InteractiveFly: GeneBrief

spinster: Biological Overview | Developmental Biology | Effects of Mutation | References

BIOLOGICAL OVERVIEW

Mutations in the spinster gene are characterized by an extraordinarily strong rejection behavior of female flies in response to male courtship. They are also accompanied by decreases in the viability, adult life span, and oviposition rate of the flies. In spin mutants, persistence of unnecessary cells results in the degeneration of oocytes in the ovaries and of neurons in the CNS. This cell death is preceded by reductions in programmed cell death of nurse cells in ovaries and of neurons in the pupal nervous system. The Spin protein is therefore postulated to function in mediating apoptotic signals conveyed from follicle cells to nurse cells or from glia to neurons. The accumulation of lipofuscin-like materials in spin mutant neurons further implies the possible function of the spin gene in regulating lysosomal turnover in nerve cells. The spin locus generates at least five different transcripts, with only two of these being able to rescue the spin behavioral phenotype; each encodes a protein with multiple membrane-spanning domains that are expressed in both the surface glial cells in the CNS and the follicle cells in the ovaries. Orthologs of the spin gene have also been identified in a number of species from nematodes to humans. Analysis of the spin mutant should provide new insights into neurodegenerative diseases and aging (Nakano, 2001).

spin mutant flies have a long abdominal ganglion. In an attempt at determining the cellular basis for the behavioral phenotypes of the spin mutant, the morphology of the CNS was examined and it was found that the abdominal ganglia in spin^P1 mutant flies of both sexes are longer than those of the wild type. The thoracic segment of the ventral nerve cord (VNC) appears normal, while the abdominal segment is abnormally long in the spin^P1 mutant. The ratio of the length of the posterior part over the total length of the VNC was estimated as follows: for wild-type males, 0.260 ± 0.006 (mean ±); for wild-type females, 0.265 ± 0.004 ; for spin males, 0.343 ± 0.016, and for spin females, 0.338 ± 0.010 (n = 23). The unshortened morphology of the VNC is still evident 2 weeks after eclosion in spin^P1 flies. Apart from the VNC, no gross morphological abnormalities were found in spin mutant flies (Nakano, 2001).

Programmed cell death (PCD) of the neurons in the VNC occurs shortly after pupariation and after the emergence of the adult; typically, the ventral abdominal ganglion cells die during metamorphosis and after eclosion. The abdominal portion of the VNC shortens after the first wave of PCD in the mid-pupal stage. The absence of condensation of the abdominal ganglia in the spin mutants implies that some cells in the VNC are prevented from undergoing PCD. To evaluate this possibility, the numbers of cells undergoing apoptosis between the spin heterozygous and homozygous VNCs were compared by selectively staining fragmented DNA using TUNEL. PCD in the VNC occurs in two phases, one during the first several hours after pupation and the other immediately after adult eclosion. No difference was found in the numbers of apoptotic cells between the spin heterozygote and homozygote after adult emergence. In contrast, the profile of PCD in the VNC of the spin homozygote is distinctly different from that in the spin heterozygote during the pupal stage: the number of cells undergoing PCD in heterozygotes is maximum at 6 h after puparium formation (APF) as reported for the wild type, whereas the number of apoptotic cells remains quite low at this developmental stage in homozygotes (Nakano, 2001).

The difference in the numbers of the cells undergoing apoptosis at 6 h APF between heterozygotes and homozygotes is statistically significant. No significant difference was found in the number of apoptotic cells between heterozygotes and homozygotes at 4 and 24 h APF. These observations suggest that many cells that will be eliminated at 6 h APF remain alive through the pupal stage in the spin mutant (Nakano, 2001).

spin mutations interfere with PCD in ovaries as well as in the nervous system. These cellular defects should contribute at least in part to behavioral phenotypes of spin mutant female flies, i.e., the reduction in oviposition and sexual receptivity. It is evident that some immature oocytes degenerate in spin mutant ovaries; thus, the total number of eggs available for oviposition decreases. However, this finding does not necessarily exclude the possibility that the spin mutant female flies are accompanied by some other deficits that hamper normal oviposition. It is important to note that the spin gene product is expressed in the follicle cells of ovaries and not in nurse cells or oocytes. This observation suggests that the Spin protein is required by follicle cells for inducing the PCD of nurse cells, a group of support cells which are crucial for the normal development of oocytes. The degeneration of immature oocytes in spin mutant female flies may thus result from the disturbance of the process of PCD of nurse cells as a consequence of the loss of Spin from follicle cells (Nakano, 2001).

The link between the PCD defect in the nervous system and enhanced mate refusal in spin mutant female flies is less obvious. By analogy with the effect on ovaries, it is conceivable that the spin mutations likely affect female sexual behavior through degeneration of cells in the nervous system. Indeed, neurodegeneration is widespread in the spin^P1 mutant nervous system, which accumulates lipofuscin-like materials. In the spin mutant, the neurodegenerative phenotype is observed in both sexes, but germ cell abnormality and abnormal sexual behavior are found to be female specific. It may be that changes in female sexual receptivity are the easiest parameter by which to detect the malfunctioning of the nervous system due to the accumulation of lipofuscin-like materials in neurons. Alternatively, the reduced receptivity to copulation in spin^P1 mutant females and the neurodegeneration observed in both sexes of the spin mutant flies might be unrelated. This issue remains to be resolved by future analysis of spin mutant flies (Nakano, 2001).

There are interesting parallels in the effect of spin mutations on ovaries and the nervous system. Like those in the ovaries, PCD defects in the nervous system are most pronounced in cells not expressing the spin gene product, i.e., neurons. In the nervous system, the spin gene is expressed exclusively in glial cells that play a number of different roles in the development and maintenance of the nervous system. The distribution of the observed autofluorescent material in the nervous system does not correlate with the distribution of the Spin-expressing cells; in addition, the time-dependent accumulation of lipofuscin-like materials is observed in most of the neuronal and glial cells in spin^P1 mutant flies. Thus, the spin gene product is expressed in follicle cells in ovaries and glial cells, particularly surface glial cells, in the CNS, which enwrap nurse cells and neurons, respectively. Loss of spin function of follicle cells and glial cells leads to suppression of PCD in nurse cells and neurons. These observations lead to the idea that the Spin protein functions in glial cells in the regulation of PCD of neurons, and the failure of PCD at the proper developmental timing leads to widespread neurodegeneration accompanied by accumulation of lipofuscin-like materials at a later stage (Nakano, 2001).

The most striking effect of spin mutations on neuronal PCD is found in the VNC at 6 h APF, at which time the number of cells undergoing PCD reaches a maximum in the wild type. In spin mutant pupae, the number of cells undergoing apoptosis is 60% less than that in wild-type pupae. Since no later or earlier peaks of PCD are observed in the spin mutant, the cells to be eliminated must remain there throughout the pupal and adult stages. The presence of supernumerary neurons would perturb the formation of adult neural circuits that are established during the pupal stage (Nakano, 2001).

In fact, the structure of the nervous system of spin mutants is aberrant even when it is observed at the level of gross anatomy. The abdominal segment of the VNC is remarkably longer in the mutant than in the wild type. This part of the VNC is shortened at the mid-pupal stage. The extent of PCD of neurons influences the shortening of the VNC. For example, the VNC does not shorten in the flies hemizygous for the H99 deficiency, in which three PCD genes, hid, rpr, and grim, are deleted, and thus PCD in the VNC is prevented. Furthermore, the VNCs of H99 hemizygous pupae contain autofluorescent material. These results support the idea that the long-abdominal-ganglion phenotype of spin mutants results from the reduced extent of PCD in the pupal VNC (Nakano, 2001).

In moth pupa, damage to glial cells interferes with the shortening of the VNC. The metamorphic shortening of the moth VNC cultured in vitro is accelerated by the insect steroid hormone ecdysteroid. Considering all these observations, it is postulated that the Spin protein functions in glial cells to induce PCD of neurons during metamorphosis, which is under the control of the humoral hormone ecdysteroid (Nakano, 2001 and references therein).

Widespread neurodegeneration and accumulation of lipofuscin-like materials are important features of the nervous systems of spin mutant adults. Although such neurodegeneration might be a secondary result of PCD defects, it provides a unique model to study the basis for some human diseases with similar phenotypes. Both histological and biochemical analyses indicate that accumulated materials in spin flies are very similar to lipofuscin. Lipofuscin was discovered as an aging pigment more than a century ago. However, the mechanism of lipofuscinogenesis has not yet been fully elucidated. In humans, the abnormal accumulation of lipofuscin has been reported for several neurodegenerative disorders such as the neural ceroid lipofuscinoses and Tay-Sachs disease. Both are categorized as lysosomal storage diseases. Although two human spin orthologs have been cloned and mapped to regions on chromosomes 16 and 17, these two loci do not match the mapped loci of the known disease holders. However, there are still many unidentified diseases that will probably be classified as lysosomal storage diseases; therefore, spin orthologs will be good candidate genes for these diseases (Nakano, 2001).

Interestingly, spin mutant flies exhibit a shortened life span. One Drosophila mutant, eggroll, has a very similar phenotype to that produced by the spin mutation; such mutants exhibit a shortened life span and have multilamellar structures in the CNS. These phenotypes are similar to some human neurodegenerative diseases such as Tay-Sachs disease and Niemann-Pick sphingomyelin storage diseases. Therefore, the study of the spin and eggroll mutants should provide a good tool for understanding the relationship between aging and lipofuscinogenesis (Nakano, 2001).

Spinster controls Dpp signaling during glial migration in the Drosophila eye

The development of multicellular organisms requires the well balanced and coordinated migration of many cell types. This is of particular importance within the developing nervous system, where glial cells often move long distances to reach their targets. The majority of glial cells in the peripheral nervous system of the Drosophila embryo is derived from the CNS and migrates along motor axons toward their targets. In the developing Drosophila eye, CNS-derived glial cells move outward toward the nascent photoreceptor cells, but the molecular mechanisms coupling the migration of glial cells with the growth of the eye imaginal disc are mostly unknown. This study used an enhancer trap approach to identify the gene spinster, which encodes a multipass transmembrane protein involved in endosome-lysosome trafficking, as being expressed in many glial cells. spinster mutants are characterized by glial overmigration. Genetic experiments demonstrate that Spinster modulates the activity of several signaling cascades. Within the migrating perineurial glial cells, Spinster is required to downregulate Dpp (Decapentaplegic) signaling activity, which ceases migratory abilities. In addition, Spinster affects the growth of the carpet cell, which indirectly modulates glial migration (Yuva-Aydemir, 2011).

During development of the nervous system, glia cell migration is tightly regulated in time and space. This study has demonstrated that, within the Drosophila visual system, glial migration requires the late endosomal/lysosomal protein Spinster to restrict a migratory stimulus provided by Dpp signaling. Spinster is crucially involved in the coordination of eye disc growth and glial migration (see Schematic view on spinster function). In spinster mutant eye discs, late endosomes accumulate and a glial overmigration phenotype develops. This migration phenotype can be modified by reducing the early to late endosomal transfer or late endosome-to-lysosome transfer through decreasing the levels of hrs. Moreover, the Dpp receptor Thickveins accumulates in spinster mutant cells. Thus, Spinster appears to antagonize Dpp signaling by facilitating the routing of Dpp receptors toward the lysosome (Yuva-Aydemir, 2011).

Retinal glial cells are born in the CNS and migrate onto the eye disc through the optic stalk. A reverse migratory direction is taken by the photoreceptor cell axons, which are born in the periphery and navigate toward the brain through the optic stalk. Peripheral glial cells generally follow axonal growth cones during their migration phase (Klämbt, 2009), and thus within the developing Drosophila visual system, glial cells have to be guided to the nascent photoreceptor axons by other means. In Drosophila, this is accomplished by the carpet cells, very large subperineurial cells that cover the entire eye field (Silies, 2007). The carpet cell shields the navigating axons within the stalk from the proliferating and migrating glial population. At the beginning of eye imaginal disc development, the carpet cell prevents precocious migration of glial cells onto the eye disc in a process requiring early eye patterning genes and Hedgehog signaling. Ablation of the carpet glia as well as disruption of, for example, early Hedgehog signaling result in an overshooting glial migration (Silies, 2007). As soon as the morphogenetic furrow is initiated, the carpet glial cell starts to grow and extends anteriorly. However, the carpet cell never reaches the morphogenetic furrow but stops to grow at a few cell rows behind where nascent photoreceptor cell axons are formed. Piggybacking on the carpet cell are the perineurial glial cells that migrate toward the anterior edge of the carpet cell. As soon as they come in contact with photoreceptor axons, they drop from the carpet cell and now follow the sensory axon toward the brain. Thus, two distinct phases of glial cell migration can be defined. Initially, the carpet cell prevents precocious migration, whereas in later phases of eye imaginal disc development the carpet cells provide a permissive substrate for the migrating perineurial glia (Yuva-Aydemir, 2011).

Spinster is involved in the regulation of both of these migratory phases and helps to coordinate the growth of the eye disc with the migration of glial cells. The most dramatic consequences of spinster mutants as seen in eye disc duplications are presumably attributable to effects on Wingless and Dpp within the eye disc. The broad glial overshooting phenotypes correlate with a failure of the carpet cell to prevent early-onset, precocious glial migration. The later glial overmigration phenotypes correlate with late, carpet cell-independent, defects in the perineurial glia. Thus, it is not surprising that, in contrast to panglial expression of Spinster, glial cell type-specific expression is not able to rescue the spinster overmigration phenotype (Yuva-Aydemir, 2011).

The size of the carpet glial cell is reduced in spinster mutants, and MARCM analysis as well as cell type-specific RNAi experiments indicate that spinster controls carpet cell growth cell autonomously. Directed expression of Hid or RicinA specifically in carpet cells resulted in a reduced cell size and a concomitant induction of ectopic glial cell migration (Silies, 2007). In contrast, single spinster mutant carpet cells did not lead to a glial migration defects, suggesting that the reduced carpet cell only contributes to the glial overmigration phenotype. How spinster controls the size of the carpet cells is currently unknown. Possibly, within the carpet cell, spinster acts via hedgehog signaling. Hedgehog bound to the Patched receptor is normally internalized and degraded in lysosomes. Moreover, Patched is expressed in glial cells and Hh signaling was suggested to prevent precocious glial cell migration through a yet-unknown pathway. However, mutations in different hedgehog signaling components did not suppress the spinster phenotype and inhibition of hedgehog signaling in glial cells did neither alter the morphology of the carpet glia nor did they affect glial migration. Likewise, activation of Hedgehog signaling through expression of PKA^dsRNA did not influence glial migration (Yuva-Aydemir, 2011).

Within the carpet cells, Spinster most likely does also not act through altered Dpp signaling, since no phospho-Mad expression was detected in the carpet cells. In contrast, enhanced Dpp signaling (Mad phosphorylation) was noted in the migrating perineurial glial cells. The analysis of additional mutations affecting different aspects of intracellular vesicle dynamics revealed the importance of endocytic processes in TGF-β signaling. The block of Rab5 function causes a reduction in the range of Dpp target gene activation, and Rab5 overexpression causes increased Dpp signaling range. Hrs, a component of ESCRT complex, has been shown to interact with Smad2 and participates in the recruitment of Smad2 to the activated receptor. Hrs seems to be involved in the constitutive ligand-independent receptor turnover since Tkv accumulates at the cell membrane even in the absence of Dpp. Hrs downregulation suppresses the spinster glial overmigration, possibly by inhibiting Tkv internalization and accumulation in the endosomes. Within the cell, endosome-to-lysosome transfer and subsequent lysosomal degradation also affects Dpp signaling. Enhanced degradation caused by expression of activated Rab7 reduces the Dpp signaling range, whereas the inhibition of lysosome function with chloroquine leads to an endosomal accumulation of ligands. In addition, pan-glial expression of a dominant-negative Tkv receptor, tkv^?GS or tkv^dsRNA, resulted in reduced glial migration and fewer glial cells. Similar results were obtained when put or mad expression were silenced using RNA interference, demonstrating that the regulation of Dpp signaling via controlling vesicle dynamics is required in glial cells to ensure normal proliferation and migration (Yuva-Aydemir, 2011).

Endocytosis and vesicle recycling is generally required in migrating cells to dynamically remodel their adhesive contacts and locate active signaling receptors to the front of the cell in response to extracellular signals. Integrins from the cell rear can be relocalized to the leading edge, and during border cell migration in the Drosophila ovary, spatial restriction of the receptor tyrosine kinase signaling by endocytosis ensures the localized intracellular response to guidance cues. Likewise, glial migration in the embryonic Drosophila PNS is regulated by the fine-tuning of Notch signaling by Numb-mediated endocytosis. Thus, endocytotic trafficking may affect cell migration through several pathways (Yuva-Aydemir, 2011).

In spin mutants, enhanced Dpp signaling is also observed in the eye imaginal disc and the most extreme eye disc phenotype appears as to be an induction of an ectopic morphogenetic furrow perpendicular to the normal furrow. Ectopic expression of the Dpp in the eye disc or expression of activated Tkv in the glial cells increases glial cell proliferation. In spin mutants, both increased Dpp expression and accumulation of Tkv are observed. Since spin controls glial cell proliferation cell autonomously, this is likely attributable to the accumulation of Tkv in glial cells. In addition, enhanced Mad phosphorylation is observed in spin mutants, especially in the optic stalk glia. In agreement with this, the increase in glial cell number is mostly noted in the optic stalk. Interestingly, the function of spinster in controlling glial differentiation is also required in the wrapping glia. Loss of spinster results in a reduced wrapping of axonal membranes, which can be rescued by reexpression of spinster in the wrapping glia. Thus, spinster function appears to be needed to extend cellular processes during migration and differentiation (Yuva-Aydemir, 2011).

In conclusion, Spinster affects glial migration on two different levels. Spinster regulates carpet glia differentiation, which indirectly affects early migration, and subsequently, Spinster acts in the perineurial glia to confer a general cell motility signal (Yuva-Aydemir, 2011).

DEVELOPMENTAL BIOLOGY

The expression pattern of the spin gene was analyzed by Northern blotting. A 3-kb transcript is observed throughout development, although the level of expression is very low in the embryonic and second-instar larval stages. The relative amount of each type of transcript was examined by RT-PCR methods. The type III and type IV transcripts are abundant, while types I and II are expressed at a moderate level and type V is very rare. No major differences were found during development between the sexes or between wild-type and spin^P1 flies in this experiment. The spatial expression of the spin transcripts was examined by whole-mount in situ hybridization using an antisense RNA or cDNA probe that was able to detect all five types of transcript. The spin transcripts are detectable at the beginning of the germ band retraction (stage 12) in a subset of cells in the ventral nerve cord and the brain, and this expression pattern continued throughout development (Nakano, 2001).

In order to establish the identity of the Spin-expressing cells, double staining was performed using an anti-Repo antibody and an antisense probe to spin mRNA. Repo is a glia-specific homeobox protein expressed in all glial cells except for the midline glial and two segmental nerve root glial cells. More than 95% of Spin-expressing cells overlapped with Repo-expressing cells in the VNC and the brain. This expression pattern was confirmed by using spin^P2, which carries a P-element with an enhancer trap reporter inserted in the middle of the first exon of the spin gene. The ß-Gal expression pattern observed in the embryos and the third-instar larval brain of the spin^P2 heterozygotes was found to correspond well with spin expression as detected by in situ hybridization. The spin gene is expressed in the surface glial cells, which include the peripheral exit glia, the subperineural glia, and the channel glia of the nervous system. In addition, ß-Gal expression in spin^P2/CyO flies in the larval and pupal stages is also observed in the trachea, gut, salivary glands, and ring gland (Nakano, 2001).

The expression of the spin gene is also observed in adult ovaries; expression is observed in the follicle cells in the manner of dorsal-ventral and anterior-posterior gradients but not in the nurse cells or the oocyte. This expression pattern has also been confirmed by in situ hybridization (Nakano, 2001).

spin is expressed in both motoneurons and muscle throughout the period of synaptic growth and development at the NMJ. The expression of spin was first assessed by embryonic in situ hybridization. Expression was observed throughout the CNS, including motoneurons. Weak expression was observed in embryonic muscle as well as other tissues. The larval expression of spin is of particular interest since this is the time period of synaptic growth. Unfortunately, RNA in situ analysis in the larval CNS and muscle is particularly difficult to interpret. Therefore, the spin expression pattern was determined in larvae by driving a GFP-tagged spin transgene (the same isoform used in the rescue experiments) with a spin promoter—GAL4 fusion (Nakano, 2001). When spin-GAL4 is used to drive expression of UAS-spin-GFP, Spin-GFP expression is observed throughout the larval CNS with pronounced expression in motoneurons. Strong expression is also observed in all body wall muscle as well as other tissues, including a subset of epithelial cells and the salivary glands. In these experiments, Spin-GFP localizes to a peri-nuclear region in both larval neurons and larval muscle. Spin-GFP fluorescence is also observed throughout the muscle and is observed in the nerves that include both sensory and motor axons. Because Spin-GFP puncta are present in both the nerve and underlying muscle, it is difficult to determine whether Spin-GFP puncta are present within the presynaptic nerve terminal in these experiments. These GFP-positive puncta are suggestive of a late endosomal localization pattern in nerve and muscle (Sweeney, 2002).

To assess the localization of the endogenous Spin protein, an antibody was raised against Spin using a combination of two peptides, one being an N-terminal peptide represented in all splice forms of the protein and the second being a peptide to a region between transmembrane domains 11 and 12 that is present in four of the five isoforms of spin. The antibody detects the same widely distributed punctate staining pattern that is observed when UAS-spin-GFP expression is driven by spin-GAL4. Spin immunoreactive puncta are observed in a peri-nuclear pattern in neurons and muscle, and these puncta are widely distributed throughout muscle fibers. In addition, Spin puncta are present within the presynaptic nerve terminal. The NMJ was co-stained with anti-Synapsin and anti-Spin. Three-dimensional optical reconstruction of individual synaptic boutons by confocal microscopy demonstrates that Spin immunoreactive puncta are present within the volume of the presynaptic Synapsin staining. To further demonstrate the presence of Spin in the presynaptic nerve terminal, animals expressing Spin-GFP presynaptically were fixed and stained and then costained with anti-Synapsin. Spin-GFP puncta are clearly present within the presynaptic nerve terminal (Sweeney, 2002).

The size and distribution of the Spin-positive puncta are suggestive of a late endosomal/lysosomal distribution. Colocalization of Spin with known endosomal and lysosomal markers was tested. Unfortunately, there is a paucity of endosomal/lysosomal markers in Drosophila. Therefore, the subcellular localization of Spin-GFP was examined in mammalian cells. Near perfect colocalization of Spin-GFP with LAMP-1 (a lysosomal marker) is found in HeLa cells, indicating that Spin is localized to the lysosome in these cells. The lysosomal localization of Spin-GFP is not influenced by where the GFP is located on the transgene. Unfortunately, anti-LAMP-1 does not recognize Drosophila lysosomes, and there are no other known lysosomal markers in the fly. However, Spin-GFP (driven by spin-GAL4) is specifically localized to a low pH compartment identified by the lysotracker vital dye. The perinuclear localization of this low pH compartment, and its scattered distribution radiating from the nucleus, is consistent with the localization of the late endosomal/lysosomal compartment in skeletal muscle as determined by electron microscopy. In these experiments, UAS-spin-GFP is driven by spin-GAL4, and the GFP localization in these animals closely matches the endogenous protein distribution detected with the antibody. The location of muscle nuclei in these experiments was determined by visualization with Nomarski optics. These data suggest that Spin is localized to a lysosomal compartment in vivo (Sweeney, 2002).

A battery of endosomal markers was tested for colocalization with Spin-GFP and anti-Spin. Partial colocalization of Spin was observed with anti-Hrs, particularly in the peri-nuclear region in neurons and muscle. It is notable that Hrs immunoreactivity in muscle is concentrated to the subsynaptic reticulum (SSR), a series of postsynaptic muscle folds of unknown function, whereas Spin does not localize to this site. Colocalization with anti-Rab5 (early endosome), anti-Hook (late endosome), and anti-Deep Orange (Dor; late endosome) was examined. Partial overlap is observed between Spin and both anti-Hook and anti-Dor. However, more frequently these markers reside in a vesicle-like compartment that appears immediately adjacent to the Spin vesicle-like compartment. No colocalization or juxtaposition of Spin with anti-Rab5 is observed. Taken together, these data support the conclusion that Spin localizes to a late endosomal/lysosomal compartment in Drosophila muscle. In motoneurons, the perinuclear localization is also consistent with a late endosomal/lysosomal localization. The identification of lysosomes at the synapse is more controversial, though lysosomes have been observed at newly formed and developing synapses and have been found to distribute down axons, being concentrated at the nodes of Ranvier. At the Drosophila NMJ, electron microscopy demonstrates that multivesicular bodies (MVB) are present within wild-type synaptic terminals at the NMJ. These MVB are sparsely distributed throughout synaptic boutons in a manner that is consistent with anti-Spin immunoreactivity. It is concluded, therefore, that Spin identifies a late endosomal/lysosomal compartment in muscle and within the presynaptic nerve terminal (Sweeney, 2002).

The localization of Spin to a late endosomal/lysosomal compartment, and the previous evidence that spin mutations are associated with accumulation of ceroid lipofuscin (Nakano, 2001) prompted an examination of the late endosomal architecture in nerve and muscle in spin mutations. Lysotracker was used to compare the late endosomal compartments in wild-type and spin mutant backgrounds. A dramatic expansion of a low pH compartment was observed in spin mutant muscle. The peri-nuclear localization and banding pattern of this low pH compartment in muscle is consistent with the localization of late endosomes/lysosomes in vertebrate skeletal muscle as determined by electron microscopy (Sweeney, 2002).

Importantly, a dramatic expansion of a low pH compartment is observed within the presynaptic nerve terminal. In these experiments, FITC-conjugated anti-HRP was used in combination with the lysotracker in a live staining protocol. FITC-anti-HRP efficiently stains extracellular epitopes on the presynaptic membrane during the time of lysotracker staining. This allows the visualization of lysotracker-positive compartments that are present within the presynaptic nerve terminal. At spin mutant NMJ, there are increased numbers of presynaptic lysotracker-positive puncta, and most notably, these puncta are significantly enlarged in size. At spin synapses, nearly all of the large boutons contain a large low pH compartment. Lysotracker staining of the wild-type synaptic terminals rarely identified such a compartment. However, on occasion these compartments were observed at one or two boutons within a wild-type synapse. Three-dimensional reconstruction of lysotracker-positive synaptic boutons demonstrates that the low pH compartments are present within the volume of the presynaptic bouton. Taken together these data demonstrate a dramatic expansion and alteration of the late endosomal compartment in both the presynaptic nerve-terminal and in muscle. Endosomal expansion has been observed in Hrs mutations in Drosophila embryos and mice. Giant endosome/lysosomes have also been observed in patients with Chediak-Higashi syndrome and in mutant beige mice. These structures are thought to arise from dysregulated homotypic fusion. It is hypothesized, therefore, that normal late endosomal function is perturbed in the spin mutant background (Sweeney, 2002).

To further investigate the expansion of the late endosomal system, the staining intensity of a variety of late endosomal markers in muscle was assessed, comparing wild-type with spin mutant muscle. Anti-Hrs, anti-Deep Orange, and anti-Cathepsin-L all have significantly elevated staining intensity in spin mutant muscle and presynaptic terminals compared to wild-type. Although the staining intensity is increased, the staining pattern is grossly normal for these proteins with the exception of anti-Dor. Anti-Dor staining is normally concentrated to the SSR at the postsynaptic side of the synapse. In spin mutant muscle anti-Dor is no longer strongly concentrated to the SSR but is now distributed throughout the muscle, giving the appearance that Dor is no longer localized to the SSR. Since Dor expression is elevated throughout the muscle, it is hypothesized that this reflects a redistribution of this protein throughout the endosomal system rather than a loss of Dor from the postsynaptic membranes. Finally, there is no change in the expression or distribution of the early endosomal protein Rab5, indicating that these changes are specific to the late endosomal compartment. These data further support the conclusion that the loss of spin causes an expansion and possibly a disruption of the late endosome compartment in Drosophila (Sweeney, 2002).

EFFECTS OF MUTATION

Mutations in the spinster gene are characterized by an extraordinarily strong rejection behavior of female flies in response to male courtship. Single male and female pairs were placed in a plastic syringe for 1 h. During this time the mating success was measured; it was found that, while 70% of the wild-type pairs copulated, only 4% of the spin^P1 mutant females that were paired with wild-type males copulate under the same conditions. Females of a revertant line, obtained by P-element excision, exhibited essentially the same level of mating success as the wild-type females. This result demonstrates that the spinster courtship phenotype is caused by the P-element insertion, since excision is able to restore normal receptivity in females. The intensity of the male courtship can be quantified by the SAPI; this index represents the percentage of time spent by the male performing unilateral wing vibration during a 10-min observation period. The SAPI is almost the same for wild-type, spin^P1, and revertant pairs, thus indicating that the females of these strains are able to elicit similar levels of courtship from the males. This means that the low mating success observed in spin^P1 females cannot be accounted for by reduced attractiveness but rather that the low mating success may reflect the unwillingness of the spin^P1 females to copulate (Nakano, 2001).

Indeed, the spin^P1 females consistently display a number of rejection responses against the courting males; these included fending, kicking, flicking, curling, punching, and decamping; ovipositor extrusion (a normal rejection behaviour, in response to courting males or males that had previously performed courtship) is rarely seen. The pattern of rejection displayed by spin^P1 females resembles that of immature wild-type virgin females rather than that of fertilized females, in that extrusion does not occur. However, the spin^P1 females do exhibit kicking and curling behavior much more frequently than the wild-type females. In response to approaching males, the spin^P1 females tend to raise their abdomens while spreading their vaginal plates. This spreading is unique to the spin mutant females and is also distinctly different from extrusion, in which the ovipositor protrudes from the female terminalia. Furthermore, the spin^P1 female often rushes toward the courting male, pushing the male's head with her forelegs; this aggressive behavior is termed punching and is rare among wild-type females. A similarly pronounced refusal of suitors is observed in heteroallelic spin^P1/spin^P2 females. spin^P1 male flies exhibit no obvious abnormality in their courtship behavior, while general locomotive activity is reduced in both sexes (12% reduction in females and 38% reduction in males 3 days old) (Nakano, 2001).

Apart from the regulation of female sexual behavior, the spin gene plays an additional vital role, since the partial loss-of-function mutation (spin^P1) reduces viability and life span and the spin^P2 mutation is lethal, yielding no adult flies. This reduction in viability caused by the spin^P1 mutation is actually more extreme in males than in females (the viability of females is 22% and that of males is 11%), resulting in an uneven sex ratio (females/males, 2:1) of the emerged homozygous adults (Nakano, 2001).

During the tissue staining of spin^P1 flies, the existence of autofluorescent material in the blue channel was observed. In the VNC, the autofluorescent material is observed mainly in the central region of the VNC, particularly in the abdominal ganglia of pupae and adults. The lethal allele, spin^P2, shows an earlier onset of the accumulation of autofluorescent materials in the CNS (from larval stages). In the brain, this material is observed in the central brain and in the optic lobe. Part of the autofluorescence overlaps with spin gene expression; however, most does not. EM analysis of the VNC cells in spin^P1 homozygotes and heterozygotes at the early pupal stage and 24 h after eclosion reveals that spin mutant samples exhibit cellular disorganization in that most of the spin mutant cells, including both the neurons and glial cells, contain multilamellate bodies and electron-dense lobulated granules; these structures are never observed in heterozygote cells. These aberrant structures are observed in the CNS from the early pupal stage and increase thereafter in spin^P1 homozygotes. The early pupal VNC contains electron-dense lobulated granules, which appear to be precursors of the multilamellate bodies seen in the adult VNC. These structures are observed in both sexes, and no differences are observed between the sexes at the cellular level. Neither structure is found in gut or muscle cells from the spin mutant; in addition, the nucleus, mitochondria, and endoplasmic reticulum all appear to be normal in the spin mutant. The aberrant structures contained within spin mutant nerve cells are found to be very similar to lipofuscin, which is known to be induced by oxidative stress, some proteinase inhibitors, inherited lysosomal storage diseases, and the normal aging process (Nakano, 2001).

In order to identify the nature of these materials, biochemical analyses were performed. A large proportion of fluorescent pigments in tissues can be extracted by a chloroform-methanol solution and a sensitive fluorometric assay is available for the measurement of fluorescent lipid peroxidation products that have accumulated in the various tissues. Lipids were extracted from the heads of spin and wild-type flies and fluorescence spectra of the lipid extracts were then measured. The lipid extracts from the heads of spin flies have an excitation maximum at 368 nm and an emission maximum at 450 nm; these are characteristic of those observed with the lipofuscin pigments. The fluorescence intensity of the spin flies was 3.1 times higher than that of wild-type flies, suggesting that the accumulation of lipid-soluble lipofuscin-like substances was significant in spin flies. The accumulation of lipid peroxides was also examined using a thiobarbituric acid assay. The amounts of thiobarbituric acid-reactive substances in the homogenates and the lipid extracts from the heads of spin flies were found to be 30.2 ± 0.17 pmol/mg of head and 104.3 ± 43.6 pmol/mg of head respectively. These values are significantly higher than those observed with wild-type flies, namely, 21.5 ± 0.24 and 51.0 ± 6.3 pmol/mg of head, respectively. These results clearly indicate that the chemical nature of the lipofuscin-like pigments that accumulates in spin flies is quite similar to that reported for lipofuscin pigments in various mammalian tissues (Nakano, 2001).

spin^P1 females not only exhibit very strong rejection behavior toward courting males, but they also rarely lay eggs. However, the distribution of motor nerve endings along the uterine muscles was found to be normal. In order to evaluate the possibility that spin^P1 mutants are defective in egg production, ovarian development in spin flies was studied. At stage 12, nurse cells are found to dump cytoplasmic components into the oocyte, their nuclei accumulate at the anterior of the oocyte, and the actin bundles are well formed. At stage 14, the dorsal appendages are well formed and nurse cell nuclei have disappeared due to PCD in heterozygous flies. In spin^P1 mutant flies, the dorsal appendages are again well formed at stage 14, but the nurse cell nuclei are still present and some oocytes are found to be degenerated; spin^P1 mutant mature ovaries exhibit an accumulation of hundreds of nurse cell nuclei near the basal stalk. The same phenotype is also observed in the EP822 line. In contrast, spin mutant males produce normal offspring; therefore, male germ cells seem to develop normally in these flies (Nakano, 2001).

The Spin mRNA is alternatively spliced into five identified transcripts (types I-V) and these show different abilities to rescue survival and behavior deficits in spin mutants. In the behavioral-rescue experiment, the type III transgene was examined in spin^P1/spin^P1 flies, while in the lethality rescue experiment, the transgene was introduced into the spin^P2/spin^P2 flies. Type I cDNA can rescue both the behavioral and lethal phenotypes. Type V cDNA, which encodes half the protein of type I, is able to rescue the behavioral phenotype; however, it is not able to rescue the lethality phenotype. Type II, III, and IV cDNAs are unable to rescue the behavioral and lethal phenotypes (Nakano, 2001).

Given that the sole difference between the type I and type II transcripts is an alternative usage of exon 4 in type I and of exon 5 in type II, the exon 4 sequence is necessary for rescuing the behavioral and lethal phenotypes. These two exons both encode the same number of amino acids and share 56% homology, and although their sequences are different, the predicted topology does not change. Since the difference between type I and type III is located in the C-terminal region, the C terminus also seems to play an important role in Spin function. These data suggest that reduced amounts of Spin proteins, particularly the type I and type V products, induce spin^P1 phenotypes (Nakano, 2001).

spinster (spin), which encodes a multipass transmembrane protein, has been identified in a genetic screen for genes that control synapse development. spin mutant synapses reveal a 200% increase in bouton number and a deficit in presynaptic release. spin is expressed in both nerve and muscle and is required both pre- and postsynaptically for normal synaptic growth. Spin has been localized to a late endosomal compartment and evidence is presented for altered endosomal/lysosomal function in spin mutants. Evidence is presented that synaptic overgrowth in spin is caused by enhanced/misregulated TGF-ß signaling. TGF-ß receptor mutants show dose-dependent suppression of synaptic overgrowth in spin. Furthermore, mutations in Dad, an inhibitory Smad, cause synapse overgrowth. A model is presented for synaptic growth control with implications for the etiology of lysosomal storage and neurodegenerative disease (Sweeney, 2002).

Mutation in spin was initially identified in a large-scale genetic screen for mutations involved in the regulation of synaptic structure and function. A single spinster mutation [EP(2)0822] referred to hereafter as spin1 was initially identified and subsequently four new P element alleles of spin were identified based on a screen of available databases. All of these alleles are predicted to be hypomorphic loss-of-functional mutations based on the P element insertion sites. In order to isolate a null allele of spin, a small deficiency (spinDelta2b was generated using the technique of male recombination from the spin1 P element. The spinDelta2b deficiency deletes approximately 5 kb of sequence including the entire first coding exon of spin, which is common to all known splice variants of this gene, as well as the majority of the first intron. A large deficiency was identified that uncovers the spin locus (Df(2)Jp4). Subsequent genetic and molecular data support the conclusion that spinDelta2b is a null mutation (Sweeney, 2002).

It was first determined that spin is necessary for viability, demonstrating that severe spin mutations cause lethality at the late pupal stage. The three P element mutations that reside within the spin transcript (spin1, spin4, and spin5) as well as the null allele, spinDelta2b, are all lethal at the pharate pupal stage. In addition, these three lethal P element insertions fail to complement the spinDelta2b and Df(2)Jp4 chromosomes with the same pupal lethal phase. RNA in situ experiments demonstrate that there is a significant maternal contribution of spin that could account for the late lethal phase. Alternatively, spin may have a particular requirement during pupal development, or may cause a progressive defect that is manifest as late pupal lethality (Sweeney, 2002).

An analysis of synaptic morphology at the third instar NMJ reveals a dramatic phenotype of synaptic overgrowth in all of the spin mutant combinations tested. The phenotype of synaptic overgrowth is highly penetrant, affecting every neuromuscular synapse examined, including muscles 6/7 and muscle 4 as well as muscles 12 and 13. Synaptic overgrowth at muscles 6/7 was quantified by counting synaptic boutons. Individual synaptic boutons within the NMJ were clearly identified by staining with the presynaptic marker anti-Synapsin. Bouton numbers were examined in wild-type as well as four genetic controls and these data were compared to quantification of seven different spin mutant combinations. Bouton numbers are increased by more than 200% in all of the strong loss-of-function mutant combinations. This remarkable synaptic overgrowth exceeds that observed in any known mutation in Drosophila, with the exception of the highwire mutation. In these experiments, bouton numbers were not normalized to muscle size as is commonly done to account for the presumed growth coupling between muscle size and presynaptic growth. spin mutant muscle fibers are slightly smaller than wild-type muscle fibers on average. Therefore, if coupling between muscle growth and presynaptic growth persists in the spin mutant background, then the increase in synapse size compared to wild-type has been significantly underestimated by not normalizing bouton counts to muscle volume (Sweeney, 2002).

There are several examples of mutations that change bouton number but do not change synapse area because altered bouton number is compensated by an opposing change in the size of individual boutons. By contrast, the dramatic increase in bouton number in spin does cause an expansion of total synaptic area. Synaptic span was measured and this parameter is increased by more than 200%. More importantly, the average bouton size is normal despite the observed 200% increase in bouton number. The two-dimensional area of individual synaptic boutons from wild-type and spin mutant synapses at muscles 6/7 were measured. The 2D bouton surface area of each individual bouton within a synapse was measured, and these numbers were then averaged across several synapses for each genotype, including several hundred boutons per genotype. Average bouton size in spin is not significantly different from wild-type. Thus, synaptic growth is enhanced at spin mutant synapses (Sweeney, 2002).

In order to determine whether spin is necessary in the neuron or the muscle for normal synaptic growth regulation, the spin mutant phenotype was rescued by overexpression of a spin cDNA (UAS-spin) using tissue-specific GAL4 drivers that express in either the nerve (elav-GAL4), the muscle (MHC-GAL4), or ubiquitously (tubulin-GAL4). The cDNA used for rescue experiments was annotated as type III by Nakano (2001) and is one of the two most abundantly expressed isoforms of the gene (Nakano, 2001). Overexpression of UAS-spin by the ubiquitous tubulin-GAL4 promoter does not alter synapse morphology, larval crawling behavior, or viability. Thus, there is no phenotype associated with Spin overexpression. However, when tubulin-GAL4 is used to overexpress UAS-spin in the spin mutant background, bouton numbers are completely rescued to wild-type. Overexpression of UAS-spin specifically on either the presynaptic (elav-GAL4) or postsynaptic (MHC-GAL4) side of the synapse achieves only partial rescue of the synaptic overgrowth phenotype. Thus, the data argue that spin is required on both sides of the synapse for normal synaptic growth regulation (Sweeney, 2002).

In order to determine whether synaptic overgrowth in spin is associated with altered synaptic function, synaptic transmission at muscles 6/7 was quantified in wild-type, spin, and rescue larvae. Despite synaptic overgrowth, quantal content is decreased in the spin mutant background by approximately 50%. There is no change in quantal size, resting potential, or muscle input resistance at spin mutant synapses. These data are consistent with a deficit in presynaptic transmitter release in the spin mutations (Sweeney, 2002).

Experiments were persued to rescue the deficit in presynaptic release with UAS-spin transgenes expressed either pre- or post-synaptically. Surprisingly, the rescue experiments demonstrate that spin is required either pre- or post-synaptically for normal presynaptic transmitter release. Expression of UAS-spin presynaptically rescues synaptic function to wild-type levels, as does expression of UAS-spin in muscle. These data are in contrast to results demonstrating that spin is required both pre- and post-synaptically for normal synaptic growth (overgrowth is only partially suppressed when spin is rescued on only one side of the synapse). One possible explanation is that the deficit in synaptic function is secondary to the dramatic synaptic overgrowth observed at spin mutant synapses. Partial rescue of synaptic overgrowth may enable normal synaptic function by bringing synaptic overgrowth below the threshold that normally causes synapse dysfunction. The alternative is that spin supplies some necessary component to functional synapse development that can be contributed from either the pre- or post-synaptic side of the synapse (Sweeney, 2002).

Importantly, the expression of spin specifically in either muscle or neurons also rescues the viability of the spin mutant animals to wild-type levels. Less than 1% of spin null mutants and only 23% of hypomorphic spin (spin⁴/spin⁵) animals are adult viable. However, when UAS-spin is expressed in either neurons (elav-GAL4 or D42-GAL4) or muscle (MHC-GAL4) adult viability in the spin⁴/spin⁵ animals is restored to 100% (Sweeney, 2002).

Protein trafficking decisions within the endosomal system can influence intracelluar signaling by specifying whether receptors are returned to the plasma membrane or targeted for degradation. Thus, a disruption of endosomal function could enhance growth factor signaling and lead to synaptic overgrowth. TGF-ß has been implicated in the regulation of synaptic growth at the Drosophila NMJ. Mutation in the type II TGF-ß receptor wishful thinking (wit) causes a significant decrease in bouton number. To test whether enhanced TGF-ß signaling causes synaptic overgrowth in the spin mutant background, a genetic analysis of TGF-ß signaling was persued at the wild-type and spin mutant synapse (Sweeney, 2002).

It was hypothesized that enhanced or unregulated growth factor signaling is the cause of overgrowth in spin, and therefore, whether enhanced TGF-ß signaling is sufficient to cause synaptic overgrowth was tested. If TGF-ß signaling is sufficient to cause enhanced synaptic growth, then a mutation in a negative regulator of TGF-ß signaling is predicted to cause an increase in bouton number. Daughters against DPP (Dad) encodes an inhibitory Smad that negatively regulates TGF-ß signaling in Drosophila and other systems. Synapse morphology was examined in a strong loss-of-function Dad mutation that is viable to third instar larvae. Dad mutant synapses reveal a dramatically altered morphology with increased numbers of clearly distinct, small boutons that sprout from what appears to be the normal synaptic process. This is a highly penetrant phenotype and is observed at muscles 6/7 and muscle 4. Quantification of total synaptic bouton number demonstrates a significant increase in bouton numbers that is nearly equivalent to that observed in the spin mutant. These data demonstrate that enhanced TGF-ß signaling can cause synaptic overgrowth (Sweeney, 2002).

To determine whether synaptic overgrowth in spin is caused by enhanced TGF-ß signaling, it was asked whether TGF-ß signaling is necessary for synaptic overgrowth in spin. The type II receptor mutation wit causes a severe decrease in bouton number at the NMJ. Type I TGF-ß receptors are known to function in concert with type II receptors, and the type I receptors tkv and sax participate in synaptic growth regulation in this system. Third instar larva mutant for sax or tkv have smaller neuromuscular synapses. This study confirms that there is a significant decrease in bouton number in wit, and that there is a similar decrease in bouton number in both tkv and sax. These receptors are shown to function in the larval motoneurons by demonstrating that pMAD staining in the cell bodies of larval motoneurons requires wit or sax. In this experiment, the larval CNS was costained with pMAD and anti-evenskipped, which labels a subset of motoneurons (Sweeney, 2002).

A genetic analysis of the TGF-ß receptor mutations wit, tkv, and sax in combination with spin demonstrates that TGF-ß signaling is necessary for synaptic overgrowth in spin. Heterozygous mutations in tkv, sax, and wit do not alter synaptic bouton numbers at the NMJ. Heterozygous mutations in tkv, sax, and wit suppress synaptic overgrowth when placed in the spin mutant background. Bouton numbers are significantly reduced in each case where a single copy of a receptor is mutated in combination with spin. Bouton numbers were quantified in each of the double mutant combinations of tkv, sax, or wit with spin. In each case, when both copies of a receptor were removed, synaptic overgrowth was suppressed in the spin mutant background further than when only a single copy of a receptor was mutated. These data demonstrate that TGF-ß receptor mutations suppress synaptic overgrowth in spin in a dose-dependent manner. Furthermore, since bouton numbers return to wild-type, or below wild-type levels, it demonstrates that TGF-ß signaling is necessary for synaptic overgrowth in spin. Taken together with the increase in bouton numbers seen in dad, these data support the conclusion that enhanced or misregulated TGF-ß signaling is a major determinant of synaptic overgrowth in spin. It is hypothesized that altered endosomal function due to loss of Spin causes enhanced TGF-ß signaling and subsequent synaptic overgrowth. Future experiments will be necessary to determine whether enhanced signaling is due to increased receptor number at the plasma membrane, or an inability to stop signaling within the late endosomal system (Sweeney, 2002).

How might endosomal malfunction be related to synaptic overgrowth? One possibility is that there is excessive or aberrant growth factor signaling from the endosome in spin mutations. It has recently become clear that signaling from activated plasma membrane receptors can continue in the endosomal compartment. Thus, the duration of any given signaling event could be controlled in the endosomal system, and a defect in this processing could dramatically prolong potent growth-related signals. Recent studies have demonstrated the importance of the late endosomal system for the regulation of intercellular signals such as wingless, Dpp, and epidermal growth factor. A variation of this model is based on well-known sorting decisions that determine whether receptors are delivered back to the plasma membrane or are degraded in the lysosome. If sorting decisions are altered in spin, it is possible that receptors are sorted back to the plasma membrane and cause enhanced growth factor signaling from the synaptic plasma membrane (Sweeney, 2002).

These models beg the question of whether synaptic overgrowth in spin is due to gross misregulation of synaptic signaling. The synaptic overgrowth observed in spin is extreme (>200% increase in bouton number) and far exceeds the effect of mutations in other synaptic signaling molecules which, in general, alter synaptic growth in the range of a 20%–50% change in bouton number. It is possible, therefore, that many different signaling molecules are altered simultaneously due to altered endosomal function in spin, thereby generating the observed synaptic overgrowth. However, if this were the case, one might predict that any disruption of the late endosomal system would cause synapse overgrowth. Previously, two synaptic endosomal proteins have also been examined for synaptic growth defects, Hook and Deep Orange. Hypomorphic mutations in these genes caused changes in synaptic growth of approximately 10%–25%, far less than that observed in spin. It is unlikely that the discrepancy between hook and dor versus spin is entirely due to differences in the severity of the mutations since even heterozygous spin larvae show an increase in synaptic growth that can exceed 30%, and hypomorphic mutations show synaptic growth that exceeds 100%. One interesting possibility is that the extraordinary overgrowth in spin mutants is caused by the misregulation of a specific signaling pathway that is not affected by Hook and Deep Orange-dependent protein trafficking (Sweeney, 2002).

Only two mutations have been identified that can cause synaptic expansion on a scale of 200% overgrowth: highwire and spin. highwire encodes a putative E3 ubiquitin ligase. Thus, both spin and highwire appear to function in protein trafficking or membrane sorting decisions, most likely in the endosomal/lysosomal system. In addition, two overexpression experiments have demonstrated this type of overgrowth; the overexpression of an ubiquitin hydrolase. These data suggest a logic for synaptic growth control. In this model, the pre- or post-synaptic release of a growth factor is the trigger for synaptic growth. However, the regulated release of growth factor is not instructive and may exceed the amount necessary for the precise growth of the synapse. Precision is achieved by sculpting excessive growth factor signaling in the endosomal/lysosomal system. Therefore, when the function of this system is perturbed, as in spin and highwire, dramatic synapse expansion ensues. An intriguing possibility is that the activity of the endosomal/lysosomal system could be modulated by intercellular signaling as well as intrinsic factors (Sweeney, 2002).

The spin phenotype is a candidate for a model of lysosomal storage disease. Strong spin mutations show a developmental defect in synaptic growth and function and are late pupal lethal. Hypomorphic, adult viable, mutations show reduced viability. Associated with these deficits is the accumulation of ceroid lipid pigment and GM2-ganglioside-like substance in neurons. In humans, there are a large number of lysosomal storage disorders that cause severe neurodegeneration, including Battens Disease, the most common childhood onset neurodegenerative disease. The parallels between spin and Battens Disease are extensive, including ceroid lipid accumulation and the presence of characteristic electron dense profiles within neurons. In addition, many known Batten Disease genes are lysosomal proteins. There are many ways to explain the cause of neurodegeneration with respect to endosomal/lysosomal malfunction, generally dealing with inappropriate protein trafficking. These data suggest that altered endosomal/lysosomal function, either pre- or post-synaptically, can have another important consequence -- the misregulation of potent and possibly diverse intercellular signaling systems (Sweeney, 2002).

Aberrant lysosomal carbohydrate storage accompanies endocytic defects and neurodegeneration in Drosophila benchwarmer

Lysosomal storage is the most common cause of neurodegenerative brain disease in preadulthood. However, the underlying cellular mechanisms that lead to neuronal dysfunction are unknown. This study reports that loss of Drosophila benchwarmer (bnch) (spinster), a predicted lysosomal sugar carrier, leads to carbohydrate storage in yolk spheres during oogenesis and results in widespread accumulation of enlarged lysosomal and late endosomal inclusions. At the bnch larval neuromuscular junction, similar inclusions were observed, and defects were found in synaptic vesicle recycling at the level of endocytosis. In addition, loss of bnch slows endosome-to-lysosome trafficking in larval garland cells. In adult bnch flies, age-dependent synaptic dysfunction and neuronal degeneration were observed. Finally, it was found that loss of bnch strongly enhances tau neurotoxicity in a dose-dependent manner. It is hypothesized that, in bnch, defective lysosomal carbohydrate efflux leads to endocytic defects with functional consequences in synaptic strength, neuronal viability, and tau neurotoxicity (Dermaut, 2005).

Although mutations in the human homologues of bnch are not a known cause of human disease, the present study suggests that the bnch mutant phenotype is very closely related to the human neurodegenerative lysosomal storage diseases (LSDs). Together with the reported subcellular lysosomal localization of Bnch, the present demonstration of progressive neurodegeneration characterized by dramatic ultrastructural lysosomal abnormalities links a primary lysosomal defect to neurodegeneration in Drosophila. Not only are the ultrastructural characteristics highly similar to the lesions observed in human LSDs, but the predicted molecular nature of the Bnch protein as a lysosomal transporter protein further suggests similarity with a subset of LSDs that are caused by defective efflux of substrates from lysosomes. Interestingly, the human protein sialin, which is defective in sialic acid storage disease, is very similar to bnch as it is an MFS transporter of the ACS subfamily (Dermaut, 2005).

Based mainly on the accumulation of autofluorescent pigments, previous studies have emphasized the similarities between bnch phenotypes and LSDs called neuronal ceroid lipofuscinoses (Sanyal, 2002). In addition, this study points out the striking similarities between bnch and human lysosomal efflux disorders. More specifically, from both a molecular mechanistic and phenotypic point of view, bnch appears similar to sialic acid storage disease, a lysosomal acid sugar efflux disorder with severe neurodegeneration characterized by neuronal accumulation of abnormal lysosomes including lipofuscin pigments and neurofibrillary tangles. Given the data suggesting a role of bnch in lysosomal carbohydrate metabolism, it is tempting to propose bnch as a candidate gene for either the molecularly undefined lysosomal neutral hexose or N-acetylhexosamine transporter systems (Dermaut, 2005).

Despite their high rates of membrane turnover, presynaptic terminals are not known to contain active lysosomal-degradative compartments. Nevertheless, the local regulation of endocytosis at the nerve terminal is crucial for the function of synapses in the nervous system due to its role in synaptic vesicle recycling. However, the extent to which endosomal compartments impinge on the synaptic vesicle cycle is not completely understood and controversial (Dermaut, 2005).

Together with the previously reported observation that bnch mutant NMJ boutons accumulate expanded acidic organelles, the current ultrastructural and physiological data indicate that nerve terminals contain degradative compartments that are important for normal synaptic function (Dermaut, 2005).

Besides severe neurodegeneration and alterations in lysosomal morphology, phenotypic analysis of bnch mutants indicates morphological and functional alterations in endocytic compartments. The evidence comes mainly from cell types that have high rates of membrane trafficking. First, in the photoreceptors, known for their high rate of membrane turnover and endocytosis, loss of bnch induces a dramatic increase in number and size of enlarged late endosomal MVBs, suggestive of a block downstream of the late endosomal compartment. Second, reduced endosome-to-lysosome trafficking was observed in bnch mutant garland cells, a cell type with high rates of fluid phase endocytosis. Third, at bnch mutant NMJ synapses, defects were observed in endocytosis and synaptic vesicle recycling, further suggesting functional abnormalities in extralysosomal membrane compartments (Dermaut, 2005).

In contrast with hrs mutants, in which a specific functional endosomal defect leads to phenotypes suggestive of increased receptor tyrosine kinase signaling, loss of bnch does not seem to result in phenotypes indicative of a downstream defect in a specific pathway. However, as bnch most likely functions at the level of lysosomal degradation, the cellular consequences of loss of bnch are expected to be highly pleiotropic, simultaneously affecting upstream autophagic and endocytic pathways that converge at the level of the lysosome. In addition, because Bnch, as a potential mobilizer of lysosomal carbohydrate reserves, is also highly expressed in glial cells, it is possible that the neurodegeneration seen in the bnch nervous system can be partially caused by lack of trophic sugar support of neurons by glia (Dermaut, 2005).

The observation that loss of bnch is a potent enhancer of tau neurotoxicity gives some clues as to how lysosomal dysfunction might lead to neurodegeneration. It is well established that tau is on the final common pathway in a wide range of human neurodegenerative diseases, and the current results give experimental in vivo evidence that alterations in lysosomal membrane compartments are able to induce tauopathy. Together with the fact that tauopathy has been reported in sialic acid storage disease and is a highly consistent finding in Niemann-Pick disease type C, the results suggest a link between lysosomal function and tau pathology (Dermaut, 2005).

Spinster is required for autophagic lysosome reformation and mTOR reactivation following starvation

Autophagy is a conserved cellular process to degrade and recycle cytoplasmic components. During autophagy, lysosomes fuse with an autophagosome to form an autolysosome. Sequestered components are degraded by lysosomal hydrolases and presumably released into the cytosol by lysosomal efflux permeases. Following starvation-induced autophagy, lysosome homeostasis is restored by autophagic lysosome reformation (ALR) requiring activation of the 'target of rapamycin' (TOR) kinase. Spinster (Spin) encodes a putative lysosomal efflux permease with the hallmarks of a sugar transporter. Drosophila spin mutants accumulate lysosomal carbohydrates and enlarged lysosomes. This study shows that defects in spin, in both mammalian cells and Drosophila, lead to the accumulation of enlarged autolysosomes. spin is essential for mTOR reactivation and lysosome reformation following prolonged starvation. Further, the sugar transporter activity of Spin is essential for ALR (Rong, 2011).

During autophagy, lysosomes fuse with autophagosomes to form autolysosomes, where contents are degraded by lysosomal hydrolases and released by lysosomal efflux transporters. The autophagic/lysosomal pathway is critical to cellular homeostasis. Defects in autophagy lead to the accumulation of damaged organelles, misfolded proteins, and toxic metabolites, and are associated with neurodegeneration and other abnormalities. Defects in specific lysosomal hydrolysis have been implicated in lysosomal storage disorders (LSDs). Loss of lysosomal protease activity can lead to the accumulation of undigested material, as well as neurodegenerative disease. In addition, defective efflux of lysosomal contents by lysosomal transporters can lead to accumulation of lysosomal substrates and defective lysosomal function (Rong, 2011).

Lysosomal efflux transporters are a family of lysosomal membrane proteins required for the export of lysosomal degradation products, such as amino acids and monosaccharides. A subset of lysosomal storage diseases has been linked to mutations found in lysosomal efflux transporters. For example, defects in Sialin, a sialic acid transporter, leads to sialic acid storage diseases (SASD), and defects in the lysosome Arginine transporter lead to Juvenile Batten Disease. Spinster (Spin) (also known as benchwarmer) is a late endosomal/lysosomal membrane protein with the amino acid sequence of a lysosomal sugar carrier in the major facilitator superfamily. Spin is a transmembrane protein containing 8-12 transmembrane domains. In Drosophila, hypomorphic mutations in spin lead to decreased adult life span, defects in courtship behavior, accumulation of autoflourescent pigments, and neurodegeneration. Drosophila spin mutants also exhibit neuromuscular synaptic overgrowth and enhanced tau-mediated toxicity. In zebrafish, loss of the spin homolog not really started (nrs) leads to embryonic lethality characterized by the accumulation of opaque substances in the yolk. Interestingly, Drosophila spin mutants exhibit endocytic defects, as well as widespread accumulation of lysosomal carbohydrates and enlarged lysosomes. Little is known, however, about the mechanism leading to the accumulation of enlarged lysosomes in spin mutants (Rong, 2011).

ALR is an evolutionarily conserved lysosome regeneration cycle that governs nutrient sensing and lysosome homeostasis following starvation-induced autophagy. In response to starvation, mTOR is inhibited, leading to the induction of autophagy. After prolonged starvation, however, mTOR is reactivated. Upon mTOR reactivation, tubules extrude from autolysosomal membranes and give rise to vesicles that ultimately mature into functional lysosomes. The degradation of autophagic cargo is required for mTOR reactivation after starvation, and inhibiting mTOR reactivation leads to the accumulation of enlarged autolysosomes. In addition, ALR requires the dissociation of the small GTPase Rab7 from autolysosomes, and overexpression of constitutively active Rab7 results in the accumulation of enlarged autolysosomes (Rong, 2011).

This study reports that loss of spin leads to the accumulation of enlarged autolysosomes that fail to degrade their contents in both mammalian cells and Drosophila. spin is required for mTOR reactivation and lysosome reformation following prolonged starvation. Interestingly, reactivation of mTOR signaling after starvation is sufficient to induce lysosome reformation even in the context of decreased spin function. Importantly, it was found that the sugar transporter activity of spin is essential for ALR. These findings elucidate the role of this lysosomal efflux transporter in ALR and reveal its contribution to LSDs (Rong, 2011).

Drosophila spin mutants have been shown to exhibit progressive neurodegeneration, and lysosomal abnormalities have been shown to contribute to neurodegeneration in this context. Nonetheless, how defects in spin lead to lysosomal abnormalities has been unclear. This study demonstrates that the structures that accumulate in spin mutants are autolysosomes, and that spin is required for ALR following prolonged starvation (Rong, 2011).

Abnormalities in lysosome function and morphology become apparent only under starvation conditions in cultured mammalian cells. This might be explained by the fact that under nutrient-rich conditions, the influx of lysosome cargo is limited, but when cells undergo autophagy, lysosome cargo influx increases, magnifying the severity of the defect. If this hypothesis is correct, then one must wonder why spin mutant flies exhibit an accumulation of slightly enlarged lysosomal-associated membrane protein 1-(Lamp1-) positive structures even when fed. One possibility is that the metabolism of flies in this respect is greater than the metabolism of mammals. Another possible explanation is that the fluctuation of nutrients in vivo is much greater than in cells grown in culture medium. Thus, a nonstarved animal could have higher basal levels of autophagy compared with cells maintained in culture. This might be particularly important in the brain because selective deletion of Atg5 in neuronal cells leads to neurodegeneration even in unstarved mice (Rong, 2011).

One question is how Spin, a lysosomal efflux sugar transporter, alters the protein degradation capacity of lysosomes. Lysosome protein degradation capacity is dependent on the lysosomal internal environment. Lysosome pH is one of the major factors regulating lysosomal degradation ability, as the optimal pH for many lysosomal proteases is around 4.5; in either lower or higher pH, lysosome protease activity is compromised. Interestingly, it was found that upon starvation, spin knock-down cells exhibit a dramatically decreased lysosomal pH. However, it is unknown how spin knock-down leads to an increase in lysosomal acidity. One possibility is that spin is a H+/sugar symporter. H+/amino acid symporters have been identified in lysosomes, for example, LYAAT1, a lysosome amino acid efflux transporter, is a H+/amino acid symporter, and the efflux transport of amino acids by LYAAT1 is driven by the H+ gradient. Similarly, cystinosin, a lysosomal cystine efflux transporter which also contains seven transmembrane domains, has been reported to be a H+/cystine symporter which uses H+ to drive cystine efflux transport. A recent structural study for FucP, a H+/Fucose symporter which also belongs to the major facilitator superfamily (MFS) of transporters further supports this hypothesis. A structural study showed that a conserved E residue must be protonated for proper Fucose transport. Interestingly, in a rescue experiment carried out in this stuyd, the E217K mutation failed to rescue ALR in spin knockdown cells. If this hypothesis is correct, it would be expected that spin knockdown would block both sugar and H+ efflux and lead to lysosomal acidification, and autolysosomal degradation defects. Further investigation will be required to explore this possibility (Rong, 2011).

There is a bidirectional regulation between autolysosomal degradation and mTOR reactivation/ALR. On one hand, degradation of autolysosomal content is required for mTOR reactivation and ALR; on the other hand, defective mTOR reactivation/ALR also causes the impairment of degradation. For example, if mTOR reactivation/ALR is blocked during starvation by either adding the mTOR inhibitor rapamycin or knocking down mTOR, the degradation of autophagy substrate LC3 is impaired. Interestingly, this study found that adding fetal calf serum rapidly, albeit partially, restores the pH and degradation capacity of spin knockdown cells, indicating that mTOR may play a role in regulating the pH, and thus the degradation capacity, of autolysosomes. These data raise the interesting possibility that mTOR may regulate ALR by affecting the degradation capacity of autolysosomes (Rong, 2011).

One important implication of the current findings is that ALR may play an important role in disease progression in LSDs. The phenotype of spin mutant flies has been long considered to be similar to certain LSDs. This study demonstrated that a spin knockdown also leads to defects in autolysosomal degradation and ALR. These data clearly demonstrate that ALR is important to the maintenance of lysosome-based cellular degradation capacity. The basal level of autophagy may be higher in vivo than cultured cells due to the greater fluctuation of nutrients during the feeding cycle. Thus, it is conceivable that a defect in ALR could cause LSD phenotypes in fed animals (Rong, 2011).

A long-lasting question in LSDs is why a mutation in a single lysosomal enzyme can cause overall lysosome degradation failure. Because the regulation between lysosomal degradation and ALR is bidirectional, the data suggest the interesting possibility that a minor defect in lysosomal degradation capacity could cause a minor defect in ALR, which could in turn amplify the lysosomal/autolysosomal degradation defect. The positive feedback nature of this regulation loop then may eventually cause the progressive pathological features of LSDs (Rong, 2011).

Oxidative stress induces overgrowth of the Drosophila neuromuscular junction

Synaptic terminals are known to expand and contract throughout an animal's life. The physiological constraints and demands that regulate appropriate synaptic growth and connectivity are currently poorly understood. Previous work has identified a Drosophila model of lysosomal storage disease (LSD), spinster (spin), with larval neuromuscular synapse overgrowth. This study identified a reactive oxygen species (ROS) burden in spin that may be attributable to previously identified lipofuscin deposition and lysosomal dysfunction, a cellular hallmark of LSD. Reducing ROS in spin mutants rescues synaptic overgrowth and electrophysiological deficits. Synapse overgrowth was also observed in mutants defective for protection from ROS and animals subjected to excessive ROS. ROS are known to stimulate JNK and fos signaling. Furthermore, JNK and fos in turn are known potent activators of synapse growth and function. Inhibiting JNK and fos activity in spin rescues synapse overgrowth and electrophysiological deficits. Similarly, inhibiting JNK, fos, and jun activity in animals with excessive oxidative stress rescues the overgrowth phenotype. These data suggest that ROS, via activation of the JNK signaling pathway, are a major regulator of synapse overgrowth. In LSD, increased autophagy contributes to lysosomal storage and, presumably, elevated levels of oxidative stress. In support of this suggestion, this study reports that impaired autophagy function reverses synaptic overgrowth in spin. These data describe a previously unexplored link between oxidative stress and synapse overgrowth via the JNK signaling pathway (Milton, 2011).

REFERENCES

Search PubMed for articles about Drosophila spinster

Dermaut, B., Norga, K. K., Kania, A., Verstreken, P., Pan, H., Zhou, Y., Callaerts, P. and Bellen, H. J. (2005). Aberrant lysosomal carbohydrate storage accompanies endocytic defects and neurodegeneration in Drosophila benchwarmer. J Cell Biol 170: 127-139. PubMed ID: 15998804

Klämbt, C. (2009). Modes and regulation of glial migration in vertebrates and invertebrates. Nat. Rev. Neurosci. 10: 769-779. PubMed Citation: 19773781

Milton, V. J., Jarrett, H. E., Gowers, K., Chalak, S., Briggs, L., Robinson, I. M. and Sweeney, S. T. (2011). Oxidative stress induces overgrowth of the Drosophila neuromuscular junction. Proc Natl Acad Sci U S A 108(42): 17521-17526. PubMed ID: 21987827

Nakano, Y., et al. (2001). Mutations in the novel membrane protein Spinster interfere with programmed cell death and cause neural degeneration in Drosophila melanogaster. Mol. Cell. Biol. 21: 3775-3788. 11340170

Rong, Y., et al. (2011). Spinster is required for autophagic lysosome reformation and mTOR reactivation following starvation. Proc. Natl. Acad. Sci. 108(19): 7826-31. PubMed Citation: 21518918

Sanyal, S. and Ramaswami, M. (2002). Spinsters, synaptic defects, and amaurotic idiocy. Neuron 36: 335-338. PubMed ID: 12408836

Silies, M., Yuva-Aydemir, Y., Franzdóttir, S. R. and Klämbt, C. (2010). The eye imaginal disc as a model to study the coordination of neuronal and glial development. Fly (Austin) 4: 71-79. PubMed Citation: 20160502

Sweeney, S. T. and Davis, G. W. (2002). Unrestricted synaptic growth in spinster -- a late endosomal protein implicated in TGF-ß-mediated synaptic growth regulation. Neuron 36: 403-416. 12408844

Yuva-Aydemir, Y., Bauke, A. C. and Klämbt, C. (2011). Spinster controls Dpp signaling during glial migration in the Drosophila eye. J. Neurosci. 31(19): 7005-15. PubMed Citation: 21562262

date revised: 20 December 2019

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