BIOLOGICAL OVERVIEW

Cell adhesion molecules (CAMs) have been universally recognized for their essential roles during synapse remodeling. However, the downstream pathways activated by CAMs have remained mostly unknown. This study used the Drosophila larval neuromuscular junction to investigate the pathways activated by Fasciclin II (FasII), a transmembrane CAM of the Ig superfamily, during synapse remodeling. The ability of FasII to stimulate or to prevent synapse formation was shown to depend on the symmetry of transmembrane FasII levels in the presynaptic and postsynaptic cell and requires the presence of the fly homolog of amyloid precursor protein (APPL). In turn, APPL is regulated by direct interactions with the PDZ (postsynaptic density-95/Discs large/zona occludens-1)-containing protein dX11/Mint/Lin-10, which also regulates synapse expansion downstream of FasII. These results provide a novel mechanism by which cell adhesion molecules are regulated and provide fresh insights into the normal operation of APP during synapse development (Ashley, 2005).

This study demonstrates that the ability of FasII to function either as a permissive or a restrictive influence on synapse growth depends on a balance of FasII levels between the presynaptic and postsynaptic cells. Furthermore, this study shows that FasII and APPL form a biochemical complex in vivo and that the ability of FasII to promote new synapse formation requires an APPL-dependent transduction cascade. Finally, this study shows that dX11 interacts with APPL in vivo and is involved in APPL/FasII-dependent new synaptic bouton formation (Ashley, 2005).

During the development of the larval NMJ, as muscles continuously increase in size, synaptic efficacy is maintained in part by the formation of new synaptic boutons. In this process, FasII plays two fundamental roles: one of maintenance, as exemplified in the absence of FasII, when synaptic boutons begin to form but later retract, and a role in bouton proliferation. Although the role of FasII in synaptic maintenance might be related to its ability to mediate cell adhesion between the presynaptic and postsynaptic membranes, its ability to regulate budding mostly depends on genetic interactions with APPL: in the absence of APPL, a symmetric increase or decrease in FasII levels has no influence or even decreases bouton number. Although APPL is not absolutely required for synaptic growth, elimination of APPL results in significantly smaller arbors (Ashley, 2005).

This study found that APPL was required for both FasII-dependent synaptic growth and for many physiological abnormalities accompanying NMJ structural defects. Although the bouton number decrease in Appl null mutants was correlated with a decreased amplitude of evoked synaptic responses, the bouton number increase in fasII^e76/+ was correlated with increased EJP amplitude. This increase was suppressed (to Appld levels) by eliminating APPL in fasII^e76/+ mutants. In [FasII]-pre-post, however, the dramatic increase in buds was correlated with an EJP amplitude decrease, possibly because these boutons were buds or immature boutons. Similarly, in [APPL]-pre, there was both an increase in buds and a decrease in EJP amplitude. Remarkably, as seen in the fasII^e76/+ mutants, the EJP phenotype is suppressed in the Appld background (again, reaching Appld levels). Thus, as in the morphological studies, many physiological abnormalities elicited by changing FasII levels depended on APPL. Interestingly, Appld does mimic many electrophysiological phenotypes reported previously in fasII^e76 homozygotes, in that both show increased mEJP frequency, increased mEJP amplitude, and decreased bouton number (Ashley, 2005).

Activation of the synapse-promoting activity of APPL by FasII depended on simultaneous changes in the presynaptic and postsynaptic cell, whereas a unilateral change in FasII in either cell alone interfered with synapse formation. This may relate to the ability of FasII to establish cis- and trans-homophilic interactions and to the exclusive presynaptic expression of APPL (Ashley, 2005).

A genetic interaction between Appl and fasII was clearly demonstrated in these studies. It was also demonstrated that both proteins form an endogenous complex at the NMJ and that this complex includes the APPL-binding protein dX11. In these interactions, it was found that FasII could independently interact with both APPL and dX11. In the absence of APPL, an interaction between dX11 and FasII was maintained, whereas in the absence of dX11, interactions between APPL and FasII were preserved. Precisely how APPL and FasII proteins interact physically remains unclear. However, it was found that, as was the case for APPL, the FasII intracellular domain was essential for the budding phenotype, suggesting that they may interact through their intracellular domains. In the case of FasII and dX11, FasII contains a PDZ-binding motif, which interacts with PDZ1-PDZ2 domains of DLG. It is possible that PDZ domains of dX11 are alternative FasII-interacting domains (Ashley, 2005).

These studies have identified a third member of the transduction cascade that promotes synaptic growth, dX11. First, dX11 was found in the same complex with APPL at the body-wall muscles, and the lack of the GYENPTY sequence of APPL or the PTB domain of dX11 suppressed or dramatically reduced this interaction, respectively. Second, a presynaptic increase of dX11 expression mimicked the effects of upregulating APPL. This effect was suppressed by deleting the PTB domain. Third, deleting the dX11-APPL interaction sequence in dX11 (dX11ΔPTB) mimicked the effect of deleting the APPL-dX11 interaction sequence (APPLΔCi) at the NMJ. Fourth, the effects of FasII gain-of-function in both the presynaptic and the postsynaptic cell were suppressed by expressing dX11ΔPTB, suggesting that the APPL and dX11 interaction is required for the effect of FasII. Finally, a hypomorphic dX11 mutant mimicked the effects of eliminating APPL during NMJ expansion (Ashley, 2005).

A variety of proteins that bind to the GYENPTY region of APP either increase APP translocation to the cell surface or alter the stabilization or cleavage of APP at the cell surface. In particular, mammalian X11/Mint is highly expressed in neurons and interacts with the APP GYENPTY sequence through its single PTB domain. Neuronal X11 also associates directly with the exocytotic protein Munc-18, which in turn interacts with syntaxin 1A. In the current studies, it was found that deleting the APPL interaction domain in dX11 resulted in large accumulations of APPL within boutons, suggesting that dX11 may be involved in transporting or facilitating the insertion of APPL into the presynaptic membrane (Ashley, 2005).

Several studies suggest that APPL behaves as a Go-protein-coupled receptor. Go has been shown to be involved in microtubule polymerization, suggesting that one of the actions of APPL during NMJ expansion might be to regulate the cytoskeleton. Recent studies show that microtubule dynamics at the Drosophila NMJ are essential for bud maturation and extension. Based on these known interactions, the following model for APPL and dX11 function can be proposed. Trans-homophilic interactions between FasII molecules localized at the presynaptic and postsynaptic cell activate the binding between the dX11 complex (containing exocytic molecules) and APPL. dX11 then transports its partners to sites of FasII-mediated cell adhesion. This results, on one hand, in the transport of the exocytic machinery and perhaps the addition of new membrane to sites of budding. In contrast, the insertion of APPL into the presynaptic membrane and its interactions with FasII activate Go, resulting in the stimulation of microtubule polymerization, which is required for bud extension (Ashley, 2005).

The notion that FasII might function not only as a cell adhesion molecule but also as a signaling molecule is not without precedence. Indeed, the mammalian FasII homolog NCAM has been shown to initiate a signal transduction cascade after activation of both nonreceptor tyrosine kinases (nRTKs) and RTKs that may influence neurite outgrowth. Interestingly, a genetic interaction between fasII and the nRTK Abelson tyrosine kinase gene (Abl) has been reported previously in flies, and, in mammals, activated Abl interacts directly with and phosphorylates the APP intracellular GYENPTY sequence. A variety of kinases are able to phosphorylate the APP cytoplasmic domain, resulting in regulation of APP metabolism and function. Phosphorylation of Thr668 of APP695 serves as a molecular switch that appears to regulate X11 binding to APP. Thus, phosphorylation of APPL could be the switch that leads to dX11 binding and subsequent translocation of APPL to the presynaptic membrane in the Drosophila NMJ (Ashley, 2005).

These studies show that an asymmetric increase in FasII at the presynaptic cell interferes with normal synaptic bouton formation. This is characterized by formation of grossly abnormal boutons containing internal membrane structures with unusual APPL deposits and microtubule tangles surrounding these deposits. These internal APPL accumulations within the boutons seem reminiscent of intraneuronal amyloid-β accumulation, which may precede extracellular amyloid plaque formation in Alzheimer's disease. This phenomenon may provide additional clues toward a mechanism by which interference with normal APP function could lead to pathological events and subsequent symptoms of Alzheimer's disease (Ashley, 2005).

In conclusion, this study demonstrated via genetic analysis that APPL, FasII, and dX11 are involved in the same pathway that regulates synaptic expansion at the Drosophila NMJ. Altogether, these results suggest that beyond a role in cell adhesion, FasII-mediated signaling depends on a precise balance of its levels of expression at the presynaptic and postsynaptic cell and is likely to activate intracellular transduction pathways that control synapse structure (Ashley, 2005).

Fasciclin 2, the Drosophila orthologue of neural cell-adhesion molecule, inhibits EGF receptor signalling

Adhesion proteins not only control the degree to which cells adhere to each other but are increasingly recognised as regulators of intercellular signalling. Using genetic screening in Drosophila, Fasciclin 2 (Fas2), the Drosophila orthologue of neural cell adhesion molecule (NCAM), has been identified as a physiologically significant and specific inhibitor of epidermal growth factor receptor (EGFR) signalling in development. Loss of fas2 genetically interacts with multiple genetic conditions that perturb EGFR signalling. Fas2 is expressed in dynamic patterns during imaginal disc development, and in the eye it was shown that this depends on EGFR activity, implying participation in a negative-feedback loop. Loss of fas2 causes characteristic EGFR hyperactivity phenotypes in the eye, notum and wing, and also leads to downregulation of Yan, a transcriptional repressor targeted for degradation by EGFR activity. No significant genetic interactions were detected with the Notch, Wingless, Hedgehog or Dpp pathways, nor did Fas2 inhibit the FGF receptor or Torso, indicating specificity in the inhibitory role of Fas2 in EGFR signalling. These results introduce a new regulatory interaction between an adhesion protein and a Drosophila signalling pathway and highlight the extent to which the EGFR pathway must be regulated at multiple levels (Mao, 2009).

These results demonstrate that the NCAM orthologue Fasciclin 2 specifically inhibits EGFR signalling activity during the normal development of the Drosophila eye, notum and wing. Interestingly, like other Drosophila EGFR inhibitors, Fas2 participates in a potential negative-feedback loop to regulate signalling, although the developmental significance of this remains to be established. The evidence for the interaction between Fas2 and EGFR relies on genetic interactions, diagnostic phenotypes of loss of function fas2 mutants, and a direct readout in fas2 clones of reduction of Yan, a transcriptional repressor targeted for degradation by EGFR activity. Furthermore, the results in the eye are supported by similar genetic logic in the developing notum and wing. Despite this, fas2 phenotypes are not identical to those of other known EGFR inhibitors. This is less surprising than it first appears, as the phenotypes of none of the known EGFR inhibitors in Drosophila (which currently include Argos, Kekkon-1, Echinoid, Sprouty, as well as some less specific proteins such as Gap-1) are as strong as constitutive activation of the receptor, and all are distinct. The explanation for the variation in strength and detail of phenotype is that each of the inhibitors has a different molecular mechanism and site of action in the pathway, as well as different sites of expression. For example, Argos is specific to the EGFR and is a diffusible molecule that sequesters ligand. By contrast, Sprouty, a cytoplasmic protein, inhibits a range of receptor tyrosine kinases, whereas Echinoid and Kekkon-1 are cell surface proteins that bind directly to the EGFR. It is evident that EGFR regulation depends on a patchwork of overlapping effects of multiple different types of modulators, each of which has greater or less importance in different developmental contexts. Presumably, this network of regulators underlies the observed precision and robustness of signalling (Mao, 2009)

Loss of Fas2 in the eye triggers at least two distinct types of extra photoreceptor recruitment. The ectopic mini-clusters appear at the same time that the normal outer photoreceptors are recruited and, by analogy with argos mutations, it is believed that they are caused by transformation of the 'mystery cells'. In normal development these form part of the precluster, but are ejected prior to the onset of photoreceptor differentiation. It is also possible that some of the mini-clusters are derived from de novo photoreceptor determination occurring in undifferentiated interommatidial cells, which is known to be triggered by excess EGFR activity. The second recognisable type of extra photoreceptors are the R7-like, Prospero-positive cells. These are presumably the product of abnormal recruitment of cone cell precursors as R7s, a switch of fates within the R7 equivalence group, which is sensitive to altered levels of receptor tyrosine kinase signalling (Mao, 2009).

The genetic data do not reveal a molecular mechanism for the inhibition of EGFR by Fas2 - that will require future biochemical analysis - but its location at the plasma membrane and the non-autonomy that was detected at the border of mutant clones point to three classes of models. (1) Fas2 reduces EGFR ligand production, presumably the TGFα homologues Spitz or Keren, for example by direct sequestration of the mature ligand. (2) Fas2 inhibits EGFR signalling, either by direct interaction with the receptor, or by indirectly downregulating its level or activity; in this case the observed non-autonomy would be indirect and caused by the well established positive feedback loop, whereby EGFR signalling activates expression of Rhomboid 1, which itself generates processed ligands. (3) Perhaps slightly less plausibly, the extracellular domain of Fas2 might be able to span the intercellular gap, thereby interacting with and inhibiting EGFR molecules on adjacent cells (Mao, 2009).

Precedence leads to a favouring of the second model. Two other adhesion proteins, Kekkon-1 and Echinoid, interact directly with the EGFR. Similarly, mammalian E-cadherin can inhibit the EGFR by direct binding. Of particular relevance to this work, it has recently been reported that mammalian EGFR can be inhibited by NCAM, the Fas2 orthologue (Povlsen, 2008). In these experiments using explanted mouse neurons combined with transfected mammalian cell lines, NCAM stimulates neurite outgrowth by blocking EGFR function. Preliminary results lead the authors to favour a mechanism of NCAM-induced downregulation of EGFR levels, although direct parallels with the current work are difficult to draw because the cytoplasmic domains of NCAM and Fas2 are not similar (Mao, 2009)

Beyond the evidence for inhibition of the EGFR described in this study and in the recent paper discussed above, Fas2/NCAM has now been implicated in several other signalling systems. The best characterised of these is an interaction with FGFR signalling, where, both in Drosophila and mammals, FGFR activity is required for Fas2/NCAM induced neurite outgrowth and direct binding of NCAM activates FGFR (Kiselyov, 2003; Christensen, 2006). By contrast, and an illustration of the context dependence of such interactions, it has also recently been reported that NCAM can inhibit FGFR activation by its ligand FGF (Francavilla, 2007). Less well studied links between NCAM and growth factors include the observation that NCAM can act as a signalling receptor for GDNF, and that it participates in the response of oligodendrocyte precursors to PDGF. The work reported in this study is the first genetic evidence to imply a role for Fas2 in the physiological inhibition of EGFR activity. It is important to set this discussion in the context of the well established role of Fas2/NCAM as a neural cell-adhesion molecule, with roles in axonal growth and pathfinding, as well as in synaptic maturation (Mao, 2009)

Overall, it is becoming clear that the EGFR pathway is regulated by multiple partially overlapping mechanisms, presumably because of the importance of regulatory precision and robustness of such a central and pleiotropic pathway. Notably, negative-feedback control is a recurring theme. Much less is known about physiologically significant regulators of EGFR signalling in mammals, and it will be interesting to determine whether feedback control is a conserved strategy. As there are many other signalling pathways and adhesion proteins that contribute to normal development, the total potential number of regulatory interactions between these key cell surface proteins is enormous and, indeed, many have been observed in vivo and in vitro. Of course, some of these might not occur in normal biological contexts, emphasising the value of a genetic approach to revealing which relationships between adhesion proteins and signalling pathways are physiologically relevant (Mao, 2009).

Characterization of Drosophila GDNF receptor-like and evidence for its evolutionarily conserved interaction with neural cell adhesion molecule (NCAM)/FasII

Glial cell line-derived neurotrophic factor (GDNF) family ligands are secreted growth factors distantly related to the TGF-β superfamily. In mammals, they bind to the GDNF family receptor α (Gfrα) and signal through the Ret receptor tyrosine kinase. In order to gain insight into the evolution of the Ret-Gfr-Gdnf signaling system, the first invertebrate Glial cell line-derived neurotrophic family receptor-like cDNA (DmGfrl) was cloned and characterized from Drosophila melanogaster, and a DmGfrl mutant allele was generated. It was found that DmGfrl encodes a large GPI-anchored membrane protein with four GFR-like domains. In line with the fact that insects lack GDNF ligands, DmGfrl mediates neither Drosophila Ret phosphorylation nor mammalian RET phosphorylation. In situ hybridization analysis revealed that DmGfrl is expressed in the central and peripheral nervous systems throughout Drosophila development, but, surprisingly, DmGfrl and DmRet expression patterns were largely non-overlapping. a DmGfrl null allele was generated by genomic FLP deletion, and it was found that both DmGfrl null females and males are viable but display fertility defects. The female fertility defect manifested as dorsal appendage malformation, small size and reduced viability of eggs laid by mutant females. In male flies DmGfrl interacted genetically with the Drosophila Ncam (neural cell adhesion molecule) homolog FasII to regulate fertility. These results suggest that Ret and Gfrl did not function as an in cis receptor-coreceptor pair before the emergence of GDNF family ligands, and that the Ncam-Gfr interaction predated the in cis Ret-Gfr interaction in evolution. The fertility defects that were describe in DmGfrl null flies suggest that GDNF receptor-like has an evolutionarily ancient role in regulating male fertility and a previously unrecognized role in regulating oogenesis. These results shed light on the evolutionary aspects of the structure, expression and function of Ret-Gfrα and Ncam-Gfrα signaling complexes (Kallijarvi, 2012).

There is ample suggestive evidence that neurons in invertebrates require trophic support similarly to vertebrate neurons, although the identification of neurotrophic ligands in e.g., Drosophila has progressed only recently. The first Drosophila homologs of vertebrate neurotrophin family proteins, Drosophila neurotrophin 1 (DNT1), DNT2 and Spätzle, were identified in silico several years ago and recently characterized in detail and shown to possess neurotrophic activity in vivo. Additionally, DmManf, the Drosophila homolog of the novel mammalian CDNF/MANF family of neurotrophic factors, is required for the development of the Drosophila embryonic nervous system (Kallijarvi, 2012 and references therein).

Glial cell line-derived neurotrophic factor (GDNF) family ligands (GFLs) are secreted growth factors distantly related to the TGF-β superfamily. GFLs are crucial for the development and maintenance of distinct populations of central and peripheral neurons, as well as for the organogenesis of the kidney, and spermatogenesis. In mammals, four different GFL-coreceptor pairs exist. They all signal intracellularly through the RET receptor tyrosine kinase. Neural cell adhesion molecule (NCAM) is an alternative signaling receptor for GDNF in mammals. NCAM binds GFRα1 and GDNF and downregulates NCAM-mediated cell adhesion, which activates cytoplasmic protein tyrosine kinase signaling in the absence of RET. Through NCAM, GDNF stimulates Schwann cell migration and axonal growth in hippocampal and cortical neurons in mouse brain (Kallijarvi, 2012).

Mammalian GDNF family alpha receptors (GFRα) contain a conserved arrangement of extracellular cysteine-rich GFRα domains and a C-terminal GPI anchor. Homologs of GFLs, RET and the four mammalian GFRα receptors exist in all vertebrates. RET homologs seem to be present in insects but not in echinoderms. The Drosophila melanogaster RET homolog is expressed in many tissues analogous to the tissues where the gene is expressed in vertebrates, suggesting similar functions in development. GFR-like proteins have been identified in silico in sea urchin, insects and worms, including D. melanogaster and C. elegans. In Drosophila, two partial mRNA sequences encoding fragments of GFR-like proteins have been identified. However, GDNF family ligand genes have not been found in invertebrates by in silico methods. To shed light on the evolutionary origin and function of invertebrate GFR-like proteins, this study set out to characterize the Drosophila melanogaster Gfr-like gene (DmGfrl) gene and protein, to investigate its interaction with the mammalian GDNF receptors and to generate a DmGfrl null allele to investigate the in vivo functions of the receptor (Kallijarvi, 2012).

At the start of this project, two Drosophila melanogaster cDNA fragments predicting amino acid sequence with similarity to the GFRα domains of mammalian GDNF receptor proteins had been annotated in Genbank. Starting from these cDNA fragments, RACE, RT-PCR and in silico sequence analysis was used to assemble what was presumed to be the full genomic structure of the gene, and altogether six transcripts produced from this locus were identified. Based on previously suggested nomenclature, this gene was named Drosophila melanogaster Glial cell line-derived neurotrophic factor family receptor-like, or DmGfrl. The two major DmGfrl transcripts (A and B) detectable on Northern blots were found to differ only in their 5' untranslated regions and the 5' coding sequence preceding the first GFRα-like domain, including the translation initiation site and a predicted signal sequence. The exons harboring the translation start sites for transcript A and B are separated in the genome by ~27 kb, which indicates that the two main transcripts are very likely to have separate promoter regions. Such differential promoter usage may serve to allow regulation of the same gene product by separate sets of transcription factors in different developmental and/or physiological contexts. Indeed, DmGfrl transcript A is predominant in embryos. Both major DmGfrl transcripts encode a protein with four cysteine-rich GFRα-like domains, which is in line with previous in silico predictions. Similarity to the mammalian GFRα receptors is restricted to these domains, which have a characteristic arrangement of 10 cysteine residues in each domain. Interestingly, a Gfr-like gene in C. elegans predicts a similarly large protein of >1000 amino acids with four GFRα-like domains. Based on gene structures a common origin has been proposed for the exons encoding D1 to D3 in insect and sea urchin Gfr-like proteins and vertebrate GFRα genes, which suggests that a protoGFRα receptor evolved before the protostome-deuterostome divergence (Kallijarvi, 2012).

Insects lack GDNF family ligands, but having cloned the Drosophila receptor homologs it was asked whether they might respond to mammalian GDNF and whether DmGfrl could mediate mammalian RET phosphorylation. Both experiments suggested that DmRet and DmGfrl are not structurally sufficiently conserved to bind to mammalian GDNF or interact with the mammalian receptor homologs. It is interesting to speculate that one of the seven Drosophila TGF-β ligands could function as a soluble ligand ('protoGDNF') for DmRet and/or DmGfrl (Kallijarvi, 2012).

During Drosophila embryogenesis, DmRet is expressed in many tissues that are functionally analogous to those in which mammalian RET is expressed, including foregut neurons, the excretory system, peripheral ganglia and the central nervous system. DmGfrl and DmRet expression in the embryonic nervous system and in the larval and adult brain was compared using in situ hybridization. The expression pattern of DmGfrl was generally concordant with the neuronal cell expression of GFRα1 and GFRα2 in mice, in which expression at both the mRNAs and proteins has been reported in several brain areas, the spinal cord and various peripheral ganglia. Interestingly, however, DmGfrl expression was detected in the Malpighian tubules, the Drosophila analog of mammalian kidney. In line with previously published in situ hybridization data, DmRet was found to be first expressed in the yolk sac and subsequently in the ventral neuroectoderm starting from embryonic stage 13. DmRet and DmGfrl expression coincided temporally but not spatially during embryogenesis. In the larval and adult brain, DmGfrl and DmRet expression patterns were also completely non-overlapping. Thus, it is concluded that DmRet and DmGfrl likely do not function as an in cis receptor-co-receptor pair as do mammalian RET and GFRα receptors. However, the data do not rule out the possibility that DmRet and DmGfrl could interact via an alternative mode, for example in trans (cell-to-cell) or by cleavage and diffusion of soluble DmGfrl. In the absence of a DmRet null allele or a suitable hypomorphic allele, a genetic interaction between DmRet and DmGfrl was sought in misexpression experiments. No evidence was found that DmGfrl coexpression could modify ectopic DmRet-induced phenotype in the eye. The ectopic expression experiment is, however, inconclusive, and progress in this direction will require the generation of a DmRet allele suitable for genetic interaction experiments (Kallijarvi, 2012).

To gain insight into the in vivo function of the DmGfrl receptor a DmGfrl null allele by was generated by FLP-mediated genomic deletion. DmGfrl null flies were grossly normal and viable. However, they displayed a severe defect in both male and female fertility. The reduced female fertility results from an oogenesis defect as the mutant females laid fewer eggs than normally and a large fraction of those were small and had abnormal dorsal appendages. The egg morphology defect was efficiently rescued by transgene expression under the widely active daughterless and actin drivers, indicating that the phenotype is specific to loss of DmGfrl expression, and likely dependent on the somatic tissue of the ovary. However, the transgene did not rescue the reduced viability of the eggs or the reduced fecundity of the females. This suggests that the reduced egg viability is either dependent on germline cells, in which this transgene should not be expressed, or is not rescued by the DmGfrlA isoform used in these experiments. Similarly to females, in DmGfrl null males a fertility defect was observed that was not fully penetrant. Because the fecundity of DmGfrl null males was much more reduced than their absolute fertility, it was reasoned that a defect in spermatogenesis is a likely cause. Dissection of the testis histology and function in the mutant flies, as well as further rescue experiments will likely clarify the mechanism of the fertility defect in DmGfrl null males. Interestingly, on the basis of a proteomics study DmRet protein is present in adult spermatozoa, which warrants studies of the putative conserved function of DmRet in spermatogenesis (Kallijarvi, 2012).

Finally, on the basis of molecular evidence from mammals, it was of interest to look if DmGfrl might interact with the Drosophila NCAM homolog FasII. In mammals NCAM binds GDNF and GFRα1 and functions as an alternative signaling receptor for GDNF, mediating neuronal migration and axonal growth. FasII is widely expressed in the embryonic VNC, making it likely that it is also expressed in the DmGfrl-expressing neurons. WA hypomorphic FasII allele was combined with the delDmGfrl allele and whether the former could modify the male fertility phenotype of DmGfrl null flies was investigated. Strikingly, the double homozygous males were completely infertile, indicating a strong genetic interaction between DmGfrl and the FasII allele. There is currently little data linking NCAM/FasII function to reproduction. Nevertheless, on the basis of in silo data both DmGfrl and FasII are expressed at low levels in the testis and ovary. There is evidence for a role of FasII in the hormonal control of the development of Drosophila male genitalia, as a FasII^spin allele has been shown to disrupt the looping of the male genitalia and spermiduct. Interestingly, in FasII^spin flies, the innervation of corpora allata in the ring gland is disrupted, which the authors suggest may lead to elevated level of juvenile hormone and eventually to the looping defect. In preliminary inspection, no rotation defect was observed in the external male genitalia in FasII^e76;;del/Df males, but this lead will be worth further study. No gross embryonic or adult neuronal phenotypes was observed in the DmGfrl null flies. As subtle developmental or behavioral phenotypes may be present this question will require careful further studies (Kallijarvi, 2012).

The strong genetic interaction between DmGfrl and FasII that was described is corroborated by data showing biochemical interaction between the ectopically expressed receptors. These data are the first to suggest that the GFRα1-NCAM interaction described in mammalian systems is evolutionarily conserved. Together with the results suggesting that DmRet and DmGfrl do not function in cis in Drosophila, which lacks GDNF ligands, these data imply that DmGfrl may be an evolutionarily ancient binding partner for NCAM/FasII. Whether or not a soluble ligand exists in Drosophila and is needed to activate the putative FasII-DmGfrl signaling complex needs to be tackled in future studies (Kallijarvi, 2012).

Drosophila full-length Amyloid precursor protein is required for visual working memory and prevents age-related memory impairment

The β-amyloid precursor protein (APP) plays a central role in the etiology of Alzheimer's disease (AD). APP is cleaved by various secretases whereby sequential processing by the β- and γ-secretases produces the β-amyloid peptide that is accumulating in plaques that typify AD. In addition, this produces secreted N-terminal sAPPβ fragments and the APP intracellular domain (AICD). Alternative cleavage by α-secretase results in slightly longer secreted sAPPalpha fragments and the identical AICD. Whereas the AICD has been connected with transcriptional regulation, sAPPalpha fragments have been suggested to have a neurotrophic and neuroprotective role.Loss of the Drosophila APP-like (APPL) protein impairs associative olfactory memory formation and middle-term memory that can be rescued with a secreted APPL fragment. This study shows that APPL is also essential for visual working memory. Interestingly, this short-term memory declines rapidly with age, and this is accompanied by enhanced processing of APPL in aged flies. Furthermore, reducing secretase-mediated proteolytic processing of APPL can prevent the age-related memory loss, whereas overexpression of the secretases aggravates the aging effect. Rescue experiments confirmed that this memory requires signaling of full-length APPL and that APPL negatively regulates the neuronal-adhesion molecule Fasciclin 2. Overexpression of APPL or one of its secreted N termini results in a dominant-negative interaction with the FASII receptor. Therefore, these results show that specific memory processes require distinct APPL products (Rieche, 2018).

Age-related memory impairment (AMI) affects all animals, and cognitive decline is one of the devastating features of Alzheimer's disease (AD). Although APP, and more specifically the β-amyloid peptide, has been connected with memory deficits in AD, the role of full-length APP and its various other fragments in AMI is unknown. Wild-type Drosophila flies display AMI at middle age (30-40 days) when tested for middle-term or long-term olfactory memory. Furthermore, Drosophila not only encodes an ortholog for APP, called amyloid precursor protein-like (APPL), but also homologs for all three types of secretases; kuzbanian (kuz) corresponds to ADAM10 considered to be an α-secretase, dBace, the fly β-secretase, and Presenilin (Psn), the catalytic subunit of γ-secretase. APPL is processed in a similar way as human APP; however, the cleavage sites of the α- and β-secretase are reversed. Therefore, cleavage by KUZ produces a shorter secreted N-terminal fragment (NTF) than processing by dBACE. Nevertheless, subsequent γ-processing of the β-cleaved C-terminal fragment (βCTF) results in a neurotoxic dAβ peptide, whereas cleavage by KUZ does not. This study asked whether the very short-term (~4 s) visual working memory in flies is also affected by AMI and whether it requires APPL or one of its proteolytic fragments. Therefore, wild-type flies and heterozygous mutants for the three secretases were aged, and their visual orientation memory was assessed (Rieche, 2018).

This working memory is tested in the detour paradigm where walking flies navigate between two inaccessible landmarks. During an approach, the targeted landmark disappears and the fly is lured toward a novel distracting landmark. This distracter disappears one second after reorientation so that the fly is now left without any landmarks. Nevertheless, wild-type Canton-S (CS) flies can recall the position of the initial landmark and try to approach it although still invisible ('positive choices'). Whereas young CS males make about 80% positive choices, aged flies showed a reduced memory when tested at 4 weeks of age and a complete memory loss when 6 weeks old. Interestingly, heterozygosity for any of the three secretases prevented AMI, with 4- and 6-week-old Psn¹⁴³/+ and kuz^e29-4/+ flies being indistinguishable from young CS flies. When using heterozygous dBace⁵²⁴³ flies, the improvement compared to age-matched CS controls did not reach significance; however, they made significantly more positive choices than chance level at 6 weeks, whereas CS did not. These findings show that visual working memory is deteriorating with age, and they suggest that reducing APPL processing can suppress AMI (Rieche, 2018).

To address whether increased processing of APPL disrupts this memory, the secretases were overexpressed in the R3 ring neurons of the ellipsoid body (using 189Y-GAL4) the seat of visual working memory. Expression of any of the secretases reduced the performance already in 3-day-old flies compared to controls, supporting a requirement of full-length flAPPL for this type of memory. On the other hand, western blot analyses using an antiserum directed against the NTFs of APPL (Ab952M) established that heterozygous secretase mutants have an overall increase especially in flAPPL which supported the hypothesis on the role of secretases and APPL processing in AMI. Furthermore, a quantitative analysis of the levels of APPL in head extracts from different ages revealed that flAPPL declines with age, whereas the NTF/flAPPL ratio increases, also suggesting that reduced levels of flAPPL are involved in AMI. To verify that APPL is indeed required for visual working memory, homozygous Appld-null mutants and transheterozygous combinations of hypomorphic Appl alleles were tested. All these mutants performed at chance level already when young, confirming that APPL is necessary for this short-termed memory. This function is dose sensitive because even young heterozygous Appld/+ showed a reduced memory that declined faster with age than in CS females (Rieche, 2018).

Next, an RNAi-mediated knockdown of Appl was introduced in the R3 neurons, which resulted in severe memory deficits already in 3- to 5-day-old flies, showing that APPL is required in these neurons for visual working memory. To identify domains in APPL that mediate this function, rescue experiments were performed expressing different APPL constructs via 189Y-GAL4 in young Appld mutant flies. This included full-length flAPPL, secretion-defective sdAPPL, specific deletion constructs, and secreted fragments. Because the exact cleavage sites in APPL are unknown, the secreted fragments are referred to as sAPPLLong (L), which comprises the N-terminal 788 amino acids and should represent the β-cleaved fragment, whereas the 758-amino-acid (aa)-long sAPPLShort (S) should represent the α-cleaved form of Drosophila APPL. In contrast to full-length wild-type APPL, neither of the secreted forms could rescue the memory deficit of Appld when induced in R3 neurons. Notably, sdAPPL very effectively rescued the memory phenotype, confirming that unprocessed flAPPL is crucial for visual working memory (Rieche, 2018).

Next, whether APPL functions as a receptor or ligand in R3 neurons was investigaged. Expression of sdAPPL that in addition lacks the intracellular C terminus (sdAPPL-ΔC) did rescue the Appld memory phenotype, which suggests that intracellular signaling is not required and that APPL does not act as a receptor. Rescue experiments with sdAPPL forms that, in addition, lack one of the two ectodomains (sdAPPL-ΔE1 and sdAPPL-ΔE2) revealed a requirement for E2 for the rescue but not for E1. To confirm this, the rescue experiments were repeated with the hypomorphic Appl4460 allele and APPL, sdAPPL, and sdAPPL-ΔE1 rescued to full extent, whereas sdAPPL-ΔE2 did not. These results suggest that membrane-bound APPL functions as a ligand in the ring neurons. This function was conserved in human APP because expression of APP695 via 189Y-GAL4 in Appld also resulted in a rescue. Using conditional expression of APPL (by combining 189Y-GAL4 with the temperature-sensitive GAL4 repressor Tub>GAL80ts) resulted in the same rescue as constitutive expression, revealing that expression in adult R3 neurons is sufficient to restore the memory in 3- to 5-day-old flies. Notably, using the same expression system to induce moderate overexpression of sdAPPL (at 25°C), it was possible to rescue the AMI in a wild-type background, demonstrating that the secretion-deficient unprocessed APPL can prevent the decline of visual working memory of aged flies. Together with the finding that the levels of endogenous flAPPL decrease with age, this suggests that a loss of flAPPL underlies the visual working memory deficits that occur during normal aging. Interestingly, an age-related increase in BACE1 activity has been described in vertebrates that could reduce levels of full-length APP (Rieche, 2018).

Most of APPL functions in synaptogenesis, neurite outgrowth, and guidance described so far required signaling via the C-terminal domain. Analyzing heterozygous Appld/+ mutant flies or inducing an adult-specific knockdown of Appl in the relevant mushroom body neurons, Preat and Goguel (2016) showed that APPL function is not required for olfactory learning, but for a 2-hr associative memory and long-term memory formation. Similar to findings in mice, overexpression of secreted sAPPLL (as well as APPL and sdAPPL) could restore the 2-hr memory in heterozygous Appld/+ flies, whereas only wild-type APPL could rescue the long-term memory deficit. Because endogenous APPL was still expressed in this rescue experiments, sAPPLL and sdAPPL might act as ligands that bind to flAPPL in mushroom body neurons. The authors therefore suggested that distinct memory phases require different forms of APPL and maybe different intracellular signaling pathways. Notably, overexpression of KUZ in the mushroom body did not affect the 2-hr olfactory memory, whereas KUZ in this study significantly reduced visual working memory. It should also be noted that unprocessed APPL can be deleterious, because expression of sdAPPL in photoreceptor cells caused cell death of lamina glia via an unknown receptor, further emphasizing that individual neuronal networks may require different APPL fragments and signaling pathways (Rieche, 2018).

Due to recent studies suggesting that output from the R3 neurons into the ellipsoid body is instrumental for visual working memory, it was hypothesized that full-length APPL is present at the axonal terminals of R3 neurons in the ellipsoid body. To analyze the sub-cellular localization of APPL and its fragments, a double-tagged version of APPL (dtAPPL) was used that carries an EGFP tag near the N terminus and RFP tag at the C terminus, resulting in yellow fluorescence of full-length dtAPPL (or co-localized fragments that have not been separated yet). Using 189Y-GAL4 to induce dtAPPL, it was observed that dtAPPL is processed differently in individual R3 neurons, whereby full-length as well as fragments of dtAPPL are found in the cell bodies and axonal/dendritic projections. That significant amounts of unprocessed APPL can be found in the R3 axons in the ellipsoid body supports the model, that full-length APPL is needed at the R3 output sites. Note that dtAPPL was able to rescue the memory deficit, as did a dtAPP695, which showed a similar distribution pattern as dtAPPL (Rieche, 2018).

Having established that full-length APPL can be found at the relevant output sides, it was asked whether proteolytic processing of APPL also changes in the R3 neurons with age. Comparing the pattern of dtAPP695 expressed with the 189Y-GAL4 driver in 3-day-old and 6-week-old flies indicated less full-length dtAPP695 in aged flies, but, when quantifying this, it did not reach significance. Therefore, another R3-neuron-specific GAL4 line (VT42759) was used that results in reduced levels of dtAPP695 with increasing age but could nevertheless be used to rescue the memory phenotype of Appld. Compared to 3-day-old flies, there was little co-localization of GFP-tagged N termini and RFP-tagged C termini in 6-week-old VT42759>dtAPP flies, revealing enhanced proteolytic processing of APP. This suggests that AMI of the visual orientation memory is caused by increased ectodomain shedding of APPL and western blot analysis of aged flies supports this notion because during aging the ratio of NTFs to flAPPL increases (Rieche, 2018).

To identify a possible receptor for APPL in visual working memory, focus was placed on the neural cell adhesion molecule Fasciclin 2 (FASII). FASII is enriched in most, if not all types of ring neurons in the ellipsoid body, and it has been shown to interact with APPL in synaptic bouton formation at the neuromuscular junction (NMJ) (Ashley, 2005). Moreover, FASII is the insect homolog of neural cell adhesion molecule (NCAM)-140, which has been demonstrated to bind APP in an E2-depending fashion. When young hemizygous mutants were tested for the strong hypomorphic FasII^e76 allele in the detour paradigm, they showed no working memory, and the same phenotype was observed when an RNAi against FasII was introduced in R3 neurons of 3- to 5-day-old flies. This phenotype could be rescued by expression of FASII in R3 neurons, which reveals that FASII is required in the same ring neuron subtype as APPL, providing a possible binding partner for APPL. At the NMJ, loss of APPL suppressed the increase in bouton number in heterozygous i>FasII^e76/+ larvae. This study therefore investigated whether removing one copy of Appl could rescue the memory deficits of homozygous i>FasII^e76 mutants. This resulted in a significant improvement in performance, as did one copy of the i>FasII^e76 mutant allele in homozygous Appld-null mutant flies. Moreover, reducing FASII expression in R3 neurons by RNAi also ameliorated the memory deficit of Appld-null mutants. Together, this suggests that APPL negatively regulates FASII in R3 neurons and that FASII acts downstream of APPL. That this negative interaction is essential to prevent AMI is demonstrated by the observation that heterozygosity for v suppresses the memory loss of 4-week-old heterozygous Appld/+ flies (Rieche, 2018).

To further investigate interactions between APPL and FASII, overexpression studies were performed using young flies, and elevated levels of flAPPL or sdAPPL were found to ameliorate the memory deficit induced by FASII overexpression. However, only wild-type APPL induced memory deficits when overexpressed without FASII. This suggested that a secreted form of APPL can induce a gain-of-function phenotype, and this study therefore overexpressed sAPPLL and sAPPLS in R3 neurons. Whereas sAPPLL had no effect, sAPPLS caused a severe impairment of visual working memory. Moreover, overexpression of sAPPLS suppressed the effects of elevated FASII levels, whereas sAPPLL did not. This shows that sAPPLS, which corresponds to the α-cleaved (KUZ) fragment, has deleterious effects when overexpressed and that these are also mediated by an interaction with FASII. Whether the lack of an effect of sAPPLL overexpression is due to a less efficient interaction with FASII or an inability to induce downstream pathways causing this gain of function remains to be determined (Rieche, 2018).

Interestingly, overexpression of the dAICD in R3 neurons also disrupted visual working memory of young flies, suggesting that α- and γ-cleavage of APPL has deleterious effects on visual working memory. This is in agreement with the finding that heterozygosity for kuz and Psn ameliorated the age-related decline in visual working memory, whereas heterozygosity for dBACE had only a modest effect. This could be explained by assuming that most of the ectodomain shedding in flies is done by KUZ activity. Therefore, reducing dBACE levels might result in a small increase of flAPPL. In addition, competitive KUZ cleavage in dBace/+ flies could result in more detrimental sAPPLS. Whereas in the case of kuz/+, the effects on memory are mediated by the interaction with FASII, in the case of Psn/+ this may be mediated by a transcriptional function of the AICD, affecting a so far unknown target (Rieche, 2018).

In summary, the results show that full-length APPL acts as a membrane-bound ligand that inhibits the FASII receptor (both acting in R3 neurons), thereby promoting visual working memory. Increased proteolytic APPL processing and therefore reduced suppression of FASII signaling then seems to cause AMI in flies. A similar interaction might also be required for working memories in vertebrates. Aging mice show reduced expression of NCAM-140 in the medial prefrontal cortex and a conditional knockout of NCAM in the forebrain promotes AMI in a delayed matching-to-place test in the Morris water maze and in a delayed reinforced alternation test in the T-maze (Rieche, 2018).

Earlier Studies

Fas2 carries out multiple functions in the Drosophila nervous system. A member of the immunoglobulin superfamily, Fas2 has been implicated in the process of fasciculation, the adherence of growing axons to one another during the process of growth cone guidance. Fas2 has also been implicated in the process of presynaptic functional plasticity, the ability of synapses to undergo long-term changes in structure. Expressed both presynaptically and postsynaptically, Fas2 controls the number and stability of neuromuscular junction synapses. In this function, Fas2 is down regulated by mutations affecting cyclic AMP levels (See the learning pathway). In addition, decreased Fas2 is necessary and sufficient to cause synaptic sprouting. Finally, Fas2 and CREB (the cyclic AMP response element binding protein) act in parallel pathways to cause increases in strength of the neuromuscular synapse.

Fas2 functions in selective fasciculation, that is, adherence of growing axons to the MP1 fascicle. The MP1 fascicle, generated by the anterior extending growth cone of the pCC axon is easily identified because a specific monoclonal antibody will stain only very specific types of growth cones. These include aCC, pCC, MP1, dMP2 and vMP2. The pCC axon pioneers the MP1 fascicle. In the absence of Fas2, growth cones do not extend in the proper rostrocaudal direction and fail to fasciculate (Grenningloh, 1991).

Subsequent studies reveal an even more complex function for Fas2. In addition to selective fasciculation, Fas2 also functions in growth cone guidance. Three major motor nerves were analyzed: the intersegmental nerve (ISN) that innerates dorsal muscles; the segmental nerve a (SNa), innervating lateral muscles, and segmental nerve b (SNb), innervating ventral muscles. High specific defects in axonal guidance occur in over- or under-expression of Fas2. These do not appear to be fasciculation phenotypes because fascicles are not formed. They are instead guidance phenotypes in which axons wander from their normally specified courses. It would appear that guidance is not taking place here solely on the basis of self adhesion, but in the context of other competing attractive and repulsive cues. In this case, cell adhesion per se is not always the sole or the decisive factor (Lin, 1994 a and b).

Muscles 7 and 6 are each innervated by two motoneurons, RP3 and MN6/7b. This innervation occurs at stage 17. The two axons form about 18 synaptic boutons by the time of the initial larval hatch, and the number increases to approximately 180 boutons by the third instar larva. Although Fas2 is initially expressed by all embryonic motor axons and their growth cones during the period of axon outgrowth, the axonic Fas2 decreases upon innervation, and Fas2 is confined to the synaptic terminal. After synapse formation, Fas2 is localized both pre-synaptically and postsynaptically, where it controls synapse stabilization. In Fas2 mutants, synapse formation is normal, but boutons then retract during larval development. Synapse elimination and the resulting lethality is rescued by transgenes that drive Fas2 expression both pre- and postsynaptically; driving Fas2 expression on either side alone is insufficient.

Fas2 can also control synaptic growth: various Fas2 alleles lead to either an increase or decrease in sprouting depending upon the level of Fas2. A 50% decrease in Fas2 can lead to a 50% increase in the number of synaptic boutons (Schuster, 1996a).

Altering the level of neuronal activity or cAMP concentration leads to increased synaptic structure and function at the neuromuscular junction. ether à go-go and Shaker code for potassium channels; mutations in both can result in increased neuronal activity and increase in synaptic structure and branching. Mutation in K+ channels display greatly enhanced nerve activity as a result of reduction in K+ currents. K+ channel mutation is thought to increase motoneuron activity and synaptic transmission by increasing the cAMP second messenger signaling. Mutations in dunce, coding for c-AMP phosphodiesterase, result in increased cAMP levels, heightened neural activation, and also result in increased axonal branching (Schuster, 1996b and references).

Increase in synaptic growth in eag, Shaker and dunce mutants is accompanied by approximately 50% reduction in synaptic levels of Fas2. This decrease in Fas2 is both necessary and sufficient for presynaptic sprouting; Fas2 mutants that decrease Fas2 levels by 50% lead to sprouting similar to eag, Shaker and dunce mutants, while Fas2 transgenetic animals that maintain synaptic Fas 2 levels suppress sprouting in eag, Shaker and dunce mutants. However, Fas2 mutants that cause a 50% increase in bouton number do not alter synaptic strength; rather, evoked release from single boutons has a reduced quantal content, suggesting that the wild-type amount of release machinery is distributed throughout more boutons. Thus these results show a requirement for the presynaptic downreguation of Fas2 in activity and cAMP-induced synaptic sprouting. It is speculated that activity or cAMP may trigger the down-regulation of synaptic Fas2 by actively removing it from the presynaptic terminal (Schuster, 1996b).

Genetic dissection of structural and functional components of synaptic plasticity. III. CREB is necessary for presynaptic functional plasticity

Since Fas2 mutants lead to an increase in the number of boutons without affecting synaptic strength, and increased cAMP in dnc mutants increases both synaptic structure and quantal content, there must be other elements downstream of cAMP, but not downstream from Fas2, that are involved in increasing quantal content. CREB, the cyclic AMP response element-binding protein, mediates the transcriptional requirement of cAMP-dependent long-term synaptic change. Thus CREB is a candidate for the cAMP target responsible for increasing quantal content. CREB acts in parallel with FAS2 to cause an increase in synaptic strength. Expression of an endogenous CREB repressor, dCREB2-b (an isoform of CREB), in dunce mutants blocks functional but not structural plasticity. Expression of the activator isoform, dCREB2-a, increases synaptic strength by increasing presynaptic transmitter release at single boutons, but only in Fas2 mutants that increase bouton number. Strong overexpression of dCREB2-a results in a significant increase in quantal content, independent of genetic background and with little effect on bouton number. Thus CREB-mediated increase in synaptic strength is due to increased presynaptic transmitter release. Expression of dCREB2-a in a Fas2 mutant background genetically reconstitutes cAMP-dependent plasticity, and cAMP initiates parallel changes in CREB and Fas2 to achieve long term synaptic enhancement (Davis, 1996).

PROTEIN STRUCTURE

Amino Acids

There is a phosphatidylinositol-linked form with 811 amino acids and a transmembrane form with 873 amino acids. They differ only in their C terminal amino acids (Grenningloh, 1991).

Structural Domains

FAS2 is a member of the immunoglobulin superfamily containing five Ig-like domains. It also contains two fibronectin-type III repeats (Grenningloh, 1991). The protein contains a signal sequence serving for protein secretion.

date revised: 20 June 2023 

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