishment of an axon scaffold necessary to direct glial migration (Dearborn, 2004).
The migratory scaffold axons were identified by their cytoplasmic
expression of ß-galactosidase from lacZ under the control of a wingless promoter. The neurons are thus residents of the Wg domains, a point additionally supported by labeling small numbers of neurons that projected their axons toward glial destinations. In total, four different wg-lacZ delineated pathways
were identified. These appear to account for all the pathways taken by optic lobe glia that have been identified as migratory by clonal studies. Separate pathways were identified for medulla neuropile glia, lobula neuropile and inner chiasm glia. A single scaffold axon pathway was observed leading to the marginal and epithelial glial layers of the lamina, suggesting that both of these glial types follow the same pathway. Perhaps these glia become separated only on the interposition of photoreceptor R1-R6 growth cones as they arrive in the lamina. Whether the epithelial and marginal glia arise from distinct precursors that migrate on the same pathway is unclear. In all cases, glia
were observed to form migratory 'chains' along axonal extensions, resembling a similar organization of migratory glia on retinal axons en route from the optic stalk to the eye field and from midline progenitor sites to destinations in the PNS. In pupal stage animals, the wg-lacZ labeled neurons were observed in
dorsal and ventral cortical locations, sending projections into neuropile
targets consistent with the patterns of glial migration. However, no axons from wg-lacZ positive neurons were observed extending into the lamina neuropile at this stage (Dearborn, 2004).
The optic lobe regions surrounding the Wg domains display complex patterns of gene expression, mainly because of the signaling activity of Wingless. Clonal analysis indicates that all five migratory glial cell types examined arise from these domains. Interestingly, the
sites from which particular glia arise are stacked on the proximal distal axis in a manner that correlates with target destinations in the developing ganglia. Thus, for example, somatic clones that label the medulla neuropile glia are located at a
position that corresponds to the medial/distal position of MNG glia relative to the lamina and lobula glia. Furthermore, on the basis of their expression of wg-lacZ, as well as clonal analysis, the neurons that extend scaffold axons arise in close proximity to the sites of glial origin. Indeed, as somatic clones induced in mid-second instar larval animals often labeled both scaffold neurons and migratory glia, the glia and neurons must share common progenitors. It is curious that all of these distinct cell types express wingless. These is no evidence that axonally transported Wg
functions in optic lobe development, as it does in the development of the
neuromuscular junction. Partial elimination of wg+ activity (by the use of a conditional wgts allele) did not result in a specific defect in glial migration in the optic lobe but other possible functions of Wg were not addressed by this
analysis (Dearborn, 2004).
These observations suggest a developmental mechanism for the control of glial
cell migration that depends on the establishment of an axon scaffold for
guidance of migrating glia. In normal development, a small number of glia migrate into the target field of the photoreceptor axon prior to the arrival of the first
photoreceptor axons.
These glia, which migrate independently of retinal innervation, may serve a necessary early role in photoreceptor axon guidance. They may be targets for the first retinal axons to arrive in the optic lobe, and provide the first signals that differentiate the outgrowth termination points of the R1-R6 and R7/8 axons. It is proposed that the first photoreceptor axons to arrive in
the optic lobe elicit outgrowth of the scaffold axons from neurons at the
dorsal and ventral margins. Subsequent migration of glia from the dorsal and
ventral margin progenitors is then both permitted and directed along the
specific pathways of the scaffold. After the erection of the migratory
scaffold, glial migration may be independent of continued photoreceptor axon ingrowth. On this point, it is noted that glia migrate into the lamina in approximately normal numbers in hh1 animals, in which ommatidial development ceases after 11-13 columns form at the posterior of the developing retina. Therefore, glial migration may not depend on continued
arrival of new retinal axons in the lamina primordium. Nonetheless, this
observation cannot rule out the alternative interpretation that retinal axons
emit a continuous attractive signal for glial migration that functions in
adjunct with the migratory axon scaffold (Dearborn, 2004).
How do photoreceptor axons control the outgrowth of axons from the Wg
domains? This does not appear to be a consequence of an affect of retinal
innervation on neuronal development in the Wg domains, which appear normal in size and organization in eyeless mutant Drosophila strains. Hedgehog,
which is brought into the brain by retinal axons, is not required for the
expression of either Wg or Dpp at the dorsal and ventral margins of the optic lobe. It seems that the induction of scaffold axon outgrowth by the photoreceptor axons may be direct, since the outgrowth occurs in the hedgehog mutant, hh1, in which the first steps of lamina neuronal development fail to occur. Thus, one might suppose that retinal axons emit a chemoattractant for scaffold axon outgrowth, either synthesized in the retina or acquired from environmental sources and redistributed by retinal axons (Dearborn, 2004).
The system for glial migration guidance permits diversified
cell types to originate from a common site, and yet target specific locations in complex neuropiles. One could imagine that as more complex and diversified
neuropiles evolved, relatively simple changes in the developmental pathway of glial progenitors and the projections of scaffold axons would deliver glial support to new structures. This system also has the feature of functioning as a developmental timing 'checkpoint' that fine-tunes the general hormonal coordination of imaginal development. Thus, upon their initial arrival in the
brain, retinal axons provide a fine level of local cellular control over the movement of glia, preparing the target field for the next steps of optic lobe development (Dearborn, 2004).
Migration and proliferation have been mostly explored in culture systems or fixed preparations. A simple genetic model, the chains of glia moving along fly wing nerves, is presented to follow such dynamic processes by time-lapse in the whole animal. Glia undergo extensive cytoskeleton and mitotic apparatus rearrangements during division and migration. Single cell labelling identifies different glia: pioneers with high filopodial, exploratory, activity and, less active followers. In combination with time-lapse, altering this cellular environment by genetic means or cell ablation has allowed the role of specific cell-cell interactions to be defined -- (1) neuron-glia interactions are not necessary for glia motility but do affect the direction of migration; (2) repulsive interactions between glia control the extent of movement; (3) autonomous cues control proliferation (Aigouy, 2004).
The fly wing contains two purely sensory nerves that navigate proximally along the so-called L1 and L3 veins, and are lined by glial cells. L3 vein contains three gliogenic sensory organs, L3.3, L3.1 and L3.v, from distal to proximal. Each sensory organ precursor (SOP) divides asymmetrically several times and produces a glial precursor (GP), which repeatedly divides and is therefore called a first order glial precursor (GPI). The GPI, initially located adjacent to the neuronal soma, subsequently moves along the underlying axon. The final number of glial cells per lineage varies stochastically from four to eight (average=six). The whole glial lineage, from GPIs to post-mitotic glial cells, expresses the Repo glial marker (Aigouy, 2004).
On L3, proliferation starts several hours after GPI birth, at around 17 hours after puparium formation (APF), and by 20-22 hours APF all GPIs have divided once. The second round of division takes place by 24 hours APF, and the third by 30 hours APF, with little glial proliferation being observed afterwards. These data are in agreement with the GPI dividing symmetrically twice, to produce two second order and four third order precursors (GPII and GPIII, respectively. The presence of up to eight glial cells suggests that GPIIIs may or may not divide, and/or that cell death takes place throughout the lineage (Aigouy, 2004).
The observation that glia migrate and proliferate to variable extents suggests a role for extrinsic signals and makes it difficult to follow the development of glial lineages in fixed tissues. Therefore an in vivo glial marker was devised by crossing the repo-Gal4 driver with a UAS-GFP reporter (repo-GFP) (Aigouy, 2004).
When L1 glia are compared with fly border cells, functioning during oogenesis, and tracheal cells, differences and similarities become apparent. Border cells rely on an asymmetric structure, the actin based-LCE (long cellular extension), which triggers movement through a grapple and pull process. By contrast, wing pioneer glial cells send out numerous filopodia that dynamically assay the environment in all directions; this is also seen in tracheal cells. This may reflect the fact that border cells form a cluster and move simultaneously, whereas, in the chain of glia, distal cells do not contact pioneer cells and follow the movement of adjacent, more proximal, cells. Based on time-lapse and ablation data, the following model is proposed. Prior to migration, pioneer cells are free to explore the proximal axonal substrate whereas follower cells are submitted to bilateral repulsive glia-glia contacts. As pioneer cells move proximally, they free space at more distal positions. Follower cells rapidly occupy such space, thus freeing even more distal regions. This domino-type of migration proceeds until homogenous axon enwrapping is reached (Aigouy, 2004).
Most exploratory activity is spatially and temporally restricted as it specifically characterises cells that move on naked axons. Whether the presence of pioneer cells at the front of migration is lineage dependent or reliant on extracellular signals remains to be determined. In the future, performing ablations throughout the L1 will help to address the issue of plasticity. The presence of LCE versus pioneer filopodia may reflect different modalities of directional migration. It is known that border cells respond to chemoattractants, whereas glial directional migration may be driven by underlying axons. Interestingly, L3 migrating and proliferating cells all show filopodia, suggesting yet a different mode of migration compared with that of cell chains (L1 glia) and clusters (border cells). Finally, glia, but not tracheal or border cells, divide as they move, suggesting that the formation of a continuous chain along the axon bundle requires both migration and proliferation. In the future, it will be crucial to determine how the two events are coordinated (Aigouy, 2004).
The different features shown by border cells, trachea and glia suggest that cell specification controls motility strategies. The role of cell specification cues is further demonstrated by the fact that, even within glial cells, different lineages display distinct features. While wing GPs divide several times, glia arising from dorsal bipolar dendritic embryonic lineages and microchaete glia do not divide, the latter dying soon after birth. Understanding the molecular pathways specifying migratory and proliferative profiles represents one of the future challenges for developmental biologists (Aigouy, 2004).
Like oligodendrocyte precursors in the rat optic nerve that are controlled by an internal clock, GPIs divide a limited number of times. Different sets of data speak in favour of an internal clock that limits the absolute number of divisions to three. (1) The average number of glia derived by different sensory organs is constant, irrespective of the number of underlying axons (L3.3 and L3.1 glia line one and four axons, respectively). Thus, the number of axons does not control proliferation. (2) Time-lapse data show that divisions within and between lineages are rather synchronous. (3) Mitotic clones lacking the Gcm (Glial cell missing) glial promoting factor in one gliogenic sensory organ result in fewer wing glia, indicating that the remaining cells do not compensate for the missing ones. (4) Ablation data demonstrate a lack of compensatory divisions within and among glial lineages. Whether vertebrate and invertebrate clocks rely on the same signals will be a matter of future studies. In addition to the internal clock, extracellular signals may be at work and may control the fine-tuning of proliferation (four to eight cells per lineage). A role of cell-cell interactions in the control of glial proliferation has also been observed in the fly embryo (Aigouy, 2004).
One of the most peculiar features of glia is that they tend to form a chain of cells. This might suggest that glial cells display affinity for axons as well as for other glial cells, the equilibrium between these affinities dictating the extent of migration and triggering the formation of a continuous glial sheath. However, even in the absence of axons (Notchts1 data), glial cells form a continuous chain, rather than staying as a cluster or moving apart from one another. Thus, axons do not trigger glial cell alignment, clearly showing that glia are endowed with an intrinsic migratory potential. The chain of glia present in Notchts1 wings is unbranched, as if the surrounding vein were providing a physical channel or instructive cues for migration. Although a participation of veins in axonal navigation and/or glia migration cannot be excluded, veinless wings still contain properly organised axons and glia. Moreover, ectopic axons present in the intervein space of hairy-wing wings carry properly lined glial cells, thus indicating that veins are not instructive for glial migration (Aigouy, 2004).
One way to reconcile all data is that different types of interactions take place. In one case, glia tend to fully occupy and enwrap naked axons, probably in response to neuronal signals. In contrast, counteracting interactions are at work between glia. Thus, while affinity between glial cells induces them to stay together, repulsive contacts prevent them from forming a cluster and trigger the formation of a chain. The observation that the cytoplasmic processes of adjacent glia largely overlap is in agreement with this hypothesis, and leads to the proposition that glial cells tend to reduce the extent of contact by sliding over each other. The equilibrium between all these forces allows the formation of a continuous chain and controls the extent of movement, compatibly with the substrate available for migration (Aigouy, 2004).
Glial cells move in a stereotyped direction and, as shown by the ablation data, do not require the presence of guide-post glia to find their way. Instead, neuron-glia interactions affect directional migration. Indeed, both in fly wings and in the zebrafish lateral line, misrouted axons result in redirected glia. Furthermore, in both systems, glial arrest is observed upon axonal arrest. The fact that glia use axons as a substrate suggests an unpredicted axonal feature. Indeed, although the polarised nature of the axon is well characterised with respect to microtubule growth, how does the axon convey directional information to the enwrapping glia?
The data clearly show that glial migration relies on complex and dynamic glia-glia and neuron-glia interactions. Establishing time-lapse protocols that simultaneously monitor neurons and glia, or aiming at simultaneously identifying the whole glial population and a subset of glia, will be crucial to gaining a better understanding of the precise nature and role of such homo- and hetero-typic interactions (Aigouy, 2004).
Most migratory cells undergo an epithelial to mesenchymal transition that implies changes in cell polarity. Similarly, glial cells originate through the apico-basal division of the IIb precursor. The newly formed GPIs wait almost ten hours before dividing, whereas GPIIs divide and migrate rapidly soon after birth. By the end of this latency period, the GPI acquires a very polarised shape, and divides along the proximodistal axis. Altogether, these results indicate that a change in cell polarity occurs in the GPI, the cell that starts migrating. Thus, latency probably serves to build up the GPI competence for proliferation and migration (Aigouy, 2004).
The fact that glial cells divide and migrate along the same axis suggests that the signalling pathways controlling cell polarity, division and motility are coordinated. The analysis of mutations affecting these processes will be fundamental for understanding the molecular bases of their integration (Aigouy, 2004).
The Drosophila CNS midline glia (MG) are multifunctional cells that ensheath and provide trophic support to commissural axons, and direct embryonic development by employing a variety of signaling molecules. These glia consist of two functionally distinct populations: the anterior MG (AMG) and posterior MG (PMG). Only the AMG ensheath axon commissures, whereas the function of the non-ensheathing PMG is unknown. The Drosophila MG have proven to be an excellent system for studying glial proliferation, cell fate, apoptosis, and axon-glial interactions. However, insight into how AMG migrate and acquire their specific positions within the axon-glial scaffold has been lacking. This study used time-lapse imaging, single-cell analysis, and embryo staining to comprehensively describe the proliferation, migration, and apoptosis of the Drosophila MG. Three groups of MG were identified that differed in the trajectories of their initial inward migration: AMG that migrate inward and to the anterior before undergoing apoptosis, AMG that migrate inward and to the posterior to ensheath commissural axons, and PMG that migrate inward and to the anterior to contact the commissural axons before undergoing apoptosis. In a second phase of their migration, the surviving AMG stereotypically migrated posteriorly to specific positions surrounding the commissures, and their final position was correlated with their location prior to migration. Most noteworthy are AMG that migrated between the commissures from a ventral to a dorsal position. Single-cell analysis indicated that individual AMG possessed wide-ranging and elaborate membrane extensions that partially ensheathed both commissures. These results provide a strong foundation for future genetic experiments to identify mutants affecting MG development, particularly in guidance cues that may direct migration. Drosophila MG are homologous in structure and function to the glial-like cells that populate the vertebrate CNS floorplate, and study of Drosophila MG will provide useful insights into floorplate development and function (Wheeler, 2012).
While cellular analysis of Drosophila MG development has been studied for greater than 20 years, novel insights were gained in this study through the use of multiple advanced imaging approaches. The most important was the use of time-lapse imaging. This allowed tracking of each MG cell between stages 12 and 17, and revealed their stereotyped migration. Development at earlier times (stages 11 to 12) was assessed by analysis of sim-Gal4 UAS-tau-GFP embryos stained for MG markers. Finally, the visualization of individual MG using argos-G1.1-Gal4 UAS-tau-GFP embryos allowed the extensive cytoplasm/membrane extensions of each AMG to be determined. In addition, the analyses of MG in sagittal view allow for a simplified and more accurate reconstruction of MG migration compared to traditional ventral views. Combined, these approaches resulted in a comprehensive cellular view of MG development that will be the basis for future genetic analyses (Wheeler, 2012).
Using multiple cell markers and confocal analysis, it was possible to accurately count the number of AMG and PMG throughout development. Overall, the numbers agree well with. There are ~8 sim+ mesectodermal cells/segment at embryonic stage 7. During stage 8, these cells synchronously divide during the mitotic cycle 14 division to give rise to ~16 cells/segment. It is hypothesized that these 16 cells are initially committed to become neural precursors, and subsequent Notch signaling acts via lateral inhibition to partition these cells into a population of neural precursors and MG. This process results in 8.8 MG/segment (5.4 AMG and 3.4 PMG) at stage 11. The number of AMG and PMG remains constant through stages 12/5 and 12/3 but between stages 12/3 and 12/0 there is an increase of 2.1 AMG/segment to 7.5, suggesting two divisions on average from existing AMG. Consistent with this view is the presence of anti-phospho-histone-H3 (PH3) positive tau-dense AMG at stage 12/0. However, it is worth noting that AMG number (and presumably divisions) can vary significantly between segments. Thus, mechanistically, AMG can be generated in two ways. First, lateral inhibition mediated by Notch signaling leads to the formation of 5.4 AMG/segment during stages 10 and 11; a recruitment step that likely occurs without cell division. Second, divisions of existing AMG occur at stage 12/0 to generate a total of 7.5 AMG/segment (Wheeler, 2012).
Both ecdysone and vein (vn) signaling may play roles in AMG cell division. Steroids are known to influence glial proliferation and apoptosis in vertebrates. Genetic and in vitro studies also indicate that high levels of ecdysteroids can inhibit proliferation and promote apoptosis of Drosophila embryonic and postembryonic MG. These results suggest that AMG have a tendency to divide, but high levels of ecdysone inhibit division. In the embryo, there is a prominent pulse of ecdysone that peaks at 8 h of development (stage 12) and gradually decreases until hatching. Thus, the presence of high levels of ecdysone may prevent AMG proliferation during late embryogenesis, but may not increase quickly enough to prevent AMG from undergoing division during stage 12. Consistent with this view, mutants of genes involved in ecdysone production (e.g. disembodied, shadow, spook) result in additional embryonic MG. While the presence of ecdysone may inhibit MG proliferation, vn signaling may be required for MG divisions. The vn gene encodes a secreted ligand that binds to Egfr and is a homolog of vertebrate neuregulin, which promotes glial division. vn was previously hypothesized to control AMG division at stage 12, since vn mutant embryos showed a reduction of 2.6 AMG/segment. This is similar to the average increase in AMG due to cell division (2.1 AMG/segment). If vn is required for AMG division throughout development, then division should be absent (regardless of ecdysone levels) when vn expression is absent. This is indeed the case as vn is expressed in the midline and surrounding cells from stages 10 to 12, but is restricted to only a small subset of lateral CNS neuroblasts and neurons after AMG divisions. Taken together, this suggests that AMG proliferation may depend on a balance between positive (vn) and negative (ecdysone) signaling (Wheeler, 2012).
AMG number peaks at stage 12/0 (7.5/segment) and declines by apoptosis to 3.3 AMG/segment by stage 17. This reduction in number has been well-studied and a model proposed as follows (Bergmann et al., 2002). AMG begin to express a pro-apoptotic gene, head involution defective (hid) beginning at stage 13. The axon commissures secrete Spitz (Spi), which is an EGF-like protein. If sufficient Spi protein binds to Egfr located on the surface of AMG, MAP kinase is activated,which then phosphorylates and inactivates Hid, thereby blocking apoptosis. Thus, it is predicted that AMG in close proximity to the axon commissures will receive sufficient Spi to survive, and those not in close contact will undergo apoptosis (Wheeler, 2012).
Time-lapse imaging provided additional insight into AMG apoptosis. This process often occurred in a two-step manner. In segments with a large number of AMG (~10) there was a rapid wave of apoptosis that yielded ~5 AMG. Further reduction of AMG to ~3 cells occurred incrementally. In segments with a smaller number of AMG (~7), all AMG underwent apoptosis incrementally with no detectable apoptotic wave. Generally, the AMG that underwent apoptosis in initial waves were those at the anterior margin of the segment, furthest from the axon commissures. This is consistent with those cells receiving less axonal Spi. While all AMG cell death is likely due to inadequate Spi signal, time-lapse imaging experiments revealed instances in which AMG in close proximity to the commissures died, were cleared, and another AMG quickly assumed its position. Thus, even cells in apparent contact with axon commissures may not receive sufficient Spi for survival, suggesting additional complexity in the regulation of AMG apoptosis (Wheeler, 2012).
Previously it was argued that there might be a positive feedback loop in which Spi signaling from axons results in increased adhesion between AMG and axons. MG and axons adhere to each other via the heterophilic adhesion membrane proteins, Wrapper (on MG) and Neurexin IV (Nrx-IV) (on axons). Ensheathment and membrane projection into axons by AMG requires wrapper function. Expression of a wrapper enhancer is upregulated when Spi is provided ectopically. This suggests that Spi could promote stronger adhesion and more elaborate contacts between AMG and axons via increased Wrapper levels. Increased adhesion and surface area contact could expose AMG to higher concentrations of Spi making survival even more likely. In this way, AMG would compete for axonal interactions, with advantage going to those with more elaborate contacts (Wheeler, 2012).
There are ~4 PMG/segment at stage 12, and no evidence was found that PMG divide after their initial recruitment and specification by Notch and Hedgehog signaling. PMG number gradually declines to zero from stage 12/5 to stage 17. The PMG furthest from the PC die first. This suggests that the PMG adjacent to the PC may transiently receive a survival signal, although they too die between stages 16 and 17. It is generally thought that: (1) PMG do not share a large surface area with commissural axons (perhaps, in part due to low levels of Wrapper), (2) their survival cannot be rescued by high levels of Spi, and (3) their apoptosis is promoted by a different combination of proapoptotic genes than AMG. However, a detailed mechanistic analysis of PMG cell death has not been carried out, but is now feasible (Wheeler, 2012).
By combining staining of early embryos with time-lapse imaging of older embryos, it was possible to define in great detail the pattern of MG migration from stages 10 to 17. The AMG at stage 11 initially are present along the anterior/posterior axis in the anterior half of the segment and lie beneath (more external to) midline neurons and their precursors. The AMG nuclei begin to delaminate and migrate inward. Some AMG migrate internally and orient in a posterior direction toward the developing axon commissure. One possibility is that AMG initiate migration in response to and toward developing commissural axons or the MP1 neurons that lie just beneath the developing commissure. If axons provide guidance cues, migration may be due to an axon-derived secreted morphogen. If so, it is unlikely to be Spi, since in embryos in which both Egfr signaling is abolished and apoptosis inhibited, MG migration appears relatively normal. Another possibility is the PDGF- and VEGF-related factors (Pvfs) that may be secreted by axons or midline neurons and activates the PDGF and VEGF-related receptor (Pvr). Alternatively, MG could survey axonal membrane proteins at a distance using gliopodia, which are filopodia-like membrane extensions that act like growth cone filopodia. PMG also migrate inwards toward the developing axons and may be responding to similar guidance cues as AMG (Wheeler, 2012).
Not all AMG orient toward the developing axons. The anterior-most AMG elongate inward, but tend to orient toward the adjacent anterior segment before undergoing apoptosis. This suggests that they are either blocked from responding to the normal AMG guidance cues, possibly by other AMG, or are intrinsically different from the AMG that migrate in the posterior direction. Regarding intrinsic differences, most AMG-expressed genes analyzed at stages 11 and 12 are expressed in all AMG, and not in AMG subsets, indicating that obvious subsets of AMG are not apparent. One exception is the medial late-differentiating AMG present at stage 11 that are runt minus. However, after stage 11, these AMG are indistinguishable from other AMG, and it is uncertain whether their migratory paths are distinct. Consequently, it remains possible that different AMG subpopulations exist with intrinsic migratory differences, but there is currently little evidence to support this view (Wheeler, 2012).
Time-lapse imaging has provided new and definitive information regarding how individual MG acquire their positions surrounding the axon scaffold. One of the key observations was that the final migratory paths and positions of AMG correlated with their location at stage 12. AMG do not move randomly, they instead migrate in specific ways around the axon commissures to take their final positions. This suggests that AMG at different locations respond to specific, yet different, cues that guide them to their final positions. In addition, some mechanism must ensure that AMG cease their migration. The signals for both guidance and the termination of migrations might include a combination of AMGñAMG interactions, as well as interactions (either diffusible or contact-mediated) between AMG and different neurons, axons, or other substrata. As demonstrated by single-cell analysis, each AMG that ensheaths the commissures has extensive membrane projections that, in principle, can interact with other AMG and potential substrates. In both Nrx-IV and wrapper mutants, MG fail to adhere to the commissures and nearby neuronal cell bodies. However, AMG still migrate inward, migrate posteriorly to surround the AC, and maintain adhesion among themselves. This suggests that different adhesive interactions are required for different aspects of AMG migration (Wheeler, 2012).
The inward migration of MG suggests that there are 3 distinct groups of MG that may undergo collective guidance (AMG that migrate inward and anterior, AMG that migrate inward and posterior, and PMG that migrate inward and anterior). While each MG within these groups likely adheres to other group members, it is unknown whether the groups collectively signal to each other and whether they respond collectively or individually. However, after their initial inward migration, surviving AMG and PMG migrate to characteristic positions, indicating a transition to more individual behavior. Nevertheless, the surviving AMG remain in close contact with each other. Most importantly, this study provides the background for genetic screens that will identify and functionally analyze genes involved in AMG migration and commissure ensheathment (Wheeler, 2012).
Through the use of time-lapse and stained embryo imaging, the data presented in this paper provide a definitive descriptive view of MG migration. These results are consistent with and greatly extend earlier studies on MG development and migration. However, previous workers proposed detailed models regarding MG specification and migration, and, at this point, it is worth comparing our current view of MG development to earlier versions (Wheeler, 2012).
One influential and detailed model has a number of features: (1) there are 3 mature pairs of MG that ensheath the commissures, and are named MGA (anterior), MGM (middle), and MGP (posterior) based on their positions along the anterior/posterior axis, (2) these MG are derived from 3 discrete MG precursors that form prior to stage 8 and divide once yielding the 6 mature MG, (3) MGP migrate in an anterior direction into the next segment where they take up residence as the most posterior MG, (4) the MGM migrate above the AC and send projections that follow along the axons of the VUM neurons (medial tract), extending between the developing AC and PC, thus separating the commissures, and (5) the MGA migrate only a short distance, residing beneath the AC and PC. Important refinements of this model proposed later by other workers established that generally ~10 MG were generated, most underwent apoptosis, and that a group of posterior-residing MG underwent apoptosis and did not contribute to the ensheathment of the commissures. These posterior glia are equivalent to PMG in the current model (Wheeler, 2012).
With respect to the position, identity, and migration of the MGA,MGM, and MGP precursors and their progeny, previous studies employed lineage tracing of enhancer trap reporters reportedly from stage 16 to stage 8. However, since expression of the MG enhancer trap lines used does not begin expression until stage 12, tracing the origins of these cells back to stage 8 could not be directly determined, and was, presumably, conjectured. Subsequent work has demonstrated that MG do not form until at least stage 10 via Notch signaling, and that Hedgehog signaling at the same stage specifies AMG and PMG cell fates. Thus, the identities and positions of specific subtypes of MG cannot be identified before stage 10. Additionally, in previous studies no molecular markers were described that allowed MGA and MGM to be distinguished, so the proposed migration pathways that each of these MG took to reach their final destinations could not have been derived from stained samples (Wheeler, 2012).
More complicated are MGP and their relationship to PMG. In some respects, these cells appear equivalent. However, all PMG undergo apoptosis, and thus are not equivalent to MGP, which were initially proposed to survive and ensheath the PC. It is also noted that the PMG delaminate, migrate, and die within the same segment. No evidence was seen that they migrate from a more posterior segment (as proposed for MGP). It is possible that, in the absence of specific AMG and PMG markers, inward migrating AMG that orient toward the anterior were mistaken for migrating MGP (Wheeler, 2012).
Finally, regarding the issue of commissure separation, the previous model proposed that MGM tracked on the medial axon tract between the commissures, resulting in their separation. The possibility that MG may track along the medial tract to separate the commissures is an interesting idea, but has not been critically tested. This study found, from single-cell analysis, that both DM region and VM region AMG extend membrane/cytoplasm between the AC and PC, indicating that more than one AMG are usually positioned between the commissures. Together, these two cells ensure that the two commissures remain distinct and separate (Wheeler, 2012).
Glia are of vital importance for all complex nervous system. One of the many functions of glia is to insulate and provide trophic and metabolic support to axons. Using glial-specific RNAi knockdown in Drosophila, this study silenced 6930 conserved genes in adult flies to identify essential genes and pathways. Among the screening hits, metabolic processes were highly represented, and genes involved in carbohydrate and lipid metabolic pathways appeared to be essential in glia. One critical pathway identified was de novo ceramide synthesis. Glial knockdown of lace, a subunit of the serine palmitoyltransferase associated with hereditary sensory and autonomic neuropathies in humans, resulted in ensheathment defects of peripheral nerves in Drosophila. A genetic dissection study combined with shotgun high-resolution mass spectrometry of lipids showed that levels of ceramide phosphoethanolamine are crucial for axonal ensheathment by glia. A detailed morphological and functional analysis demonstrated that the depletion of ceramide phosphoethanolamine resulted in axonal defasciculation, slowed spike propagation, and failure of wrapping glia to enwrap peripheral axons. Supplementing sphingosine into the diet rescued the neuropathy in flies. Thus, this RNAi study in Drosophila identifies a key role of ceramide phosphoethanolamine in wrapping of axons by glia (Ghosh, 2013).
Serine palmitoyltransferase catalyzes the condensation of serine and palmitoyl-CoA to generate 3-ketosphinganine, the rate-limiting step in de novo sphingolipid synthesis. Mutations in the two human subunits of the serine palmitoyltransferase are associated with hereditary sensory and autonomic neuropathy. A common feature of the mutations is the loss of canonical enzyme activity and the generation of toxic lipid intermediates. Glial inhibition of lace function (repo>mCD8-GFP/lace RNAi) resulted in glial bulging and in an alteration of the axonal packing in all eight pairs of abdominal nerves in all larval PNS examined. The bulging of glia was localized to focal regions, but appeared randomly along the entire peripheral nerves with diameters ranging from 10 μm to 30 μm. Nerves of repo-GAL4/+ control flies were straight and packed in bundles with a uniform diameter of 5-8 μm. In contrast, knockdown of lace in neurons did not result in any visible alterations of axonal morphology . The average cross-section area of the nerve were similar in the knockdown (elav-GAL4/lace RNAi) and in the elav-GAL4/+ control flies (Ghosh, 2013).
Notably, the ensheathment defect was not due to a compromised blood-nerve-barrier as has for example been observed in null fray mutants. In addition, the number of glial cells in the peripheral nerves was comparable to control. It is important to note glial cell death affects neuronal survival and results in embryonic lethality. The absence of embryonic lethality and the comparable glial cell number suggested that glial cell death did not occur at the larval stage after knockdown of lace. The expression of lace in glia was confirmed by double immunolabeling of lace5(LacZ enhancer trap line) with anti-β-galactosidase and anti-repo in L3 larval peripheral nerves. In addition, by RT-PCR analysis of the fly brain and PNS lace transcript was identified in the nervous system of both male and female flies (Ghosh, 2013).
Two independent RNAi lines (Transformant ID 21803 and 110181, VDRC) against lace showed identical swelling and wrapping defects. In addition, hypomorphic lace mutant (lace2/lace5) resulted in axonal defasciculation, ruling out off-target effects of the RNAi lines. As in the lace-RNAi knockdown, the average cross-section area of the nerves was increased in the hypomorphic lace mutant animals. The mutant phenotypes appeared to be subtle, which is not surprising as complete loss of lace during the development is lethal, while this hypomorphic combination are viable even into adulthood. However, 100% penetrance of the phenotype was observed both for the RNAi knockdownand the hypomorphic mutants. Importantly, the lace mutant phenotype was rescued by expressing UAS-lace specifically in the glial cells (repo-GAL4), pointing to an essential function of lace in glia (Ghosh, 2013).
Next, an analysis was performed to see in which of the different glial subtype lace was required. Glia subtype specific GAL4 drivers were used to silence lace function. A phenotype was only observed when lace was depleted in wrapping glia (Nrv2-GAL4). Quantification of GFP signal intensity revealed that membrane area was significantly reduced as compared to control (Nrv2>mCD8GFP/lace RNAi versus Nrv2>mCD8GFP). Similar results were observed when the Nrv2>mCD8-mcherry driver line was used to knockdown lace in the wrapping glia. In contrast, knockdown of lace in the two other glial subtypes, the subperineurial (gliotactin-GAL4) and the perineurial (NP6293-GAL4), did not lead to any visible changes (glial swellings or decrease in the GFP signal intensity) in glia or in axons (-G), suggesting a predominant role of lace in the encapsulation of peripheral nerves (Ghosh, 2013).
To examine the ultrastructure in more detail electron microscopy was performed. Electron micrographs clearly showed that knockdown of lace in glia (repo/lace RNAi) severely impaired axonal enwrapping compared to control (repo/+). Notably, also in the non-swollen regions (A2-A3 segment) of the nerve much less glial processes covered the axons. Quantification demonstrated a significant increase in the number of completely unwrapped axons in this region. A similar phenotype was observed when lace was knocked down in wrapping glia (Nrv2/lace RNAi). Again, a clear increase in the completely unwrapped axons was detected as compared to the controls. Importantly, TUNEL assay could not detect any apoptotic glial nuclei (Nrv2>laceRNAi) suggesting that loss of axonal ensheathment is not because of dying wrapping glial cells. Together, these results indicate that sphingolipids or intermediates of the sphingolipid pathway are necessary for membrane expansion of wrapping glia (Ghosh, 2013).
In order to search for the specific sphingolipid (SL) species required by the wrapping glia to mediate axonal ensheathment, a genetic dissection study was performed by expressing RNAi against all known SL metabolic enzymes selectively in glia. Out of 12 genes, knockdown of Spt-I, schlank, Des1 and Pect in glia (repo>mCD8-GFP/RNAi) with two different RNAi lines (except Des1 due to unavailability) phenocopied the glial swelling and axonal defasciculation as observed upon loss of lace function. Interestingly, all four genes that show 100% penetrance are known to be involved in the biosynthesis of ceramide-phosphoethanolamine (CerPE). The specificity of the effect was demonstrated by the absence of any visible phenotype after neuronal specific knockdown of Spt-I, schlank, Des1 and Pect. Additionally, glial specific knockdown of different ceramide derivative synthesizing enzymes (GlcT1, CGT, CerK) and PE synthesizing enzyme (bbc) did not show any visible defects of axon or glial morphology. Moreover, when Spt-I, schlank, Des1 or Pect were knocked down specifically in wrapping glia, wrapping defects similar to the lace phenotype were observed . The quantification revealed that the GFP signal intensity was significantly reduced in all four experiments as observed after lace knockdown. Ultrastructural analysis by transmission electron microscopy also showed that wrapping glia failed to extend their membrane around the axons; and consequently there was an increase of the completely unwrapped axons. Hence, the data strongly suggests an essential function of glial CerPE in axonal ensheathment by wrapping glia (Ghosh, 2013).
In order to analyze whether knockdown of lace, schlank, Des1 and Pect resulted in depletion of CerPE levels, a detailed lipidomics analysis of the nervous system was performed. This is particularly important in RNAi studies targeting enzymes, because residual enzyme activity due to inefficient RNAi-silencing is often sufficient for their function. Since the nervous system of Drosophila only contains 10% of glia, the RNAi was expressed both in neurons and glia using repo-GAL4 and elav-GAL4 drivers to deplete the enzymes in the entire nervous system. L3 larval brain and peripheral nerves were dissected and lipidomics analysis was performed with high-resolution shotgun mass spectrometry. Importantly, lipidomics analysis confirmed that knockdown of lace, schlank, Des1 and Pect reduced CerPE levels significantly, whereas triacylglycerol (TAG) and diacylglycerol (DAG) and sterol levels were unaltered. Ceramide levels were reduced upon downregulation of lace and Des1, whereas knockdown of Pect lead to increased ceramide levels consistent with its function as a phosphoethanolamine cytidylyltransferase. Phosphatidylcholines (PC) and Phosphatidylethanolamine (PE) levels were slightly changed possibly due to compensatory mechanisms (Ghosh, 2013).
Next, tests were performed to see whether it was possible to rescue the morphological phenotype induced by knockdown of lace in glia by supplementing sphingosine (re-converted to ceramide by condensation with a fatty-acylCoA catalyzed by the various ceramide synthases) into the diet of the flies. Indeed, the phenotype of glia-specific knockdown of lace was efficiently rescued by the exogenous addition of sphingosine (300 μM) to the food. Double Immunolabelling of glia and neuronal membrane reveals that the glial bulging and axonal unpacking was rescued upon addition of sphingosine to the diet. Orthogonal projections and the quantification demonstrated the rescue of the neuropathy like phenotype in flies. It was furthermore observed with the ultrastructural analysis that the glial enwrapment defect was recovered upon sphingosine addition to the diet. Quantitative analysis of the peripheral nerves using the confocal and electron microscopy showed that the oral administration of sphingosine can restore the enwrapping defect and the neuropathy-like phenotype (Ghosh, 2013).
Sphingolipids have both structural and signalling functions in cells. CerPE is a relatively low abundant lipid constituting only around 1% of the total fly lipidome. Interestingly, CerPE appears to be enriched in the fly brain membrane lipidome (4%). In mammals, CerPE is only found in trace amounts, since sphingolipids are in general built on ceramide phosphatidylcholine in higher organisms. There are different possibilities of how CerPE could exert its function in glia. CerPE might be required for signal transduction pathways that control membrane synthesis in wrapping glia. Recently, a mutation in egghead, an enzyme that extends the glycosphingolipids (GSLs) in flies, causes the proliferation and overgrowth of subperineurial glia mediated by aberrant activation of phosphatidylinositol 3-kinase-Akt pathway. CerPE may also increase the packing density of the lipids in the membrane, thereby helping to build up an efficient barrier for the electrical insulation of the axons. In vertebrates, a related sphingolipid, galactocylceramide, is critical for the formation of an insulating myelin sheath in oligodendrocytes. Galactosylceramide and/or its sulphated form are required for the tight sealing of the glial paranodal membrane to the axon. Interestingly, mice lacking ceramide synthase 2, a vertebrate homolog of schlank, have myelination defects (Ghosh, 2013).
Alterations of enzyme function or enzyme deficiencies do not only result in a reduction in the amount of an essential product, but can also lead to the accumulation of a toxic intermediate, or the production of a toxic side-product. For example, mutations in human serine palmitoyltransferase result in a loss of normal enzyme function causing a shift in the substrate specificity, which increase the accumulation of atypical, toxic lipid products. Thus, gain-of-toxic-function is another possibility of how knockdown of lace may cause the axonal ensheathment defects (Ghosh, 2013).
Interestingly, supplementing sphingosine to the diet restored the ability of wrapping glia to extend their membrane around axons. How diets affect the distribution of lipids in cells and thereby modulate biological processes will be an important question for future investigations. Drosophila is an ideal system to pursue such studies because of the short life span and the powerful genetics, which enable rapid and detailed analysis. In summary, the current study illustrates that a large-scale screen in Drosophila, in combination with concomitant morphological and electrophysiological analysis has the potential to dissect the basic mechanisms of neuron-glia communication. Detailed knowledge of neuron-glia interactions is a pre-requirement for the rational design of treatment strategies for neuropathies or other diseases in the future (Ghosh, 2013).
Midline glia are a source of cues for neuronal navigation and differentiation in the Drosophila CNS. Despite their importance, how glia and neurons communicate during development is not fully understood. This study examined dynamic morphology of midline glia and assessed their direct cellular interactions with neurons within the embryonic CNS. Midline glia extend filopodia-like 'gliopodia' from the onset of axogenesis through the near completion of embryonic neural development. The most abundant and stable within the commissures, gliopodia frequently contact neurites extending from the neuropil on either side of the midline. Misexpression of Rac1N17 in midline glia not only reduces the number of gliopodia but also shifts the position of neuropils towards the midline. Midline-secreted signaling protein Slit accumulates along the surface of gliopodia. Mutant analysis supports the idea that gliopodia contribute to its presentation on neuronal surfaces at both the commissures and neuropils. It is proposed that gliopodia extend the range of direct glia-neuron communication during CNS development (Vasenkova, 2005).
Axonal and dendritic growth cones are guided into commissures, longitudinal fascicles and neuropils within the embryonic CNS. Throughout this period, midline glia extend gliopodia not only into commissures near the midline but also at the medial region of longitudinal neuropils several cell diameters away from their edges. While navigating within the CNS, neuronal growth cones also extend filopodia that span several cell diameters. Put together, the distance of direct filopodial contact between midline glia and neurons expands dramatically. As a result, the embryonic CNS displays a 'filopodial web' in which a majority of neurons could contact midline glia directly. The main effect of gliopodial presence during CNS development could be to extend the reciprocal influence of specialized midline cells over, and/or sensitivity toward, outgrowing neurites that lie at a distance. Whether or not a similarly extensive 'filopodial web' exists in embryonic nervous systems of larger animals is yet to be determined. However, it is interesting to note that the floorplate cells at the midline in the vertebrates have been documented to extend 'side-arms' (Vasenkova, 2005).
Midline glia in Drosophila are generally thought to serve a role analogous to floorplate cells of the vertebrate CNS. Unlike other glia that migrate along the medial-lateral axis of the embryonic nervous system, midline glia remain at the midline of the CNS throughout development. Yet, the distance over which midline glia-derived factors exerts their influence reaches at least the neuropil. Although a subset of axons and dendrites cross the midline through commissures, the remaining majority stays within the neuropil. Axons from the peripheral sensory neurons terminate here as well, without crossing the midline. The positioning of these CNS neurites and PNS terminals is apparently under the influence of Slit, a midline glia-derived signal protein. This study shows that midline signaling protein Slit is enriched extracellularly along gliopodia. Thus, Slit, which has been previously thought to reach neuropil through diffusion, could be also 'hand-delivered' (or foot-delivered?) onto the neuropilar surface by gliopodia (Vasenkova, 2005).
Slit is perhaps the easiest endogenous midline-derived signaling molecule to be examined for its redistribution in vivo through immunocytochemistry. This is because in the CNS, unlike other molecules such as Netrins that are also expressed by surrounding neurons, Slit is exclusively supplied by midline glia. In theory, however, other midline glia-derived molecules could also be carried through gliopodia. Vesicular transport of synaptic vesicle proteins has been observed along neuronal filopodia. Similarly, vesicles containing midline-signaling molecules could travel along gliopodia to neuropils where simple diffusion of the cue might not be effective or sufficiently discrete to mediate cell-specific interactions. The data suggest that gliopodia could serve as a vehicle for cue delivery to axons and dendrites that develop within commissures and neuropil of the embryonic CNS (Vasenkova, 2005).
In addition to delivering secreted molecules, gliopodia could also allow membrane-associated molecules to reach over a long distance from their source. Through dynamic gliopodial behavior, even membrane-bound ligands could create a local gradient of concentration, or availability, similar to the situation with diffusible ligands. However, unlike diffusible molecules, such molecules convey information regarding the identity of cells that produce them by virtue of being integrated into that cell's membrane. They can therefore serve as cell-recognition molecules that neurons detect along the whole length of gliopodia. In contrast, some gliopodia-bound molecules can also serve as remote-sensing receptors for the glia. Thus, discrete two-way communication through cell-bound molecules, often thought of as being an exclusive privilege of short-distance cellular encounters, is possible even for long-distance communication among glia and neurons. This extended molecular communication through the “filopodial web” could bear large impact on the course of embryogenesis in which the initial size of its emerging nervous system is small (Vasenkova, 2005).
The development of nervous systems involves reciprocal interactions between neurons and glia. In the Drosophila olfactory system, peripheral glial cells arise from sensory lineages specified by the basic helix-loop-helix transcription factor, Atonal. These glia wrap around the developing olfactory axons early during development and pattern the three distinct fascicles as they exit the antenna. In the moth Manduca sexta, an additional set of central glia migrate to the base of the antennal nerve where axons sort to their glomerular targets. In this work, whether similar types of cells exist in the Drosophila antenna has been investigated.
Different P(Gal4) lines were used to drive Green Fluorescent Protein (GFP) in distinct populations of cells within the Drosophila antenna. Mz317::GFP, a marker for cell body and perineural glia, labels the majority of peripheral glia. An additional ~30 glial cells detected by GH146::GFP do not derive from any of the sensory lineages and appear to migrate into the antenna from the brain. Their appearance in the third antennal segment is regulated by normal function of the Epidermal Growth Factor receptor and small GTPases. These distinct populations of cells have been denoted as Mz317-glia and GH146-glia respectively. In the adult, processes of GH146-glial cells ensheath the olfactory receptor neurons directly, while those of the Mz317-glia form a peripheral layer. Ablation of GH146-glia does not result in any significant effects on the patterning of the olfactory receptor axons. This study has demonstrated the presence of at least two distinct populations of glial cells within the Drosophila antenna. GH146-glial cells originate in the brain and migrate to the antenna along the newly formed olfactory axons. The number of cells populating the third segment of the antenna is regulated by signaling through the Epidermal Growth Factor receptor. These glia share several features of the sorting zone cells described in Manduca (Sen, 2005).
Studies in Manduca, have described three types of glia associated with the developing olfactory neuropil and nerve: (1) peripheral glia that arises from the antenna and ensheath olfactory axons; (2) lobe neuropil glia that surround and stabilize olfactory glomeruli; (3) sorting zone glia which are central in origin and migrate to the base of the antennal nerve where they serve to segregate axons that target independent glomeruli. In Drosophila, previous work has described the direct equivalents of peripheral antennal and the neuropil glia. This paper identifies an addition set of glia marked by GH146::GFP that share common features with sorting zone glia. A model suggests that these glia arise in the brain and migrate along the ORNs which have newly connected to the brain. Cells proliferate as they migrate along the nerve and enter the third segment of the antenna. Coincident with GH146-glia arrival at the periphery, the Ato-derived glia marked by Mz317::GFP (that are already present on the olfactory axons) move to wrap around the cell bodies of the sensory organs. The GH146-glial processes tightly ensheath the olfactory fascicles and form a layer below that of the Mz317-glial projections. In all 36 hrs APF preparations examined, ~30 GH146-glia were detected suggesting there must exist mechanisms to regulate the number of cells terminating in the antenna (Sen, 2005).
What is the function of GH146-glia during olfactory development? Although these cells share many of the properties of sorting zone glia described in the moth, it was not possible to implicate them in any guidance function. In Manduca, Fasciclin II (FasII), the insect ortholog of N-CAM, is expressed in a subset of ORNs scattered throughout the antennal nerve. Upon reaching the glial rich sorting zone, these projections segregate from the non-expressing axons and terminate in distinct glomeruli. In Drosophila, the presence of FasII protein on the ORNs during axon projection could not be detected using the available antibodies. When the GH146-glia were completely absent within the third antennal segment, no significant abnormalities were detected in ORN fasciculation within the antenna. The processes of the GH146-glia extend into the antennal nerve to ensheath axon bundles. This segregation is not based on Or gene type and its functional significance is obscure. A similar migration of glial cells from the centre into the periphery has been described during development of the Drosophila compound eye (Sen, 2005).
What are the factors that induce migration of glia from the brain towards the periphery? Cell migration in insect glia is well known to be mediated by signaling through the Fibroblast Growth Factor Receptors (FGFRs). Preliminary observations appear to rule out a requirement for FGFR signaling during GH146-glial migration. The downstream effector of FGF (Dof), is not expressed in these cells at any time during their development. Further, expression of dominant negative forms of Drosophila FGF receptors does not affect glial cell number in the antenna (Sen, 2005).
It is suggested that GH146-glial migration is mediated by EGF signaling. The ligand for Egfr, Vein, has been shown to be known to be expressed in the antennal epidermis at a time consistent with the arrival of cells from the center. The mechanisms involved in triggering this homing of cells into the antenna require extensive investigation (Sen, 2005).
Glial cells are crucial for the proper development and function of the nervous system. In the Drosophila embryo, the glial cells of the peripheral nervous system are generated both by central neuroblasts and sensory organ precursors. Most peripheral glial cells need to migrate along axonal projections of motor and sensory neurons to reach their final positions in the periphery. This paper studied the spatial and temporal pattern, the identity, the migration, and the origin of all peripheral glial cells in the truncal segments of wildtype embryos. The establishment of individual identities among these cells is reflected by the expression of a combinatorial code of molecular markers. This allows the identification of individual cells in various genetic backgrounds. Furthermore, mutant analysis of two of these marker genes, spalt major and castor, reveal their implication in peripheral glial development. Using confocal 4D microscopy to monitor and follow peripheral glia migration in living embryos, it was shown that the positioning of most of these cells is predetermined with minor variations, and that the order in which cells migrate into the periphery is almost fixed. By studying their lineages, the origin of each of the peripheral glial cells was uncovered and they were linked to identified central and peripheral neural stem cells (von Hilchen, 2008).
This study has characterized the expression of a collection of cell-specific molecular markers, which allows to identify and distinguish all glial cells in the embryonic peripheral nervous system. The reproducibility with which enhancer-trap lines and marker genes are expressed in the individual peripheral glial cells, indicates that these cells display unique identities. Furthermore, the spatial and temporal pattern of migration and the final arrangement of these cells are relatively stereotypic. This suggests that the specification of the unique identity of each cell does not only define a specific combination of genes to be expressed, but also includes the information about the timing of migration, the nerve tract it is associated with, and to some degree the final position to be occupied along the respective nerve. How the cell receives this information is still unknown. The individual characteristics could be determined (1) by lineage or (2) during migration by cell-cell interactions (between the glial cells or between the glia and other closely associated cells, e.g. neurons, tracheae), or (3) by a combination of both (von Hilchen, 2008).
The master regulatory gene glial cells missing (gcm) is required to induce the glial cell fate. Gcm as a transcription factor switches on downstream target genes, of which the gene encoding for the homeobox transcription factor Reversed polarity (Repo) is well described. As this cascade of gene activation is required for all glial cells in the Drosophila embryo (except the midline glia), it is unlikely to contribute to cell fate diversification among the glia. Whereas central glial cells migrate over rather short distances, in literally any possible direction, to finally occupy stereotypic positions within the CNS, the peripheral glial cells behave differently as they have to migrate over remarkable distances into the periphery. It has been recently shown that the migration of PGs depends on Notch signalling. In Notch mutants or in mutants where Notch signalling is altered in PGs, the migration is impaired or even completely blocked. However, this signalling does not appear to supply the cells with characteristics of their fate apart from the onset and/or maintenance of the migration itself. Sepp (2000) described the developmental dynamics and morphology of a subset of peripheral glial cells and could show that a signalling cascade mediated by the small GTPases RhoA and Rac1 influences the actin cytoskeleton of migrating PGs. Sepp further showed, that, within the analysed population of cells, a 'leading glia expresses filopodia-like structures whereas the follower cells do not. Similar results were reported by Aigouy (2004). Aigouy established a 4D microscopy technique to record and analyse the developmental dynamics and migratory behaviour of PNS glia during pupal stages in the developing fly wing. In this system, differences between 'leading' and 'follower' glia cells were also observed. The glial cells in the wing PNS migrate along wing veins in a chain with one 'leading' cell in front. If this chain is interrupted by laser ablation of either the leading or intermediate cells, a new 'leading cell starts to form filopodia and explores the surrounding. Once this new 'leading' cell catches up with the previous chain or reaches its target area, the filopodia disappear and the cells' morphology changes again. Hence, these differences in glial cell morphology and behaviour in the wing PNS are based on interactions of the glial cells with each other rather than on a predetermined intrinsic cell fate (von Hilchen, 2008).
Findings for the embryonic PNS glia suggest that these cells are predetermined at least to a certain extent. The 4D microscopy approach allowed tracing of the migration of individually identified PGs in living embryos from the moment they leave the CNS until they reach their final position. Apart from the dorsal SOP-derived cells, which never change their position or behaviour, it is always the ePG9 that leaves the CNS first and 'leads' the track. This cell expresses filopodia-like structures, while the following cells do not, although it remains to be experimentally shown whether they can take over the leading function in the absence of ePG9. It is worth mentioning that the SOP-derived ePG12 migrates along trajectories of the ISN prior to ePG9. It is not clear whether ePG12 has any leading function for ePG migration or functions as a guidepost cell for axonal projections. It is the only cell, though, that swaps nerve tracts and finally associates with the TN. Most likely, cell-cell communication between ePG12 and axonal projections and/or neighbouring cells is required for proper pathfinding and positioning. It is always the ePG4 that migrates along and finally enwraps the segmental nerve. As this cell is the only cell associated with the distal part of the segmental nerve, it functions as 'leading' glia for this nerve and expresses filopodia-like structures at least in later stages when it enwraps the SN. This enwrapment occurs in a bidirectional fashion, i.e. the filopodia occur at both ends of the glial cell (von Hilchen, 2008).
Lineage analysis revealed that the PGs mentioned above originate from the CNS neuroblast NB 1-3 and a ventrally located SOP. Interestingly, the two NB 2-5 derived PGs (ePG6 and ePG8) differ from these cells with respect to both identity and behaviour. They express fewer of the analysed PG-specific markers (cas-Gal4 and mirr-lacZ) and it is not possible to distinguish between these two cells so far. Whether the lack of identifying markers is a consequence of or a prerequisite for their different identity and behaviour is not yet clear. The cells migrate along the ISN independently of the NB 1-3- and SOP-derived PGs and frequently overtake them (and occasionally even one another). The correlation of such characteristics with the different origin of these three subpopulations of PGs lends support to the hypothesis that some aspects of cell fate diversification among the PGs may be predetermined by lineage. It is likely, that such predetermined characteristics include the competence to respond to specific external signals that guide the respective cell along the correct nerve to its target position (von Hilchen, 2008).
One incidence for lineage-dependent cell fate determination comes from the analysis of the ladybird homeobox genes. The ladybird genes are expressed in the developing CNS in only few NBs including NB 5-6. The NB 5-6 lineage produces one of the proximal PGs (ePG2) which expresses the Ladybird early (Lbe) protein. It has been shown that a loss of ladybird gene function results in a loss of the ePG2 in a third of all analysed hemisegments, accompanied with a higher number of medially located glial cells in the CNS. An opposite phenotype with excessive cells in the transition zone was observed by ectopic expression of the ladybird genes throughout the CNS. Using an anti-Repo antibody as well as a subset specific reporter transgene (K-lacZ), De Graeve (2004) provided evidence suggesting that the ladybird genes play a role in glial subtype specification in particular NB lineages. Another factor shown to be required for the specification of a lineage-specific set of glial cells (NB1-1-derived subperineurial glia) is Huckebein, which interacts with Gcm to amplify its expression specifically in these cells (von Hilchen, 2008).
Furthermore, in cas mutants, it was shown that the two NB 2-5-derived glia (ePG6 and ePG8) do not migrate into the periphery but most likely stay at their place of birth, although they acquire glial cell fate (as can be deduced from Repo stainings). Thus, similar to Ladybird and Huckebein, Cas seems to be involved in lineage-dependent glial subtype specification rather than determination of glial fate in general. In contrast to ladybird (De Graeve, 2004), though, Cas is not sufficient to ectopically induce glial cell fate or PG subtype specification (von Hilchen, 2008).
This study shows that salm is a likely candidate participating in the control of glial development. Embryos homozygous for salm445 show a pleiotropic and variable phenotype affecting not only glial cells but also PNS neurons, sensory organs, and other tissues. Yet, nearly all ventrally derived PGs stall in the transition zone between CNS and PNS and do not migrate properly into the periphery. In about 50% of the analysed hemisegments, a variable number of one to three PGs are missing, even though these cells could remain in the CNS. salm-lacZ is expressed in the two ventral SOP-derived ePG4 and ePG5, as well as in the dorsal SOP-derived ePG11 along the DLN, and in some of the ligament cells of the lateral chordotonal organ. In salm445 mutants the ePG4 cell can sometimes be detected at its wildtypic position along the SN. If ePG4 is missing along the SN, it could well be a secondary effect, as the SN itself is affected with the SNc shortened or occasionally missing. The ePG5 however, cannot be unambiguously identified in Repo-staining within the group of cells stalling in the transition zone (von Hilchen, 2008).
It needs to be further shown whether the differences between the PGs derived from certain progenitor cells result in functional differences in the larva. The peripheral nerves of the larva are ensheathed by two distinct types of glial cells, the perineurial and the subperineurial glial cells. The subperineurial glia build septate junctions with each other (or themselves) and thereby form the blood-nerve barrier, whereas the perineurial glia form an outer layer and secrete the neural lemma. In order to allow proper electrical conductance, the peripheral nerves must be enwrapped and insulated at the end of embryogenesis when hatching of the larva requires coordinated muscle contractions. It is not known to date which of the embryonic PGs will become perineurial or subperineurial glia, or what other functions they might fulfill (von Hilchen, 2008).
The comprehensive description of the ancestry, identity and dynamics of the developing embryonic peripheral glia, and the molecular markers at hand, provide a crucial basis for further clarification of the mechanisms controlling development, migration, and function of peripheral glia on a single cell level (von Hilchen, 2008).
Glial cells exist throughout the nervous system, and play essential roles in various aspects of neural development and function. Distinct types of glia may govern diverse glial functions. To determine the roles of glia requires systematic characterization of glia diversity and development. In the adult Drosophila central brain, five different types of glia were identified based on its location, morphology, marker expression, and development. Perineurial and subperineurial glia reside in two separate single-cell layers on the brain surface, cortex glia form a glial mesh in the brain cortex where neuronal cell bodies reside, while ensheathing and astrocyte-like glia enwrap and infiltrate into neuropils, respectively. Clonal analysis reveals that distinct glial types derive from different precursors, and that most adult perineurial, ensheathing, and astrocyte-like glia are produced after embryogenesis. Notably, perineurial glial cells are made locally on the brain surface without the involvement of gcm (glial cell missing). In contrast, the widespread ensheathing and astrocyte-like glia derive from specific brain regions in a gcm-dependent manner. This study documents glia diversity in the adult fly brain and demonstrates involvement of different developmental programs in the derivation of distinct types of glia. It lays an essential foundation for studying glia development and function in the Drosophila brain (Awasaki, 2008).
Two layers of distinct glial cells surround the entire adult Drosophila brain. The inner one, possibly composed of a fixed number of large sheet-like subperineurial glial cells since larval hatching, constitutes the fly blood-brain barrier in both larval and adult brains. In contrast, the outer layer consists of numerous small oblong perineurial glial cells that proliferate through postembryonic development of the brain and may remain immature until formation of the adult brain. Given its more external location and apparent adherence to the subperineurial layer, the perineurial glia possibly helps make up the adult blood-brain barrier. However, these two types of surface glia likely play nonoverlapping, complementary roles in the regulation of the permeability to the CNS, as implied from their different enhancer trap expression patterns (Awasaki, 2008).
The other three types of glia form independent networks inside the brain. Cortex glia enwrap individual neuronal cell bodies in the brain cortex. Each cortex glial cell send mesh-like processes to surround multiple neuronal cell bodies locally. Cortex glia may provide metabolic support for neurons and have the potential for modulating neuronal cell body functions in a spatially controlled manner. Further inside the brain lie two types of neuropil glia. The ensheathing glia outlines individual neuropils and their subcompartments, while the astrocyte-like glia extend processes into the neuropils where synapses form (Awasaki, 2008).
The glial boundary established by the ensheathing glia may provide the necessary insulation to prevent lateral communication between adjacent neuropils, and provide septa when a neuropil is composed of multiple independent subcompartments. In contrast, astrocyte-like glial cells potentially associate with synapses. Several electron microscopic studies have shown that glial processes are located around synapses of insect neuropils. In an analogous manner, mammalian astrocytes target processes to synapses. They express excitatory amino-acid transporters (EAAT-1 and -2), and can recover excess extracellular glutamate or aspartate to keep the local environment suitable for the next synaptic transmission. In addition, mammalian astrocytes may release gliotransmitters in response to synaptic activity to modulate synaptic functions. Whereas in the vertebrate nervous system synapses are formed not only on the dendrites and axon terminals but also on the surface of the neural cell bodies, in the invertebrate brain no synapses are formed on the neuronal cell bodies. Accordingly, the cortex glia and neuropil glia are associated exclusively to the neural cell bodies and synaptic neuropils, respectively. Although Drosophila glia also expresses EAAT-1 (dEAAT-1) that is concentrated in neuropils, it remains to be determined which function of the vertebrate astrocytes is achieved by which type of insect glial cells (Awasaki, 2008).
In the adult brain or ventral nerve cord of other insects, two types of glial cells associated with neuropil have been identified; type II and type III glia or interface and neuropilar glia in Musca, interface and neuropil glia in Acheta and neuropil cover type and neuropil glia or simple and complex neuropil glia in Manduca. One type is localized cortex/neuropil interface or borders among neuropils and ensheaths neuropil, and another type extends glial processes inside the neuropils, like the ensheathing and astrocyte-like glia of adult Drosophila brain, respectively. It is, therefore, these two subtypes of neuropil glia would commonly exist in insects (Awasaki, 2008).
These distinct types of adult fly brain glia arise independently from each other. The proliferation of subperineurial glia is probably restricted to embryogenesis, and any postembryonic increase in the cortex glial cell number should be much less significant than the expansion of the other three types of adult glia, which primarily occurs during larval development. Furthermore, distinct populations of precursors give rise to perineurial, ensheathing, or astrocyte-like glia. Notably, perineurial glia precursors distribute around the brain surface and make clones that expand locally as the brain grows in size through development. In contrast, ensheathing and astrocyte-like glial cells are derived from specific proliferation centers and do not migrate throughout the brain until metamorphosis. Most somatic clones of neuropil glia exclusively consist of ensheathing or astrocyte-like glial cells. However, the identity of the neuropil clones could not be unambiguously determined until late pupal stages, making it unclear whether the progenitors of the ensheathing and astrocyte-like glia are mixed in those proliferation centers. Perineurial glial precursors make clones of similar sizes at a given developmental stage, suggesting that they retain the same proliferation potential through development. In contrast, the clones of neuropil glia derived at the same stage vary in size, raising the possibility that neuropil glial precursors undergo stochastic proliferation, possibly, in response to some limited local cue(s). Supply of Vein, a neuregulin-like trophic factor, from neurons has been shown to promote local proliferation of the embryonic longitudinal glia. It remains to be determined whether similar mechanisms govern the postembryonic expansion of the adult neuropil glia. Finally, all the postembryonic glial precursors apparently divide symmetrically to produce two daughters following each division; intriguingly, the final round of mitosis consistently takes place around pupal formation. This suggests a stage-specific signal may trigger the final glia-producing mitoses (Awasaki, 2008).
The involvement of different developmental programs in the derivation of different types of adult glia is further evidenced in the differential temporal requirement for gcm. GCM promotes all embryonic gliogenesis except for midline glia. GCM suppresses neural differentiation through expression of Tramtrack and confers glial identity via induction of Repo and other glial genes. Transient expression of gcm also occurs in the postembryonic development of both medulla and thoracic glial lineages, and inhibition of GCM function by expression of a dominant-negative form of GCM suppresses differentiation of their glial development. In addition, this study shows that precursors of neuropil glia express gcm transiently and the postembryonic induction of the gcm mutant clone effectively arrest the development of neuropil glia. These observations indicate that GCM plays analogous roles in promoting glial fate at different developmental stages. It is possible that the precursors of neuropil glia do not acquire full glial identity until gcm is expressed at an intermediate stage of development. In contrast, gcm is dispensable for postembryonic development of perineurial glia. Unlike midline glia in which Repo is never expressed, perineurial glial cells are strongly positive for Repo, a direct target for GCM, making it likely that GCM is involved in the development of perineurial glia. It appears more plausible that like neuropil glia transient expression of gcm also occurs in the perineurial glial precursors but at an earlier stage, and that they have already acquired glial cell fate before larval hatching (Awasaki, 2008).
While most glial types appear to be conserved in flies, the microglia type of glia is not obvious in the adult fly brain. It is possible that not all major types of glia were identified because of the restriction of of the analysis to only Repo-positive cells. Nevertheless, evidence is accumulating that neuropil glia may share microglia-type functions to engulf degenerating axons in the process of programmed axon pruning or following injury of neurites. Additional glial diversity has been suggested in vertebrates. Many subtypes of glial cells in the Drosophila embryonic ventral ganglion has also been revealed through detailed analysis of lineage as well as gene expression. The identification of five major types of adult fly brain glia and the analysis of their developmental origins pave a way for further use of the Drosophila as a model system for study of glial diversity, development, and function (Awasaki, 2008).
The study of adult neural cell production has concentrated on neurogenesis. The mechanisms
controlling adult gliogenesis are still poorly understood. This
study provides evidence for a homeostatic process that maintains the population of glial cells in
the Drosophila adult brain. Flies lacking microRNA miR-31a start adult life with a normal
complement of glia, but transiently lose glia due to apoptosis. miR-31a expression identifies
a subset of predominantly gliogenic adult neural progenitor cells. Failure to limit expression of
the predicted E3 ubiquitin ligase, Rchy1,
in these cells results in glial loss. After an initial decline in young adults, glial numbers
recovered due to compensatory overproduction of new glia by adult progenitor cells, indicating an
unexpected plasticity of the Drosophila nervous system. Experimentally induced ablation of glia was
also followed by recovery of glia over time. These studies provide evidence for a homeostatic
mechanism that maintains the number of glia in the adult fly brain (Foo, 2017a).
Despite the critical role that glia play in the proper functioning of neurons, study of adult neural cell production has focused mainly on neurons. Much less is known about gliogenesis in the adult brain. This report provide several lines of evidence for ongoing gliogenesis in the adult. These data include labelling the newly synthesized DNA of newly born cells in the adult showed the production of glia as well as neurons. Genetic mosaic analysis to produce clones of marked cells in the adult brain showed the production of new neurons and new glia from progenitor cells expressing Insc-Gal4 as well as a newly identified population of progenitors that express the miR-31a microRNA. Interestingly, the miR-31a-expressing progenitors appear to be mainly gliogenic, while those expressing Insc-Gal4 are mainly neurogenic. Given that both progenitors can make neurons and glia, it is suggested that the miR-31a-expressing progenitor cells may be a specialized subset of Insc-Gal4-expressing neuroglial progenitors. This is supported by the observation of miR-31a sensor activity in a subset of Insc-Gal4 cells (Foo, 2017a).
In the absence of miR-31a expression, glial number is affected. It is noted that the number of glia is normal in young adult miR-31a mutants, drops sharply over several days and then recovers. This pattern implies an active process of glial turnover in the young adult brain, which is impacted by the absence of the miRNA in the progenitor population. Evidence is provided that this defect is due to the requirement for the miRNA in progenitor cells, but not in mature glia or neurons (Foo, 2017a).
The findings show that miR-31a acts through regulation of Rchy1, a predicted E3 ubiquitin ligase. Although many transcripts are misregulated in the mutant, restoring expression of Rchy1 towards normal levels effectively suppressed the glial defect in the mutant. This implies that the overexpression of Rchy1 contributes to the failure of these cells to produce sufficient glia during the remodelling process that was observed in the young brain. In miR-31a mutants, Rchy1 protein was detected at elevated levels in the progeny of the miR-31a progenitor cells. Excess Rchy1 appears to be detrimental to the survival of the glial progeny, in that loss of glia can be suppressed by blocking apoptosis. There was only a small change in the number of neurons in adult brains of miR-31a mutants compared to wild-type animals. But, it is noted that the miR-31a progenitor cells are few in number compared to the predominantly neurogenic Insc-Gal4-expressing progenitors. Therefore, an effect of excess Rchy1 in these cells on production of neurons cannot be ruled out (Foo, 2017a).
Ubiquitin-mediated protein turnover has been shown to play several important roles during CNS development in Drosophila. Ubiquitination and proteosomal degradation of glial cells missing allow embryonic glia to exit the cell cycle and begin differentiation. Additionally, neural cell fate can be controlled by ubiquitin-dependent regulation of protein translation. This study shows that a microRNA is responsible for regulating the level of a predicted E3 ubiquitin ligase in a neural progenitor cell, which affects the viability of its progeny. Identification of the targets of Rchy1 in neural progenitor cells may provide new insights into the control of their differentiation and survival (Foo, 2017a).
Loss of glia in the miR-31a mutant points to a previously unidentified plasticity of the young adult brain in Drosophila. These findings imply ongoing replacement of glia in the young adult brain. Furthermore, they provide evidence that the brain can detect when there are too few glia and that this can trigger progenitor cells to increase production of new glia, by increasing the proportion of their progeny that differentiate into glia. How the number of glia is monitored in the brain will be a topic for future study (Foo, 2017a).
Approximately half of the glia affected in the miR-31a mutant were astrocytes. In this context, parallels to the mammalian brain may be interesting. In mammals, most astrocytes contact a blood vessel. Since astrocytes rely on the vasculature for survival, it is speculated that there is a matching of astrocytes to blood vessels. The fly does not have a closed circulatory system, so there are no blood vessels in the brain. There must be other mechanisms by which astrocyte number is monitored. An obvious possibility is that the need for astrocytes (or their survival) is linked to the number of neurons with which they make contact (Foo, 2017a).
Two recent studies have shown that mammalian glia, specifically oligodendrocyte precursor cells (OPC) and microglia, can repopulate the brain after induced loss of the entire populations of these two cell types. It has been demonstrated that OPC differentiation to oligodendrocytes occurs throughout life. As such, the homeostatic response to maintain OPCs in the brain likely reflects the need to replace the OPCs that have either differentiated or died. Their data also demonstrate that there is a continuous turnover of oligodendrocytes in the adult. Astrogenesis has been observed to occur in the prefrontral cortex of mice in response to voluntary exercise. The current findings provide evidence that this normal turnover of astrocytes also occurs in the Drosophila brain and that specific neural progenitor cells maintain an ongoing homeostatic control of astrocyte numbers in the adult Drosophila brain. It will be interesting to learn whether a comparable progenitor population exists in mammals to support astrocyte turnover (Foo, 2017a).
The neural stem cells that give rise to the neural lineages of the brain can generate their progeny directly or through transit amplifying intermediate neural progenitor cells (INPs). The INP-producing neural stem cells in Drosophila are called type II neuroblasts, and their neural progeny innervate the central complex, a prominent integrative brain center. This study used genetic lineage tracing and clonal analysis to show that the INPs of these type II neuroblast lineages give rise to glial cells as well as neurons during postembryonic brain development. These data indicate that two main types of INP lineages are generated, namely mixed neuronal/glial lineages and neuronal lineages. Genetic loss-of-function and gain-of-function experiments show that the gcm gene is necessary and sufficient for gliogenesis in these lineages. The INP-derived glial cells, like the INP-derived neuronal cells, make major contributions to the central complex. In postembryonic development, these INP-derived glial cells surround the entire developing central complex neuropile, and once the major compartments of the central complex are formed, they also delimit each of these compartments. During this process, the number of these glial cells in the central complex is increased markedly through local proliferation based on glial cell mitosis. Taken together, these findings uncover a novel and complex form of neurogliogenesis in Drosophila involving transit amplifying intermediate progenitors. Moreover, they indicate that type II neuroblasts are remarkably multipotent neural stem cells that can generate both the neuronal and the glial progeny that make major contributions to one and the same complex brain structure (Viktorin, 2011).
This study used a combination of genetic lineage tracing, clonal MARCM techniques and molecular labeling to study the developmental mechanisms that give rise to the glial cells of the amplifying type II neuroblast lineages. A novel mode of neurogliogenesis in these lineages was uncovered that involves transit amplifying INPs, which can generate glial cells as well as neurons. This analysis also shows that lineal INP-derived glial and neuronal cells both make major contributions to the central complex. INP-derived neurons project into the neuropile compartments of the central complex and INP-derived glial cells surround and delimit these compartments while undergoing clonal expansion through local proliferation (Viktorin, 2011).
The majority of the glial cells in the adult brain are generated postembryonically. Hitherto unidentified neuroglioblasts have been postulated to give rise to the bulk of these adult-specific glial cells, although some of these arise via proliferative glial cell divisions. This paper reports the identification of type II neuroblasts as neural stem cells with neuroglioblast function in postembryonic development of the central brain. Previous work has shown that these amplifying type II neuroblasts augment proliferation through the generation of INPs resulting in the generation of remarkably large neuronal lineages. These data indicate that type II neuroblasts also generate glial progeny through INPs and, hence, reveal a novel form of CNS neurogliogenesis that involves transit amplifying cells (Viktorin, 2011).
Although type II neuroblasts represent a new type of neuroglioblast in Drosophila brain development, the six identified type II neuroblasts are heterogeneous in terms of their gliogenic activity. While four of these neuroblasts generate comparable numbers of glial cells, a fifth neuroblast (DM4) gives rise to only a few glial cells and the sixth (DM6) rarely generates glia during larval development. This heterogeneity in gliogenic activity is also reflected in the INPs of these type II lineages. Thus, while mixed glial/neuronal INP clones were recovered for most type II lineages, all of the INP clones that contained glia in the DM5 lineage were purely glial, and no glial INP clones were recovered for DM6. Despite this heterogeneity, the generation of INP-derived glial cells in all of these type II lineages appears dependent on the gcm gene. Indeed, this seems to be a feature common to most glial progenitors in the embryonic and postembryonic CNS (Viktorin, 2011).
While glia can derive from the very first larval INPs produced by DM neuroblasts, glia differentiate late in the progression of the lineage, at a time when neuroblast markers are no longer present in the INP. At that time, lineages already contain large amounts of differentiating cells, positive for Prospero and/or the neural differentiation marker Elav. Interestingly, both Prospero and Elav are also found in many young glia, some of them verified to be part of Type II-derived lineages. This co-expression is reminiscent of embryonic glia that have been reported to transiently express Elav, which may thus be a common feature of glial cells also in the larval brain. In addition, there may be Prospero-positive, gliogenic ganglion mother cells that could divide symmetrically to contribute to the variable number of glia observed in late larval INP lineages (Viktorin, 2011).
Neural stem cells in the mammalian brain, notably the radial glia of the cortex, also represent mixed progenitors that can give rise to both neuronal and glial cells in different proliferative modes, and one of these proliferative modes involves transit amplifying INPs. Moreover, some of the amplifying INPs involved in mammalian neocortex development are thought to give rise to neuronal progeny while others give rise to glial progeny. The amplification of proliferation through INPs has been postulated to be fundamental for the increase in cortical size during evolution. The fact that a very comparable mode of INP-dependent proliferation operates in the generation of complex brain architecture in Drosophila suggests that this might represent a general strategy for increased size and complexity in brain development and evolution (Viktorin, 2011).
Previous work has shown that a large subset of the neurons generated by type II neuroblasts contributes to the development of the central complex. The data presented in this study indicate that the INP-derived glial cells from the type II lineages are also involved in central complex development. INP-derived glial cell bodies associate with the larval primordium and, throughout pupal development; they surround and delimit all of the compartments of the differentiating central complex neuropile. Thus neural and glial cells from the same neuroblast lineage participate in the development of the same complex brain neuropile. This reveals a remarkable multipotential nature of the type II neuroblasts; they are neural stem cells that have the potential to generate both neural and glial cells of one and the same complex brain structure (Viktorin, 2011).
Neuroblast lineages have been viewed as 'units of projection' in that the neurons of a given lineage often project their axons along a common trajectory and contribute to the formation of a common neuropile; this is exemplified by the four neuroblast lineages that give rise to the intrinsic cells of the mushroom body neuropile. The neurons of the type II neuroblast lineages do not strictly conform to this notion, since subsets of neurons in the lineage project to different parts of the brain. However, for the large subset of neurons in the type II lineages that project to the developing central complex, the notion of a lineal 'unit of projection' is valid. Indeed this concept can be expanded to include the lineally related glial cells that also contribute to the development of the same neuropile compartment (Viktorin, 2011).
In type II neuroblast clones, most of the INPs, as well as the cell bodies of the neurons that they produce, remain clustered together in the peripheral cell body layer of the brain. Although a few of the INP-derived glial cells are also found in these clusters, most are not. During larval and pupal development, the majority of the INP-derived glial cells are found in or near midline commissural structures such as the central complex precursor (late larva) or the developing central complex neuropile (pupa) (Viktorin, 2011).
One reason for this is that INP-derived glial cells probably migrate away from their site of origin to a different site of final differentiation. Migration of glial cells during CNS development is a common feature in many species and has been studied in detail in the developing ventral nerve cord and optic lobes of Drosophila. Migration of glial cells has also been reported in postembryonic development of the central brain, and in some cases the migrating cells appear to form clusters suggesting that they might derive from common progenitors (Viktorin, 2011).
Another reason for the fact that so many INP-derived glial cells are found in or near the developing central complex is that they proliferate locally. This implies that INP-derived glial cells can undergo clonal expansion in the neuropile. Clonal expansion has been described for the perineurial glial cells localized on the surface of the brain and has also been postulated to take place during the postembryonic development of neuropile glial cells. Since INP-derived glial cells undergo substantial (at least fourfold) proliferative clonal expansion, it will be important to determine what controls their mitotic activity. In view of the vulnerability of the amplifying type II neuroblast lineages to overproliferation and brain tumor formation, a tight control of self-renewing glial cell proliferation in these lineages is likely to be essential (Viktorin, 2011).
The function of a complex nervous system depends on an intricate interplay between neuronal and glial cell types. One of the many functions of glial cells is to provide an efficient insulation of the nervous system and thereby allowing a fine tuned homeostasis of ions and other small molecules. This study presents a detailed cellular analysis of the glial cell complement constituting the blood-brain barrier in Drosophila. Using electron microscopic analysis and single cell-labeling experiments, different glial cell layers at the surface of the nervous system, the perineurial glial layer, the subperineurial glial layer, the wrapping glial cell layer, and a thick layer of extracellular matrix, the neural lamella, were characterized. To test the functional roles of these sheaths a series of dye penetration experiments were performed in the nervous systems of wild-type and mutant embryos. Comparing the kinetics of uptake of different sized fluorescently labeled dyes in different mutants led to the conclusion that most of the barrier function is mediated by the septate junctions formed by the subperineurial cells, whereas the perineurial glial cell layer and the neural lamella contribute to barrier selectivity against much larger particles (i.e., the size of proteins). The requirements of different septate junction components were compared for the integrity of the blood-brain barrier, and evidence is provided that two of the six Claudin-like proteins found in Drosophila are needed for normal blood-brain barrier function (Stork, 2008).
Fast neuronal conductance requires a tight electrical insulation of the axons and in the mammalian nervous system, myelin and saltatory conductance evolved. Arthropods have not evolved saltatory conductance, but they are nevertheless in need for fast electrical conductance. In this respect it is not surprising that in marine shrimps myelin-like structures have been described . Drosophila follows two different and seemingly independent strategies to ensure fast conductance. In some central neuronal networks large caliber axons develop, whereas in the peripheral nervous system axons are insulated by several glial sheaths to ensure insulation. Initially, at the beginning of larval life, the different sensory and motor axons are kept as separate fascicles within the segmental nerves, suggesting there might be some degree of electrical cross talk within the different modalities. As the larva matures, the inner wrapping glia starts to grow around single axons, which may allow more sophisticated movements of the wandering larvae (Stork, 2008).
Whereas the wrapping glia insulates individual axons, do perineurial and subperineurial glia insulate the entire nervous system and set up the blood-brain-barrier? Genetic experiments and ultrastructural studies have long indicated that septate junctions provide the most effective part of this barrier. Indeed, the subperineurial cell are formed early in development and these cells are connected by septate junctions from late embryonic stages onwards. Using Gal4 driver strains specific to the subperineurial cells as well as in vivo septate junction markers, this study confirms that during larval life the subperineurial cells do not divide but grow enormously large in size. Septate junctions formed by the subperineurial cells are mostly found in interdigitated zones of cell-cell contact. Cell division would likely require disintegration of septate junctions and thus result in a temporal opening of the blood-brain barrier, which would be deleterious for the animal. This is in agreement with previous findings that Gliotactin expressing cells, forming septate junctions, do not divide during larval live (Stork, 2008).
The outermost glial cell layer is formed by the perineurial cells. Although these cells have long been described, their origin is still a matter of debate. In EM micrographs of late embryonic staged peripheral nerves some perineurial cells can be detected apically to the subperineurial cells. These glial cells divide during larval life and generate a large number of fine cell protrusions that cover the subperineurial cells. One function of the perineurium might be to influence the development and/or the tightness of the subperineurial layer. A comparable cellular function has been attributed to the astrocytes in the mammalian nervous system. Alternatively, the perineurial glial cells might provide a cellular basis for the response to injury. Unfortunately, to date there is no specific driver strains that allow manipulation of this glial cell population. Interestingly, a reverse relationship between subperineurial and perineurial cells has been suggested previously as subperineurial expression of activated Ras or PI3K (phosphoinositide 3-kinase) resulted in an thickening of the perineurial sheath (Stork, 2008).
Additionally, the fray gene has been shown to be required for normal axonal ensheathment. Interestingly the mutant phenotype could be rescued by expressing fray using three different Gal4 drivers. After the analysis of the specificity of these drivers (Mz317, subperineurial glia and weak wrapping glia; Mz709, all glial cell types; gliotactinGal4, subperineurial glia), it is concluded that fray is expressed in subperineurial glia and controls axonal ensheathment of wrapping glia in a noncell autonomous manner (Stork, 2008).
Given the different cellular barriers described in this report, questions arise concerning the functional contributions of the different layers. Kinetic studies were performed that supported the importance of the septate junctions in particular for small components. Animals lacking septate junctions are as leaky to a 10 kDa dextran as animals lacking all glial cell layers. However, when it comes to larger molecules, the relevance of the other cell layers becomes obvious. Although a 500 kDa dextran can easily penetrate into the nervous system of a glial cells missing embryo, its leakage into the nervous system of a neurexinIV mutant lacking septate junctions is greatly reduced. Thus, the other layers contribute to the function of the blood-brain barrier. Because a continuous perineurium is not fully formed in first instar larvae, the barrier function has to be assigned to the neural lamella and the inner glial layer. There are several reports showing that the neural lamella can act as an efficient filter for heavy metal ions. Possibly, large molecules such as the 500 kDa dextran are also trapped in this ECM. Alternatively, large particles are stopped by the diffusion barrier established by the normal cell-cell contacts between subperineurial cells and inner glial cell types like wrapping glia in the peripheral nerves and cortex and neuropile glia in the CNS (Stork, 2008).
The diffusion barrier provided by glial cells or epithelial sheaths is generated by special junctional complexes that help to tightly associate the involved cells. Drosophila epithelia as well as glial cells are characterized by septate junctions. Quite similar structures are also found at the mammalian paranodal junctions, which provide the structural basis for the tight electrical insulation of the nerve. A core component of the mammalian axoglial septate junctions is the NeurexinIV homolog Caspr that together with its binding partners, Contactin and Neurofascin155, sets up a tripartite adhesion complex at the paranode (Stork, 2008).
The function of this complex appears conserved in Drosophila, although there are some notable differences. The Caspr homolog NeurexinIV is expressed by glial cells as are Contactin and the Neurofascin155 homolog Neuroglian. As a consequence, in the fly septate junctions are formed between glial cells, whereas they are formed between neuronal and glial membranes in the mammalian system. The Caspr/Contactin/Neurofascin155 complex seals the paranodal junction and a similar function has been attributed to this protein complex in the invertebrate blood-brain barrier. This study found a less pronounced function of Contactin compared with NeurexinIV for the blood-brain barrier establishment, corroborating findings made in embryonic epithelia (Stork, 2008).
Another prominent component of the junctional complexes are the Claudin proteins. In mammals, members of these four transmembrane domain proteins are associated with tight junctions that are often considered to be functionally equivalent to the invertebrate septate junctions. In Drosophila two Claudin-like proteins have been described to be required for formation of normal epithelial barrier formation. This study shows that both Sinuous and Megatrachea are also needed for the establishment of normal blood-brain barrier formation. Similarly, it was shown that mammalian claudin5 is a major component of tight junctions of brain endothelial cells. claudin5 mutant mice show no structural or ultrastructural deficits, but have an impaired blood-brain barrier. The association of Claudins integrated in opposing membranes is thought to provide pores that can control the paracellular diffusion of small molecules. Although Drosophila Sinuous and Megatrachea clearly contribute to the barrier function, it is inconceivable that fly Claudins traverse the 20 nm wide septate gap to form a Claudin pore as it is discussed for the vertebrate Claudins. It has also been suggested that invertebrate Claudins might have lost their pore-like functions and exert only signaling function to establish the barrier (Stork, 2008).
Such a signaling function may control the size selectivity of the barrier and indeed sinuous mutants show only a weak barrier phenotype comparable with moody mutants, correlating with reduced septate junctions. This study demonstrates that a loss of septate junctions associated with neurexinIV mutants results in breakdown of the blood-brain barrier comparable with what is observed in animals lacking all glial cells. However, additional mechanisms are in place to control the paracellular diffusion of larger particles. A 500 kDa dextran can easily penetrate the nervous system of a glial cells missing embryo but cannot enter a nerve cord only lacking septate junctions (Stork, 2008).
Neurons and glia are the two major cell types in the nervous system and work closely with each other to program neuronal interplay. Traditionally, neurons are thought to be the major cells that actively regulate processes like synapse formation, plasticity, and behavioral output. Glia, on the other hand, serve a more supporting role. To date, accumulating evidence has suggested that glia are active participants in virtually every aspect of neuronal function. Despite this, fundamental features of how glia interact with neurons, and their spatial relationships, remain elusive. This study describes the glial cell population in Drosophila adult brains. Glial cells extend and tightly associate their processes with major structures such as the mushroom body (MB), ellipsoid body (EB), and antennal lobe (AL) in the brain. Glial cells are distributed in a more concentrated manner in the MB. Furthermore, subsets of glia exhibit distinctive association patterns around different neuronal structures. Whereas processes extended by astrocyte-like glia and ensheathing glia wrap around the MB and infiltrate into the EB and AL, cortex glia stay where cell bodies of neurons are and remain outside of the synaptic regions structured by EB or AL (Ou, 2016).
Glial cells play important roles in the developing brain during axon fasciculation, growth cone guidance, and neuron survival. In the Drosophila brain, three main classes of glia have been identified including surface, cortex, and neuropile glia. While surface glia ensheaths the brain and is involved in the formation of the blood-brain-barrier and the control of neuroblast proliferation, the range of functions for cortex and neuropile glia is less well understood. This study used the nirvana2-GAL4 driver to visualize the association of cortex and neuropile glia with axon tracts formed by different brain lineages and to selectively eliminate these glial populations via induced apoptosis. The larval central brain consists of approximately 100 lineages. Each lineage forms a cohesive axon bundle, the secondary axon tract (SAT). While entering and traversing the brain neuropile, SATs interact in a characteristic way with glial cells. Some SATs are completely invested with glial processes; others show no particular association with glia, and most fall somewhere in between these extremes. The results demonstrate that the elimination of glia results in abnormalities in SAT fasciculation and trajectory. The most prevalent phenotype is truncation or misguidance of axon tracts, or abnormal fasciculation of tracts that normally form separate pathways. Importantly, the degree of glial association with a given lineage is positively correlated with the severity of the phenotype resulting from glial ablation. Previous studies have focused on the embryonic nerve cord or adult-specific compartments to establish the role of glia. This study provides, for the first time, an analysis of glial function in the brain during axon formation and growth in larval development (Spindler, 2009).
Secondary neurons, which are born during the larval period, form SATs that have to extend over relatively long distances, finding their way amidst a complex array of (primary) axons, dendrites, and glia. The association of glia and SATs varies for different lineages. SATs either (1) remained wrapped within the neuropile or joined other tracts that were then ensheathed as a larger tract system, (2) encountered strands of glial condensations, or (3) had no association with glial sheaths. The association between individual SATs and glia was highly invariant. Thus, if SAT A joined SAT B to form a larger tract system that became wrapped by glia, the same densities of glia could be observed in other brains for the corresponding SATs (Spindler, 2009).
To address the role of glia during SAT fasciculation, growth, and guidance, cortex and neuropile glia, the two glial types in contact with growing SATs, were selectively eliminate. Expression of the pro-apoptotic proteins Hid and Rpr were effective in inducing apoptosis of most cortex and neuropile glia by the early or mid larval stage. It can be assumed that the primary axon tract formation is not disrupted, given that expression of the
nrv2-GAL4 driver line does not set in prior to stage 12, primary axon tract (PAT) patterning is complete before glia invade the neuropile, and the first signs of apoptosis appear after hatching. In addition, the surface glia remain intact, providing general ensheathment around the brain, a functional blood–brain barrier, and potential signaling molecules used to control neuroblast proliferation (Spindler, 2009).
Upon the elimination of glia, frequent abnormalities are seen in the pattern of SATs. Importantly, the strength of an SAT phenotype appears to correlate with the degree of glial association of that SAT in a wild-type brain. Of all SATs analyzed, the mushroom body (associated with a complete glial sheath) exhibits the most severe defects, including complete SAT misguidance and aberrant fasciculation of neurites from adjacent SATs. In contrast, SATs that were less endowed with glia in the wild type typically had a normal projection pattern (Spindler, 2009).
This study does not differentiate between a role for glia in producing chemo-attractant signals or acting as a physical scaffold for guidance. In previous studies, ectopic expression of dominant-negative Drosophila E-cadherin in either cortex/neuropile glia or SATs themselves results in non-radial trajectories of SATs into the neuropile. This suggests a requirement for SAT-glia adhesion as secondary axons project toward the cortex-neuropile boundary. However, the direction of neuroblast division is also disrupted with DE-cadherin knock-down, thus aberrant SAT trajectories
may be a secondary effect of abnormal cell body layering within the cortex. In support of a signaling role for glia, the midline guidance defect of the CP1 SAT (anteromedially, crossing the peduncle and entering the diagonal commissure) is reminiscent of the robo-slit phenotypes in the
Drosophila ventral nerve cord. Whether robo/slit signaling is also used between SATs and glia in the brain is a question that warrants further investigation (Spindler, 2009).
The situation that SATs in part require glia for pathway guidance in the neuropile is different than the embryonic brain in which pioneer axons and PATs are formed in the absence of extensive glial processes. Why are SATs different? One can imagine the scenario in which the PATs generate the neuropile de novo, forcing cell body movement outwards as the central neuropile grows. By first instar, a full neuropile is established, and SATs must guide through a dense maze of neurites, glia, and trachea. It therefore
appears that: (1) PATs establish the initial connectivity of the brain; (2) glia grow in around this initial scaffolding, and finally (3) SATs use both the PAT scaffolding and the glial boundaries for guidance into and around the neuropile (Spindler, 2009).
Insects have long been used to evaluate glial-neuronal interactions from embryonic to adult stages. An important focus of these studies was whether or not glia or axon tracts appear first, and in how far axonal pathfinding is disrupted if glia is ablated. In this regard, clear developmental differences have been found between distinct regions of the nervous system, as well as between insect species for homologous nervous system domains (Spindler, 2009).
In the Drosophila ventral nerve cord, two subpopulations of neuropile glia were studied in the context of axonal pathfinding: midline glia and longitudinal glia. Both types of glial cells appear around the same stage when pioneer neurons extend their axons. The proper number and positioning of midline glia is clearly required for the formation of commissural axon tracts. The loss of longitudinal glia (by ablation and in embryos mutant for the
gcm gene) primarily affects the defasciculation and fasciculation events of longitudinal pioneer tracts, subsequently affecting the follower neuron trajectories. Note that gcm is required for all classes of glia, including surface glia; thus, in very late gcm mutant embryos, severe disruptions of the entire neuropile result from the fact that, with the onset of embryonic movement, the CNS lacking surface glia is literally 'shredded to pieces' (Spindler, 2009).
In the embryonic Drosophila peripheral nervous system, ablation of the peripheral glia (i.e. exit glia) via targeted overexpression of the cell death genes grim and ced-3 lead to aberrant pathfinding of both motor and sensory axons as they exited the CNS; although the motor neurons eventually overcame the absence of peripheral glia finding correct muscle targets, suggesting a limited role for the peripheral glia in the initial trajectory of motor neurons. In grasshopper, ablation of the cell-segment boundary glial guidepost cell lead to more severely aberrant axon trajectories. Ablation of glia surrounding the antennal lobe of adult Manduca generates olfactory axon de-fasciculation and misguidance. Finally, guidance phenotypes have also been observed with disruption of the Drosophila lamina glia; R1-R6 photoreceptor axons show aberrant guidance past the lamina into the medulla of the optic lobe (Spindler, 2009 and references therein).
An important aspect of the developmental role of glia added by this study is the focus on the correlation between closeness of axon tract/glia association, and axon tract abnormality in the absence of glia. In other words: the nervous system is formed by a large number of fascicles, and these fascicles vary in their degree of glial wrapping. It would be misleading when carrying out a genetic study to only focus on a single fascicle (or small subset of fascicles), and extrapolate from the phenotype observed for this fascicle onto the brain as a whole. In this analysis, SATs of several lineages, notably those that in normal brains have little glia covering them in the neuropile, show few abnormalities in glia-less brains. By contrast, other lineages were affected in the majority of cases, and typically, these lineages also were the ones whose SATs were associated more closely with glia (Spindler, 2009).
Neuropile compartments are formed by terminal branches of axons and dendrites and their synapses. For example, the antennal lobe of insects consists of the axonal terminals of sensory neurons located in the antenna, and dendritic terminals of antennal projection neurons and local interneurons located in the deuterocerebrum, aside from a relatively small number of other modulatory neurons. Sensory neurons expressing the same olfactory receptor all converge onto the dendrites of a small number of projection neurons
to form an olfactory glomerulus. In many insects, notably Manduca, olfactory glomeruli are individually compartmentalized by neuropile glia, and it has been shown that glia plays a prominent role in establishing the glomeruli organization. According to the prevailing view, glomeruli are initially ordered by the specialized endings of sensory terminals into protoglomeruli; however, glial processes soon invade the space in between protoglomeruli and restrict the arborization of receptor axons. Therefore, in Manduca antennal lobes, glia is required for early maintenance of the glomerular map (Spindler, 2009 and references therein).
A recent analysis of the time course of glial development in the Drosophila antennal lobe suggested that in this species, glia plays an even lesser role, since glomeruli are only incompletely, and at a late time point, wrapped by glia. While glia were never ablated in those studies, later work found that elimination of Neuroglian from midline glia resulted in an inability of ORN axons to cross through the antennal commissural tract to the contralateral lobe. In this study, most adult brains lacking cortex and neuropile glia still form antennal lobes with glomeruli, however in some samples the glomeruli are poorly defined, and cannot be identified by their position and shape in the antennal lobe. The variability in phenotype penetrance likely stems from larvae containing the most glia surviving to eclosion; therefore, a relatively normal looking adult brain could stem from a weak glial apoptosis early in development. Whether the antennal lobe
disorganization is a secondary defect due to a lack of definition normally provided by the presence of glia, a result of SATs that normally contribute to the AL misguiding or truncating early, or a maintenance defect in which axons begin inappropriately intermingling among glomeruli is not clear. Perhaps the Drosophila antennal lobe glia is required for glomeruli maintenance even after glomeruli organization is established, and future studies will hopefully address this possibility (Spindler, 2009).
In the post-embryonic midline of Drosophila, ablation of the transient interhemispheric fibrous ring (TIFR), a transient population of midline glia, by ectopic expression of the pro-death gene hid, generates defects in the adult central complex. What is unclear, however, is the cellular event that is effected to cause the defects. This study suggests that morphological abnormalities in adult compartments from glial manipulation are due to the misguidance of larval SATs to the correct neuropile compartment in the brain, affecting the formation of adult neuropile structures. This study presents evidence that glia is an important mediator of axon guidance in the Drosophila larval brain; the mechanism for glia-neuron communication during this process is an exciting area for future investigation (Spindler, 2009).
Proper development requires coordination in growth of the cell types composing an organ. Many plant and animal cells are polyploid, but how these polyploid tissues contribute to organ growth is not well understood. This study found the Drosophila subperineurial glia (SPG) to be polyploid, and ploidy is coordinated with brain mass. Inhibition of SPG polyploidy caused rupture of the septate junctions necessary for the blood-brain barrier. Thus, the increased SPG cell size resulting from polyploidization is required to maintain the SPG envelope surrounding the growing brain. Polyploidization likely is a conserved strategy to coordinate tissue growth during organogenesis, with potential vertebrate examples (Unhavaithaya, 2012).
How different tissues coordinate their growth to form the final, properly sized organ is not well understood. SPG envelop neurons via septate junctions to create the blood-brain barrier during Drosophila development. This strategy creates a developmental conundrum, because neurons continue to divide and increase in mass, while SPG need to both maintain a tight seal and increase cell size to accommodate neuronal growth. This study has demonstrate that by increasing cell size via increases in ploidy, SPG can keep the cell number constant and maintain the septate junctions unperturbed by cytokinesis. The experiments suggest that there exists feedback between the polyploidizing SPG tissue and the mitotic neuronal tissue to coordinate SPG ploidy with brain lobe size. Consistent with this model, the SPG of the brain lobes, which must cover a larger surface area than those in the ventral cord, attain a higher level of ploidy and cell size. It remains to be determined why some SPG are multinucleated and others have a single polyploid nucleus and why this is restricted to SPG in the brain. This study presents a novel mechanism for how different tissues are scaled to size in a developing brain, as well as establishing a new paradigm for the use of polyploidization to coordinate growth of different tissues during organogenesis (Unhavaithaya, 2012).
The strategy of using polyploidization to coordinate tissue growth appears to be conserved in vertebrates and to function in other organ systems. Examples are the polyploidizing trophoblasts that surround the mitotic blastula during embryo implantation, as well as the polyploid keratinocytes situated atop the dividing basal cell layer in the skin. Both trophoblasts and keratinocytes form tight junctions. In trophoblasts, these are proposed to shield embryos from the potential harmful factors in the maternal environment, whereas in keratinocytes, the tight junctions provide a barrier for the skin epithelia. Polyploidy is thus a likely strategy by which these polyploid tissues achieve growth while maintaining tight junctions. The signaling pathways these tissues and the SPG use for growth coordination remain to be defined. The coordination of SPG and brain growth in Drosophila reveals a mechanism of organogenesis that may have evolved to act also in vertebrate tissues with polyploid cells to control proper development of the tissue layers (Unhavaithaya, 2012).
Glia perform diverse and essential roles in the nervous system, but the mechanisms that regulate glial cell numbers are not well understood. This study identified and characterized a requirement for the Hippo pathway and its transcriptional co-activator Yorkie in controlling Drosophila retinal and central brain glia proliferation. Yorkie is both necessary for normal glial cell numbers and, when activated, sufficient to drive glial over-proliferation. Yorkie activity in glial cells is controlled by a Merlin-Hippo signaling pathway, whereas the upstream Hippo pathway regulators Fat, Expanded, Crumbs and Lethal giant larvae have no detectable role. Functional characterization of Merlin-Hippo signaling was extended by showing that Merlin and Hippo can be physically linked by the Salvador tumor suppressor. Yorkie promotes expression of the microRNA gene bantam in glia, and bantam promotes expression of Myc, which is required for Yorkie and bantam-induced glial proliferation. These results provide new insights into the control of glial growth, and establish glia as a model for Merlin-specific Hippo signaling. Moreover, as several of the genes studied have been linked to human gliomas, the results suggest that this linkage could reflect their organization into a conserved pathway for the control of glial cell proliferation (Reddy, 2011).
Merlin was first identified as the product of a human tumor suppressor gene, NF2, loss of which in peripheral glial cells results in benign tumors. Merlin has also been identified as an inhibitor of gliomas. Observations in this study indicate that the role of Merlin as a negative regulator of glial cell proliferation is conserved from humans to Drosophila and, thus, that Drosophila can serve as a model for understanding Merlin-dependent regulation of glial growth (Reddy, 2011).
Studies in Drosophila imaginal discs first linked Merlin to Hippo signaling, and Merlin was subsequently linked to Hippo signaling in mammalian cells, including its role in meningioma. However, the tumor suppressor activity of Merlin has also been linked to other downstream effectors in mammals, including Erb2, Src, ras, rac, TORC1 (CRTC1 - Human Gene Nomenclature Database; see Drosophila CRTC) and CRL4 (IL17RB - Human Gene Nomenclature Database), creating some uncertainty regarding the general importance of the linkage of Merlin to Hippo in growth control. It was found that depletion of Merlin, depletion of other tumor suppressors in the Hippo pathway, or expression of an activated form of Yki, all result in similar glial overgrowth phenotypes. Moreover, depletion of Merlin increased nuclear localization of Yki, and depletion of Yki suppressed the overgrowth phenotype of Merlin. Together, these observations clearly establish that the glial overgrowth phenotype associated with Merlin depletion in Drosophila is mediated through the Hippo signaling pathway (Reddy, 2011).
A noteworthy feature of Hippo signaling in Drosophila glial cells is that Merlin appears to be uniquely required as an upstream regulator of Hippo signaling, as the Fat-dependent, Ex-dependent and Lgl-dependent branches have no detectable role. Glia might, thus, provide an ideal model for mechanistic investigations of the Merlin branch of Hippo signaling. Fat-Hippo signaling employs Fat as a transmembrane receptor and Dachsous as its transmembrane ligand, whereas Ex-Hippo signaling appears to employ Crumbs as a transmembrane receptor and ligand. By contrast, Drosophila transmembrane proteins that mediate extracellular signaling and interact with Merlin have not yet been identified. Distinct mechanisms might also be involved in signal transduction downstream of Merlin. Although there is evidence that Ex and Merlin can both influence Hippo activity, Ex, but not Mer, can directly associate with Hpo. Conversely, Merlin, but not Ex, can interact directly with Salvador, and Merlin, Salvador and Hippo can form a trimeric complex. Moreover, the kibra loss-of-function phenotype is weaker than expanded in imaginal discs, but comparable to Merlin, and it was found that depletion of kibra also has a significant effect on glial cell proliferation. Kibra is highly expressed in mammalian brain, and alleles of KIBRA (WWC1 - Human Gene Nomenclature Database) have been linked to human memory performance. The role of kibra in regulating glial cell numbers in Drosophila thus raise the possibility that the influence of KIBRA on human memory might reflect a role in glial cells (Reddy, 2011).
Finally, it is noted that although Hippo signaling has been investigated in several different organs in Drosophila, including imaginal discs, ovarian follicle cells, neuroepithelial cells and intestinal cells, these all involve roles in epithelial cells, in which upstream regulators of the pathway (e.g., Fat, Ex, Mer) all have a distinctive localization near adherens junctions. The identification of a requirement for Hippo signaling in glia is the first time in Drosophila that a role for the pathway has been identified in non-epithelial cells. Indeed, in previous studies it was found that Hippo signaling influences proliferation of neuroepithelial cells, but other neuronal cell types, including neuroblasts, ganglion mother cells and neurons, are insensitive to Yki (Reddy, 2011).
Considerable attention has been paid to genes for which mutation or inappropriate activation can cause over-proliferation of glial cells, resulting in glial tumors. However, less is known about the mechanisms required for normal glial growth. Through loss-of-function studies, several genes were identified that are essential for normal glial cell numbers, including yki, sd, ban, mad and myc. The requirement for yki, mad and sd, together with epistasis studies, identifies a requirement for active Yki in glial growth. This in turn implies that downregulation of Hippo signaling is important for normal glial growth. Understanding how this is achieved will provide further insights into the regulation of glial cell numbers (Reddy, 2011).
A requirement for Mad, together with its upstream regulator Thickveins (Tkv), in promoting retinal glial cell proliferation has been described previously. The current studies of glial cells, together with recent work in imaginal discs, emphasize that in mediating the growth-regulating activity of Hippo signaling, Yki utilizes multiple DNA-binding partners (i.e. Mad and Sd) in the same cells at the same time to regulate distinct downstream target genes required for tissue growth (Reddy, 2011).
Although Yki activity influenced glial cell numbers throughout the nervous system, direct analysis of cell proliferation by Ethynyl deoxyuridine (EdU) labeling revealed that retinal glia were more sensitive to Yki activation at late third instar than central brain glia, and significant induction of central brain glial cell proliferation was only observed when Yki activation was combined with Myc over-expression. Further studies will be required to define the basis for this differential sensitivity, but the implication that the proliferative response to Yki is modulated by developmental stage and/or glial cell type has important implications for diseases associated with both excess and deficits of glial cells (Reddy, 2011).
These studies in Drosophila delineate functional relationships among genes involved in the control of glial cell proliferation. Mammalian homologs of Merlin, Yki and Myc have been implicated in glioma. Although a mammalian homolog of ban has not been described, other miRNAs have also been linked to glioma. The current observations imply that these genes can be placed into a pathway, in which Merlin, through Hippo signaling, regulates Yki, Yki regulates ban, and ban regulates Myc. However, as expression of Myc alone did not lead to substantial overgrowth of glia, Yki and ban must also have other downstream targets important for the promotion of glial cell proliferation. Moreover, the observations indicate that a Yki-Sd complex is also required for glial growth. In addition to the well characterized downstream target Diap1, Yki-Sd complexes in glial cells might regulate Myc directly, as suggested by studies in imaginal disc, and might regulate cell cycle genes in conjunction with E2F1 (Reddy, 2011).
The influence of activated-Yki on a ban-GFP sensor, together with the observations that yki is not required for ban-mediated overgrowth, whereas ban is required for Yki-mediated overgrowth, position ban downstream of Yki. This is consistent with studies of Hippo signaling in imaginal discs, in which ban has also been identified as a target of Yki for growth regulation. The placement of Myc downstream of Yki and ban is supported by the observation that Myc levels can be increased by expression of ban or activated-Yki, and by genetic tests that indicate that Myc is required for Yki- and ban-promoted glial overgrowth. A mechanism by which ban can regulate Myc levels, involving downregulation of a ubiquitin ligase that negatively regulates Myc, was identified recently in imaginal discs, and might also function in glial cells. Myc has been reported to downregulate Yki expression in imaginal discs and, although whether a similar negative-feedback loop exists in glial cells has not been investigated, the synergistic enhancement of glial cell proliferation observed when Yki and Myc were co-expressed is consistent with this possibility, as the expression of both genes under heterologous promoters could bypass negative regulation of Yki by Myc (Reddy, 2011).
The Myc proto-oncogene is de-regulated or amplified in several human cancers, including gliomas. The sensitivity of Yki/ban-induced overgrowth to reduced Myc levels parallels studies of glioma models involving other signaling pathways. For example, Myc is upregulated by EGFR, and is limiting for EGFR-PI3K-induced glial cell overgrowth in a Drosophila glioma model, and p53 and Pten-driven glioma in mouse models is also Myc dependent. Considering the evidence linking Merlin and Yap to glial growth in mammals, and the identification of Myc as a downstream target of Yap in cultured cells, it is likely that Yap could also influence glial growth in mammals, in part, through regulation of Myc (Reddy, 2011).
Glial cells are essential for the development and function of the nervous system. In the mammalian brain, vast numbers of glia of several different functional types are generated during late embryonic and early fetal development. However, the molecular cues that instruct gliogenesis and determine glial cell type are poorly understood. During post-embryonic development, the number of glia in the Drosophila larval brain increases dramatically, potentially providing a powerful model for understanding gliogenesis. Using glial-specific clonal analysis this study found that perineural glia and cortex glia proliferate extensively through symmetric cell division in the post-embryonic brain. Using pan-glial inhibition and loss-of-function clonal analysis it was found that Insulin-like receptor (InR)/Target of rapamycin (TOR) signalling is required for the proliferation of perineural glia. Fibroblast growth factor (FGF) signalling is also required for perineural glia proliferation and acts synergistically with the InR/TOR pathway. Cortex glia require InR in part, but not downstream components of the TOR pathway, for proliferation. Moreover, cortex glia absolutely require FGF signalling, such that inhibition of the FGF pathway almost completely blocks the generation of cortex glia. Neuronal expression of the FGF receptor ligand Pyramus is also required for the generation of cortex glia, suggesting a mechanism whereby neuronal FGF expression coordinates neurogenesis and cortex gliogenesis. In summary, this study has identified two major pathways that control perineural and cortex gliogenesis in the post-embryonic brain and has shown that the molecular circuitry required is lineage specific (Avet-Rochex, 2012). The correct control of gliogenesis is crucial to CNS development and the Drosophila post-embryonic nervous system is a powerful model for elucidating the molecular players that control this process. This study has identified two separate glial populations that proliferate extensively and have defined the key molecular players that control their genesis and proliferation. Perineural and cortex glia both use insulin and FGF signalling in a concerted manner, but the requirements for these pathways are different in each glial type. The data suggest a model that describes the molecular requirements for post-embryonic gliogenesis in each of these glial types in the brain (Avet-Rochex, 2012). The results show that Pyramus is expressed by perineural glia to activate FGF signalling in adjacent glia and acts in parallel to InR/TOR signalling (activated by the expression of Dilp6). These two pathways act synergistically to generate the correct complement of perineural glia. The results also show that cortex glia proliferation is controlled by FGF signalling through FGFR (Htl) and the Ras/MAPK pathway. Pyr expression is required from both glia and neurons and acts non-cell-autonomously. Neuronal Pyr expression activates the FGFR on adjacent cortex glia, thereby coordinating neurogenesis and glial proliferation. InR is also partially required in cortex glia and is likely to signal through the Ras/MAPK pathway (Avet-Rochex, 2012). Using both pan-glial inhibition and LOF clonal analysis this study has shown that the InR/TOR pathway is required for perineural glia proliferation. InR/TOR signalling has widespread roles in nervous system development and a role has been demonstrated for this pathway in the temporal control of neurogenesis (Bateman, 2004; McNeill, 2008). InR can be activated by any one of seven DILPs encoded by the Drosophila genome, which can act redundantly by compensating for each other. dilp6 is expressed in most glia during larval development, including perineural and cortex glia, and that dilp6 mutants have reduced gliogenesis. The dilp6 phenotype is weaker than that associated with the inhibition of downstream components of the InR/TOR pathway, suggesting that other DILPs might be able to compensate for the absence of dilp6 expression in glia (Gronke, 2010). Pan-glial inhibition and clonal analysis also demonstrated that the FGF pathway is required for normal levels of perineural glia proliferation. FGF signalling is activated in perineural glia by paracrine expression of Pyr. Inhibition of either the InR/TOR or FGF pathway reduced perineural glia proliferation by about half, so tests were performed to see whether these two pathways act together. The data demonstrate that inhibition of both pathways simultaneously has a synergistic effect, suggesting that these two pathways act in parallel, rather than sequentially, and that their combined activities generate the large numbers of perineural glia found in the adult brain (Avet-Rochex, 2012). Cortex glia employ a molecular mechanism distinct from that of perineural glia to regulate their proliferation. Cortex glia have a clear requirement for InR, as InR mutant cortex clones are significantly reduced in size. The early events in post-embryonic gliogenesis are poorly understood, but FGF signalling is likely to be required during this stage as LOF clones for components of this pathway almost completely block cortex gliogenesis. These data suggest that InR acts in parallel to FGF signalling in these cells, as loss of InR combined with activation of FGF signalling only partially rescues the InR phenotype. Interestingly, the PI3K/TOR pathway is not required in cortex glia, suggesting that InR signals through the Ras/MAPK pathway to control cortex glia proliferation (Avet-Rochex, 2012). The FGF pathway in cortex glia responds to paracrine Pyr expression from both glia and neurons. Expression from both glia and neurons is required to activate the pathway and stimulate cortex gliogenesis. Neuronal regulation of glial FGF signalling enables cortical neurogenesis to modulate the rate of gliogenesis, so that the requisite number of glia are generated to correctly enwrap and support developing cortical neurons. Recent studies have also identified a mechanism by which DILP secretion by glia controls neuroblast cell-cycle re-entry in the Drosophila early post-embryonic CNS. Thus, neurons and glia mutually regulate each other's proliferation to coordinate correct brain development (Avet-Rochex, 2012). This study has shown that two major glial populations in the larval brain, perineural and cortex glia, are generated by glial proliferation rather than differentiation from neuroglioblast or glioblast precursors. Differentiation of most embryonic glia from neuroglioblasts in the VNC requires the transcription factor glial cells missing (gcm), which is both necessary and sufficient for glial cell fate. In the larval brain the role of gcm is much more restricted and it is not expressed in, nor required for, generation of perineural glia. Thus, the developmental constraints on gliogenesis in the embryonic and larval CNS are distinct. The larval brain undergoes a dramatic increase in size during the third instar, which might favour a proliferative mode, rather than continuous differentiation from a progenitor cell type (Avet-Rochex, 2012). Glial dysfunction is a major contributor to human disease. The release of toxic factors from astrocytes has been suggested to be a contributory factor in amyotrophic lateral sclerosis and astrocytes might also play a role in the clearance of toxic Aβ in Alzheimer's disease. Rett syndrome is an autism spectrum disorder caused by LOF of the transcription factor methyl-CpG-binding protein 2 (MeCP2). Astrocytes from MeCP2-deficient mice proliferate slowly and have been suggested to cause aberrant neuronal development. This hypothesis was recently confirmed by astrocyte-specific re-expression of Mecp2 in MeCP2-deficient mice, which improved the neuronal morphology, lifespan and behavioural phenotypes associated with Rett syndrome. Characterisation of the molecular control of gliogenesis during development might lead to a better understanding of such diseases (Avet-Rochex, 2012). During development, differentiation is often initiated by the activation of different receptor tyrosine kinases (RTKs), which results in the tightly regulated activation of cytoplasmic signaling cascades. In the differentiation of neurons and glia in the developing Drosophila eye, this study found that the proper intensity of RTK signaling downstream of fibroblast growth factor receptor (FGFR) or epidermal growth factor receptor require two mutually antagonistic feedback loops. A positive feedback loop was identified mediated by the Ras association (RA) domain-containing protein Rau (CG8965) that sustains Ras activity and counteracts the negative feedback loop mediated by Sprouty. Rau has two RA domains that together show a binding preference for GTP (guanosine 5'-triphosphate)-loaded (active) Ras. Rau homodimerizes and is found in large-molecular weight complexes. Deletion of rau in flies decreases the differentiation of retinal wrapping glia and induces a rough eye phenotype, similar to that seen in alterations of Ras signaling. Further, the expression of sprouty is repressed and that of rau is increased by the COUP transcription factor Seven-up in the presence of weak, but not constitutive, activation of FGFR. Together, these findings reveal another regulatory mechanism that controls the intensity of RTK signaling in the developing neural network in the Drosophila eye (Sieglitz, 2013).
During development, often single bursts of RTK activity suffice to direct important cellular decisions. In other cases, multiple rounds of RTK activation are required to trigger a certain reaction profile, and in yet other cases, such as in the developing eye imaginal disc, a sustained low level of EGFR activity is needed. This study identified the Drosophila RA domain containing protein Rau, which constitutes the first cell-autonomous positive feedback regulator acting on both EGFR- and FGFR-induced signaling. In the developing fly compound eye, it was found that sustained RTK activity is modulated through a positive feedback loop initiated by Rau, which is counterbalanced by the negative regulator Sprouty. The balance of these two regulatory mechanisms ensures the correct activity of EGFR- and FGFR-dependent signaling pathways in the developing eye (Sieglitz, 2013).
Within the RTK signaling pathway, different positive and negative feedback mechanisms have been identified. A prominent negative feedback mechanism is triggered by the secreted protein Argos. Argos expression is induced by RTK activation, and secreted Argos protein can sequester the activating ligand Spitz. In addition, intracellular proteins have been identified to exert a negative feedback function. Sprouty is the most prominent inhibitor of RTK activity and was shown in this study to act downstream of FGFR signaling as well as downstream of EGFR signaling. However, the precise point at which Sprouty intercepts RTK signaling is variable. In the developing fly eye, Sprouty acts upstream of Ras, whereas in the developing wing, Sprouty functions at the level of Raf. An additional negative feedback loop is mediated by the cell surface protein Kekkon, which is specific to EGFR signaling (Sieglitz, 2013).
Positive feedback loops are less frequent and may act through the transcriptional activation of genes that encode activating ligands. This is found in the ventral ectoderm of Drosophila embryos or in follicle epithelium, where the activity of the EGFR pathway is amplified by induction of the expression of its ligand Vein. In addition, the expression of rhomboid may be triggered, which subsequently facilitates the release of activating ligands. Together, these mechanisms ensure a paracrine-mediated amplification of the RTK signal and are thus likely not as effective in regulating RTK activity in single cells (Sieglitz, 2013).
Rau is a previously unidentified positive regulator of RTK signaling that acts within the cell. This study found that Rau function sustains both EGFR and FGFR signaling activity. Rau is a small 51-kD protein that harbors two RA domains, which are found in several RasGTP effectors such as guanine nucleotide–releasing factors. Pull-down experiments demonstrated that Rau preferentially binds GTP-loaded (activated) Ras. The Rau protein is characterized by two RA domains. Although both RA domains are able to bind Ras individually, single RA domains do not show any selectivity toward the GTP-bound form of Ras. Thus, the clustering of two RA domains promotes the selection of RasGTP. In agreement with this notion, it was found that Rau can form dimers or, possibly, multimers. In lysates from embryos, Rau is found in high–molecular weight protein complex, suggesting that it could interact with other components of the RTK signalosome. This way, RA domains are further clustered, and thus, GTP-loaded Ras may be sequestered. In addition, this may also contribute to the clustering of Raf, which is more active in a dimerized state. Moreover, it was recently shown that Ras signaling depends on the formation of nanoclusters at the membrane. This local aggregation may further promote interaction of Ras with Son of sevenless, which can trigger additional activation of the RTK signaling cascade. In addition, Rau harbors a class II PDZ-binding motif, suggesting that Rau can integrate further signals to modulate RTK signaling (Sieglitz, 2013).
The activity of the EGFR and the FGFR signaling cascades is conveyed in part through the transcription factor Pointed. Heterozygous loss of pointed significantly increases the rough eye phenotype evoked by loss of Rau function. Moreover, upon overexpression of the constitutively active PointedP1, rau expression was also increased. In line with this notion, CG8965/rau was also identified in a screen for receptor tyrosine signaling targets. Thus, the data suggest that Rau activation occurs after initial RTK stimulation through direct transcriptional activation through Pointed, which is similar to the activation of the secreted EGFR antagonist Argos (Sieglitz, 2013).
This study has dissected the role of Rau in differentiating glial cells of the fly retina. These glial cells are borne out of the optic stalk and need to migrate onto the eye imaginal disc where some of these cells differentiate into wrapping glial cells upon contacting axonal membranes. The development of these glial cells is under the control of FGFR signaling. Initially, low activity of FGFR signaling in these glial cells is permissive for expression of seven-up, which encodes an orphan nuclear receptor of the COUP-TF (COUP transcription factor) family that suppresses sprouty, but not rau, expression. Activation of Rau requires greater activity of FGFR, which is achieved only through interaction with axons. High activation of FGFR signaling also inhibits seven-up expression and thus relieves the negative regulation of sprouty. This negative regulation of COUP-TFII transcription factors by RTKs is also seen during photoreceptor development in the fly eye and appears to be conserved during evolution (Sieglitz, 2013).
In conclusion, the Rau/Sprouty signaling module provides effective means to sustain a short RTK activation pulse, for example, during cellular differentiation. It is proposed that Rau dimers or multimers assemble a scaffold that favors the recruitment of RasGTP, which then could more efficiently activate the MAPK cascade. Thus, ultimately, Rau may promote the formation of Raf dimers, which might confer robustness and increased signaling intensity. Future studies will reveal the precise conformations and complexes that enable Rau to modulate RTK signaling in fly development (Sieglitz, 2013).
Animals generally have either compound eyes, which have evolved repeatedly in different invertebrates, or camera eyes, which have evolved many times across the animal kingdom. Both eye types include two important kinds of cells: photoreceptor cells, which can be excited by light, and non-neuronal support cells (SupCs), which provide essential support to photoreceptors. Only a handful of studies, primarily on the compound eyes of Drosophila melanogaster, have demonstrated molecular similarities in SupCs. D. melanogaster SupCs (Semper cells and primary pigment cells) are specialized eye glia that share several molecular similarities with certain vertebrate eye glia, including Müller glia. This led to speculation as to whether there are conserved molecular signatures of SupCs, even in functionally different eyes such as the image-forming larval camera eyes of the sunburst diving beetle Thermonectus marmoratus. To investigate this possibility, an in-depth comparative whole-tissue transcriptomics approach was used. Specifically, the larval principal camera eyes were dissected into SupC- and retina-containing regions and the respective transcriptomes were generated. This analysis revealed several conserved features of SupCs including enrichment of genes that are important for glial function (e.g. gap junction proteins such as innexin 3), glycogen production (glycogenin), and energy metabolism (glutamine synthetase 1 and 2). To evaluate the extent of conservation, the transcriptomes were compared with those of fly (Semper cells) and vertebrate (Müller glia) eye glia as well as respective retinas. T. marmoratus SupCs were found to have distinct genetic overlap with both fly and vertebrate eye glia. These results provide molecular evidence for the deep conservation of SupCs in addition to photoreceptor cells, raising essential questions about the evolutionary origin of eye-specific glia in animals (Rathore, 2023).
Glial cells play structural and functional roles central to the formation, activity and integrity of neurons throughout the nervous system. In the retina of vertebrates, the high energetic demand of photoreceptors is sustained in part by Muller glia, an intrinsic, atypical radial glia with features common to many glial subtypes. Accessory and support glial cells also exist in invertebrates, but which cells play this function in the insect retina is largely undefined. Using cell-restricted transcriptome analysis, this study shows that the ommatidial cone cells (aka Semper cells) in the Drosophila compound eye are enriched for glial regulators and effectors, including signature characteristics of the vertebrate visual system. In addition, cone cell-targeted gene knockdowns demonstrate that such glia-associated factors are required to support the structural and functional integrity of neighboring photoreceptors. Specifically, this study shows that distinct support functions (neuronal activity, structural integrity and sustained neurotransmission) can be genetically separated in cone cells by down-regulating transcription factors associated with vertebrate gliogenesis (pros/Prox1, Pax2/5/8, and Oli/Olig1,2, respectively). Further, specific factors critical for glial function in other species are also critical in cone cells to support Drosophila photoreceptor activity. These include ion-transport proteins (Na/K+-ATPase, Eaat1, and Kir4.1-related channels) and metabolic homeostatic factors (dLDH and Glut1). These data define genetically distinct glial signatures in cone/Semper cells that regulate their structural, functional and homeostatic interactions with photoreceptor neurons in the compound eye of Drosophila. In addition to providing a new high-throughput model to study neuron-glia interactions, the fly eye will further help elucidate glial conserved "support networks" between invertebrates and vertebrates (Charlton-Perkins, 2017).
Astrocytes are critically important for neuronal circuit assembly and function. Mammalian protoplasmic astrocytes develop a dense ramified meshwork of cellular processes to form intimate contacts with neuronal cell bodies, neurites, and synapses. This close neuron-glia morphological relationship is essential for astrocyte function, but it remains unclear how astrocytes establish their intricate morphology, organize spatial domains, and associate with neurons and synapses in vivo. This study characterized a Drosophila glial subtype that shows striking morphological and functional similarities to mammalian astrocytes. The Fibroblast growth factor (FGF) receptor Heartless was demonstrated to autonomously control astrocyte membrane growth, and the FGFs Pyramus and Thisbe direct astrocyte processes to ramify specifically in CNS synaptic regions. It was further shown that the shape and size of individual astrocytes are dynamically sculpted through inhibitory or competitive astrocyte-astrocyte interactions and Heartless FGF signaling. The data identify FGF signaling through Heartless as a key regulator of astrocyte morphological elaboration in vivo (Stork, 2014).
Astrocytes are among the most abundant cell types in the mammalian CNS and fulfill diverse functions in brain development and physiology. In the mature brain, astrocytes buffer ions and pH, metabolically support neurons, and clear neurotransmitters. Astrocytes can sense neuronal activity, react with transient increases of intracellular calcium ion concentration, and in turn modulate neuronal activity. The diverse homeostatic and modulatory roles for astrocytes are essential for neuronal function, and evidence is mounting that this tight physiological relationship between astrocytes and neurons is highly regulated and provides astrocytes with the capacity to exert powerful and dynamic control over neuronal circuits (Stork, 2014).
Astrocytic functions are critically dependent on the intimate spatial relationship between astrocytes and neurons, and accordingly astrocytes exhibit a highly ramified morphology. Primary cellular extensions radiate from the soma of gray matter astrocytes, which then branch into hundreds of increasingly finer cellular processes, ultimately forming a dense meshwork in the brain that associates closely with synapses, neuronal cell bodies, and the brain vasculature. Intriguingly, individual mature mammalian astrocytes occupy unique spatial domains within the brain, apparently 'tiling' through a mechanism akin to dendritic tiling, such that the processes of neighboring astrocytes exhibit very limited overlap. Whether these unique spatial domains are functionally important remains a point of speculation (Stork, 2014).
Despite recent advances in understanding the molecular basis of astrocyte fate specification, control of synapse formation, and neuronal signaling, pathways regulating astrocyte morphogenesis in vivo remain poorly understood. While there appears to be a spatial restriction of astrocyte subtypes to particular regions of the vertebrate CNS, it is not clear whether astrocytes selectively associate with predetermined subsets of neurons. The morphology of individual mammalian astrocytes is quite variable, suggesting that sculpting of their morphology may be stochastic and shaped by cell-cell interactions (Stork, 2014).
This study characterizes a glial cell type in Drosophila remarkably similar to mammalian protoplasmic astrocytes. Drosophila astrocytes dynamically and progressively invade the synaptic neuropil late in embryonic development, associate closely with synapses throughout the CNS, and tile with one another to establish unique spatial domains. The Heartless FGF receptor signaling pathway was identified as a key mediator of astrocyte outgrowth into synaptic regions and the size of individual astrocytes. Through ablation studies, it was demonstrated that individual astrocytes have a remarkable potential for growth, and the establishment of astrocyte spatial domains is mediated by astrocyte-astrocyte inhibitory and/or competitive interactions. This work provides insights into cell-cell interactions governing astrocyte growth in vivo and demonstrates that the requirement for astrocytes is an ancient feature of the nervous system of complex metazoans (Stork, 2014).
Drosophila astrocytes form a highly ramified and dense meshwork of processes that infiltrate the entire neuropil and associate closely with synapses. This close spatial relationship is reminiscent of the mammalian 'tripartite synapse,' thought to be critical for neurotransmitter clearance and the modulation of synaptic activity during complex behaviors. In the L3 VNC, the majority of synapses were in close proximity to astroglial processes, although not directly ensheathed. Nevertheless, using the iGluSnFR reporter, it was demonstrated that local increases in extracellular glutamate readily reached astrocyte membranes, indicating that they are within the functional range of synapses (Stork, 2014).
Functional roles of Drosophila astrocytes also appear well conserved when compared to mammals. The glutamate transporter EAAT1 is expressed in Drosophila astrocytes and is essential for coordinated locomotor activity in larvae and prevention of excitotoxicity in the adult. This study demonstrates astrocyte-specific expression the GABA transporter Gat and partial loss of Gat impeded larval locomotion. GABA transporter inhibitors also impair larval coordinated locomotion, and Manduca and Trichoplusia Gat homologs are high-affinity GABA transporters, supporting the notion that gat-depleted animals experience disruption of GABA neurotransmitter clearance. Despite apparently normal CNS morphology, gat null animals die as late embryos. Astrocytic Gat is therefore essential for viability, and it is proposed that Gat plays a central role for astrocyte-mediated GABA clearance even before animal hatching (Stork, 2014).
Ca2+ microdomain signaling in mammalian astrocytes is emerging as a key mechanism by which astrocytes respond to and regulate neuronal activity. Drosophila cortex glia, cells closely associated with neuronal cell bodies, also exhibit microdomain Ca2+ oscillations, and glial Ca2+ signaling events can modulate fly circadian behavior and seizure activity. Interestingly, this study found Drosophila astrocytes exhibit spontaneous, local Ca2+ transients in vivo and seem to be coupled with respect to Ca2+ signaling: laser-induced injury of a single astrocyte in the larva induced an increase in intracellular calcium in the injured cell, which subsequently spread into neighboring astrocytes (Stork, 2014).
These data taken together argue strongly that Drosophila astrocytes will prove an excellent in vivo system in which to study many fundamental aspects of astrocyte biology and astrocyte-neuron interactions (Stork, 2014).
This study has shown that Drosophila astrocytes are critically important for animal survival. Partial ablation of mouse astrocytes during development also led to death at birth. Interestingly, astrocyte depletion by ~30% in selected spinal cord domains led to atrophy and loss of neuropil and synapses. In Drosophila larvae lacking the majority of astrocytes, gross CNS morphology was surprisingly normal. Therefore, fly astrocytes may not be strictly required for neuronal survival, although earlier ablation or ablations in the adult could yield different results. Alternatively, other subtypes of CNS glia (e.g., ensheathing or cortex glia) might functionally substitute for astrocytes and promote neuronal survival (Stork, 2014).
Depletion of astrocytes from large regions of the mammalian CNS did not lead to a repopulation of these zones by astrocytes from neighboring domains, suggesting that astrocytes possess a high regional specificity and low invasive behavior (Tsai, 2012). However, while dramatic movement of populations of astrocytes was not observed, it is less clear whether astrocytes at the border of astrocyte-depleted regions react more locally with increased growth. Regional astrocyte ablation studies in mammals followed by the use of markers that highlight single-cell astrocyte morphology will be essential to definitively resolve these question (Stork, 2014).
It has been proposed that astrocyte domain organization and association with specific subsets of neurons has an important role in the proper function of neuronal networks. While Drosophila astrocytes are quite stereotyped in cell number and cell body position, the domains of the neuropil covered by astrocyte processes show variability in size and shape. It therefore seems unlikely that individual astrocytes are genetically programmed to associate with particular regions of the brain or specific synapses (Stork, 2014).
Astrocytes appear to harbor a massive growth potential but exert a strong growth-inhibiting effect on one another. First, when adjacent cells are ablated, astrocytes expand their territories while tiling where they are in contact with other astrocytes. Second, when htl or dof mutant clones that failed to infiltrate the neuropil, the space adjacent to these clones was efficiently infiltrated by other astrocytes. Finally, while enhancing Htl signaling increased domain size, neighboring cells still 'tiled' and the overlap of astrocytic domains did not increase noticeably. How tiling of astrocytes occurs remains unclear but could be accomplished through contact-dependent growth inhibition or competition for neuropil growth factors. Nevertheless, based on the multiple lines of evidence presented in this study, it is proposed that astrocyte morphology is shaped dynamically during development by neuron-astrocyte and astrocyte-astrocyte interactions (Stork, 2014).
Finally, while the relative overlap of neighboring astrocytes appears to be higher in Drosophila compared to mammalian astrocytes, it is important to note from a mechanistic perspective that the size of a Drosophila astrocyte is smaller compared to mouse and that the absolute overlap of astrocyte processes in mouse and fly seem comparable. This discovery of tiling behavior in Drosophila suggests that fly and mammalian astrocytes may share common molecular mechanisms by which neighboring cells define their territories (Stork, 2014).
Loss of the FGF receptor Htl, its ligands Pyr and Ths, or the downstream signaling molecule Dof/Stumps blocked the infiltration of astrocyte processes into the neuropil, demonstrating that the Htl signaling pathway is critical for effective astrocytic growth into the synapse-rich neuropil. The level of Htl signaling is also critically involved in the regulation cell and domain size of astrocytes, with increased Htl signaling leading to increased astrocyte volume. Expression data, clonal analysis, and astrocyte-specific rescue experiments all indicate that Htl and Dof function autonomously in glia. Precisely where the FGF ligands Pyr and Ths are generated during development was more difficult to determine. However, based on its expression pattern and the ability to rescue astrocyte outgrowth when expressed in neurons, it is proposed that at least Ths is primarily derived from neurons (Stork, 2014).
Ectopic expression of Pyr or Ths away from the neuropil or astrocytic expression of a constitutively active form of Htl is able to partially restore astrocyte infiltration. These observations suggest a permissive role for the Htl signaling pathway in astroglial growth. However, expression of Pyr or Ths is also able to promote the outgrowth of ectopic astroglial branches outside of the neuropil, indicating that these ligands can provide directional cues for astrocyte outgrowth. Pyr and Ths appear different in their signaling abilities: single neuron expression revealed Pyr was unable to promote extension of astrocyte processes, while Ths drove robust astrocytic process outgrowth, suggesting that the promotion of outgrowth by Ths can act at a short range (Stork, 2014).
How can Pyr and Ths direct astrocyte process growth into the neuropil even when ectopically expressed? FGF signaling is critically dependent on heparan sulfate proteoglycans (HSPGs) in vivo. Two of the four HSPGs in Drosophila, Dally-like and Syndecan, have been reported to be prominently enriched in the embryonic neuropil, where they have been shown to act in Slit-dependent axon guidance. Expression of Sdc in the neuropil was confirmed, ectopic Sdc expression was found to be sufficient to redirect astrocyte membranes outside of the neuropil, and loss of Sdc led to a defect in the ventral migration of astrocyte cell bodies and, to a lesser extent, problems in early neuropil infiltration. Based on these observations it is speculated that Sdc plays a modulatory role in the development of astrocytes by concentrating the FGFs Pyr and Ths in the neuropil to drive directional infiltration even when the ligands are provided ectopically. Finally, Pyr and Ths might act redundantly with additional unidentified neuropil-restricted factors that can provide directional information for astrocytic process outgrowth (Stork, 2014).
ths null mutants showed a slight decrease in the number of astrocytes in late embryos and L3 larvae, while embryonic htlAB42 mutants did not show a reduction in cell counts. sdc mutants also showed a similar slight reduction in total cell number in L3 larvae. These data suggest that astrocytes are generated in the embryo at normal numbers in FGF-pathway mutants but that individual cells might be outcompeted by neighbors during process outgrowth, resulting in death of individual cells. Since it was not possible to uniquely identify the presumptive ventral cell among the dorsally located cells, it is not clear whether the nonmigrating presumptive ventral cells preferentially die or whether cell death is stochastic among all astrocytes. While the mechanism of such adjustment of cell numbers through cellular competition remains poorly understood, it might be based on competition for trophic factors or a more active form of cell killing by 'winning' neighbors. The data deepen understanding of the diverse roles FGF signaling plays in insect glial development, where FGFs have been shown to also regulate glial proliferation, survival, migration and ensheathment of axons, and glial wrapping of FGF2-coated beads in grasshopper (Stork, 2014).
FGF signaling has also been implicated in mammalian astrocyte development. Mammalian FGFs can act as mitogens for glial precursors and potentiate the ability of secreted factors CNTF and LIF to promote astroglial fate in neural progenitors. In addition, FGF application can induce maturation of astroglia in cell culture by controlling morphological stellation in two dimensions and the expression of GFAP and glutamine synthetase. FGF receptors 1-3 are expressed in astrocytes and their precursors. In particular, FGFR3 is highly enriched in the radial precursor cells in the ventricular zone and immature and mature astrocytes and in FGFR3 and other FGF pathway mutants, GFAP expression in astrocytes is perturbed in vivo. Furthermore FGFR1/2 mutants show a reduction in GFAP-positive astrocytes in the cortex and impaired Bergmann glia morphology in the cerebellum. The exact roles of mammalian FGFRs and their ligands in astrocyte ramification, association with neurons and synapses, and establishment of astrocytic domain size, however, remain to be tested. Observations of an essential requirement for FGF signaling in astrocyte development in vivo in Drosophila suggests that a detailed analysis of FGF signaling pathways in mammalian astrocyte development should prove fruitful. FGF signaling is known to be perturbed in glioma, and this study's observations of the key role for FGFs in astrocyte process outgrowth may ultimately provide insight into the highly invasive nature of glioma cells in the brain (Stork, 2014).
Neuronal information processing requires a large amount of energy, indicating that sugars and other metabolites must be efficiently delivered. However, reliable neuronal function also depends on the maintenance of a constant microenvironment in the brain. Therefore, neurons are efficiently separated from circulation by the blood-brain barrier, and their long axons are insulated by glial processes. At the example of the Drosophila brain, this study addressed how sugar is shuttled across the barrier to nurture neurons. Glial cells of the blood-brain barrier specifically take up sugars and that their metabolism relies on glycolysis, which, surprisingly, is dispensable in neurons. Glial cells secrete alanine and lactate to fuel neuronal mitochondria, and lack of glial glycolysis specifically in the adult brain causes neurodegeneration. This work implies that a global metabolic compartmentalization and coupling of neurons and glial cells is a conserved, fundamental feature of bilaterian nervous systems independent of their size (Volkenhoff, 2015).
The amyloid precursor protein (APP) is a structurally and functionally conserved transmembrane protein whose physiological role in adult brain function and health is still unclear. Because mutations in APP cause familial Alzheimer's disease (fAD), most research focuses on this aspect of APP biology. This study investigated the physiological function of APP in the adult brain using the fruit fly Drosophila melanogaster, which harbors a single APP homologue called APP Like (APPL). Previous studies have provided evidence for the implication of APPL in neuronal wiring and axonal growth through the Wnt signaling pathway during development. However, like APP, APPL continues to be expressed in all neurons of the adult brain where its functions and their molecular and cellular underpinnings are unknown. This study reports that APPL loss of function (LOF) results in the dysregulation of endolysosomal function in neurons, with a notable enlargement of early endosomal compartments followed by neuronal cell death and the accumulation of dead neurons in the brain during a critical period at a young age. These defects can be rescued by reduction in the levels of the early endosomal regulator Rab5, indicating a causal role of endosomal function for cell death. Finally, this study shows that the secreted extracellular domain of APPL interacts with glia and regulates the size of their endosomes, the expression of the Draper engulfment receptor, and the clearance of neuronal debris in an axotomy model. It is proposes that APP proteins represent a novel family of neuroglial signaling factors required for adult brain homeostasis (Kessissoglou, 2020).
Neuronal and glial progenitor cells exist in the adult Drosophila brain. The primarily glial progenitor cells rely on a microRNA, mir-31a, to inhibit the expression of a predicted E3 ubiquitin ligase, CG16947. Erroneous inheritance of CG16947 by the progeny when the neural progenitor cell divides leads to death of the progeny, however how CG16947 achieves glial cell death is unknown. This study has identified the interacting partner of CG16947 to be Cdk9. Reduction of cdk9 expression in glia causes glial loss; highlighting the importance of cdk9 in mediating the survival of glia. Further, glial loss observed in mir-31a mutants was prevented with adult-specific expression of cdk9 in glia. Biochemical evidence is provided that the binding of CG16947 to Cdk9 causes its degradation. Taken together, this data shows that cdk9 plays a role in the survival of adult glia in the Drosophila brain. Thus, a fine balance exists between mir-31a and CG16947 expression in the progenitor cells that in turn regulates the levels of cdk9 in the progeny. This serves to allow the progenitor cells to regulate the number of glia in the adult brain (Foo, 2017b).
CG16947 is expressed in the progenitor cells and mir-31a serves to limit its expression in the progenitor cells. When this interaction between the microRNA and the CG16947 transcript is abolished, CG16947 is aberrantly translated at higher levels, leading to increased inheritance by the progeny (Foo, 2017b).
This paper shows that depletion of cdk9 alone in adult glia can reduce the number of glia in the adult brain. Thus, cdk9 is necessary for adult glial survival. Additionally, this study shows that in the condition where there are fewer glia in the brain; as in the mir-31a mutants, the adult-specific overexpression of cdk9 in glia prevents glial loss. This shows that it is loss of cdk9 in adult glia that is the likely reason for the glial loss observed in mir-31a mutants (Foo, 2017b).
Using co-immunoprecipitation, evidence is provided that CG16947 binds to and interacts directly with cdk9 leading to its degradation. Taken together, the genetic results and the biochemistry suggest that the subsequent ubiquitination and degradation of cdk9 due to excessive inheritance of CG16947 by the progeny in mir-31a mutants, is the likely cause for glial cell death in these mutants. This study does not exclude the possibility that cdk9 is degraded by other mechanisms, but do show that cdk9 is necessary for glial cell survival. Given the role of cdk9 in controlling RNA-polymerase II-mediated transcription, it is possible that cdk9 works by facilitating the transcription of pro-survival genes (Foo, 2017b).
As an increase in glial number was not observed in the brain when cdk9 was overexpressed in adult glia, it suggests that cdk9 is not sufficient to generate more glial cells unless in aberrant conditions where glia number is reduced, as is in the case of the mir-31a mutants (Foo, 2017b).
Knocking down of CG16947 in progenitor cells did not lead to an increase in glia numbers in the brain. Thus, it suggests that glial numbers are very tightly regulated in the adult brain. The rational being that since high levels of CG16947 inherited by glia from the progenitor cells is detrimental to the survival of glia then, in the wildtype setting, if more glia than necessary are made, then knocking down of CG16947 in progenitor cells, would lead to increased survival of glia and more glia observed. But as more glia is not observed in either setting, when CG16947 is knocked down or cdk9 is overexpressed, it suggests that the number of glia that are made is very tightly regulated and no more than necessary glia are made by the progenitors in the adult central brain of flies (Foo, 2017b).
In mammals, there are several examples of the number of glia in the mature brain being tightly regulated by contact with a target. For instance, mammalian astrocytes are thought to be matched to blood vessels, mediated by the limited secretion of trophic factors from the vasculature. A similar reliance of oligodendrocytes on their target axons for survival has also been observed. This study shows that the number of glia can be in part, regulated from the progenitor cell itself, through controlled expression of CG16947, rather than reliance on the target of the glial cell. These results do not exclude the possibility that glial number in the Drosophila adult brain is regulated by another mechanism (Foo, 2017b).
The mechanism by which mir-31a expression in the progenitor cell is regulated and in turn CG16947 expression is regulated, has yet to be uncovered, however, this study provides evidence that misregulation of this fine balance between mir-31a with CG16947 in the progenitor cells and subsequent excessive degradation of cdk9 in the progeny can alter the number of glia in the adult Drosophila brain. Thus providing evidence for a mechanism by which the progenitor cell itself can regulate the number of progeny that are generated. This study does not exclude the possibility that cdk9 can be regulated in a mir-31a-CG16947-independent manner, but illustrates one way in which cdk9 expression can be regulated in glia (Foo, 2017b).
Chromatin remodeling plays a critical role in gene regulation and impacts many biological processes. However, little is known about the relationship between chromatin remodeling dynamics and in vivo cell lineage commitment. This study revealed the patterns of histone modification change and nucleosome positioning dynamics and their epigenetic regulatory roles during the in vivo glial differentiation in early Drosophila embryos. The genome-wide average H3K9ac signals in promoter regions are decreased in the glial cells compared to the neural progenitor cells. However, H3K9ac signals are increased in a group of genes that are up-regulated in glial cells and involved in gliogenesis. There occurs extensive nucleosome remodeling including shift, loss, and gain. Nucleosome depletion regions (NDRs) form in both promoters and enhancers. As a result, the associated genes are up-regulated. Intriguingly, NDRs form in two fashions: nucleosome shift and eviction. Moreover, the mode of NDR formation is independent of the original chromatin state of enhancers in the neural progenitor cells (Ye, 2016).
Stem cells reside in specialized microenvironments known as niches. During Drosophila development, glial cells provide a niche that sustains the proliferation of neural stem cells (neuroblasts) during starvation. This study finds that the glial cell niche also preserves neuroblast proliferation under conditions of hypoxia and oxidative stress. Lipid droplets that form in niche glia during oxidative stress limit the levels of reactive oxygen species (ROS) and inhibit the oxidation of polyunsaturated fatty acids (PUFAs). These droplets protect glia and also neuroblasts from peroxidation chain reactions that can damage many types of macromolecules. The underlying antioxidant mechanism involves diverting PUFAs, including diet-derived linoleic acid, away from membranes to the core of lipid droplets, where they are less vulnerable to peroxidation. The study reveals an antioxidant role for lipid droplets that could be relevant in many different biological contexts (Bailey, 2015).
Animals have evolved multiple mechanisms to protect themselves from the cumulative effects of age-related cellular damage. This study revealed an unexpected link between the TNF (tumour necrosis factor) inflammatory pathway, triggered by the metalloprotease ADAM17/TACE, and a lipid droplet (LD)-mediated mechanism of protecting retinal cells from age-related degeneration. Loss of ADAM17, TNF and the TNF receptor Grindelwald in pigmented glial cells of the Drosophila retina leads to age-related degeneration of both glia and neurons, preceded by an abnormal accumulation of glial LDs. The glial LDs initially buffer the cells against damage caused by glial and neuronally generated reactive oxygen species (ROS), but in later life the LDs dissipate, leading to the release of toxic peroxidated lipids. Finally, this study demonstrates the existence of a conserved pathway in human iPS-derived microglia-like cells, which are central players in neurodegeneration. Overall, this study has discovered a pathway mediated by TNF signalling acting not as a trigger of inflammation, but as a cytoprotective factor in the retina (Muliyil, 2020).
This paper reports a previously unrecognised role of ADAM17 and TNF in protecting Drosophila retinal cells from age- and activity-related degeneration. Loss of ADAM17 and TNF signalling in retinal glial cells causes an abnormal accumulation of LDs in young glial cells. These LDs disperse by about 2 weeks after eclosion (middle age for flies), and their loss coincides with the onset of severe glial and neuronal cell death. By 4 weeks of age, no intact glia or neurons remain. Cell death depends on neuronal activity: retinal degeneration and, to a lesser extent, LD accumulation are rescued in flies reared fully in the dark. LD accumulation does not merely precede, but is actually responsible for subsequent degeneration, because preventing the accumulation of LDs fully rescues cell death. The data indicate that Eiger/TNF released by ADAM17 acts specifically through the Grindelwald TNF receptor. Loss of ADAM17-mediated TNF signalling also leads to elevated production of mitochondrial ROS in glial cells, causing activation of the JNK pathway and elevated lipogenic gene expression. Together, these changes trigger cell death through the production of toxic peroxidated lipids. Importantly, toxicity is also contributed to by ROS generated by normal activity of neighbouring neurons. Finally, this study shows that a similar signalling module is conserved in mammalian cells: when ADAM17 is inhibited in human iPSC-derived microglial-like cells, the same series of events is seen: LD accumulation, elevated mitochondrial ROS and high levels of toxic peroxidated lipids (Muliyil, 2020).
It is proposed that TNF is an autocrine trophic factor that protects retinal pigmented glial cells from age-related cumulative damage caused by the ROS that are normal by-products of neuronal activity. This ADAM17/TNF protection system is located specifically in retinal glial cells, but its role is to protect both glia and neighbouring neurons. In the absence of this TNF cytoprotective pathway, severe early-onset retinal neurodegeneration is seen. The data imply that cells die by being overwhelmed by toxic peroxidated lipids when abnormal accumulations of LDs disperse. This occurs in Drosophila middle age, when LDs stop accumulating and begin to disperse, triggering the cytotoxic phase of the ADAM17-/- phenotype. It is important to emphasise that despite the ADAM17/TNF protection system being located specifically in retinal glial cells, there is neuronal involvement. Not only does TNF indirectly protect against neurodegeneration, but photoreceptor neurons are also significant sources of the ROS that generate the toxic peroxidated lipids in glia. More generally, this work provides a model for investigating more widely the functional links between ageing, cellular stress, lipid droplet accumulation and neurodegeneration. Indeed, in the light of the discovery that the pathway discovered in Drosophila is conserved in human microglia-like cells, it is significant that lipid droplets have been reported to accumulate in human microglia, cells that are increasingly prominent in the pathology of Alzheimer's disease and other neurodegenerative conditions (Muliyil, 2020).
In a Drosophila model of neuronal mitochondrionopathies, abnormal neuronal ROS production led to elevated neuronal lipid production, followed by transfer of the lipids to the PGCs, where LDs accumulated. In that work, lipase expression in neurons suppressed LD accumulation; in contrast, suppression was only observed when lipase was expressed in PGCs, not neurons, suggesting that in the case of ADAM17 mutants, the primary source of accumulating lipids is the glial cells. Despite not being able to detect a role for neuronal lipid production in ADAM17-/- mutants, it was found that photoreceptor neurons are significant sources of the ROS that generate the toxic peroxidated lipids in glia. Despite these differences between this work and what has been previously reported, a growing body of work points to a close coupling between ROS, the JNK pathway, lipid droplets and cellular degeneration, a relationship conserved in mammals. This study did not investigate the involvement of SREBP in mediating LD accumulation caused by ADAM17 loss, but its well-established connection with stress-induced and JNK-mediated lipid synthesis suggests that it is a likely additional shared component of this conserved regulatory axis (Muliyil, 2020).
It has become clear that LDs are much more than simply passive storage vessels for cellular lipids; they have multiple regulatory functions. Indeed, although this study highlighted a developing picture of an LD/ROS-dependent trigger of cell death, in other contexts LDs have protective functions against oxidative damage, both in flies and mammals. This may occur by providing an environment that shields fatty acids from peroxidation by ROS and/or by sequestering toxic peroxidated lipids. Although this superficially appears to contradict the theme of LD/ROS toxicity, it is important to recall that in LD-related cell death is not simultaneous with LD accumulation. In fact, degeneration temporally correlates with the dispersal of LDs in middle age, rather than their earlier accumulation. Together, the strands of evidence from several studies suggest that it is the combination of elevated ROS and the dispersal of abnormally high quantities of lipids from previously accumulated LDs that trigger death. This suggests that cells die by being overwhelmed by toxic peroxidated lipids when abnormal accumulations of LDs break down in the presence of high levels of ROS. Experiments with Brummer lipase are consistent with this idea: the Brummer lipase was expressed from early in development, thereby preventing abnormal LD accumulation, and this protected against cell death. This sequence of events implies the existence of a metabolic switch, when LDs stop accumulating and begin to disperse, triggering the toxic phase of ADAM17 loss. It will be interesting in the future and may provide insights into the normal ageing process, to understand the molecular mechanism of this age- and/or activity-dependent change (Muliyil, 2020).
ADAM17 is one of the most important ≈, it has been the focus of major pharmaceutical efforts, with a view to treating inflammatory diseases and cancer. It is therefore surprising that it has been very little studied in Drosophila. This is the first report of Drosophila ADAM17 mutants. This study also confirmed for the first time that Drosophila ADAM17 is indeed an active metalloprotease, able to shed cell surface proteins including the Drosophila TNF homologue Eiger. The only other description of Drosophila ADAM17 function is mechanistically consistent with the data, despite relating to a different physiological context. In that case, ADAM17 was shown to cause the release of soluble TNF from the fat body so that it can act as a long range adipokine (Agrawal, 2016). Other ADAM17 substrates in different developmental or physiological contexts cannot be ruled out, although the relatively subtle phenotype of null mutant flies implies that ADAM17 does not have essential functions that lead to obvious defects when mutated. Moreover, wdevelopmental defects or LD accumulation were not observed in any neuronal or non-neuronal ADAM17-/- larval tissues, suggesting that the mechanism reported in this study is both age and tissue specific (Muliyil, 2020).
Although TNF is sometimes viewed as a specific cell death-promoting signal, and the pathways by which it activates caspase-induced apoptosis have been studied extensively in flies and mammals, the response to TNF is in fact very diverse, depending on the biological context. Indeed, its most well-studied role in mammals is as the primary inflammatory cytokine, released by macrophages and other immune cells, and triggering the release of other cytokines, acting as a chemoattractant, stimulating phagocytosis, and promoting other inflammatory responses. However, TNF has not previously been shown to have trophic activity, protecting cells in the nervous system from stress-induced damage, although a link with the Nrf2/Keap1 redox pathway in cardiomyocytes provides an interesting parallel (Muliyil, 2020).
In conclusion, this work highlights three important biological concepts. The first is to identify a new function for the ADAM17/TNF pathway in a cytoprotective role that protects Drosophila retinal cells against age- and activity-dependent degeneration. This contrasts with its well-established roles in inflammation and cell death. Secondly, the existence is highlighted of a glia-centric cellular pathway by which the breakdown of accumulated LDs and ROS together participate in promoting stress-induced and age-related cell death. Finally, this study has shown that the core phenomenon of the ADAM17 protease, acting to regulate the homeostatic relationship between ROS and LD biosynthesis, is conserved in human microglial cells, which themselves are intimately involved in neuroprotection (Muliyil, 2020).
The mammalian brain contains many subtypes of glia that vary in their morphologies, gene expression profiles, and functional roles; however, the functional diversity of glia in the adult Drosophila brain remains poorly defined. This study defines the diversity of glial subtypes that exist in the adult Drosophila brain, show they bear striking similarity to mammalian brain glia, and identify the major phagocytic cell type responsible for engulfing degenerating axons after acute axotomy. Beuropil regions were found to contain two different populations of glia: ensheathing glia and astrocytes. Ensheathing glia enwrap major structures in the adult brain, but are not closely associated with synapses. Interestingly, it was found that these glia uniquely express key components of the glial phagocytic machinery (e.g., the engulfment receptor Draper, and dCed-6), respond morphologically to axon injury, and autonomously require components of the Draper signaling pathway for successful clearance of degenerating axons from the injured brain. Astrocytic glia, in contrast, do not express Draper or dCed-6, fail to respond morphologically to axon injury, and appear to play no role in clearance of degenerating axons from the brain. However, astrocytic glia are closely associated with synaptic regions in neuropil, and express excitatory amino acid transporters, which are presumably required for the clearance of excess neurotransmitters at the synaptic cleft. Together these results argue that ensheathing glia and astrocytes are preprogrammed cell types in the adult Drosophila brain, with ensheathing glia acting as phagocytes after axotomy, and astrocytes potentially modulating synapse formation and signaling (Doherty, 2009).
In an effort to identify distinct morphological subtypes of glial cells in the mature Drosophila brain, the MARCM system was used to generate small clones of glial cells labeled with membrane-tethered GFP. Larvae containing a hs-flipase allele and a wild-type chromosome arm for recombination were subjected to a short heat shock (37°C) and glial cells within clones were visualized by use of the pan-glial driver, repo-Gal4. The analysis of glial subtype morphology was focused mainly on the adult antennal lobe region owing to its well defined histology and accessibility to genetic manipulation. In the adult brain clones were identified resembling each of the three main types of glial cells found in embryos and larvae: (1) cortex glia, which resided outside the neuropil in regions housing neuronal cell bodies, ramified dramatically to surround individual cell bodies; (2) surface glia, which appeared as large flat cells enveloping the surface of the brain, did not extend any processes into the brain; and (3) neuropil glia, which were closely associated with the neuropil, extended membranes into synaptic regions, and surrounded large bundles of axons. As in the embryo and larva, glial cell bodies were not found within the neuropil, rather they resided at the edge of the neuropil (neuropil glia), in the cortex (cortex glia), or at the surface of the brain (surface glia) (Doherty, 2009).
Interestingly, the single-cell resolution provided by MARCM analysis allowed further subdivision of neuropil glia into two distinct morphological classes, 'ensheathing glia' and 'astrocytic glia.' Ensheathing glia appeared as flattened cells that lined the borders of the neuropil and subdivided regions of the brain by isolating neuropil from the surrounding cortex. Within the antennal lobe, ensheathing glial membranes surrounded individual glomeruli (the functional units of the antennal lobe) but did not extend into the synaptic regions of the glomeruli. In addition, an astrocyte-like cell type was identified that extended membrane processes deeply into the neuropil and ramified profusely in synaptic-rich regions. This latter cell type is referred to as the fly 'astrocyte', based on its striking morphological similarity to mammalian astrocytes, as well as the conserved expression of a number of molecular markers used to identify astrocytes in the mammalian brain. Mammalian astrocytes remove excess amounts of extracellular glutamate through the high-affinity excitatory amino acid transporters (EAATs), GLAST and GLT-1, which transports the glutamate into glial cells where it is then converted into glutamine by glutamine synthetase. It was found that Drosophila astrocytes also express the transporter EAAT1. While the adult antennal lobe was used as the primary model tissue in this study, the morphological glial subtypes described above were observed in all brain regions examined, suggesting that the results are generally applicable to glial populations throughout the adult Drosophila brain (Doherty, 2009).
Attempts were made to identify Gal4 driver lines that would allow unique labelling and and manipulation of these glial populations, with a major focus being to genetically subdivide neuropil glia (i.e., ensheathing glia versus astrocytes). To accomplish this, UAS-mCD8::GFP
was crossed to a previously described collection of embryonic and larval glial drivers (Ito, 1995), as well as a number of drivers generated for this study. The adult antennal lobe was studied to examine the morphology and spatial distribution of cell types marked by these drivers in a background with glial nuclei ({alpha}-Repo) and the neuropil ({alpha}-nc82) also labeled. The repo-Gal4 driver labeled all Repo+ glial subtypes in the adult brain, as evidence by {alpha}-Repo immunostaining in the nuclei of GFP+ cells. Membrane processes from Repo+ cells are found throughout the adult brain, and together they constitute the diverse collection of glial subtypes identified in single-cell MARCM analysis. Upon examination of a single glomerulus within the antennal lobe, membranes from Repo+ cells were found to both surround and invade glomeruli. All GFP expression in the adult brain driven by the repo-Gal4 driver can be suppressed by coexpression of Gal80 (a Gal4 inhibitor) under control of the repo promoter (repo-Gal80), arguing that repo-Gal80 can efficiently block Gal4-mediated activation of UAS-reporters in all adult brain glia (Doherty, 2009).
Two drivers, mz0709-Gal4 and alrm-Gal4, appeared to show very specific expression in ensheathing glia and astrocytes, respectively. Glial processes labeled by mz0709-Gal4 were found at the edge of the antennal lobe and extended deeply into the neuropil region. These flattened glial processes surrounded, but did not invade, individual glomeruli, and did not extend into the cortex region. With the exception of variable expression in a small number of neurons, all mz0709-Gal4-induced expression was suppressed by repo-Gal80, indicating that mz0709-Gal4 is largely specific to ensheathing glia. The generation of MARCM clones labeled with the mz0709-Gal4 driver resulted in the consistent labeling of ensheathing glia, but not astrocytes, within the antennal lobes. Reciprocally, alrm-Gal4 was found to be expressed exclusively in astrocytes. All cellular processes from cells labeled with alrm-Gal4 extended into the neuropil, showed a highly branched or tufted morphology, invaded individual glomeruli, and all alrm-Gal4-driven expression was suppressed by repo-Gal80. Additionally, single cell MARCM clones were found labeled with the alrm-Gal4 driver resulted in the consistent labeling of astrocytes, but not ensheathing glia. Together, these drivers are excellent tools to manipulate and functionally distinguish different subtypes of glia in the adult Drosophila brain (Doherty, 2009).
What are the functional roles for each glial subtype in the adult brain? Is each subtype responsible for a unique collection of tasks, or are all glial subtypes functionally equivalent? As a first step to determining the in vivo functional differences between adult brain glial subtypes the cell autonomy of glial phagocytic function was explored. Severing olfactory receptor neuron (ORN) axons by surgical ablation of maxillary palps leads to axon degeneration (termed Wallerian degeneration), recruitment of glial membranes to fragmenting axons, and glial engulfment of axonal debris. These glial responses are mediated by Draper, the Drosophila ortholog of the C. elegans cell corpse engulfment receptor cell death defective-1 (CED-1). In draper null mutants, glia fail to extend membranes to degenerating ORN axons and axonal debris is not removed from the CNS. Thus, Draper function should be autonomously required in phagocytic glial subtypes and Draper expression is predicted to act as a molecular marker for glial cells capable of performing engulfment functions (Doherty, 2009).
To define the precise cell types that express Draper, all glial membranes were labeled with mCD8::GFP driven by repo-Gal4, stained with α-Draper antibodies, and assayed for colocalization of Draper and GFP. Extensive overlap of Draper and GFP was found in this background. Draper and GFP signals overlapped at the edge of the neuropil, in membranes surrounding antennal lobe glomeruli, and in all cortex glia. This labeling was specific to Draper since expression of a UAS-draperRNAi construct with repo-Gal4 led to the elimination of all Draper immunoreactivity in the adult brain. Thus, the entire population of cortex glia appear to express Draper and are likely to be phagocytic. However, cortex glia do not extend membranes into the antennal lobe neuropil, even after ORN axon injury. Therefore, cortex glia are not likely responsible for clearing severed ORN axonal debris from the antennal lobe neuropil (Doherty, 2009).
Interestingly, when mCD8::GFP was driven by mz0709-Gal4 extensive overlap of Draper and GFP was observed in neuropil-associated ensheathing glia. A high-magnification view of the antennal lobe revealed Draper and mz0709-Gal4 labeled membranes colocalizing and surrounding, but not innervating individual glomeruli. Moreover, expression of UAS-draperRNAi in ensheathing glia with mz0709-Gal4 led to a dramatic reduction in Draper immunoreactivity in the neuropil, but the weaker Draper immunoreactivity in the cortex remained unchanged. Conversely, no overlap was observed between Draper and GFP when astrocytic membranes were labeled using the alrm-Gal4 driver. Furthermore, driving the expression of UAS-draperRNAi in astrocytes had no obvious effect on Draper expression in the brain. These results indicate that Draper is expressed in cortex glia and ensheathing glia but not in astrocytes (Doherty, 2009).
The specific expression of Draper in antennal lobe ensheathing glia suggests that this glial subset is the phagocytic cell type responsible for engulfing degenerating axonal debris after ORN axotomy. To explore this possibility, it was asked whether ensheathing glia or astrocytes extend membranes to severed axons after injury, and in which cell type Draper was required for clearance of axonal debris from the CNS. To assay extension of glial membranes to severed axons, glial membranes were labeled with mCD8::GFP, maxillary palp axons were severed, and colocalization of Draper and GFP was assayed in glomeruli housing severed ORN axons. Within 1 d after injury, Repo+ glial membranes were found to localize to glomeruli housing severed maxillary palp axons and these membranes were decorated with Draper immunoreactivity. Similarly, mz0709+ glial membranes also localized to severed axons and colocalized with intense Draper immunoreactivity 1 d after injury. Knockdown of Draper with UAS-draperRNAi using repo-Gal4 or mz0709-Gal4 completely suppressed the recruitment of both Draper and glial membranes to severed axons. In contrast, when astrocyte membranes were labeled with GFP no colocalization of GFP and Draper immunoreactivity was observed 1 d after axotomy. In addition, knockdown of Draper in astrocytes with UAS-draperRNAi did not suppress the recruitment of Draper to severed axons. In an effort to identify any indirect role for astrocytes during the injury response, the morphology of astrocytes was examined both before and after injury to determine whether they exhibited any overt changes in morphology or retracted their membranes from the site of injury to accommodate the recruitment of ensheathing glial membranes. However, no obvious changes were detected in morphology or in the positions of the astrocyte glial cells in response to axon injury. Together, these data indicate that Draper is required in ensheathing glia for recruitment of glial membranes and accumulation of Draper on severed ORN axons, and suggest that Drosophila astrocytic glia do not undergo any dramatic changes in morphology in response to ORN axotomy (Doherty, 2009).
From the above data it is predicted that ensheathing glia would act as phagocytes to engulf degenerating ORN axonal debris from the CNS. To test this a subset of maxillary palp ORN axons was labeled with mCD8::GFP using the OR85e-mCD8::GFP transgene, Draper function was knocked down in glial subsets using subset-specific driver lines, maxillary palp ORN axons were severed, and clearance of axons was assayed 5 d after injury. GFP-labeled axons were first severed in control animals with each driver and GFP+ axonal debris was found to be efficiently cleared from the CNS within 5 d after injury, confirming that glial phagocytic function is not affected in the driver lines. Strikingly, RNA interference (RNAi) knockdown of Draper using UAS-draperRNAi in a background with repo-Gal4 or mz0709-Gal4 completely blocked clearance of GFP-labeled axonal debris from the CNS, while RNAi knockdown of Draper in astrocytes with alrm-Gal4 had no effect on axon clearance. Thus, Draper is required autonomously in ensheathing glia for the clearance of degenerating ORN axonal debris from the CNS. In addition, knockdown of Draper in ensheathing glia with mz0709-Gal4 had no measurable effect on Draper expression in cortex glia, arguing that cortex glia are not capable of compensating for the loss of phagocytic activity in ensheathing glia during the clearance of axonal debris from the antennal lobe neuropil after axotomy. From these data on morphogenic responses to injury and phagocytic function, it is concluded that astrocytic, cortex, and ensheathing glia represent functionally distinct subsets of glial cells in the adult Drosophila brain (Doherty, 2009).
The Draper signaling pathway is a central mediator driving glial engulfment of neuronal cell corpses, and axons undergoing Wallerian degeneration in Drosophila. These studies show that within the adult brain neuropil, ensheathing glial cells are the only cell type that expresses Draper. Draper expression overlaps precisely with ensheathing glial membranes (when labeled with GFP driven by the ensheathing glia-specific driver mz0709-Gal4), and RNAi for draper in ensheathing glia leads to the elimination of Draper immunoreactivity in the neuropil. Similarly, it was found that dCed-6, the fly ortholog of C. elegans CED-6, a PTB-domain binding protein that functions genetically downstream of worm CED-1, is expressed in the adult brain in a pattern indistinguishable from Draper. dCed-6 immunoreactivity is strongly reduced in the neuropil through mz0709-Gal4-mediated knockdown (with UAS-dCed-6RNAi), and eliminated from the entire brain when dCed-6RNAi treatment is performed with the pan-glial driver repo-Gal4. Thus, in the adult brain dCed-6 appears to be expressed exclusively in glia, including cortex glia and ensheathing glia of the neuropil (Doherty, 2009).
Components of the Draper signaling pathway are required specifically in ensheathing glia for glial membrane recruitment to severed ORN axons, and clearance of degenerating axonal debris from the brain. Knocking down either draper or shark in ensheathing glia is sufficient to block recruitment of glial membranes and Draper to severed axons and clearance of degenerating axonal debris from the CNS. It is suspected that dCed-6 is also required in ensheathing glia for a number of reasons. First, dCed-6 and Draper show perfect overlap in expression in the neuropil, and Draper is only expressed in ensheathing glia. Second, ensheathing glia are recruited to degenerating axon injury, and dCed-6, like Draper, is specifically recruited to degenerating maxillary palp ORN axons after maxillary palp ablation. Third, dCed-6 expression is dramatically increased in ensheathing glia surrounding the antennal lobe after antennal ablation, similar to what has been found for Draper. Surprisingly, RNAi knock down of dced-6 in ensheathing glia (with mz0709-Gal4) failed to suppress the recruitment of Draper to severed ORN axons or the clearance of axonal debris from the brain. It is suspected that this is because mz0709-Gal4-mediated knockdown of dced-6 is incomplete in ensheathing glia, based on reduced but not eliminated staining in this background. Nevertheless, the observations that dCed-6 is localized with Draper in immunostains, is required in glia by RNAi knockdown with repo-Gal4, and that a null allele of dced-6 genetically interacts with draper null mutations in axon engulfment, argues strongly for a role for dCed-6 in ensheathing glia (Doherty, 2009).
Moreover, knockdown of draper in ensheathing glia, or suppressing glial engulfing activity by blocking endocytic function with Shibirets fully suppresses the recruitment of Draper to severed axons and clearance of degenerating axonal debris from the brain. In contrast, astrocytes do not express Draper (or dCed-6), fail to respond morphologically to axon injury, and knockdown of draper in astrocytes has no effect on the recruitment of Draper to severed axons or clearance of axonal debris from the CNS. Such a separation of phagocytic function in neuropil glial cells suggests similarities to the assigned functional roles in mammalian glia, with ensheathing glia as resident phagocytes, engulfing the majority or all axonal debris and astrocytes perhaps playing a less important role in phagocytosis of degenerating axons (Doherty, 2009).
It is concluded that ensheathing glia are the phagocytes of the central brain, responsible for engulfing degenerating ORN axons after axotomy. Based on their expression of Draper and dCed-6 it is proposed that cortex glia play a similar role in the cortex, perhaps engulfing degenerating axons in this tissue, or cell corpses generated during neuronal development or after brain injury. Drosophila astrocytes, in contrast, are in close association with synapse rich regions of the brain and it is speculated they likely play an important role in neural circuit and synapse physiology. This work represents the first functional dissection of glial subtypes in the central brain of adult Drosophila, and lays the foundation for future functional studies of these diverse classes of glia (Doherty, 2009).
Many aspects of glial development are regulated by extracellular signals, including those from the extracellular matrix (ECM). Signals from the ECM are received by cell surface receptors, including the integrin family. Previous studies have shown that Drosophila integrins form adhesion complexes with Integrin-linked kinase and talin in the peripheral nerve glia and have conserved roles in glial sheath formation. However, integrin function in other aspects of glial development is unclear. The Drosophila eye imaginal disc (ED) and optic stalk (OS) complex is an excellent model with which to study glial migration, differentiation and glia-neuron interactions. The roles of the integrin complexes was studied in these glial developmental processes during OS/eye development. The common β subunit βPS and two α subunits, αPS2 and αPS3, are located in puncta at both glia-glia and glia-ECM interfaces. Depletion of βPS integrin and talin by RNAi impaired the migration and distribution of glia within the OS resulting in morphological defects. Reduction of integrin or talin in the glia also disrupted photoreceptor axon outgrowth leading to axon stalling in the OS and ED. The neuronal defects were correlated with a disruption of the carpet glia tube paired with invasion of glia into the core of the OS and the formation of a glial cap. These results suggest that integrin-mediated extracellular signals are important for multiple aspects of glial development and non-autonomously affect axonal migration during Drosophila eye development (Xie, 2014).
This study found that OS glia express integrin complexes that play a role in the development of the glia and axons of the ED and OS. βPS integrin is located in puncta at the glial membrane and associates with Talin and ILK. These focal adhesion markers are found between the perineural glia (PG) and ECM, plus at the interfaces of the PG-CG (carpet glia) and CG-CG layers. A different distribution was found for the αPS2 and αPS3 integrins, which were concentrated at the periphery and interior of the OS, respectively. The results from RNAi-mediated knockdown revealed that these complexes play important roles in OS glial development, as knockdown led to disruption of PG and CG morphology. Specifically, the loss of βPS integrin or talin caused PG to aggregate in the distal half of the OS, resulting in an accumulation of glia in the OS. The PG formed clusters instead of a surrounding monolayer, suggesting that PG make integrin-mediated associations that maintain their distribution (Xie, 2014).
The PG migrate between the CG and the basal ECM, and loss of focal adhesions led to a disruption in PG migration, suggesting that integrin complexes on one or both surfaces play a role in mediating glia migration. The αPS2/βPS heterodimer binds ligands containing the tripeptide RGD sequence and αPS3/βPS binds laminins, so either or both could mediate adhesion of the PG to the ECM. However, it appears that depletion of the integrin complex in apposing glial layers is necessary to disrupt glial migration into the OS, as MARCM clones within the PG alone did not disrupt migration. Integrin function is conserved in mediating glial cell migration either on ECM or neighboring glial surfaces. For example, vertebrate glial studies found that integrins are involved in astrocyte, oligodendrocyte precursor and Schwann cell migration on various ECM molecules. Loss of β1-integrin in Bergmann glia leads to mislocalization, ectopic migration and disruption of process growth within the vertebrate cerebellum. ILK and CDC42 within Bergmann glia are required for the β1-integrin-dependent control of process outgrowth (Xie, 2014).
Reduction of focal adhesions disrupted the CG sheath and the integrity of the blood-nerve barrier, suggesting that maintenance of the CG tube also requires integrin-mediated adhesion. The disruption of the CG tube is similar to observations made in vertebrates, in which glial tubes are necessary for chain migration of neuroblasts along the rostral migratory stream; β1-integrin plays a role in both chain migration and maintenance of the glial tubes. However, the link between the integrin complex and the formation or stabilization of the CG tube is currently unknown (Xie, 2014).
The integrin complex appears to play a limited role in the migration of the WG into the OS. After knockdown of βPS or talin, WG with normal bipolar membrane processes were observed and WG mys1 MARCM clones had morphologies similar to control clones, suggesting that integrin signaling is not required for migration in differentiated WG. TEM analysis suggests that the WG failed to properly ensheath and segregate the bundles of photoreceptor axons, a phenotype consistent with that observed in vertebrate glia, although it is also possible that the lack of WG ensheathment is a secondary effect of axon stalling (Xie, 2014).
Loss of integrin complexes resulted in a failure of photoreceptor axons to exit the ED, navigate the OS or correctly target the optic lobe. Previously, blocking glial migration from the OS using dominant-negative Ras1 (Ras85D -- FlyBase) resulted in photoreceptor axons stalling in the ED but not the OS. The phenotype suggested that photoreceptor axons require physical contact with retinal glia to exit the ED. However, this mechanism does not seem to apply to the stalling phenotype observed with knockdown of the integrin complex. In the majority of samples, glia were still present in the ED and around the axonal stalling region, suggesting that the axon stalling phenotypes are likely to be due to a different mechanism (Xie, 2014).
It is possible that axon stalling results from a combination of glial changes affecting the multiple subtypes of OS glia and disruption of both the βPS/αPS2 and βPS/αPS3 adhesion complexes. Only the simultaneous loss of both αPS2 and αPS3 triggered the axonal phenotypes. Similarly, knockdown of βPS or talin within individual glial subtypes does not trigger axon stalling, whereas disruption of adhesion complexes in all glial subtypes does. It might be that only the repo-GAL4 driver is sufficiently strong or expressed early enough for the effective knockdown of integrin or talin to trigger the axon stalling phenotypes. However, the axon stalling phenotype can be effectively produced by delaying the expression of the RNAi with the repo-GAL4 driver until the second instar, suggesting that early expression is not key. Overall, the results suggest that it is the combined loss of the focal adhesion complex in multiple glial layers that led to the axon stalling phenotype, although the underlying mechanism is not known. It is possible that the simultaneous aggregation of the stalled PG and disruption of the CG sheath triggers axon stalling by allowing ectopic PG to enter the center of the OS or form the glial cap. The ectopic PG within the axon stalling area and in the glial cap were likely to be PG given their expression of LanB2, Apt and the lack of the WG Gli-lacZ marker. Normally, the PG migrate into the ED and differentiate into WG in the presence of photoreceptor axons. However, in the RNAi-treated OS many of the Apt-positive glia also expressed Gli-lacZ, suggesting a change in the normal differentiation pathway, perhaps owing to the premature and ectopic contact of the PG with the photoreceptors within the OS. Loss of integrins throughout the entire glial population could also lead to global changes to the ECM, as loss of integrins can alter the deposition of ECM components during epithelial morphogenesis. Although loss of integrins in the PNS does not lead to changes in the neural lamella of the peripheral nerve, it is possible that ECM changes in terms of structural integrity or the ability to recruit protein components could result in the multiple glial morphological changes (Xie, 2014).
In summary, this study has shown that glia in the OS and ED express integrins and Talin, through which they receive external signals important for PG migration, organization and CG barrier formation. The combined impact of integrin complexes on the morphology and development of both glial layers is crucial for proper axonal outgrowth through the OS and targeting in the brain (Xie, 2014).
The Drosophila antennal lobe is organized into glomerular compartments, where olfactory receptor neurons synapse onto projection neurons. Projection neuron dendrites also receive input from local neurons, which interconnect glomeruli. This study investigated how activity in this circuit changes over time when sensory afferents are chronically removed in vivo. In the normal circuit, excitatory connections between glomeruli are weak. However, after receptor neuron axons projecting to a subset of glomeruli were chronically severed by removal of antennae, it was found that odor-evoked lateral excitatory input to deafferented projection neurons was potentiated severalfold. This was caused, at least in part, by strengthened electrical coupling from excitatory local neurons onto projection neurons, as well as increased activity in excitatory local neurons. Merely silencing receptor neurons was not sufficient to elicit these changes, implying that severing receptor neuron axons is the relevant signal. When the neuroprotective gene Wallerian degeneration slow (WldS; Hoopfer, 2006) was expressed in receptor neurons before severing their axons, this blocked the induction of plasticity. Because expressing WldS prevents severed axons from recruiting glia, this result suggests a role for glia. Consistent with this, it was found that blocking endocytosis in ensheathing glia blocked the induction of plasticity. In sum, these results reveal a novel injury response whereby severed sensory axons recruit glia, which in turn signal to central neurons to upregulate their activity. By strengthening excitatory interactions between neurons in a deafferented brain region, this mechanism might help boost activity to compensate for lost sensory input (Kazama, 2011).
The results demonstrate that when all the ORN afferents to a subset of glomeruli are removed, excitatory interactions between glomeruli become stronger. As a result, deafferented PNs acquire robust responses to odors. Whereas normal PNs respond selectively to different odor stimuli, deafferented PNs respond nonselectively. This presumably reflects the fact that each excitatory LN (eLN; excitatory input to projection neurons) arborizes in most or all glomeruli. Thus, these PNs likely pool indirect excitatory input from all surviving ORNs (Kazama, 2011).
The key finding of this study is that that removing ORN input causes an upregulation of excitatory connections between glomeruli. Previously, it was shown that overstimulating one ORN type causes an upregulation of inhibitory input to a glomerulus (Sachse, 2007). Both of these phenomena may be seen as forms of compensatory plasticity. Compensatory plasticity also occurs in the mammalian olfactory bulb at several synaptic sites (Kazama, 2011).
Silencing electrical activity in ORNs was not sufficient to induce the same functional changes produced by severing ORN axons. This implies that the trigger is not the loss of electrical activity, but rather a molecular signal that is produced by severed axons. Mis-expressing WldS in ORNs blocks induction, and this implies that WldS suppresses the signal that severed axons produce. Suppressing endocytosis in ensheathing glia also blocks induction. This suggests that the signal produced by severed axons acts on glial receptors that require endocytosis for signal transduction. It is interesting that blocking endocytosis in astrocytes had no effect, because astrocytes interact with neurons in other systems. It is possible that astrocytes are involved in this process, but astrocytic endocytosis is not required (Kazama, 2011).
It is notable that both the manipulations that blocked the induction of plasticity (mis-expressing WldS in ORNs, or blocking endocytosis in ensheathing glia) also block the recruitment of ensheathing glia into deafferented glomeruli after ORNs are removed. This would appear to suggest that the same signal triggers both neural plasticity and morphological changes in glia. However, these signaling cascades clearly diverge: the recruitment of glial membranes to degenerating neurons is blocked by mutating the glial transmembrane receptor draper, whereas draper is not required for the plasticity described in this study. Interestingly, removing only one antenna was not sufficient to induce plasticity in glomerulus VM2 PNs. This manipulation kills half the ORNs that target these PNs. It should be noted that removing both palps kills fourfold fewer ORNs than removing one antenna, and this manipulation also affects fewer glomeruli, yet this was sufficient to induce plasticity in palp PNs. Removing both palps is also sufficient for glial mobilization and phagocytosis in the palp glomeruli (Doherty, 2009). The current results argue that the relevant factor is not the total number of afferents that are killed, but the proportion of live and dead axons in a given glomerulus. However, it also seems that killing all the ORNs that target a single glomerulus is not sufficient. This conclusion arises from the finding that removing the ipsilateral antenna did not produce potentiation in glomerulus V PNs, which receive strictly ipsilateral antennal input. This result implies that some minimum number of glomeruli must be completely deafferented to trigger the described phenomenon (Kazama, 2011).
The results indicate that after some ORNs are chronically removed, several changes occur in the antennal lobe circuit over time. First, depolarization propagates more effectively from eLNs to PNs. This could reflect increased gap junctional conductance from eLNs onto PNs. However, the possibility cannot be excluded that it is the result of a change in the intrinsic properties of eLNs that produces better propagation of voltages from the eLN soma to the site of the eLN-PN gap junctions. In this latter scenario, there would not necessarily be a change in gap junction conductance. Because good voltage clamp in eLNs cannot be achieved, these alternatives could not be evaluated directly, but two pieces of evidence argue for a change in the gap junction itself. First, the gap junction subunit composition of these electrical connections is evidently changed, because it was observed that electrical coupling from eLNs onto PNs is no longer completely dependent on the ShakB.neural subunit. Whereas in normal flies odor-evoked lateral excitation is abolished by the shakB2 mutation, which eliminates ShakB.neural, odor-evoked lateral excitation is not abolished in mutant antennal PNs after chronic antennal removal. Second, no significant change was found in any intrinsic properties of eLNs, including input resistance, resting potential, or excitability (Kazama, 2011).
A second change that occurs in chronically deafferented PNs is that spontaneous membrane potential fluctuations are larger in these PNs compared with acutely deafferented PNs. This may result from the increased input from eLNs onto PNs (Kazama, 2011).
A third change is that odors elicit stronger depolarization in eLNs. The intrinsic excitability of eLNs does not significantly increase, and therefore this change is likely caused by increased synaptic drive to eLNs. This potentiated synaptic drive may originate from PNs: because odor responses in deafferented PNs become larger after the induction of plasticity, and because PNs make chemical as well as electrical synapses onto eLNs, a net increase in the synaptic drive that PNs provide onto eLNs would be expected. In addition, it is possible that ORN-to-eLN synapses are potentiated (Kazama, 2011).
In sum, the net effect of these changes is to produce more robust activity in chronically deafferented PNs, compared with acutely deafferented PNs. These findings also help explain why plasticity is expressed globally rather than locally: if eLNs are responding more robustly to odors, and each eLN innervates all glomeruli, then this increased excitation should propagate across the antennal lobe (Kazama, 2011).
Whereas normal PNs are selective for odor stimuli, the potentiated odor responses of deafferented PNs are comparatively nonspecific. This presumably reflects the fact that each eLN arborizes in most or all glomeruli and so likely pools input from all surviving ORN types. Nevertheless, the odor responses of deafferented PNs may still be useful from the perspective of higher olfactory brain regions. Because acutely deafferented PNs regain normal levels of activity over time, this type of plasticity should tend to restore normal levels of activity in higher olfactory regions. This might help maintain the sensitivity of these regions to sensory signals, or maintain tropic support to these regions (Kazama, 2011).
More broadly, it is speculated that the phenomenon describe in thes study might reflect a general injury response in the Drosophila nervous system, and perhaps also a phenomenon that occurs during normal nervous system development. By triggering the upregulation of specific interactions between surviving neurons following the death of other neurons, this mechanism might help increase the number of neurons that are driven by active afferents. This could be a generally useful adaptation to neuronal death because it should tend to maintain total neural activity within a normal dynamic range (Kazama, 2011).
The reorganization of central sensory representations following changes in sensory input is generally thought to reflect changes in the strength of chemical synapses. The results suggest that central electrical synapses can also be persistently altered following sensory deafferentation. It is well known that neuromodulators can produce short-term changes in the strength of electrical synapses, as illustrated by studies in the vertebrate retina and crustacean stomatogastric ganglion. There are fewer examples of long-term changes in electrical synapse strength, but a growing literature suggests that this may be a fundamental mechanism of neural plasticity (Kazama, 2011).
The reorganization of central sensory representations following sensory deafferentation is sometimes assumed to be triggered by reduced electrical activity, not cell death. However, there is growing evidence that changes in electrical activity may produce synaptic plasticity via signaling pathways that are also linked to injury and inflammation. Thus, changes in electrical activity can produce synaptic plasticity by 'co-opting' signaling systems that are involved in injury responses. The results show that, in the Drosophila antennal lobe, some functional rearrangements following deafferentation can be specific responses to cell death signals, and are not necessarily induced by electrical silencing. In this study, reduced electrical activity was disambiguated from cell death because genetic tools were used to create 'undead' severed axons. The results are reminiscent of studies in vertebrates showing that sensory afferent death can produce changes in target brain regions that are not mimicked by electrical silencing using pharmacological manipulations (Kazama, 2011).
Finally, these findings provide a new window on neural-glial interactions. In mammals, there is good evidence that glia can modulate synaptic transmission and neural excitability. In both mammals and in Drosophila, glia also play important roles following injury. In particular, there are many instances of sensory afferent injury causing morphological changes in glia and glial proliferation in target brain regions . However, it is not entirely clear how such glial responses might affect neuronal physiology and sensory codes in these brain regions. The results illustrate specific cellular and synaptic changes in a sensory circuit that result from glial responses to sensory afferent injury. More broadly, the results illustrate the power of Drosophila as a genetically tractable model for studying neural-glial interactions in vivo (Kazama, 2011).
Axon pruning is involved in establishment and maintenance of functional neural circuits. During metamorphosis of Drosophila, selective pruning of larval axons is developmentally regulated by ecdysone and caused by local axon degeneration. Previous studies have revealed intrinsic molecular and cellular mechanisms that trigger this pruning process, but how pruning is accomplished remains essentially unknown.
Detailed analysis of morphological changes in the axon branches of Drosophila mushroom body (MB) neurons revealed that during early pupal stages, clusters of neighboring varicosities, each of which belongs to different axons, disappear simultaneously shortly before the onset of local axon degeneration. At this stage, bundles of axon branches are infiltrated by the processes of surrounding glia. These processes engulf clusters of varicosities and accumulate intracellular degradative compartments. Selective inhibition of cellular functions, including endocytosis, in glial cells via the temperature-sensitive allele of shibire both suppresses glial infiltration and varicosity elimination and induces a severe delay in axon pruning. Selective inhibition of ecdysone receptors in the MB neurons severely suppresses not only axon pruning but also the infiltration and engulfing action of the surrounding glia.
These findings strongly suggest that glial cells are extrinsically activated by ecdysone-stimulated MB neurons. These glial cells infiltrate the mass of axon branches to eliminate varicosities and break down axon branches actively rather than just scavenging already-degraded debris. It is therefore proposed that neuron-glia interaction is essential for the precisely coordinated axon-pruning process during Drosophila metamorphosis (Awasaki, 2004).
The dorsal and medial axon branches of the larval γ neurons have many varicosities along its trajectory that are clearly visualized when a single γ neuron is labeled with cytoplasmic-GFP (cGFP) reporter combined with the mosaic analysis with a repressible cell marker (MARCM) system and the enhancer-trap strain GAL4-201Y. These axon branches are also labeled strongly with the anti-Synaptotagmin antibody, which detects synaptic vesicle protein accumulated in the presynaptic sites. Thus, the larval γ neurons seem to be extensively interconnected with other neurons through synapses. Because synaptic terminals are condensed in the varicosities, the fate of these varicosity structures were first examined during the time course of axon pruning (Awasaki, 2004).
The number of varicosities on a dorsal axon branch was counted at various developmental stages. A single branch contained 20.7 and 21.4 varicosities on average in the brains of late third-instar larvae (L3) and in pupae just after puparium formation (0 hr APF), respectively. The number of varicosities was reduced by approximately 35% at 6 hr APF (an average of 13.6 varicosities per axon branch). To compare the timing of local axon degeneration and reduction of varicosity numbers, the frequency of disconnected axons was simultaneously examined. Only a very few axon branches were disconnected at 6 hr APF (2 of 60 observed axons) (Awasaki, 2004).
At 12 hr APF, approximately 90% of varicosities disappeared (an average of 2.0 varicosities per branch) and 75% of axons were disconnected (30 of 40). At 18 hr APF, more than 95% of varicosities disappeared (an average of 0.9 varicosities per branch) and 90% of axons were disconnected (48 of 53). In many cases, distal portions of disconnected axons remained at 12 hr APF, and only distal tips of axons frequently remained at 18 hr APF, suggesting that the shaft region of the axon branch tends to degenerate first. At 12 hr and 18 hr APF, the number of varicosities was drastically reduced even on the axons that were not yet disconnected (an average 2.8 [n = 10] and 1.4 [n = 5] varicosities per branch, respectively). These data suggest that disappearance of varicosities precedes local axon degeneration (Awasaki, 2004).
Pruning of axons is triggered by ecdysone through the complex of ecdysone receptor type B1 (EcR-B1) and Ultraspiracle (USP). If the disappearance of varicosities is associated with programmed axon pruning, the disappearance should be suppressed by inhibition of ecdysone signaling. To address this, axon branches of the clones of usp mutant γ neurons were examined. Both disappearance of varicosities and axon disconnection were significantly suppressed. Thus, these phenomena were regulated cell autonomously by the ecdysone-induced stimulation of γ neurons (Awasaki, 2004).
There are more than 2000 neurons that form the larval MB, and these results have indicated that disappearance of the varicosities proceeds gradually during the first 18 hr of the pupal stage. How do the varicosities disappear in these masses of axon branches? Do they disappear randomly, one by one, or in a coordinated process? To examine this, the detailed structure of the dorsal MB lobe was observed by using the enhancer-trap strain GAL4-201Y and the anti-Fasciclin II (FasII) antibody, both of which label axons of γ neurons from the larva to adult stage (Awasaki, 2004).
In the dorsal lobe of late larvae (L3), a mass of γ neuron axons was labeled uniformly with cGFP driven by GAL4-201Y and with anti-FasII antibody. At 6 hr APF, however, many hole-like structures of unlabeled regions appeared in cross-sections. Considering that the number of varicosities was significantly reduced at this stage whereas most axons had not been disconnected, it is highly likely that the hole-like structures correspond to the vestiges of disappeared varicosities (Awasaki, 2004).
To confirm this, lobes labeled with the targeted expression of the presynapse marker n-Synaptobrevin::GFP (nSyb::GFP) and the membrane bound reporter mCD8::GFP (mGFP) were compared. Like cGFP, nSyb::GFP strongly labels varicosities along the axon branches. Images of the sections at 6 hr APF clearly reveal the hole-like structures. In contrast, mGFP labels voluminous varicosities much less intensively than cGFP, because these subcellular regions contain less membrane surface per volume. mGFP labels the hole-like structures much less clearly than does cGFP. This further supports the hypothesis that the unlabeled regions are associated with the disappearance of varicosities (Awasaki, 2004).
Each hole-like structure was considerably larger than the size of a single varicosity. The diameter of many holes was more than 5 μm, whereas the size of the largest varicosity was 1.8 μm. This indicates that each unlabeled hole corresponds to the vestige of multiple varicosities. Because the varicosities of a single neuron were scattered along its axon branch, it is unlikely that a single hole corresponds to the varicosities that belong to a single neuron. Rather, neighboring varicosities from different neurons must simultaneously disappear in a cluster (Awasaki, 2004).
The larval neuropil of the central nervous system of Drosophila is covered by sheaths of neuropil-associating glial cells. Three-dimensional reconstruction of the confocal serial sections revealed a smooth surface of the MB dorsal lobe in the larval brain. At 6 hr APF, however, the surface appeared eroded from the outside. This suggests that glial cells surrounding the MB lobe might erode this neuropil structure. The morphologic changes of glial cells were therefore traced by using the pan-glial driver GAL4-repo and the cGFP reporter (repo>cGFP). In larvae, glial cells surrounded the lobe, but the axon branches inside the lobe were devoid of glial processes. At 6 hr APF, glial processes had infiltrated into the lobes and formed many lumps. The positions of these lumps correspond to the hole-like structures that were unlabeled with anti-FasII antibody (Awasaki, 2004).
Close observation of the glial processes revealed various patterns of infiltration at 6 hr APF. In some sections, thin glial processes surround parts of axon branches labeled with anti-FasII antibody. Glial processes also spread into the surrounded mass, forming glial lumps. Vesicle-like structures in these glial lumps contained small dots that were FasII-positive. In the specimens with massive glial infiltration, glial processes formed large lumps that occupied whole regions unlabeled with anti-FasII antibody (Awasaki, 2004).
Considering that dorsal lobes are initially filled with FasII-positive axon mass and hole-like structures unlabeled with anti-FasII antibody are eventually filled with glial lumps labeled with repo>cGFP, images like the ones described are best interpreted as being intermediate steps. Hypothetically it would seem that at first, infiltrating glial processes surround clusters of varicosities, which are still labeled with anti-FasII antibody. Next, glial processes further penetrate between varicosities to form lumps. FasII-positive signals observed in the glial lumps might be parts of varicosities engulfed by glia. Intensity of the FasII signal within glia is remarkably reduced compared to the signals observed outside of glia. This might be because engulfed FasII protein is subject to intracellular degradation. Finally, the FasII signal totally disappears, and glial lumps replace the vestiges of varicosities (Awasaki, 2004).
If this hypothesis has validity, infiltrated parts of glia must exhibit evidence of high activity in the intracellular degradation process. To examine whether this is the case, vital staining was performed with LysoTracker, which labels acidic organelles such as late endosomes and lysosomes. In the larval lobes, no detectable signal was found with the LysoTracker staining. In the dorsal lobe at 6 hr APF, however, the LysoTracker-positive signals were constantly found. In most cases, the signals were localized in the lumps of the infiltrated glia. This finding strongly suggests that varicosities are engulfed by glia and degraded in the endosome-lysosome pathway (Awasaki, 2004).
Though most of the varicosities disappear by 12 hr APF, glial infiltration was still observed at 12 hr and 18 hr APF. Glial processes at these stages frequently form columnar structures lying along the axon branches. These columnar structures are often observed in the shaft region of the dorsal lobes. The disconnection of axons also occurs frequently in the shaft region rather than in the tip region at 12 hr APF. Thus, the formation of the columnar structures was spatially and temporally correlated with the occurrence of axon degeneration. This suggests that infiltrated glia are involved in the disconnection and degeneration of axon branches as well as the disappearance of varicosities (Awasaki, 2004).
These results so far suggested that varicosities and axon branches are engulfed and degraded in the glial lumps. There are two possibilities for the role of glia during this process: (1) axon branches of γ neurons might be degenerated by intrinsic mechanisms and infiltrating glia might just scavenge the already degraded debris; (2) degradation of the axon branches of the γ neurons may not be due to intrinsic mechanisms alone and may require infiltrating glia to actively break down the axon branches. To determine the most likely possibility, the pruning process was observed while glial cell function was conditionally inhibited by the targeted expression of the temperature-sensitive allele of shibire (shits1). The shibire gene is the Drosophila homolog of dynamin, which is involved in cellular membrane functions, including endocytosis, phagocytosis, and membrane growth. Because Shits1 protein acts as a dominant-negative at a restrictive temperature, targeted expression of Shits1 in glial cells would inhibit endocytosis and other membrane-related functions (Awasaki, 2004).
Larvae expressing Shits1 in glial cells by using the GAL4-repo driver (repo>shits1) were raised at a permissive temperature (19°C) and then placed in the restrictive temperature (29°C) from 0 hr APF to either 6 hr or 18 hr APF. Raising repo>shits1 pupae at the restrictive temperature results in an abnormal morphology of the glial cells. The most striking abnormality of these glia is that they did not extend their processes into the lobe at all. Whereas glial processes in a normal condition have infiltrated into lobes intensely already at 6 hr APF, dorsal lobes of repo>shits1 in the restrictive temperature were devoid of any glial processes even at 18 hr APF. This demonstrates that Shits1 almost completely suppresses the infiltration of glial processes (Awasaki, 2004).
Anti-FasII labeling of dorsal lobes revealed hole-like structures of unlabeled regions at 6 hr APF, and severe loss of axon branches at 18 hr APF. Neither holes of unlabeled regions nor loss of axon branches were observed in the lobes of the repo>shits1 animal in the restrictive temperature at 6 hr and 18 hr APF. The phenotypes were qualitatively indistinguishable among different individuals. These indicate that axon pruning including both disappearance of clustered varicosities and local axon degeneration were severely suppressed in these pupae (Awasaki, 2004).
Because inhibition of glial infiltration caused severe suppression of axon pruning, it is highly likely that infiltrating glia actively break down and remove axon branches rather than just scavenging already degraded debris. This result, together with the engulfment and degradation of clustered varicosities by glial lumps, suggests that infiltrating glia actively eliminate clustered varicosities at the early phase of pruning (Awasaki, 2004).
If the pruning of γ neurons is suppressed by the targeted disruption of glial function, do the unpruned axon branches remain in the adult MB? The repo>shits1 pupae incubated at the restrictive temperature did not survive until adult stage; therefore, glial function could not be disrupted throughout pupal development. When the temperature was shifted from the restrictive temperature to 25°C at 24 hr APF, however, a small number of survivors eclosed. In these animals (n = 10), abnormal axon branches expressing abnormal FasII-positive signals were observed shortly after hatching beside the α and β lobes, which are adult-specific branches labeled with anti-FasII antibody. Such abnormal FasII-positive branches were observed only in the distal tip of the lobes, because continuous fibers could not be traced from them to the shaft region of the lobe. Moreover, the abnormal branches were no longer visible in the brains more than 7 days after eclosion. It is therefore likely that these FasII-positive branches were the remnants of the γ neuron axons that were not pruned at 18 hr APF but gradually degenerated thereafter. Thus, transient inhibition of glial function in early pupae does not completely block axon pruning but severely delays the time course of axon pruning (Awasaki, 2004).
Axon pruning is triggered by a surge of ecdysone, which occurs just before puparium formation. The γ neurons express the ecdysone receptor EcR-B1 strongly in late third-instar larvae. The surrounding glial cells express EcR-B1 faintly at this stage. The expression of EcR-B1 in the glial cells, however, is unlikely to be associated with axon pruning. Although EcR-B mutation causes complete suppression of axon pruning, this suppression is rescued by expression of EcR-B1 only in the γ neurons. How, then, is the glial activation controlled? Because expression of EcR-B1 in γ neurons is sufficient for axon pruning, the glial cells must be activated via ecdysone stimulation of γ neurons. To test this hypothesis, expression of a dominant-negative form of the ecdysone receptor was induced in γ neurons by using the 201Y driver (201Y>EcR-DN), which drives expression in γ neurons, but not in the surrounding glia. If the activation of glia is dependent on the ecdysone-induced stimulation of γ neurons, glial infiltration would be suppressed in these pupae. Conversely, if the glial activation is independent of γ neurons, infiltration would not be disturbed. The results strongly suggest that ecdysone-induced stimulation of γ neurons is indispensable for the infiltrating and engulfing action of glial cells. Once γ neurons receive ecdysone by EcR-B1, these neurons would induce glial infiltration and engulfment. Thus, the neuron-glia interaction is essential for coordinated axon pruning (Awasaki, 2004).
The neuron-glia interactions for selective infiltration and engulfment resemble the interactions between phagocytes and apoptotic cells. It is not likely, however, that axon pruning and apoptosis use identical molecular mechanisms to activate glial cells and phagocytes. Mutation of Drosophila apoptosis activator genes (grim, hid, and rpr) and overexpression of apoptosis inhibitor genes (p35 and DIAP1) in γ neurons have no effect on axon pruning. However, Draper (Drpr), the homolog of the C. elegans cell corpse engulfment molecule CED-1, accumulates on the cell membrane of infiltrating glial processes. Engulfment of apoptotic neurons by glial cells is suppressed in the central nervous system (CNS) of drpr mutant embryos. This raises the possibility that sensing of apoptotic cells by phagocytes and sensing of unwanted axon branches by glial cells share a common molecular mechanism. Although the drpr mutant is isolated in Drosophila, it is embryonic lethal. Furthermore, clonal analysis with a flippase-mediated MARCM system does not work reliably in the larval and pupal glial cells. Analysis of the function of drpr in the engulfing glial cells with more sophisticated techniques to overcome the above problem, together with the analyses of the involvement of phosphatidylserine and lysophosphatidylcholine in the axon pruning of γ neurons, might reveal correlated mechanisms between removal of the cell corpse and unwanted axons (Awasaki, 2004).
Inhibition of glial function almost completely suppresses axon pruning until 18 hr APF, but the remaining axons still undergo degeneration in the adult. There is therefore a possibility that cell-autonomous mechanisms can prune axon branches without glial involvement. The time course of degeneration, however, is severely delayed (Awasaki, 2004).
Rapid degeneration of γ neuron axons seems vital for proper remodeling of the MB neural circuit. In the late larval brain, the α'/β' neurons extend their axons into the core of the axon bundle of γ neurons. As γ neuron axons are pruned during the early pupal stage, α'/β' neurons claim the region previously occupied by γ neurons. When pruning of γ neurons is suppressed by ectopic expression of yeast ubiquitin protease, UBP2, the unpruned axons remain in the vicinity of axon branches of α'/β' neurons in the adult. The morphology of α'/β' axon branches was distorted in this case. This suggests that proper elimination of larval axon branches might be important for the correct formation of adult axon branches. Rapid pruning with the help of the engulfing action of glia would be advantageous in such a situation (Awasaki, 2004).
Although there are many examples of axon pruning, the underlying mechanisms have not been identified except for Drosophila γ neurons and the mossy fiber pathway of the mouse hippocampal neurons. While axon pruning in Drosophila γ neurons is mediated by the degeneration of axons, which occurs within approximately 18 hr, the axon pruning of the mossy fiber pathway requires the retraction of axons over 500 μm in length, which occurs over more than 30 days. Further identification of the cellular and molecular mechanisms that mediate degeneration and retraction of axons might elucidate the differences and similarities in the strategy of each type of axon pruning (Awasaki, 2004).
Larval motor neurons remodel during Drosophila neuro-muscular junction dismantling at metamorphosis. This study describes the motor neuron retraction as opposed to degeneration based on the early disappearance of β-Spectrin and the continuing presence of Tubulin. By blocking cell dynamics with a dominant-negative form of Dynamin, this study shows that phagocytes have a key role in this process. Importantly, the presence of peripheral glial cells is shown close to the neuro-muscular junction that retracts before the motor neuron. In muscle, expression of EcR-B1 encoding the steroid hormone receptor required for postsynaptic dismantling, is under the control of the ftz-f1/Hr39 orphan nuclear receptor pathway but not the TGF-β signaling pathway. In the motor neuron, activation of EcR-B1 expression by the two parallel pathways (TGF-β signaling and nuclear receptor) triggers axon retraction. This study interrupted TGF-β signaling in motor neurons using expression of dominate negative Wishful thinking. It is proposed that a signal from a TGF-β family ligand is produced by the dismantling muscle (postsynapse compartment) and received by the motor neuron (presynaptic compartment) resulting in motor neuron retraction. The requirement of the two pathways in the motor neuron provides a molecular explanation for the instructive role of the postsynapse degradation on motor neuron retraction. This mechanism insures the temporality of the two processes and prevents motor neuron pruning before postsynaptic degradation (Boulanger, 2012).
It is a general feature of maturing brains, both in vertebrates and in invertebrates, that neural circuits are remodeled as the brain acquires new functions. In holometabolous insects, the difference in lifestyle is particularly apparent between the larval and the adult stages. These insects possess two distinct nervous systems at the larval and adult stages. A class of neurons is likely to function in both the larval and the adult nervous systems. The neuronal remodeling occurring during this developmental period is expected to be necessary for the normal functioning of the new circuits (Boulanger, 2012).
The pruning of an axon can involve a retraction of the axonal process, its degeneration or both a retraction and degeneration. The MB γ axon is pruned through a local degeneration mechanism. In contrast, axons may retract their cellular processes from distal to proximal in the absence of fragmentation and this mechanism is called retraction. Interestingly, the two mechanisms can occur sequentially in the same neuron, as in the case of the dendrites of the da neurons, where branches degenerate and the remnant distal tips retract (Boulanger, 2012).
This study provides evidence that the motor neuron innervating larval muscle 4 (NMJ 4) is pruned predominantly through a retraction mechanism. The first morphological indication of motor neuron retraction is the absence of fragmentation observed with anti-HRP staining at the level of the presynapse in all the developmental stages analyzed, together with a decrease in perimeter size observed after 2 h APF. The continuity of this HRP staining is in contrast to the pronounced interruptions between blebs observed with an antibody against mCD8 in γ axons. A molecular indication of motor neuron retraction in these studies is the fact that β-Spectrin disappears at the synapse 5 h APF, before motor neuron pruning takes place. Indeed, it has been shown using an RNA interference approach that loss of presynaptic β-Spectrin leads to presynaptic retraction and synapse elimination at the NMJ during larval stages. The modifications of the microtubule morphology that were observed, such as an increase in microtubule thickness and withdrawal, provide additional evidence of axonal retraction during NMJ remodeling. Finally, a strong argument in favor of a motor neuron retraction mechanism is the fact that Tubulin is present at the NMJ throughout all stages of axonal pruning at the start of metamorphosis (0-7 h APF). This stands in clear contrast to the abolition of Tubulin expression observed before the first signs of γ axon degeneration. It is also interesting to note that the motor neuron retraction observed in this study at metamorphosis and at larval stages are morphologically different. During metamorphosis, retraction bulbs or postsynaptic footprints, which have been reported at larval stages, were never visualized. The fact that the postsynapse dismantles at metamorphosis before motor neuron retraction might explain these discrepancies. Worth noting is the mechanistic correlation between accelerated debris shedding observed here for NMJ pruning at the start of metamorphosis and axosome shedding occurring during vertebrate motor neuron retraction (Boulanger, 2012).
In vertebrates, glia play an essential role in the developmental elimination of motor neurons. In Drosophila, the role of glia in sculpting the developing nervous system is becoming more apparent. Clear examples of a role for engulfing glial cells in axon pruning are well documented during the MB γ axon degeneration at metamorphosis. Also, glia are required for clearance of severed axons of the adult brain. A distinct protective role of glia has been recently discovered during the patterning of dorsal longitudinal muscles by motor neurobranches. This study describes the presence of glia processes close to the end of the pupal NMJ. The observations suggest that the glial extensions retract at 5 h APF, just before motor neuron retraction is observed. When the glial dynamic is blocked, the NMJ dismantling might be also blocked. It is hypothesized that during development in larvae and early pupae, glial processes have a protective role and aid in the maintenance of the NMJ. Then, between 2 and 5 h APF, glial retraction would be a necessary initial step that allows NMJ dismantling. In accordance with this hypothesis, glia play a protective role in the maintenance of NMJ during pruning of second order motor neuron branches 31 h APF (Boulanger, 2012).
Disruption of shi function specifically in glial cells results in an unpruned mushroom body γ neuron phenotype and prevents glial cell infiltration into the mushroom body (Awasaki, 2004). One can note that at the NMJ the role of the glia is proposed to be essentially opposite from its role in MB γ axons pruning but in both cases blocking the glia dynamics results in a similar blocking of the pruning process (Boulanger, 2012).
In vertebrates, phagocytes are recruited to the injured nerve where they clear, by engulfment, degenerating axons. In Drosophila, phagocytic blood cells engulf neuronal debris during elimination of da sensory neurons. This study shows that blocking phagocyte dynamics with shi produces a strong blockade of the NMJ dismantling process. One possibility is that phagocytes attack and phagocytose the postsynaptic material, a process blocked by compromising shi function resulting in postsynaptic protection. In accordance, it has been shown that phagocytes attack not only the da dendrites to be pruned, but also the epidermal cells that are the substrate of these dendrites (Boulanger, 2012).
During NMJ dismantling, the muscle has an instructive role for motor neuron retraction. In all the situations where postsynapse dismantling is blocked, the corresponding presynaptic motor neuron retraction is also blocked. Therefore, it is sufficient to propose that both glial cells and phagocytes affect only the postsynaptic compartment. Nevertheless, one cannot rule out that these two cell types both act directly at the pre and at the postsynapse (Boulanger, 2012).
ECR-B1 is highly expressed and/or required for pruning in remodeling neurons of the CNS. MB γ neurons and antennal lobe projection neurons remodeling require both the same TGF-β signaling to upregulate EcR-B1. In the MBs only neurons destined to remodel show an upregulation of EcR-B1. At least two independent pathways insure EcR-B1 differential expression. The TGF-β pathway and the nuclear receptor pathway are thought to provide the necessary cell specificity of EcR-B1 transcriptional activation. This study shows that in the motor neuron pruning these two pathways are also necessary to activate EcR-B1. Noteworthy, showing an analogous requirement of ftz-f1/Hr39 pathway in two different remodeling neuronal systems unravels the fundamental importance of this newly described pathway (Boulanger, 2012).
The following model is proposed for the sequential events that are occurring during NMJ dismantling at early metamorphosis. First, EcR-B1 is expressed in the muscle under the control of FTZ-F1. FTZ-F1 activates EcR-B1 and represses Hr39. This repression is compulsory for EcR-B1 activation. Importantly, TGF-β/BMP signaling does not appear to be required for EcR-B1 activation in this tissue, however, a result of EcR-B1 activation in the muscle would be the production of a secreted TGF-β family ligand. Then, this secreted TGF-β family ligand reaches the appropriate receptors and activates the TGF-β signaling in the motor neuron. Finally, TGF-β signaling in association with the nuclear receptor pathway activates EcR-B1 expression resulting in motor neuron retraction. Since glial cells and phagocytes are required for the dismantling process, it is possible that a TGF-β/BMP family ligand(s) be produced by one or both of these cell types and not by the postsynaptic compartment. Noteworthy, a recent study shows that glia secrete myoglianin, a TGF-β ligand, to instruct developmental neural remodeling in Drosophila MBs (Awasaki, 2011). Nevertheless, one can note that the requirement of the two pathways in the motor neuron provides a simple molecular explanation of the instructive role of postsynapse degradation on motor neuron retraction. This mechanism insures the temporality of the two processes and prevents motor neuron pruning before postsynaptic degradation. It was proposed that in the MBs, the association of these two pathways provides the cell (spatial) specificity of pruning. In this paper, this association is proposed to provide the temporal specificity of the events. Future studies will be necessary to understand how EcR-B1 controls the production of a TGF-β/BMP ligand(s) in the muscle, the reception of this signal by the motor neuron and the ultimate response by the motor neuron to initiate retraction. These steps will be necessary to unravel the molecular mechanisms underlying the NMJ dismantling process and related phenomenon in vertebrate NMJ development and disease. Interestingly, it appears that TGF-β ligands on the one hand are positive regulators of synaptic growth during larval development and on the other hand, they are positive regulators of synaptic retraction, at the onset of metamorphosis. In both situations signaling provides a permissive role, sending a signal from the target tissue to the neuron. The consequence of this signal would be dependent on developmental timing thus, on a change in context (Boulanger, 2012).
Maintaining long, energetically demanding axons throughout the life of an animal is a major challenge for the nervous system. Specialized glia ensheathe axons and support their function and integrity throughout life, but glial support mechanisms remain poorly defined. This study identified a collection of secreted and transmembrane molecules required in glia for long-term axon survival in vivo. The majority of components of the TGFβ superfamily are required in glia for sensory neuron maintenance but not glial ensheathment of axons. In the absence of glial TGFβ signaling, neurons undergo age-dependent degeneration that can be rescued either by genetic blockade of Wallerian degeneration or caspase-dependent death. Blockade of glial TGFβ signaling results in increased ATP in glia that can be mimicked by enhancing glial mitochondrial biogenesis or suppressing glial monocarboxylate transporter function. It is proposed that glial TGFβ signaling supports axon survival and suppresses neurodegeneration through promoting glial metabolic support of neurons (Lassetter, 2023).
Precise neural circuit assembly is achieved by initial overproduction of neurons and synapses, followed by refinement through elimination of exuberant neurons and synapses. Glial cells are the primary cells responsible for clearing neuronal debris, but the cellular and molecular basis of glial pruning is poorly defined. This study shows that Drosophila larval astrocytes transform into phagocytes through activation of a cell-autonomous, steroid-dependent program at the initiation of metamorphosis and are the primary phagocytic cell type in the pupal neuropil. The developmental elimination was examined of two neuron populations-mushroom body (MB) gamma neurons and vCrz+ neurons (expressing Corazonin [Crz] neuropeptide in the ventral nerve cord [VNC])-where only neurites are pruned or entire cells are eliminated, respectively. MB gamma axons were found to be engulfed by astrocytes using the Draper and Crk/Mbc/dCed-12 signaling pathways in a partially redundant manner. In contrast, while elimination of vCrz+ cell bodies requires Draper, elimination of vCrz+ neurites is mediated by Crk/Mbc/dCed-12 but not Draper. Intriguingly, it was also found that elimination of Draper delayed vCrz+ neurite degeneration, suggesting that glia promote neurite destruction through engulfment signaling. This study identifies a novel role for astrocytes in the clearance of synaptic and neuronal debris and for Crk/Mbc/dCed-12 as a new glial pathway mediating pruning and reveals, unexpectedly, that the engulfment signaling pathways engaged by glia depend on whether neuronal debris was generated through cell death or local pruning (Tasdemir-Yilmaz, 2013).
Astrocytes serve as regulators of synapse formation and function and are generally supportive of neural circuits. Based on several lines of evidence, this study showed that astrocytes transform morphologically and functionally into phagocytes at pupariation and engulf significant amounts of neural debris. Phagocytic astrocytes take on a highly vacuolated appearance, up-regulate the engulfment molecule Draper, contain cytoplasmic vacuoles filled with debris that stains for synaptic or axonal markers, and exhibit high levels of lysosomal activity. Activation of this phagocytic program depends on cell-autonomous signaling through the EcR, since blockade of EcR in even single astrocytes suppressed their transformation by all morphological and molecular criteria. Blockade of astrocyte phagocytic function by multiple methods (e.g., EcRDN or Shits) suppressed the clearance of synapses throughout the CNS, axons of MB γ neurons in the central brain, and the neurites of vCrz+ cells in the VNC. This latter observation defines vCrz+ neurons as a new system to explore astrocyte-neuron interactions during neuronal apoptosis and neurite or synapse elimination in the CNS (Tasdemir-Yilmaz, 2013).
While roles for Drosophila glia in the engulfment of pruned MB γ axons have been previously described, it was unexpected that astrocytes would be the phagocytic cell type. In the adult Drosophila brain, a second type of neuropil glial cells, ensheathing glia, is responsible for engulfing injured axonal debris, while astrocytes fail to respond in a detectable way morphologically or molecularly (e.g., by Draper up-regulation). The stark difference in glial subtypes executing phagocytic function in the pupa versus the adult could result from differences in glial genetic programs during development versus in mature glia or indicate a key difference in the molecular nature of pruned neurites compared with those undergoing injury-induced axon degeneration (Tasdemir-Yilmaz, 2013).
The extent to which mammalian astrocytes are phagocytes in vivo has remained unclear until recently. In culture, astrocytes can be phagocytic and engulf apoptotic cells or amyloid-β. In vivo, the post-laminar optic nerve head myelination transition zone (MTZ) astrocytes express Mac-2, a molecule implicated in phagocytic activity, and internalize axonal evulsions, and this event appears to be increased in frequency in glaucoma models. Most impressively, it has been recently demonstrated that astrocytes engulf synaptic material in vivo and that this event is mediated by MEGF10 (mouse Draper) and the engulfment receptor MERTK. Thus, Draper/MEGF10-dependent engulfment of pruned synapses appears to be an ancient astrocytic mechanism; whether the same is true for the Crk/Mbc/dCed-12 pathway remains to be determined. Transcriptome data from purified mouse forebrain astrocytes support this notion, since molecular pathways for engulfment are highly enriched, including, in addition to MEGF10, Gulp1 (dCed-6), and in addition to Crk, Dock1 (Mbc) and Elmo (dCed-12). It will be important to explore whether these additional pathways have similar roles in astrocytes for the pruning of mammalian neural circuits during development and whether astrocyte engulfment activity is modified in neurological diseases involving axonal, dendritic, or synaptic loss (Tasdemir-Yilmaz, 2013).
Two types of neurons that undergo different types of developmental reorganization were examined. MB γ neurons prune their medial and dorsal axon branches and dendrites, their cell bodies remain viable, and, at midpupal stages, they re-extend medial axon branches to establish adult-specific connectivity. In contrast, vCrz+ neurons exhibit complete neurite degeneration, and cell bodies undergo apoptotic death and eventually are completely eliminated. In addition to there being remarkable differences in the patterns of fragmentation exhibited by these subsets of neurons, there are also critical differences in the engulfment signaling pathways used to promote their initial destruction and clearance (Tasdemir-Yilmaz, 2013).
Previous work has revealed a role for Draper in MB γ neuron pruning, with draper-null mutants exhibiting a delay of ~2 d in the clearance of pruned MB γ neuron axonal debris. This study provides evidence supporting a key role for the Crk/Mbc/dCed-12 complex in the clearance of MB γ neuron axonal debris and demonstrates that Crk/Mbc/dCed-12 knockdown and draper mutants have additive phenotypes, indicating that these signaling pathways act in a partially redundant fashion in astrocytes to promote clearance of pruned MB γ neuron axons. While these phenotypes are additive, it is noted that draper mutants exhibit a stronger delay in MB γ neuron pruning, suggesting that Draper signaling plays a more prominent role in this brain region than signaling through the Crk/Mbc/dCed-12 complex. However, the possibility that a Crk, mbc, or dCed-12 mutant might have a stronger phenotype than the RNAi lines used in this study cannot be excluded (Tasdemir-Yilmaz, 2013).
vCrz+ neuronal clearance appears to involve both Draper signaling and the Crk/Mbc/dCed-12 complex, with the former primarily promoting clearance of vCrz+ cell bodies, and the latter driving clearance of degenerating vCrz+ neurites. In draper-null mutants at 18 h APF, the majority of neuronal cell bodies remained at the edge of the neuropil, while neurite debris was largely cleared. Reciprocally, astrocytic knockdown of dCed-12 suppressed neurite clearance, while vCrz+ cell bodies were promptly eliminated. The lack of additivity of the phenotypes for either neurite or cell body clearance in draper-null mutants with astrocytic dCed-12RNAi suggests that elimination of cell bodies is primarily driven by the Draper pathway, while neurite clearance is largely accomplished by signaling through the Crk/Mbc/dCed-12 complex (Tasdemir-Yilmaz, 2013).
This study reveals that the genetic pathways engaged by glia to engulf pruned neuronal material versus apoptotic neurons are context-dependent and correlate with the type of destructive program. Interestingly, the molecular pathways that mediate axonal degeneration during axon pruning versus apoptosis are also distinct in mammalian cultured neurons. Local withdrawal of NGF in the axon activates Caspase 6-dependent axon degeneration, which is not sensitive to XIAP1 inhibition. In contrast, whole-cell NGF withdrawal leads to Caspase 6-independent apoptotic cell death and degeneration of axons, which is then sensitive to XIAP1. These observations argue that context matters when neurites and cell bodies are being destroyed. It is speculated that neuron-glia signaling during engulfment events might also be compartmentalized, with neurites and cell bodies generating different types of 'eat me' cues for clearance by glia (Tasdemir-Yilmaz, 2013).
Do engulfing glial cells actively promote the destruction of target neurons? The intriguing observation was made that loss of a single copy of draper is sufficient to dominantly suppress the elimination of vCrz+ cell bodies and neurites at 6 h APF. Moreover, loss of two copies results in the retention of nearly all vCrz+ cell bodies and significant parts of the vCrz+ scaffold, and many regions of the scaffold appeared morphologically intact, suggesting a delay of neurite fragmentation. Previous work has shown that expression of the anti-apoptotic molecule P35 is sufficient to suppress the pruning of vCrz+ neurites for at least 1 d; this study extended this observation and found this to be true for at least 2 d . Thus, blocking apoptosis in vCrz+ is sufficient to significantly delay neurite degeneration. This observation, coupled with the known role for CED-1 in actively promoting the death of engulfment targets, suggests that engulfing nonastrocyte glia may promote neuronal apoptosis through Draper signaling. This in turn would promote degeneration of the neurite scaffold after cell body death. If this underlies the delay that was observed in neurite degeneration in draper mutants, it would argue that glia actively sculpt neural networks by promoting the destruction of selected subsets of neurons. Subsequent work exploring the survival of cell bodies preserved in draper-null animals will be essential to explore this exciting possibility (Tasdemir-Yilmaz, 2013).
Axon pruning is an evolutionarily conserved strategy used to remodel neuronal connections during development. The Drosophila mushroom body (MB) undergoes neuronal remodeling in a highly stereotypical and tightly regulated manner, however many open questions remain. Although it has been previously shown that glia instruct pruning by secreting a TGF-beta ligand, myoglianin, which primes MB neurons for fragmentation and also later engulf the axonal debris once fragmentation has been completed, which glia subtypes participate in these processes as well as the molecular details are unknown. This study shows that, unexpectedly, astrocytes are the major glial subtype that is responsible for the clearance of MB axon debris following fragmentation, even though they represent only a minority of glia in the MB area during remodeling. Furthermore, astrocytes both promote fragmentation of MB axons as well as clear axonal debris and that this process is mediated by ecdysone signaling in the astrocytes themselves. In addition, this study found that blocking the expression of the cell engulfment receptor Draper in astrocytes affects only axonal debris clearance. Thereby this study uncoupled the function of astrocytes in promoting axon fragmentation to that of clearing axonal debris after fragmentation has been completed. This study finds a novel role for astrocytes in the MB and suggests two separate pathways in which they affect developmental axon pruning (Hakim 2014).
Nervous system injury or disease leads to activation of glia, which govern postinjury responses in the nervous system. Axonal injury in Drosophila results in transcriptional upregulation of the glial engulfment receptor Draper; there is extension of glial membranes to the injury site (termed activation), and then axonal debris is internalized and degraded. Loss of the small GTPase Rac1 from glia completely suppresses glial responses to injury, but upstream activators remain poorly defined. Loss of the Rac guanine nucleotide exchange factor (GEF) Crk/myoblast city (Mbc)/dCed-12 has no effect on glial activation, but blocks internalization and degradation of debris. This study shows that the signaling molecules Downstream of receptor kinase (DRK) and Daughter of sevenless (DOS) (mammalian homologs, Grb2 and Gab2, respectively) and the GEF Son of sevenless (SOS) (mammalian homolog, mSOS) are required for efficient activation of glia after axotomy and internalization/degradation of axonal debris. At the earliest steps of glial activation, DRK/DOS/SOS function in a partially redundant manner with Crk/Mbc/dCed-12, with blockade of both complexes strongly suppressing all glial responses, similar to loss of Rac1. This work identifies DRK/DOS/SOS as the upstream Rac GEF complex required for glial responses to axonal injury, and demonstrates a critical requirement for multiple GEFs in efficient glial activation after injury and internalization/degradation of axonal debris (Lu, 2014).
Anatomical, molecular, and physiological interactions between astrocytes and neuronal synapses regulate information processing in the brain. The fruit fly Drosophila melanogaster has become a valuable experimental system for genetic manipulation of the nervous system and has enormous potential for elucidating mechanisms that mediate neuron-glia interactions. This study shows the first electrophysiological recordings from Drosophila astrocytes and characterizes their spatial and physiological relationship with particular synapses. Astrocyte intrinsic properties were found to be strongly analogous to those of vertebrate astrocytes, including a passive current-voltage relationship, low membrane resistance, high capacitance, and dye-coupling to local astrocytes. Responses to optogenetic stimulation of glutamatergic pre-motor neurons were correlated directly with anatomy using serial electron microscopy reconstructions of homologous identified neurons and surrounding astrocytic processes. Robust bidirectional communication was present: neuronal activation triggered astrocytic glutamate transport via Eaat1, and blocking Eaat1 extended glutamatergic interneuron-evoked inhibitory post-synaptic currents in motor neurons. The neuronal synapses were always located within a micron of an astrocytic process, but none were ensheathed by those processes. Thus, fly astrocytes can modulate fast synaptic transmission via neurotransmitter transport within these anatomical parameters (MacNamee, 2016).
Understanding neuronal function at the local and circuit level requires understanding astrocyte function. This study provides a detailed analysis of astrocyte morphology and territory in the Drosophila third-instar ventral nerve cord where there already exists considerable understanding of the neuronal network. Astrocyte shape varies more than previously reported; many have bilaterally symmetrical partners, many have a high percentage of their arborization in adjacent segments, and many have branches that follow structural features. Taken together, these data are consistent with, but not fully explained by, a model of a developmental growth process dominated by competitive or repulsive interactions between astrocytes. The data suggest that the model should also include cell-autonomous aspects, as well as the use of structural features for growth. Variation in location of arborization territory for identified astrocytes was great enough that a standardized scheme of neuropil division among the six astrocytes that populate each hemi-segment is not possible at the third instar. The arborizations of the astrocytes can extend across neuronal functional domains. The ventral astrocyte in particular, whose territory can extend well into the proprioceptive region of the neuropil, has no obvious branching pattern that correlates with domains of particular sensory modalities, suggesting that the astrocyte would respond to neuronal activity in any of the sensory modalities, perhaps integrating across them. This study sets the stage for future studies that will generate a robust, functionally oriented connectome that includes both partners in neuronal circuits-the neurons and the glial cells, providing the foundation necessary for studies to elucidate neuron-glia interactions in this neuropil (Hernandez, 2020).
How the brain controls the need and acquisition of recovery sleep after prolonged wakefulness is an important issue in sleep research. The monoamines serotonin and dopamine are key regulators of sleep in mammals and in Drosophila. This study found that the enzyme arylalkylamine N-acetyltransferase 1 (AANAT1) is expressed by Drosophila astrocytes and specific subsets of neurons in the adult brain. AANAT1 acetylates monoamines and inactivates them, and it was found that AANAT1 limited the accumulation of serotonin and dopamine in the brain upon sleep deprivation. Loss of AANAT1 from astrocytes, but not from neurons, caused flies to increase their daytime recovery sleep following overnight sleep deprivation. Together, these findings demonstrate a crucial role for AANAT1 and astrocytes in the regulation of monoamine bioavailability and homeostatic sleep (Davla, 2020).
Characteristic features of sleep are conserved among species, and from humans to insects sleep is influenced by neural circuits involving monoamines such as serotonin and dopamine. Glial cells are known to take up and metabolize monoamines and they have been increasingly implicated in mechanisms of baseline and homeostatic sleep regulation in mammals and flies, but it remains unknown whether and how glia might influence monoaminergic control of sleep. Sleep is regulated by circadian clocks and a homeostatic drive to compensate for prolonged wakefulness, and growing evidence suggests that neural mechanisms controlling homeostatic sleep can be discriminated from those controlling baseline sleep. In Drosophila, mutants of arylalkylamine N-acetyltransferase 1 (AANAT1lo) were reported to have normal baseline amounts of sleep and motor activity, but increased recovery sleep ('rebound') following deprivation. AANAT1 corresponds to speck, a long-known mutation characterized by a darkly pigmented region at the wing hinge. AANAT1 can acetylate and inactivate monoamines in vitro, but the role of AANAT1 in vivo remains poorly understood (Davla, 2020).
This paper reports that AANAT1 is expressed in astrocytes and subsets of neurons in the adult Drosophila brain, with levels in astrocytes first rising then declining markedly overnight. In sleep deprived AANAT1 mutant flies, heightened recovery sleep is accompanied by increased serotonin and dopamine levels in the brain. With cell type selective AANAT1 knockdown, this study found that AANAT1 functions in astrocytes, but not neurons, to limit the amount of recovery sleep that flies take in response to sleep deprivation (SD). These findings identify a critical role for astrocytes in the regulation of monoamine bioavailability and calibration of the response to sleep need (Davla, 2020).
These findings illustrate a newly discovered role for astrocytes in the control of monoamine bioavailability and homeostatic sleep drive, where they are specifically engaged to catabolize monoamines whose levels are elevated by overnight SD. Drosophila astrocytes also express the enzyme Ebony, which couples dopamine to N-β-alanine, and a receptor for octopamine and tyramine, reinforcing how they are well-equipped to metabolize monoamines, and to monitor and respond to monoaminergic neuronal activity. Neither gene expression studies nor RNA sequencing databases provide evidence for monoamine-synthesizing enzymes in Drosophila astrocytes, so it appears likely that monoamines inactivated by AANAT1 in astrocytes are brought into these cells by an unidentified transporter. Astrocytes are particularly well-suited for regulating sleep in this way because they have ramified processes that infiltrate neuropil regions to lie in close proximity to synapses. SD can seemingly reduce the degree of contact between astrocytes and neurons in the fly brain, and so it is possible that these structural changes could influence monoamine uptake and inactivation by astrocytes (Davla, 2020).
In neurons, AANAT1 may function to limit sleep consolidation at night, but evidence for this came from only one of the two RNAi lines used in this study and was not observed in AANAT1lo mutants. Further studies are needed to characterize sleep-control functions of AANAT1 in neurons, if any, to understand better how cellular context can impact AANAT1 function in sleep regulation. In light of this, it is noted that loss of the related enzyme aanat2 in zebrafish larvae decreases baseline sleep, which could be attributed to a loss of melatonin since the AANAT1 product N-acetylserotonin is an intermediate in the synthesis of melatonin in vertebrates. Clearly, the appropriate balance and cellular context of AANAT activity is critical for the regulation of sleep, and this study shows in Drosophila that astrocytes are an important contributor to this balance. Interestingly, astrocytes in rodents express the monoamine transporters and receptors for dopamine and serotonin, raising the possibility that astrocytes in mammals might also participate in mechanisms of sleep regulation involving monoaminergic neural signaling (Davla, 2020).
Sleep is under homeostatic control, whereby increasing wakefulness generates sleep need and triggers sleep drive. However, the molecular and cellular pathways by which sleep need is encoded are poorly understood. In addition, the mechanisms underlying both how and when sleep need is transformed to sleep drive are unknown. Using ex vivo and in vivo imaging, this study shows in Drosophila that astroglial Ca(2+) signaling increases with sleep need. This signaling is dependent on a specific L-type Ca(2+) channel and is necessary for homeostatic sleep rebound. Thermogenetically increasing Ca(2+) in astrocytes induces persistent sleep behavior, and this phenotype was exploited to conduct a genetic screen for genes required for the homeostatic regulation of sleep. From this large-scale screen, TyrRII, a monoaminergic receptor required in astrocytes for sleep homeostasis, was identifed. TyrRII levels rise following sleep deprivation in a Ca(2+)-dependent manner, promoting further increases in astrocytic Ca(2+) and resulting in a positive-feedback loop. Moreover, these findings suggest that astrocytes then transmit this sleep need to a sleep drive circuit by upregulating and releasing the interleukin-1 analog Spätzle, which then acts on Toll receptors on R5 neurons. These findings define astroglial Ca(2+) signaling mechanisms encoding sleep need and reveal dynamic properties of the sleep homeostatic control system (Blum, 2020).
Although astrocytes have been implicated in the homeostatic regulation of sleep, their specific role and the underlying mechanisms are unresolved. The current data support a role for astrocytes as sensors of sleep need and define signaling mechanisms within these cells that mediate the integration and transmission of this information to a downstream homeostatic sleep circuit. In this model, neural activity is sensed by astrocytic processes, leading to an increase in Ca2+ levels, which depends at least in part on specific L-type voltage-gated Ca2+ channels (VGCC). Although astrocytes have been shown to exhibit hyperpolarized membrane potentials with small depolarizations, this particular subtype of L-type VGCC can be activated at substantially lower membrane potentials than other members of this channel family. Interestingly, two recent studies in mice found that intracellular Ca2+ levels in astrocytes vary across sleep/wake states and that Ca2+ signaling in these cells is required for normal sleep architecture and responses to sleep deprivation. These observations suggest a conserved role for astroglial Ca2+ signaling in sleep homeostasis (Blum, 2020).
The current model further suggests that, as the increased neural activity persists, Ca2+-mediated transcription of TyrRII is induced in astrocytes. TyrRII is relatively unstudied, but in vitro data suggest that it responds non-specifically to multiple monoamines. Thus, its upregulation in astrocytes should sensitize these cells to signaling via monoamines, which are intimately associated with wakefulness. The requirement for monoamines in this pathway may provide a logic gate for the system, imparting specificity to the signaling mechanism acting downstream of neural activity, whose semantic properties may be too broad. TyrRII itself is required for further Ca2+ elevations, forming a positive-feedback loop (Blum, 2020).
The data suggest that this amplification of astrocytic Ca2+ signals results in transcriptional upregulation of spz, the fly analog of IL-1. There is an accumulating body of evidence implicating IL-1 in sleep homeostasis in mammals, and the current findings demonstrating a functional role for astrocytic Spz in sleep homeostasis demonstrate that these mechanisms are conserved from invertebrates to vertebrates. In this model, Spz is released from astrocytes under conditions of strong sleep need and transmits this information by signaling to a central sleep drive circuit (the R5 neurons) to promote homeostatic sleep rebound. It is worth noting that fly astrocytes likely possess multiple output mechanisms to regulate sleep, as they not only activate sleep-promoting neurons (R5 neurons) but also inhibit arousal-promoting neurons (l-LNvs) (Blum, 2020).
From a broader perspective, this model draws attention to a fundamental, yet poorly understood, aspect of sleep homeostasis-how a highly dynamic input (i.e., neural activity operating on the millisecond timescale) is integrated and transformed to generate a sleep homeostatic force that functions on a significantly slower timescale. Although the precise identity of the signals embodying sleep need remain unclear, there is substantial experimental and conceptual support for the notion that neural activity increases with wakefulness and is a key trigger for this process. Yet the dynamic mechanisms by which this neural activity and, by extension, sleep need are transformed to sleep drive are unknown. The homeostatic regulation of processes and behaviors involving bistable states, such as sleep versus wakefulness, requires a prominent delay between the detection of the disturbance and the generation of the response. In addition, the stability and switching between such bistable states can be facilitated by positive-feedback loops. It is speculated that the transcription/translation of TyrRII, coupled with the generation of a positive-feedback loop, provide a timing delay followed by a more rapid elevation in astroglial Ca2+ after reaching a set threshold, thus enabling a non-linear response to the continual sampling of sleep need. The transcriptional/translation upregulation of Spz could represent an additional layer of delay (Blum, 2020).
Tetrad synapses are formed between the retina photoreceptor terminals and postsynaptic cells in the first optic neuropil (lamina) of Drosophila. They are remodelled in the course of the day and show distinct functional changes during activity and sleep. These changes result from fast degradation of the presynaptic scaffolding protein Bruchpilot (BRP) by Cryptochrome (CRY) in the morning and depend on BRP-170, one of two BRP isoforms. This process also affects the number of synaptic vesicles, both clear and dense-core, delivered to the presynaptic elements. In cry01 mutants lacking CRY and in brpΔ170, the number of synaptic vesicles is lower in the morning peak of activity than during night-sleep while in wild-type flies the number of synaptic vesicles is similar at these two time points. CRY may also set phase of the circadian rhythm in plasticity of synapses. The process of synapse remodelling stimulates the formation of clear synaptic vesicles in the morning. They carry histamine, a neurotransmitter in tetrad synapses and seem to be formed from glial capitate projections inside the photoreceptor terminals. In turn dense-core vesicles probably carry synaptic proteins building the tetrad presynaptic element (Damulewicz, 2020).
The results confirmed earlier studies that the presynaptic element (T-bar) of tetrad synapses is remodelled during the day and night and this rhythm is regulated by light and circadian clock (Gorska-Andrzejak, 2013; Woznicka, 2015). In the present study, it was found additionally that cyclic changes occur in the T-bar ultrastructure and its volume. Ultrastructural changes in T-bars and synaptic vesicles were possible because of using high resolution electron microscope tomography (EMT). In the present study it was also found that the number of synaptic vesicles cycles during the day, but this rhythm is masked by light. This result indicates that in the case of tetrad synapses, which are sites of fast neurotransmission between photoreceptors and the first order interneurons, intense neurotransmission occurs during the morning peak of locomotor activity, when more synaptic vesicles are attached to the T-bar platform than during sleep (ZT16) and are transported from capitate projections located next to the T-bars. At ZT16, tetrad synapses are ready for neurotransmission, since the total number of synaptic vesicles near tetrad synapses is similar at ZT1 and ZT16, but it is in a standby mode with lower frequency of synapses and vesicles, which are not attached to the T-bar platform and are not delivered from glial cells, respectively. However, transmission can be activated and is efficient because synapses during sleep have larger volume, and synaptic vesicles can be transported to the T-bar platform, if necessary, in response to an unexpected light pulse. Cyclic remodelling of tetrad presynaptic sites depends on BRP, which must be delivered to T-bars and degraded after light exposure after binding to CRY. This fast remodelling of synapses affects the number of vesicles transported to the presynaptic element (Damulewicz, 2020).
The present study was carried out only in light/dark (LD 12:12) conditions because in earlier studies it was found that the rhythm of the changes in BRP abundance in tetrad synapses of D. melanogaster is circadian. The rhythm is maintained in constant darkness (DD) and abolished in the per01 clock null mutant. On the basis of these results, it is assumed that rhythmic changes during the 24 h cycle reported is this paper are also circadian; however, in DD, the morning peak in BRP is not present because it depends on light (Damulewicz, 2020).
Electron microscope tomography (EMT) used in this study showed that synaptic vesicles are attached to the platform of the T-bar with filamentous proteins that are reduced in brpΔ170 and brpΔ190 mutants, which have fewer synaptic vesicles compared with wild-type flies. Two types of vesicles, clear and dense-core were detected. Dense-core synaptic vesicles were less numerous than clear ones. It is known that synaptic vesicles of tetrad synapses contain histamine, while the content of dense-core vesicles is unknown. It is possible that they carry presynaptic proteins to the T-bar. The comparison of ultrastructure of tetrad synapses in the morning peak of activity (ZT1) and during sleep (ZT16) indicates that more vesicles are attached to the T-bar platform and to capitate projections at ZT1, but this pattern is not present in brpΔ170, brpΔ190 and cry01 . This result confirmed an earlier study that both high motor activity in the morning and light exposure increase activity of the visual system. In the morning, there is intense transport of synaptic vesicles to T-bars and delivery of histamine in clear vesicles from the epithelial glial cells through capitate projections. The evidence for a role of capitate projections in neurotransmitter recycling has already been reported and now this study showed, using EMT, that vesicles are produced from capitate projections and directly delivered to T-bars. This intense transport is damaged in all mutants studied, suggesting that both BRP isoforms and CRY are needed for this process. In addition, in the brpΔ190 mutant lacking BRP-190, the T-bar structure is less dense than in the other strains studied. In a previous study, it was found that there are approximately 50% fewer synapses in brpΔ190 than in Canton S and brpΔ170. Another study reported that BRP isoforms are important for the formation of T-bars in neuromuscular junctions, and in brp mutants T-bars are smaller than in controls. T-bar height was reduced in brpΔ190, whereas the widths of pedestal and platform were reduced in both mutants. They also decrease transmission since the active zone was smaller in both mutants and the number of synaptic vesicles was reduced. These ultrastructural changes are correlated with cell physiology since the amplitude of evoked excitatory junctional current was decreased in both mutants with a stronger effect in brpΔ190 (Damulewicz, 2020).
The obtained reconstructions of tetrad T-bars from TEM serial sections of the lamina showed that although there were fewer synapses during the night (ZT16), the volume of the T-bar was larger at that time than in the morning (ZT1), while the total number of synaptic vesicles was similar. In contrast, a day/night difference (ZT1 vs. ZT16) in the number of vesicles was observed in brpΔ170 and cry01 . This suggests that the CRY protein and BRP-170 are responsible for an increase in the number of synaptic vesicles during the morning peak of activity. Since CRY co-localizes with BRP and is involved in BRP degradation in the morning, CRY is probably also involved in the degradation of other proteins of synaptic vesicle organization in the morning since the number of vesicles in cry01 was low in the morning but high at night (ZT16). It seems that in cry01, synaptic vesicles are not delivered to the photoreceptor terminals from capitate projections and tethered to the cytomatrix in the morning. It is also interesting that the daily rhythm in the number of vesicles is not maintained in BRP mutants, which confirms an earlier study, and the BRP N-terminus, which lacks brpΔ190, is necessary to maintain daily remodelling of the T-bar structure. Although both isoforms participate in building the cytomatrix their functions seem to be different in the course of the day. It is also possible that CRY is not only responsible for degradation of synaptic proteins but also as a protein, what is known, affecting the clock. In another cell types, in clock neurons l-LNvs, CRY, except interaction with TIM, is responsible for blue light response and firing of the l-LNvs. In the lamina it was found that in cry01 mutant the daily rhythm in synapse number and their remodelling was delayed in phase and the day/night difference in Canton S increased when peaks in the number of synapses were shift to ZT4 and ZT16. In result the difference between ZT1 and ZT16 was increased in cry01 (Damulewicz, 2020).
BRP is also responsible for rapid remodelling of the presynaptic active zone (AZ), and as reported in Drosophila NMJ, presynaptic homeostatic potentiation increases the number of BRP molecules and other AZ proteins, Unc13A and RBP, within minutes (Damulewicz, 2020).
When synaptic vesicles were counted at two different distances from the T-bar, to 200 nm and above 200 nm, there were differences between clear vesicles containing histamine and dense-core ones located in both areas. More clear vesicles were located near the T-bar and fewer above 200 nm. In the case of dense-core vesicles, their number was similar in both areas. This difference was not so striking in mutants in the case of vesicles located next to the platform, but in brpΔ170 and cry01, there were more dense-core vesicles at ZT16 in the distance above 200 nm than closer to the T-bar. This result indicates that BRP-170 and CRY are important for the distribution of clear synaptic vesicles next to the T-bar as well as dense-core vesicles located above 200 nm from the presynaptic element. It is possible that dense-core vesicles contain T-bar proteins, probably BRP. When transport along the actin cytoskeleton is disrupted, the number of tetrad synapses decreases, and rapid AZ remodelling also fails (Damulewicz, 2020).
The above mentioned ultrastructural changes depend on the level of the presynaptic scaffolding protein BRP, which changes in abundance during the day and night. These changes are controlled by the daily expression of CRY, which seems to have many functions in photoreceptors in addition to being the circadian clock photoreceptor. In an earlier study, it was found that CRY interacts with BRP but only during light exposure and leads to the degradation of BRP during the day/light phase of the 24 h cycle. This seems to be responsible for the decrease in BRP level in the middle of the day after its peak at the beginning of the day. The lack of CRY in cry01 mutants changes the pattern of the tetrad presynaptic profile frequency during the day and the size of the T-bar. However, the rhythm is not completely abolished, which indicates that other proteins are also involved in the daily remodelling of tetrad synapses. Since CRY plays several functions in photoreceptors, changes in the number of tetrad synapse and T-bar size in cry01 may result from different processes and lack of interactions of CRY with TIM and BRP. CRY is a component of the molecular clock and interacts with TIM, and this may affect daily changes in the number and size of T-bars. In addition, light-activated CRY binds BRP and targets it to degradation. Previous work showed that flies with constitutively active CRY have low BRP level. In turn, cry01 mutants show changes in the pattern of BRP expression, with higher BRP level during the day (ZT4), at the time when wild-type flies have minimum of BRP expression. The pattern of BRP expression is similar to the pattern of daily changes in tetrad synapse number, so it is possible that the number of synapses or T-bar size is directly dependent on the amount of BRP which oscillates during the day. However, CRY in the retina photoreceptors binds also actin and is involved in the organization of phototransduction cascade of proteins in rhabdomeres38. This may be also involved in the regulation of T-bar structure. The differences in T-bar size of cry01 are shown at time when in Canton S CRY is active (during the day) or its level increases (ZT16). At the beginning of the night the level of CRY is very low, so there was no effect on T-bar structure and no difference between CS and cry01 was observed (Damulewicz, 2020).
The epithelial glial cells are important for many processes during phototransduction and in recycling neurotransmitters and other compounds during the night. Glia take up histamine and metabolize it to carcinin, which is next delivered to the photoreceptor terminals, and capitate projections are involved in this process. Activity of glial cells is also controlled by the circadian clock. During the night, glial cells seem to be more active than neurons to recycle neurotransmitters, and many proteins, including proteins of ion pumps, are found at higher concentrations at that time. The high number of synaptic vesicles near the tetrad T-bar during the morning peak of activity in Drosophila seems to depend on capitate projections invaginating from the epithelial glia to the photoreceptor terminals in the lamina of Drosophila (Damulewicz, 2020).
Although the presynaptic cytomatrix can be rapidly remodelled with transmission strength, it is also affected by motor and visual system activity, external factors, such as light in the case of the visual system, and the circadian clock, showing plasticity and correlation to changes in behaviour during the day/night cycle. As was shown in the present study synaptic plasticity and synapse remodelling during the day is a complex process which involves presynaptic proteins of the T-bar as well as two types of synaptic vesicles, clear and dense-core, and glial cells. It was also found that fast degradation of proteins involved in transmission is as important as pre- and postsynaptic protein synthesis (Damulewicz, 2020).
When the brain is in a pathological state, the content of lipid droplets (LDs), the lipid storage organelles, is increased, particularly in glial cells, but rarely in neurons. The biology and mechanisms leading to LD accumulation in astrocytes, glial cells with key homeostatic functions, are poorly understood. Fluorescently labeled LDs were imaged by microscopy in isolated and brain tissue rat astrocytes and in glia-like cells in Drosophila brain to determine the (sub)cellular localization, mobility, and content of LDs under various stress conditions characteristic for brain pathologies. LDs exhibited confined mobility proximal to mitochondria and endoplasmic reticulum that was attenuated by metabolic stress and by increased intracellular Ca(2+) , likely to enhance the LD-organelle interaction imaged by electron microscopy. When de novo biogenesis of LDs was attenuated by inhibition of diacylglycerol O-acyltransferases DGAT1 and DGAT2 enzymes, the astrocyte cell number was reduced by ~40%, suggesting that in astrocytes LD turnover is important for cell survival and/or proliferative cycle. Exposure to noradrenaline, a brain stress response system neuromodulator, and metabolic and hypoxic stress strongly facilitated LD accumulation in astrocytes. The observed response of stressed astrocytes may be viewed as a support for energy provision, but also to be neuroprotective against the stress-induced lipotoxicity (Smolic, 2021).
Critical periods (brief intervals during which neural circuits can be modified by activity) are necessary for proper neural circuit assembly. Extended critical periods are associated with neurodevelopmental disorders; however, the mechanisms that ensure timely critical period closure remain poorly understood. This study defined a critical period in a developing Drosophila motor circuit and identified astrocytes as essential for proper critical period termination. During the critical period, changes in activity regulate dendrite length, complexity and connectivity of motor neurons. Astrocytes invaded the neuropil just before critical period closure, and astrocyte ablation prolonged the critical period. Finally, a genetic screen was used to identify astrocyte-motor neuron signalling pathways that close the critical period, including Neuroligin-Neurexin signalling. Reduced signalling destabilized dendritic microtubules, increased dendrite dynamicity and impaired locomotor behaviour, underscoring the importance of critical period closure. Previous work defined astroglia as regulators of plasticity at individual synapses. This study shows that astrocytes also regulate motor circuit critical period closure to ensure proper locomotor behaviour (Ackerman, 2021).
Critical periods are brief windows during which neural circuit activity can modify the morphological properties of neurons, producing permanent changes to circuit structure and function. Critical periods integrate multiple forms of plasticity to modify neural circuits. 'Homeostatic plasticity' encompasses changes to synapse number, structure and function across an entire neuron, as well as changes to long-range connectivity. Whereas homeostatic plasticity can occur in the adult brain, substantial activity-dependent remodelling peaks in early development. Indeed, failure to terminate critical period plasticity is linked to neurodevelopmental disorders such as autism and epilepsy. Although putative critical period disorders present with motor defects, the field has largely focused on sensory circuits. To that end, this study developed a novel critical period model in a developing motor circuit (Ackerman, 2021).
This study focused on two well-characterized Drosophila motor neurons, aCC and RP2, which are segmentally repeated in the central nervous system. These motor neurons are susceptible to activity-induced remodelling, although pioneering studies used chronic activity manipulations and did not define an end point for homeostatic plasticity. This study expressed the anion channelrhodopsin GtACR215 specifically in the aCC-RP2 motor neurons using the Gal4-upstream activation system (UAS) system and delivered acute 1-h windows of silencing, terminating at progressively later times in development. Silencing motor neurons for the last hour of embryogenesis (stage 17) increased aCC-RP2 dendritic volume at 0 h after larval hatching (ALH), whereas silencing for 1 h at later stages showed progressively less of an effect, with no remodelling occurring at 8 h ALH or beyond. By contrast, acute 1-h windows of activation using the channelrhodopsin Chrimson resulted in significant loss of motor neuron dendrites at 0 h ALH; activation at 8 h ALH and beyond had little or no effect. Activity-induced changes to dendrite length for single-cell RP2 clones [using the MultiColor FlpOut (MCFO) system] showed similar results Note that these experiments used far shorter periods of tonic activation than past studies. Although Tonic activity manipulations were primarily used, identical results were observed using 600 ms:400 ms pulses of activation or silencing, as well as thermogenetics to activate (via TrpA1) or silence [using the temperature-sensitive shibire gene (shibirets)] motor neurons. Notably, dendrite loss following acute activation could be rescued by a 22-h period of dark rearing, indicating that activity induces dendrite plasticity and not excitotoxicity. Together, these experiments define a critical period for activity-dependent motor dendrite plasticity represent the first analyses of motor circuit critical period closure within the central nervous system (Ackerman, 2021).
In vertebrates, homeostatic plasticity functions on a slow timescale, from hours to days. To determine the timescale for motor neuron dendrite expansion following GtACR2 silencing, aCC-RP2 motor neurons were silenced for 15 min, 1 h or 4 h in stage 17 embryos, terminating silencing at 0 h ALH. Larvae were then immediately dissected and dendritic morphology was assessed in single, well-spaced RP2 neurons using MCFO17. Increased dendritic arbor size and complexity following 1 h and 4 h of silencing were used. These results were confirmed using shibirets. By contrast, embryonic Chrimson activation resulted in decreased dendrite length and complexity at 0 h ALH after as little as 15 min of activation. Furthermore, using live imaging, significant dendrite retraction was observed within 12 min of Chrimson activation. The fact that silencing required more time to show an effect is not surprising, as extension requires generation of new membrane. It is concluded that activity-induced remodelling of Drosophila motor neurons occurs within minutes, much more quickly than previously documented for homeostatic plasticity in mammals (Ackerman, 2021).
This study showed that motor neurons scale dendrite length according to activity. An important question is whether these morphological changes are accompanied by changes in excitatory or inhibitory synaptic inputs. The excitatory cholinergic neuron A18b and inhibitory GABAergic neuron A23a were examined that are synaptically coupled to aCC-RP2 dendrites in a larval transmission electron microscopy (TEM) reconstruction. To quantify excitatory and inhibitory synapse number by light microscopy, a functionally inactive pre-synaptic marker, Bruchpilotshort::Cherry (Brp), was expressed in excitatory cholinergic neuron A18b or inhibitory GABAergic neuron A23a using the complementary LexA-LexAop binary expression system. A23a-inhibitory GABAergic synapses onto aCC-RP2 dendrites were examined, quantifying cell-type specific Brp puncta overlapping with aCC-RP2 dendritic membrane (putative synapses) using published standards. All critical period manipulations terminated at 4 h ALH (stage matched to the TEM data). It was found that 1 h of motor neuron silencing reduced the number of inhibitory synapses between A23a and aCC-RP2 dendrites. Silencing for a longer period (4 h) also yielded a significant increase in A18b excitatory synapses. Decreasing motor neuron activity thus leads to a compensatory reduction of inhibitory inputs and a corresponding increase in excitatory inputs to rebalance network activity. A18b excitatory cholinergic synapse numbers onto aCC-RP2 dendrites were quantified after activation or silencing. Motor neuron activation was found to significantly decreased numbers of A18b excitatory synapses onto aCC-RP2 dendrites following 1 h and 4 h manipulations. A significant increase in inhibitory synapse number following extended motor neuron activation was observed, possibly owing to insufficient dendritic membrane after activity-induced dendrite retraction. Increasing motor neuron activity thus leads to a compensatory reduction of excitatory pre-synaptic inputs. Finally, a functionally inactive reporter of excitatory post-synaptic densities (Drep2::GFP or Drep2::mStrawberry) was observed, specifically in aCC-RP2, and scaling of synapses was observed accross the entire dendritic arbor in response to altered activity—reduced excitatory post-synapses followed motor neuron activation, whereas increased excitatory post-synapses followed motor neuron silencing during the critical period. Of note, homeostatic scaling of motor neuron synapses did not occur after critical period closure. In sum, motor neurons scale excitatory and inhibitory inputs relative to their level of activity during the critical period (Ackerman, 2021).
The mechanisms that close critical periods remain poorly defined. Drosophila astrocytes infiltrate the neuropil at late embryogenesis and progressively envelop motor neuron synapses as the critical period closes. To test whether astrocytes promote critical period closure, all astrocytes were genetically ablated and optogenetics was used to assay for extension of critical period plasticity at 8 h ALH. Astrocyte elimination was confirmed by loss of the astrocyte marker Gat3. As expected, controls closed the critical period by 8 h ALH. By contrast, astrocyte ablation extended dendrite plasticity following Chrimson activation or GtACR2 silencing up to 8 h ALH. This effect was not observed at earlier stages, indicating that astrocytes do not constitutively dampen plasticity. Additionally, it was found that control motor dendrites were less dynamic after critical period closure, but that astrocyte ablation extends dendrite filopodial dynamicity. It is concluded that astrocytes are required for the transition from dynamic to stable filopodia and concurrent critical period closure (Ackerman, 2021).
To determine how astrocytes close the critical period, the astrocyte-specific alrm-gal4 was used to perform a targeted UAS RNA-mediated interference (RNAi) knockdown screen. Flies were assayed for critical period extension following 1 h of Chrimson activation from 7-8 h ALH. Four genes were identified that were required in astrocytes for timely critical period closure: gat (regulates excitatory-inhibitory balance), chpf [synthesizes chondroitin sulfate proteoglycans (CSPGs)] and the Neuroligins (Nlg) 4 and 2 (Ackerman, 2021).
Neuroligins are cell-adhesion proteins that are known to regulate astrocyte morphogenesis. In Drosophila, astrocyte-specific knockdown of nlg2 (the mouse orthologue is known as Nlgn1) had no effect on astrocyte volume or tiling, suggesting a more specific defect in astrocyte-motor neuron signalling. Knockdown of the remaining critical period regulators had variable effects on astrocyte morphology but all extended the critical period. Neuroligins bind cell adhesion proteins called Neurexins. RNAi against nrx-1 was used, which is known to bind both Nlg2 and Nlg4, specifically in aCC-RP2 motor neurons, and critical period extension was observed; this is consistent with astrocyte Nlg2 and motor neuron Nrx-1 acting in a common pathway to close the critical period. Motor neuron-specific RNAi knockdown of the CSPG receptor Lar also extended critical period plasticity. Notably, while Nrx-1 is often pre-synaptic, there is evidence for dendritic localization of these receptors. Furthermore, antibody staining for endogenous Nrx-1 and Nlg2 revealed localization of this receptor-ligand pair on motor dendrites and astrocytes, respectively. Finally, cell-type-specific overexpression of Nrx-1 and Nlg2 could induce precocious critical period closure (assayed by Chrimson activation from 3-4 h ALH). It is concluded that Nlg2-Nrx-1 ligand-receptor signalling between astrocytes and motor neurons is required for timely critical period closure (Ackerman, 2021).
How does Nlg2-Nrx-1 signalling close the critical period? The balance of excitatory to inhibitory synapses in neural circuits can instruct critical period timing. Additionally, numbers of excitatory synapses are decreased following astrocyte-specific knockout of neuroligins in mouse. This study observed no significant changes in excitatory-inhibitory balance following knockdown of nlg2 in astrocytes, suggesting that critical period closure is not dependent on Nlg2-mediated excitatory-inhibitory synapse balance (Ackerman, 2021).
Alternatively, Nrx-1 can promote microtubule stability in axons of motor neurons, suggesting a mechanism for critical period closure involving microtubule stabilization. To test this hypothesis, Chrimson::mVenus was used to activate and visualize aCC-RP2 dendrite membranes at 0 h ALH (peak critical period), and Cherry::Zeus to visualize stable microtubules during and after dendritic retraction. In live preparations, dendrites showed a reduction in Cherry::Zeus intensity immediately preceding activity-dependent retraction, suggesting that microtubule collapse in distal branches can induce dendrite retraction. In fixed preparations, this study found that proximal dendrites with the highest levels of stable microtubules were protected from activity-dependent retraction. Of note, overexpression of Nrx-1 was sufficient to increase both stable microtubules and stable dendrites at 4 h ALH. It is proposed that Nlg2 in astrocytes binds Nrx-1 in motor neurons to stabilize dendritic microtubules and close the critical period (Ackerman, 2021).
In mammals, inappropriate critical period extension has long-term effects on nervous system function. Indeed, this study observed persistent changes in motor neuron connectivity at least 24 h following acute motor neuron activation at the end of the critical period, which lead to an assay for long-term effects on behaviour. The critical period was transiently extended until 12 h ALH (4 h beyond control critical period closure), and then behaviour was assayed 1.5 days later. Control larvae showed persistent linear locomotion; by contrast, larvae with extended critical periods due to transient knockdown of motor neuron genes showed excessive turning, leading to abnormal spiralling behaviour. Similar but less severe effects were seen in larvae following knockdown of astrocyte genes. It is concluded that a modest extension of the critical period can, in some cases, lead to long-lasting alteration in locomotor behaviour (Ackerman, 2021).
Astrocytes regulate synaptogenesis, synaptic pruning and synaptic efficacy. Within critical periods, astrocyte signalling can tune neuronal plasticity, but its role in critical period closure was not known. This study identified astrocytes as promoting closure of a motor critical period, and defined a series of astrocyte-motor neuron signalling pathways required to close the critical period. Based on previous literature, it is hypothesized that astrocytes could modify critical period closure through regulation of excitatory-inhibitory balance or extracellular matrix composition. Consistent with mammalian studies, it was found that perturbing excitatory-inhibitory balance through astrocyte-specific RNAi of the sole GABA transporter gat was sufficient to extend critical period plasticity. Furthermore, it was found that decreasing signalling from inhibitory extracellular matrix CSPGs through RNAi knockdown of Chondroitin polymerizing factor (Chpf) in astrocytes extended critical period plasticity. Thus, astrocytes use similar strategies in Drosophila and mammals to regulate critical period timing. Unexpectedly, this study also identified astrocyte-derived Neuroligins and their neuronal partner Nrx-1 as instrumental for critical period closure. In sum, this study have identified a key role of astrocytes in closure of a motor critical period required for locomotor function (Ackerman, 2021).
Neural circuit formation and function require that diverse neurons are specified in appropriate numbers. Known strategies for controlling neuronal numbers involve regulating either cell proliferation or survival. This study used the Drosophila visual system to probe how neuronal numbers are set. Photoreceptors from the eye-disc induce their target field, the lamina, such that for every unit eye there is a corresponding lamina unit (column). Although each column initially contains ~6 post-mitotic lamina precursors, only 5 differentiate into neurons, called L1-L5; the 'extra' precursor, which is invariantly positioned above the L5 neuron in each column, undergoes apoptosis. This study showed that a glial population called the outer chiasm giant glia (xg(O)), which resides below the lamina, secretes multiple ligands to induce L5 differentiation in response to epidermal growth factor (EGF) from photoreceptors. By forcing neuronal differentiation in the lamina, it was uncovered that though fated to die, the 'extra' precursor is specified as an L5. Therefore, two precursors are specified as L5s but only one differentiates during normal development. It was found that the row of precursors nearest to xg(O) differentiate into L5s and, in turn, antagonise differentiation signalling to prevent the 'extra' precursors from differentiating, resulting in their death. Thus, an intricate interplay of glial signals and feedback from differentiating neurons defines an invariant and stereotyped pattern of neuronal differentiation and programmed cell death to ensure that lamina columns each contain exactly one L5 neuron (Prasad, 2022).
Some types of glia play an active role in neuronal signaling by modifying their activity although little is known about their role in sensory information signaling at the receptor level. In this research, a functional role is reported for the glia that surround the soma of the olfactory receptor neurons (OSNs) in adult Drosophila. Specific genetic modifications have been targeted to this cell type to obtain live individuals who are tested for olfactory preference and display changes both increasing and reducing sensitivity. A closer look at the antenna by Ca(2+) imaging shows that odor activates the OSNs, which subsequently produce an opposite and smaller effect in the glia that partially counterbalances neuronal activation. Therefore, these glia may play a dual role in preventing excessive activation of the OSNs at high odorant concentrations and tuning the chemosensory window for the individual according to the network structure in the receptor organ (Calvin-Cejudo, 2023).
Drosophila Toll-9 is most closely related to mammalian Toll-like receptors; however, physiological functions of Toll-9 remain elusive. This study examined the roles of Toll-9 in fly brains in aging and neurodegeneration. Toll-9 mRNA levels were increased in aged fly heads accompanied by activation of nuclear factor-kappa B (NF-kB) and stress-activated protein kinase (SAPK; Jun-N-terminal Kinase pathway) signaling, and many of these changes were modulated by Toll-9 in glial cells. The loss of Toll-9 did not affect lifespan or brain integrity, whereas it exacerbated hydrogen peroxide-induced lethality. Toll-9 expression was also induced by nerve injury but did not affect acute stress response or glial engulfment activity, suggesting Toll-9 may modulate subsequent neurodegeneration. In a fly tauopathy model, Toll-9 deficiency enhanced neurodegeneration and disease-related tau phosphorylation with reduced SAPK activity, and blocking SAPK enhanced tau phosphorylation and neurodegeneration. In sum, Toll-9 is induced upon aging and nerve injury and affects neurodegeneration by modulating stress kinase signaling (Sakakibara, 2023).
Glia have an emergent role in brain aging and disease. In the Drosophila melanogaster brain, ensheathing glia function as phagocytic cells and respond to acute neuronal damage, analogous to mammalian microglia. Changes in glia composition over the life of ants and fruit flies have been reported, including a decline in the relative proportion of ensheathing glia with time. How these changes influence brain health and life expectancy is unknown. This study shows that ensheathing glia but not astrocytes decrease in number during Drosophila melanogaster brain aging. The remaining ensheathing glia display dysregulated expression of genes involved in lipid metabolism and apoptosis, which may lead to lipid droplet accumulation, cellular dysfunction, and death. Inhibition of apoptosis rescued the decline of ensheathing glia with age, improved the neuromotor performance of aged flies, and extended lifespan. Furthermore, an expanded ensheathing glia population prevented amyloid-beta accumulation in a fly model of Alzheimer's disease and delayed the premature death of the diseased animals. These findings suggest that ensheathing glia play a vital role in regulating brain health and animal longevity (Sheng, 2023).
Modification by sialylated glycans can affect protein functions, underlying mechanisms that control animal development and physiology. Sialylation relies on a dedicated pathway involving evolutionarily conserved enzymes, including CMP-sialic acid synthetase (CSAS) and sialyltransferase (SiaT) that mediate the activation of sialic acid and its transfer onto glycan termini, respectively. In Drosophila, CSAS and DSiaT genes function in the nervous system, affecting neural transmission and excitability. These genes were found to function in different cells: the function of CSAS is restricted to glia, while DSiaT functions in neurons. This partition of the sialylation pathway allows for regulation of neural functions via a glia-mediated control of neural sialylation. The sialylation genes were shown to be required for tolerance to heat and oxidative stress and for maintenance of the normal level of voltage-gated sodium channels. The results uncovered a unique bipartite sialylation pathway that mediates glia-neuron coupling and regulates neural excitability and stress tolerance (Scott, 2023).
Protein glycosylation, the most common type of posttranslational modification, plays numerous important biological roles, and regulates molecular and cell interactions in animal development, physiology, and disease. The addition of sialic acid (Sia), i.e., sialylation, has prominent effects due to its negative charge, bulky size, and terminal location of Sia on glycan chains. Essential roles of sialylated glycans in cell adhesion, cell signaling, and proliferation have been documented in many studies. Sia is intimately involved in the function of the nervous system. Mutations in genes that affect sialylation are associated with neurological symptoms in human, including intellectual disability, epilepsy, and ataxia due to defects in sialic acid synthase (N-acetylneuraminic acid synthase [NANS]), sialyltransferases (ST3GAL3 and ST3GAL5), the CMP-Sia transporter (SLC35A1), and CMP-Sia synthase (CMAS). Polysialylation (PSA) of NCAM, the neural cell adhesion molecule, one of the best studied cases of sialylation in the nervous system, is involved in the regulation of cell interactions during brain development. Non-PSA-type sialylated glycans are ubiquitously present in the vertebrate nervous system, but their functions are not well defined. Increasing evidence implicates these glycans in essential regulation of neuronal signaling. Indeed, N-glycosylation can affect voltage-gated channels in different ways, ranging from modulation of channel gating to protein trafficking, cell surface expression, and recycling/degradation. Similar effects were shown for several other glycoproteins implicated in synaptic transmission and cell excitability, including neurotransmitter receptors. Glycoprotein sialylation defects were also implicated in neurological diseases, such as Angelman syndrome and epilepsy. However, the in vivo functions of sialylation and the mechanisms that regulate this posttranslational modification in the nervous system remain poorly understood (Scott, 2023).
Drosophila has recently emerged as a model to study neural sialylation in vivo, providing advantages of the decreased complexity of the nervous system and the sialylation pathway, while also showing conservation of the main biosynthetic steps of glycosylation. The final step in sialylation is mediated by sialyltransferases, enzymes that use CMP-Sia as a sugar donor to attach Sia to glycoconjugates (see Schematic of the sialylation pathways in vertebrate and Drosophila. Unlike mammals that have 20 different sialyltransferases, Drosophila possesses a single sialyltransferase, DSiaT, that has significant homology to mammalian ST6Gal enzymes. The two penultimate steps in the biosynthetic pathway of sialylation are mediated by sialic acid synthase (also known as NANS) and CMP-sialic acid synthetase (CSAS, also known as CMAS), the enzymes that synthesize sialic acid and carry out its activation, respectively. These enzymes have been characterized in Drosophila and found to be closely related to their mammalian counterparts. In vivo analyses of DSiaT and CSAS demonstrated that Drosophila sialylation is a tightly regulated process limited to the nervous system and required for normal neural transmission. Mutations in DSiaT and CSAS phenocopy each other, resulting in similar defects in neuronal excitability, causing locomotor and heat-induced paralysis phenotypes, while showing strong interactions with voltage-gated channels. DSiaT was found to be expressed exclusively in neurons during development and in the adult brain. Intriguingly, although the expression of CSAS has not been characterized in detail, it was noted that its expression appears to be different from that of DSiaT in the embryonic ventral ganglion, suggesting a possibly unusual relationship between the functions of these genes. This study tested the hypothesis that CSAS functions in glial cells, and that the separation of DSiaT and CSAS functions between neurons and glia underlies a novel mechanism of glia-neuron coupling that regulates neuronal function via a bipartite protein sialylation (Scott, 2023).
In vertebrates, phosphorylated sialic acid is produced by N-acetylneuraminic acid synthase (Neu5Ac-9-P synthase, or NANS) from N-acetyl-mannosamine 6-phosphate (ManNAc-6-P), converted to sialic acid (Scott, 2023).
Glial cells have been recognized as key players in neural regulation. Astrocytes participate in synapse formation and synaptic pruning during development, mediate the recycling of neurotransmitters, affect neurons via Ca2+ signaling, and support a number of other essential evolutionarily conserved functions. Studies of Drosophila glia have revealed novel glial functions in vivo. Drosophila astrocytes were found to modulate dopaminergic function through neuromodulatory signaling and activity-regulated Ca2+ increase. Glial cells were also shown to protect neurons and neuroblasts from oxidative stress and promote the proliferation of neuroblasts in the developing Drosophila brain. The metabolic coupling between astrocytes and neurons, which is thought to support and modulate neuronal functions in mammals, is apparently conserved in flies. Indeed, Drosophila glial cells can secrete lactate and alanine to fuel neuronal oxidative phosphorylation. In the current work, a novel mechanism id described of glia-neuron coupling mediated by a unique compartmentalization of different steps in the sialylation pathway between glial cells and neurons in the fly nervous system. This study explored the regulation of this mechanism and demonstrate its requirement for neural functions (Scott, 2023).
Neuronal function is highly energy demanding and thus requires efficient and constant metabolite delivery by glia. Drosophila glia are highly glycolytic and provide lactate to fuel neuronal metabolism. Flies are able to survive for several weeks in the absence of glial glycolysis. This study examined how Drosophila glial cells maintain sufficient nutrient supply to neurons under conditions of impaired glycolysis. Glycolytically impaired glia were shown to rely on mitochondrial fatty acid breakdown and ketone body production to nourish neurons, suggesting that ketone bodies serve as an alternate neuronal fuel to prevent neurodegeneration. In times of long-term starvation, glial degradation of absorbed fatty acids is essential to ensure survival of the fly. Further, it was shown that Drosophila glial cells act as a metabolic sensor and can induce mobilization of peripheral lipid stores to preserve brain metabolic homeostasis. This study gives evidence of the importance of glial fatty acid degradation for brain function, and survival, under adverse conditions in Drosophila (McMullen, 2023).
The astrocyte-neuron lactate shuttle hypothesis posits that glial-generated lactate is transported to neurons to fuel metabolic processes required for long-term memory. Although studies in vertebrates have revealed that lactate shuttling is important for cognitive function, it is uncertain if this form of metabolic coupling is conserved in invertebrates or is influenced by age. Lactate dehydrogenase (Ldh) is a rate limiting enzyme that interconverts lactate and pyruvate. This study genetically manipulated expression of Drosophila melanogaster Lactate dehydrogenase (dLdh) in neurons or glia to assess the impact of altered lactate metabolism on invertebrate aging and long-term courtship memory at different ages. Survival, negative geotaxis, brain neutral lipids (the core component of lipid droplets) and brain metabolites were also examined. Both upregulation and downregulation of dLdh in neurons resulted in decreased survival and memory impairment with age. Glial downregulation of dLdh expression caused age-related memory impairment without altering survival, while upregulated glial dLdh expression lowered survival without disrupting memory. Both neuronal and glial dLdh upregulation increased neutral lipid accumulation. Evidence is provided that altered lactate metabolism with age affects the tricarboxylic acid (TCA) cycle, 2-hydroxyglutarate (2HG), and neutral lipid accumulation. Collectively, these findings indicate that the direct alteration of lactate metabolism in either glia or neurons affects memory and survival but only in an age-dependent manner (Frame, 2023).
Glial phagocytic activity refines connectivity, though molecular mechanisms regulating this exquisitely sensitive process are incompletely defined. This study developed the Drosophila antennal lobe as a model for identifying molecular mechanisms underlying glial refinement of neural circuits in the absence of injury. Antennal lobe organization is stereotyped and characterized by individual glomeruli comprised of unique olfactory receptor neuronal (ORN) populations. The antennal lobe interacts extensively with two glial subtypes: ensheathing glia wrap individual glomeruli, while astrocytes ramify considerably within them. Phagocytic roles for glia in the uninjured antennal lobe are largely unknown. Thus, this study tested whether Draper regulates ORN terminal arbor size, shape, or presynaptic content in two representative glomeruli: VC1 and VM7. Glial Draper was found to limits the size of individual glomeruli and restrains their presynaptic content. Moreover, glial refinement is apparent in young adults, a period of rapid terminal arbor and synapse growth, indicating that synapse addition and elimination occur simultaneously. Draper has been shown to be expressed in ensheathing glia; unexpectedly, it was found to be expressed at high levels in late pupal antennal lobe astrocytes. Surprisingly, Draper plays differential roles in ensheathing glia and astrocytes in VC1 and VM7. In VC1, ensheathing glial Draper plays a more significant role in shaping glomerular size and presynaptic content; while in VM7, astrocytic Draper plays the larger role. Together, these data indicate that astrocytes and ensheathing glia employ Draper to refine circuitry in the antennal lobe before the terminal arbors reach their mature form and argue for local heterogeneity of neuron-glia interactions (Jindal, 2023).
In the developmental remodeling of brain circuits, neurons are removed by glial phagocytosis to optimize adult behavior. Fragile X mental retardation protein (FMRP) regulates neuron-to-glia signaling to drive glial phagocytosis for targeted neuron pruning. This study finds that FMRP acts in a mothers against decapentaplegic (Mad)-insulin receptor (InR)-protein kinase B (Akt) pathway to regulate pretaporter (Prtp) and amyloid precursor protein-like (APPL) signals directing this glial clearance. Neuronal RNAi of Drosophila fragile X mental retardation 1 (dfmr1) elevates mad transcript levels and increases pMad signaling. Neuronal dfmr1 and mad RNAi both elevate phospho-protein kinase B (pAkt) and delay neuron removal but cause opposite effects on InR expression. Genetically correcting pAkt levels in the mad RNAi background restores normal remodeling. Consistently, neuronal dfmr1 and mad RNAi both decrease Prtp levels, whereas neuronal InR and akt RNAi increase Prtp levels, indicating FMRP works with pMad and insulin signaling to tightly regulate Prtp signaling and thus control glial phagocytosis for correct circuit remodeling. Neuronal dfmr1 and mad and akt RNAi all decrease APPL levels, with the pathway signaling higher glial endolysosome activity for phagocytosis. These findings reveal a FMRP-dependent control pathway for neuron-to-glia communication in neuronal pruning, identifying potential molecular mechanisms for devising fragile X syndrome treatments (Song, 2023).
Neuron-to-glia communication has critical roles in controlling brain circuit remodeling. Neuronal signaling induces glial phagocytosis from synapses to whole neurons, crucial in normal brains and in neurodevelopmental disorder conditions. Disruption of neuron-to-glia communication causes aberrant neuronal pruning, resulting in defects ranging from defective synaptic transmission to impaired circuit wiring to transient neuroinflammation. A key case is fragile X syndrome (FXS), a leading intellectual disability and autism spectrum disorder, typically caused by the epigenetic loss of fragile X mental retardation protein (FMRP) owing to expanded CGG repeats in the 5'
-untranslated region of fragile X mental retardation 1 (fmr1). In neurons, FMRP binds to specific transcripts to regulate protein translation during brain circuit development and later plasticity, including synaptic connectivity remodeling and intercellular signaling mechanisms In the Drosophila FXS disease model, FMRP loss blocks removal of the developmentally transient pigment-dispersing factor (PDF)-Tri peptidergic neurons from the juvenile brain. Cell-specific RNAi studies show that FMRP is required only in neurons, not glia, to transcellularly activate glial phagocytosis driving PDF-Tri neuron clearance. Thus, FMRP-dependent neuron-to-glia communication drives targeted neuron pruning, but the molecular mechanisms remain largely unknown (Song, 2023).
FMRP regulates bone morphogenic protein (BMP) and insulin-like peptide (ILP) signaling. Activated Drosophila BMP receptors phosphorylate Mothers against decapentaplegic (pMad) to control gene transcription, including the Insulin receptor (InR). InRs phosphorylate protein kinase B (pAkt) to suppress dendrite pruning. Importantly, mouse FMRP binds smad messenger ribonucleic acid (mRNA) (Drosophila mad homologue), and Drosophila FMRP regulates pMad signaling levels in neurons. Moreover, FMRP loss elevates InR-dependent signaling. In neuron-to-glia communication, pretaporter (Prtp) and amyloid precursor protein like (APPL) from neurons both activate glial phagocytosis. Drosophila Prtp traffics to the neuron surface to bind the glial phagocytotic receptor Draper (Drpr). Loss of neuronal FMRP decreases glial Drpr expression, consistent with FMRP-dependent Prtp signaling. Drosophila APPL has a cleavable N terminus, and APPL release from neurons activates glial phagocytosis. Glia take up secreted APPL to maintain Drpr expression and up-regulate Rab GTPases, activating the glial endolysosomal network for the neuron clearance mechanism. Taken together, these studies suggest that neuronal FMRP interacts with neuronal InR, pMad, and pAkt signaling cascades to tightly regulate Prtp and APPL neuron-to-glia communication controlling the glial phagocytosis of target neurons during circuit remodeling in the juvenile brain (Song, 2023).
This study used Drosophila brain PDF-Tri neuron removal via glial phagocytosis to study the neuron-to-glia communication remodeling mechanism, assaying both the early pruning steps at 1 day post-eclosion (dpe) and end-stage clearance (5 dpe). It was discovered that neuronal FMRP binds Mad mRNA to restrict pMad signaling in neurons. Surprisingly, however, it was found that both neuronal Drosophila Fragile X mental retardation 1 (Dfmr1) and Mad RNAi similarly block PDF-Tri neuron removal, indicating a more complex regulatory mechanism. Consistently, it was found that pMad is a positive transcription factor for InRs driving downstream pAkt signaling but that pMad also indirectly inhibits pAkt, inducing the phenocopy between neuronal dfmr1 and Mad RNAi conditions. Both neuronal dfmr1 and Mad RNAi similarly decrease Prtp neuron-to-glia signaling, resulting in reduced glial phagocytic activity and a block of PDF-Tri neuron clearance, whereas loss of neuronal InR and pAkt has that opposite phenotype of elevating Prtp to accelerate neuronal removal. It was also discovered that the FMRP-pMad-InR-pAkt pathway positively regulates neuronal APPL signaling to induce glial Rab7-driven endolysomal activation for PDF-Tri neuron clearance. Taken together, it is concluded that neuronal FMRP-pMad and InR-pAkt cascades coordinate an integrated regulatory decision network governing neuron-to-glia communication via neuronal Prtp and APPL signaling ligands that drive glial phagocytosis for targeted neuron pruning from brain circuits (Song, 2023).
This study has discovered an integrated mechanism of neuronal FMRP-dependent network signaling that regulates neuron-to-glia communication to drive the glial phagocytic removal of targeted neurons from an otherwise maintained brain circuit. Specifically, within the neurons, RNA-binding FMRP restricts the translation of bound Mad transcripts to limit phosphorylated Mad (pMad) signaling, which, in turn, inhibits phosphorylated Akt (pAkt) to promote glial phagocytosis for neuron removal. In parallel, the neuronal insulin receptor (InR) regulates pAkt signaling in a second intersecting cascade controlling the neuronal clearance mechanism. This bone morphogenic protein (BMP) and insulin-like peptide (ILP) neural decision-making network controls neuron-to-glia communication regulating glial phagocytosis function for targeted neuron removal. Neuronal pretaporter (Prtp) is a ligand for the Draper (Drpr) engulfment receptor on glia. The FMRP-pMad pathway promotes neuron-to-glia Prtp signaling to induce glial phagocytosis for neuron clearance, whereas the InR-pAkt pathway suppresses Prtp signaling to repress glia-mediated neuron removal. Neuronal amyloid precursor protein like (APPL) is released via a cleavable N terminus to activate Rab7 GTPase endolysomes in glia. The FMRP-pMad regulatory pathway promotes this neuron-to-glia APPL signaling, consistent with inducing glial phagocytosis for neuron clearance, and the intersecting InR-pAkt pathway also up-regulates this signaling, suggesting additional roles in neuron removal. Overall, these findings indicate neuronal FMRP coordinates signal transduction cascades to provide cross talk downstream of two signaling inputs (BMP and ILP), which provides output in the form of two neuron-to-glia signaling ligands (Prtp and APPL) that regulate glial phagocytosis for the clearance of targeted neurons from the juvenile brain (Song, 2023).
It is suggested that RNA-binding FMRP limits pMad signaling levels by reducing the number of Mad transcripts available for translation. FMRP is established to maintain protein translational homeostasis by modulating RNA target stability. Ribosome profiling and transcriptome sequencing demonstrate imbalanced FMRP-targeted mRNA levels are common in the mouse FXS model brain due to reduced stabilization. In mouse RNA immunoprecipitation (RIP) sequencing, FMRP binds Smad (Drosophila Mad homologue) transcripts widely in the 5'
-UTR, within the coding region, and in the 3'-UTR. With both RIP and qPCR measurements, this study found Drosophila FMRP likewise binds Mad mRNA and that FMRP loss increases both mad transcripts and pMad protein levels within brain neurons. It is therefore suggest a causal effect correlation, although other indirect regulatory mechanisms are also possible. Loss of neuronal pMad decreases InR levels but elevates InR-dependent pAkt signaling, suggesting cross talk inhibition from pMad to pAkt. The pMad transcription factor positively regulates InR expression. Downstream of the InR, pAkt regulates numerous targets, including target of rapamycin complexes and the transcription factor forkhead box O, to regulate cell fate decisions. In the brain neuronal fate decision, it is suggested that pMad inhibits pAkt signaling as a major cross talk regulation within the neuron-to-glia signaling network. With western blot assays, this study found reducing neuronal pMad levels causes no changes in Akt protein levels but a approximately twofold increase in pAkt signaling. Thus, FMRP-dependent pMad signaling modulates InR-pAkt signaling at two levels: as a positive transcription factor regulating InR expression and as an inhibitor of downstream pAkt signaling. It is concluded that this cross talk provides critical regulation for targeted neuron pruning from the juvenile brain (Song, 2023).
Neuron removal from brain circuits is a normal mechanism in neurodevelopment, often mediated by glial phagocytosis. During the clearance process, glia engulf neurons and degrade internalized debris in endolysosomes. In glia, the engulfed neuronal debris is first sorted in Rab5 GTPase early endosomes, then trafficked from early-to-late endosomes with accompanying increased intravacuolar acidification, and finally delivered to Rab7 GTPase lysosomes where acid hydrolases complete the degradation process. In both rodent and Drosophila FXS disease models, this glial phagocytosis mechanism is severely impaired, resulting in a failure to prune neurons in brain circuits. In the Drosophila FXS model, loss of neuronal FMRP decreases the glial Drpr engulfment receptor, and loss of glial Drpr blocks glial phagocytosis to prevent neuron removal. This previous study generated the hypothesis that the impaired brain circuit neuron removal in the FXS disease model is caused by the loss of neuron-to-glia communication driving glial phagocytosis. With neuron-specific RNAi trials, this study found the neuronal FMRP-pMad pathway triggers Drpr ligand Prtp signaling from neurons to mediate glial engulfment and phagocytosis, whereas the neuronal InR-pAkt pathway represses Prtp to oppose the glial removal mechanism. Furthermore, targeted knockdown of neuronal FMRP, pMad, and Prtp impedes glial endolysosomal activation with reduced Rab7 expression, showing the neuronal FMRP-dependent network is required for glial phagocytosis function. These findings are consistent with the impaired glia-mediated neuron pruning in FXS disease models and provide insight into the molecular mechanisms of this defect. Clearly, the decision to eliminate neurons by glial phagocytosis rests on multiple, distinct signaling inputs and is executed via parallel avenues of neuron-to-glia communication (Song, 2023).
Loss of either neuronal Prtp or APPL was found to hampers glial phagocytosis neuron removal from the juvenile brain circuit but that loss of either signal alone causes a less severe impairment than blocking the neuronal FMRP-pMad pathway. These results suggest that Prtp and APPL act combinatorially downstream of FMRP-pMad, consistent with both neuronal Prtp and APPL driving the glial phagocytic clearance of targeted neurons. Prtp is a transmembrane protein trafficked from the endoplasmic reticulum (ER) to the cell surface of a neuron thus marked for removal, where it binds the Drpr engulfment receptor on glia. Through this mechanism, Prtp surface presentation downstream of the neuronal FMRP-pMad pathway is proposed to directly signal glial engulfment. In contrast, the cleaved extracellular domain of transmembrane APPL from neurons activates the glial endolysosomal network. This provides a separable function for APPL, suggesting why two signaling ligands mediate neuron removal by the glial phagocytosis mechanism. The Prtp signaling changes closely match the neuron clearance phenotypes of all neuronal dfmr1, mad, InR, and akt RNAi experiments. Likewise, the APPL signaling changes are consistent with FMRP-pMad regulation but not the InR-pAkt pathway. It id therefore proposed that Prtp and APPL coregulate glial phagocytic activity with different strengths or independently participate in glial phagocytosis via other regulatory mechanisms. To test the possibility that Prtp interacts with APPL in this clearance mechanism, this study could test the putative cross talk interaction at molecular and/or genetic levels. Conversely, neuronal Prtp and APPL may be independent signals in neuron-to-glial communication linked predominantly or solely through upstream FMRP regulation. Future studies will dissect these combinatorial signals downstream of inputs targeting the clearance of neurons from the juvenile brain (Song, 2023).
The pigment-dispersing factor (PDF) brain circuit mediates circadian clock functions, with the developmentally transient central PDF-Tri neurons presumably driving eclosion timing. Multiple signals appear to coordinate the targeted clearance of PDF-Tri neurons from the juvenile brain, including insulin-like peptide (ILP) signaling . The insulin receptor (InR) is present in both neurons and glia, acting to coordinate intercellular communication and neuronal remodeling. For glial signaling, InRs are proposed to be activated by secreted ILPs from the remodeling neurons to stimulate transduction cascades promoting glial phagocytosis. Glial InR phosphorylation triggers pAkt production, which, in turn, elevates Drpr engulfment receptor levels to drive glial phagocytosis. Consistently, genetically increasing glial InR levels elevates glia-dependent neuron removal of the PDF-Tri neurons. In the Drosophila FXS disease model, loss of FMRP represses InR phosphorylation in glia, whereas glial InR activation restores PDF-Tri neuron pruning. For neuron signaling, it. is proposed that InR-mediated pAkt signal transduction regulates neuron-to-glia communication in this same remodeling process. Similarly, InR signaling in Drosophila dendrite arborization neurons inhibits their developmental pruning. This work shows neuronal InR loss elevates Prtp signaling to drive glial phagocytosis, thus accelerating PDF-Tri neuron clearance from the juvenile brain. Neuronal InRs may be responding to autocrine ILP signaling from the remodeling neurons or ILPs from another source which is coordinating neuron and glia functions to properly regulate the glial phagocytosis neuron removal process. Future work will investigate how the different intercellular signals act on both neurons and glia to ensure the exact targeting and timing of the coordinated PDF-Tri neuron clearance from the juvenile brain (Song, 2023).
The Drosophila systemic immune response against many Gram-positive bacteria and fungi is mediated by the Toll pathway. How Toll-regulated effectors actually fulfill this role remains poorly understood as the known Toll-regulated antimicrobial peptide (AMP) genes are active only against filamentous fungi and not against Gram-positive bacteria or yeasts. Besides AMPs, two families of peptides secreted in response to infectious stimuli that activate the Toll pathway have been identified, namely Bomanins and peptides derived from a polyprotein precursor known as Baramicin A (BaraA). Unexpectedly, the deletion of a cluster of 10 Bomanins phenocopies the Toll mutant phenotype of susceptibility to infections. This study demonstrates that BaraA is required specifically in the host defense against Enterococcus faecalis and against the entomopathogenic fungus Metarhizium robertsii, albeit the fungal burden is not altered in BaraA mutants. BaraA protects the fly from the action of distinct toxins secreted by these Gram-positive and fungal pathogens, respectively, Enterocin V and Destruxin A. The injection of Destruxin A leads to the rapid paralysis of flies, whether wild type (WT) or mutant. However, a larger fraction of wild-type than BaraA flies recovers from paralysis within 5 to 10 h. BaraAs' function in protecting the host from the deleterious action of Destruxin is required in glial cells, highlighting a resilience role for the Toll pathway in the nervous system against microbial virulence factors. Thus, in complement to the current paradigm, innate immunity can cope effectively with the effects of toxins secreted by pathogens through the secretion of dedicated peptides, independently of xenobiotics detoxification pathways (Hyang, 2023).
Neuronal information conductance often involves the transmission of action potentials. The spreading of action potentials along the axonal process of a neuron is based on three physical parameters: the axial resistance of the axon, the axonal insulation by glial membranes, and the positioning of voltage-gated ion channels. In vertebrates, myelin and channel clustering allow fast saltatory conductance. This study shows that in Drosophila melanogaster voltage-gated sodium and potassium channels, Para and Shal, co-localize and cluster in an area resembling the axon initial segment. The local enrichment of Para but not of Shal localization depends on the presence of peripheral wrapping glial cells. In larvae, relatively low levels of Para channels are needed to allow proper signal transduction and nerves are simply wrapped by glial cells. In adults, the concentration of Para increases and is prominently found at the axon initial segment of motor neurons. Concomitantly, these axon domains are covered by a mesh of glial processes forming a lacunar structure that possibly serves as an ion reservoir. Directly flanking this domain glial processes forming the lacunar area appear to collapse and closely apposed stacks of glial cell processes can be detected, resembling a myelin-like insulation. Thus, Drosophila development may reflect the evolution of myelin which forms in response to increased levels of clustered voltage-gated ion channels (Rey, 2023).
In the vertebrate nervous system, saltatory conductance allows very fast spreading of information. This requires localized distribution of voltage-gated ion channels and concomitantly, the formation of the myelin sheath. The evolution of this complex structure is unclear. This study reports glial-dependent localization of voltage-gated ion channels at an axon initial segment (AIS)-like domain of peripheral Drosophila larval motor axons. As more channels accumulate in adults, a lacunar system and adjacent myelin-like structures are formed by central tract glia and peripheral wrapping glia (Rey, 2023).
In myelinated axons of vertebrates, voltage-gated Na+ and K+ channels are clustered at the AIS and the nodes of Ranvier. In invertebrate neurons, the AIS corresponds to the spike initiation zone located distal to the soma and distal to the dendrite branching point. Such segments were found in Caenorhabditis elegans and have been previously postulated for Drosophila neurons due to the localization of a giant ankyrin, which in all systems appears to be an important scaffolding protein at the AIS, as well as the presence of voltage-gated ion channels. Moreover, recent modeling approaches at the example of the pioneering aCC motor neuron predicted the localization of voltage-gated ion channels at the CNS/PNS boundary, which very well matches the localization of the voltage-gated ion channels Para and Shal, as reported in this study. Interestingly, in Drosophila para mRNA expression as well as Para protein localization depend on the presence of peripheral wrapping glia. In glia ablated nerves, Para expression is increased and decorates the entire axonal membrane. This loss of a clustered distribution may contribute to the pronounced reduction in axonal conductance velocity noted earlier in such glia ablated animals. In addition, an increased para mRNA expression was found. How glial cells control Para localization and how this is then transduced to an increased expression of para remains to be further studied. Since alterations in glial differentiation caused by manipulation of FGF-receptor signaling specifically in peripheral wrapping glia does not cause a change in Para expression or localization, proteins secreted by wrapping glia might be needed for the correct positioning of voltage-gated ion channels (Rey, 2023).
In the adult nervous system, the AIS-like domain is embedded in glial lacunar regions formed by wrapping glial cell processes.
The increased expression of Para within the AIS-like segments of adult brains is expected to generate strong ephaptic coupling forces. These are caused by ion flux through open channels which generate an electric field that is able to influence the gating of ion channels in closely neighboring axons. Ephaptic coupling helps to synchronize firing axons, but is also detrimental to the precision of neuronal signaling in closely apposed axons (Rey, 2023).
Ephaptic coupling is counteracted by the glial lacunar system, that spatially separates axons and adds more levels of wrapping. Furthermore, it was postulated that the lacunar system provides a large extracellular ion reservoir. Given the tight apposition of axonal and glial membranes with most parts of the nerve, which is in the range of 20 nm, only a very small interstitial fluid volume is normally present. Thus, action potential generation would deplete sodium and potassium ions very fast, and would prevent sustained neuronal activity. The development of lacunar structures might therefore provide sufficient amount of ion and at the same time physically separates axons to reduce the likelihood of ephaptic coupling. It will be interesting to test this hypothesis in the future (Rey, 2023).
Close to the lacunar structures myelin-like structures were detected. It appears that the glial processes that form the lacunae collapse to form compact myelin-like membrane sheets. Interestingly, myelin-like structures are not formed at the lateral borders of the lacunae but rather form at its distal end. This indicates that insulation is likely not a key function of the myelin-like structures, but rather these structures originate as a consequence of the collapsed lacunar system. Concomitant with the occurrence of the myelin-like differentiations a decrease is noted in the Para ion channel density. At the same time, the need for a large ion reservoir decreases, favoring the formation of myelin-like structures (Rey, 2023).
A hallmark of vertebrate myelin is the spiral growth of the insulating glial membrane. This is generally not observed in large fly nerves where glial membrane sheets rather fold back than spirally grow around a single axon. Compared to myelinated vertebrate axons, this provides the disadvantage that axons are not entirely insulated. However, spiral growth can be seen in small nerves where less extensive wrapping is noted. An additional unique feature of vertebrate myelin is its compact organization which is mediated by the MBP (Nave and Werner, 2021). In contrast to vertebrate myelin where extra- and intercellular space is removed, fly myelin-like structures only show a compaction of the extracellular space, which is expected to increase resistance as the number of freely moving ions is diminished. A fully compact myelin state would require MBP-like proteins which have not been identified in the fly genome (Rey, 2023).
In conclusion, the evolution of myelin appears reflected in the different developmental stages of Drosophila. First, voltage-gated ion channels are clustered at the AIS with the help of Drosophila glia. Second, upon increased expression of such ion channels in the adult nervous system, an ion reservoir might be formed by the lacunar system. The collapse of glial processes in the non-lacunar regions then provides the basis of myelin formation. In the future, it will be interesting to identify glial-derived signals that ensure channel positioning and determine how neuronal signaling adjusts channel expression and triggers formation of myelin (Rey, 2023).
The Pallidin protein is a central subunit of a multimeric complex called biogenesis of lysosome-related organelles complex 1 (BLOC1) that regulates specific endosomal functions and has been linked to schizophrenia. Downregulation of Pallidin and other members of BLOC1 in the surface glia, the Drosophila equivalent of the blood-brain barrier, reduces and delays nighttime sleep in a circadian-clock-dependent manner. In agreement with BLOC1 involvement in amino acid transport, downregulation of the large neutral amino acid transporter 1 (LAT1)-like transporters JhI-21 and mnd, as well as of TOR (target of rapamycin) amino acid signaling, phenocopy Pallidin knockdown. Furthermore, supplementing food with leucine normalizes the sleep/wake phenotypes of Pallidin downregulation, and this study identified a role for Pallidin in the subcellular trafficking of JhI-21. Finally, evidence is provided that Pallidin in surface glia is required for GABAergic neuronal activity. These data identify a BLOC1 function linking essential amino acid availability and GABAergic sleep/wake regulation (Li, 2023).
Biogenesis of lysosome-related organelles complex 1 (BLOC1) is an octameric complex linked to endosomal compartments and the cytoskeleton (Cheli, 2010). The genes coding for the 8 subunits in mice (Pallidin, dysbindin, BLOS1, BLOS2, BLOS3, cappuccino, muted, and snapin) are broadly expressed within the brain and in peripheral tissues. The complex regulates the trafficking of various receptors and transporters and appears to play a prominent role in the biogenesis of recycling endosomes. Mice bearing severe or complete loss-of-function mutations in BLOC1 genes are viable and fertile. They display common phenotypes, originating from defects in highly specialized lysosome-related organelles, such as reduced pigmentation due to impaired retinal and epidermal melanosomes or extended bleeding times resulting from the lack of dense granules in platelets. In humans, mutations in BLOC1 genes and other functionally related genes are found in the Hermansky-Pudlak syndrome (Li, 2023).
In addition, genetic studies have identified variants of the dysbindin gene and other BLOC1 genes as risk factors for developing schizophrenia. Although the latter results are debated, several postmortem studies have reported reduced levels of dysbindin mRNA and protein in the brain of schizophrenics. Furthermore, genome-wide association studies also reported a link between dysbindin genetic variants and cognitive abilities (Li, 2023).
These findings have led to investigations aiming at deciphering the role of BLOC1 in neuronal function using not only mouse models defective for individual BLOC1 gene function but also the Drosophila model, in which the complex is well conserved. These studies confirmed the involvement of BLOC1 in behavior and memory. They identified potential cellular and molecular mechanisms such as abnormal glutamatergic, GABAergic, and dopaminergic transmission. However, the vast majority of these studies have been carried out in animals with spontaneous or artificial mutations. This approach makes the interpretation of the resulting phenotypes challenging, given the broad expression of BLOC1 in many cell types. Furthermore, the complex is expressed during development, when it can be involved in neurodevelopmental diseases such as autism spectrum disorders (Li, 2023).
Apart from one recent report (Lee, 2018) the involvement of BLOC1 in sleep/wake regulation has not been investigated. Interestingly, it has been found that Pallidin is upregulated in a somnolent mouse model with defective histaminergic transmission (Seugnet, 2023).
Thus, further investigation is required given the critical implication of sleep in brain function, and in particular in schizophrenia, in which sleep disruption could be in part a consequence of the pathology, or an aggravating factor as seen in human
and rodent models. This study investigated the potential role of BLOC1 in sleep/wake regulation in Drosophila. Neurotransmission systems, ion channels, and glial functions in Drosophila and mammals are globally conserved (Li, 2023).
There are differences regarding sleep/wake in insects compared with mammals, such as the absence of slow wave and paradoxical sleep. Nevertheless, the evidence for conserved regulatory principles and functions is compelling.
The flexibility and short generation time of the Drosophila model is an asset in molecular genetic studies, providing hypotheses that can be tested in rodents and ultimately used for therapy. A conditional knockdown strategy was used to target Pallidin, a major component of BLOC1. The pool of Pallidin in the cell is associated almost entirely with the complex, and this protein plays a central role through its interactions with dysbindin, BLOS1, and cappuccino (Cheli, 2010), with its loss leading to their degradation (Li, 2023).
This study focused on glia, as a previous results suggest a function for Pallidin in non-neuronal cells. Glial cells are increasingly proven to play very significant roles in the control of sleep/wake and circadian rhythms both in Drosophila and in rodent models. Glia-dependent neurotransmitter reuptake, neurotransmitter metabolism, and glial calcium transients critically influence neuronal networks that control sleep timing and sleep homeostasis. Neuroglia signaling pathways have been shown to modulate sleep-deprivation-induced learning impairments. Recent reports have demonstrated the prominent role of glial cells at the interface between the brain and the circulating fluids during the sleep/wake cycle (Li, 2023 and references therein).
This study provides evidence in Drosophila that Pallidin and other BLOC1 components regulate the initiation of sleep in the early part of the night, at the level of surface glial (SG) cells. In Drosophila, SG cells express tight junctions and molecular components in common with the mammalian BBB. As in the BBB this organization prevents the entry of macromolecules and tightly regulates the exchanges of solutes between the circulatory system (the hemolymph in Drosophila) and the brain, thus maintaining the particular interstitial fluid composition necessary for neuronal activity. This function relies on intense transporter activity. The data support a mechanism whereby BLOC1 regulates LAT1-like transporters subcellular trafficking in both perineurial and subperineurial cells, leading to adequate essential amino acid supply and GABAergic neuronal activity promoting sleep, in a circadian-clock-dependent manner. This study observed that downregulating Pallidin using a pan-neuronal driver also resulted in reduced night sleep, suggesting that SG is not the only cell type in the brain where Pallidin is required to regulate sleep (Li, 2023).
Blocking the cycling of the molecular clock by placing flies in a per0 mutant background completely abolishes the Pallidin knockdown sleep/wake phenotype, suggesting that the circadian clock is implicated. Accordingly, it was found that hyperpolarization of PDF-expressing clock neurons is sufficient to normalize the main Pallidin knockdown phenotype: increased sleep latency at night. This raises the possibility that Pallidin affects clock-dependent sleep/wake regulatory networks and in particular, those under the control of the large PDF-expressing neurons (lLNv). In lLNv neurons, GABAergic input is modulated in a circadian manner, resulting in higher inhibition in the early night, promoting sleep, while a disruption of this input results in delayed and fragmented sleep (Li, 2023).
The GABAergic neurons responsible for lLNv inhibition are so far unidentified. The delayed and reduced night sleep observed upon Pallidin knockdown in SG is also similar to the phenotype previously reported with pan-GABAergic neuron inhibition.
Conversely, feeding flies the GABA agonist THIP65
or increasing GABAergic neuronal firing by expressing TrpA1 can potently induce sleep. The latter manipulation cannot promote sleep when Pallidin is downregulated in SG, suggesting that the gene is required to allow sustained GABAergic neuronal activity. Alternatively, Pallidin function could be regulated directly by the clock present in perineurial cells, which modulates the efflux properties of subperineurial cells in a circadian manner (Li, 2023).
Downregulation of several members of BLOC1 in SG phenocopies the knockdown of Pallidin, strongly suggesting that the whole complex is involved in this Pallidin-dependent sleep/wake regulation. This is consistent with the observation that the pool of Pallidin and dysbindin present in the cell is almost entirely associated with the complex and that the lack of one subunit leads to a destabilization of the others (Li, 2023).
The lack of sleep alteration with snapin manipulation may result from lower efficiency of the genetic tools used for downregulation. Alternatively, it may also reflect different gene dosage sensitivity and functionality for the different members of the complex, as previously reported, or the existence of multiple subunits within the complex2
or different complexes containing a subset of the subunits (Li, 2023).
In Drosophila larval neuromuscular junction, Snapin has, for example, been shown to regulate synaptic homeostasis (Dickman, 2012),
while Pallidin does not seem to affect baseline neurotransmission and is required during sustained neuronal activity to replenish the pool of releasable synaptic vesicles (Chen, 2017). BLOC1 has been shown to be required for the biogenesis of recycling endosomes through its interactions with sorting endosomes and the cytoskeleton (Delevoye, 2016).
These BLOC1 functions and the regulation of sleep by BLOC1 members reported in this study are in agreement with a recent report showing bidirectional interactions between sleep and endocytosis in SG (Lee, 2018).
Intriguingly, the endocytosis in SG was reported to be the most intense in the early part of the night,
when sleep is the deepest and the phenotype of Pallidin downregulation is the most pronounced. In addition, the activity of the recycling endosome associated small GTPase Rab11 appears to play a prominent role in this context.
Consistent with these findings, extensive protein-protein interaction analyses identified rab11 as the best interacting partner for BLOC1 in both Drosophila and humans.5
Thus, BLOC1 may facilitate the high endocytic activity of SG during the early part of the night, notably the biogenesis of recycling endosomes, while having a less prominent role at other times during the day. In line with the results, Pallidin mutant mice display reduced sleep amount during the light phase of the day, the primary sleep period in rodents, and shorter average sleep-bout duration (Li, 2023).
However, BBB-specific and conditional knockout models in mice would be necessary to determine whether the model outlined in this study applies to mammals. In humans, the incidence of sleep/wake disorders in the rare Hermansky-Pudlak syndrome, bearing deficits in BLOC1 or other functionally related complexes, is unknown but deserves scrutiny. In contrast, sleep disruption is common among patients suffering from schizophrenia, another pathology associated with BLOC1 deficits. Interestingly, one of the prominent sleep abnormalities in schizophrenia patients is a reduction in sleep spindle density, which strongly relies on GABAergic activity, and the latter is also affected in this pathology. Accordingly, previous studies in dysbindin mutant mice have shown that BLOC1 can disrupt GABAergic activity (Li, 2023).
Thus, a potential role for amino acid transport at the BBB in those contexts would deserve investigation given these results. Early studies of Pallidin function pointed to a role in amino acid import into the brain transport, as suggested by lower sensitivity to intraperitoneal injections of the LAT1 substrates l-DOPA and tryptophan. The LAT1 transporter plays a major role in the import of these amino acids and in the import of large neutral essential amino acids such as leucine, isoleucine, and histidine (Li, 2023).
A conditional knockout of LAT1 in brain endothelial cells confirmed the crucial role of LAT1 in the regulation essential amino acid abundance within the brain, its impact on GABAergic transmission, and its potential implication in autism spectrum disorders.
Intriguingly, the relative abundance of amino acids in the brain of Pallidin mutant mice resembles those found in mice with a BBB specific knockout LAT1 (Li, 2023).
This study provides evidence that Pallidin function in SG is linked to essential amino acid supply and to LAT1-like transporter activity. First, downregulation of the LAT1-like transporters and of TOR signaling phenocopy knock down, to a large extent or completely, the BLOC1 genes. Second, supplementing the food with leucine can normalize the JhI-21, Pallidin, and Blos2 phenotypes. Interestingly, the phenotype of the JhI-21 transporter knockdown could be rescued by valine and tryptophan supplementation, while Pallidin knockdown could be rescued only by leucine. This suggests that Pallidin does not solely modulate LAT1-like transporter function and has complex effect on multiple transport systems, as previously suggested (Li, 2023).
JhI-21 and minidisc are the Drosophila closest homologs of LAT1 light-chain.
JhI-21 is expressed broadly in the Drosophila brain, with most of the signal in perinuclear punctae that colocalizes partially with the lysosomal marker ATG8. The subcellular trafficking of JhI-21 in SG is abnormal following Pallidin downregulation, with a substantial fraction of the transporter localized in the nucleus. This abnormal trafficking is likely to reduce the functionality of the transporter, explaining the similar sleep/wake phenotypes of JhI-21 and Pallidin downregulation in SG and their normalization by essential amino acid supplementation. A nuclear localization has already been reported in glioma cell lines for the solute carrier (SLC) Eaat1 and Eaat2 transporters, and for the K+ inwardly rectifying channel. In both cases, this unusual subcellular localization was associated with an overall reduced functionality of the protein in the cell. Aside transporters, several full-length transmembrane receptors have been repeatedly reported to be localized in the nucleus where some of them may act as a transcription factors (Li, 2023).
The mechanisms underlying this phenomenon are mostly unknown. In this study, it was not possible to detect obvious changes in autophagy, suggesting that Pallidin downregulation does not induce major changes in lysosomal activity and rather affects more specifically particular cargos, as previously suggested. Interestingly, in addition to their cytoplasmic localization, a nuclear localization has previously been reported for two BLOC1 subunits: BLOS2102
and, in particular, dysbindin. ATG8 is also found both in lysosomes and in the nucleus where it can regulate gene expression in association with transcription factors. Such lysosomal-nuclear connections open the possibility of trapping a diversity of unexpected proteins in the nucleus in normal as well as in abnormal conditions (Li, 2023).
In Drosophila, protein intake, threonine intake, and D-serine levels play a role in sleep/wake regulation. Although the changes in amino acid intake presumably change global free amino acid levels within the brain, the effect on sleep/wake regulation seems to originate from specific sleep/wake regulatory networks or neurotransmission systems. In humans, amino-acid-supplemented diets have been designed to improve sleep and wakefulness, on the basis of the principle that this will increase the synthesis of monoamines. The results point to a model independent from these previously identified mechanisms: higher recycling endosome activity in the early part of the night, mediated by BLOC1, would lead to high LAT1-like activity and essential amino acid import in the brain. The TOR signaling appears also to be required and may facilitate LAT1-like transporter function. This assessment suggests that Pallidin-dependent sleep regulation does not involve a modulation of monoamine levels, in contrast with previous reports. However, in these reports the conclusions were reached using complete knock out and not by downregulation in specific cell types, making comparison with the present results difficult. For example, Drosophila Dysbindin has been suggested to regulate global brain dopamine level by modulating its recycling through glial cells (Li, 2023).
In contrast, the current data suggest that essential amino acid import to the brain facilitated by Pallidin would enhance GABAergic transmission required for sleep. This study showed that a large number of GABAergic neurons in the adult fly brain are in direct cellular contacts with SG. Although the significance and the function of these contacts remain to be determined, these observations fit the idea that a significant subset of these neurons have particular metabolic needs (Li, 2023).
How could amino acid transport regulate GABAergic transmission? Amino acids are at the interplay among many processes, being involved in protein synthesis, energy metabolism, neurotransmitter synthesis, and degradation. Amino acid transporters may further intertwine these processes: as a prime example, LAT1 is an antiporter that can associate the import of leucine to the export of glutamine, two amino acids that are crucial in the glutamate/GABA/glutamine cycle in the brain (Li, 2023).
Pharmacological experiments have indeed suggested that LAT1 in the BBB regulates GABA homeostasis in the interstitial fluid. Branched-chain amino acids
(BCAA) and leucine in particular are thought to be critical providers of nitrogen for glutamate synthesis through transamination,
affecting glutamate/GABA/glutamine cycling. Enzymes for BCAA metabolism are conserved in Drosophila, suggesting similar metabolic regulation in this model organism. An interesting example is Drosophila mutants for GABA transaminase (GABAT), which sleep 2–3 h longer than their genetic controls because of impaired GABA degradation. For survival, these mutants require that food include glutamate or BCAAs (leucine and valine), whose transamination can provide glutamate in the cells. These data indicate that GABA metabolism and BCAAs as a source of glutamate play a substantial role in sleep/wake regulation and brain energy metabolism. Interestingly, this study found that the GABA/glutamate ratio tends to be decreased in Pallidin knockdown flies, while the glutamate/aspartate ratio is increased compared with controls. These results suggest that BLOC1 function in SG modulates neurotransmitter and brain amino acid metabolism on a global level and may control the inhibitory/excitatory balance (Li, 2023).
The data presented in this study emphasize the implication of circulating amino acids, in particular BCAA, in sleep/wake regulation, corroborating several recent metabolomics studies in mammalian models and in humans, in which branched-chain amino acid (BCAA) levels have been repeatedly shown to be modulated by the circadian clock and/or the sleep homeostat. For example, a recent study identified in insomniac patients prominent changes in the levels of several circulating BCAA, including increased levels of leucine during the night.
Food supplemented with BCAA can correct sleep disorders in a mouse model with chronic sleep disruption. It is worth noting that genes involved in amino acid transport are among those commonly disrupted in autism and schizophrenia. In conclusion, this study provides potential mechanisms at the blood-brain interface that may be relevant to both sleep disruption and psychosis and emphasizes the possibility of diet-based therapies (Li, 2023).
One limitation of this study has been the inability to monitor Pallidin protein expression using immunofluorescence. A previously published antibody
produced a non-specific signal in the adult brain, and attempts to generate a new antibody were unsuccessful. This precluded determining the subcellular localization of the protein and assessing the local efficiency of the knockdown constructs. The lack of appropriate drivers prevented more precise manipulation of the neuronal targets affected by Pallidin function: the GABAergic neurons presynaptic to lLNv neurons are unknown, and therefore there is no identified driver making it possible to monitor or control their activity, and in addition, there are no available LexA construct specifically expressed in lLNv neurons. For future investigations, it will be important to develop an efficient conditional system, independent of temperature, to limit the knockdown of Pallidin to the adult stage. Finally, further work is required to elucidate the involvement of the TOR signaling pathway in this context and the Pallidin-JhI-21-dependent interactions between SG and GABAergic neurons (Li, 2023).
glia display diverse morphologies, both across and within glial classes. This study explored the relationship between glial morphology and transcriptional signature using the Drosophila CNS, where glia are categorised into 5 main classes (outer and inner surface glia, cortex glia, ensheathing glia, and astrocytes). Single-cell RNA sequencing data of Drosophila glia were analysedin 2 well-characterised tissues from distinct developmental stages containing distinct circuit types: the embryonic ventral nerve cord (VNC) (motor) and the adult optic lobes (sensory). This analysis identified a new morphologically and transcriptionally distinct surface glial population in the VNC. However, many glial morphological categories could not be distinguished transcriptionally, and indeed, embryonic and adult astrocytes were transcriptionally analogous despite differences in developmental stage and circuit type. While extensive within-class transcriptomic diversity was detected for optic lobe glia, this could be explained entirely by glial residence in the most superficial neuropil (lamina) and an associated enrichment for immune-related gene expression. In summary, this study generated a single-cell transcriptomic atlas of glia in Drosophila, and this extensive in vivo validation revealed that glia exhibit more diversity at the morphological level than was detectable at the transcriptional level (Lago-Baldaia, 2023).
Recent studies have demonstrated that astrocytes cooperate with neurons of the brain to mediate circadian control of many rhythmic processes including locomotor activity and sleep. Transcriptional profiling studies have described the overall rhythmic landscape of the brain, but few have employed approaches that reveal heterogeneous, cell-type specific rhythms of the brain. Using cell-specific isolation of ribosome-bound RNAs in Drosophila, the first circadian "translatome" for astrocytes was generated. This analysis identified 293 "cycling genes" in astrocytes, most with mammalian orthologs. A subsequent behavioral genetic screen identified a number of genes whose expression is required in astrocytes for normal sleep behavior. In particular, this study showed that certain genes known to regulate fly innate immune responses are also required for normal sleep patterns (You, 2021).
Astrocytes play key roles in regulating multiple aspects of neuronal function from invertebrates to humans and display Ca(2+) fluctuations that are heterogeneously distributed throughout different cellular microdomains. Changes in Ca(2+) dynamics represent a key mechanism for how astrocytes modulate neuronal activity. An unresolved issue is the origin and contribution of specific glial Ca(2+) signaling components at distinct astrocytic domains to neuronal physiology and brain function. The Drosophila model system offers a simple nervous system that is highly amenable to cell-specific genetic manipulations to characterize the role of glial Ca(2+) signaling. This study has identified a role for ER store-operated Ca(2+) entry (SOCE) pathway in perineurial glia (PG), a glial population that contributes to the Drosophila blood-brain barrier. PG cells display diverse Ca(2+) activity that varies based on their locale within the brain. Ca(2+) signaling in PG cells does not require extracellular Ca(2+) and is blocked by inhibition of SOCE, Ryanodine receptors, or gap junctions. Disruption of these components triggers stimuli-induced seizure-like episodes. These findings indicate that Ca(2+) release from internal stores and its propagation between neighboring glial cells via gap junctions are essential for maintaining normal nervous system function (Weiss, 2021).
Developmental neural activity is a common feature of neural circuit assembly. Although glia have established roles in synapse development, the contribution of neuron-glia interactions to developmental activity remains largely unexplored. This study shows that astrocytes are necessary for developmental activity during synaptogenesis in Drosophila. Using wide-field epifluorescence and two-photon imaging, it was shown that the glia of the central nervous system participate in developmental activity with type-specific patterns of intracellular calcium dynamics. Genetic ablation of astrocytes, but not of cortex or ensheathing glia, leads to severe attenuation of neuronal activity. Similarly, inhibition of neuronal activity results in the loss of astrocyte calcium dynamics. By altering these dynamics, this study showed that astrocytic calcium cycles can influence neuronal activity but are not necessary per se. Taken together, these results indicate that, in addition to their recognized role in the structural maturation of synapses, astrocytes are also necessary for the function of synapses during development (Bajar, 2022).
A precise balance between sleep and wakefulness is essential to sustain a good quality of life and optimal brain function. GABA is known to play a key and conserved role in sleep control, and GABAergic tone should, therefore, be tightly controlled in sleep circuits. This study examined the role of the astrocytic GABA transporter (Gat) in sleep regulation using Drosophila melanogaster. A hypomorphic Gat mutation (Gat33-1) increased sleep amount, decreased sleep latency, and increased sleep consolidation at night. Interestingly, sleep defects were suppressed when Gat33-1 was combined with a mutation disrupting wide-awake (wake), a gene that regulates the cell-surface levels of the GABA(A) receptor Resistance to dieldrin (Rdl) in the wake-promoting large ventral lateral neurons (l-LNvs). Moreover, RNAi knockdown of Rdl and its modulators dnlg4 and wake in these circadian neurons also suppressed Gat33-1 sleep phenotypes. Brain immunohistochemistry showed that GAT-expressing astrocytes were located near RDL-positive l-LNv cell bodies and dendritic processes. It is concluded that astrocytic GAT decreases GABAergic tone and RDL activation in arousal-promoting LNvs, thus determining proper sleep amount and quality in Drosophila (Chaturvedi, 2022).
Anastasis is a recently described process in which cells recover after late-stage apoptosis activation. The functional consequences of anastasis for cells and tissues are not clearly understood. Using Drosophila, rat and human cells and tissues, including analyses of both males and females, this study presents evidence that glia undergoing anastasis in the primary astrogliopathy Alexander disease subsequently express hallmarks of senescence. These senescent glia promote non-cell autonomous death of neurons by secreting interleukin family cytokines. These findings demonstrate that anastasis can be dysfunctional in neurologic disease by inducing a toxic senescent population of astroglia (Wang, 2022).
Huntington disease (HD) is a neurodegenerative disorder caused by expanded CAG repeats in the huntingtin gene that alters cellular homeostasis, particularly in the striatum and cortex. Astrocyte signaling that establishes and maintains neuronal functions are often altered under pathological conditions. In this study single-nuclei RNA-sequencing was performed on human HD patient-induced pluripotent stem cell (iPSC)-derived astrocytes and on striatal and cortical tissue from R6/2 HD mice to investigate high-resolution HD astrocyte cell state transitions. Altered maturation and glutamate signaling were observed in HD human and mouse astrocytes. Human HD astrocytes also showed upregulated actin-mediated signaling, suggesting that some states may be cell-autonomous and human specific. In both species, astrogliogenesis transcription factors may drive HD astrocyte maturation deficits, which are supported by rescued climbing deficits in HD Drosophila with NFIA knockdown. Thus, dysregulated HD astrocyte states may induce dysfunctional astrocytic properties, in part due to maturation deficits influenced by astrogliogenesis transcription factor dysregulation (Reyes-Ortiz, 2023).
The remodeling of neurons is a conserved fundamental mechanism underlying nervous system maturation and function. Astrocytes can clear neuronal debris and they have an active role in neuronal remodeling. Developmental axon pruning of Drosophila memory center neurons occurs via a degenerative process mediated by infiltrating astrocytes. However, how astrocytes are recruited to the axons during brain development is unclear. Using an unbiased screen, the gene requirement of orion/CG2206, encoding for a chemokine-like protein, was identified in the developing mushroom bodies. Functional analysis shows that orion is necessary for both axonal pruning and removal of axonal debris. orion performs its functions extracellularly and bears some features common to chemokines, a family of chemoattractant cytokines. It is proposed that orion is a neuronal signal that elicits astrocyte infiltration and astrocyte-driven axonal engulfment required during neuronal remodeling in the Drosophila developing brain (Boulanger, 2021).
Neuronal remodeling is a widely used developmental mechanism, across the animal kingdom, to refine dendrite and axon targeting necessary for the maturation of neural circuits. Importantly, similar molecular and cellular events can occur during neurodevelopmental disorders or after nervous system injury. A key role for glial cells in synaptic pruning and critical signaling pathways between glia and neurons has been identified. In Drosophila, the mushroom body (MB), a brain memory center, is remodeled at metamorphosis and MB γ neuron pruning occurs by a degenerative mechanism. Astrocytes surrounding the MB have an active role in the process: blocking their infiltration into the MBs prevents remodeling. MB γ neuron remodeling relies on two processes: axon fragmentation and the subsequent clearance of axonal debris. Importantly, it has been shown that astrocytes are involved in these two processes and that these two processes can be decoupled. Altering the ecdysone signaling in astrocytes, during metamorphosis, results both in a partial axon pruning defect, visualized as either some individual larval axons or as thin bundles of intact larval axons remaining in the adults, and also in a strong defect in clearance of debris, visualized by the presence of clusters of axonal debris. Astrocytes have only a minor role in axon severing as evidenced by the observation that most of the MB γ axons are correctly pruned when ecdysone signaling is altered in these cells. When astrocyte function is blocked, the γ axon-intrinsic fragmentation process remains functional and the majority of axons degenerate (Boulanger, 2021).
It has been widely proposed that a 'find-me/eat-me' signal emanating from the degenerating γ neurons is necessary for astrocyte infiltration and engulfment of the degenerated larval axons. However, the nature of this glial recruitment signal is unclear (Boulanger, 2021).
This study has identified a gene (orion), not previously described, by screening for viable ethyl methanesulfonate (EMS)-induced mutations and not for lethal mutations in MB clones as was done previously. This allowed the identification of genes involved in glial cell function by directly screening for defects in MB axon pruning. It was found that orion1, a viable X-chromosome mutation, is necessary for both the pruning of some γ axons and removal of the resulting debris. orion is secreted from the neurons, remains near the axon membranes where it associates with infiltrating astrocytes, and is necessary for astrocyte infiltration into the γ bundle. This implies a role for an as-yet-undefined orion receptor on the surface of the astrocytes. orion bears some chemokine features, for example, a CX3C motif, three glycosaminoglycan (GAG) binding consensus sequences that are required for its function. Altogether, these results identify a neuron-secreted extracellular messenger, which is likely to be the long-searched-for signal responsible for astrocyte infiltration and engulfment of the degenerated larval axons and demonstrate its involvement for neuronal remodeling (Boulanger, 2021).
Adult orion1 individuals showed a clear and highly penetrant MB axon pruning phenotype as revealed by the presence of some adult unpruned vertical γ axons as well as the strong presence of debris (100% of mutant MBs). Astrocytes, visualized with alrm-GAL4, are the major glial subtype responsible for the clearance of the MB axon debris. The presence of γ axon debris is a landmark of defective astrocyte function, as has been described, and is also further shown in this study. The unpruned axon phenotype was particularly apparent during metamorphosis. At 24 h after puparium formation (APF), although γ axon branches were nearly completely absent in the wild-type control, they persisted in the orion1 mutant brains, where a significant accumulation of debris was also observed. The number of unpruned axons at this stage is lower in orion1 than in Hr39C13 where the γ axon-intrinsic process of pruning is blocked. In addition, the MB dendrite pruning was clearly affected in orion1 individuals (Boulanger, 2021).
The orion1 EMS mutation was localized by standard duplication and deficiency mapping as well as by whole-genome sequencing. The orion gene (CG2206) encodes two putatively secreted proteins: orion-A [664 amino acid (a.a.)] and orion-B (646 a.a.), whose messenger RNAs (mRNAs) arise from two different promoters. These two proteins differ in their N-terminal domains and are identical in the remainder of their sequences. The EMS mutation is a G to C nucleotide change inducing the substitution of the glycine (at position 629 for orion-A and 611 for orion-B) into an aspartic acid. The mutation lies in the common shared part and therefore affects both orion-A and -B functions. Both isoforms display a signal peptide at their N termini, suggesting that they are secreted. Interestingly, a CX3C chemokine signature is present in the orion common region. Chemokines are a family of chemoattractant cytokines, characterized by a CC, CXC, or CX3C motif, promoting the directional migration of cells within different tissues. Mammalian CX3CL1 (also known as fractalkine) is involved in, among other contexts, neuron-glia communication. Mammalian Fractalkines display conserved intramolecular disulfide bonds that appear to be conserved with respect to their distance from the CX3C motif present in both orion isoforms. Fractalkine and its receptor, CX3CR1, have been recently shown to be required for post-trauma cortical brain neuron microglia-mediated remodeling in a mouse whisker lesioning paradigm. This study observed that the change of the CX3C motif into CX4C or AX3C blocked the orion function necessary for the MB pruning. Similarly, the removal of the signal peptide also prevented pruning. These two results indicate that the orion isoforms likely act as secreted chemokine-like molecules. Three CRISPR/Cas9-mediated mutations in the orion gene were generated that either delete the common part (orionΔC), the A-specific part (orionΔA), or the B-specific part (orionΔB). Noticeably, orionΔC displayed the same MB pruning phenotype as orion1, which is also the same in orion1/Deficiency females, indicating that orion1 and orionΔC are likely null alleles for this phenotype. In contrast, orionΔA and orionΔB have no MB phenotype by themselves, indicating the likelihood of functional redundancy between the two proteins in the pruning process (Boulanger, 2021).
Using the GAL4/UAS system, this study found that expression of wild-type orion in the orion1 MB γ neurons (201Y-GAL4) fully rescued the MB mutant phenotype (100% of wild-type MBs n = 387), although wild-type orion expression in the astrocytes (alrm-GAL4) did not rescue. repo-GAL4 could not be used because of lethality when combined with UAS-orion. This supports the hypothesis that orion is produced by axons and, although necessary for astrocyte infiltration, not by astrocytes. Both UAS-orion-A and UAS-orion-B rescued the orion1 pruning phenotype indicating again a likely functional redundancy between the two proteins at least in the pruning process. Complementary to the rescue results, this study found that the expression of an orion-targeting RNA interference (RNAi) in the MBs produced unpruned axons similar to that in orion1, although the debris is not apparent likely due to an incomplete inactivation of the gene expression by the RNAi. The expression of the same RNAi in the glia had no effect. Using the mosaic analysis with a repressible cell marker (MARCM), it was found that orion1 homozygous mutant neuroblast clones of γ neurons, in orion1/+ phenotypically wild-type individuals, were normally pruned. Therefore, orion1 is a non-cell-autonomous mutation that is expected if the orion proteins are secreted. orion proteins secreted by the surrounding wild-type axons rescue the pruning defects in the orion mutant clones (Boulanger, 2021).
From genetic data, orion expression is expected in the γ neurons. The fine temporal transcriptional landscape of MB γ neurons was recently described and a corresponding resource is freely accessible. Noteworthy, orion is transcribed at 0 h APF and dramatically decreases at 9 h APF with a peak at 3 h APF. The nuclear receptor EcR-B1 and its target Sox14 are key transcriptional factors required for MB neuronal remodeling. orion was found to be a likely transcriptional target of EcR-B1 and Sox14. This is also consistent with earlier microarray analysis observations. Noticeably, forced expression of UAS-EcR-B1 in the MBs did not rescue the orion mutant phenotype and EcR-B1 expression, in the MB nuclei, and is not altered in orion1 individuals. Furthermore, the unpruned axon phenotype produced by orion RNAi is rescued by forced expression of EcR-B1 in the MBs. Therefore, the genetic interaction analyses support orion being downstream of EcR-B1 (Boulanger, 2021).
Further molecular and cellular work focused on orion-B alone since a functional redundancy between the two isoforms was apparent. The orion-B protein was expressed in the γ neurons using an UAS-orion-B-Myc insert and the 201Y-GAL4 driver. orion-B was present along the MB lobes and extracellularly present as visualized by anti-Myc staining. Indeed, anti-Myc staining was particularly strong at the tip of the lobes indicating the presence of extracellular orion-B. Synaptic terminals are condensed in the γ axon varicosities that disappear progressively during remodeling and hole-like structures corresponding to the vestiges of disappeared varicosities can be observed at 6 h APF. The presence of Myc-labeled orion-B was noticed inside these hole-like structures. The secretion of the orion proteins should be under the control of their signal peptide and, therefore, orion proteins lacking their signal peptide (ΔSP) should not show this 'extracellular' phenotype. When UAS-orion-B-Myc-ΔSP was expressed, orion-B was not observed outside the axons or in the hole-like structures. The possibility that this 'extracellular' phenotype was due to some peculiarities of the Myc labeling was excluded by using a UAS-drl-Myc construct. Drl is a membrane-bound receptor tyrosine kinase and Drl-Myc staining, unlike orion-B, was not observed outside the axons or in the hole-like structures. In addition, the presence of Myc-labeled orion-B protein not associated with green fluorescent protein (GFP)-labeled axon membranes can be observed outside the γ axon bundle in 3D reconstructing images. Nevertheless, these signals are possibly located inside the glial compartments and not as freely diffusing orion protein. Finally, supporting the hypothesis that orion acts as a secreted protein, it has been reported to be present in biochemically purified exosomes, indicating that it may act on the glia via its presence on or in exosomes (Boulanger, 2021).
Since glial cells are likely directly involved in the orion1 pruning phenotype, their behavior early during the pruning process was examined. At 6 h APF the axon pruning process starts and is complete by 24 h APF, but the presence of glial cells in the vicinity of the wild-type γ lobes is already clearly apparent at 6 h APF9. Glial cells visualized by a membrane-targeted GFP (UAS-mGFP) under the control of repo-GAL4 were examined, and the γ axons were co-stained with anti-Fas2. At 6 h APF, a striking difference was noted between wild-type and orion1 brains. Unlike in the wild-type control, there is essentially no glial cell invasion of the γ bundle in the mutant. Interestingly, glial infiltration as well as engulfment of the degenerated larval axons was not observed in orion1 neither at 12 h APF nor at 24 h APF, suggesting that glial cells never infiltrate MBs in mutant individuals. The possibility that this lack of glial cell activity was due to a lower number of astrocytes in mutant versus wild-type brains was ruled out (Boulanger, 2021).
The proximity was examined between MB orion-Myc and astrocytes, as inferred from the shape of the glial cells, labeled with the anti-Drpr antibody at 6 h APF. The distribution was examined along the vertical γ lobes (60 μm of distance) of orion-B-Myc (wild-type protein) and of orion-B-ΔSP-Myc (not secreted), in an otherwise wild-type background. Quantification was performed only from images where an astrocyte sat on the top of the vertical lobe. A peak of orion-Myc localization was always found in the axonal region close to the astrocyte (<7 μm) when wild-type orion-B-Myc was quantified. However, this was not the case (n = 9) when orion-B-ΔSP-Myc was quantified. This strongly suggests that astrocytic processes may be 'attracted' by secreted orion (Boulanger, 2021).
Moreover, it was observed that extracellularly present orion stays close to axon membranes. Protein (in particular chemokine) localization to membranes is often mediated by GAGs, a family of highly anionic polysaccharides that occurs both at the cell surface and within the extracellular matrix. GAGs, to which all chemokines bind, ensure that these signaling proteins are presented at the correct site and time in order to mediate their functions. Three consensus sequences for GAG linkage were identified in the common part of orion. These sequences were mutated individually, and the mutant proteins were examined for their ability to rescue the orion1 pruning deficit in vivo. The three GAG sites are required for full orion function, although mutating only GAG3 produced a strong mutant phenotype (Boulanger, 2021).
These findings imply a role for an as-yet-undefined orion receptor on the surface of the glial cells. The glial receptor draper (drpr) seemed an obvious candidate, although Drpr ligands unrelated to orion have been identified. The MB remodeling phenotypes in orion1 and drprΔ5 are, however, different with orion mutant phenotype being stronger than the drpr one. The use of an UAS-mGFP driven by 201Y-GAL4, instead of anti-Fas2, where the labeling of αβ axons often masks individual unpruned γ axons, allowed occasionally observation of unpruned axons in drprΔ5 1-week-old post-eclosion brains in addition to uncleared debris. This indicates a certain degree of previously undescribed unpruned axon persistence in the mutant background. Nevertheless, only orion mutant displayed a 100% penetrant phenotype of both unpruned axons and debris (strong category) in adult flies, which are still present in old flies. On the contrary, the weaker drpr mutant phenotype strongly decreases throughout adulthood. This suggests that Drpr is not an, or at least not the sole, orion receptor (Boulanger, 2021).
Independently of the possible role of Drpr as an orion receptor, it was of interest to test if orion could activate the drpr signaling pathway as it is the case for neuron-derived injury released factors and Spätzle ligands, which bind to glial insulin-like receptors and Toll-6, respectively, upregulating in turn the expression of drpr in phagocytic glia. These ligands are necessary for axonal debris elimination and act as a find-me/eat-me signal in injury and apoptosis, as orion is doing for MB pruning. The data indicate that orion does not modify either Drpr expression nor the level of the drpr transcriptional activator STAT92E in astrocytes. Consequently, orion does not seem to induce the Drpr signaling pathway in astrocytes (Boulanger, 2021).
This study has uncovered a neuronally secreted chemokine-like protein acting as a 'find-me/eat-me' signal involved in the neuron-glia crosstalk required for axon pruning during developmental neuron remodeling. Chemokine-like signaling in insects was not described previously and, furthermore, the results point to an unexpected conservation of chemokine CX3C signaling in the modulation of neural circuits. Thus, it is possible that chemokine involvement in neuron/glial cell interaction is an evolutionarily ancient mechanism (Boulanger, 2021).
The oxidative-stress-induced impairment of autophagy plays a critical role in the pathogenesis of Parkinson's disease (PD). This study investigated whether the alteration of Nrf2 in astrocytes protected against 6-OHDA (6-hydroxydopamine)- and rotenone-induced PD-like phenotypes, using 6-OHDA-induced rat PD and rotenone-induced Drosophila PD models. In the PD rat model, Nrf2 expression was significantly higher in astrocytes than in neurons. CDDO-Me (CDDO methyl ester, an Nrf2 inducer) administration attenuated PD-like neurodegeneration mainly through Nrf2 activation in astrocytes by activating the antioxidant signaling pathway and enhancing autophagy in the substantia nigra and striatum. In the PD Drosophila model, the overexpression of Nrf2 in glial cells displayed more protective effects than such overexpression in neurons. Increased Nrf2 expression in glial cells significantly reduced oxidative stress and enhanced autophagy in the brain tissue. The administration of the Nrf2 inhibitor ML385 reduced the neuroprotective effect of Nrf2 through the inhibition of the antioxidant signaling pathway and autophagy pathway. The autophagy inhibitor 3-MA partially reduced the neuroprotective effect of Nrf2 through the inhibition of the autophagy pathway, but not the antioxidant signaling pathway. Moreover, Nrf2 knockdown caused neurodegeneration in flies. Treatment with CDDO-Me attenuated the Nrf2-knockdown-induced degeneration in the flies through the activation of the antioxidant signaling pathway and increased autophagy. An autophagy inducer, rapamycin, partially rescued the neurodegeneration in Nrf2-knockdown Drosophila by enhancing autophagy. These results indicate that the activation of the Nrf2-linked signaling pathways in glial cells plays an important neuroprotective role in PD models (Guo, 2021).
Over the course of a four-day period of metamorphosis, the Drosophila larval nervous system is remodeled. In peripheral nerves in the abdomen, five pairs of abdominal nerves (A4-A8) fuse to form the terminal nerve trunk. This reorganization is associated with selective remodeling of four layers that ensheath each peripheral nerve. The neural lamella (NL), is the first to dismantle; its breakdown is initiated by 6 hours after puparium formation, and is completely removed by the end of the first day. This layer begins to re-appear on the third day of metamorphosis. Perineurial Glial (PG) cells situated just underneath the NL, undergo significant proliferation on the first day of metamorphosis, and at that stage contribute to 95% of the glial cell population. Cells of the two inner layers, Sub-Perineurial Glia (SPG) and Wrapping Glia (WG) increase in number on the second half of metamorphosis. Induction of cell death in perineurial glia via the cell death gene reaper and the Diptheria toxin (DT-1) gene, results in abnormal bundling of the peripheral nerves, suggesting that perineurial glial cells play a role in the process. A significant number of animals fail to eclose in both reaper and DT-1 targeted animals, suggesting that disruption of PG also impacts eclosion behavior. These studies will help establish the groundwork for further work on cellular and molecular processes that underlie the co-ordinated remodeling of glia and the peripheral nerves they ensheath (Subramanian, 2017).
ACOX1 (acyl-CoA oxidase 1) encodes the first and rate-limiting enzyme of the very-long-chain fatty acid (VLCFA) beta-oxidation pathway in peroxisomes and leads to H2O2 production. Unexpectedly, Drosophila ACOX1 is mostly expressed and required in glia, and loss of ACOX1 leads to developmental delay, pupal death, reduced lifespan, impaired synaptic transmission, and glial and axonal loss. Patients who carry a previously unidentified, de novo, dominant variant in ACOX1 (p.N237S) also exhibit glial loss. However, this mutation causes increased levels of ACOX1 protein and function resulting in elevated levels of reactive oxygen species in glia in flies and murine Schwann cells. ACOX1 (p.N237S) patients exhibit a severe loss of Schwann cells and neurons. However, treatment of flies and primary Schwann cells with an antioxidant suppressed the p.N237S-induced neurodegeneration. In summary, both loss and gain of ACOX1 lead to glial and neuronal loss, but different mechanisms are at play and require different treatments (Chung, 2020).
Lipids are critical for neuronal development, synaptic plasticity, and function. Abnormal lipid metabolism contributes to the pathogenesis of several neurodegenerative disorders, including Alzheimer's disease, Parkinson's disease, and various diseases associated with glial dysfunction. Mitochondria and peroxisomes have been implicated in some of these neurodegenerative diseases, but the molecular events that underlie the demise of neurons vary widely. Some neurodegenerative diseases have been associated with defects in the degradation of fatty acids by β-oxidation, and these disorders share some common features. In yeast and plants, peroxisomes are the only site of β-oxidation. However, in vertebrates and insects, peroxisomes have more restricted functions as they are the site of β-oxidation of VLCFA, whereas long and medium chain fatty acids are processed in mitochondria (Chung, 2020).
Peroxisomal ACOX1 (acyl‐CoA oxidase 1) is the first and rate‐limiting enzyme in fatty acid β‐oxidation of VLCFA and a major producer of hydrogen peroxide (H2O2). ACOX1 deficiency (OMIM #264470) in humans is an autosomal recessive disorder that causes a rapid and severe loss of nervous system function. Newborns with ACOX1 deficiency exhibit hypotonia and seizures, then they experience a gradual loss of learning and speaking skills, usually beginning between the ages of 1 and 3. As the condition worsens, children develop exaggerated reflexes, have an increase in VLCFA, increased muscle tone, severe and recurrent seizures, and loss of vision and hearing. Most children with peroxisomal ACOX1 deficiency do not survive past early childhood (Chung, 2020).
Previous work has shown that loss of various peroxisomal proteins and enzymes causes severe neuronal defects in flies (Di Cara, 2019, Mast, 2011, Wangler, 2017). Exactly how loss of peroxisomes contributes to severe neurologic phenotypes in flies, mice, and humans is still poorly explored. This study reports that dACOX1 is expressed in glia and that loss of dACOX1 severely affects lifespan, increases VLCFA, causes loss of vision, and leads to glial loss and reduced neuronal survival in flies. In contrast, a novel variant, p.N237S, identified in three patients with progressive ataxia and hearing loss, stabilizes ACOX1 as an active dimer, acts as a gain-of-function mutation, produces elevated levels of reactive oxygen species (ROS) in insulating glia, does not alter VLCFA levels, and leads to the demise of wrapping glia in flies and primary Schwann cells in mouse. A potent antioxidant, N-acetyl cysteine amide (NACA), strongly suppresses these effects. These data show that proper activity of peroxisomal β-oxidation is essential for glial survival, and both loss and gain of ACOX1 severely affect glial function in flies and humans, albeit via different pathways (Chung, 2020).
Flies have no myelin, and this has often been raised as a concern for modeling glial disease in flies. However, the current data indicate striking parallels between wrapping glia and Schwann cells. Myelin is rich in VLCFA and ELOVL1, an enzyme that synthesizes VLCFA from LCFA, and VLCFA are required for myelin maintenance. Indeed, VLCFA are incorporated in glycerophospholipids and most VLCFAs in mammals are present in sphingolipids, lipids that are abundant in the outer leaflet of the plasma membrane, especially in lipid rafts and myelin where they reduce the fluidity of membranes. The ratio of C24 sphingolipids/total sphingolipids varies in different tissues. In mice, C24 sphingomyelin accounts for 25% of the sphingomyelin in the brain, and VLCFA are converted to C24 sphingolipids by ceramide synthase 2 (CerS2), which is required for myelin maintenance. Loss of CerS2 leads to an 80% reduction of myelin basic protein. Hence, VLCFAs are critical for myelin maintenance (Chung, 2020).
Interestingly, a form of the progressive neuropathy Charcot-Marie-Tooth disease (CMT1A) is caused by a mutation in PMP2, which encodes Fatty Acid Binding Protein 8 that is abundantly expressed in Schwann cells and binds LCFA. Glia in flies do not contain myelin, but the wrapping glia that insulate axons are rich in the ceramide phosphoethanolamine (CPE), a lipid that is very similar to sphingomyelin. Depletion of CPE results in defasciculation and failure of wrapping glia to enwrap peripheral axons reminiscent of defasciculation observed in CMT. Previous work has shown that VLCFAs are present in CPE in flies (Lin, 2018); based on these data it is estimated that 67% of all CPEs contain VLCFAs above C22 in length and 19% contain FA longer than C24. The data indicate that an increase in VLCFA upon loss of ACOX1 also causes a defasciculation and failure of wrapping glia to ensheath axons. Similarly, patients with loss of ACOX1 also exhibit demyelinating peripheral and central neuropathies. Hence, not being able to break down VLCFA is toxic and most likely severely affects the membrane composition of wrapping glia and myelin, drawing striking parallels between myelin in vertebrate glia and CPE in wrapping glia in insects. The current findings that bezafibrate, a drug that inhibits the synthesis of VLCFA, strongly suppresses the phenotypes in ACOX1-deficient flies clearly supports the evidence that elevated levels of VLCFA in glia may be a key trigger for glial and axonal loss (Chung, 2020).
The data show that ACOX1 is abundant in glia and poorly expressed in neurons in both flies and mice. This finding is in agreement with previous observations in vertebrates that peroxisomes are abundant in Schwann cells, but absent in the axons they enwrap. The breakdown of myelin leads to the production of VLCFAs, which, through β-oxidation in peroxisomes, produce LCFA that can be used as an energy source in the mitochondria of glial cells. Glial cells are known to provide lactate to neurons as an energy source in vertebrates. Kassmann (2014) proposed that LCFA produced in glia may correspond to a second energy source for axons. Previous work has shown that lipids can be transferred from neurons to glia, requiring fatty acid transport protein and apolipoprotein D (Dourlen, 2012, Liu, 2017, Liu, 2015). A similar export pathway may transport fatty acids from glia to neurons (Kis, 2015). Hence, the reduction in LCFA in glia upon loss of ACOX1 may also be detrimental for axons and deprive them of an energy source (Chung, 2020).
In mammals, peroxisomes are observed in astrocytes, oligodendrocytes, microglia, and Schwann cells. Although mice that lack the peroxisomal proteins Pex5 or Pex11 display severe neurological defects, removal of Pex5 specifically from neurons or from astrocytes does not cause obvious phenotypes. In contrast, removal of Pex5 from oligodendrocytes (CNP-Pex5 knockout) induces demyelination and severe axonal loss, suggesting that axonal loss is caused by peroxisome-deficient oligodendrocytes. In addition, mice that lack Mfp2, a VLCFA metabolizing enzyme that acts downstream of Acox1, also exhibit severe loss of axons. Again, these defects are not observed when Mfp2 is only absent from neurons or astrocytes. However, mice deficient for Mfp2 in oligodendrocytes do not show prominent pathology in the first year of life. This is inconsistent with observations in flies and humans, suggesting some redundancy for Mfp2 in mice. Finally, loss of Acox1 in mice has not been associated with axonal loss in the first 6 months of life, nor has a neurodegenerative phenotype been documented yet. Hence, humans and flies share similar phenotypes in the absence of ACOX1 that have not yet been observed in mice. It is proposed that this may be due to redundancy with murine Acox2 and Acox3 (Chung, 2020).
Apoptotic cell clearance by phagocytes is a fundamental process during development, homeostasis and the resolution of inflammation. However, the demands placed on phagocytic cells such as macrophages by this process, and the limitations these interactions impose on subsequent cellular behaviours are not yet clear. This study sought to understand how apoptotic cells affect macrophage function in the context of a genetically tractable Drosophila model in which macrophages encounter excessive amounts of apoptotic cells. Loss of the glial-specific transcription factor Repo prevents glia from contributing to apoptotic cell clearance in the developing embryo. This leads to the challenge of macrophages with large numbers of apoptotic cells in vivo. As a consequence, macrophages become highly vacuolated with cleared apoptotic cells, and their developmental dispersal and migration is perturbed. It was also shown that the requirement to deal with excess apoptosis caused by a loss of repo function leads to impaired inflammatory responses to injury. However, in contrast to migratory phenotypes, defects in wound responses cannot be rescued by preventing apoptosis from occurring within a repo mutant background. In investigating the underlying cause of these impaired inflammatory responses, it was demonstrated that wound-induced calcium waves propagate into surrounding tissues, including neurons and glia of the ventral nerve cord, which exhibit striking calcium waves on wounding, revealing a previously unanticipated contribution of these cells during responses to injury. Taken together, these results demonstrate important insights into macrophage biology and how repo mutants can be used to study macrophage-apoptotic cell interactions in the fly embryo. Furthermore, this work shows how these multipurpose cells can be 'overtasked' to the detriment of their other functions, alongside providing new insights into which cells govern macrophage responses to injury in vivo (Armitage, 2020).
Glial engulfment of neuron-derived debris after trauma, during development, and in neurodegenerative diseases supports nervous system functions. However, mechanisms governing the efficiency of debris degradation in glia have remained largely unexplored. This study showed that LC3-associated phagocytosis (LAP), an engulfment pathway assisted by certain autophagy factors, promotes glial phagosome maturation in the Drosophila wing nerve. A LAP-specific subset of autophagy-related genes is required in glia for axon debris clearance, encoding members of the Atg8a (LC3) conjugation system and the Vps34 lipid kinase complex including UVRAG and Rubicon. Phagosomal Rubicon and Atg16 WD40 domain-dependent conjugation of Atg8a mediate proper breakdown of internalized axon fragments, and Rubicon overexpression in glia accelerates debris elimination. Finally, LAP promotes survival following traumatic brain injury. These results reveal a role of glial LAP in the clearance of neuronal debris in vivo, with potential implications for the recovery of the injured nervous system (Szabo, 2023).
Elucidating signal transduction mechanisms of innate immune pathways is essential to defining how they elicit distinct cellular responses. Toll-like receptors (TLR) signal through their cytoplasmic TIR domains which bind other TIR domain-containing adaptors. dSARM/SARM1 is one such TIR domain adaptor best known for its role as the central axon degeneration trigger after injury. In degeneration, SARM1's domains have been assigned unique functions: the ARM domain is auto-inhibitory, SAM-SAM domain interactions mediate multimerization, and the TIR domain has intrinsic NAD+ hydrolase activity that precipitates axonal demise. Whether and how these distinct functions contribute to TLR signaling is unknown. This study shows divergent signaling requirements for dSARM in injury-induced axon degeneration and TLR-mediated developmental glial phagocytosis through analysis of new knock-in domain and point mutations. Intragenic complementation was demonstrated between reciprocal pairs of domain mutants during development, providing evidence for separability of dSARM functional domains in TLR signaling. Surprisingly, dSARM's NAD+ hydrolase activity is strictly required for both degenerative and developmental signaling, demonstrating that TLR signal transduction requires dSARM's enzymatic activity. In contrast, while SAM domain-mediated dSARM multimerization is important for axon degeneration, it is dispensable for TLR signaling. Finally, dSARM functions in a linear genetic pathway with the MAP3K Ask1 during development but not in degenerating axons. Thus, it is proposed that dSARM exists in distinct signaling states in developmental and pathological contexts (Herrmann, 2022).
Mechanisms of TLR signal transduction are many, but a common feature is that intracellular TIR domains of TLR receptors engage TIR domain-containing adaptors. In TLR pathways, TIR domains have been considered solely as protein-protein interaction domains. Recently a glial function has been defined for the TIR adaptor dSARM downstream of a TLR that promotes engulfment of neuronal corpses during development. It was asked to what extent dSARM's contribution to TLR signaling is related to its function in pathological axon degeneration. To address this question, a series of dSARM domain and point mutants were generated via CRISPR/Cas9-mediated genome engineering. The behavior of these new dSARM alleles in development and degeneration was compared, and while dSARM's enzymatic activity is essential in both contexts, SAM domain-mediated multimerization is critical for axon degeneration but dispensable for glial TLR signaling. Conversely, the MAP3K Ask1 is required for glial TLR signaling, but not axon degeneration. These findings align well with the independent companion manuscript from the DiAntonio lab (Brace, 2022). Together, these studies expand the repertoire of TLR signal transduction mechanisms to include deployment of the dSARM NADase for Ask1 activation. The dichotomous functions of dSARM in developmental and degenerative settings is discussed (Herrmann, 2022).
No difference was found in the protection afforded to either olfactory receptor neuron (ORN) or wing axons by deletion of dSARM or mutation of a key glutamic acid residue in dSARM's active site (E1170A. Thus, the NAD+ hydrolase activity is strictly required for axon degeneration in these two in vivo paradigms. These results differ from a recently published study (Hsu, 2021) that independently generated a dSARME1170A knock-in allele. These authors found that dSARME1170A mutant clones exhibit a weaker phenotype than dSARM nulls. In a wing axotomy assay, these dSARME1170A mutant clones provided only 50% protection of severed axons at 7 DPI relative to full protection in dSARM nulls. This discrepancy may stem from distinct CRISPR strategies or, alternatively, differences in genetic background. To validate the current approach, both studies sequenced the entire dSARM locus in this background and demonstrated that recombination of the wild-type sequence into the founder chromosome fully rescues viability and fertility. The finding of an essential requirement for dSARM's NAD+ hydrolase activity in axon degeneration in Drosophila is also in excellent agreement with data from mammalian models (Herrmann, 2022).
Unexpectedly, the behavior of the dSARMTIR allele differed between ORN and wing sensory axon paradigms. In uninjured ORN axons, dSARMTIR mutant clones exhibit spontaneous degeneration over the course of days arguing that SAM-mediated multimerization is not essential for NAD+ hydrolysis in the absence of the ARM domain. It is proposed that free TIR monomers have low-level constitutive activity leading to NAD+ loss, metabolic failure, and cellular demise. Consistent with unregulated activity of isolated TIR domains, the lethal phase of homozygous dSARMTIR animals is significantly earlier than that of dSARMKO homozygotes: early L1 for dSARMTIR, wandering L3 for dSARMKO. Surprisingly, dSARMTIR mutant ORN clones not only exhibited slow, injury-independent degeneration, but were also capable of timely injury-induced degeneration. It is suggested that dSARMTIR clones are primed to degenerate given ongoing TIR domain activity, and that in this case, TIR monomers can support rapid axon destruction following axotomy. In contrast, dSARMTIR behaved equivalently to the null allele in wing sensory axons. No evidence was detected of spontaneous axon degeneration in these neurons and mutant clones were fully protected for at least 7 days following axotomy (Herrmann, 2022).
It is possible that this difference reveals an underlying differential genetic sensitivity to NADase activity in these two neuronal populations. However, it is hypothesized that the differential behavior of this allele may reflect a difference in timing of clone induction (Herrmann, 2022).
The generation of MARCM clones relies on enhancer-driven Flippase (FLP) activity that recombines Flippase Recombination Targets (FRTs) on a chromosomal arm. ORN clones are induced by ey-FLP in the eye-antennal imaginal disc: ey activity starts in the eye-disc primordium (stage 15 embryo) and is maintained until the late third instar larvae. In contrast, wing sensory neuron clones are induced by ase-FLP activity in the wing imaginal disc. The activity of ase initiates in sensory organ precursors, which develop in third instar larvae and the first 10 h after puparium formation. Thus, a likely explanation for the differential dSARMTIR observation is that ORN clones are generated significantly earlier relative to wing sensory neuron clones, which might result in relatively higher levels of dSARMTIR in adult neuron clones (Herrmann, 2022).
This study uncovered a differential requirement for dSARM's SAM domain in injury-induced degeneration and glial TLR signaling. dSARM/SARM1 assembles into an octamer via SAM-SAM mediated interactions, which is required for axon degeneratio. A dSARM allele was generated lacking only the SAM domains, and it was found to behave as a null in axon degeneration. Thus, SAM domain-mediated dSARM multimerization is essential for axon degeneration in vivo. In contrast, dSARMARM-TIR homozygotes display normal glial TLR signaling, demonstrating that the SAM domains are not required in this context. It is proposed that in TLR signal transduction, dSARM's TIR domains heterodimerize with TIR domains on TLR receptors. In this model, TIR-TIR interactions between TLRs and dSARM support the NADase activity of dSARM, which is consistent with the finding that catalytically inactive dSARM variants retain the TIR-TIR interactions leading to NADase activation. These findings raise the possibility that dSARM exists in at least two distinct signaling complexes: a dSARM homomultimer that drives pathological axon degeneration and a dSARM-TLR heteromultimer that promotes signal transduction. It is alternatively possible that TLR activation drives dSARM homodimerization and NADase activity. It will be interesting to determine whether there are distinct pools of dSARM dedicated for each signaling state. While dSARM's SAM domains are dispensable for glial TLR signaling, they must be required for other developmental functions of dSARM since dSARMARM-TIR homozygotes die at the L3/pupal transition. The SAM domains of TIR-1/SARM1 are proposed to regulate its synaptic localization in C. elegans, suggesting that dSARM's SAM domains may likewise promote synaptic functions in motorneurons (Herrmann, 2022).
Evidence from multiple groups now argues for a dSARM/SARM1-Ask1 signaling cassette. It is not yet known how dSARM activates Ask1, but it was demonstrated that dSARM's NADase activity is required for activation of Ask1 in glia. Ask1 orthologs are widely implicated in ROS-mediated signaling. Specifically, they are ROS-activated by Thioredoxin (Trx), the first identified Ask1-binding protein and its major cellular inhibitor. ROS signaling activates Ask1 because local oxidation relieves Trx-mediated repression and drives Ask1 activation. Ask1 proteins lacking the Trx-binding domains (Ask1ΔN) can be constitutively active. Indeed, this study found that glial expression of Ask1ΔN activates TLR signaling, arguing that Trx may contribute to Ask1 activation in this context. It is conceivable that NAD+ hydrolysis mediated by dSARM could interfere with the maintenance of a reduced Trx pool, thus promoting activation of Ask1. Taken together with the companion study, these findings indicate that activation of the dSARM NAD+ hydrolase does not necessarily drive irreversible axon destruction, but rather that NAD+ hydrolysis is deployed for signaling in both neurons and glia (Herrmann, 2022).
Recruitment of stem cells is crucial for tissue repair. Although stem cell niches can provide important signals, little is known about mechanisms that coordinate the engagement of disseminated stem cells across an injured tissue. In Drosophila, adult brain lesions trigger local recruitment of scattered dormant neural stem cells suggesting a mechanism for creating a transient stem cell activation zone. This study found that injury triggers a coordinated response in neuro-glial clusters that promotes the spread of a neuron-derived stem cell factor via glial secretion of the lipocalin-like transporter Swim. Strikingly, swim is induced in a Hif1-α-dependent manner in response to brain hypoxia. Mammalian Swim (Lcn7) is also upregulated in glia of the mouse hippocampus upon brain injury. These results identify a central role of neuro-glial clusters in promoting neural stem cell activation at a distance, suggesting a conserved function of the HIF1-α/Swim/Wnt module in connecting injury-sensing and regenerative outcomes (Simoes, 2022).
Injury is known to stimulate diverse forms of plasticity, which serve to restore organ function. Many tissues harbor a small number of undifferentiated adult stem cells that are engaged in tissue turnover or become activated following injury to replace damaged cells. Some tissues, such as muscle or brain, contain mainly dormant stem cells that are not dividing and reside in a reversible state of quiescence. Niche cells in intimate contact with quiescent stem cells have been found to provide activating cues upon tissue damage. However, little is known how the activation of multiple dispersed stem cell units is coordinated to establish an adequate stem cell response zone across an injured tissue (Simoes, 2022).
Quiescent progenitor cells in muscle and the brain respond to injury in mammals, but also in fruit flies (Drosophila). This allows to harness the extensive genetic tools available in Drosophila to dissect injury-dependent stem cell activation. Although still unclear, the presence of dormant stem cells in short-lived insects indicates that these cells may play a beneficial role for tissue plasticity or repair upon predator attacks or inter-species aggressions (Simoes, 2022).
In the adult fly brain, experimental stab lesions to the optic lobes (OLs) or the central brain trigger a proliferative response resulting in local neurogenesis several days after injury (AI), which has been linked to activation of normally quiescent neural progenitor cells (qNPs). qNPs have also been found to promote adult brain plasticity in contexts unrelated to injury. On the other hand, stab lesions can also trigger glial divisions shortly after injury (Simoes, 2022).
Despite extensive knowledge on neural stem cell proliferation during fly development, the signals governing qNP activation in response to injury are unknown. A ubiquitous pulse of Drosophila Myc (dMyc) overexpression has been previously shown to promote qNP division, but the signals detected by qNPs remained enigmatic (Simoes, 2022).
In mammals, a wide variety of signals are known to regulate quiescent neural stem cells (qNSCs) in homeostatic conditions, whereas their response to tissue damage is less well understood. qNSCs are located in two main niches, the subventricular zone and the dentate gyrus of the hippocampus, buried within the brain. Upon brain injury, qNSCs only partially enter an activated state, and neuroblast recruitment to infarcted brain regions and local neurogenesis is limited (Simoes, 2022).
Strikingly, the initial consequences triggered by brain injury, which include neural cell death, upregulation of antioxidant defense, and c-Jun N-terminal kinase (JNK) stress signaling, are very conserved in flies and mice suggesting that injury sensing of qNSCs/qNPs may rely on common principles.
In this work, injury-induced changes were studied in the adult fly brain leading to recruitment of isolated qNPs near the injury site. A crucial role was identified of damage-responsive neuro-glial clusters (DNGCs), which enable proliferation of distant qNPs by promoting an enlarged stem cell activation zone. Evidence is provided that these multicellular units orchestrate the spatial and temporal availability of an essential, but localized stem cell factor for qNPs via injury-stimulated secretion of the transport protein Swim. As Swim production is dependent on the injury-sensitive transcription factor HIF1-α, the identified mechanism may serve to spatially and temporary adjust the stem cell activation zone to the extent of damage suffered in a given tissue area, resulting in locally calibrated pulses of stem cell activity (Simoes, 2022).
How tissue damage is sensed and how the recruitment of multiple stem cell units is coordinated in response to local, heterogeneous tissue damage represents a fundamental question. By investigating how dispersed qNPs are locally recruited to injury, we have identified a mechanism that creates a defined zone of stem cell activation in the adult fly brain. The process is dependent on DNGCs, which depending on their size and possibly composition may regulate the extent by which a localized stem cell factor such as Wg/Wnt can travel to rare qNPs in the vicinity. Whereas the neuronal cells provide Wg/Wnt, the glial component supplies the carrier protein Swim, thereby promoting the dispersion of the signal. This cooperative interaction of two different cell types to gain long range function of Wg/Wnt is rather unique (Simoes, 2022).
At the cellular level, a model is proposed whereby injury-sensitive HIF1-α directs Swim synthesis in glial cells. Swim transporters diffuse and facilitate the spread of localized neural-derived Wg ligands, probably by binding to and shielding the lipid-residues of Wg/Wnt in the aqueous extracellular space. Mobile Wg-Swim complexes are consequently able to reach and activate qNPs in the injured brain domain. Wg/Wnt signal transduction and downstream upregulation of dmyc is shown to be crucial for the proliferation of this novel cell type. Overall, it is proposed that the described mechanism provides a means to match recruited stem cell activity to the spatial and temporal persistence of damage in the injured brain.
Activation of dormant neural progenitors by high levels of Wg/Wnt
Wg/Wnt signaling is probably one of the most universal pathways driving stem cell proliferation. Nevertheless, an understanding of Wg/Wnt signals for dormant stem cells has only recently emerged. Dormant muscle stem cells, for example, maintain quiescence by raising their threshold for Wnt transduction via cytoplasmic sequestration of &betal-catenin, and qNSC in the hippocampus do not rely on Wnt signaling under homeostasis but display a high capability to respond to Wnt in a graded manner when exposed. Similarly, the results demonstrate that qNPs start proliferating when high Wg/Wnt levels are provided in an autocrine fashion (Simoes, 2022).
Overall, the results suggest that activation of qNPs in the adult fly brain is mainly prevented by the low availability of Wg/Wnt ligands under homeostatic conditions. Although Wnt signaling normally occurs between adjacent cells, this study provides evidence that Wg functions at a tissue scale in the injured fly brain (Simoes, 2022).
This study describes the property of Swim to extend the signaling range of Wg/Wnt. Further research will be required to determine whether other stem cell-relevant factors can be transported by Swim.
In zebrafish, reduced levels of Swim/Lcn7 produce craniofacial defects due to compromised Wnt3 signaling, highlighting a different context of Wnt/Swim interaction. A Wg/Swim interaction has previously been proposed in developing epithelia in flies, although the effect was not observed in a more recent study (Simoes, 2022).
Swim::mCherry is strongly expressed in the adult ovary germline of flies, in agreement with data from the recently published Fly Cell Atlas. Remarkably, swim KO flies showed reduced fertility, a phenotype which has also been reported for lcn7/tinagl1 KO mice (Takahashi, 2016). Interestingly, Swim expression in the germarium strongly overlapped with Wg::GFP, in line with previous findings describing a requirement of extensive Wg travel from the niche to distant follicular stem cells (Simoes, 2022).
Finally, this study elucidated how the Swim/Wg stem cell-activating signal is connected to damage sensing in the injured brain. Both in flies and mice, swim/lcn7 induction occurs in glial cells in response to brain injury. Remarkably, stroke-induced lcn7 induction is not observed in mouse brains, in which Hif1-α has been deleted from mature neurons and glial cells. This suggests that HIF1-α-dependent swim regulation is conserved in mammals (Simoes, 2022).
According to the current model, the damage responsiveness of stem cells is strongly gated by the availability of stable HIF1-α during acute hypoxia. Such a limited activation pulse would effectively restrict the mitotic effect of Swim/Wg complexes to the acute phase of repair, acting as a safeguard mechanism against overgrowth. Moreover, the hypoxia-dependent secretion of Swim would allow to temporally and locally fine-tune the realm of the stem cell activation zone to injury.
Local oxygen concentrations modulate the activity of adult stem cells in different niches. In the fly larval OL, Dpn-expressing neural progenitors proliferate in a pronounced hypoxic environment, which bears parallels to the situation following brain injury (Simoes, 2022).
In the mammalian brain, injury-induced Wnt ligands may not efficiently reach qNSCs in distant neurogenic niches, resulting in poor stem cell activation. As such, Wnt pathway stimulating approaches hold promise as possible treatment for brain injury as they are known to support regeneration at several levels including qNSC activation, neurogenesis, and axon outgrowth. Increasing the mobility or stability of Wg/Wnts by Swim-like transporters may therefore represent a successful strategy to engage endogenous progenitors into regeneration. Given the fact that Wg/Wnts can support tissue renewal and regeneration in numerous tissues, the properties of Swim to transform a restricted tissue area into a temporary stem cell-activating zone, uncovered in this study may have important applications in regenerative medicine (Simoes, 2022).
Although the current experiments have revealed an impaired distribution of Wg in the injured brain in the absence of Swim transporters, it cannot be completely rule out that Swim may alter Wg function by other means than physical binding and direct transport. Ideally, the injury-induced formation of Wg-Swim complexes should be observable in the extracellular space. Although colocalization of Swim and Wg signals was detected, it was not possible to image Wg-Swim complexes at high resolution due to elevated background of Wg and mCherry antibodies when performing extracellular stainings. Overcoming these current limitations with overexpression systems or optimized immunodetection should allow to capture the dynamics of Wg-Swim interactions in injured brain tissue in the future (Simoes, 2022).
How glia control axon regeneration remains incompletely understood. This study investigated glial regulation of regenerative ability differences of closely related Drosophila larval sensory neuron subtypes. Axotomy elicits Ca(2+) signals in ensheathing glia, which activates regenerative neurons through the gliotransmitter adenosine and mounts axon regenerative programs. However, non-regenerative neurons do not respond to glial stimulation or adenosine. Such neuronal subtype-specific responses result from specific expressions of adenosine receptors in regenerative neurons. Disrupting gliotransmission impedes axon regeneration of regenerative neurons, and ectopic adenosine receptor expression in non-regenerative neurons suffices to activate regenerative programs and induce axon regeneration. Furthermore, stimulating gliotransmission or activating the mammalian ortholog of Drosophila adenosine receptors in retinal ganglion cells (RGCs) promotes axon regrowth after optic nerve crush in adult mice. Altogether, the findings demonstrate that gliotransmission orchestrates neuronal subtype-specific axon regeneration in Drosophila and suggest that targeting gliotransmission or adenosine signaling is a strategy for mammalian central nervous system repair (Wang, 2023).
Axon regeneration failure is a major hurdle for functional recovery after CNS lesion. In addition, Wallerian axon degeneration is associated with neurodegenerative diseases, hence promoting that axon regeneration in these diseases is of great interest. By parallel investigation of closely related Drosophila neuronal subtypes, gliotransmission is proposed as a previously unknown mechanism by which glia could control axon regeneration. These findings expand the existing glial roles in axon regeneration. In a working model that summarizes these Drosophila studies, ensheathing glia actively respond to axotomy and the resulting glial Ca2+ spikes lead to the release of ATP/adenosine. Although adenosine could interact with all sensory neurons, the AdoR expression pattern ensures that C4da, but not C3da, neurons respond to adenosine. This glia-to-neuron signaling then mounts pro-regenerative programs in C4da neurons, including burst firing, Ca2+ spikes, and Ras activity to promote axon regrowth of C4da neurons. Moreover, ectopic AdoR expression or activation of those regenerative programs in C3da neurons is sufficient to induce their axon regeneration (Wang, 2023).
How could axotomy elicit glial Ca2+ signals in Drosophila larvae? Glia are known to express neurotransmitter receptors, allowing them to "listen to" neuronal activity and provide feedback modulation of neuronal activity through gliotransmitters. To assess whether the neuronal activity could have an effect on glial Ca2+ signals, AITC was applied to activate C4da neurons. However, Allyl isothiocyanate (AITC) did not evoke Ca2+ signals in ensheathing glia, arguing against the neuronal activity as a trigger of glial Ca2+ signals. These results support unidirectional signaling from glia to sensory neurons. They also suggest that glial Ca2+ spikes are produced either cell-autonomously or alternatively through a cell-non-autonomous mechanism that involves glial interactions with non-neuronal cells.
Burst firing observed in Drosophila larval C4da neurons is reminiscent of the ectopic discharges of injured dorsal root ganglion (DRG) neurons, the mammalian counterpart of Drosophila larval PNS sensory neurons. As a hallmark of neuropathic pain, the ectopic discharges of DRG neurons are also regulated by gliotransmission. This suggests that burst firing of C4da neurons could reflect nociceptive hypersensitivity after axotomy. Hence, one possible role of gliotransmission could be to sensitize injured C4da neurons. Taken together, gliotransmission appears to be related to both axon regeneration and nociceptive hypersensitivity. Future work is needed to determine whether gliotransmission could regulate DRG axon regeneration (Wang, 2023).
By stimulating Drosophila larval C3da neurons with different firing patterns, this study provides direct evidence that firing patterns, instead of excitability, dictate the axon regenerative strength. A possible mechanism is that neuronal activity patterns are associated with different Ca2+ signals. For example, burst, but not tonic, firing evokes strong Ca2+ signals in C3da neurons, likely due to prolonged depolarization associated with burst firing. Previous studies indicate that the stimulation of neuronal activity promotes mammalian axon regeneration, but the reported effects are variable. One possibility could be that the activity patterns are not controlled in these studies. In the future, it will be of interest to deliver precisely controlled neuronal activity patterns to determine their effects on axon regeneration in other experimental models (Wang, 2023).
It is proposed that the Ca2+ spikes in Drosophila larval C4da neurons are pro-regenerative signal with the following characteristics: they are neuronal-type-specific, correlate with burst firing, exhibit delayed onset, persist throughout axon regeneration, and are evoked by axonal but not dendritic injury. Optogenetic stimulation of C3da neurons starting at ~6 hpa (i.e., after immediate Ca2+ transients) further indicates that Ca2+ spikes are sufficient to induce axon regeneration. Because Ca2+ signals with different spatiotemporal patterns activate diverse neuronal pathways, it is likely that Ca2+ spikes and immediately Ca2+ transients regulate different aspects of axon regeneration. Although immediate Ca2+ transients could regulate growth cone formation, persistent Ca2+ spikes could maintain Ras activity at a high level for sustained axon regrowth. It will be interesting to determine whether axotomy could trigger Ca2+ spikes in other systems, and if so, their roles in axon regeneration (Wang, 2023).
Unlike CNS, mammalian PNS neurons such as DRG can regenerate their axons. Therefore, mammalian CNS and PNS neurons could be broadly defined as two neuronal groups bearing axon regenerative differences, although each group is highly heterogeneous and contains multiple neuronal subtypes. In this regard, the differences between mammalian CNS and PNS are much larger than those between Drosophila C4da and C3da neurons. To explore whether Adora2b signaling could underlie the regenerative differences of mammalian CNS and PNS neurons, the published single-cell RNA-seq dataset was search, but it was found that Adora2b was expressed at low levels in both CNS and DRG neurons. Hence, other mechanisms must exist to explain their regenerative differences. One such mechanism could be related to intrinsic growth abilities. For example, the injury of mouse RGCs causes a reduction of mTOR activity; by contrast, no reduction of mTOR activity was found after the injury of DRG neurons (Wang, 2023).
Glial cells in the mammalian retina could release ATP, glutamate, and D-serine. The mouse study indicates that the stimulation of the gliotransmitter release alone by designer receptor exclusively activated by designer drugs (DREADDs) is ineffective, but combining glial DREADD stimulation and neuronal Adora2b expression greatly enhanced axon regeneration. Because Adora2b is barely expressed in the mouse retina, these results suggest that engineering RGCs to respond to the gliotransmitter adenosine is a prerequisite for the observed effect. Similarly, Drosophila C3da neurons do not express AdoR, and engineering them to respond to adenosine induces axon regeneration. These analyses suggest that stimulation of gliotransmission could promote axon regeneration across species if Adora2b orthologs are expressed in neurons. Notably, Adora2b overexpression in RGCs alone moderately increased axon regeneration, suggesting that extracellular adenosine exists in the injured retina. These results further suggest that in the absence of glial DREADD stimulation, axotomy could trigger basal adenosine release (Wang, 2023).
Adenosine exerts protective functions in different mammalian tissues including the nervous system, by triggering diverse signaling events. Extracellular adenosine is normally low but is rapidly increased after injury. As a classic neuromodulator, adenosine controls essential homeostatic functions such as sleep. However, whether adenosine could play a role in axon regeneration is largely unknown. Pharmacological Adora3 activation is shown to promote neurite outgrowth of cultured RGCs. In the current study, Adora2b was pharmacologically activated, and this was found to promote axon regeneration and survival of RGCs in adult mice. The magnitude of RGC axon regeneration induced by Adora2b activation was comparable with that after PTEN knockdown, and combining Adora2b activation with PTEN knockdown further enhanced axon regeneration. These findings suggest that targeting Adora2b signaling, either alone or in combination with other pro-regenerative pathways, is likely a strategy for mammalian CNS repair (Wang, 2023).
In the mouse study, DREADDs were used to stimulate the gliotransmitter release. However, the spatiotemporal dynamics of Ca2+ elevations and gliotransmitter release are yet unknown and will be the focus of future studies. Moreover, the current study has focused on mouse retinal glial cells that are positive for GFAP, which is normally found in astrocytes. Future studies are needed to determine whether the stimulation of gliotransmission from other retinal glial types such as Müller glia or microglia could have a similar role in RGC axon regeneration (Wang, 2023).
Nervous system injury and disease have broad effects on the functional connectivity of the nervous system, but how injury signals are spread across neural circuits remains unclear. This study explored how axotomy changes the physiology of severed axons and adjacent uninjured "bystander" neurons in a simple in vivo nerve preparation. Within hours after injury, suppression of axon transport was observed in all axons, whether injured or not, and decreased mechano- and chemosensory signal transduction was observed in uninjured bystander neurons. Unexpectedly, it was found the axon death molecule Sterile alpha and Armadillo motif (dSarm), but not its NAD(+) hydrolase activity, was required cell autonomously for these early changes in neuronal cell biology in bystander neurons, as were the voltage-gated calcium channel Cacophony (Cac) and the mitogen-activated protein kinase (MAPK) signaling cascade. Bystander neurons functionally recovered at later time points, while severed axons degenerated via α/Armadillo/Toll-interleukin receptor homology domain (dSarm)/Axundead signaling, and independently of Cac/MAPK. Interestingly, suppression of bystander neuron function required Draper/MEGF10 signaling in glia, indicating glial cells spread injury signals and actively suppress bystander neuron function. This work identifies a new role for dSarm and glia in suppression of bystander neuron function after injury and defines two genetically and temporally separable phases of dSarm signaling in the injured nervous system (Hsu, 2020).
Nervous system injury or neurodegenerative disease can lead to profound alterations in neural circuit function. The precise cellular basis is poorly defined in any context, but disruption of circuit signaling is generally thought to occur as a result of a loss of physical connectivity between damaged neurons. Indeed, axon and synapse degeneration are among the best correlates of functional loss in patients with a variety of brain injuries or neurological diseases. But whether, and the extent to which, an injured or diseased neuron might also alter the functional properties of neighboring healthy 'bystander' neurons (i.e., those not damaged or expressing disease-associated molecules) is an important and open question. If the physiology of bystander neurons is radically altered by their damaged neighbors, this would force reconsideration of the simple loss-of-physical-connectivity model as the appropriate explanation for functional loss in neural circuits after trauma (Hsu, 2020).
It is well documented that bystander neurons can change their physiology in response to their neighbors being injured. For instance, mouse L5 spinal nerve transection results in the degeneration of distal L5 afferents in sciatic nerve alongside intact L4 C fiber afferents. Within 1 day after L5 lesion, L4 C fibers develop spontaneous activity that lasts for at least 1 week and appears to mediate injury-induced pain and hyperalgesia behaviors. Bystander effects have also been observed in the central nervous system (CNS). In a mouse model of mild traumatic brain injury (TBI), 1 day after injury, pyramidal neurons with severed axons and intact bystander neurons both exhibited injury-induced changes in action potential firing and afterhyperpolarization. Injured neurons failed to recover, while bystander neurons ultimately exhibited a return to normal firing properties. How injured neurons or surrounding glia signal to bystander neurons, or how bystander neurons receive this signal, is not known, but the similar electrophysiological changes observed in axotomized and intact dorsal root ganglion neurons have been proposed to be associated with Wallerian degeneration (Hsu, 2020).
Recent work has begun to illuminate the mechanisms by which damaged axons autonomously drive their own degeneration during Wallerian degeneration. A forward genetic screen in Drosophila identified the sterile α/Armadillo/Toll-interleukin receptor homology domain (dSarm) molecule as essential for axon auto-destruction, as loss of dSarm completely blocked Wallerian degeneration (Osterloh, 2012). All known dSarm pro-degenerative function requires the BTB and BACK domain molecule Axundead (Axed), another powerful regulator of axon degeneration (Neukomm, 2017). dSarm function in axon degeneration after injury is conserved in mouse: Sarm1-/- mutants block Wallerian degeneration, and loss of Sarm1 also suppresses axon degeneration in mouse models of TBI and peripheral neuropathy. Sarm1 inhibition is thus an exciting potential approach for blocking axon loss and neuroinflammation in human disease (Hsu, 2020).
dSarm/Sarm1 has been studied primarily in the nervous system as a positive regulator of axonal degeneration. In mammals, axotomy leads to the depletion of the labile NAD+ biosynthetic enzyme Nmnat2 and a decrease in NAD+ in severed axons. Nmnat2 loss somehow activates Sarm1 (Gilley, 2015), which is proposed to lead to further NAD+ depletion and metabolic catastrophe in the severed axon (Gerdts, 2015) through a Sarm1-intrinsic NAD+ hydrolase activity (Essuman, 2017). The Sarm1 NAD+ hydrolase activity appears to be activated directly by the NAD+ precursor, NMN, presumably through allosteric conformational changes in Sarm1 upon NMN binding. This NAD+ depletion model has been proposed as the primary mechanism by which Sarm1 drives axon loss, and to explain the mechanistic basis of protection by several other neuroprotective molecules (Gerdts, 2016). For instance, the slow Wallerian degeneration molecule (WldS), which includes the highly stable NAD+ biosynthetic enzyme Nmnat1, is thought to protect axons by substituting for the labile Nmnat2 molecule, thereby reducing NMN levels and avoiding NAD+ depletion. Similarly, the protective effects of loss of the E3 ubiquitin ligase Highwire/Phr1 is thought to result from blockade of its direct role in degrading Nmnat2, such that in hiw/phr1 mutants Nmnat2 is stabilized and continues to maintain NAD+ levels (Hsu, 2020).
Elegant genetic studies in C. elegans demonstrated that TIR-1 (the worm homolog of dSarm/Sarm1) is part of a signaling cascade downstream of the voltage-gated calcium channel UNC-36 and CamK-II and signals via the mitogen-activated protein kinase (MAPK) signaling cascade. Based on this work, MAPK signaling was examined for roles in Wallerian degeneration but met with mixed results. Changes in MAPK signaling (i.e., phosphorylation of MAPK pathway members) were found in axons within 15-30 min after axotomy, were Sarm1 dependent and suppressed by Nmnat overexpression, and partial suppression of axon degeneration was observed after simultaneous blockade of multiple MAPK components. But how MAPK signaling modulates axon degeneration, particularly in the context of Sarm1 signaling, remains controversial, as one study proposed MAPK signals downstream of Sarm1, while another argued Sarm1 was upstream of MAPK signaling, and the neuroprotective phenotypes resulting from MAPK blockade do not approach levels afforded by loss of Sarm1 in vivo (Hsu, 2020).
This study used a partial nerve injury model to examine early changes in the physiology of severed axons and neighboring uninjured bystander neurons. Axotomy of even a small subset of neurons was shown to leads to inhibition of cargo transport in all axons within the nerve and suppression of sensory signal transduction in bystander neurons. Surprisingly, this early blockade of axon transport and sensory signal transduction required dSarm in both severed and uninjured bystander neurons, where it signaled via the conserved UNC-36/MAPK signaling pathway. Early suppression of axon transport and bystander neuron function did not require dSarm NAD+ hydrolase function or Axed, was not modulated by NMN, and was not induced by depletion of dNmnat. This suggests it is mechanistically different from later events in axon death, where dSarm drives axon degeneration with Axed. Intriguingly, this study found that this early spreading of injury signals to bystander neurons required the Draper receptor in surrounding glia, indicating that glial cells actively signal to inhibit the function of bystander neurons in vivo. This work identifies new roles for dSarm and glia in modifying neurophysiology early after injury, assigns the NAD+ hydrolase function exclusively to later axon degenerative events, and reveals a new role for UNC-36/MAPK signaling in promoting these dSarm-dependent changes early after an injury has occurred in the nervous system. It is proposed that two temporally and genetically separable phases of dSarm signaling exist that mediate these distinct injury-induced changes in neurophysiology and axon degeneration (Hsu, 2020).
This study shows that relatively small injuries can lead to the rapid and efficient spreading of injury signals across nerves that potently suppress axon transport throughout the nerve, and broadly inhibit neurophysiology in uninjured bystander neurons. Surprisingly, the same molecule was found to be required to drive explosive axon degeneration in severed axons at later stages, dSarm/Sarm1, is required for this early suppression of neuronal function, although the signaling mechanisms at each stage appear to be different. The data support a model whereby early (i.e., 1-3 h after injury) dSarm signals with Cac and MAPK components, but independent of its NAD+ hydrolase activity, to suppress axon transport and neurophysiology, while at later stages (8-12 h), dSarm signals with Axed to promote explosive axon degeneration. This significantly expands the role for dSarm/Sarm1 in regulating nervous system responses to injury to include even uninjured bystander neurons. Furthermore, a critical role was discoverd for glial cells, through Draper, in signaling to bystander neurons to inhibit their axon transport and neurophysiology. Together, this work suggests that a significant amount of functional loss after neural trauma is a result of not only frank degeneration but also more widespread changes in neuronal function, and it occurs in uninjured neurons through glial spreading of injury signals (Hsu, 2020).
The data support the notion that widespread signaling occurs between cells in injured neural tissues immediately after injury and that injury signals can radically alter neuronal function. Severing even a small number of axons led to a suppression of axon transport within hours in all axons in the adult wing nerve, even in uninjured bystander neurons. Beyond axon transport, local uninjured bystander sensory neurons also exhibited a disruption of mechano- and chemosensory signal transduction, which was partially reversible within a few hours. These observations suggest that beyond simple breakage of connectivity, a significant part of functional loss after brain injury or in neurodegenerative disease may also be occurring in healthy, intact neurons that have received function-suppressing signals from nearby damaged neurons (Hsu, 2020).
Surprisingly, dSarm was found to be required cell autonomously in bystander neurons to alter axon transport and nerve function in response to injury, and this role did not require its NAD+ hydrolase activity. Reception of this injury signal in the bystander neuron (and severed axons) requires the VGCC Cac and the MAPK signaling cascade, similar to Tir-1 signaling in C. elegans, but not Axed. Reciprocally, Cac and MAPK components are not required for Wallerian degeneration at later stages. Explosive axon degeneration requires dSarm, its NAD+ hydrolase activity, and Axed. Based on the timing of these different events (i.e., changes in neuronal function versus frank degeneration) with the genetic studies indicating they are separable, a two-phase model is proposed for dSarm signaling in injured neural tissues: early dSarm-dependent changes in axon biology and neurophysiology that occur within hours after injury are mediated by the Cac/dSarm/MAPK signaling cascade (phase I), while late-stage axon degeneration is driven by dSarm signaling through Axed (phase II). The existence of these temporally distinct phases of dSarm signaling likely explain previous results that seemed in conflict, where MAPK signaling was proposed to act both upstream (Yang, 2015) and downstream (Walker, 2017) of Sarm1 after axotomy. According to the current model, both of these assertions would be correct, with dSarm/Sarm1 acting upstream of MAPK early (phase I) and independent but ultimately downstream of MAPK later to drive dSarm/Axed-dependent axon degeneration (phase II)(Hsu, 2020).
To date, dSarm/Sarm1 has been thought of primarily as a cell-autonomous regulator of explosive axon degeneration, but the current work shows that dSarm can also drive important changes in circuit function through altering neuronal cell biology and neurophysiology. That bystander neurons recover and remain viable also demonstrates that activation of dSarm after injury does not necessarily lead to axon death. It is suspected that recovery occurs in large part because bystander neurons have not been severed, which is an extreme injury, and depends on their connection to the cell body, which is a source of axon survival factors like Nmnat2. Connection to the cell body may also explain why axon transport was less severely suppressed in the bystander neurons; additional transport factors can still be continuously supplied to the distal axon from the soma. Defining how dSarm activity is regulated in each of these contexts to interact with Cac/MAPKs versus Axed, and why the first phase does not require NAD+ hydrolase function, are key questions for the future (Hsu, 2020).
A compelling case exists for the NAD+ depletion hypothesis for dSarm/Sarm1 function in axon degeneration (Essuman, 2017; Gerdts, 2015, 2016), although arguments have been made this dSarm/Sarm1 signaling is likely more complex (Neukomm, 2017). In this model, depletion of Nmnat2 via Hiw/Phr1 results in the accumulation of NMN, which functions as an activator of Sarm1, with Sarm1 NAD+ hydrolase activity driving metabolic catastrophe. This study provides several lines of evidence that the above, newly described early dSarm signaling events (i.e., suppression of axon transport and neurophysiology) are mechanistically distinct but are nevertheless also regulated by some axon-death-associated molecules. First, while NMNd can suppress axon degeneration in flies and other species, it cannot block early suppression of axon transport or changes in bystander neuron function. This argues that NMN is not a driving force for dSarm activation in the early phase. Second, although limited to tagged versions of dNmnat for this analysis, no depletion of dNmnat was observed within the time frame of 6 h after injury. Previous studies in SCG or DRG cultures in vitro suggest Nmnat2 depletion takes 4-6 h and NAD+ depletion begins ~2-3 h after axotomy, which is slightly later than the bystander effect was observed in vivo. Because full axon degeneration is prolonged in vivo compared to in vitro studies, the timing of Nmnat2 loss and NAD+ depletion is likely also prolonged in vivo, further suggesting this likely happens after cessation of axon transport. Third, Axed, which is genetically downstream of dSarm during axon degeneration (Neukomm, 2017), is not required for early suppression of nerve responses to injury in either severed or intact neurons, only later axon degenerative events in the severed axons. Finally, this study shows that while the NAD+ hydrolase function of dSarm is required in vivo for efficient axon degeneration, it is dispensable for early suppression of axon transport (Hsu, 2020).
Despite these clear molecular and genetic differences between early- and late-phase signaling events, WldS or dNmant expression or hiw mutants are capable of suppressing early changes in axon transport and neurophysiology, even in bystander neurons. This could be interpreted as evidence for similarity in signaling mechanisms at early and late stages of dSarm signaling (i.e., that they act by maintaining NAD+). However, the alternative possibility is favored that these data point to an important role for dNmnat in mediating early dSarm signaling events during suppression of bystander neuron function. Loss of Axed does not affect the bystander effect, and axon transport is suppressed. However, this study found that loss of dNmnat in axed null backgrounds (which allows for preservation of neuronal integrity despite loss of dNmnat) blocked the ability of injury to induce the bystander effect. This result reveals a paradoxical, positive role for dNmnat in promoting the bystander effect early. It is suspected that dNmnat exerts this effect through modulating MAPK signaling, whose interactions are complex: loss of Nmant has been shown to suppress MAPK signaling, while increased Nmnat activity can also potently block the activation of MAPK signaling within the first few hours after axotomy. It is proposed that dNmnat activity is required early for the bystander effect and that dNmnat levels need to be precisely tuned for proper signaling at each phase (Hsu, 2020).
Glial cells are well positioned to rapidly spread signals to all axons in the wing nerve. Much like Remak bundles in mammals, the Drosophila L1 wing nerve has glial cells that appear to wrap axons individually, which would imply that axon-to-axon signals must pass through glia. The observation that selective elimination of Draper signaling in glia is sufficient to inhibit the spreading of injury signals to bystander neurons is consistent with an axon->glia->bystander neuron signaling event, although it is also possible that glia are directly injured by the axotomy and signal to bystander neurons without input from the severed axons. Given the similarities in the response of severed axons and those of bystanders (i.e., both block axon transport on the same timescale), and the selective effects of Draper on the bystander neuron axons, the former model is favored rather than the latter (Hsu, 2020).
Draper signals to bystander neurons through a transcriptional JNK/dAP-1 cascade, likely through activating MMP-1. Nerve injury also rapidly activates JNK/c-Jun signaling in mammalian Schwann cells, where JNK/c-Jun mediate most aspects of Schwann cell injury responses and reprogramming events. This conserved glial response likely occurs in Schwann-cell-like wrapping glia present in the Drosophila L1 wing nerve, although it may be activated in the subperineurial glia, which can act in a partially redundant fashion with wrapping glia. The involvement of Mmp-1 is intriguing given its well-known role in neuroinflammatory responses to brain injury in mammals, where it functions to break down the extracellular matrix and has been proposed to promote diffuse axon injury. Other key components of the Draper signaling pathway (dCed-6 in particular, which is required for Draper signaling in all other known contexts) were not required for suppression of bystander neuron neurophysiology (Hsu, 2020).
How bystander neurons receive injury signals and respond has remained unclear, although injury- or disease-induced effects on bystander neurons is well documented. In most cases, this has been explored in the context of bystander neuron cell death driven by neuroinflammatory cells. For instance, release of C1q, interleukin-1α (IL-1α), tumor necrosis factor (TNF) from microglia following brain injury drives the formation of neurotoxic astrocytes, which can promote the death of neurons through release of yet-to-be-identified toxins. Bystander neuronal cell death is also driven by brain-infiltrating inflammatory monocytes in viral encephalitis, in a way that is mediated by calpains, which are also important regulators of axon degeneration. Secondary axon degeneration (i.e., that occurring in neurons not damaged by the initial injury) can be driven in a way that requires intracellular Ca2+ release through IP3Rs and ryanodine receptors. These represent extreme cases of bystander effects, where cells undergo apoptosis or their axons degeneration. Whether dSarm/Sarm1 is involved in these effects is an open question. The model employed by this study is likely most relevant to partial nerve injury, where non-autonomous changes in bystander neurons have been well documented. Uninjured bystander neurons in mild TBI models are certainly altered physiologically in a reversible way. The molecular basis of any of these signaling events remains unknown, but this study points to dSarm/Sarm1 as a candidate mediator. It is interesting to note that in contrast to control mice, which show significant behavioral defects for hours after mild TBI, Sarm1-/- animals exhibited almost immediate recovery, and this was at a time point long before diffuse axon injury is observed in TBI models. It is plausible that this early loss of function is mediated in part by the bystander effect (Hsu, 2020).
In summary, this study defines two genetically separable phases of dSarm signaling, places dSarm/Sarm1 at the heart of neuronal injury signaling throughout neural tissues, identifies new signaling partners for dSarm, and expands its role to regulating the responses of uninjured neurons to local tissue injury (Hsu, 2020).
Glial phagocytosis of apoptotic neurons is crucial for development and proper function of the central nervous system. Relying on transmembrane receptors located on their protrusions, phagocytic glia recognize and engulf apoptotic debris. Like vertebrate microglia, Drosophila phagocytic glial cells form an elaborate network in the developing brain to reach and remove apoptotic neurons. However, the mechanisms controlling creation of the branched morphology of these glial cells critical for their phagocytic ability remain unknown. This study demonstrated that during early embryogenesis, the Drosophila fibroblast growth factor receptor (FGFR) Heartless (Htl) and its ligand Pyramus are essential in glial cells for the formation of glial extensions, the presence of which strongly affects glial phagocytosis of apoptotic neurons during later stages of embryonic development. Reduction in Htl pathway activity results in shorter lengths and lower complexity of glial branches, thereby disrupting the glial network. This work thus illuminates the important role Htl signaling plays in glial subcellular morphogenesis and in establishing glial phagocytic ability (Ayoub, 2023).
Damage in the nervous system induces a stereotypical response that is mediated by glial cells. This study used the eye disc of Drosophila melanogaster as a model to explore the mechanisms involved in promoting glial cell response after neuronal cell death induction. These cells rapidly respond to neuronal apoptosis by increasing in number and undergoing morphological changes, which will ultimately grant them phagocytic abilities. This glial response is controlled by the activity of Decapentaplegic (Dpp) and Hedgehog (Hh) signalling pathways. These pathways are activated after cell death induction, and their functions are necessary to induce glial cell proliferation and migration to the eye discs. The latter of these 2 processes depend on the function of the c-Jun N-terminal kinase (JNK) pathway, which is activated by Dpp signalling. Evidence is presented that a similar mechanism controls glial response upon apoptosis induction in the leg discs, suggesting that these results uncover a mechanism that might be involved in controlling glial cells response to neuronal cell death in different regions of the peripheral nervous system (PNS) (Velarde, 2021).
Glioblastoma (GBM), a very aggressive and incurable tumor, often results from constitutive activation of EGFR (epidermal growth factor receptor) and of phosphoinositide 3-kinase (PI3K). To understand the role of autophagy in the pathogenesis of glial tumors in vivo, an established Drosophila melanogaster model of glioma was used based on overexpression in larval glial cells of an active human EGFR and of the PI3K homolog Pi3K92E/Dp110. Interestingly, the resulting hyperplastic glia express high levels of key components of the lysosomal-autophagic compartment, including vacuolar-type H(+)-ATPase (V-ATPase) subunits and ref(2)P (refractory to Sigma P), the Drosophila homolog of SQSTM1/p62. However, cellular clearance of autophagic cargoes appears inhibited upstream of autophagosome formation. Remarkably, downregulation of subunits of V-ATPase, of Pdk1, or of the Tor (Target of rapamycin) complex 1 (TORC1) component raptor prevents overgrowth and normalize ref(2)P levels. In addition, downregulation of the V-ATPase subunit VhaPPA1-1 reduces Akt and Tor-dependent signaling and restores clearance. Consistent with evidence in flies, neurospheres from patients with high V-ATPase subunit expression show inhibition of autophagy. Altogether, these data suggest that autophagy is repressed during glial tumorigenesis and that V-ATPase and MTORC1 components acting at lysosomes could represent therapeutic targets against GBM (Formica, 2021).
Insects, as poikilotherms, have adaptations to deal with wide ranges in temperature fluctuation. Allelic variations in the foraging gene that encodes a cGMP dependent protein kinase, were discovered to have effects on behavior in Drosophila by Dr. Marla Sokolowski in 1980. This single gene has many pleiotropic effects and influences feeding behavior, metabolic storage, learning and memory and has been shown to affect stress tolerance. PKG regulation affects motoneuronal thermotolerance in Drosophila larvae as well as adults. While the focus of thermotolerance studies has been on the modulation of neuronal function, other cell types have been overlooked. Because glia are vital to neuronal function and survival, this study determine if glia play a role in thermotolerance as well. In this investigation, a novel calcium wave was discovered at the larval NMJ and the wave's dynamics and the potential mechanism underlying the wave prior was characterized to determining what effect, if any, PKG modulation has on the thermotolerance of glia cells. Using pharmacology, it was determined that calcium buffering mechanisms of the mitochondria and endoplasmic reticulum play a role in the propagation of the novel glial calcium wave. By coupling pharmacology with genetic manipulation using RNA interference (RNAi), it was found that PKG modulation in glia alters thermoprotection of function as well as glial calcium wave dynamics (Krill, 2021).
Juvenile hormone (JH) is one of the most important hormones in insects since it is essential for insect development. The mechanism by which JH affects the central nervous system still remains a mystery. This study demonstrates that one of the JH receptors, Methoprene-tolerant (Met), is important for the control of neurite development and sleep behavior in Drosophila. With the identification of Met-expressing glial cells, the mechanism that Met negatively controls the mushroom body (MB) β lobes fusion and positively maintains pigment-dispersing factor sLNvs projection pruning has been established. Furthermore, despite the developmental effects, Met can also maintain nighttime sleep in a development-independent manner through the α/β lobe of MB. Combining analyses of neuronal morphology and entomological behavior, this study advances understanding of how the JH receptor regulates the nervous system (Wu, 2021).
Infection-induced aversion against enteropathogens is a conserved sickness behaviour that can promote host survival. The aetiology of this behaviour remains poorly understood, but studies in Drosophila have linked olfactory and gustatory perception to avoidance behaviours against toxic microorganisms. Whether and how enteric infections directly influence sensory perception to induce or modulate such behaviours remains unknown. This study shows that enteropathogen infection in Drosophila can modulate olfaction through metabolic reprogramming of ensheathing glia of the antennal lobe. Infection-induced unpaired cytokine expression in the intestine activates JAK-STAT signalling in ensheathing glia, inducing the expression of glial monocarboxylate transporters and the apolipoprotein glial lazarillo (GLaz), and affecting metabolic coupling of glia and neurons at the antennal lobe. This modulates olfactory discrimination, promotes the avoidance of bacteria-laced food and increases fly survival. Although transient in young flies, gut-induced metabolic reprogramming of ensheathing glia becomes constitutive in old flies owing to age-related intestinal inflammation, which contributes to an age-related decline in olfactory discrimination. These findings identify adaptive glial metabolic reprogramming by gut-derived cytokines as a mechanism that causes lasting changes in a sensory system in ageing flies (Cai, 2021).
Olfactory perception influences nutrition and promotes physiological and mental well-being. In flies, a dedicated olfactory circuit elicits avoidance behaviours towards geosmin-a volatile compound that is released by mould and some bacteria. Olfactory receptors also mediate an initial attraction to food that contains certain enteropathogens. After infection with these pathogens, however, an avoidance behaviour is triggered by immune receptors in the brain, gustatory bitter neurons, and the neuropeptide leukokinin. Whether changes in olfactory perception contribute to this behavioural switch from attraction to avoidance remains unclear (Cai, 2021).
In Drosophila, odorants are sensed by olfactory receptor neurons in the head, the antenna and the maxillary palp. Olfactory receptor neurons synapse into projection neurons at the antennal lobe (AL), where the signal is converted into a spatiotemporal code in 50 glomerular compartments. Projection neurons axons project to higher olfactory centres to instruct innate and learned behaviour. In this system, glia and neurons operate as a tightly coupled unit to maintain olfactory sensitivity (Cai, 2021).
In ageing flies, olfactory perception of both aversive and attractive odours declines, but the mechanism(s) of this decline remain unclear. Olfactory perception and other neurological processes also decline in ageing mammals, often influenced by gastrointestinal signals (Cai, 2021).
This study investigated the communication between the gut and the brain, and how it influences infection-induced avoidance behaviour, infection tolerance, and olfactory decline during ageing (Cai, 2021).
A modified capillary feeder (CAFE) assay was used to measure choice between food that did or did not contain Erwinia carotovora carotovora 15 (Ecc15), a non-lethal enteropathogen that causes intestinal inflammation. Consistent with recent reports, naive flies consumed more Ecc15-containing food than normal food. However, when orally infected with Ecc15 for 24 h before the feeding assay, flies developed a distinct aversion to food that contained Ecc15. To assess whether this involved changes in olfactory perception, the 'preference index' for attractive (such as putrescine) or aversive (such as 3-octanol) odours was determined in T-maze assays. Preference or aversion for attractive or aversive odours, respectively, declined after infection, which indicates that infection causes a non-selective decline in olfactory discrimination. This was transient, as olfactory discrimination recovered 5 days after infection, coincident with the clearance of bacteria and epithelial regeneration in the intestine. Olfactory discrimination was not influenced by starvation or exposure to heat-killed Ecc15. Consistent with their reported role in sensing pathogenic bacteria, the CO2 receptor Gr63a or the odorant receptor co-receptor Orco was required for the attraction to Ecc15 food: Orco1 and Gr63a1 mutants ingested less Ecc15 food under naive conditions, and when infected, failed to further reduce ingestion of Ecc15-containing food. Together, these observations suggest that after an initial odorant-mediated attraction, flies develop aversion to enteropathogens, through a concerted activation of gustatory and immune receptors and suppression of olfaction (Cai, 2021).
After oral infection with Ecc15, damaged intestinal enterocytes produce the inflammatory IL-6-like cytokines Unpaired 2 and 3 (Upd2 and Upd3) to stimulate intestinal stem-cell proliferation and epithelial regeneration. Proteins of the Upd family activate the JAK-STAT signalling pathway through the receptor Domeless (Dome) and the JAK homologue Hopscotch (Hop). Using the 2xSTAT::GFP reporter for JAK-STAT pathway activity, this study found upregulated GFP expression in the brain 4 h after oral Ecc15 infection, as well as after oral infection with the more lethal enteropathogen Pseudomonas entomophila (PE) that damages the gut epithelium. JAK-STAT activity was observed in a sparse population of cells of the brain that stained positive for the glial marker Repo. Subtype-specific Gal4 drivers revealed that among the five subtypes of Drosophila glia (astrocytes, ensheathing, perineural, subperineural and cortex glia), ensheathing glia (EG) were the main population that upregulate STAT activity in response to Ecc15 infection. This was confirmed using four different Gal4 drivers to label EG and by flow cytometry. Infection did not influence numbers and membranous processes (labelled using UAS::mCD4GFP) of EG at the AL, and glomerular compartmentalization in the AL and lobe size remained unaffected. JAK-STAT activation in EG was sufficient and required for infection-induced changes in olfactory discrimination, as overexpression of constitutively active Hop (hoptuml) in EG reduced olfactory discrimination, whereas loss of Dome or STAT in all glia (repo::Gal4), or specifically in EG (GMR56F03::Gal4), rescued the decline of olfactory discrimination caused by Ecc15 infection. Overexpression of hoptuml in EG also reduced ingestion of Ecc15-containing food and promoted survival of flies fed PE-containing food, whereas knockdown of Dome or STAT in EG increased ingestion of Ecc15-containing food in infected flies and increased mortality on PE-containing food. It is proposed that the corresponding changes in ingestion of PE-laced food contribute to reduced mortality, but it is possible that additional genetic background conditions influence mortality, as seen, for example, in Orco-mutant flies, which ingest less bacteria but show increased susceptibility to PE (Cai, 2021).
To test whether gut-derived Upd proteins directly contribute to the infection-induced activation of JAK-STAT signalling in EGs, intestinal enterocyte-specific perturbations were performed using Mex1::Gal4, an enterocyte driver with no expression in the brain. Indeed, JAK-STAT activation in glia at the AL could be triggered in naive flies or prevented in infected flies by overexpression or knockdown, respectively, of Upd2 and Upd3 in enterocytes. Consistently, enterocyte-derived Upd2 and Upd3 were sufficient and required for the modulation of olfactory discrimination caused by infection. Knockdown of Upd2 or Upd3 did not affect olfaction in naive flies, and perturbing these ligands in fatbody (cg::Gal4) or haemocytes (hml::Gal4), tissues that are sources for Upd proteins in other contexts, did not significantly affect STAT activity in glia at the AL (Cai, 2021).
Loss of olfactory sensitivity is an early sign of normal ageing and neurodegeneration. In ageing Drosophila, olfactory perception has been reported to deteriorate before vision, a decline that was possible to recapitulate in T-maze assays. Glomerular compartments in the AL became less organized and less distinct in geriatric (60-70-day-old) flies and AL size increased with age. This correlates with a reduction in the number of EG and of glial membranous processes, changes that are expected to affect AL structure, and thus probably contribute to the age-related decline in olfaction (Cai, 2021).
Ageing in Drosophila is accompanied by the development of intestinal inflammation, and is associated with the constitutive expression and release of Upd cytokines. Consistently, JAK-STAT activity in the AL of EG was increased in old flies, and knockdown of Dome or STAT by RNA interference (RNAi) in EG specifically or in all glia rescued the decline of olfactory discrimination in old flies. The loss of Dome in EG also rescued the age-related decline of EGs and restored the size of the AL. JAK-STAT activation in the EG of old flies is a consequence of intestinal Upd release, as knocking down Upd2 and Upd3 in enterocytes alleviated STAT activation in the AL, and prevented the age-related decline of olfactory discrimination (Cai, 2021).
This age-related decline of olfactory discrimination was independent of the microbiota, as germ-free old flies still exhibited reduced olfaction sensitivity, increased JAK-STAT signalling in the AL, decreased numbers of EG, loss of glial cellular processes, and an enlarged AL. These results are consistent with the observation that the age-related increase in Upd released from the gut is also independent of the microbiota (Cai, 2021).
To understand why EG but not other glia selectively respond to Upd ligands and activate JAK-STAT signalling during ageing or infection, single-cell RNA sequencing (scRNA-seq) was performed on purified glia from young and old flies. Either all glia (labelled using repo::Gal4) or EG selectively (labelled using GMR56F03::Gal4) were profiled using Smart-seq2. The expression of dome was significantly higher in EG than in other glia, consistent with the specific upregulation of socs36E, a known target of JAK-STAT signalling, in EG but not in other glia during ageing. These results are supported by a similar upregulation of Socs36E in EG of old flies observed in a previous scRNA-seq dataset (Cai, 2021).
Bulk RNA sequencing analysis on glia (repo::Gal4, UAS::tdTomato) purified from central brains of flies expressing a 10xSTAT::GFP reporter revealed that the transcriptomes of STAT::GFP+ glia from Ecc15 infected and uninfected flies were more similar to each other than to STAT::GFP- glia of either condition, which indicates that JAK-STAT induction has a stronger influence on glial transcriptomes than other infection-related changes. Differentially expressed genes (866 genes using a cut-off of twofold change, P < 0.001, false discovery rate (FDR) < 0.01 and reads > 0.5) were significantly enriched in genes that encode proteins involved in lipid metabolism and carbohydrate transmembrane transport. These included the lipid binding protein Glial lazarillo (GLaz, a homologue of apolipoprotein D in mammals), which facilitates lipid transport from neurons to glia in flies; the lipid droplet surface binding proteins Lsd-1 and Lsd-2; the diacylglycerol O-acyltransferase Midway, which is a central regulator of triacylglycerol biosynthesis, and Coatomer, which is responsible for protein delivery to lipid droplets (LDs). This induction of lipid storage genes was coupled with induction of the monocarboxylate transporter (MCT) Outsiders (Out), and the MCT accessory protein Basigin (Bsg), sugar transporters (Tret1-1 and Tret1-2), and 17 enzymes involved in β-oxidation (Cai, 2021).
Glial MCTs promote lipid production in neurons and LD accumulation in glia by establishing a neuron and glia 'lactate shuttle'. To test a potential role for STAT signalling in influencing this shuttle at the AL, the accumulation of LDs was assessed at the AL in infected young flies using a combination of a neutral lipid probe (LipidTox, deep red) and a lipid peroxidation probe (C11-Bodipy, 581/591). A transient accumulation of LDs was observed 24 h after infection that decreased 4 days after infection, possibly owing to increased levels of β-oxidation. Overexpression of hoptuml in the EG of young flies also promoted LD accumulation, whereas knocking down Dome or STAT rescued infection-induced accumulation. GLaz and Out were required for LD accumulation after infection, and overexpression of Upd2 and Upd3 in the gut induced LD accumulation at the AL, whereas knockdown of Upd2 or Upd3 alleviated LD accumulation in infected flies. Infection or JAK-STAT perturbation did not influence lipid peroxidation in LDs in young flies (Cai, 2021).
During neuronal stress, neurons can preferentially transfer fatty acids to glia, causing lipid accumulation and increasing fatty acid β-oxidation in glia. This study observed a significant induction of LDs specifically in EG at the AL in old flies, phenocopying hoptuml overexpression. As fatty acid β-oxidation is a source of reactive oxygen species (ROS) that can result in lipid peroxidation, and lipid peroxidation in pigment cells (glia of the retina) promotes the demise of photoreceptors in the retina (whereas oxidative stress contributes to age-related dysfunction of cholinergic projection neurons within the olfactory circuit) it was reasoned that overall levels of ROS might increase in glia with age. Various genetically encoded ROS sensors were expressed in all glia (repo::Gal4) or in EG only (GMR56F03::Gal4) to measure levels of hydrogen peroxide (H2O2; measured by RoGFP2_Orp1) or the glutathione redox potential (measured by RoGFP2_Grx1) within the mitochondria or cytosol, respectively. Cytosolic levels of H2O2 were increased in the EG of old flies (both cytosolic and mitochondrial H2O2 levels were increased in all glia), whereas the cytosolic glutathione redox potential remained unchanged. In contrast to acute intestinal infection in young flies, lipids were peroxidated in LDs of old flies. Knocking down STAT specifically in EG, or knocking down Upd2 and Upd3 in gut enterocytes, inhibited LD accumulation and alleviated lipid peroxidation in old flies (Cai, 2021).
Olfactory discrimination was partially rescued in old and young infected flies after knockdown of GLaz and Out in EG. Knockdown of GLaz and Out also led to more Ecc15 food consumption, increased mortality after PE exposure, and reduced LD accumulation in the glia of old flies (Cai, 2021).
To confirm that infection or ageing-induced metabolic changes in EG affect neuron or glia metabolic coupling at the AL, the consequences of perturbing projection neurons directly were assessed using GH146::Gal4. Knocking down Out but not lactate dehydrogenase (Ldh) in projection neurons rescued olfactory discrimination of infected or aged flies, whereas food preference or mortality was not influenced. Overexpression of lipase 4 (Lip-4) in projection neurons, or knockdown of the neuronal lipid binding protein Neural lazarillo (NLaz), significantly improved olfactory discrimination in infected or old flies, and overexpression of Lip-4 increased Ecc15 food consumption and increased mortality after PE exposure (Cai, 2021).
This work suggests that gut-derived inflammatory cytokines modulate the metabolic coupling of glia and neurons in the brain of Drosophila to induce an adaptive temporary halt of olfactory discrimination after intestinal infection, but also contribute to age-related olfactory decline. It is proposed that gut-derived Upd2 and Upd3 reprogram lipid metabolism in EG, increasing lactate and lipid transport between glia and olfactory neurons, resulting in LD accumulation and upregulation of mitochondrial β-oxidation, potentially a source of increased ROS production. Chronic activation of this metabolic shift in old flies results in the accumulation of peroxidated lipids in EG, promoting their decay and contributing to the previously described functional decline of olfactory neurons. Detailed characterization of this metabolic reprogramming, and further exploration of the role of lipid synthesis in projection neurons for glial lipid accumulation and for olfactory discrimination are important avenues for further study (Cai, 2021).
These findings further determine the regulation of avoidance behaviour against enteropathogens in insects. In addition to gustatory bitter neurons and immune receptors in octopaminergic neurons, Upd proteins constitute a direct endocrine signal from the damaged intestinal epithelium in this complex but essential behaviour. It is proposed that Upd-mediated suppression of olfactory discrimination is required to prevent olfaction-mediated attraction to a food source after pathogenicity has been established and aversion is induced by gustatory neurons. It remains unclear, however, whether gustatory neurons are also affected by JAK-STAT signalling in EG. Whether similar mechanisms are conserved and control infection-induced loss of sensory perception in vertebrates including humans will be interesting to explore (Cai, 2021).
The blood brain barrier (BBB) has the essential function to protect the brain from potentially hazardous molecules while also enabling controlled selective uptake. How these processes and signaling inside BBB cells control neuronal function is an intense area of interest. Signaling in the adult Drosophila BBB is required for normal male courtship behavior and relies on male-specific molecules in the BBB. This study shows that the dopamine receptor D2R is expressed in the BBB and is required in mature males for normal mating behavior. Conditional adult male knockdown of D2R in BBB cells causes courtship defects. The courtship defects observed in genetic D2R mutants can be rescued by expression of normal D2R specifically in the BBB of adult males. Drosophila BBB cells are glial cells. These findings thus identify a specific glial function for the DR2 receptor and dopamine signaling in the regulation of a complex behavior (Love, 2023).
Glia and neurons face different challenges in aging and may engage different mechanisms to maintain their morphology and functionality. This study reports that adult-onset downregulation of a Drosophila gene CG32529/GLAD led to shortened lifespan and age-dependent brain degeneration. This regulation exhibited cell type and subtype-specificity, involving mainly surface glia (comprising the BBB) and cortex glia (wrapping neuronal soma) in flies. In accordance, pan-glial knockdown of GLAD disrupted BBB integrity and the glial meshwork. GLAD expression in fly heads decreased with age, and the RNA-seq analysis revealed that the most affected transcriptional changes by RNAi-GLAD were associated with upregulation of immune-related genes. Furthermore, a series of lifespan rescue experiments indicated that the profound upregulation of immune and related pathways was not the consequence but cause of the degenerative phenotypes of the RNAi-GLAD flies. Finally, GLAD encoded heterochromatin-associating protein bound to the promoters of an array of immune-related genes and kept them silenced during the cell cycle. Together, these findings demonstrate a previously unappreciated role of heterochromatic gene silencing in repressing immunity in fly glia, which is required for maintaining BBB and brain integrity as well as normal lifespan (Shu, 2023).
Glial cells play important roles during neurogenesis and in maintaining complex functions of the nervous system. This study reports the characterization of a gene, Sdr, which contains a putative insulin-like growth factor receptor domain and is required to maintain critical nervous system functions in Drosophila. Sdr is expressed in glial cells during embryonic and larval stages of development, but its role in adult flies is poorly understood. As insulin signaling is important throughout the lifespan in human, this study investigated the Sdr's role in adult flies. Ther results demonstrate that Sdr is expressed on surface glial cells that surround the nervous system. Mutation of Sdr did not affect development but caused defects in locomotion and lifespan. Sdr mutants also showed increasingly severe defects in the blood-brain- and blood-retina-barriers as they aged. Therefore, a novel role of Sdr in maintaining the integrity of the blood-brain- and blood-retina-barriers in adult flies is suggested (Kim, 2023).
This study used a synthetic genetic system based on ligand-induced intramembrane proteolysis to monitor cell-cell contacts in animals. Upon ligand-receptor interaction in sites of cell-cell contact, the transmembrane domain of an engineered receptor is cleaved by intramembrane proteolysis and releases a protein fragment that regulates transcription in the interacting partners. The system can be used to regulate gene expression between interacting cells, both in vitro and in vivo, in transgenic Drosophila. The system allows for detection of interactions between neurons and glia in the Drosophila nervous system. In addition, it was observed that when the ligand is expressed in subsets of neurons with a restricted localization in the brain it leads to activation of transcription in a selected set of glial cells that interact with those neurons. This system will be useful to monitor cell-cell interactions in animals, and can be used to genetically manipulate cells that interact with one another (Huang, 2016).
Receptor Tyrosine Kinase (RTK) signaling controls key aspects of cellular differentiation, proliferation, survival, metabolism, and migration. Deregulated RTK signaling also underlies many cancers. Glycosphingolipids (GSL) are essential elements of the plasma membrane. By affecting clustering and activity of membrane receptors, GSL modulate signal transduction, including that mediated by the RTK. GSL are abundant in the nervous system, and glial development in Drosophila is emerging as a useful model for studying how GSL modulate RTK signaling. Drosophila has a simple GSL biosynthetic pathway, in which the mannosyltransferase Egghead controls conversion of glucosylceramide (GlcCer) to mactosylceramide (MacCer). Lack of elongated GSL in egghead (egh) mutants causes overgrowth of subperineurial glia (SPG), largely due to aberrant activation of phosphatidylinositol 3-kinase (PI3K). However, to what extent this effect involves changes in upstream signaling events is unresolved. This study shows that glial overgrowth in egh is strongly linked to increased activation of insulin and Fibroblast Growth Factor receptor (FGFR). Glial hypertrophy is phenocopied when overexpressing gain-of-function mutants of the Drosophila Insulin Receptor (InR) and the FGFR homolog Heartless (Htl) in wild type SPG, and is suppressed by inhibiting Htl and InR activity in egh. Knockdown of GlcCer synthase in the SPG fails to suppress glial overgrowth in egh nerves, and slightly promotes overgrowth in wild type, suggesting that RTK hyperactivation is caused by absence of MacCer and not by GlcCer accumulation. It is concluded that an early product in GSL biosynthesis, MacCer, prevents inappropriate activation of Insulin and Fibroblast Growth Factor Receptors in Drosophila glia (Gerdoe-Kristensen, 2016).
The Drosophila Ntan1 gene encodes an N-terminal asparagine amidohydrolase that is highly conserved throughout evolution. Protein isoforms share more than 72% of similarity with their human counterparts. At the cellular level, this gene regulates the type of glial cell growth in Drosophila larvae by its different expression levels. The Drosophila Ntan1 gene has 4 transcripts that encode 2 protein isoforms. This study describes that although this gene is expressed at all developmental stages and adult organs tested (eye, antennae and brain) there are some transcript-dependent specificities. Therefore, both quantitative and qualitative cues could account for gene function. However, widespread developmental stage and organ-dependent expression could be masking cell-specific constraints that can be explored in Drosophila by using Gal4 drivers. A new genetic driver within this gene, Mz317-Gal4, is reported that recapitulates the Ntan1 gene expression pattern in adults. It shows specific expression for perineural glia in the olfactory organs but mixed expression with some neurons in the adult brain. Memory and social behavior disturbances in mice and cancer and schizophrenia in humans have been linked to the Ntan1 gene. Therefore, these new tools in Drosophila may contribute to greater understanding of the cellular basis of these alterations (Castaneda-Sampedro, 2022).
Although, glial cells have well characterized functions in the developing and mature brain, it is only in the past decade that roles for these cells in behavior and plasticity have been delineated. Glial astrocytes and glia-neuron signaling, for example, are now known to have important modulatory functions in sleep, circadian behavior, memory and plasticity. To better understand mechanisms of glia-neuron signaling in the context of behavior, cell-specific, genome-wide expression profiling was conducted of adult Drosophila astrocyte-like brain cells and RNA interference (RNAi)-based genetic screens were performed to identify glial factors that regulate behavior. Importantly, these studies demonstrate that adult fly astrocyte-like cells and mouse astrocytes have similar molecular signatures; in contrast, fly astrocytes and surface glia-different classes of glial cells-have distinct expression profiles. Glial-specific expression of 653 RNAi constructs targeting 318 genes identified multiple factors associated with altered locomotor activity, circadian rhythmicity and/or responses to mechanical stress (bang sensitivity). Of interest, one of the relevant genes encodes a vesicle recycling factor, four encode secreted proteins and three encode membrane transporters. These results strongly support the idea that glia-neuron communication is vital for adult behavior (Ng, 2016).
During development, many epithelia are formed by a mesenchymal-epithelial transition (MET). This
study examined the major stages and underlying mechanisms of MET during blood-brain barrier (BBB)
formation in Drosophila. Contact with the basal lamina was shown to be essential for the growth of
the barrier-forming subperineurial glia (SPG). Septate junctions (SJs), which provide insulation of the
paracellular space, are not required for MET, but are necessary for the establishment of polarized
SPG membrane compartments. In vivo time-lapse imaging reveals that the Moody GPCR signalling pathway regulates SPG cell growth and shape,
with different levels of signalling causing distinct phenotypes. Timely, well-coordinated SPG growth
is essential for the uniform insertion of SJs and thus the insulating function of the barrier. This
is the first dynamic in vivo analysis of all stages in the formation of a secondary epithelium and
of the key role trimeric G protein signalling plays in this important morphogenetic process (Schwabe, 2017).
This study of Drosophila BBB development represents the first dynamic in vivo study of MET and secondary epithelium formation. The data shed particular light on the roles of the basal lamina and of the insulating SJs, as well as on the function of GPCR signaling in this important morphogenetic process.
Once SPG reach the CNS surface, contact with the basal lamina is essential for the extensive growth of the SPG during epithelium formation. Previous in vitro studies have shown that adhesion to basal lamina components is necessary for cell spreading and proliferation, however this study is the first to demonstrate in vivo that attachment to the basal lamina is essential for non-proliferative cell growth and ensheathment. Attachment to the ECM occurs primarily through focal adhesions and integrins, which in turn can activate MAPK signaling, triggering cell proliferation and growth. In addition, adhesion to the ECM has been shown to provide traction, which facilitates cell spreading. Contact to the ECM may thus provide the SPG with both growth signals and attachment sites. Being highly expressed on the basal lamina facing side of SPG, Dystroglycan (Dg) is an excellent candidate for mediating ECM attachment. However, zygotic mutants of Dg show no BBB defects and germline clones could not be analyzed due to Dg's role in oogenesis (Schwabe, 2017).
Beyond supporting SPG growth, contact with the basal lamina likely provides an important cue for polarizing the cells, as judged by their strong enrichment of Dg at the basal lamina facing (basal) membrane compartment. Previous studies have shown that Dg and its ligand Pcan/Trol are required for the establishment of polarity in follicle cells. However, when the basal lamina and thus its ligand Pcan are depleted, Dg is still expressed and polarized in the SPG, suggesting that glial polarity can be supported by the residual basal lamina or that additional polarizing signals exist (Schwabe, 2017).
Once SJs have formed, the GPCR Moody and the Mdr65 transporter are asymmetrically distributed within the SPG, further demonstrating that these cells possess distinct apical and basal membrane compartments. This polarized distribution is coincident with and dependent on the presence of SJs, demonstrating for the first time that SJs serve a function in cell polarity. By acting as a fence and preventing diffusion of membrane proteins across the lateral compartment, the SJs maintain asymmetric protein distributions, which could result from polarized exocytosis or endocytosis. Intriguingly, a separate study has identified PKA as a crucial antagonistic effector of Moody signaling. PKA has been shown to regulate polarized exocytosis at the trans-Golgi network in different types of epithelia. Apical-basal polarity plays an important morphogenetic role in the continued growth of the SPG epithelium during larval stages and in the function of the BBB (Schwabe, 2017).
Signaling by the GPCR Moody plays a critical role both in regulating the growth of individual SPG and in synchronizing this process across the entire SPG cell population. In Moody pathway mutants, glial growth behavior is more erratic, and more variable between cells. This increased variability of glial cell shape, size, and growth causes a significant delay of epithelial closure of up to 1.5 hrs. This delay is not caused by an earlier delay in glial migration or by a delay in SJ formation (Schwabe, 2017).
The detailed dynamic analysis reveals that, in moody and loco mutants, the spatio-temporal coordination of cell spreading is impaired. Spreading cells, like other motile cells, show fluctuating exploratory motions of the leading edge visible as cycles of protrusion and retraction. This complex process can be broken down into discrete steps: actin protrusion of the leading edge, adhesion to the ECM, and myosin-driven contraction against adhesions. Time-lapse recordings indicate that Moody signaling has its most pronounced effect on the stabilization of protrusions, as evidenced by an increase in the ratio of retractions to extensions, and the marked shift of cell contours over time. The destabilization of protrusions might be due to weaker integrin-mediated interaction of focal adhesions with the ECM, but also due to impaired stress-mediated maturation of focal adhesions. The fact that both under- and overactivity of the Moody pathway impair protrusion stabilization may be due to the feedback between actin-myosin and focal adhesion, which also causes the well-known biphasic response of migration speed to adhesion strength of migrating cells. While the loss of moody has no significant effects on the other parameters that were measured, the loss of loco also reduces the frequency and size of protrusions, suggesting that actin polymerization may be specifically affected by increased GPCR signaling activity. Cumulatively, these impairments in protrusion/retraction behavior lead to retarded, non-isometric growth of SPG and to the irregular cell shapes observed in moody and loco mutants (Schwabe, 2017).
Interestingly, PKA, Rho1 and MLCK have been identified as important downstream effectors of Moody signaling. All three factors are well known to control actin-myosin contraction; Rho1 and MLCK as positive regulators, PKA as a negative regulator. More recently, Rho1 activity has been shown to also drive actin polymerization at the leading edge, and a PKA-RhoGDI-Rho1 regulators feedback loop has been suggested to act as a pacemaker of protrusion- retraction cycles (Schwabe, 2017).
The role of Moody pathway signaling in directed and well-coordinated cell growth is strikingly similar to the function of trimeric G protein signaling in other contexts. In Dictyostelium, G protein signaling is essential for directed cell migration: When all G protein signaling is abolished, cells are still mobile and actively generate protrusions, however these protrusions form in random directions, with the result that the cells lose their directionality. During gastrulation in Drosophila, signaling by the G12 ortholog Concertina and the putative GPCR ligand Folded Gastrulation synchronizes apical actin-myosin constrictions of mesodermal precursor cells, and thereby effects their concerted invagination. Thus, a major role of G protein signaling during development may be to modulate basic cellular behaviors such as cell growth, protrusion, or contraction, and reduce variability within cells and between neighboring cells, with the goal of generating uniform patterns and behaviors (Schwabe, 2017).
Synchronized growth behavior of SPG is not only important for rapid epithelial closure but, ultimately, also for generating an evenly sealed BBB. All the evidence supports the notion that the defects in SJ organization that are responsible for the BBB failure are a secondary consequence of the morphogenetic function of the GPCR pathway. Cell contacts precede and are necessary for SJ formation, and the growth of cell contacts and SJ accumulation are strongly correlated. Delayed and more erratic cell- cell contact formation, as is the case in Moody pathway mutants, is likely to result in uneven circumferential distribution of SJ material later on; conversely, the timing of SJ formation per se is not affected by the pathway, arguing against a direct effect. Since the insulating function of SJs depends on their length, a decrease in the length in some local areas will result in insulation defects. Moreover, since SJs are known to form very static complexes, any irregularity in SJ distribution may be retained for long periods of time (Schwabe, 2017).
Although under- and overactivity of the Moody pathway lead to globally similar outcomes, impaired epithelium formation and failure of BBB insulation, the data point to subtly different subcellular effects of the two types of pathway modulation. During MET, loco mutants (which this study confirms as inducing pathway overactivity) show predominantly retarded growth, presumably as a result of curtailed protrusive activity, while moody mutants show severe fluctuation and variability in growth. It will be very interesting to investigate these distinct outcomes of Moody pathway misregulation in greater detail (Schwabe, 2017).
During development tissue growth is mediated by either cell proliferation or cell growth, coupled with polyploidy. Both strategies are employed by the cell types comprising the Drosophila blood-brain barrier. During larval growth, the perineurial glia proliferate, whereas the subperineurial glia expand enormously and become polyploid. This study shows that the level of ploidy in subperineurial glia is controlled by the N-terminal asparagine amidohydrolase homolog Obek, where high Obek levels are required to limit replication. In contrast, perineurial glia express moderate levels of Obek, and increased expression blocks their proliferation. Interestingly, other dividing cells are not affected by alteration of Obek expression. In glia, Obek counteracts FGF- and Hippo-signaling to differentially affect cell growth and number. A mechanism is proposed where growth signals are integrated differentially in a glia-specific manner through different levels of Obek protein to adjust cell proliferation versus endoreplication in the blood-brain barrier (Zulbahar, 2018).
Glial regulation of extracellular potassium (K(+)) helps to maintain appropriate levels of neuronal excitability. While channels and transporters mediating K(+) and water transport are known, little is understood about upstream regulatory mechanisms controlling the glial capacity to buffer K(+) and osmotically obliged water. This study identified salt-inducible kinase (SIK3) as the central node in a signal transduction pathway controlling glial K(+) and water homeostasis in Drosophila. Loss of SIK leads to dramatic extracellular fluid accumulation in nerves, neuronal hyperexcitability, and seizures. SIK3-dependent phenotypes are exacerbated by K(+) stress. SIK promotes the cytosolic localization of HDAC4, thereby relieving inhibition of Mef2-dependent transcription of K(+) and water transport molecules. This transcriptional program controls the glial capacity to regulate K(+) and water homeostasis and modulate neuronal excitability. HDAC was identified as a candidate therapeutic target in this pathway, whose inhibition can enhance the K(+) buffering capacity of glia, which may be useful in diseases of dysregulated K(+) homeostasis and hyperexcitability (Li, 2019).
Neuronal excitability is tightly coupled to complex ion dynamics in the nervous system. As ions move, so too must osmotically obliged water molecules move. Following bursts of action potentials, ionic gradients are restored via active transport. Defects in water and ion homeostasis disrupt neuronal firing and can result in edema. Potassium (K+) homeostasis is particularly important for maintaining the physiological function of neurons. Repolarization of the axonal membrane during action potentials transfers K+ ions into the extracellular space. With synchronous activity, the bulk extracellular [K+] rises, and this K+ must be rapidly buffered via glial and/or neurons lest axons depolarize, disrupting neuronal firing (Li, 2019).
Glial cells are essential for potassium and water homeostasis. Glia remove K+ ions from the extracellular space and redistribute them to areas with lower [K+] to maintain ionic gradients. Glia also express aquaporin water channels to functionally couple K+ clearance and water transport, thereby relieving osmotic stress. A number of K+ and water transport molecules mediate glial regulation of extracellular [K+], including K+ channels, Na+-K+-Cl−cotransporter 1, Na+-K+-ATPase, and aquaporin. Defects in glial transporters can result in build-up of ions in the extracellular space between axons and glia, leading to extracellular fluid accumulation. Indeed, mouse and Drosophila melanogaster mutants with defective K+transport exhibit dramatic and strikingly similar edema phenotypes in their peripheral nerves. These swellings correlate with seizure sensitivity and offer an elegant readout for identifying molecules that are required to maintain ionic and water homeostasis and a healthy level of neuronal excitability (Li, 2019).
While much is known about the key transporters and channels that mediate the flux of water and ions, the mechanisms by which glial cells regulate expression of the relevant transporters are not well understood. Delineating such regulatory mechanisms could identify approaches to leverage glial K+ and water buffering as a therapeutic strategy for neuroprotection against K+ stress-related damage. A glial-specific screen was conducted for this study in Drosophila, and a signal transduction pathway required for glial regulation of water and ion homeostasis was identified. This screen uncovered a central role for salt-inducible kinase (SIK3), a highly conserved AMP-activated protein kinase (AMPK)-family kinase that links signal sensing to changes in cellular response. Loss of SIK3 in glia results in nerve edema, neuronal hyperexcitability, and increased seizure susceptibility, all phenotypes that are commonly associated with human genetic disorders disrupting glial water and ion homeostasis. This swelling phenotype is critically and selectively sensitive to K+ stress. Moreover, this study demonstrates that SIK functions via regulation of a downstream HDAC4/Mef2 transcriptional program that controls expression of relevant ion and water transporters. HDAC is a critical negative regulator in the pathway, and pharmacological inhibition of HDAC potently suppresses the edema, hyperexcitability, and seizure phenotypes of SIK mutants. Hence, this study identifies a druggable pathway controlling the glial capacity to buffer K+ and water and a candidate therapeutic approach to achieve the long-standing goal of targeting glia for the control of hyperexcitability (Li, 2019).
Glia remove K+ from the extracellular space to maintain ionic and water homeostasis in the nervous system. Disrupting K+ buffering leads to nerve edema, neuronal hyperexcitability, and increased seizure susceptibility. In Drosophila, loss of glial K+ transport function results in characteristic focal peripheral nerve swellings due to accumulation of extracellular fluid. To identify signaling pathways required for glial regulation of K+ and water homeostasis, an in vivo glial-specific screen was performed in Drosophila. The pan-glial driver Repo-GAL was used to express a library of ~RNAi lines targeting signaling molecules, and were genes screened for that are required to prevent swelling of larval peripheral nerves. From the screen a single hit was found: glial knockdown of the gene for SIK3 results in pronounced nerve swellings (Li, 2019).
Glia modulate neuronal excitability and seizure sensitivity by maintaining potassium and water homeostasis. A salt inducible kinase 3 (SIK3)-regulated gene expression program controls the glial capacity to buffer K(+) and water in Drosophila, however upstream regulatory mechanisms are unknown. This study identified an octopaminergic circuit linking neuronal activity to glial ion and water buffering. Under basal conditions, octopamine functions through the inhibitory octopaminergic G-protein-coupled receptor (GPCR) OctβR to upregulate glial buffering capacity, while under pathological K(+) stress, octopamine signals through the stimulatory octopaminergic GPCR OAMB1 to downregulate the glial buffering program. Failure to downregulate this program leads to intracellular glia swelling and stress signaling, suggesting that turning down this pathway is glioprotective. In the eag shaker Drosophila seizure model, the SIK3-mediated buffering pathway is inactivated. Reactivation of the glial buffering program dramatically suppresses neuronal hyperactivity, seizures, and shortened life span in this mutant. These findings highlight the therapeutic potential of a glial-centric therapeutic strategy for diseases of hyperexcitability (Li, 2021).
K+ homeostasis in the nervous system is required to maintain a healthy level of neuronal activity. Neurons release K+ ions as they repolarize during action potentials. If this K+ builds up in the extracellular space, it can disrupt neuronal firing and set the stage for neuronal hyperexcitability. Conventional treatments for conditions of hyperexcitability mostly target neuronal channels. However, more than one-third of epilepsy patients suffer from medically intractable seizures, while others experience debilitating side effects as neuron-targeting treatments often interfere with healthy neuronal functions. Hence, there is an urgent need for innovative therapeutic approaches that improve seizure control. This study explored glial mechanisms that regulate K+ balance to modulate neuronal excitability and seizure sensitivity (Li, 2021).
Glia buffer K+ stress by taking in excess K+ ions and osmotically obliged water molecules from the extracellular space. This function allows glia to control the extracellular level of K+ and thereby modulate neuronal excitability, raising the hope that glia may be targeted to restore K+ homeostasis and suppress hyperexcitability in epilepsy and stroke. Glial-centric therapeutic strategies for these conditions have not been possible, largely due to a lack of understanding of mechanisms controlling the glial K+ buffering capacity. Previous work discovered a signal transduction pathway that controls the glial capacity to buffer K+ and water in Drosophila (Li, 2019). SIK3 (salt inducible kinase 3) is an AMPK family kinase that sequesters histone deacetylase 4 (HDAC4) in the cytoplasm, thereby promoting myocyte enhancer-factor 2 (Mef2)-dependent expression of K+ and water transport molecules, including the Drosophila orthologs of aquaporin-4 (Drip) and SPAK (Frayed) a kinase necessary for activating the Na+/K+ transporter NKCC1 Ncc69. This glial program suppresses extracellular nerve edema and prevents neuronal hyperexcitability and seizure in Drosophila, suggesting that it plays an important role in glial maintenance of K+ and water homeostasis and is required for a healthy level of neuronal activity. This glial program is best characterized in wrapping glia, which ensheathe axons in the Drosophila peripheral nervous system (PNS) and are akin to vertebrate non-myelinating Schwann cells, and so are well positioned to regulate the ionic composition around PNS axons (Li, 2021).
While this study has elucidated downstream effectors by which SIK3 regulates K+ and water buffering, the upstream signals that work through the SIK3 pathway to control glial buffering capacity are unknown. In other contexts, SIK3 integrates signals from different signal transduction pathways, coupling extracellular signals to changes in cellular responses (Choi, 2015; Wang, 2011; Wein, 2018). One of these pathways is G-protein-coupled receptor (GPCR)-cyclic AMP (cAMP)-protein kinase A (PKA) signaling, in which PKA directly inhibits SIK3 activity (Wang, 2011). A better understanding of upstream signaling mechanisms that regulate the SIK3 pathway and extracellular cues that modulate glial K+ buffering capacity will inform approaches to leverage this glial function for therapeutic benefits in diseases of hyperexcitability (Li, 2021).
This study identified an octopaminergic circuit linking neuronal activity to glial K+ and water buffering. Octopamine regulates the glial buffering program via GPCR-dependent regulation of PKA, which inhibits SIK3. At baseline, octopamine acts through the inhibitory octopaminergic GPCR OctβR to activate the glial SIK3 pathway to promote K+ and water buffering and healthy levels of excitability. Under pathological K+ stress, in contrast, octopamine functions through the stimulatory octopaminergic GPCR OAMB1 to inhibit SIK3 and downregulate the glial capacity to buffer K+ and water. While loss of the SIK3 pathway leads to extracellular edema, constitutive activation of the pathway results in intracellular glial swelling and activation of stress signaling, suggesting that turning down this pathway protects glia. In eag shaker, a classic Drosophila hyperexcitable mutant with defective K+ channels, the SIK3-mediated glial buffering program is turned off, likely as a self-protective mechanism for glia. However, reactivation of this glial K+ and water buffering program dramatically suppresses neuronal hyperexcitability, seizures, and shortened life span in eag shaker. Therefore, the maintenance of a robust glial K+ and water buffering program in response to extreme neuronal excitability is beneficial to the organism despite the risks posed to glial health. Taken together, this study identifies a neuromodulatory circuit that links neuronal activity to glial K+ buffering and highlights the promise of a glial-centric therapeutic strategy that restores K+ and water homeostasis for control of hyperexcitability in diseases such as epilepsy and stroke (LiH, 2021).
K+ dysregulation drives edema and neuronal hyperexcitability and can lead to brain damage in patients with epilepsy and stroke. Glia buffer K+ stress by taking up excess K+ ions and water to maintain a healthy extracellular environment for neuronal excitability. Previous work identified SIK3 as the central node of a signal transduction pathway that controls the glial capacity to buffer K+ and water in Drosophila (Li, 2019). This study uncovered upstream regulatory mechanisms controlling the SIK3 pathway and demonstrated that activation of this glial program can dramatically ameliorate the pathological consequences of neuronal hyperexcitability. An octopaminergic circuit couples neuronal activity to glia buffering, exerting a dual effect on SIK3-mediated K+ buffering: low levels of octopamine enhance the glial K+ buffering capacity, while high levels of octopamine inhibit this buffering program, likely to protect glia against extreme K+ stress. Glial-specific inhibition of HDAC4, a central repressor of SIK3 signaling, results in a constitutively active glial buffering program that dramatically suppresses seizure and extends life span in a classic epilepsy model. Hence, augmenting glial K+ and water buffering holds promise as a therapeutic approach for the treatment of seizures (Li, 2021).
The glial SIK3/HDAC4/Mef2 pathway regulates expression of both fray/SPAK, a kinase controlling ion transporter activity, and aquaporin 4, a water channel essential for volume regulation (Leiserson, 2011; Li, 2019; Papadopoulos, 2004). The original analysis of SIK3 LOF mutants demonstrated that glial SIK3 inhibits extracellular edema, presumably by promoting the uptake of ions and water into glial cells. This is an essential activity of glia, since accumulation of K+ in the extracellular space around axons leads to hyperexcitability and heightened seizure susceptibility. Since peripheral nerves must continuously fire, this raised the question of why expression of fray and aquaporin 4 would be regulated by SIK3, which is best understood to integrate opposing signals such as feeding and fasting to control the levels of downstream transcriptional targets. If ion and water buffering were so essential, then why is expression of essential buffering proteins not constitutive? The findings of this study suggest that the downregulating glial K+ buffering capacity is a protective response that prevents pathological glial swelling. Activity-dependent glial swelling is a commonly observed physiological phenomenon, but when taking in an excess load of ions and water, glia may swell to the point of cytolysis. This study has shown that glia with enhanced K+ buffering capacity undergo cell swelling that is exacerbated by extreme K+ stress. These glia also exhibit enhanced JNK pathway activity, which responds to a variety of stressors, including mechanical stress caused by cell stretching. JNK plays a critical role in the regulation of apoptosis, suggesting that pathological swelling that results from enhanced K+ buffering could promote cell death. Taken together, these findings demonstrate that overactivity of glial SIK3 activity leads to intracellular edema while underactivity of glial SIK3 signaling leads to extracellular edema. Hence, SIK3 balances the need for robust extracellular buffering with the health of the glial cell. It is suggested that the ability of the SIK3 pathway to integrate disparate upstream signals is important for maintaining this balance between an extracellular milieu conducive to neuronal firing with intracellular volume regulation that maintains glial health (Li, 2021).
Norepinephrine is a stress hormone that can exert anticonvulsant effects in the mammalian nervous system. Moreover, norepinephrine regulates astrocyte transporter activity. In Drosophila, octopamine is structurally and functionally analogous to norepinephrine and regulates a variety of stress responses, including aggression, alertness, and starvation. Octopamine is also similar to norepinephrine in that it exerts dual effects on cellular responses by signaling through receptors with antagonistic functions. In this study, it is proposed that octopamine bidirectionally controls SIK3-mediated glial K+ buffering in response to changes in neuronal excitability (Li, 2021).
Like its mammalian counterpart, octopamine is normally maintained at low levels and is released upon physiological stimuli to help the system mount a stress response. Octopamine release can be enhanced by a surge in the level of extracellular K+, suggesting that its concentration correlates with the level of K+ stress in the nervous system. This study proposes a model in which octopamine release links activity-dependent K+ stress to glial K+ buffering. This model is supported by a number of key findings. First, octopamine is required for SIK3 signaling and this regulation occurs through the inhibitory octopaminergic GPCR Octβ1R. Second, elevated levels of either octopamine or K+ each inhibit the glial SIK3 pathway, and both of these effects require the stimulatory octopaminergic GPCR OAMB. Hence, elevated K+ works through octopaminergic signaling to regulate the SIK3 pathway and glial buffering. Finally, the glial SIK3 pathway is inhibited in the hyperexcitable eag,Sh mutant. It is proposed that basal levels of octopamine promote SIK3-dependent glial K+ buffering to cope with physiological K+ stress and maintain a healthy level of neuronal activity. When neurons become hyperexcitable, as would occur under pathological conditions, levels of extracellular K+ rise and more octopamine is released. High levels of octopamine will inhibit SIK3 activity and turn down glial K+ buffering, likely to protect glia from pathological cell swelling (Li, 2021).
This model posits that different levels of octopamine differentially activate Oct&beta1R and OAMB to exert these dual effects on glial K+ buffering. Octβ1R couples to the inhibitory Gαi protein, whereas OAMB can function through the excitatory Gαs protein . How might octopamine differentially signal through both Octβ1R and OAMB when they bind to the same ligand, reside in the same cell, and have antagonistic functions? Potentially, Octβ1R and OAMB could have different binding affinities for octopamine, with Octβ1R having a higher affinity such that Octβ1R is preferentially activated at low levels of octopamine. Inhibitory Gαi-coupled Octβ1R would decrease cAMP levels and inhibit PKA activity, thereby relieving the inhibition on SIK3 to upregulate glial K+ buffering capacity. Conversely, octopamine would bind to the lower affinity OAMB when octopamine levels are high, as would occur during hyperexcitability. High levels of octopamine signaling through the excitatory Gαs-coupled OAMB would enhance PKA activity and downregulate the glial capacity to buffer K+ stress. Hence, different binding affinities would allow Octβ1R and OAMB to be activated by different octopamine concentrations. A similar differential regulation via opposing octopaminergic receptors residing in the same cell is described at the larval NMJ, where octopamine signals through inhibitory Octβ1R and excitatory Octβ2R to bidirectionally control synaptic function (Li, 2021).
This study has defined an octopaminergic circuit that links neuronal activity to glial K+ buffering to maintain K+ homeostasis and neuronal excitability. Neuromodulators such as octopamine are useful for integrating synaptic activity over time and space rather than signaling rapidly and locally like classical neurotransmitters. Moreover, altering the glial transcriptional response will be reasonably slow to change physiological function since such changes require transcription and translation. Hence, this system is likely tuned to respond to average neuronal activity rather than to transient changes in neuronal firing. This pathway may work in concert with additional mechanisms for rapid tuning of glial ion transporter function as described in mammalian astrocytes (Li, 2021).
This model for octopaminergic regulation of the glial SIK3-dependent ionic buffering program leaves open a number of important questions. First, is the octopamine released locally or does release from the central octopaminergic neurons diffuse widely to set the glial tone for ionic buffering? Second, while this study showed that a glial octopamine receptor is necessary for pathway modulation by octopamine, it would also be interesting to test whether direct activation of octopaminergic neurons is sufficient to regulate the glial SIK3 pathway. Similarly, is direct inhibition of octopaminergic neurons capable of rescuing the whole animal phenotypes of the eag, Sh mutants? Third, while this study has focused on octopaminergic regulation, in other systems the SIK3 pathway is regulated by additional upstream signals including the kinase Lkb1 and the insulin receptor. Input from these additional pathways would allow for regulation of glial ion and water buffering in locations that may not be accessible for octopaminergic neuromodulation. Finally, this analysis focused on peripheral nerves and peripheral glia. In future studies it will be important to assess the role of this SIK3 buffering program in the cortex glia that buffer ions around neuronal cell bodies (Li, 2021).
With hyperexcitability, waves of action potentials can lead to increases in extracellular K+ that, if not properly buffered, will lead to neuronal depolarization and further hyperexcitability. This study has explored the role of glial buffering in a paradigmatic Drosophila seizure mutant, eag,Sh, that exhibits dramatic neuronal hyperexcitability, seizure behaviors, and shortened life span. Although it would be expected that eag,Sh larvae have an increased need for glial buffering, this study found that the glial SIK3 signaling pathway is downregulated, likely as part of the glial protective response described above. Since downregulating the glial SIK3 pathway is sufficient to cause hyperexcitability and seizures, whether this inhibition of SIK3 could be exacerbating the eag,Sh phenotypes was tested. HDAC4 is the key repressor of SIK3-regulated glial K+ buffering, and so SIK3 signaling was activated by glial knockdown of HDAC4. Reactivation of the glial buffering program dramatically suppressed neuronal hyperexcitability, seizures, and shortened life span in eag,Sh. This result is quite remarkable, since the neurons are still deficient for two extremely important K+ channels. Nonetheless, the pathological consequences for the organism are almost wholly mitigated by enhanced glial buffering. This highlights the potential of glial-centric therapeutic strategies for diseases of hyperexcitability (Li, 2021).
The nervous system is surrounded by an extracellular matrix composed of large glycoproteins, including perlecan, collagens, and laminins. Glial cells in many organisms secrete laminin, a large heterotrimeric protein consisting of an α, β, and γ subunit. Prior studies have found that loss of laminin subunits from vertebrate Schwann cells causes loss of myelination and neuropathies, results attributed to loss of laminin-receptor signaling. This study demonstrates that loss of the laminin γ subunit (LanB2) in the peripheral glia of Drosophila melanogaster results in the disruption of glial morphology due to disruption of laminin secretion. Specifically, knockdown of LanB2 in peripheral glia results in accumulation of the β subunit (LanB1), leading to distended endoplasmic reticulum (ER), ER stress, and glial swelling. The physiological consequences of disruption of laminin secretion in glia included decreased larval locomotion and ultimately lethality. Loss of the γ subunit from wrapping glia resulted in a disruption in the glial ensheathment of axons but surprisingly did not affect animal locomotion. It was found that Tango1, a protein thought to exclusively mediate collagen secretion, is also important for laminin secretion in glia via a collagen-independent mechanism. However loss of secretion of the laminin trimer does not disrupt animal locomotion. Rather, it is the loss of one subunit that leads to deleterious consequences through the accumulation of the remaining subunits (Petley-Ragan, 2016).
The role of laminin and the ECM in glial wrapping has been investigated in vertebrate systems by examining the gross morphological and molecular changes induced by knocking out individual laminin subunits in glia. A decrease in glial wrapping, proliferation, and survival was attributed to a lack of laminin binding to various receptors, including integrins, dystroglycan, and syndecans. However, it was found that knock-out of laminin resulted in more severe phenotypes than the additive phenotypes of multiple-receptor knock-out studies. This paper prresents evidence that eliminating one laminin subunit causes cellular changes that go beyond the loss of receptor-ligand signaling and contribute to glial mutant phenotypes (Petley-Ragan, 2016).
This study has demonstrated that glial cells expressing RNAi against three different laminin subunits result in morphological defects. Knockdown of the laminin γ subunit LanB2 results in extensively swollen glia, accumulation of LanB1 in the ER, and expanded ER, leading to ER stress with disrupted intracellular morphology, including vacuole-like structures. In laminin assembly, the β-γ dimer is formed followed by α chain recruitment where trimer formation is necessary for transport through the Golgi and secretion. Studies in vitro and in C. elegans found intracellular accumulation of the γ laminin subunit in the absence of the β subunit. However, the subcellular localization and physiological consequences of the intracellular laminin accumulations was not determined. Analysis of the knockdown of the γ subunit (LanB2) in all glia extended this line of study and found that loss of LanB2 led to β subunit (LanB1) accumulations, ER expansion, and ER stress. Glial swelling due to LanB2 knockdown was correlated with excess LanB1, and the swollen glial phenotype was partially rescued by introducing a heterozygous LanB1 deficiency in the background, effectively halving the amount of LanB1 available for translation. The partial rescue in glial swelling due to LanB1 heterodeficiency supports the hypothesis that unbound LanB1 is acting as a misfolded protein in the ER to induce the unfolded protein response and ER stress. In further support, overexpression of LanB1 alone was found to result in ER aggregates and vacuoles in glia. Knockdown of Tango1, a protein known to mediate ER exit of collagen, resulted in LanB1 and LanB2 accumulations in the ER. Tango1 knockdown results in laminin retention in Drosophila ovary follicle cells. However, the subcellular localization of laminin was not demonstrated. The current results refine these observations as both LanB2 and Tango1 knockdown result in LanB1 accumulations specifically in the expanded ER. However, while LanB2-RNAi led to severe glial swelling and locomotion defects, expression of Tango1 RNAi did not. In the case of Tango1 RNAi, this is possibly due to Tango1 knockdown inhibiting secretion of assembled laminin heterotrimers. Previously, Tango1 has been implicated primarily in collagen secretion, and this study found Tango1 mediates laminin secretion in the absence of collagen IV in perineurial glia. The findings imply that there may be a Tango1-specific binding site on laminin or that Tango1 may play a broader role in protein secretion than previously thought. Together, these findings suggest that the glial defects observed after LanB2 knockdown are due to excess amounts of unbound LanB1 monomers in the ER causing ER stress. Alternatively, the increased LanB1 and ribosomal content in these areas may indicate increased translation due to glia sensing inadequate amounts of external laminin (Petley-Ragan, 2016).
The glial defects observed are likely due to disruption of laminin secretion, but it is possible that the defects observed were in part due to loss of laminin from the extracellular space and loss of ECM receptor signaling, such as integrin signaling. The first explanation is favored as blocking the secretion of the laminin trimer by Tango1 RNAi did not result in glial swelling, suggesting that the reduction in laminin secretion did not lead to glia disruption. Loss of integrins and metalloproteinase-mediated degradation of the surrounding ECM does not lead to glial swelling or formation of vacuoles, but rather triggers a loss of perineurial glia wrapping of the axon. In Drosophila, the fat body and hemocytes contribute to the deposition of ECM proteins in the basement membrane surrounding the nervous system, explaining why the loss of laminin secretion from glia in Tango1 does not lead to the same phenotypes as integrin knockdown (Petley-Ragan, 2016).
This raises the interesting question of why Drosophila glia secrete laminin and not other ECM proteins. Similarly, why do Drosophila wrapping glia, which do not contact a basement membrane, express laminin? Wrapping glia were morphologically affected by LanB2 knockdown. However. accumulations of LanB1 within wrapping glia were not seen, implying that wrapping glia express low levels of laminin. With the available antibodies, it is difficult to detect and assess the levels of laminin between axons and wrapping glia or between wrapping and subperineurial glia. The presence of laminin interior to the nerve is supported by previous experiments where integrin knockdown in wrapping glia resulted in a lack of process extension similar to that seen after LanB2 knockdown, suggesting that integrin may bind to laminin present between axons and wrapping glia or between wrapping and subperineurial glia. Although LanB2 knockdown in wrapping glia resulted in morphological defects, including decreased wrapping, third-instar larvae exhibited no mobility defects and even survived to adulthood and this is also true of integrin knockdown in the wrapping glia (data not shown). The surprising finding that full contact between wrapping glia and peripheral axons is unnecessary for locomotion is consistent with mutants in the Na-K-Cl cotransporter, Ncc69. Ncc69 mutant larvae have significant osmotic swelling that inhibits direct contact between axons and the wrapping glia and yet these larvae had normal action potential activity and survived to adult stages. Apoptosis-induced death of the wrapping glia did, however, result in a total lack of mobility and lethality, indicating that either the wrapping glia or other nrv2-GAL4-expressing cells are essential to survival and locomotion (Petley-Ragan, 2016).
Larval survival and mobility were affected by knockdown of LanB2 in perineurial glia, a glial subtype that does not directly contact axons. Perineurial glia are born late in embryogenesis, surround the CNS and PNS during larval stages, and have been implicated in ECM remodeling during nervous system growth. This study demonstrates that perineurial glia secrete laminin, but not collagen IV, and contribute to nervous system function. The decreased mobility seen in 46F>LanB2-RNAi larvae provides support for the role of the outer layer of perineurial glia and the ECM in structurally supporting the larval nervous system as these glia do not contact either neuronal cell bodies or axons (Petley-Ragan, 2016).
Ultimately, the current findings suggest that loss of receptor-ligand signaling is not the sole cause of glial morphological and physiological defects due to loss of laminins. Due to the conserved structure and function of glia across the animal kingdom, it is likely that Schwann cells lacking one subunit of laminin or with a mutation in one subunit affecting laminin trimerization could also demonstrate morphological and functional defects due to the accumulation of unbound laminin subunits leading to ER stress. This could have significant implications as ER stress is important in the pathogenesis of various diseases affecting myelination in the CNS and PNS. This hypothesis has not yet been investigated in vertebrate glia, but it may provide insight into the reasons decreased glial wrapping, proliferation, and survival are seen in vertebrates after knock-out or with inherited mutations in laminin subunits. Schwann cell-specific deletion of Lamc1 (the γ-1 subunit) leads to a loss of α and β subunit expression and disruption of basement membrane secretion. Loss of γ-1 leads to defects in radial sorting and myelination by myelinating Schwann cells, as well as to loss of proliferation and blocked differentiation in the premyelination stage. Similarly loss of γ-1 leads to the absence of nonmyelinating Schwann cells and the loss of Remak bundles. The wrapping glia of Drosophila are most similar to nonmyelinating Schwann cells and in these cells loss of the γ subunit led to wrapping defects and ER stress. Whether the loss of the vertebrate γ-1 subunit leads to ER stress that results in the loss of nonmyelinating Schwann cells has not been determined. However, the current findings may represent a broader trend in the cellular response to unequal expression of related subunits that together comprise a multimeric protein, such as laminin. Future studies should focus on the effects of ER stress due to mutations in individual subunits of laminin in Schwann cells and other tissues (Petley-Ragan, 2016).
Phosphatidylserine (PS) is an integral component of eukaryotic cell
membranes and organelles. The Drosophila genome contains a single PS
synthase (PSS)-encoding gene (Pss)
homologous to mammalian PSSs. Flies with Pss loss-of-function alleles
show a reduced life span, increased bang sensitivity, locomotor defects,
and vacuolated brain, which are the signs associated with
neurodegeneration. Defective mitochondria were observed in mutant adult
brain, as well as elevated production of reactive oxygen species, and an
increase in autophagy and apoptotic cell death. Intriguingly, glial-specific
knockdown or overexpression of Pss alters synaptogenesis and axonal
growth in the larval stage, causes developmental arrest in pupal stages,
and neurodegeneration in adults. This is not observed with pan-neuronal
up- or down-regulation. These findings suggest that precisely regulated
expression of Pss in glia is essential for the development and
maintenance of brain function. A mechanism is proposed that underlies
these neurodegenerative phenotypes triggered by defective PS metabolism (Park, 2021).
Hyccin/FAM126A mutations are linked to hypomyelination and congenital cataract disease (HCC), but whether and how Hyccin/FAM126A deficiency causes hypomyelination remains undetermined. This study shows Hyccin/FAM126A expression was necessary for the expression of other components of the PI4KIIIalpha complex in Drosophila. Knockdown of Hyccin/FAM126A in glia reduced the enrichment of glial cells, disrupted axonal sheaths and visual ability in the visual system, and these defects could be fully rescued by overexpressing either human FAM126A or FAM126B, and partially rescued by overexpressing a plasma membrane-targeting recombinant mouse PI4KIIIα. Additionally, PI4KIIIα knockdown in glia phenocopied Hyccin/FAM126A knockdown, and this was partially rescued by overexpressing the recombinant PI4KIIIα, but not human FAM126A or FAM126B. This study establishes an animal model of HCC and indicates that Hyccin/FAM126A plays an essential role in glial enrichment and axonal sheath in a cell-autonomous manner in the visual system via controlling the expression and stabilization of the PI4KIIIα complex at the plasma membrane (Hyccin/FAM126A deficiency reduces glial enrichment and axonal sheath, which are rescued by overexpression of a plasma membrane-targeting PI4KIIIα in Drosophila (Zhang, 2022).
This study shows that the dietary lipid cholesterol, which is required as a component of cell membranes and as a substrate for steroid biosynthesis, also governs body growth and maturation in Drosophila via promoting the expression and release of insulin-like peptides. This nutritional input acts via the nutrient sensor TOR, which is regulated by the Niemann-Pick-type-C 1 (Npc1) cholesterol transporter, in the glia of the blood-brain barrier and cells of the adipose tissue to remotely drive systemic insulin signaling and body growth. Furthermore, increasing intracellular cholesterol levels in the steroid-producing prothoracic gland strongly promotes endoreduplication, leading to an accelerated attainment of a nutritional checkpoint that normally ensures that animals do not initiate maturation prematurely. These findings, therefore, show that a Npc1-TOR signaling system couples the sensing of the lipid cholesterol with cellular and systemic growth control and maturational timing, which may help explain both the link between cholesterol and cancer as well as the connection between body fat (obesity) and early puberty (Texada, 2022).
Animal growth and development depend upon nutrient availability. Therefore, specialized cells and tissues have arisen that sense nutritional inputs and adjust growth and developmental programs via systemic hormonal pathways. In most eumetazoans, these include the conserved insulin-like peptide and steroid-hormone signaling systems. These become dysfunctional when nutrient levels exceed their physiologically normal range. Overloading of the insulin system leads to obesity, metabolic syndrome, insulin resistance, and other pathophysiologies, and overnutrition also leads to precocious puberty associated with childhood obesity (Texada, 2022).
The early life of many animals is a nonreproductive stage of rapid growth, terminated at some nutritional threshold that signals readiness to become a reproductively fit adult. In animals as diverse as humans and insects, this transition is driven by steroid hormones - gonadal steroids including testosterone and estrogen trigger mammalian puberty, and insect metamorphosis is initiated by ecdysone, produced in the prothoracic gland (PG). Similar neuroendocrine cascades regulate insect and mammalian steroidogenesis, including the orthologous neuropeptides Allatostatin A/Kisspeptin and Corazonin/gonadotropin-releasing hormone (GnRH) as well as analogous steroid-feedback circuits. These axes are clearly linked to the metabolic state of the animal, including attainment of a certain critical size. However, the mechanisms of body-size estimation and the effects of nutritional status are not completely understood. Recent work in Drosophila suggests that progression to adulthood is gated by a checkpoint system monitoring tissue growth and nutritional status. When animals reach a 'critical weight' (CW), they become committed to completing their development and maturation, irrespective of further nutritional inputs, whereas animals starved before this checkpoint is satisfied halt their progression to adulthood. This suggests that the CW checkpoint assesses the animal's nutritional state, but the specific nutrients required, and the mechanisms by which their levels are sensed, are incompletely defined (Texada, 2022).
In Drosophila, nutritional input drives growth and maturation through the insulin pathway. Nutrient intake, of amino acids in particular, is sensed via the fat body (analogous to mammalian adipose tissue) and the glia of the blood-brain barrier (BBB). These tissues release factors that regulate the expression and release of Drosophila insulin-like peptides (ILPs) from the insulin-producing cells (IPCs) within the brain, which share functional and developmental homologies with mammalian pancreatic beta cells. These ILPs promote systemic growth through the conserved insulin-receptor/PI3K/Akt pathway. Insulin also promotes PG ecdysone production, linking nutrition directly to developmental progression (Texada, 2022).
Human puberty-triggering CW appears to be linked to body-fat stores, which may explain the link between childhood obesity and early puberty. Despite this, the mechanism by which adiposity affects puberty initiation is unclear. Furthermore, the role of cholesterol has not been considered, even though adipose tissue is a major cholesterol storage depot, especially in obesity. Sterols such as cholesterol have membrane-structural functions but also play important signaling roles, and sterols are required as substrates for steroid-hormone production. Insects, including Drosophila, have lost the ability to synthesize sterols de novo and thus must acquire them through feeding. Mammals are cholesterol prototrophs, but most intracellular cholesterol still comes from low-density-lipoprotein (LDL)-mediated cellular uptake of dietary cholesterol. In both taxa, consumed sterols are transported in the circulatory system bound within lipoprotein particles (LPPs such as mammalian LDL/HDL), and target tissues take them up through a variety of mechanisms including receptor-mediated endocytosis. LPP-bound sterols are extracted in the lysosome and inserted into the lysosomal membrane by membrane-integral transport proteins. The primary such protein, Niemann-Pick-type-C 1 (Npc1), underlies the Niemann-Pick type C lysosomal storage disorder; without Npc1 function, cholesterol accumulates in endosomal-lysosomal compartments, leading to increased intracellular cholesterol signaling. Thus, Npc1 seems to be part of a mechanism by which cells regulate cholesterol signaling (Texada, 2022).
This study set out to determine whether, and the routes by which, cholesterol might regulate Drosophila larval growth. The findings show that dietary cholesterol dose-responsively promotes growth and accelerates development by increasing insulin signaling. Cholesterol sensing is mediated by the target of rapamycin (TOR) pathway in the cells of the fat body and the glia of the BBB, which remotely induce the expression and release of ILPs from the IPCs. Enhancing cholesterol signaling in the PG also promotes TOR activity, drives endoreduplication, and leads to premature attainment of the CW checkpoint. Thus, dietary cholesterol accelerates growth through insulin signaling and leads to early maturation through effects on steroidogenesis, effects which are mediated by promoting TOR activity in sensing tissues (Texada, 2022).
Nutrition is one of the most important influences on developmental growth and maturation. Malnutrition or disease can impair growth and delay puberty, whereas obese children enter puberty early. Similarly, Drosophila larvae exposed to poor nutrition, tissue damage, or inflammation delay their development, whereas rich conditions promote rapid growth and maturation. These environmental factors are coupled to the appropriate gating of steroid production via internal checkpoints, one of which is a nutrition-dependent CW required to initiate the maturation process. This suggests that signals reflecting nutritional state and body-fat storage play a key role in activating the neuroendocrine pathways that trigger puberty. Although studies suggest that the adipokine leptin may be involved, the mechanisms linking body fat to puberty are poorly defined, and the potential involvement of lifestyles associated with excessive accumulation of cholesterol, one of the most important lipids, has not been considered. In humans, white adipose tissue is the main site of cholesterol storage and can contain over half the body's total cholesterol in obesity. The results show that dietary cholesterol intake promotes systemic body growth through insulin-dependent pathways and that animals raised on high dietary cholesterol initiate maturation earlier. Cholesterol is sensed through an Npc1-regulated TOR-mediated mechanism in the fat body and the glial cells of the BBB, which relay information to the IPCs within the brain to promote insulin expression and release, thus coupling growth and maturation with cholesterol status (Texada, 2022).
Insect CW likely evolved as a mechanism ensuring that maturation will not occur unless the animal has accumulated adequate nutrient stores to survive the nonfeeding metamorphosis period and has completed sufficient growth to produce an adult of proper size and thus of maximal fitness. Likewise, the link between body fat and maturation in humans probably ensures adequate stores of fat before maturation onset to support pregnancy and reproductive success. In Drosophila, insulin signaling plays a critical role in coordinating steroidogenesis with nutritional conditions. Insulin acts upon the PG and induces a small ecdysone peak early in L3 that is correlated with CW attainment. In combination with nutrition-related signaling mediated via insulin, nutrient availability is also assessed directly in the PG and is coupled to irreversible endoreduplication that permits ecdysone production at CW. the current findings show that accumulation of cholesterol in the PG, induced by the loss of Npc1a, drives a remarkable TOR-dependent increase in endoreduplication and leads to inappropriate attainment of CW. Taken together, These findings indicate that cholesterol sensed by the BBB glia and the fat body promotes growth through insulin signaling and that cholesterol sensed by the PG accelerates maturation through ecdysone signaling (Texada, 2022).
Loss of Npc1 function in humans leads to the Niemann-Pick lysosomal storage disorder, marked by intracellular cholesterol accumulation. Although neurodegeneration is the hallmark of NPC disease, including in a Drosophila model, alterations in glial, adipose, hepatic, and endocrine systems are also components of NPC syndrome. In humans, Npc1 itself is strongly expressed in glia and in adipose tissues, especially in obese individuals, and variants in Npc1 are associated with obesity, type-2 diabetes, and hepatic lipid dysfunction. These findings link glial and adipose-tissue cholesterol sensing through Npc1 to systemic growth and metabolic control through effects on insulin signaling. It was also found that intracellular cholesterol accumulation driven by Npc1 loss leads to hyperactivation of TOR that drives increases in DNA replication and cell growth. TOR activity is frequently upregulated in cancer, and the results therefore provide mechanistic insight for understanding the emerging link between cholesterol and a range of cancers (Texada, 2022).
As the coupling of nutrition with growth and maturation is ancient and highly conserved, this work provides a foundation for understanding how cholesterol is coupled to developmental growth and maturation initiation in humans. These findings link a high concentration of this particular lipid in adipose tissues to the neuroendocrine initiation of maturation, which may explain the critical link between obesity (body fat) and early puberty (Texada, 2022).
Glia are crucial for the normal development and functioning of the nervous system in many animals. Insects are widely used for studies of glia genetics and physiology. Drosophila melanogaster surface glia (perineurial and subperineurial) form a blood-brain barrier in the central nervous system and blood-nerve barrier in the peripheral nervous system. Under the subperineurial glia layer, in the cortical region of the central nervous system, cortex glia encapsulate neuronal cell bodies, whilst in the peripheral nervous system, wrapping glia ensheath axons of peripheral nerves. This study shows that the expression of the evolutionarily conserved swiss cheese gene is important in several types of glia. swiss cheese knockdown in subperineurial glia leads to morphological abnormalities of these cells. The number of subperineurial glia nuclei is reduced under swiss cheese knockdown, possibly due to apoptosis. In addition, the downregulation of swiss cheese in wrapping glia causes a loss of its integrity. Transcriptome changes were revealed under swiss cheese knockdown in subperineurial glia and in cortex + wrapping glia; the downregulation of swiss cheese in these types of glia provokes reactive oxygen species acceleration. These results are accompanied by a decline in animal mobility measured by the negative geotaxis performance assay (Ryabova, 2021).
The adaptable transcriptional response to changes in food availability not only ensures animal survival, but also lets progressing with embryonic development. Interestingly, the central nervous system is preferentially protected to periods of malnutrition, a phenomenon known as 'brain sparing'. However, the mechanisms that mediates this response remains poorly understood. To get a better understanding of this, Drosophila melanogaster was used as a model, analysing the transcriptional response of neural stem cells (neuroblasts) and glia of the blood-brain barrier (BBB), from larvae of both sexes, during nutrient restriction using targeted DamID. Differentially expressed genes were found in both neuroblasts and glia of the BBB, although the effect of nutrient deficiency was primarily observed in the BBB. The function of a nutritional sensitive gene expressed in the BBB, the serine protease homologue, scarface (scaf), was characterized. Scaf is expressed in subperineurial glia in the BBB in response to nutrition. Tissue-specific knockdown of scaf increases subperineurial glia endoreplication and proliferation of perineurial glia in the blood-brain barrier. Furthermore, neuroblast proliferation is diminished upon scaf knockdown in subperineurial glia. Interestingly, re-expression of Scaf in subperineurial glia is able to enhance neuroblast proliferation and brain growth of animals in starvation. Finally, this study shows that loss of scaf in the blood-brain barrier increases the sensitivity to drugs in adulthood suggesting a physiological impairment. It is proposed that Scaf integrates the nutrient status to modulate the balance between neurogenesis and growth of the BBB, preserving the proper equilibrium between the size of the barrier and the brain (Contreras, 2021).
The blood brain barrier (BBB) forms a stringent barrier that protects the brain from components in the circulation that could interfere with neuronal function. At the same time, the BBB enables selective transport of critical nutrients and other chemicals to the brain. Beyond these functions, another recently recognized function is even less characterized, specifically the role of the BBB in modulating behavior by affecting neuronal function in a sex-dependent manner. Notably, signaling in the adult Drosophila BBB is required for normal male courtship behavior. Courtship regulation also relies on male-specific molecules in the BBB. Previous studies have demonstrated that adult feminization of these cells in males significantly lowered courtship. In this study microarray analysis was carried out of BBB cells isolated from males and females. Findings revealed that these cells contain male- and female-enriched transcripts, respectively. Among these transcripts, nuclear receptor Hr46/Hr3 was identified as a male-enriched BBB transcript. Hr46/Hr3 is best known for its essential roles in the ecdysone response during development and metamorphosis. This study demonstrated that Hr46/Hr3 is specifically required in the BBB cells for courtship behavior in mature males. The protein is localized in the nuclei of sub-perineurial glial cells (SPG), indicating that it might act as a transcriptional regulator. These data provide a catalogue of sexually dimorphic BBB transcripts and demonstrate a physiological adult role for the nuclear receptor Hr46/Hr3 in the regulation of male courtship, a novel function that is independent of its developmental role (Lama, 2022).
The blood-brain barrier (BBB) of Drosophila comprises a thin epithelial layer of subperineural glia (SPG), which ensheath the nerve cord and insulate it against the potassium-rich hemolymph by forming intercellular septate junctions (SJs). Previous work identified a novel Gi/Go protein-coupled receptor (GPCR), Moody, as a key factor in BBB formation at the embryonic stage. However, the molecular and cellular mechanisms of Moody signaling in BBB formation and maturation remain unclear. This study identified cAMP-dependent protein kinase A (PKA) as a crucial antagonistic Moody effector that is required for the formation, as well as for the continued SPG growth and BBB maintenance in the larva and adult stage. PKA is enriched at the basal side of the SPG cell, and this polarized activity of the Moody/PKA pathway finely tunes the enormous cell growth and BBB integrity. Moody/PKA signaling precisely regulates the actomyosin contractility, vesicle trafficking, and the proper SJ organization in a highly coordinated spatiotemporal manner. These effects are mediated in part by PKA's molecular targets MLCK and Rho1. Moreover, 3D reconstruction of SJ ultrastructure demonstrates that the continuity of individual SJ segments, and not their total length, is crucial for generating a proper paracellular seal. It is proposed that Moody/PKA signaling plays a central role in controlling the cell growth and maintaining BBB integrity (Li, 2021).
Previous studies implicated a novel GPCR signaling pathway in the formation of the Drosophila BBB in late embryos. This work also revealed that besides the GPCR Moody two heterotrimeric G proteins (Gαiβγ, Gαoβγ5) and the RGS Loco participate in this pathway. This study provides a comprehensive molecular and cellular analysis of the events downstream of G protein signaling using a candidate gene screening approach. New, more sensitive methods for phenotypic characterization are presented, and the analysis was extended to beyond the embryo into larval stages. This work identifies, together with some of its targets, as crucial antagonistic effectors in the continued cell growth of SPG and maintenance of the BBB sealing capacity. This role is critical to ensure proper neuronal function during BBB formation and maturation (Li, 2021).
Multiple lines of evidence demonstrate a role of PKA for proper sealing of the BBB: loss of PKA activity leads to BBB permeability defects, irregular growth of SPG during epithelium formation, reduced membrane overlap, and a narrower SJ belt at SPG cell-cell contacts. The role of PKA as an effector of the Moody signaling pathway is further supported by dominant genetic interaction experiments, which show that the dye penetration phenotype of PkaC1 heterozygous mutant embryos was partially rescued by removing one genomic copy of Gβ13F or loco. Moreover, the analysis of the larval phenotype with live SJ and cytoskeleton markers shows that PKA gain of function behaved similarly to Moody loss of function. Conversely, PKA loss of function resembled the overexpression of GαoGTP, which mimics Moody gain-of-function signaling (Li, 2021).
The results from modulating PKA activity suggest that the total cell contact and SJ areas are a major function of PKA activity: low levels of activity cause narrow contacts, and high levels give rise to broad contacts. Moreover, the analysis of various cellular markers (actin, microtubules, SJs, vesicles) indicates that the circumferential cytoskeleton and delivery of SJ components respond proportionately to PKA activity. This, in turn, promotes the changes in cell contact and junction areas coordinately at the lateral side of SPG. These experiments demonstrate that the modulation of the SPG membrane overlap by PKA proceeds, at least in part, through the regulation of actomyosin contractility, and that this involves the phosphorylation targets MLCK and Rho1. This suggests that crucial characteristics of PKA signaling are conserved across eukaryotic organisms (Li, 2021).
At the ultrastructural level, ssTEM analysis of the larval SPG epithelium clarifies the relationship between the inter-cell membrane overlaps and SJ organization and function. Across different PKA activity levels, the ratio of SJ areas to the total cell contact area remained constant at about 30%. This proportionality suggests a mechanism that couples cell contact with SJ formation. The primary role of Moody/PKA appears in this process to be the control of membrane contacting area between neighboring cells. This is consistent with the results of a temporal analysis of epithelium formation and SJ insertion in late embryos of WT and Moody pathway mutants, which shows that membrane contact precedes and is necessary for the appearance of SJs. The finding that the surface area that SJs occupy did not exceed a specific ratio, irrespective of the absolute area of cell contact, suggests an intrinsic, possibly steric limitation in how much junction can be fitted into a given cell contact space. While most phenotypic effects are indeed a major function of Moody and PKA activity, the discontinuity and shortening of individual SJ strands is not. It occurred with both increased and decreased signaling and appears to cause the leakiness of the BBB in both conditions. ssTEM-based 3D reconstruction thus demonstrates that the total area covered by SJs and the length of individual contiguous SJ segments are independent parameters. The latter appears to be critical for the paracellular seal, consistent with the idea that Moody plays a role in the formation of continuous SJ stands (Li, 2021).
The asymmetric localization of PKA that was observed sheds further light on the establishment and function of apical-basal polarity in the SPG epithelium. Prior to epithelium formation, contact with the basal lamina leads to the first sign of polarity. Moody becomes localized to the apical surface only after epithelial closure and SJ formation, suggesting that SJs are required as a diffusion barrier and that apical accumulation of Moody protein is the result of polarized exocytosis or endocytosis. This study now shows that the intracellular protein PKA catalytic subunit-PkaC1 accumulates on the basal side of SPG, and that this polarized accumulation requires (apical) Moody activity. Such an asymmetric, activity-dependent localization has not previously been described for PKA in endothelium, and while the underlying molecular mechanism is unknown, the finding underscores that generating polarized activity along the apical-basal axis of the SPG is a key element of Moody pathway function (Li, 2021).
An intriguing unresolved question is how increased SPG cell size and SJ length can keep up with the expanding brain without disrupting the BBB integrity during larva growth. The SJ was found to grow dramatically in length (0.57 ± 0.07 μm vs. 2.16 ± 0.14 μm, about 3.7-fold) from the late embryo to third instar larva, which matches the increased cell size of SPG (about fourfold). During the establishment of the SPG epithelium in the embryo, both increased and decreased Moody signaling resulted in asynchronous growth and cell contact formation along the circumference of SPG, which in turn led to irregular thickness of the SJ belt. Therefore, a similar relationship may exist during the continued growth of the SPG epithelium in larvae, with the loss of continuity of SJ segments in Moody/PKA mutants resulting from unsynchronized expansion of the cell contact area and an ensuing erratic insertion of SJ components. Since SJs form relatively static complexes, any irregularities in their delivery and insertion may linger for extended periods of time. The idea that shortened SJ segments are a secondary consequence of unsynchronized cell growth is strongly supported by the finding that disruption of actomyosin contractility in MLCK and Rho1 mutants compromises BBB permeability (Li, 2021).
Collectively, these data suggest the following model: polarized Moody/PKA signaling controls the cell growth and maintains BBB integrity during the continuous morphogenesis of the SPG secondary epithelium. On the apical side, Moody activity represses PKA activity (restricting local cAMP level within the apial-basal axis in SPG) and thereby promotes actomyosin contractility. On the basal side, which first adheres to the basal lamina and later to the PNG sheath, PKA activity suppresses actomyosin contractility via MLCK and Rho1 phosphorylation and repression. Throughout development, the SPG grow continuously while extending both their cell surface and expanding their cell contacts. The data suggest that the membrane extension occurs on the basolateral surface through insertion of plasma membrane and cell-adhesive proteins, with similar behavior in epithelial cell, but regulated by a distinct polarized Moody/PKA signaling in SPG. In analogy to motile cells, the basal side of the SPG would thus act as the 'leading edge' of the cell, while the apical side functions as the 'contractile rear'. According to this model, Moody/Rho1 regulate actomyosin to generate the contractile forces at the apical side to driving membrane contraction, which directs the basolateral insertion of new membrane material and SJs. In this way, differential contractility and membrane insertion act as a conveyor belt to move new formed membrane contacts and SJ from the basolateral to apical side. Loss of Moody signaling leads to symmetrical localization of PKA and to larger cell contact areas between SPG due to diminished apical constriction. Conversely, loss of PKA causes smaller cell contact areas due to increased basal constriction (Li, 2021).
These results may have important implications for the neuron-glia interaction in the nervous system and the development and maintenance of the BBB in vertebrates. SJs have several structural and functional components in common with paranodal junctions, which join myelinating glial cells to axons in the vertebrate nervous system, and they share similar regulation mechanisms. The vertebrate BBB consists of a secondary epithelium with interdigitations similar to the ones between the Drosophila SPG. While the sealing is performed by TJs, it will be interesting to investigate whether there are similarities in the underlying molecular and cellular mechanisms that mediate BBB function (Li, 2021).
Temporal patterning of neural progenitors leads to the sequential production of diverse neurons. To understand how extrinsic cues influence intrinsic temporal programs, Drosophila mushroom body progenitors (neuroblasts) were studied that sequentially produce only three neuronal types: γ, then α'β', followed by αβ. Opposing gradients of two RNA-binding proteins Imp and Syp comprise the intrinsic temporal program. Extrinsic activin signaling regulates the production of α'β' neurons but whether it affects the intrinsic temporal program was not known. This study shows that the activin ligand Myoglianin from glia regulates the temporal factor Imp in mushroom body neuroblasts. Neuroblasts missing the activin receptor Baboon have a delayed intrinsic program as Imp is higher than normal during the α'β' temporal window, causing the loss of α'β' neurons, a decrease in αβ neurons, and a likely increase in γ neurons, without affecting the overall number of neurons produced. These results illustrate that an extrinsic cue modifies an intrinsic temporal program to increase neuronal diversity (Rossi, 2020).
The building of intricate neural networks during development is controlled by highly coordinated patterning programs that regulate the generation of different neuronal types in the correct number, place and time. The sequential production of different neuronal types from individual progenitors, i.e. temporal patterning, is a conserved feature of neurogenesis. For instance, individual radial glia progenitors in the vertebrate cortex sequentially give rise to neurons that occupy the different cortical layers in an inside-out manner. In Drosophila, neural progenitors (called neuroblasts) also give rise to different neuronal types sequentially. For example, projection neurons in the antennal lobe are born in a stereotyped temporal order and innervate specific glomeruli. In both of these examples, individual progenitors age concomitantly with the developing animal (e.g., from embryonic stages 11-17 in mouse and from the first larval stage (L1) to the end of the final larva stage (L3) in Drosophila). Thus, these progenitors are exposed to changing environments that could alter their neuronal output. Indeed, classic heterochronic transplantation experiments demonstrated that young cortical progenitors placed in an old host environment alter their output to match the host environment and produce upper-layer neurons (Rossi, 2020).
The adult Drosophila central brain is built from ~100 neuroblasts that divide continuously from L1 to L3. Each asymmetric division regenerates the neuroblast and produces an intermediate progenitor called ganglion mother cell (GMC) that divides only once, typically producing two different cell types. Thus, during larval life central brain neuroblasts divide 50-60 times, sequentially producing many different neuronal types. All central brain neuroblasts progress through opposing temporal gradients of two RNA-binding proteins as they age: IGF-II mRNA binding protein (Imp) when they are young and Syncrip (Syp) when they are old. Loss of Imp or Syp in antennal lobe or Type II neuroblasts affects the ratio of young to old neuronal types. Imp and Syp also affect neuroblast lifespan. Thus, a single temporal program can affect both the diversity of neuronal types produced and their numbers (Rossi, 2020).
Since central brain neuroblasts produce different neuronal types through developmental time, roles for extrinsic cues have recently garnered attention. Ecdysone triggers all the major developmental transitions including progression into the different larval stages and entry in pupation. The majority of central brain neuroblasts are not responsive to ecdysone until mid-larval life when they begin to express the Ecdysone Receptor (EcR). Expressing a dominant-negative version of EcR (EcR-DN) in Type II neuroblasts delays the Imp to Syp transition that normally occurs ~60 hr after larval hatching (ALH). This leads to many more cells that express the early-born marker gene Repo and fewer cells that express the late-born marker gene Bsh (Rossi, 2020).
To further understand how extrinsic signals contribute to temporal patterning, Drosophila mushroom body neuroblasts were studied because of the deep understanding of their development. The mushroom body is comprised of ~2000 neurons (Kenyon cells) that belong to only three main neuronal types that have unique morphologies and play distinct roles in learning and memory. They receive input mainly from ~200 projection neurons that each relays odor information from olfactory receptor neurons. Each projection neuron connects to a random subset of Kenyon cells and each Kenyon cell receives input from ~7 different projection neurons. This connectivity pattern requires a large number of mushroom body neurons (~2,000) to represent complex odors. To produce this very large number of neurons, mushroom body development is unique in many respects. Mushroom body neurons are born from four identical neuroblasts that divide continuously (unlike any other neuroblast) from the late embryonic stages until the end of pupation (~9 days for ~250 divisions each). Furthermore, the two neurons born from each mushroom body GMC are identical. The neuronal simplicity of the adult mushroom body makes it ideal to study how extrinsic cues might affect diversity since the loss of any single neuronal type is obvious given that each is represented hundreds of times (Rossi, 2020).
The three main neuronal types that make up the adult mushroom body are produced sequentially during neurogenesis: first γ, followed by α'β', and then αβ neurons (see α'β' neurons are not generated from babo mutant neuroblasts), representing the simplest lineage in the central brain. The γ temporal window extends from L1 (the first larval stage) until mid-L3 (the final larval stage) when animals attain critical weight and are committed to metamorphosis; the α'β' window from mid-L3 to the beginning of pupation, and the αβ window from pupation until eclosion (the end of development). Like all other central brain neuroblasts Imp and Syp are expressed by mushroom body neuroblasts, but in much shallower gradients through time, which accounts for their extended lifespan. Imp and Syp are inherited by newborn neurons where they instruct temporal identity. Imp positively and Syp negatively regulate the translation of chronologically inappropriate morphogenesis (chinmo), a gene encoding a transcription factor that acts as a temporal morphogen in neurons. The first-born γ neurons are produced for the first ~85 cell divisions, when Imp levels in neuroblasts, and thus Chinmo in neurons, are high. α'β' neurons are produced for the next ~40 divisions, when Imp and Syp are at similar low levels that translate into lower Chinmo levels in neurons. Low Chinmo then regulates the expression in neurons of maternal gene required for meiosis (mamo), which encodes a transcription factor that specifies the α'β' fate and whose mRNA is stabilized by Syp. αβ neurons are generated for the final ~125 neuroblast divisions, when Syp levels are high, Imp is absent in neuroblasts, and thus Chinmo and Mamo are no longer expressed in neurons (Rossi, 2020).
Extrinsic cues are known to have important roles in regulating neuronal differentiation during mushroom body neurogenesis. The ecdysone peak that controls entry into pupation regulates γ neuron axonal remodeling. Ecdysone was also proposed to be required for the final differentiation of α'β' neurons. EcR expression in γ neurons is timed by activin signaling, a member of the TGFβ family, from local glia. Activin signaling from glia is also required for the α'β' fate (Marchetti, 2019): Knocking-down the activin pathway receptor Baboon (Babo) leads to the loss of α'β' neurons. It was proposed that activin signaling in mushroom body neuroblasts regulates the expression of EcR in prospective α'β' neurons and that when the activin pathway is inhibited, it leads to the transformation of α'β' neurons into later-born pioneer-αβ neurons (a subclass of the αβ class) (Marchetti and Tavosanis, 2019) (Rossi, 2020).
Although there is strong evidence that extrinsic cues have important functions in neuronal patterning in the Drosophila central brain, it remains unknown how extrinsic temporal cues interface with the Imp and Syp intrinsic temporal program to regulate neuronal specification. This question was addressed using the developing mushroom bodies. Activin signaling from glia was shown to be required for α'β' specification. However, this study also showed that activin signaling lowers the levels of the intrinsic factor Imp in mushroom body neuroblasts to define the mid-α'β' temporal identity window. Removing the activin receptor Babo in mutant clones leads to the loss of α'β' neurons, to fewer last-born αβ neurons, and to the likely generation of additional first-born γ neurons without affecting overall clone size. This appears to be caused by a delayed decrease in Imp levels, although the intrinsic temporal clock still progresses even in the absence of activin signaling. This study also demonstrated that ecdysone signaling is not necessary for the specification of α'β' neurons, although it might still be involved in later α'β' differentiation. These results provide a model for how intrinsic and extrinsic temporal programs operate within individual progenitors to regulate neuronal specification (Rossi, 2020).
Mushroom body neurogenesis is unique and programmed to generate many copies of a few neuronal types. During the early stages of mushroom body development, high Imp levels in mushroom body neuroblasts are inherited by newborn neurons and translated into high Chinmo levels to specify γ identity. As in other central brain neuroblasts, as development proceeds, inhibitory interactions between Imp and Syp help create a slow decrease of Imp and a corresponding increase of Syp. However, at the end of the γ temporal window (mid-L3), activin signaling from glia acts to rapidly reduce Imp levels in mushroom body neuroblasts without significantly affecting Syp, establishing a period of low Imp (and thus low Chinmo in neurons) and also low Syp. This is required for activating effector genes in prospective α'β' neurons, including Mamo, whose translation is promoted by Syp (Liu, 2019). The production of αβ identity begins when Imp is further decreased and Syp levels are high during pupation (see Model of how activin signaling defines the α'β' temporal identity window.). Low Chinmo in αβ neurons is also partly regulated by ecdysone signaling through the activation of Let-7-C, which targets chinmo for degradation. Based on this model, α'β' neurons could not be rescued by knocking-down Imp in babo clones, since low Imp is required for α'β' specification while the knockdown reduces its level below this requirement. It would be expected to rescue α'β' neurons if Imp levels were specifically reduced to the appropriate levels at L3. However, reducing Imp levels might not be the only function of activin signaling, which may explain why α'β' neurons are not simply made earlier (e.g., during L1-L2) when Imp is knocked-down (Rossi, 2020).
In babo mutant clones, it is speculated that additional γ neurons are produced at the expense of α'β' neurons since Imp levels in neuroblasts (as well as Chinmo in neurons) are higher for a longer time during development; There was also a significant decrease in the total number of αβ neurons in babo mutant clones that contrasts with a recent report by Marchetti (2019) that instead concluded that additional pioneer-αβ neurons are produced. It is believed that there is both an increase in the number of γ neurons and of the pioneer-αβ neuron subclass because pioneer-αβ neurons are the first of the αβ class to be specified (when Imp is still present at very low levels) during pupation. It is speculated that pioneer-αβ neurons are produced during the extended low Imp window that was detected during pupation in babo clones. However, this does not leave the time for the remaining population of αβ neurons to be formed, which explains why their number is reduced (Rossi, 2020).
This study has focused on the three main classes of mushroom body neurons although at least seven subtypes exist: 2 γ, 2 α'β' and 3 αβ. The subtypes are specified sequentially suggesting that each of the three broad mushroom body temporal windows can be subdivided further, either by fine-scale reading of the changing Imp and Syp gradients, by additional extrinsic cues, or perhaps by a tTF series as in other neuroblasts (Rossi, 2020).
Postembryonic central brain neuroblasts are long-lived and divide on average ~50 times. Unlike in other regions of the developing Drosophila brain, rapidly progressing series of tTFs have not yet been described in these neuroblasts. Instead, they express Imp and Syp in opposing temporal gradients. Conceptually, how Imp and Syp gradients translate into different neuronal identities through time has been compared to how morphogen gradients pattern tissues in space. During patterning of the anterior-posterior axis of the Drosophila embryo, the anterior gradient of the Bicoid morphogen and the posterior Nanos gradient are converted into discrete spatial domains that define cell fates. Since gradients contain unlimited information, differences in Imp and Syp levels through time could translate into different neuronal types. Another intriguing possibility is that tTF series could act downstream of Imp and Syp, similarly to how the gap genes in the Drosophila embryo act downstream of the anterior-posterior morphogens. This study has shown that another possibility is that temporal extrinsic cues can be incorporated by individual progenitors to increase neuronal diversity. In mushroom body neuroblasts activin signaling acts directly on the intrinsic program, effectively converting two broad temporal windows into three to help define an additional neuronal type. It is proposed that subdividing the broad Imp and Syp temporal windows by extrinsic cues may be a simple way to increase neuronal diversity in other central brain neuroblasts (Rossi, 2020).
This study has also shown that activin signaling times the Imp to Syp transition for mushroom body neuroblasts, similar to the function of ecdysone for other central brain neuroblasts. In both cases however, the switch still occurs, indicating that a separate independent clock continues to tick. This role for extrinsic cues during Drosophila neurogenesis is reminiscent of their roles on individual vertebrate progenitors. For example, hindbrain neural stem cells progressively produce motor neurons followed by serotonergic neurons before switching to producing glia. The motor neuron to serotonergic neuron switch is fine-tuned by TGFβ signaling. It would be interesting to determine if hindbrain neuronal subtypes are lost in TGFβ mutants, similar to how α'β' identity is lost in the mushroom bodies in babo mutants (Rossi, 2020).
The specification of α'β' neurons begins at mid-L3 with the onset of Mamo expression. In contrast, high levels of EcR are detected in mature mushroom body neurons starting at late L3. At this stage, both γ and α'β' neurons already exist and new α'β' neurons are still being generated. Thus, Mamo expression precedes EcR expression. These non-overlapping expression patterns suggest that ecdysone signaling does not regulate Mamo and therefore cannot control the specification of α'β' neurons. Furthermore, expression of UAS-EcR-RNAi or mutants for usp do not lead to the loss of α'β' neurons. It is noted that usp results contradict the loss of α'β' neuron reported by Marchetti (2017) in usp clones. However, α'β' neurons were seen in these clones based on the morphology of these neurons but the remodeling defect of γ neurons makes α'β' neurons difficult to identify. Nevertheless, ecdysone might still function later during α'β' differentiation, particularly during pupation when all mushroom body neurons express EcR (Rossi, 2020).
This study and that of Marchetti both show that expression of UAS-EcR-DN leads to the loss of α'β' neurons by acting in mushroom body neurons but not in neuroblasts. However, EcR must be first be expressed in the target cells of interest in order to make any conclusions about ecdysone function using UAS-EcR-DN. Since this study could not detect EcR protein in Mamo+ cells at L3, but expressing UAS-EcR-DN inhibits Mamo in those cells, it is concluded that EcR-DN artifactually represses Mamo and leads to the loss of α'β' neurons. This explains why expressing UAS-EcR-B1 does not rescue α'β' neurons in babo clones. However, Marchetti did rescue babo-RNAi by expressing EcR (Marchetti, 2019). This is likely because the current experiments were performed using babo MARCM clones in which the loss of α'β' neurons is much more severe than with babo-RNAi used in their experiments. Indeed, when attempts were made to eliminate α'β' neurons using a validated UAS-babo-RNAi construct, γ neurons did not remodel but there was only a minor (but significant) decrease in the number of α'β' neurons. This indicates that knocking-down babo with mb-Gal4 that is only weakly expressed in neuroblasts and newborn neurons is not strong enough to inhibit α'β' specification. Thus, it is speculated that the LexA line used by Marchetti (GMR26E01-LexA) may not be a reliable reporter for α'β' neurons upon babo knockdown, and that it might be ecdysone sensitive later in α'β' differentiation. Since EcR expression in all mushroom body neurons at L3 may be dependent on activin signaling directly in neurons, as it is in γ neurons for remodeling, expressing UAS-EcR-B1 together with UAS-babo-RNAi using OK107-Gal4 might both reduce the effectiveness of the RNAi while also allowing for the re-expression of GMR26E01-LexA (Rossi, 2020).
Glia are a source of the activin ligand myo, which is temporally expressed in brain glia starting at L3 to initiate the remodeling of mushroom body γ neurons (Awasaki, 2011) and α'β' specification (this study and Marchetti, 2019). However, knocking-down Myo from glia is not as severe as removing Babo from mushroom body neuroblasts. This might be due to incomplete knockdown of myo or to other sources of Myo, potentially from neurons. For example, in the vertebrate cortex, old neurons signal back to young neurons to control their numbers. It is also possible the Babo is activated by other activin ligands, including Activin and Dawdle. An intriguing hypothesis is that the temporal expression of myo in glia beginning at mid-L3 is induced by the attainment of critical weight and rising ecdysone levels. It would be interesting to determine whether blocking ecdysone signaling in glia leads to the loss of α'β' specification, similar to how blocking ecdysone reception in astrocytes prevents γ neuron remodeling (Rossi, 2020).
It is well established that extrinsic cues play important roles during vertebrate neurogenesis, either by regulating temporal competence of neural stem cells or by controlling the timing of temporal identity transitions. Competence changes mediated by extrinsic cues were demonstrated in classic heterochronic transplantation studies that showed that young donor progenitors produce old neuronal types when placed in older host brains. Recent studies show that the reverse is also true when old progenitors are placed in a young environment (Rossi, 2020).
Mechanisms of intrinsic temporal patterning are also conserved. For example, vertebrate retinal progenitor cells use an intrinsic tTF cascade to bias young, middle, and old retinal fates. Two of the factors (Ikaros and Casz1) used for intrinsic temporal patterning are orthologs to the Drosophila tTFs Hb and Cas. tTF series might also exist in cortical radial glia progenitors and even in the spinal cord. Recent results also show the importance of post-transcriptional regulation in defining either young or old cortical fates, which can be compared to the use of post-transcriptional regulators that are a hallmark of neuronal temporal patterning in Drosophila central brain neuroblasts. These studies highlight that the mechanisms driving the diversification of neuronal types are conserved (Rossi, 2020).
Discoidin domain receptor regulates ensheathment, survival, and caliber of peripheral axons
Most invertebrate axons and small caliber axons in mammalian peripheral nerves are unmyelinated but still ensheathed by glia. This study used Drosophila wrapping glia to study the development and function of non-myelinating axon ensheathment, which is poorly understood. Selective ablation of these glia from peripheral nerves severely impaired larval locomotor behavior. In an in vivo RNAi screen to identify glial genes required for axon ensheathment, the conserved receptor tyrosine kinase Discoidin domain receptor (Ddr) was identified. In larval peripheral nerves, loss of Ddr resulted in severely reduced ensheathment of axons and reduced axon caliber, and a strong dominant genetic interaction was found between Ddr and the type XV/XVIII collagen Multiplexin (Mp), suggesting Ddr functions as a collagen receptor to drive axon wrapping. In adult nerves, loss of Ddr decreased long-term survival of sensory neurons and significantly reduced axon caliber without overtly affecting ensheathment. These data establish essential roles for non-myelinating glia in nerve development, maintenance, and function, and identify Ddr as a key regulator of axon-glia interactions during ensheathment and establishment of axon caliber (Corty, 2022).
Non-myelinating ensheathment of axons is a conserved but understudied feature of the PNS. Although this type of multi-axonal ensheathment has been less studied compared with myelination, a growing body of evidence indicates it is important for the health and function of neurons and axons in the periphery. For example, Schwann cell-specific loss of the transmembrane receptor LDL receptor related protein-1 (LRP1) causes both thin myelin and abnormal Remak bundle structure. These conditional knockout animals also showed a lowered pain threshold, suggesting that the physiology of nociceptor neurons is impaired when Remak ensheathment is disrupted. Disrupting metabolism in Schwann cells causes progressive axon loss, with small unmyelinated fibers dying first, before myelinated fibers begin to show signs of degeneration. In the fly, disruption of axonal wrapping leads to uncoordinated behavioral responses that hint at aberrant ephaptic coupling between neighboring axons in nerves when not properly separated (Kottmeier, 2020). Such coupling could cause the inappropriate activation of sensory or nociceptive neurons underlying peripheral neuropathies. Previous studies lab have shown that wrapping glia are required to clear neuronal debris after nerve injury and mediate injury signaling between injured and intact 'bystander' neurons, which might be important for functional recovery after nerve trauma. These and other findings suggest that Remak-type ensheathment and axon-glia signaling of unmyelinated fibers play a variety of underappreciated roles in peripheral nerve physiology that contribute to the pathophysiology of a number of PNS disorders, including debilitating peripheral neuropathies and responses to nerve injury (Corty, 2022).
To gain insight into non-myelinating ensheathment, the Drosophila peripheral nerves were used to identify a molecular pathway important for the development and function multi-axonal ensheathment. A new Split-Gal4 intersectional driver was generated to target wrapping glia more specifically for functional and behavioral studies in order to improve understanding of whether and how wrapping glia support axon health, physiology and, ultimately, circuit function. Finally, this study uncovered roles for glia in mediating long-term neuronal survival and driving increased axon caliber that are separable from overt effects on wrapping, demonstrating that non-myelinating ensheathing glia perform crucial, previously unappreciated, roles in nervous system development, maintenance and function (Corty, 2022).
A main advantage of Drosophila is the ability to conduct large-scale in vivo screens. Use was made of available UAS-RNAi libraries to carry out a broad screen for regulators of axonal ensheathment in intact nerves. This morphological screen was sensitive enough to identify genes previously implicated in wrapping glia development, including vn, LanB1 and mys, validating the approach. Moreover, in the case of Ddr, it was possible to identify an important regulator of ensheathment that a simple behavioral or lethality screen would have missed in light of follow-up behavioral testing. Knockdown of Ddr in wrapping glia resulted in reduced glial membrane coverage in nerve cross-sections by fluorescence microscopy. Similar phenotypes were observed in Ddr loss-of-function animals and could be rescued by resupplying Ddr specifically in wrapping glia, confirming the specificity of the RNAi results. TEM clearly showed that reduced glial membrane coverage at the light level corresponds to decreased axon wrapping (Corty, 2022).
Although neither of the vertebrate homologs, Ddr1 and Ddr2, has been explicitly implicated in glial development, several lines of evidence suggests that Ddr1 may have a conserved role in vertebrate glial development or function. Ddr1 is highly expressed in the mouse oligodendrocyte lineage starting from when the cells begin to associate with axons, is upregulated in newly formed oligodendrocytes after cuprizone treatment, and is expressed in both myelinating and Remak Schwann cells. Moreover, DDR1 is expressed in human oligodendrocytes and myelin, and variants in the human gene have been correlated with abnormal white matter and schizophrenia (Corty, 2022).
Vertebrate Ddr1 and Ddr2 are potently activated by collagens in vitro, prompting an investigation of whether collagens were involved with Ddr function in fly nerves. Knockdown of the Drosophila collagen Mp specifically in wrapping glia but not in neurons was found to disrupted ensheathment. Together with the established roles for vertebrate Ddr1 and Ddr2 as collagen receptors, the strong genetic interaction observed between Ddr and Mp is consistent with a model in which Mp acts as a collagen ligand for Ddr during axonal ensheathment. Although the Mp-GFP protein trap shows diffuse Mp expression throughout the nerve, it remains unclear precisely which cell type(s) within the nerve are producing it. Previous reports indicate that Mp can be expressed in the outer peripheral glia layers, so they may provide some Mp to the wrapping glia. However, the strong ensheathment defect seen when Mp is knocked down exclusively in wrapping glia indicates that wrapping glia themselves are likely to be the primary, relevant source of the Mp required for their own morphogenesis. Schwann cells similarly rely on components of their own basal lamina to regulate their development. For example, laminin-211 serves as a ligand for GPR126 to promote myelination. Mp is the sole Drosophila homolog of collagen types XV/XVIII, containing a central helical collagen region with a cleavable N-terminal thrombospondin-like domain and C-terminal endostatin-like domain. Collagen 15a1 and 18a1 are expressed in mouse peripheral nerves and Col15a1 mutants have radial-sorting defects, suggesting that the role of Mp in promoting axon wrapping is likely conserved. In fact, Mp appears to play multiple roles in nerve biology. For example, Mp secreted by the outer glia layers acts via its cleaved endostatin domain to modulate homeostatic plasticity at motor neuron synapses. How Ddr activation within wrapping glia ultimately drives axon wrapping still remains to be determined, but Ddr joins two other receptor tyrosine kinases - EGFR and FGFR - as important and conserved regulators of axon ensheathment. As a non-canonical collagen receptor, Ddr may also interact with other collagen receptors, such as integrins (known to play roles in wrapping glia development), to sense and remodel the extracellular matrix and permit extension of glia processes between axons, similar to its roles in promoting tumor metastasis (Corty, 2022).
The nrv2-Gal4 driver has been the standard method to genetically target wrapping glia for morphological studies, but it is imperfect for manipulation of wrapping glia in ablation or behavioral assays owing to its expression in several subtypes of CNS glia. This study generated a new Split-Gal4 intersectional driver that drives exclusively in wrapping glia. This allowed performing of precise ablation of wrapping glia that led to severely impaired larval locomotion, indicating that the wrapping glia are essential for basic crawling circuit function. This phenotype was particularly striking in light of that fact that no clear crawling defect was observed in Ddr mutant larvae, even though wrapping was severely impaired. It may be possible that non-contact-mediated mechanisms, such as one or more secreted factors, constitute the essential contribution of wrapping glia to axon health and physiology. Alternatively, perhaps even a small amount of direct glia-axon contact may be sufficient to support neuron health and axon function. This would be consistent with the lack of overt behavioral defects in newly hatched first instar larvae, which have poor wrapping compared with later stages, and even in wild-type third instar larvae, in which not every axon is individually wrapped. It is also consistent with our findings that many nerves in WG-ablated larvae seem to be missing axons, whereas this was not observed in Ddr mutant nerves. These results are also similar to what has been recently reported using a different approach to ablate wrapping glia, where only minor behavioral defects were observed upon FGFR signaling disruption but profound crawling defects were seen upon ablation. As with all ablation studies, it is not possible to strictly rule out unexpected negative side effects of the ablation itself; however, using a genetic approach should limit collateral damage (compared with laser or toxin approaches). Together, these data support the conclusion that even limited wrapping or simply some degree of glia-axon contact is sufficient to support axon survival and nerve function compared with no glia at all at least for the first ~5 days of larval life (Corty, 2022).
Previous studies of oligodendrocytes and Schwann cells have found that impairing glial function can result in seemingly normal wrapping and circuit function in young animals, with deficits only appearing when the system is stressed or aged. Studying wrapping in adult Drosophila allows for aging and maintenance studies that the short larval period precludes. Adult peripheral nerves are encased in a transparent but hard cuticle that allows for live imaging but makes fixation challenging. Because of the resolution limits of light microscopy, a reliable method was developed to study their ultrastructure using TEM. Ensheathment in the adult wing nerve was found to differ from that of the larva, as all axons appear to be separated by glial membranes. Surprisingly, wrapping was not obviously impaired in adult nerves of Ddr knockdown or mutant animals. One difference between larval and adult wrapping glia is the territory size of each cell. In larvae, one wrapping glia cell covers the majority of the nerve from the VNC to the muscle field. This wrapping glial cell must therefore undergo tremendous growth to keep up with nerve elongation as the animal grows, as well as radial growth to ensheathe axons. A single cell can end up covering from ~750 μm to 2.5 mm of nerve length, depending on the segment, whereas in the wing there are ~13 wrapping glia along the region of the L1 nerve that was analyzed; This is is ~400 μm long. In larval wrapping glia, there are three receptor tyrosine kinases (EGFR, the FGFR Heartless, and now Ddr) that are each required for normal ensheathment, and thus cannot fully compensate for one another. It is hypothesized that in the larva the cell is pushed to its growth limits and any perturbation in pro-wrapping signaling has a strong effect on morphology, whereas in the adult nerve the system is robust and redundant enough to withstand perturbations of single genes. Future studies of double and triple mutants may be able to test this hypothesis (Corty, 2022).
Loss of Ddr led to an increase in spontaneous neurodegeneration in the nerve as animals naturally aged. Such an uncoupling of neuron health from overt effects on myelination has been demonstrated previously. For example, Cnp1 (Cnp) mutant mice show severe age-dependent neurodegeneration, although they have grossly normal myelin with only subtle changes in myelin ultrastructure. Loss of the proteolipid PLP results in axon degeneration despite having largely normal myelin. It was found that the number of VGlut+ neurons was reduced in aged wings of Ddr knockdown animals, indicating that wrapping glial Ddr is important for long-term neuronal survival. When Ddr whole animal mutants were analyzed by TEM a small but significant reduction was found in axon profile number, which should correspond to the number of surviving neurons. Together with the increased variability observed, this suggests that absence of Ddr signaling increases the susceptibility of subpopulations of neurons to insult or injury that may underlie age-related degeneration (Corty, 2022).
Myelination can directly affect the structure and function of the axons they wrap, including controlling caliber. In general, myelination increases caliber. For example, dysmyelinated Trembler mice have reduced axon calibers compared with controls, and in the PNS caliber along a single axon can vary with reduced caliber at points without direct myelin contact, such as nodes of Ranvier. Axon caliber is an important determinant of conduction velocity but varies widely between neuronal subtypes, so achieving and maintaining appropriate caliber is crucial for proper circuit function. How non-myelinating ensheathment impacts axon caliber is not understood. This study found glial Ddr promotes increased axon caliber. This study focused on the distal twin sensilla of the margin (dTSM) neuron, so it was possible to directly compare the caliber of an identifiable axon between conditions. The reductions in caliber were similar between Ddr mutants and glial-specific DdrRNAi, supporting a non-cell-autonomous role for glial Ddr in regulating axon caliber. The effect is considerable: nearly a 50% reduction in axon caliber at 5 dpe. We hypothesize that by this time point, wild-type dTSM axons have reached their mature caliber, as it is comparable between 5 dpe and 28 dpe in comparable genetic backgrounds. In Ddr mutants, however, we observe that the relative size compared with controls changes over time, suggesting that in Ddr mutants (or knockdowns) the axon continues to increase its caliber, perhaps in an effort to achieve the optimal size, although the axons still remain ~25% smaller than wild-type axons at 28 dpe (Corty, 2022).
Two proteins, MAG, which acts to increase the caliber of myelinated axons, and CMTM6, which restricts the caliber of myelinated and unmyelinated axons, are the only proteins reported to non-cell-autonomously affect the caliber of vertebrate axons, and both do so without overtly affecting myelin. In the fly, it has been shown that a shift in the average size of axons in larval nerves when wrapping glia are absent or severely disrupted, supporting a general role for wrapping glia in promoting axon size. In the adult, this study showed that Ddr is still required for increased axon caliber even when wrapping appears intact. The exact molecular mechanism by which Ddr may promote increased caliber size remains unclear as the control of axon caliber, generally, is not well understood. Genes involved in the general regulation of cell size have been implicated as cell-autonomous determinants. For example, in the fly, S6 kinase signaling is a positive regulator of motor neuron size, including axon caliber. In mammalian axons, the phosphorylation state of neurofilaments and microtubules determines their spacing to determine caliber. Determining how glial Ddr activity ultimately influences the axonal cytoskeleton is an important next step. A 25-50% reduction in caliber would be predicted to impact conduction velocity along the dTSM axon. Given that campaniform sensilla provide essential rapid sensory feedback to fine-tune movement, it will be of interest to test conduction velocity and flight behavior in Ddr mutant animals to see how the proprioceptive circuit might be affected (Corty, 2022).
Taken together, these studies identify Ddr as an important regulator of wrapping glia development and function in the fly, with distinct roles in larval and adult wrapping glia. Ddr is essential for the normal morphological development of axon wrapping in the larvae, and also mediates important axon-glia communication that controls axon caliber growth and affects neuronal health and survival. Given its expression pattern in vertebrate oligodendrocytes and Schwann cells, it seems likely that these essential functions are conserved in vertebrates. Further study into how Ddr functions in both fly and vertebrate glia promises to increase understanding of axon ensheathment in health and disease (Corty, 2022).
To study the influence of adult glial cells in ageing flies, a genetic screen was performed in Drosophila using a collection of transgenic lines providing conditional expression of micro-RNAs (miRNAs). This study describes a methodological algorithm to identify and rank genes that are candidate to be targeted by miRNAs that shorten lifespan when expressed in adult glia. Four different databases were used for miRNA target prediction in Drosophila but little agreement was found between them, overall. However, top candidate gene analysis shows potential to identify essential genes involved in adult glial functions. One example from the top candidates' analysis is gartenzwerg (garz). It was established that garz is necessary in many glial cell types, that it affects motor behaviour and, at the sub-cellular level, is responsible for defects in cellular membranes, autophagy and mitochondria quality control. The remarkable conservation of functions between garz and its mammalian orthologue, GBF1, was verified validating the use of Drosophila as an alternative 3Rs-beneficial model to knock-out mice for studying the biology of GBF1, potentially involved in human neurodegenerative diseases (Goncalves-Pimentel, 2020).
The retinal basal glia (RBG) is a group of glia that migrates from the optic stalk into the third instar larval eye disc while the photoreceptor cells (PR) are differentiating. The RBGs are grouped into three major classes based on molecular and morphological characteristics: surface glia (SG), wrapping glia (WG) and carpet glia (CG). The SGs migrate and divide. The WGs are postmitotic and wraps PR axons. The CGs have giant nucleus and extensive membrane extension that each covers half of the eye disc. This study used lineage tracing methods to determine the lineage relationships among these glia subtypes and the temporal profile of the lineage decisions for RBG development. The CG lineage was found to segregate from the other RBG very early in the embryonic stage. It has been proposed that the SGs migrate under the CG membrane, which prevented SGs from contacting with the PR axons lying above the CG membrane. Upon passing the front of the CG membrane, which is slightly behind the morphogenetic furrow that marks the front of PR differentiation, the migrating SG contact the nascent PR axon, which in turn release FGF to induce SGs' differentiation into WG. Interestingly, it was found that SGs are equally distributed apical and basal to the CG membrane, so that the apical SGs are not prevented from contacting PR axons by CG membrane. Clonal analysis reveals that the apical and basal RBG are derived from distinct lineages determined before they enter the eye disc. Moreover, the basal SG lack the competence to respond to FGFR signaling, preventing its differentiation into WG. Thes findings suggest that this novel glia-to-glia differentiation is both dependent on early lineage decision and on a yet unidentified regulatory mechanism, which can provide spatiotemporal coordination of WG differentiation with the progressive differentiation of photoreceptor neurons (Tsao, 2020).
Glial cells are essential for proper nervous system development and function. To understand glial development and function, glial cells were comprehensively annotated in a single-cell mRNA-sequencing (scRNAseq) atlas of the developing Drosophila visual system. This allowed study of their developmental trajectories, from larval to adult stages, and to understand how specific types of glia diversify during development. For example, neuropil glia that are initially transcriptionally similar in larvae, split into ensheathing and astrocyte-like glia during pupal stages. Other glial types, such as chiasm glia change gradually during development without splitting into two cell types. The analysis of scRNA-seq led to the discovery that the transcriptome of glial cell bodies can be distinguished from that of their broken processes. The processes contain distinct enriched mRNAs that were validated in vivo. Therefore, this study have identified most glial types in the developing optic lobe and devised a computational approach to identify mRNA species that are localized to cell bodies or cellular processes (Ferreira, 2023).
Spalt-like transcription factor 1 (SALL1) is a critical regulator of organogenesis and microglia identity. This study demonstrates that disruption of a conserved microglia-specific super-enhancer interacting with the Sall1 promoter results in complete and specific loss of Sall1 expression in microglia. By determining the genomic binding sites of SALL1 and leveraging Sall1 enhancer knockout mice, evidence is provided for functional interactions between SALL1 and SMAD4 required for microglia-specific gene expression. SMAD4 binds directly to the Sall1 super-enhancer and is required for Sall1 expression, consistent with an evolutionarily conserved requirement of the TGFβ and SMAD homologs Dpp and Mad for cell-specific expression of Spalt in the Drosophila wing. Unexpectedly, SALL1 in turn promotes binding and function of SMAD4 at microglia-specific enhancers while simultaneously suppressing binding of SMAD4 to enhancers of genes that become inappropriately activated in enhancer knockout microglia, thereby enforcing microglia-specific functions of the TGFβ-SMAD signaling axis (Fixsen, 2023).
Central sensitization is defined by nociceptive and somatosensory circuitry changes in the spinal cord leading to dysfunction of antinociceptive gamma-aminobutyric acid (GABA)ergic cells, amplification of ascending nociceptive signals, and hypersensitivity. Astrocytes are key mediators of the neurocircuitry changes that underlie central sensitization and neuropathic pain, and astrocytes respond to and regulate neuronal function through complex Ca (2+) signaling mechanisms. Ca (2+) release from astrocyte endoplasmic reticulum (ER) Ca (2+) stores via the inositol 1,4,5-trisphosphate receptor (IP(3)]R) is required for centrally mediated neuropathic pain; however recent evidence suggests the involvement of additional astrocyte Ca (2+) signaling mechanisms. Therefore this study investigated the role of astrocyte store-operated Ca (2+) entry (SOCE), which mediates Ca (2+) influx in response to ER Ca (2+) store depletion. Using an adult Drosophila melanogaster model of central sensitization based on thermal allodynia in response to leg amputation nerve injury, it was shown that astrocytes exhibit SOCE-dependent Ca (2+) signaling events three to four days following nerve injury. Astrocyte-specific suppression of Stim and Orai, the key mediators of SOCE Ca (2+) influx, completely inhibited the development of thermal allodynia seven days following injury, and also inhibited the loss of ventral nerve cord (VNC) GABAergic neurons that is required for central sensitization in flies. It was lastly shown that constitutive SOCE in astrocytes results in thermal allodynia even in the absence of nerve injury. These results collectively demonstrate that astrocyte SOCE is necessary and sufficient for central sensitization and development of hypersensitivity in Drosophila, adding key new understanding to the astrocyte Ca (2+) signaling mechanisms involved in chronic pain (Prokhorenko, 2023).
Astrocytes are essential for synapse formation, maturation, and plasticity; however, their function during developmental neuronal remodeling is largely unknown. To identify astrocytic molecules required for axon pruning of mushroom body (MB) γ neurons in Drosophila, astrocytes were profiled before (larva) and after (adult) remodeling. Focusing on genes enriched in larval astrocytes, 12 astrocytic genes were identified that are required for axon pruning, including the F-actin regulators Actin-related protein 2/3 complex, subunit 1 (Arpc1) and formin3 (form3). Interestingly, perturbing astrocytic actin dynamics does not affect their gross morphology, migration, or transforming growth factorβ (TGF-β) secretion. In contrast, actin dynamics is required for astrocyte infiltration into the axon bundle at the onset of pruning. Remarkably, decreasing axonal adhesion facilitates infiltration by Arpc1 knockdown (KD) astrocytes and promotes axon pruning. Conversely, increased axonal adhesion reduces lobe infiltration by wild-type (WT) astrocytes. Together, these findings suggest that actin-dependent astrocytic infiltration is a key step in axon pruning, thus promoting understanding of neuron-glia interactions during remodeling (Marmor-Kollet, 2023).
The increasing age of global populations highlights the urgent need to understand the biological underpinnings of ageing. To this end, inhibition of the insulin/insulin-like signalling (IIS) pathway can extend healthy lifespan in diverse animal species, but with trade-offs including delayed development. It is possible that distinct cell types underlie effects on development and ageing; cell-type-specific strategies could therefore potentially avoid negative trade-offs when targeting diseases of ageing, including prevalent neurodegenerative diseases. The highly conserved diversity of neuronal and non-neuronal (glial) cell types in the Drosophila nervous system makes it an attractive system to address this possibility. This study has thus investigated whether IIS in distinct glial cell populations differentially modulates development and lifespan in Drosophila. Glia-specific IIS inhibition, using several genetic means, delays development while extending healthy lifespan. The effects on lifespan can be recapitulated by adult-onset IIS inhibition, whereas developmental IIS inhibition is dispensable for modulation of lifespan. Notably, the effects observed on both lifespan and development act through the PI3K branch of the IIS pathway and are dependent on the transcription factor FOXO. Finally, IIS inhibition in several glial subtypes can delay development without extending lifespan, whereas the same manipulations in astrocyte-like glia alone are sufficient to extend lifespan without altering developmental timing. These findings reveal a role for distinct glial subpopulations in the organism-wide modulation of development and lifespan, with IIS in astrocyte-like glia contributing to lifespan modulation but not to developmental timing. These results enable a more complete picture of the cell-type-specific effects of the IIS network, a pathway whose evolutionary conservation in humans make it tractable for therapeutic interventions. These findings therefore underscore the necessity for cell-type-specific strategies to optimise interventions for the diseases of ageing (Woodling, 2020).
Aging is a complex process involving various systems and behavioral changes. Altered immune regulation, dysbiosis, oxidative stress, and sleep decline are common features of aging, but their interconnection is poorly understood. Using Drosophila, this study discovered that IM33, a novel immune modulator, and its mammalian homolog, secretory leukocyte protease inhibitor (SLPI), are upregulated in old flies and old mice, respectively. Knockdown of IM33 in glia elevates the gut reactive oxygen species (ROS) level and alters gut microbiota composition, including increased Lactiplantibacillus plantarum abundance, leading to a shortened lifespan. Additionally, dysbiosis induces sleep fragmentation through the activation of insulin-producing cells in the brain, which is mediated by the binding of Lactiplantibacillus plantarum-produced DAP-type peptidoglycan to the peptidoglycan recognition protein LE (PGRP-LE) receptor. Therefore, IM33 plays a role in the glia-microbiota-neuronal axis, connecting neuroinflammation, dysbiosis, and sleep decline during aging. Identifying molecular mediators of these processes could lead to the development of innovative strategies for extending lifespan (Xu, 2023).
Glia and neurons face different challenges in aging and may engage different mechanisms to maintain their morphology and functionality. This study reports that adult-onset downregulation of a Drosophila gene CG32529/GLAD led to shortened lifespan and age-dependent brain degeneration. This regulation exhibited cell type and subtype-specificity, involving mainly surface glia (comprising the BBB) and cortex glia (wrapping neuronal soma) in flies. In accordance, pan-glial knockdown of GLAD disrupted BBB integrity and the glial meshwork. GLAD expression in fly heads decreased with age, and the RNA-seq analysis revealed that the most affected transcriptional changes by RNAi-GLAD were associated with upregulation of immune-related genes. Furthermore, a series of lifespan rescue experiments was conducted and the results indicated that the profound upregulation of immune and related pathways was not the consequence but cause of the degenerative phenotypes of the RNAi-GLAD flies. Finally, this study showed that GLAD encoded a heterochromatin-associating protein that bound to the promoters of an array of immune-related genes and kept them silenced during the cell cycle. Together, these findings demonstrate a previously unappreciated role of heterochromatic gene silencing in repressing immunity in fly glia, which is required for maintaining BBB and brain integrity as well as normal lifespan (Shu, 2023).
Aging is thought to be associated with decreased immune functions (immune-paralysis) and increased pro-inflammatory activity (inflamm-aging). Several cellular processes are considered contributing factors to the age-related changes in immunity and inflammation. For example, (1) the adaptive immune responses decrease with age, leading to accumulation of pathogens and cellular stress, which activate inflammatory responses such as the JAK/STAT pathway; (2) senescent cells accumulate in aging, which secrete a range of inflammatory cytokines, and chemokines; (3) the microbial load and pathogen diversity that one is exposed to grow with age; and, (4) AMPs of the IMD pathway are upregulated in the brain of aged animals even when reared in germ-free conditions, suggesting a pathogen-independent mechanism underlying the age-associated immune overactivation in the nervous system during aging (Shu, 2023).
The findings of this study add a new paradigm for age-related dysregulation of the innate immunity-the decline of heterochromatic silencing of immune-related genes in fly glia. This study shows that the Drosophila gene GLAD encodes a BAH domain-containing protein, which is localized to the heterochromatin and binds to the promotors of an array of immune-related genes to keep them silenced. With age, GLAD expression and GLAD-mediated heterochromatic silencing decrease. Meanwhile, the innate immunity including the expression of AMP genes is increased in the brain of aged flies. A progressive heterochromatin loss also led to deregulation of the genes involved in immune responses in the gut of aged flies. Thus, it is possible that the heterochromatin-mediated immune silencing may be an underappreciated mechanism that is employed by other tissues and systems to keep immunity on a leash as well. In addition, it will be interesting to investigate how GLAD affects the cell number of glia in the fly brain in the future. This could not be done because the strong deleterious effect of RNAi-GLAD made it difficult to accurately assess the cell number of glia or the effect of GLAD on the cell cycle in vivo in this study (Shu, 2023).
It should be pointed out that, although this work focuses on glia and immunity, GLAD does not function 'only' in glia. Nor does it 'only' repress immune-related genes. With that said, the RNA-seq analysis of the TubGS > RNAi-GLAD fly heads indeed indicates that the most affected molecular pathways are enriched in immune and related functions. It is interesting to note that the immune genes in the Drosophila genome display a significant excess of clustering in the chromatin. And, some heterochromatin domains are enriched for genes involved in immunity in both fly and mammalian cells. Similarly, the genes of the GstD family are also clustered in the fly genome. Heterochromatin is spatially more complex and dynamic than previously thought, and a network of subdomains can regulate diverse heterochromatin functions. Thus, the chromosomal arrangement of the fly genome may facilitate the recruitment and repression of selected heterochromatin subdomains enriched for immune-related genes. In addition, several upstream PGRP genes (e.g., PGRP-SD and PGRP-SA) are upregulated in the TubGS > RNAi-GLAD flies, which may enhance the overactivation of the IMD and Toll immune pathways, thereby further boosting the upregulation of the downstream effector AMPs (Shu, 2023).
Widespread, age-dependent BBB disintegration is associated with physiological aging and may be an early pathological hallmark of several human diseases. In particular, the BBB is vulnerable to systemic overactivation of immune and inflammation in neurological disorders. This study shows that local overactivation of the innate immunity in fly glia, by either KD of GLAD or OE of Imd, is sufficient to cause BBB breakdown. With the leaky BBB, immune and inflammatory factors released from glia and immune cells may leak out of the nervous system and get into the circulation system (and vice versa), which may further augment local and systemic immune and inflammatory responses to accelerate aging. Indeed, glial KD of GLAD or OE of Imd leads to age-dependent brain degeneration and dramatically reduces longevity in flies. Of note, although the anatomy of the fly BBB is different from that of mammals, they are organized and function in a similar manner. For example, the mammalian BBB is formed by a tightly sealed monolayer of brain endothelial cells that are connected by tight junctions (TJs); whereas in the fly BBB, the SPGs are connected to each other by septate junctions (SJs). The SJs function as the paracellular barrier like the mammalian TJs and disruption of SJs impairs the fly BBB. Moreover, Claudin is a key component of the mammalian TJs and the fly SJs contains Claudin-like molecules, suggesting the evolutionary conserved molecular organization of the TJs and the SJs. Therefore, it would be interesting to investigate how the GLAD-regulated, age-associated effector AMPs and cytokines and their mammalian counterparts affect BBB permeability in the future, which may provide new mechanistic insights about immune overactivation and BBB disintegration in aging and neurodegenerative diseases (Shu, 2023).
Finally, Bahd1 is the putative mammalian homologue of GLAD, which encodes a core protein of the heterochromatin-repressive complex. BAHD1 silences IFN-stimulated genes and modulates innate immune defense in response to bacterial infection. The Bahd1 null mutation causes significant perinatal death in mice, whilst the Bahd1+/− heterozygotes exhibit anxiety-like behaviors suggesting a potential role of BAHD1 in the rodent nervous system. Future research on GLAD and BAHD1 is warranted to reveal the evolutionarily conserved and the species-specific mechanisms in heterochromatin-mediated immune gene silencing and how they impact on brain aging and longevity (Shu, 2023).
Axons in the mature central nervous system (CNS) fail to regenerate after axotomy, partly due to the inhibitory environment constituted by reactive glial cells producing astrocytic scars, chondroitin sulfate proteoglycans, and myelin debris. This study investigated this inhibitory milieu, showing that it is reversible and depends on glial metabolic status. Glia can be reprogrammed to promote morphological and functional regeneration after CNS injury in Drosophila via increased glycolysis. This enhancement is mediated by the glia derived metabolites: L-lactate and L-2-hydroxyglutarate (L-2HG). Genetically/pharmacologically increasing or reducing their bioactivity promoted or impeded CNS axon regeneration. L-lactate and L-2HG from glia acted on neuronal metabotropic GABA(B) receptors to boost cAMP signaling. Local application of L-lactate to injured spinal cord promoted corticospinal tract axon regeneration, leading to behavioral recovery in adult mice. These findings revealed a metabolic switch to circumvent the inhibition of glia while amplifying their beneficial effects for treating CNS injuries (Li, 2020).
Drosophila neuroblasts (NBs) are neural stem cells whose maintenance relies on Notch activity. NBs proliferate throughout larval stages to generate a large number of adult neurons. Their proliferation is protected under conditions of nutrition restriction. As amino acid transporters (Solute Carrier transporters, SLCs), such as SLC36, have important roles in coupling nutrition inputs to growth pathways, they may have a role in this process. For example, an SLC36 family transporter Pathetic (Path) that supports body size and neural dendrite growth in Drosophila, was identified as a putative Notch target in genome-wide studies. This study examined expression and regulation of Path in the Drosophila larval brain. Path function in NB proliferation and overall brain growth was investigated under different nutrition conditions by depleting it from specific cell types in the CNS, using mitotic recombination to generate mutant clones or by directed RNA-interference. Path is expressed in both NBs and glial cells in the Drosophila CNS. In NBs, path is directly targeted by Notch signalling via Su(H) binding at an intronic enhancer, PathNRE. This enhancer is responsive to Notch regulation both in cell lines and in vivo. Loss of path in neural stem cells delayed proliferation, consistent with it having a role in NB maintenance. Expression from pathNRE (Notch responsive element) was compromised in conditions of amino acid deprivation although other Notch regulated enhancers are unaffected. However, NB-expressed Path was not required for brain sparing under amino acid deprivation. Instead, it appears that Path is important in glial cells to help protect brain growth under conditions of nutrient restriction. This study has identified a novel Notch target gene path that is required in NBs for neural stem cell proliferation, while in glia it protects brain growth under nutrition restriction (Feng, 2020).
Drosophila Neuroblasts (NBs) are neural stem cells that divide to give progeny, which differentiate into neurons and glia that later constitute the adult brain. NBs arise from neuroectoderm during embryonic development and enter quiescence at the end of the embryonic stage, until they are reactivated upon feeding during larval stages. After reactivation, around 350 NBs reside on the surface of the brain and constitute the stem cell pool, undergoing multiple rounds of asymmetric cell divisions. During division, each NB generates one larger daughter cell that retains stem cell identity and one smaller daughter cell that divides further to generate progeny that differentiate into certain types of neurons and glia. At the time of metamorphosis, the central nervous system (CNS) contains about 30,000 neurons and 10,000 glial cells. The glial cells fulfil supporting and nurturing function to neurons. Importantly they also ensure NBs receive the correct growth signal at the correct times. For example, signals from glia are necessary for NBs to exit quiescence upon feeding, and to remain proliferative during nutrition deprivation once the larva passes the critical weight time-point (Feng, 2020).
Notch signalling is one of the key regulators in maintaining NSCs and performs a similar function in both Drosophila and vertebrate NSCs. Notch depletion causes loss of NB lineages while Notch over-activation inhibits NBs from differentiating and induces brain tumours. In the canonical Notch signalling model, upon Notch ligand binding to the receptor, the Notch intracellular domain (NICD) is cleaved and released into the nucleus. The nuclear NICD interacts with the DNA-binding protein known as Suppressor of Hairless (Su(H)) in flies, to activate the expression of target genes. The functions of Notch are very context-dependent, making it important to identify the Notch regulated genes in different processes including stem cell maintenance (Feng, 2020).
The brain, like other organs, needs to translate changing nutrition inputs into cell growth decisions. An emerging role of amino acid transporters, especially the SLC family, in coupling the nutrition signalling and growth pathways has been revealed in recent years. SLC38A9 acts as an amino acid sensor in the process of mTORC-activation in mammalian cell lines. Similarly, SLC36A4 helps to promote proliferation in colorectal cancer cells through its interaction with mTORC1. A requirement for SLC36A4 in mice retinal pigmented epithelial cells also involved mTORC-activation. However, where and how might these transporters function in other cases of nutrient sensing, such as the Drosophila NBs, remains unknown. Also, it is unclear whether the growth-promoting role of these amino acid transporters would be adaptive to starvation. For example, the sparing mechanism in nutrient deprived NBs somehow bypasses the Tor pathway (Feng, 2020).
Pathetic (Path) is the Drosophila orthologue of SLC36A4, having the characteristics of a broad specificity transporter with multiple transmembrane domains. It interacts with Tor (Target of apamycin) pathway components in regulating eye growth and body growth of Drosophila and promotes dendritic growth in C4da neurons (Lin, 2015). path also exhibited the hall marks of a Notch regulated gene in a genome-wide study of genes upregulated during Notch-induced NB hyperplasia. This study has followed up on this observation by analysing the role and regulation of Path in NBs under normal and abnormal nutrition conditions. path was shown to be a novel direct Notch target in NBs, and it is required for NB proliferation. Further its role in brain sparing was characterised, and it was found to be required to fully protect the CNS from nutrient restriction. However, the evidence indicates that glial-expressed Path is important for protecting brain growth under nutrient restriction, rather than its activity in the NBs themselves (Feng, 2020).
Previously, Path was found to be required for overall body growth and extreme dendrite growth, potentially through interacting with the PI3K/Tor pathway and protein synthesis pathways. This study identified an autonomous role of path in maintaining NB proliferation, which is in line with previous findings that path promotes growth. Path expression in NBs is partially dependant on Notch, as it contains an intronic enhancer, which is directly regulated by the pathway. Thus its regulation and involvement in NB proliferation argues that Path contributes to the normal function of Notch in NBs, although it remains to be established whether it has a similar essential role in Notch induced tumours. Furthermore, the striking reduction in pathNRE driven expression under nutrient restriction (NR) suggests that the NBs are sensitive to changes in the internal milieu of the animal. This argues that, while glial cells may shield NBs from many environmental effects, the NBs are nevertheless able to detect altered nutrient levels and may harbour addition pathways that contribute to brain-sparing (Feng, 2020).
Although this study found that Path is required for brain sparing (the maintenance of brain size) during NR, this appears to rely on its expression in Glial cells rather than NBs, despite the fact that the intronic pathNRE enhancer is sensitive to nutrient deprivation. Alk/Jeb is the major pathway that has been linked to brain growth under NR conditions, and glial knockdown of Jeb (repo-gal4 > jebRNAi) resulted in smaller NB-clone size as well as lineage number. It is possible that Path at the membrane of surface glial cells could detect the environmental amino acid levels and in turn regulate Alk/Jeb expression. Path is a potential amino acid transporter with multiple transmembrane domains, which exhibited high affinity for alanine and glycine with low transporting capacity when expressed in Xenopus oocytes. The closest mammalian relatives, proton-assisted transporter 4 (PAT4 or hPAT4), have a high affinity for proline and tryptophan. Although it remains to be fully determined which are the main substrates for Path in vivo, a recent study suggests that Path is required for scavenging proline to promote tumour growth in conditions of obesity enhanced tumorigenesis in Drosophila. In these conditions, Path regulation was also involved with nutrient sensing mechanisms albeit the levels of sugars were elevated rather than restricted. Further studies will be needed to determine whether proline is the primary substrate for Path in all conditions and the extent that path expression is differentially regulated by changes in nutrient levels according to the tissue and its proliferative state (Feng, 2020).
The Tor pathway is involved in NB reactivation, growth and proliferation. However, in L3, NB growth is regulated by the Alk/Jeb pathway which activates downstream PI3K/Akt signalling, but appears independent of Tor. One model to explain the current results is that Path functions as a mediator between Alk and the Tor pathway. Alk is required for and acts through the PI3K/Akt pathway. In L3 larvae brains, most Tor pathway components are dispensable for brain growth and brain size is not changed significantly when levels of Insulin-like peptides (Ilp) are manipulated. In contrast, the Tor pathway is activated in younger brains suggesting there may be a mechanism to bypass or switch-off the Tor pathway at later stages. One hypothesis, considering pathNRE is upregulated after NB reactivation, is that path helps to keep activity of the Tor pathway at restricted levels at later stages. In this case, the effects of path in NBs would be through nutrient sensing and protein synthesis, similar to its function in C4da neurons (Feng, 2020).
The regulation and expression of Path in the NBs suggests that, in this context, Path promotes cell growth and lineage size. Further studies will be needed to characterise what the consequences from Path up-regulation are in different circumstances. For example, whether Path has the same role in Notch induced NB tumours, and whether this would differ depending on nutrient status, remains to be established. Likewise, this study has investigated its role in so-called Type I NBs. Not mutant clones were obtained to distinguish whether it makes the same contribution in Type II NBs, which produce a highly proliferative transit amplifying intermediary, or whether its activity there differs. Finally, it is striking that pathNRE-GFP expression is decreased in neuroblasts under nutrient restriction, when neuroblast proliferation becomes spared. Whether this reflects a physiological response, whereby NB Path levels are reduced while glial cell Path persists under NR, or whether it reflects a regulatory feature, whereby a different enhancer becomes activated in these conditions, remains to be established. Nevertheless, these data highlight the importance of distinguishing the different regulatory inputs to Path, and the extent that they are dependent on different nutrient and/or amino acid (Feng, 2020).
Brain expression of Path, which encodes a broad specificity transporter, occurs in both NBs and glial cells where its functions appear to differ. NB expression of path, is partially dependent on Notch activity and is required for NB lineage proliferation but not for brain sparing under nutrient restrictions. In contrast Glial expression of Path appears to be independent of Notch and is essential for protecting the brain growth under nutrition restriction. In conclusion, this study has demonstrate different roles of Path in distinct parts of the brain that would together enable the larval brain to proliferate and grow in both normal and NR conditions (Feng, 2020).
Neural stem cells (NSCs) reside in a defined cellular microenvironment, the niche, which supports the generation and integration of newborn neurons. The mechanisms building a sophisticated niche structure around NSCs and their functional relevance for neurogenesis are yet to be understood. In the Drosophila larval brain, the cortex glia (CG) encase individual NSC lineages in membranous chambers, organising the stem cell population and newborn neurons into a stereotypic structure. CG were found to wrap around lineage-related cells regardless of their identity, showing that lineage information builds CG architecture. It was then discovered that a mechanism of temporally controlled differential adhesion using conserved complexes supports the individual encasing of NSC lineages. An intralineage adhesion through homophilic Neuroglian interactions provides strong binding between cells of a same lineage, while a weaker interaction through Neurexin-IV and Wrapper exists between NSC lineages and CG. Loss of Neuroglian results in NSC lineages clumped together and in an altered CG network, while loss of Neurexin-IV/Wrapper generates larger yet defined CG chamber grouping several lineages together. Axonal projections of newborn neurons are also altered in these conditions. Further, the loss of these 2 adhesion complexes specifically during development is linked to locomotor hyperactivity in the resulting adults. Altogether, these findings identify a belt of adhesions building a neurogenic niche at the scale of individual stem cell and provide the proof of concept that niche properties during development shape adult behaviour (Banach-Latapy, 2023).
The second most commonly mutated gene in primary microcephaly (MCPH) patients is wd40-repeat protein 62 (wdr62), but the relative contribution of WDR62 function to the growth of major brain lineages is unknown. This study used Drosophila models to dissect lineage-specific WDR62 function(s). Interestingly, although neural stem cell (neuroblast)-specific depletion of WDR62 significantly decreased neuroblast number, brain size was unchanged. In contrast, glial lineage-specific WDR62 depletion significantly decreased brain volume. Moreover, loss of function in glia not only decreased the glial population but also non-autonomously caused neuroblast loss. It was further demonstrated that WDR62 controls brain growth through lineage-specific interactions with master mitotic signaling kinase, AURKA (Aurora A). Depletion of AURKA in neuroblasts drives brain overgrowth, which was suppressed by WDR62 co-depletion. In contrast, glial-specific depletion of AURKA significantly decreased brain volume, which was further decreased by WDR62 co-depletion. Thus, dissecting relative contributions of MCPH factors to individual neural lineages will be critical for understanding complex diseases such as microcephaly (Lim, 2017).
Genome-wide exome sequencing of microcephaly (MCPH) patients identified wd40-repeat protein 62 (wdr62) as the second most commonly mutated gene. WDR62 is a ubiquitously expressed cytoplasmic protein in interphase and localizes to the spindle pole in mitosis. A feature of many WDR62 MCPH-associated alleles is an inability to localize to the mitotic spindle pole, and wdr62 depletion is also associated with defects in spindle and centrosomal integrity, mitotic delay, and reduced brain growth in rodents. The neural stem cell (NSC) population gives rise to all neuronal cells in the adult brain. NSC behavior is governed by both cell intrinsic factors and extrinsic factors from the supporting stem cell niche, including the glial lineage, which acts non-autonomously to control stem cell renewal and differentiation of daughters (Lim, 2017).
The connection between NSCs and their niche, and the importance of spindle integrity to asymmetric division, has been best defined for Drosophila NSCs/neuroblasts (NB). WDR62 scaffolds kinases that are important mitotic regulators including c-Jun N-terminal kinase (JNK) members of the mitogen-activated protein kinase superfamily and Aurora kinase A (AURKA). In flies, AURKA regulates NB proliferation and is required for the localization of mitotic NB polarity complex protein Bazooka (mammalian Par3) to the apical Par complex (comprising the Par anchor, Inscuteable [Insc] adaptor protein, and Gαi/Pins/Mud complex). This establishes the apical-basal NB axis essential for self-renewal and differentiation. As a consequence, mutant aurka causes NB overproliferation and tissue overgrowth. The WDR62 ortholog in Drosophila (dWDR62) is required for brain growth (Nair, 2016), but whether signaling between WDR62 and AURKA modulates brain development has not been reported (Lim, 2017).
In addition to the NB lineage, Drosophila studies suggest that the glial lineage governs overall brain volume through regulation of cell-cycle re-entry and neuroepithelial expansion of NBs. However, potential contribution(s) of individual brain lineage(s) (NB or glia) to the defective brain growth associated with global depletion of wdr62 or aurka is currently unclear. This study confirmed that WDR62 is required for spindle orientation in NBs (Nair, 2016), however, wdr62 depletion specifically in NBs does not significantly retard brain growth. Rather, control of brain growth predominantly depends upon glial lineage function, as depletion of either aurka or wdr62 specifically in the glial lineage significantly reduces brain volume. Moreover, although wdr62 depletion suppressed brain overgrowth associated with aurka depletion in NBs, wdr62 knockdown specifically in the glial lineage enhanced the small brain phenotype associated with aurka depletion. Collectively, these data suggest that WDR62 function is negatively regulated by AURKA in NBs but positively regulated by AURKA in glia, and thus demonstrates that lineage-specific signaling functions of AURKA-WDR62 in Drosophila orchestrate larval brain growth and development (Lim, 2017).
In the mammalian brain, radial glia behave as NSCs that are supported by outer radial glia through cell-cell contact and secretion of growth factors required for maintenance of a stem cell niche. Another class of glial cells, the microglia population, regulates neural precursor cell numbers to govern final neuronal numbers residing in the cortex. However, whether MCPH genes such as wdr62 are important for glial cell fate is unclear. This study dissected the lineage-specific contribution of WDR62 to brain development and revealed that loss of WDR62 function specifically in the glial, but not the NB lineage, profoundly altered brain growth. Moreover, wdr62 depletion in glia likely impairs brain growth autonomously (i.e., through depletion of glia), and also results in NSC loss, suggesting that a focus on WDR62 function in glia will be integral to elucidating how wdr62 loss of function contributes to MCPH. That depletion of wdr62 in the NB lineage was not associated with reduced brain volume, despite reducing NB number, also provides a likely explanation for the recent observation that NB defects associated with global wdr62 depletion fail to account for reduced brain size (Lim, 2017).
Hypomorphic wdr62 mutant mice have reduced brain size, with associated mitotic defects and an overall decrease in neural progenitor cells. In Drosophila, spindle orientation defects following wdr62 loss of function likely underlie the G2 delay and increased mitotic figures in NBs. This phenotype is also reminiscent of the cleavage plane misorientation observed in NSCs in wdr62-depleted rat brains. In Drosophila NBs, WDR62 regulates the interphase localization of Centrosomin (CNN, mammalian CDK5RAP2) to the apical centrosome, and thus centrosomal maturation and positioning. Interestingly, CNN is also an AURKA target that governs spindle orientation independently from cortical polarity establishment during mitosis. Similar to the phenotype observed for wdr62 depletion in NBs, cnn loss of function is associated with spindle orientation defects and reduced NB number. Thus, it is tempting to speculate that WDR62 and CNN function in the same AURKA-dependent signaling complex during mitosis (Lim, 2017).
Ex vivo studies have demonstrated that AURKA phosphorylation of WDR62 promotes spindle pole localization during mitosis. Mouse models suggest AURKA and WDR62 interact in vivo to control brain growth. Compound heterozygous wdr62+/-;aurka+/- mice have a much smaller body size than single heterozygotes but, although the mitotic index of the cerebral cortex was significantly increased and NSCs were reduced, consistent with a mitotic delay radial glia, potential changes to brain volume were not measured. This study, demonstrates that the brain overgrowth associated with aurka depletion specifically in NBs was suppressed by co-depletion of wdr62, bringing brain volume to within the control range. In contrast, the small brain phenotypes, due to glial-specific depletion of either aurka or wdr62, were further reduced by co-knockdown. Thus, in the context of normal brain development, AURKA likely acts to promote WDR62-dependent glial proliferation, but antagonizes WDR62 function in the NB lineage (Lim, 2017).
These findings indicate that WDR62 likely functions in AURKA-mediated regulation of spindle orientation but not in the establishment of cortical polarity. One reason for the differential output of AURKA regulation of WDR62 (between NB and glia) could stem from the symmetrical nature of glial division, where there is no evidence for cortical polarization. In contrast to the in vivo mammalian studies and previous Drosophila studies, which employed global depletion strategies for wdr62, these studies have enabled dissection of the relative contribution of wdr62 loss-of-function from each of the major brain lineages. In particular, the observation that depletion in either the NB or glial lineage is associated with reduced cell number, but an overall reduction in brain size was only observed when wdr62 was reduced in glia, places great interest in examining the relative contribution of glial-specific depletion of wdr62 in mice to brain size. Moreover, future studies of the pathogenic wdr62 mutations, and identified AURKA phosphorylation sites on WDR62, in the glial lineage are likely to inform on the contribution of this lineage to impaired brain growth in microcephaly (Lim, 2017).
The receptor tyrosine kinase (RTK) pathway plays an essential role in development and disease by controlling cell proliferation and differentiation. This study profiled the Drosophila larval brain by single cell RNA-sequencing and identified Amalgam (Ama), encoding a cell adhesion protein of the immunoglobulin IgLON family, that regulates the RTK pathway activity during glial cell development. Depletion of Ama reduces cell proliferation, affects glial cell type composition and disrupts the blood-brain barrier (BBB) that leads to hemocyte infiltration and neuronal death. Ama depletion lowers RTK activity by upregulating Sprouty (Sty), a negative regulator of RTK pathway. Knockdown of Ama blocks oncogenic RTK signaling activation in the Drosophila glioma model and halts malignant transformation. Finally, knockdown of a human ortholog of Ama, LSAMP, results in upregulation of SPOUTY2 in glioblastoma cell lines suggesting that the relationship between Ama and Sty is conserved (Ariss, 2020).
The RTK signaling pathway is critical in a plethora of glial cell functions, such as migration, differentiation, proliferation, neurodegeneration and locomotion. This study identified Ama as a regulator of RTK signaling in glial cells and uncover an essential function of Ama in the maintenance of the BBB. This study underscores the power of scRNA-seq profiling to explore a knockdown phenotype and led to the identification of Sty as a major Ama target during regulation of RTK signaling (Ariss, 2020).
Profiling cells by scRNA-seq identified distinct changes in gene expression profiles across multiple glial cell types in Ama-depleted brain that otherwise would have not been possible using conventional approaches. First, it was found that Ama depletion affects gene expression in surface glia, which in turn leads to a disruption in the BBB. Second, the increase in hemocyte numbers in repo>AmaRNAi scRNA-seq dataset enabled the discovery of infiltrating blood cells in Ama-depleted brains. Third, the increase in Sty and decrease in PntP1 levels following Ama knockdown in glia encouraged exploration of the impact of Ama in RTK signaling (Ariss, 2020).
scRNA-seq identifies and clusters cells based on similarity in gene expression profile to uncover the cellular heterogeneity in a tissue. In this study, scRNA-seq of the normal fly brains identified all the glial cell types in addition to a Fas cluster that appeared to encompass multiple glial cell types. This cell cluster displayed high expression levels of cell adhesion proteins, which is a hallmark of glial cells, while also supporting the observation that scRNA-seq not only clusters cells by type but also by similar biological features. This underlies the robustness of scRNA-seq, since it uncovers complex cellular dynamics related to certain stimuli and continuous temporal differentiation processes, as well as the spatial arrangement of cells. Notably, the cellular perturbations in clustering that were observed following Ama depletion were validated experimentally by genetic analysis, immunofluorescence and other assays. This type of single-cell data validation is essential as it addresses the concern of a batch effect between scRNA-seq samples. Thus, this work highlights the power of scRNA-seq to profile a knockdown or mutant phenotype (Ariss, 2020).
The results revealed that Ama is critical for maintaining the BBB, as its depletion results in discontinuous surface glia (SG) membranes, suggesting a lack of tight junctions or organization that leads to disruption of the barrier. This in turn exposes the larval brains to the high potassium content of the hemolymph, which damages neurons. Ama knockdown decreases overall glial cell proliferation, which can affect SG cells and the BBB in two additional ways. First, lack of proliferation in PG cells can potentially affect the secretion of metabolites, which is important to prevent neurodegeneration. Second, Ama depletion can alter subperineurial glia (SPG) growth by reducing endoreplication and endomitosis, as evident by reduced expression of cell cycle genes in repo>AmaRNAi, and, therefore, hinder the ability of SPG to accommodate the growing brain during late larval stages (Ariss, 2020).
The conventional way to determine the intactness of the BBB function is by labeling brains with a fluorescent dextran dye. If the barrier is permissible to large molecules, such as the dye, then the BBB is considered to be broken. This study presents an alternative approach to monitor a disruption in the BBB by measuring the increase in infiltrating hemocytes in larval brains both by scRNA-seq and through staining with a hemocyte-specific antibody. Although SG protect neurons from the hemolymph, penetration of hemocytes into the brain through the damaged BBB has not been previously reported. Whether infiltrating hemocytes have a role in inflicting the damage in the brain is unknown but raises the possibility that they might have a function in that context (Ariss, 2020).
Ama and Lachesin are part of the IgLON family, with Lachesin also shown to be required for the BBB maintenance. Since IgLONs are also expressed in the BBB in mammals, this suggests that the immunoglobin superfamily may indeed also have an evolutionarily conserved BBB function in humans. These findings highlight an important role of IgLONs in neurodegenerative diseases that result in BBB breakdown (Ariss, 2020).
Through scRNA-seq and measuring P-ERK and PntP1 levels, this study found a drastic reduction in the level of RTK signaling in Ama-depleted brains. It is suggested that Ama regulates the RTK pathway since its depletion increases Sty levels, a general inhibitor of the pathway, and, conversely, overexpression of Ama has an opposite effect. Sty in repo>AmaRNAi brains predominantly localizes to the nuclear and plasma membranes. Intriguingly, in mammalian cells, SPRY2 localization to the membrane has been shown to be crucial for its phosphorylation and inhibitory effect. The results of this study therefore suggest that, while depletion of Ama increases Sty levels, there might additionally be an effect in the cellular localization or post-translational modification of Sty. Genetic experiments indicate that Sty is the key target of Ama and that activated ERK can partially rescue the repo>AmaRNAi phenotype, whereas activated EGFR in the glioma fly model cannot. These epistatic interactions support the model that Ama, acts through Sty, and is downstream of EGFR but upstream of ERK in the RTK signaling pathway (Ariss, 2020).
Although knockdown of Sty in Ama-depleted brains partially rescues the glial cell numbers and brain size, it fails to suppress the neuronal apoptosis in repo>AmaRNAi brains. Since Ama can act non-cell-autonomously, it cannot be excluded that Ama might also affect neurons non-cell-autonomously and, therefore, the expression of Sty in glial cells fails to rescue neurons. This is noteworthy since LSAMP, the human counterpart of Ama, was reported to control neurite growth (Akeel, 2011). The precise mechanism of how Ama affects Sty is unknown and requires further investigation. However, SPRY2 and EGFR in human cell lines have been shown to compete with c-Cbl (a ubiquitin ligase) binding. In this context, the ubiquitin ligase attenuates the inhibitory effect of SPRY2 and vice versa. Since Ama and Sty localize to cell membranes, one possible explanation is that they may interact with each other, whereby loss of Ama releases Sty to bind to RTK receptors and promotes inhibition of the signaling pathway (Ariss, 2020).
Finally, this study shows that the role of Ama in controlling Sty levels is conserved in human glioblastoma (GBM) cell lines. Additionally, GBM patients with EGFR mutations display a significant increase in survival when they have low expression levels of LSAMP, suggesting a potential role for the IgLON family member in this cancer. Previous work has shown that LSAMP either promotes growth or acts as a tumor suppressor (Kresse, 2009). Intriguingly, SPRY2 inhibits or activates RTK signaling based on the context and cell type and also acts either as an oncogene or tumor suppressor (Masoumi-Moghaddam, 2014). These results suggest a potential connection between LSAMP and SPRY2 that may help to explain the role of LSAMP in tumorigenesis and, thus, point to LSAMP as a potential therapeutic target (Ariss, 2020).
Drosophila circadian behavior relies on the network of heterogeneous groups of clock neurons. Short- and long-range signaling within the pacemaker circuit coordinates molecular and neural rhythms of clock neurons to generate coherent behavioral output. The neurochemistry of circadian behavior is complex and remains incompletely understood. This study demonstrates that the gaseous messenger nitric oxide (NO) is a signaling molecule linking circadian pacemaker to rhythmic locomotor activity. mutants lacking nitric oxide synthase (NOS) have behavioral arrhythmia in constant darkness, although molecular clocks in the main pacemaker neurons are unaffected. Behavioral phenotypes of mutants are due in part to the malformation of neurites of the main pacemaker neurons, s-LNvs. Using cell-type selective and stage-specific gain- and loss-of-function of NOS, this study also demonstrated that NO secreted from diverse cellular clusters affect behavioral rhythms. Furthermore, perineurial glia, one of the two glial subtypes that form the blood-brain barrier, as the major source of NO that regulates circadian locomotor output. These results reveal for the first time the critical role of NO signaling in the Drosophila circadian system and highlight the importance of neuro-glial interaction in the neural circuit output (Kozlov, 2020).
It is rather surprising that the lack of NOS enzyme is not lethal as NO is part of various developmental processes. NOSΔ mutants are nonetheless strongly arrhythmic in DD and have reduced morning anticipation in LD. The data suggest that both congenital impairments and lack of NO signaling in adulthood contribute to the behavioral phenotype of the mutants. Axonal terminals of the master pacemaker, s-LNvs, in NOS mutants are profoundly disordered, suggesting the wrong or absent synaptic connections with the downstream partners. However, molecular rhythms in the pacemaker neurons are unaffected. During adulthood, NOS activity in the perineurial glia is required for producing free-running locomotor rhythms but not for maintaining PDF rhythms and structure of the s-LNvs. This finding indicates that NO produced in the perineurial glia is necessary for proper the functioning of circadian locomotor output circuits. Taken together, these results demonstrate that NO signaling is essential for establishing and controlling circadian output circuit (Kozlov, 2020).
The functional isoform dNOS1 shows a circadian variation of its RNA levels throughout the day, which suggest that levels of NO could cycle at least in LD. However, dNOS is likely to be regulated by its truncated isoforms in a stage- and cell-type-specific manner, which lays an additional complexity to the regulation of NO production and probably leads to the heterogeneous and context-specific variations of NO. Hyperproduction of NO modulates molecular clockwork, albeit modestly, and is generally detrimental to locomotor rhythms. Therefore, the level and potentially the rhythms of NO production should be tightly controlled in wild-type flies (Kozlov, 2020).
In an NOS RNAi mini screen, two optic lobe-specific drivers, GMR79D04 and GMR85B12, reduced locomotor rhythmicity in DD. This phenotype was observed when NOS was downregulated constitutively in these cells but not when knockdown was restricted to adulthood. These results reinforce the idea that NO is necessary for a proper establishment of neuronal circuits. A low rhythmicity phenotype caused by NOS knockdown with the pan-neuronal driver GMR57C10-GAL4 is congruent with the above findings. Intriguingly, however, in addition to the low rhythmicity, GMR57C10 > NOS RNAi in adulthood resulted in an extended period. What might be the neuronal subsets that produce NO and regulate period length of locomotor activity? A recent study by Aso (2019), NO was shown to act as a co-transmitter in a subset of dopaminergic neurons, specifically in some of the PAMs, PPL1s and PPL2abs. It is thus possible that dopamine signaling modulated by NO is involved in the control of the locomotor activity period. It is also noteworthy that NO-mediated signaling has a profound neuromodulatory effect on spinal motor networks and regulates frequency and amplitude of motor activity in various vertebrate species. NO-mediated regulation of circadian locomotor output in flies might involve a similar mechanism (Kozlov, 2020).
DAR4-M staining showed an enrichment of NO in glial cells, including the surface glia. Targeting glial cells leads to the strongest and most persistent phenotype in locomotor activity both for gain- and loss-of-function of NOS. Among glial subpopulations, the perineurial glia appears to be the major site of NOS activity that regulates locomotor rhythms. The importance of glia in circadian rhythms have been recognized, especially those containing the molecular clocks and exert reciprocal communication with the pacemaker neural circuit. It has been shown that the perineurial glial cells harbor molecular clocks, which drive daily rhythms in the blood-brain barrier permeability but are not required for locomotor activity rhythms. This study is the first to identify NO as a signaling molecule produced in glia and mediates part of the role of glia, independently of their molecular clocks, in Drosophila circadian rhythms (Kozlov, 2020).
It has been shown that in mammals NO mediates light-induced phase-shifts through the cGMP pathway. It is an interesting parallel to note that forced production of NO in the s-LNvs caused phase shift rather than amplitude dampening. Since high levels of NO inhibit E75/UNF dimerization and E75/UNF normally enhances per transcription, it is speculated that NO-induced phase-shift may be partly mediated by the inhibition of E75/UNF (Hormone receptor 51) heterodimerization. It will be interesting to test this hypothesis in future studies. Mammalian clocks contain E75 homologs REV-ERB α/β, which repress Bmal1 transcription. Analogous to the notion in flies, NO is thought to decrease REV-ERB α/β activity. Consistently, in vitro studies in mammalian cell culture showed that excessive presence of NO increases the production of Bmal1 mRNA. These findings altogether point out that NO is an evolutionarily conserved regulator of circadian rhythms (Kozlov, 2020).
In line with recent studies, this research expands the view on the factors that participate in neuronal and molecular mechanisms of circadian rhythmicity. The finding that gaseous messenger NO contributes to the various aspects of circadian rhythmicity emphasizes that non-cell-autonomous, systemic regulation is integral to the circadian circuit operation. These results set a foundation for future studies addressing the mechanism by which NO signaling modulates the state of the pacemaker circuit and its output (Kozlov, 2020).
Membraneless RNA-protein granules play important roles in many different cell types and organisms. In particular, granules found in germ cells have been used as a paradigm to study large and dynamic granules. These germ granules contain RNA and proteins required for germline development. This study unexpectedly identified large granules in specific subtypes of glial cells ("glial granules") of the adult Drosophila brain which contain polypeptides with previously characterized roles in germ cells including scaffold Tudor, Vasa, Polar granule component and Piwi family proteins. Interestingly, super-resolution microscopy analysis shows that in the glial granules, these proteins form distinct partially overlapping clusters. Furthermore, it was shown that glial granule scaffold protein Tudor functions in silencing of transposable elements and in small regulatory piRNA biogenesis. Remarkably, the data indicate that the adult brain contains a small population of cells, which express both neuroblast and germ cell proteins. These distinct cells are evolutionarily conserved and expand during aging suggesting the existence of age-dependent signaling. This work uncovers previously unknown glial granules and indicates the involvement of their components in the regulation of brain transcriptome (Tindell, 2020).
Glial cells are early sensors of neuronal injury and can store lipids in lipid droplets under oxidative stress conditions. This study investigated the functions of the RNA-binding protein, SPEN/SHARP, in the context of Parkinson's disease (PD). Using a data-mining approach, it was found that SPEN/SHARP is one of many astrocyte-expressed genes that are significantly differentially expressed in the substantia nigra of PD patients compared with control subjects. Interestingly, the differentially expressed genes are enriched in lipid metabolism-associated genes. In a Drosophila model of PD, it was observed that flies carrying a loss-of-function allele of the ortholog split-ends (spen) or with glial cell-specific, but not neuronal-specific, spen knockdown were more sensitive to paraquat intoxication, indicating a protective role for Spen in glial cells. It was also found that Spen is a positive regulator of Notch signaling in adult Drosophila glial cells. Moreover, Spen was required to limit abnormal accumulation of lipid droplets in glial cells in a manner independent of its regulation of Notch signaling. Taken together, these results demonstrate that Spen regulates lipid metabolism and storage in glial cells and contributes to glial cell-mediated neuroprotection (Girard, 2020).
The final size and function of the adult central nervous system (CNS) are determined by neuronal lineages generated by neural stem cells (NSCs) in the developing brain. In Drosophila, NSCs called neuroblasts (NBs) reside within a specialised microenvironment called the glial niche. This study explored non-autonomous glial regulation of NB proliferation. Lipid droplets (LDs) which reside within the glial niche were shown to be closely associated with the signalling molecule Hedgehog (Hh). Under physiological conditions, cortex glial Hh is autonomously required to sustain niche chamber formation. Upon FGF-mediated cortex glial overgrowth, glial Hh non-autonomously activates Hh signalling in the NBs, which in turn disrupts NB cell cycle progression and its ability to produce neurons. Glial Hh's ability to signal to NB is further modulated by lipid storage regulator lipid storage droplet-2 (Lsd-2) and de novo lipogenesis gene fatty acid synthase 1 (Fasn1). Together, these data suggest that glial-derived Hh modified by lipid metabolism mechanisms can affect the neighbouring NB's ability to proliferate and produce neurons (Dong, 2021).
Peripheral nerves contain sensory and motor neuron axons coated by glial cells whose interplay ensures function, but molecular details are lacking. SNARE-proteins mediate the exchange and secretion of cargo by fusing vesicles with target organelles, but how glial SNAREs contribute to peripheral nerve function is largely unknown. This study identified non-neuronal Synaptobrevin (Syb) as the essential vesicular SNARE in Drosophila peripheral glia to insulate and metabolically supply neurons. Tetanus neurotoxin light chain (TeNT-LC), which potently inhibits SNARE-mediated exocytosis from neurons, also impairs peripheral nerve function when selectively expressed in glia, causing nerve disintegration, defective axonal transport, tetanic muscle hyperactivity, impaired locomotion, and lethality. While TeNT-LC disrupts neural function by cleaving neuronal Synaptobrevin (nSyb), it targets non-neuronal Synaptobrevin (Syb) in glia, which it cleaves at low rates: Glial knockdown of Syb (but not nSyb) phenocopied glial TeNT-LC expression whose effects were reverted by a TeNT-LC-insensitive Syb mutant. This study linked Syb-necessity to two distinct glial subtypes: Impairing Syb function in subperineurial glia disrupted nerve morphology, axonal transport, and locomotion, likely, because nerve-isolating septate junctions (SJs) could not form as essential SJ components (like the cell adhesion protein Neurexin-IV) were mistargeted. Interference with Syb in axon-encircling wrapping glia left nerve morphology and locomotion intact but impaired axonal transport. This study identifies crucial roles of Syb in various glial subtypes to ensure glial-glial and glial-neural interplay needed for proper nerve function, animal motility, and survival (Bohme, 2021).
The insulin-producing cells (IPCs), a group of 14 neurons in the Drosophila brain, regulate numerous processes, including energy homeostasis, lifespan, stress response, fecundity, and various behaviors, such as foraging and sleep. Despite their importance, little is known about the development and the factors that regulate morphological and functional differentiation of IPCs. This study describes the use of a new transgenic reporter to characterize the role of the Drosophila L1-CAM homolog Neuroglian (Nrg), and the transmembrane Semaphorin-1a (Sema-1a) and its receptor Plexin A (PlexA) in the differentiation of the insulin-producing neurons. Loss of Nrg results in defasciculation and abnormal neurite branching, including ectopic neurites in the IPC neurons. Cell-type specific RNAi knockdown experiments reveal that Nrg, Sema-1a and PlexA are required in IPCs and glia to control normal morphological differentiation of IPCs albeit with a stronger contribution of Nrg and Sema-1a in glia and of PlexA in the IPCs. These observations provide new insights into the development of the IPC neurons and identify a novel role for Sema-1a in glia. In addition, this study shows that Nrg, Sema-1a and PlexA in glia and IPCs not only regulate morphological but also functional differentiation of the IPCs and that the functional deficits are likely independent of the morphological phenotypes. The requirements of nrg, Sema-1a, and PlexA in IPC development and the expression of their vertebrate counterparts in the hypothalamic-pituitary axis, suggest that these functions may be evolutionarily conserved in the establishment of vertebrate endocrine systems (Clements, 2021).
Understanding how injury to the central nervous system induces de novo neurogenesis in animals would help promote regeneration in humans. Regenerative neurogenesis could originate from glia and glial neuron-glia antigen-2 (NG2) may sense injury-induced neuronal signals, but these are unknown. This study used Drosophila to search for genes functionally related to the NG2 homologue kon-tiki (kon), and identified Islet Antigen-2 (Ia-2), required in neurons for insulin secretion. Both loss and over-expression of ia-2 induced neural stem cell gene expression, injury increased ia-2 expression and induced ectopic neural stem cells. Using genetic analysis and lineage tracing, this study demonstrated that Ia-2 and Kon regulate Drosophila insulin-like peptide 6 (Dilp-6) to induce glial proliferation and neural stem cells from glia. Ectopic neural stem cells can divide, and limited de novo neurogenesis could be traced back to glial cells. Altogether, Ia-2 and Dilp-6 drive a neuron-glia relay that restores glia and reprogrammes glia into neural stem cells for regeneration (Harrison, 2021).
The central nervous system (CNS) can regenerate after injury in some animals, and this involves de novo neurogenesis. Newly formed neurons integrate into functional neural circuits, enabling the recovery of function and behaviour, which is how CNS regeneration is measured. The human CNS does not regenerate after injury. However, in principle it could, as we continue to produce new neurons throughout life that integrate into functional circuits. Through understanding the molecular mechanisms underlying natural regenerative neurogenesis in animals, it might be possible to provoke de novo neurogenesis in the human CNS to promote regeneration after damage or neurodegenerative diseases. Regenerative neurogenesis across animals may reflect an ancestral, evolutionarily conserved genetic mechanism, which manifests itself to various degrees in regenerating and non-regenerating animals. Accordingly, it may be possible to discover molecular mechanisms of injury-induced neurogenesis in the fruit-fly Drosophila, which is a powerful genetic model organism (Harrison, 2021).
Regenerative neurogenesis could occur through activation of quiescent neural stem cells, de-differentiation of neurons or glia, or direct conversion of glia to neurons. Across many regenerating animals, new neurons originate mostly from glial cells. In the mammalian CNS, radial glial cells behave like neural stem cells to produce neurons during development. Remarkably, whereas NG2-glia (also known as oligodendrocyte progenitor cells, OPCs) produce only glia (oligodendrocytes and astrocytes) in development, they can also produce neurons in the adult and upon injury, although this remains controversial. Discovering the molecular mechanisms of a neurogenic response of glia is of paramount urgency (Harrison, 2021).
NG2-glia are progenitor cells in the adult human brain, constituting 5-10% of total CNS cells, and remain proliferative throughout life. In development, NG2-glia are progenitors of astrocytes, OPCs, and oligodendrocytes, but postnatally and upon injury they can also produce neurons. They can also be directly reprogrammed into neurons that integrate into functional circuits. The diversity and functions of NG2-glia are not yet fully understood, but they are particularly close to neurons. They receive and respond to action potentials generating calcium signals, they monitor and modulate the state of neural circuits by regulating channels and secreting chondroitin sulphate proteoglycan perineural nets, and they also induce their own proliferation to generate more NG2-glia, astrocytes that sustain neuronal physiology, and oligodendrocytes that enwrap axons. NG2-glia have key roles in brain plasticity, homeostasis, and repair in close interaction with neurons, but to what extent this depends on the NG2 gene and protein, is not known (Harrison, 2021).
NG2 (also known as chondroitin sulphate proteoglycan 4, CSPG4) is expressed by NG2-glia and pericytes, but not by oligodendrocytes, neurons, or astrocytes. NG2 is a transmembrane protein that can be cleaved upon neuronal stimulation to release a large secreted extracellular domain and an intracellular domain. The intracellular domain (ICD, NG2ICD) is mostly cytoplasmic, and it induces protein translation and cell cycle progression (Nayak, 2018). NG2ICD lacks a DNA binding domain and therefore does not function as a transcription factor, but it has a nuclear WW4 domain and nuclear localisation signals and can regulate gene expression. It is thought that NG2 functions as a receptor, triggering nuclear signalling in response to ligands or partners (Sakry, 2014; Sakry and Trotter, 2016). NG2 protein is abundant in proliferating NG2-glia and glioma. It is also required for OPC proliferation and migration in development and in response to injury. Given the close relationship of NG2-glia with neurons, it is anticipated that key partners of NG2 are produced from neurons, but these remain largely unknown (Harrison, 2021).
The fruit-fly Drosophila is particularly powerful for discovering novel molecular mechanisms. The Drosophila NG2 homologue is called kon-tiki (kon) or perdido. Kon functions in glia, promotes glial proliferation and glial cell fate determination in development and upon injury, and promotes glial regeneration and CNS injury repair. Kon works in concert with the receptor Notch and the transcription factor Prospero (Pros) to drive the glial regenerative response to CNS injury. It is normally found in low levels in the larval CNS, but injury induces a Notch-dependent increase in kon expression in glia. Together, Notch signalling and Kon induce glial proliferation. Kon also initiates neuropile glial differentiation and pros expression, and Pros maintains glial cell differentiation. This glial regenerative response to injury is homeostatic and time-limited, as two negative feedback loops halt it: Kon represses Notch, and Pros represses kon expression, preventing further cell division. The relationship between these genes is also conserved in the mouse, where the homologue of pros, Prox1, is expressed together with Notch1 in NG2-glia. Following cell division, Prox1 represses NG2-glia proliferation and promotes oligodendrocyte differentiation. Together, Notch, Kon, and Pros form a homeostatic gene network that sustains neuropile glial integrity throughout life and drives glial regeneration upon injury. As Kon is upregulated upon injury and provokes glial proliferation and differentiation, it is the key driver of the glial regenerative response to CNS injury (Harrison, 2021).
A critical missing link to understand CNS regeneration was the identification of neuronal partners of glial NG2/Kon that could induce regenerative neurogenesis. Injury to the Drosophila larval CNS also resulted in spontaneous, yet incomplete, repair of the axonal neuropile. This strongly suggested that injury might also induce neuronal events, such as axonal regrowth or generation of new neurons. Thus, this study asked whether Kon may interact with neuronal factors that could contribute to regenerative neurogenesis after injury. Relay of insulin signalling involving neuronal Ia-2 and glial Kon drives in vivo reprogramming of neuropile glia into neural stem cells (Harrison, 2021).
NG2-glia are abundant progenitor cells present throughout life in the adult human brain and can respond to injury. Thus, they are the ideal cell type to manipulate to promote regeneration. However, whether NG2-glia can give rise to neurons is highly debated, and potential mechanisms remained unknown. Using Drosophila in vivo functional genetic analysis this study has identified neuronal Ia-2 as a genetic interactor of the NG2 homologue Kon and shows that it can induce a neurogenic response from glial cells via insulin signalling (Harrison, 2021).
Evidence is provided that Ia-2, Kon, and Dilp-6 induce a regenerative neurogenic response from glia (Ia-2 and Dilp-6 drive a regenerative neurogenic response to central nervous system (CNS) injury). In the un-injured CNS, Kon and Ia-2 are restricted to glia and neurons, respectively (Ia-2 and Dilp-6 drive a regenerative neurogenic response to central nervous system (CNS) injury). Ia-2 is required for neuronal Dilp-6 secretion, Dilp-6 is produced by some neurons and mostly glia, and its production depends mostly on Kon regulated glia. Alterations in Ia-2 levels, increased Dilp-6, and concerted activation of Ras or PI3Kinase downstream of insulin signalling induced ectopic neural stem cells from glia. Both loss and gain of ia-2 function induced ectopic Dpn cells. Ia-2 depends on Pros and in turn negatively regulates Pros. Pros controls the switch from neural stem cell to progenitor state. In this way, cell-cell interactions involving Ia-2 can influence neural progenitor cell fate. ia-2 loss of function would also cause a decrease in Dilp-6 secretion from neurons, but not from glia, as kon mRNA levels were unaffected, and dilp-6 expression depends mostly on glial kon. As neuronal Ia-2 and glial Kon mutually exclude each other, perhaps loss of ia-2 function might increase kon-dependent Dilp-6 production. As Ia-2 is required for Dilp-6 secretion, ia-2 GOF would increase Dilp-6 release triggering the Dilp-6 amplification loop. Conceivably, either way Dilp-6 increased and this induced Dpn. Upon injury, levels of kon and ia-2 expression increased. Ia-2 drives secretion of Dilp-6 from neurons, Dilp-6 is received by glia, and a positive feedback amplification loop drives the further Kon and InR dependent production of Dilp-6 from cortex glia. Dilp-6 can then both promote glial proliferation to generate more glia and induce the neural stem cell marker Dpn in neuropile glia -- the subset known as 'Drosophila astrocytes' and midline glia. Ectopic Dpn+ cells were induced from glia both upon injury and genetic manipulation of Ia-2, Dilp-6, Ras, and PI3Kinase. Importantly, these glial-derived neural stem cells could divide, as revealed by the S-phase marker PCNA-GFP and the mitotic marker pH3, and could generate neurons, albeit to a rather limited extent. Altogether, Dilp-6 is relayed from neurons to cortex and then to neuropile glia. This neuron-glia communication relay could enable concerted glio- and neuro-genesis, matching interacting cell populations for regeneration. Interestingly, Dilp-6 is also involved in non-autonomous relays between distinct CNS cell populations to activate neural stem cells and induce neuronal differentiation in development (Harrison, 2021).
This study has demonstrated that ectopic neural stem cells originate from glia. Regenerative neurogenesis could occur via direct conversion of glia into neurons, glial de-differentiation, or neuronal de-differentiation. Neuronal de-differentiation occurs both in mammals and in Drosophila. However, in most animals, neural stem cells in the adult CNS and upon injury are generally distinct from developmental ones, and can originate from hemocytes, but most often, glial cells. In the mammalian brain, radial glia in the hippocampus respond to environmental challenge by dividing asymmetrically to produce neural progenitors that produce neurons; and astrocytes and NG2-glia can generate neurons, particularly in response to stroke, excitoxic injury, and genetic manipulations. Furthermore, genetic manipulation can lead to the direct conversion of NG2-glia into neurons. The findings that Dilp-6 and InR signalling can induce dpn expression are reminiscent of their functions in the induction of neural stem cells from quiescent progenitors in development. However, the Dpn+ cells induced upon injury and after development are distinct from the developmental neural stem cells normally induced by Dilp-6 in multiple ways. Firstly, in injuries carried out in third instar larvae, the induced neural stem cells were more numerous than normal neural stem cells. Secondly, in injuries carried out late in wandering larvae, Dpn+ cells were found after normal developmental neural stem cells have been eliminated through apoptosis. Thirdly, Dpn+ cells were found in dorsal ectopic locations not normally occupied by developmental neural stem cells. In all injury and genetic manipulation experiments involving overexpression of either ia-2, dilp-6, or PI3K, ectopic Dpn+ cells were located along the midline and surrounding the neuropile, in positions normally occupied by glia. Remarkably, concerted overexpression of ras and dilp-6 induced Dpn in potentially all glial cells and more, consistently with further Dpn+ cell proliferation. Consistent with the current findings, ectopic neuroblasts were also observed upon co-expression of activated rasV12 and knock-down of PTEN in glia, within glioma models in Drosophila. This study has demonstrated that ectopic Dpn+ originated from glia, most particularly neuropile glia (midline glia and 'Drosophila astrocytes'). Firstly, ectopic Dpn+ cells did not have Ia-2YFP, which is expressed in all neurons. Secondly, overexpression of ia-2 or dilp-6, alone or in combination with ras and PI3K, in glia dramatically increased Dpn levels, meaning that insulin signalling induces dpn expression in glia. Thirdly, ectopic Dpn+ cells surrounding the neuropile occupied positions of astrocytes and had the pan-glial marker Repo, and Repo- Dpn+ along the midline had the midline glia marker Wrp. Fourthly, the glial origin of the ectopic Dpn+ cells was demonstrated using two cell-lineage tracing methods (G-TRACE and glial activation of the actin promoter) whereby the expression initiated from the glia repo promoter was turned permanent despite cell state transitions. Consistently with these findings, TRAP-RNA analysis of the normal third instar larva revealed expression of dpn and multiple genes involved in neuroblast polarity, asymmetric cell division, neuroblast proliferation, and neurogenesis in glia. And single cell RNAseq analysis of the larval CNS revealed that in normal larvae some Repo+ glial cells can express dpn, or other neuroblast markers like wor and ase. The current findings show that basal or potential expression of neuroblast genes in glia is switched on and amplified by insulin signalling. It is concluded that Ia-2 and Dilp-6 could reprogramme glial cells in vivo into neural stem cells (Harrison, 2021).
The data showed that the ectopic ia-2 and dilp-6 induced neural stem cells could divide and generate neurons. In fact, concomitant overexpression of dilp-6 and PI3K, and most prominently dilp-6 and ras, dramatically increased Dpn+ cell number. Dilp-6 induced glial-derived Dpn+ cells could express the S-phase marker PCNA-GFP, and Ia-2 induced Wrp+ Dpn+ cells that were pH3+ in mitosis. No mitotic cells surrounding the neuropile were detected, but mitosis is brief, and could have easily been missed. The Dilp-6 induced ectopic Dpn+ cells could generate neurons that could be traced with GFP expression from their glial origin. Thus, ectopic neural stem cells induced by Dilp-6 can divide and produce neuronal progeny cells. However, the clusters of GFP+ cells originating from the in vivo reprogrammed glial cells were rather small, indicating that although neurogenesis was possible in late larvae, it was extremely constrained. This could be due to the fact that in the third instar larva, time is rather limited by pupariation. Injury and genetic manipulation in late larvae may not allow sufficient time for cell lineages to progress, before pupariation starts. Pupariation and metamorphosis bring in a different cellular context, which could interfere with regenerative neuronal differentiation. Alternatively, Ia-2 and Dpn may not be sufficient to carry neurogenesis through either. For instance, gain of ia-2 function resulted only in Dpn+ but not Pros+ or Eve+ cells, suggesting that Ia-2 and Dpn are not sufficient for neuroblasts to progress to GMCs and neurons. In fact, ectopic Dpn+ cells still had Repo. Furthermore, other ectopic neuroblast markers, such as Wor or Ase were not detected in glia. Nevertheless, RNA seq data revealed expression of neuroblast markers, including dpn, wor, and ase in some glia in normal larval CNS, meaning they could potentially be further regulated. Still, to generate neurons, glia may not only require the expression of neural stem cell markers like dpn, but also perhaps receive other yet unknown signals. In mammals, injury creates a distinct cellular environment that prompts glial cells to generate different cell types than in the un-injured CNS. For instance, elevated Sox-2 is sufficient to directly reprogramme NG2-glia into neurons, but only upon injury. Whereas during normal development NG2-glial cells may only produce oligodendrocyte lineage cells, upon injury they can also produce astrocytes and neurons. This suggests that there are injury-induced cues for neuronal differentiation. In the future, it will be compelling to find out what signals could enhance neurogenesis from glial cells reprogrammed in vivo by insulin signalling (Harrison, 2021).
This work has revealed a novel molecular mechanism driving a regenerative neurogenic response from glia, involving Kon/NG2 and insulin signalling. Ia-2 induces an initial secretion of Dilp-6 from neurons, Dilp-6 is received by glia, and a positive feedback loop amplifies the Kon-dependent production of Dilp-6 by cortex glia, Dilp-6 is then relayed to neuropile glia, resulting in the in vivo reprogramming of glial cells into neural stem cells. This mechanism can induce both glial regeneration and neural stem cells from glia, potentially also neurons, matching interacting neuronal and glial cell populations. The incidence of neuropile glia conversion to Dpn+ cells was variable, meaning the process is stochastic. However, all glia converted when activated Ras or PI3K were combined with Dilp-6, meaning levels of insulin signalling matter. Such a mechanism may also operate in mammals. In fact, Ia-2 has universal functions in dense core vesicles to release insulin. Insulin-like growth factor 1 (IGF-1) induces the production of astrocytes, oligodendrocytes, and neurons from progenitor cells in the adult brain, in response to exercise. The transcription factor Sox-2 that can switch astrocytes to neural stem cells and produce neurons is a downstream effector of InR/AKT signalling). NG2 also interacts with downstream components of the InR signalling pathway (e.g., PI3K-Akt-mTOR) to promote cell cycle progression and regulate the expression of its downstream effectors in a positive feedback loop. Together, all of these findings indicate that Ia-2, NG2/Kon, and insulin signalling have a common function across animals in reprogramming glial cells into becoming neural stem cells (Harrison, 2021).
Intriguingly, dpn was mostly induced in neuropile associated glial cells and was only induced in other glial types with overexpression of active RasV12 together with Dilp-6. Thus, perhaps prominently neuropile glia have neurogenic potential. Of the neuropile glia, Drosophila 'astrocytes' and midline glia express Notch, pros, and kon, as well as InR. The cells frequently called 'astrocytes' share features with mammalian NG2-glia. In mammals, the combination of Notch1, Prox1, and NG2 is unique to NG2-glia and is absent from astrocytes. Perhaps Ia-2 and Dilp-6 can only induce neural stem cells from NG2-like glia bearing this combination of factors. Notch activates glial proliferation and kon expression in Drosophila, and in the mammalian CNS, Notch promotes NG2-glia proliferation and maintains the progenitor state. In Drosophila, Notch and Pros also regulate dpn expression: Notch activates dpn expression promoting stemness, and Pros inhibits it, promoting transition to GMC and neuron. Thus, only glial cells with Notch and Pros may be poised to modulate stemness and neuronal differentiation. This study showed that InR is expressed in neuropile glia, which was confirmed by publically available single cell RNAseq data. Insulin signalling represses FoxO, which represses dpn, and thus ultimately activates dpn expression. As Notch and insulin signalling positively regulate dpn expression, and injury induces a Notch-dependent upregulation of Kon, which enables dilp-6 expression, and of Ia-2, which secretes Dilp-6, the data indicate that Notch-Kon/NG2-insulin synergy triggers the activation of dpn expression. Importantly, no evidence was found that Kon functions in neural stem cells. Thus, perhaps induced neural stem cells can generate only glia from daughter cells that inherit Kon, on which Repo and glial cell fate depend, or generate neurons, from daughter cells that lack Kon, but have Pros, on which Ia-2 depends. Thus, upon injury, Notch, Pros, Kon/NG2, Ia-2, and insulin signalling function together to enable the regenerative production of both glial cells and neural stem cells from glia. Intriguingly, developmental neural stem cells are thought to be eliminated through upregulation of Pros, induction of cell cycle exit, and terminal differentiation into glia. The current findings imply that such termination may not be final (Harrison, 2021).
To conclude, a neuron-glia communication relay involving Ia-2, Dilp-6, Kon, and InR is responsible for the induction of neural stem cells from glia, their proliferation, and limited neurogenesis. Neuronal Ia-2 and Dilp-6 trigger two distinct responses in glia: (1) in cortex glial cells, insulin signalling boosts Kon-dependent amplification of Dilp-6, glial proliferation, and glial regeneration. (2) In neuropile-associated NG2-like glial cells, insulin signalling unlocks a neurogenic response, inducing neural stem cell fate. As a result, these genes can drive the production of both glial cells and neurons after injury, enabling the matching of interacting cell populations, which is essential for regeneration (Harrison, 2021).
Idiopathic Parkinson's disease is the second most common neurodegenerative disease and is estimated to be approximately 30% heritable. Genome wide association studies have revealed numerous loci associated with risk of development of Parkinson's disease. The majority of genes identified in these studies are expressed in glia at either similar or greater levels than their expression in neurons, suggesting that glia may play a role in Parkinson's disease pathogenesis. The role of individual glial risk genes in Parkinson's disease development or progression is unknown, however. It was hypothesized that some Parkinson's disease risk genes exert their effects through glia. A Drosophila model of α-synucleinopathy was developed in which gene expression can be individually expressed in neurons and glia. Human wild type α-synuclein is expressed in all neurons, and these flies develop the hallmarks of Parkinson's disease, including motor impairment, death of dopaminergic and other neurons, and α-synuclein aggregation. In these flies, a candidate genetic screen was performed, using RNAi to knockdown 14 well-validated Parkinson's disease risk genes in glia, and the effect on locomotion was measured in order to identify glial modifiers of the &alpha-synuclein phenotype. Four modifiers were identified: aux, Lrrk, Ric, and Vps13, orthologs of the human genes GAK, LRRK2, RIT2, and VPS13C, respectively. Knockdown of each gene exacerbated neurodegeneration as measured by total and dopaminergic neuron loss. Knockdown of each modifier also increased α-synuclein oligomerization. These results suggest that some Parkinson's disease risk genes exert their effects in glia and that glia can influence neuronal α-synuclein proteostasis in a non-cell-autonomous fashion. Further, this study provides proof of concept that this novel Drosophila α-synucleinopathy model can be used to study glial modifier genes, paving the way for future large unbiased screens to identify novel glial risk factors that contribute to PD risk and progression (Olsen, 2021).
Synaptic homeostatic plasticity is a foundational regulatory mechanism that maintains the stability of synaptic and neural functions within the nervous system. Impairment of homeostatic regulation has been linked to synapse destabilization during the progression of Alzheimer's disease (AD). Various glial cell types play critical roles in modulating synaptic functions both during the aging process and in the context of AD. This study investigated the impact of glial dysregulation of histone acetylation and transcriptome in AD on synaptic homeostatic plasticity, using computational analysis combined with electrophysiological methods in Drosophila. By integrating snRNA-seq and H3K9ac ChIP-seq data from the same AD patient cohort, this study pinpointed cell type-specific signature genes that were transcriptionally altered by histone acetylation. The role of these glial genes in regulating presynaptic homeostatic potentiation was subsequently investigated in Drosophila. Remarkably, nine glial-specific genes, which were identified through computational methods as targets of H3K9ac and transcriptional dysregulation, were found to be crucial for the regulation of synaptic homeostatic plasticity at the NMJ in Drosophila. This genetic evidence connects abnormal glial transcriptomic changes in AD with the impairment of homeostatic plasticity in the nervous system. In summary, these integrative computational and genetic studies highlight specific glial genes as potential key players in the homeostatic imbalance observed in AD (Cai, 2023)
Diabetic peripheral neuropathy (DPN) is the most common chronic, progressive complication of diabetes mellitus. The main symptom is sensory loss; the molecular mechanisms are not fully understood. This study found that Drosophila fed a high-sugar diet, which induces diabetes-like phenotypes, exhibit impairment of noxious heat avoidance. The impairment of heat avoidance was associated with shrinkage of the leg neurons expressing the Drosophila transient receptor potential channel Painless. Using a candidate genetic screening approach proteasome modulator 9b (CG9588)/ was identified as one of the modulators of impairment of heat avoidance. It waS further showN that proteasome inhibition in the glia reversed the impairment of noxious heat avoidance, and heat-shock proteins and endolysosomal trafficking in the glia mediated the effect of proteasome inhibition. These results establish Drosophila as a useful system for exploring molecular mechanisms of diet-induced peripheral neuropathy and propose that the glial proteasome is one of the candidate therapeutic targets for DPN (Suzuki, 2023).
Accumulation of cytoplasmic inclusions of TAR-DNA binding protein 43 (TDP-43) is seen in both neurons and glia in a range of neurodegenerative disorders, including amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD) and Alzheimer's disease (AD). Disease progression involves non-cell autonomous interactions among multiple cell types, including neurons, microglia and astrocytes. This study investigated the effects in Drosophila of inducible, glial cell type-specific TDP-43 overexpression, a model that causes TDP-43 protein pathology including loss of nuclear TDP-43 and accumulation of cytoplasmic inclusions. TDP-43 pathology in Drosophila is sufficient to cause progressive loss of each of the 5 glial sub-types. But the effects on organismal survival were most pronounced when TDP-43 pathology was induced in the perineural glia (PNG) or astrocytes. In the case of PNG, this effect is not attributable to loss of the glial population, because ablation of these glia by expression of pro-apoptotic reaper expression has relatively little impact on survival. To uncover underlying mechanisms, cell-type-specific nuclear RNA sequencing was used to characterize the transcriptional changes were identified that were induced by pathological TDP-43 expression. Numerous glial cell-type specific transcriptional changes. Notably,SF2/SRSF1 levels were found to be decreased in both PNG and in astrocytes. Further knockdown of SF2/SRSF1 in either PNG or astrocytes lessens the detrimental effects of TDP-43 pathology on lifespan, but extends survival of the glial cells. Thus TDP-43 pathology in astrocytes or PNG causes systemic effects that shorten lifespan and SF2/SRSF1 knockdown rescues the loss of these glia, and also reduces their systemic toxicity to the organism (Krupp, 2023).
A single adenosine receptor gene (dAdoR) has been detected in Drosophila melanogaster. However, its function in different cell types of the nervous system is mostly unknown. Therefore, this study overexpressed or silenced the dAdoR gene in eye photoreceptors, all neurons, or glial cells and examined the fitness of flies, the amount and daily pattern of sleep, and the influence of dAdoR silencing on Bruchpilot (BRP) presynaptic protein. Furthermore, the dAdoR and brp gene expression was examined in young and old flies. A higher level of dAdoR was found in the retina photoreceptors, all neurons, and glial cells negatively influenced the survival rate and lifespan of male and female Drosophila in a cell-dependent manner and to a different extent depending on the age of the flies. In old flies, expression of both dAdoR and brp was higher than in young ones. An excess of dAdoR in neurons improved climbing in older individuals. It also influenced sleep by lengthening nighttime sleep and siesta. In turn, silencing of dAdoR decreased the lifespan of flies, although it increased the survival rate of young flies. It hindered the climbing of older males and females, but did not change sleep. Silencing also affected the daily pattern of BRP abundance, especially when dAdoR expression was decreased in glial cells. The obtained results indicate the role of adenosine and dAdoR in the regulation of fitness in flies that is based on communication between neurons and glial cells, and the effect of glial cells on synapses (Bhattacharya, 2023).
Synthesis of cytochrome c oxidase (Scox) is a Drosophila homolog of human SCO2 encoding a metallochaperone that transports copper to cytochrome c, and is an essential protein for the assembly of cytochrome c oxidase in the mitochondrial respiratory chain complex. SCO2 is highly conserved in a wide variety of species across prokaryotes and eukaryotes, and mutations in SCO2 are known to cause mitochondrial diseases such as fatal infantile cardioencephalomyopathy, Leigh syndrome, and Charcot-Marie-Tooth disease, a neurodegenerative disorder. These diseases have a common symptom of locomotive dysfunction. However, the mechanisms of their pathogenesis remain unknown, and no fundamental medications or therapies have been established for these diseases. This study demonstrated that the glial cell-specific knockdown of Scox perturbs the mitochondrial morphology and function, and locomotive behavior in Drosophila. In addition, the morphology and function of synapses were impaired in the glial cell-specific Scox knockdown. Furthermore, Scox knockdown in ensheathing glia, one type of glial cell in Drosophila, resulted in larval and adult locomotive dysfunction. This study suggests that the impairment of Scox in glial cells in the Drosophila CNS mimics the pathological phenotypes observed by mutations in the SCO2 gene in humans (Kowada, 2021).
