InteractiveFly: GeneBrief

Rab-protein 3: Biological Overview | References

BIOLOGICAL OVERVIEW

Synaptic transmission requires the localization of presynaptic release machinery to active zones. Mechanisms regulating the abundance of such synaptic proteins at individual release sites are likely determinants of site-specific synaptic efficacy. A role for the small GTPase Rab3 has been identified in regulating the distribution of presynaptic components to active zones. At Drosophila rab3 mutant NMJs, the presynaptic protein Bruchpilot, calcium channels, and electron-dense T bars are concentrated at a fraction of available active zones, leaving the majority of sites devoid of these key presynaptic release components. Late addition of Rab3 to mutant NMJs rapidly reverses this phenotype by recruiting Brp to sites previously lacking the protein, demonstrating that Rab3 can dynamically control the composition of the presynaptic release machinery. While previous studies of Rab3 have focused on its role in the synaptic vesicle cycle, these findings demonstrate an additional and unexpected function for Rab3 in the localization of presynaptic proteins to active zones (Graf, 2009).

Individual neurons can form thousands of discrete synaptic connections with their postsynaptic partners. Each synapse comprises tightly apposed pre- and post-synaptic membranes, a postsynaptic cluster of neurotransmitter receptors, and a presynaptic complex of proteins that promotes neurotransmitter release. For a synapse to function, the proper complement of proteins must localize to the presynaptic release machinery, and the protein composition at the release site is a likely determinant of its synaptic efficacy. While the general properties of synapses formed by a single axon are similar, the release probability of such synapses can vary dramatically. This presynaptic heterogeneity is likely due to mechanisms that control synapse specific plasticity and may represent one aspect of the molecular basis of learning and memory. Thus, identifying mechanisms that control the protein composition and presynaptic release properties of individual synapses will provide insights into plasticity mechanisms in the brain (Graf, 2009).

The Drosophila neuromuscular junction (NMJ) is an excellent system for identifying mechanisms that regulate the protein composition of individual active zones. A single Drosophila motoneuron and single muscle cell form an NMJ comprising hundreds of individual release sites, or presynaptic active zones, each apposed to a postsynaptic glutamate receptor (GluR) cluster. Each release site is akin to a single mammalian central nervous system synapse, and like CNS synapses, there is heterogeneity in their release properties. Drosophila contains orthologs of all of the major vertebrate presynaptic proteins with the exception of Bassoon and Piccolo. Among these, Bruchpilot (Brp), the Drosophila ortholog of CAST, plays an essential role in organizing the presynaptic release machinery. This role is similar in mammals where CAST acts as a molecular scaffold within the cytomatrix at the active zone, interacting with Piccolo, Bassoon, Rim1α, and α-liprins/SYD-2 and in C. elegans where the Brp homolog ELKS-1 acts with SYD-2/α-liprin to promote the assembly of presynaptic active zone components (Dai, 2006; Patel, 2009). In Drosophila, Bruchpilot localizes to every active zone, but its distribution is heterogeneous, and the abundance of Brp at an active zone appears to correlate with the release probability of that site. Brp is not required for active zone formation per se, but is an integral component of T bars, electron-dense active zone specializations that likely promote transmitter release, and is required for the continuous accumulation of Ca²⁺-channels at active zones during synapse maturation. These findings with Brp imply that mechanisms exist to (1) ensure that Brp is present at each release site and (2) regulate the level of Brp at each site. Such mechanisms would likely impact site-specific release probability by controlling the protein composition of the release machinery at each site. To identify such mechanisms, a large-scale genetic screen was performed to identify genes required for the proper localization of Brp to active zones (Graf, 2009).

This study found that the small GTPase Rab3 functions to influence the distribution of Brp and other crucial presynaptic active zone components to release sites. In the absence of Rab3, key constituents of the presynaptic release machinery are concentrated at a fraction of available sites, resulting in the formation of a small number of super sites with enhanced release probability and a larger number of sites devoid of key presynaptic release proteins. Rab3 can rapidly recruit Brp to active zones, demonstrating that the protein composition of the release machinery is under dynamic control and that Rab3 is well positioned to participate in synapse-specific plasticity mechanisms. Whereas previous studies have implicated Rab3 in the cycling and docking of synaptic vesicles (Sudhof, 2004), this study reports a role for Rab3 in influencing the protein composition of the presynaptic release apparatus at individual active zones (Graf, 2009).

To identify mechanisms that control the molecular composition of individual release sites, a collection of Drosophila mutants was screened for those with defects that differentially affect presynaptic active zones within an NMJ. An anatomical genetic screen was performed on a collection of ∼1500 lines that carry unique insertions of transposable elements in or near genes on the second chromosome. Third-instar homozygous mutant larvae were dissected from each line and stained for the presynaptic active zone protein Bruchpilot (Brp) and the essential glutamate receptor subunit DGluRIII. The immunostained NMJs were stained with fluorescence microscopy and mutants were identified with altered active zones, including changes in Brp puncta size, number, or intensity, as well as those with defects in the apposition of presynaptic Brp and postsynaptic DGluRIII puncta. Within this group, one line, P{SUPor-P}KG07292, was identified that has an active zone phenotype. In this mutant, there is a dramatic loss of Brp-positive active zones, yet the morphology and number of DGluRIII clusters appears grossly normal. As such, most GluR clusters are unapposed to a Brp-positive active zone. The remaining Brp puncta are apposed to GluR clusters, and these Brp puncta are significantly larger than in wild-type. Due to the large number of unapposed GluR clusters, this mutant was named running-unapposed (rup) (Graf, 2009).

While Brp morphology is altered in rup, the gross morphology of the mutant NMJ is normal, and the synaptic terminal area is not significantly different than in wild-type. Staining with an antibody against the vesicular glutamate transporter (DVGLUT) demonstrates that synaptic vesicles are distributed throughout the NMJ, and co-staining for the postsynaptic scaffolding protein Discs-large (Dlg) reveals that the presynaptic terminal is apposed to the postsynaptic specialization across its length. Hence, the presence of unapposed GluR clusters is not due to synaptic retraction of synaptic boutons or branches; instead, affected synapses distribute in a salt-and-pepper pattern throughout the synaptic terminal, suggesting that the defect occurs at the level of individual synapses. Glutamate receptors colocalize with the serine-threonine kinase Pak at the Drosophila NMJ. Pak distribution appears normal in the rup mutant, and like GluR clusters, most Pak clusters are unapposed to Brp-positive active zones. Hence, in rup postsynaptic morphology is relatively normal and the primary morphological defect is likely presynaptic. Despite their abnormal active zones, rup mutant animals are viable and fertile (Graf, 2009).

To investigate the mechanism underlying the defective active zones in rup, it was necessary to identify the responsible gene. Although rup was found in a collection of insertional mutants, the phenotype does not map to the P{SUPor-P}KG07292 transposable element. Instead, rup is a second-site mutation fortuitously present on the chromosome. rup was roughly mapped by meiotic recombination to position 43-48 on the right arm of the second chromosome and a deficiency chromosome (Df(2R)ED2076) was identified that fails to complement the mutation. This deficiency deletes 26 predicted genes in the region between 47A10 and 47C1. The list of candidates was narrowed to 21 by complementation testing with known null mutants in the region. The coding regions of candidate genes was sequenced, ultimately identifying a five base pair deletion near the 3' end of the rab3 gene. This deletion throws rab3 out of frame and would lead to a deletion of the last 35 amino acids of the protein, including the final CXC motif that in other systems is required for lipid modification, the binding of Rab3 to synaptic vesicles, and proper Rab3 localization (Iwasaki, 1997; Johnston, 1991; Graf, 2009 and references therein).

A single ortholog of Drosophila rab3 was previously cloned and demonstrated to be highly conserved (Johnston, 1991). It was further shown to be expressed throughout the fly nervous system (DiAntonio, 1993); however, no functional studies were performed. To investigate the localization of Rab3 protein and the nature of the mutant allele, a polyclonal antibody was generated to a peptide epitope in the unique C-terminal region of Drosophila Rab3. This antibody stains synaptic terminals of wild-type NMJs in a pattern similar to synaptic vesicle markers such as synapsin and DVGLUT. However, unlike synapsin and DVGLUT, Rab3 staining is further concentrated in a punctate pattern at active zones, as visualized by costaining with Brp. This punctate localization of Rab3 at active zones is not observed in brp mutant NMJs (brp⁶⁹/Df(2R)BSC29) even though the synaptic vesicle-like distribution of Rab3 staining remains. In addition, the antibody recognizes a single band of the predicted size on immunoblots from wild-type larvae. Both the synaptic staining at the NMJ and the band on the immunoblot are absent in the rup mutant, demonstrating that the antibody is specific for Rab3 and that the rup mutant does not express wild-type Rab3 protein. Since the mutation in rup is located in the C-terminal region of rab3 just upstream of the epitope, it is possible that a truncated protein could be expressed. While such a mutant protein could have residual function, the active zone phenotype of rup homozygotes and transheterozygotes of rup and Df(2R)ED2076 are similar in terms of the percentage of GluR clusters apposed to Brp and the average area of individual Brp punctum. Therefore, rup behaves as a genetic null or a very strong hypomorph (Graf, 2009).

This study shows that the small GTPase Rab3 controls the protein composition and release probability of individual active zones at the Drosophila neuromuscular junction. In a rab3 mutant, key constituents of the presynaptic release machinery are enriched at a subset of active zones while the remaining release sites are apparently devoid of these proteins. Expression of Rab3 rapidly and reversibly rescues this altered protein distribution. Physiological studies are consistent with these morphological findings, demonstrating an increase in release probability from an apparently decreased number of release sites. Mechanistic studies indicate that Rab3 functions to increase the probability that the essential synaptic organizing molecule Bruchpilot will cluster at an active zone. This Rab3-dependent regulation of active zone protein composition and release probability provides a potential mechanism for the synapse-specific control of synaptic efficacy (Graf, 2009).

The Drosophila NMJ consists of a motoneuron axon terminal arranged as a chain of synaptic boutons closely associated with the postsynaptic muscle membrane. Within each string of boutons are hundreds of individual synapses, discrete sites of neurotransmitter release where a presynaptic active zone is directly apposed to a postsynaptic glutamate receptor cluster. Such a synapse comprises (1) the site where the axon and muscle membranes are in closest proximity, likely tethered by trans-synaptic cell adhesion molecules; (2) the presynaptic release apparatus that influences the Ca²⁺-mediated release of the neurotransmitter-filled vesicles; and (3) the neurotransmitter receptors and scaffolding and signaling proteins of the postsynaptic density. This study demonstrates that disrupting rab3 alters the distribution of proteins that make up the presynaptic release machinery without grossly disturbing the other two components of the synapse (Graf, 2009).

In the absence of Rab3, a subset of synapses contain increased amounts of the active zone protein Bruchpilot, higher levels of the calcium channel Cacophony, and more electron dense T bars at the active zone. Since Brp is a component of T bars and influences Ca²⁺-channel accumulation, the altered distribution of these components is likely a direct consequence of changes in Brp distribution. The creation of additional active zone markers will be necessary to determine the full extent of this altered distribution. However, since all three components examined influence the probability of evoked vesicle release, the active zones where they accumulate likely are sites of enhanced vesicle release. Conversely, the remaining sites that are devoid of these components likely exhibit impaired evoked release. Two lines of evidence support this conclusion. First, glutamate receptors preferentially cluster opposite sites with the highest release probability. In the rab3^rup mutant, GluR clusters are larger at Brp-positive than Brp-negative sites, suggesting that those active zones containing Brp have a higher release probability. Second, facilitation resulting from short stimulus trains is reduced in the mutant, consistent with an increased release probability (p). However, since quantal content and quantal size are unchanged, the increase in p must be balanced by a decrease in the number of sites that are firing. Hence, both the morphological and electrophysiological data are consistent with the model that Rab3 controls the distribution of active zone proteins to influence the efficacy of individual release sites (Graf, 2009).

Other Drosophila mutants have active zone phenotypes, but none have the combination of phenotypes described in this study. Mutations in synaptojanin, neurexin, and spectrin affect the size and spacing of the entire array of active zones. Mutations in the Unc-51 kinase and the protein phosphatase PP2A have differential affects on active zones, resulting in a subset of glutamate receptors unapposed to Bruchpilot puncta as in the rab3^rup mutant (Viquez, 2009; Wairkar, 2009). However, in the unc-51 and PP2A mutants, the remaining Brp puncta are not enlarged and there is no increase in the proportion of active zones with multiple T bars. Such phenotypes are consistent with defects in active zone formation, rather than in the distribution of proteins across active zones. Finally, GluR clusters unapposed to Brp puncta occurs following synaptic retraction, but in such mutants the active zone defects are secondary to the loss of the entire presynaptic terminal. Hence, Rab3 participates in a previously undescribed mechanism that differentially regulates active zones within an NMJ (Graf, 2009).

These findings demonstrate that Rab3 plays a central role in the localization of Bruchpilot to individual active zones. In the absence of Rab3, approximately 70% of active zones are devoid of Brp while the other 30% contain an excess of Brp. What is the function of Rab3 such that its loss leads to this altered Brp distribution? It is suggested that Brp is present in two pools: one fraction bound in complexes at active zones and a second mobile fraction in the cytosol. It is further suggested that Brp is dynamic and may alternate between these two pools by associating with or dissociating from the active zone complex. As such, unbound Brp in the cytosol may either nucleate a cluster at an active zone, creating a new Brp punctum, or add to a pre-existing Brp punctum making it larger. Given this scenario, the rab3 phenotype may be explained by two alternative models of Rab3 function: (1) Rab3 limits Brp puncta size, or (2) Rab3 increases the ability of Brp to nucleate new Brp clusters at active zones. If Rab3 functions to limit the addition of Brp to already existing sites, disruption of Rab3 would allow Brp clusters to grow to a maximal size, reducing the availability of cytosolic Brp to create new puncta and consequently constraining the number of puncta formed. In such a model, it would be predicted that Brp puncta size would be large at rab3^rup mutant NMJs regardless of Brp expression levels. Instead, decreasing Brp levels in the rab3^rup mutant decreases the size of Brp puncta. Even more telling, increasing Brp levels in the rab3^rup mutant also reduces the size of Brp puncta. These results are inconsistent with the model that primary function of Rab3 is to limit the size of Brp puncta (Graf, 2009).

Instead, it is suggested that Rab3 functions to increase the probability that Brp will nucleate a new cluster at an active zone. The presence of Brp at some active zones demonstrates that Rab3 is not absolutely required for Brp localization. Why then is Rab3 required for Brp localization to the 70% of active zones that are bereft of Brp? Rather than posit that these two classes of active zones are fundamentally different in the rab3^rup mutant, it is suggested that in the absence of Rab3, Brp is much less likely to nucleate a cluster at an active zone (Graf, 2009).

The data presented in this study are consistent with this model. First, late rescue with rab3 leads to the rapid addition of new, small Brp puncta and, on a slower time scale, a decrease in the size of the large Brp puncta. This demonstrates that Brp is dynamic and can move into and out of active zones. Second, reducing the levels of Brp at wild-type synapses leads to a decrease in both the number of Brp puncta formed as well as their size. An increase in Brp expression at a wild-type synapse cannot increase the number of puncta since essentially 100% of active zones already contain a Brp puncta, but it does lead to an increase in the size of the puncta. Hence, Brp levels affect both the likelihood of forming a Brp puncta at an active zone as well as the ultimate size of the Brp puncta. Third, increased Brp expression enhances the ability of Brp to cluster at active zones, overcoming the absence of Rab3 and leading to the formation of more Brp puncta in the rab3^rup mutant. This demonstrates that these mutant active zones do have the capacity to cluster Brp, but that it requires the stronger driving force provided by the additional Brp to overcome the absence of Rab3. Finally, when Brp is overexpressed in the rab3^rup mutant the Brp puncta are smaller than when Brp is expressed at wild-type levels. This apparent paradox suggests that Brp puncta compete for unbound Brp and that the large increase in the number of Brp puncta provides more sites for unbound Brp and so ultimately results in smaller puncta. Hence, this model explains the variation in the number and size of Brp puncta present in the various genetic backgrounds tested above, and highlights a novel role for Rab3 in controlling the protein composition of active zones (Graf, 2009).

Brp appears to play a prominent role in the mechanism by which Rab3 regulates the distribution of active zone components to release sites. However, there is no evidence that Rab3 interacts directly with Brp, and such a direct interaction between Rab3 and members of the CAST/ERC family, of which Brp is an ortholog, has not previously been reported. Other proteins could mediate the interaction between Rab3 and Brp. In other species, Rab3 is known to interact with proteins involved in the Rab3 GTPase cycle such as Rab3-GEF, Rab3-GAP, and GDI, as well as the putative Rab3 effectors Sec15, Rabphilin, and Rim. Among the Rab3 effectors, Rabphilin is an unlikely candidate because Rabphilin knockout mice and worms have no observable morphological or physiological synaptic defects. Rim is a more plausible candidate because it is a constituent of the presynaptic release apparatus and binds to many other presynaptic active zone proteins including orthologs of Brp (Schoch, 2002). Alternatively, Rab3 may act on a yet unidentified target to regulate the molecular properties of Brp. Understanding Rab3 function at the fly NMJ will require the identification of the protein(s) Rab3 interacts with to distribute active zone components among release sites (Graf, 2009).

Previous studies of rab3 knockouts in other organisms suggest that Rab3 is involved in regulating vesicle cycling, docking, and exocytosis (Sudhof, 2004). While Rab3 may play a direct role in vesicle dynamics and release at the Drosophila NMJ, this study also suggesta that Rab3 plays a second, separate role in influencing the distribution of the presynaptic release apparatus. Defects at the active zone in the rab3^rup mutant are unlikely to be secondary to altered synaptic vesicle release because (1) other mutants affecting release do not disrupt the composition of the active zone and (2) neither increasing nor decreasing activity in the rab3^rup mutant exacerbates or ameliorates the active zone phenotype (Graf, 2009).

If Rab3 controls the protein composition of the active zone, then why have genetic analyses of rab3 in mice and C. elegans not identified structural abnormalities at the synapse? In Drosophila, loss of rab3 results in a very specific ultrastructural phenotype. The active zone, visualized as an electron dense thickening of tightly apposed pre- and postsynaptic membranes, is normal in Drosophila rab3^rup mutants as it is in worms and mice. However, some synapses, including Drosophila NMJ synapses, contain prominent electron-dense specializations such as T bars that are thought to promote transmitter release. It is the distribution of these T bars that is altered in the Drosophila rab3^rup mutant, which would not be apparent at, for example, hippocampal synapses, where such dense bodies are not readily observed. While structural defects have not been detected in other organisms, the electrophysiological findings in mice show interesting parallels to the fly phenotype. The quadruple knockout of Rab3A, Rab3B, Rab3C, and Rab3D in mice demonstrates that Rab3 increases the release probability of a subset of vesicles in the readily releasable pool (Schluter, 2006). The authors of this study propose two hypotheses to explain these findings. The first stays within the traditional vesicle-centric framework for Rab3, suggesting that Rab3 docks specific vesicles to sites of high release probability. The second hypothesis posits that Rab3 recruits additional proteins to the release machinery at certain synapses, thereby making Ca²⁺-mediated release more efficient (Schluter, 2006). This second possibility is consistent with the findings in Drosophila that Rab3 regulates the distribution of release apparatus proteins to control the efficacy of individual sites (Graf, 2009).

Many neurons differentially regulate the release properties of individual release sites along their axonal lengths through presynaptic, synapse-specific mechanisms. These include the regulation of Ca²⁺-channel localization and function and the selective accumulation of group III metabotropic glutamate receptors to specific presynaptic active zones (Pelkey, 2007). It is suggested that Rab3 is well positioned to participate in such synapse-specific plasticity mechanisms. The finding that late expression of Rab3 can rapidly reverse the apposition phenotype of the mutant and redistribute Brp to active zones that previously lacked the protein indicates that (1) Brp is highly mobile and (2) Rab3 can rapidly modulate its distribution among individual sites. Multiple proteins control Rab3 function via its GTPase cycle (Sudhof, 2004), so mechanisms that locally activate or inhibit Rab3 could lead to rapid and local changes in active zone structure and function. Thus, Rab3 is a candidate to participate in plasticity mechanisms that regulate the protein composition and efficacy of individual release sites (Graf, 2009).

Rab3-GAP controls the progression of synaptic homeostasis at a late stage of vesicle release

Homeostatic signaling systems stabilize neural function through the modulation of neurotransmitter receptor abundance, ion channel density, and presynaptic neurotransmitter release. Molecular mechanisms that drive these changes are being unveiled. In theory, molecular mechanisms may also exist to oppose the induction or expression of homeostatic plasticity, but these mechanisms have yet to be explored. In an ongoing electrophysiology-based genetic screen, 162 new mutations were tested for genes involved in homeostatic signaling at the Drosophila NMJ. This screen identified a mutation in the rab3-GAP gene. This study shows that Rab3-GAP is necessary for the induction and expression of synaptic homeostasis. Evidence is provided that Rab3-GAP relieves an opposing influence on homeostasis that is catalyzed by Rab3 and which is independent of any change in NMJ anatomy. These data define roles for Rab3-GAP and Rab3 in synaptic homeostasis and uncover a mechanism, acting at a late stage of vesicle release, that opposes the progression of homeostatic plasticity (Müller, 2011).

The function of Rab3-GAP and Rab3 have been analyzed extensively, both biochemically and genetically, in systems ranging from yeast to the mammalian central nervous system, and there are several (~10 on average) copies of Rab3-GTP on an individual synaptic vesicle (Takamori, 2006). It has also been shown that Rab3 binds to several presynaptic proteins, most often in its GTP-bound form (see Kanno, 2010). Rab3-GAP is required to promote hydrolysis of Rab3-GTP to Rab3-GDP. It is unknown precisely when and where Rab3-GAP acts upon Rab3-GTP, but evidence suggests that this interaction may occur at the synapse. For example, Rab3-GTP is on the vesicle (Takamori, 2006) and delivered to the synapse, where it is found bound to the active zone associated protein ">RIM (Wang, 1997). Biochemical data indicate that clathrin-coated vesicles lack Rab3-GTP (Fischer von Mollard, 1991). In combination, these data place Rab3-GAP activity at or near the release site (Müller, 2011).

The data presented in this study are consistent with a model in which Rab3-GTP acts, directly or indirectly, to inhibit the progression of synaptic homeostasis at a late stage of vesicle release, and that Rab3-GAP functions to inactivate this action of Rab3-GTP. Loss of Rab3-GAP was shown to block both the rapid induction and sustained expression of synaptic homeostasis. Rab3 mutations alone do not block synaptic homeostasis and synaptic homeostasis proceeds normally in the rab3–rab3-GAP double mutant. Genetically, these data indicate that the presence of Rab3 is required for the block of synaptic homeostasis observed in the rab3-GAP mutant. Thus, Rab3 is likely to be the cognate GTPase for Rab3-GAP. Furthermore, in genetic terms, Rab3 functions to oppose the progression of synaptic homeostasis and, when it is removed, homeostasis proceeds (Müller, 2011).

The possibility is considered that homeostasis proceeds in the absence of Rab3 because another, redundant Rab takes the place of Rab3. In yeast membrane trafficking, there is evidence for semiredundant Rab function (Grosshans, 2006). However, this seems to be an exception because Rabs are hypothesized to have unique binding affinities for downstream effector proteins that are essential for their ability to define discrete membrane domains within membrane trafficking and secretory systems (Grosshans, 2006; Wickner, 2008). In C. elegans, Rab3 and Rab27 are both involved in synaptic vesicle release and they can be activated by a common exchange factor (Mahoney, 2006). However, based upon available genetic data, these Rabs do not appear to function redundantly during release (Mahoney, 2006). In Drosophila, loss of Rab3 causes a dramatic change in active zone size and organization (Graf, 2009). Thus, a redundant Rab would have to selectively and completely replace Rab3 function during synaptic homeostasis without rescuing synapse development, and this seems unlikely. Finally, since Rab3 is required for the complete block of synaptic homeostasis observed in the rab3-GAP mutant, it is concluded that Rab3 itself participates in mechanisms that determine whether or not synaptic homeostasis will proceed (Müller, 2011).

Next, the possibility is considered that Rab3 accumulates in a GTP-bound form at the synapse in the rab3-GAP mutant background, and this accumulation could block homeostatic plasticity. This model is attractive because it could explain why loss of Rab3 does not block homeostatic plasticity. However, a direct test of this model failed to provide supporting evidence. A constitutively active rab3 transgene was expressed in the rab3 mutant background. This experiment should mimic the accumulation of Rab3-GTP in a rab3-GAP mutant background. It was found that the constitutively active rab3 transgene (rab3^CA) is trafficked to the NMJ and localizes in a manner that is indistinguishable from overexpressed wild-type Rab3. Furthermore, rab3^CA has activity at the synapse because it rescues the defects in active zone organization that are caused by loss of rab3. In addition, constitutively active Rab3A was shown to biochemically interact with Rab3-GAP. However, the expression of rab3^CA did not disrupt synaptic homeostasis. Therefore, aberrant accumulation of Rab3-GTP is not the cause of impaired synaptic homeostasis in the rab3-GAP mutant and another model should therefore be considered (Müller, 2011).

Another activity of Rab3-GAP that could be relevant to synaptic homeostasis is its ability to physically bind Rab3-GTP (Clabecq, 2000). It is known that Rab3-GTP can bind several synaptic proteins including RIM and Rabphillin (Kanno, 2010). Moreover, this study provides evidence that Rab3's GTPase activity is not limiting during synaptic homeostasis. Therefore, it is proposed that Rab3-GAP competes for Rab3-GTP binding with another protein. If this other protein inhibits synaptic homeostasis when bound to the synaptic vesicle, then displacement by Rab3-GAP binding would be a required step for synaptic homeostasis to proceed. This model can explain all of the experimental data. First, Rab3-GAP would be necessary for synaptic homeostasis. Second, synaptic homeostasis would proceed normally in the rab3 mutant because the homeostatic inhibitor would no longer localize to the synaptic vesicle. Third, overexpression of rab3^CA in the rab3 mutant background would not block synaptic homeostasis because Rab3-GAP would still be able to compete for Rab3-GTP binding, displace the homeostatic inhibitor, and allow homeostatic plasticity to proceed (Müller, 2011).

This model is consistent with a conserved function of Rab proteins throughout the membrane trafficking and secretory pathways of organisms ranging from yeast to mammals. Rab proteins, in their GTP-bound state, function to nucleate the assembly of 'effector' protein complexes that define membrane microdomains (Grosshans, 2006; Wickner, 2008). However, throughout the literature, more is known about the assembly of these Rab-dependent complexes than is known about their disassembly (Nottingham, 2009). The model assumes that the binding of Rab3-GAP to Rab3 and the Rab3^CA mutant protein is sufficient to disrupt effector binding (including the proposed homeostatic inhibitor). It is generally believed that Rab-GAPs interact with their cognate GTPases with lower affinity than the effector proteins. However, although Rab3-GAP has a relatively low affinity for Rab3A, Rab3-GAP effectively competes with an effector (Rabphillin) for binding to Rab3A and this is true even when Rab3A harbors the Q81L mutation (Clabecq, 2000). These data support the possibility that Rab3-GAP could compete for effector binding and disrupt a Rab3-GTP dependent scaffold. This is not the only manner in which Rab3-GAP differs from other Rab-GAPs. Rab3-GAP is somewhat unique in that it does not contain additional protein-protein interaction motifs. Thus, unlike IQ-GAP proteins for instance, it seems unlikely that Rab3-GAP has unique functions that are independent of Rab3, consistent with the double-mutant analysis of rab3 and rab3-GAP (Müller, 2011).

Finally, the possibility is considered that the rab3-GAP mutation could create a ceiling effect where baseline transmission is normal but release cannot be potentiated under any condition. Several pieces of data argue against this possibility. First, by elevating extracellular calcium, quantal content can be increased in the rab3-GAP mutant, indicating that there is no restriction on the absolute number of quanta that can be released. Second, during a stimulus train (20 Hz, 0.4 mM extracellular calcium), the rab3-GAP mutant plateaus at a higher EPSP amplitude compared to wild-type. Again, there is no evidence for a ceiling effect in rab3-GAP. Finally, it has been demonstrated that mutations that cause a severe defect in baseline transmission can still undergo homeostatic compensation (Dickman, 2009). Taken together, these data argue against a simple ceiling effect and support the conclusion that Rab3-GAP is directly involved in the mechanisms of synaptic homeostasis (Müller, 2011).

A question that cannot be addressed is which step in the model is modified during the induction of synaptic homeostasis. One interesting possibility is that the interaction of Rab3-GTP with the homeostatic repressor is regulated. For example, if this interaction is stabilized, then homeostatic plasticity would be opposed and, conversely, if the interaction is weakened, then homeostasis would be allowed to proceed. A recent study in C. elegans (Simon, 2008) provided evidence that an unknown retrograde signal, from muscle to motoneuron, causes increased expression of YFP-Rab3 at the presynaptic terminal. Although no evidence was found for a similar phenomenon at the Drosophila NMJ during synaptic homeostasis, these data support the possibility that the Rab3/Rab3-GAP signaling complex could be a downstream, regulated target of a homeostatic, retrograde signal at the NMJ (Müller, 2011).

Interpreting the current data requires consideration of a recent study examining the effects of a rab3 mutation on synapse organization at the Drosophila NMJ (Graf, 2009). In the Graf study, it was discovered that a rab3 mutation causes a dramatic accumulation of both the active zone-associated protein Bruchpilot (Brp, T-bars, the Drosophila homolog of CAST/ELKS) and presynaptic calcium channels at a subset of active zones (Graf, 2009). Based on these and other data it was suggested that Rab3 promotes the nucleation of new active zones, and without this activity, active zones coalesce (Graf, 2009). It was possible to clearly dissociate any morphological reorganization of the NMJ from a blockade of synaptic homeostasis. The rab3 mutants have altered NMJ morphology, but normal synaptic homeostasis, whereas rab3-GAP mutants have normal NMJ morphology and a defect in synaptic homeostasis) (Müller, 2011).

It is also important to consider why rab3 mutants do not show excessive homeostatic compensation. It is predicted that Rab3 and Rab3-GAP will not control the magnitude of the homeostatic response, just whether or not it is allowed to proceed. Additional negative feedback signaling mechanisms would be responsible for determining the magnitude of the homeostatic response. This would explain why no excessive homeostatic compensation was observed in the absence of Rab3. Thus, Rab3 and Rab3-GAP provide an additional layer of control on synaptic homeostasis, ensuring that modulation of release probability only occurs when, and perhaps where, appropriate (Müller, 2011).

Ultimately, it would be important to understand how presynaptic vesicle release is modulated during homeostatic plasticity. It is known from previously published data that a homeostatic increase in vesicle release is due to a change in presynaptic release probability without a change in active zone number (Frank, 2006; Frank, 2009; Dickman, 2009). Mechanistically, the full functionality of presynaptic calcium channels is necessary for synaptic homeostasis (Frank, 2006; Frank, 2009). However, it remains unknown whether synaptic homeostasis involves a change in calcium channel number versus calcium channel function. Given these prior data, one possibility is that the homeostatic signaling system, identified in this study, acts upon presynaptic calcium channels to prevent a change in calcium influx. In this respect, the involvement of RIM and RIM binding protein are intriguing since RIM binds to Rab3-GTP and has been proposed to influence calcium-channel function (Kiyonaka, 2007; Hibino, 2002; Müller, 2011 and references therein).

Rab3-GAP is the first protein to be implicated in the homeostatic modulation of presynaptic release that directly interacts with a resident synaptic vesicle protein. This fact, and analysis of baseline synaptic transmission in the rab3-GAP mutant raise the possibility that the homeostatic modulation of presynaptic release also includes mechanisms that are independent of increased calcium influx. For example, a defect was observed in presynaptic release probability in the rab3-GAP mutant that occurs only when recording is performed in low extracellular calcium. This defect could reveal a function of Rab3-GAP during vesicle release, or it could reflect the activity of the proposed homeostatic repressor on baseline synaptic transmission. One possibility that could explain the decrease in release probability is that synaptic vesicles reside at a greater physical distance from the calcium channel in the rab3-GAP mutant. When recording in low extracellular calcium, the calcium microdomains at the active zone would not effectively trigger the release of these more distant vesicles. This model would suggest that enhanced coupling of the synaptic vesicle and the calcium channel is part of the homeostatic modulation of presynaptic release. By extension, the action of the homeostatic repressor would be to prevent a tight association of the synaptic vesicle with the calcium channel. It is interesting to speculate that the homeostatic repressor could be Rabphillin. It has been shown that Rabphillin can compete with Rab3-GAP for binding to Rab3-GTP (Clabecq, 2000). Rabphillin has two C2 domains that could confer calcium-dependence to this protein-protein interaction and, by extension, homeostatic plasticity. Ultimately, a molecular change that influences the functionality of the calcium sensor for vesicle fusion cannot be ruled out. Regardless, the data identify a homeostatic mechanism that functions at a late stage of vesicle release to modulate presynaptic release probability (Müller, 2011).

In combination with a previously published genetic screen (Dickman, 2009), thirteen mutations have now been identified that disrupt the expression of synaptic homeostasis without severely altering baseline synaptic transmission. Among these genes are rab3-GAP and dysbindin (Dickman, 2009). The mutant phenotypes forrab3-GAP and dysbindin are remarkably similar. In both cases, loss of function mutations have little effect on baseline transmission under standard recording conditions (0.4 mM extracellular calcium). However, decreasing extracellular calcium reveals a significant decrease in release probability. In agreement, an increase was also observed in short-term synaptic facilitation in both mutations. Furthermore, neither mutation has an effect on synapse morphology or active zone number. In dysbindin mutants, these effects were shown to be downstream or independent of presynaptic calcium influx. It is tempting to place Dysbindin into the proposed model for homeostatic plasticity. One possibility is that Dysbindin functions to stabilize the close association of synaptic vesicles with the presynaptic calcium channel. The absence of Dysbindin would therefore phenocopy the rab3-GAP mutant but function through a different set of molecular interactions on the synaptic vesicle. The similarity between the phenotypes of dysbindin and rab3-GAP are also interesting because dysbindin has been linked to schizophrenia in human. The intriguing possibility that Dysbindin interacts with Rab3-Rab3-GAP signaling will be the subject of future studies (Müller, 2011).

Optical quantal analysis of synaptic transmission in wild-type and rab3-mutant Drosophila motor axons

Synaptic transmission from a neuron to its target cells occurs via neurotransmitter release from dozens to thousands of presynaptic release sites whose strength and plasticity can vary considerably. An in vivo imaging method is described that monitors real-time synaptic transmission simultaneously at many release sites with quantal resolution. This method was applied to the model glutamatergic system of the Drosophila larval neuromuscular junction. It was found that, under basal conditions, about half of release sites have a very low release probability, but these are interspersed with sites with as much as a 50-fold higher probability. Paired-pulse stimulation depresses high-probability sites, facilitates low-probability sites, and recruits previously silent sites. Mutation of the small GTPase Rab3 substantially increases release probability but still leaves about half of the sites silent. These findings suggest that basal synaptic strength and short-term plasticity are regulated at the level of release probability at individual sites (Peled, 2011).

This study has combined a new postsynaptically targeted Ca²⁺ sensor, SynapGCaMP2, with fast-scanning confocal microscopy to image glutamatergic transmission in the D. melanogaster larval NMJ. The method provides simultaneous quantal resolution for hundreds of active zones in dozens of boutons along a long axon in vivo. The simultaneous information on the release properties of such a large collection of release sites makes it possible to examine how both spontaneous miniature release events and events evoked by a single action potential are distributed in time and space along the axon, to compare the release properties of the two kinds of events, and to study the changes in release in a paradigm of short-term plasticity (Peled, 2011).

in vivo observations confirm earlier deductions from less direct experimental techniques that transmitter release properties and short-term facilitation can differ between the release sites of a single axon, that there is a correlation between the probability of evoked release and the frequency of spontaneous release, and that, in the D. melanogaster NMJ, transmission is stronger at distal boutons. The optical quantal analysis of these imaging and analysis methods enabled examination of the collection of hundreds of release sites that make up the NMJ and determine the release probability and amplitude of each one of them. In wild-type NMJs, it was found that release sites can differ in their release probabilities and transmission amplitudes, although the difference in transmission amplitudes is only significant for a small number of sites of exceptionally high release probabilities. Additionally, it was found that the short-term modulation of overall synaptic strength is achieved by a change in the balance of release probabilities that leads to an altered spatial arrangement of strong and weak release sites and not by changes in the amount of neurotransmitter released when a site is active (Peled, 2011).

In hippocampal neurons, release probability is correlated with active-zone size. In the D. melanogaster NMJ, more glutamate receptors cluster opposite larger presynaptic active zones, suggesting that the same correspondence between active-zone area and release probability could hold. However, no clear relation was found between active-zone size and basal release probability in wild-type NMJs, suggesting that other source or sources of variability dominate. Indeed, of the many hundreds of active zones present along an axon branch only a small minority has a high basal release probability, and these are disproportionately concentrated at distal boutons, despite uniformity in the density of presynaptic active zones (Peled, 2011).

The observations show that a substantial number of release sites have such low basal release probability that no activity is detected, that is, they are effectively silent. Some of these sites become active when presented with a second stimulation pulse a short time after the first stimulation, confirming that they are functional and within detection capabilities. These observations are consistent with earlier evidence indicating that whereas some release sites are more active, releasing transmitter in response to single action potentials, others are reluctant, participating in synaptic activity only under more demanding conditions that involve high-frequency presynaptic activity (Peled, 2011).

SynapGCaMP2 was targeted to the muscle postsynapse to report Ca²⁺ influx via glutamate receptors. This postsynaptic measure seems to give an accurate representation of presynaptic neurotransmitter release in that the number of detected ΔF spots (measured mEPSPs) agreed with the calculated quantal content in wild-type preparations and the altered presynaptic organization of the rab3 mutants matched the pattern of postsynaptically imaged activity. Some transmission events could be too small, because of a paucity of postsynaptic receptors, to be detected by the method and, therefore, fall into the group of 44% of sites that were assign as functionally silent. However, a previous study found that glutamate receptor subunit composition and numbers are not correlated with average postsynaptic transmission strength in wild-type NMJs. If there is a postsynaptic mechanism that affects the amount of postsynaptic Ca²⁺ influx it would therefore probably involve post-translational modifications that affect glutamate receptor function but are not necessarily detected by immunohistochemistry. One such mechanism could be the suggested PKA regulation of the function of the D. melanogaster glutamate receptor subunit DGluRIIA (Peled, 2011).

It remains to be determined how different release properties are established. This work demonstrates that the mechanisms involved in this process operate differentially even for closely spaced release sites within the same bouton. Yet the resulting arrangement of release properties along the axonal branch is not random, as more high-probability release sites are located at distal boutons. These results suggest the existence of both local intrabouton regulation at the level of single release sites and global interbouton regulation at the level of the entire axon (Peled, 2011).

The small GTPase Rab3 is involved in the regulation of neurotransmitter release. Recently, a study in D. melanogaster has shown that in rab3-mutant NMJs essential presynaptic proteins are concentrated in a subset of the available active zones (Graf, 2009) suggesting either a separate additional role of Rab3 in presynaptic organization, or that the effect of Rab3 on release probability is achieved via its recruitment of presynaptic proteins to active zones. This study found that in rab3-mutant NMJs transmission occurs exclusively at the presynaptically enriched active zones, which are elevated in both release probability and transmission amplitude. Notably, the absence of Rab3 not only alters the size and release properties of individual active zones but also affects the distribution of release probabilities along the axon branch. In contrast to wild-type NMJs, where there are more high-probability release sites in distal boutons, the distribution of high-probability sites is equal throughout the axon branch in the mutant NMJs. This is not because of an inhomogeneity in Rab3 distribution in wild-type axons, but perhaps it reflects a spatial inhomogeneity in Rab3 regulation, for example in the regulation of the GTPase activity of Rab3 (Peled, 2011).

Search PubMed for articles about Drosophila Rab3

Clabecq, A., Henry, J. P. and Darchen, F. (2000). Biochemical characterization of Rab3-GTPase-activating protein reveals a mechanism similar to that of Ras-GAP. J. Biol. Chem. 275: 31786-31791. PubMed ID: 10859313

Dai, Y., et al. (2006). SYD-2 Liprin-alpha organizes presynaptic active zone formation through ELKS. Nat. Neurosci. 9: 1479-1487. PubMed ID: 17115037

DiAntonio, A. R., et al. (1993). Identification and characterization of Drosophila genes for synaptic vesicle proteins. J. Neurosci. 13: 4924-4935. PubMed ID: 8229205

Dickman, D. K. and Davis, G. W. (2009). The schizophrenia susceptibility gene dysbindin controls synaptic homeostasis. Science 326: 1127-1130. PubMed ID: 19965435

Fischer von Mollard, G., Südhof, T. C. and Jahn, R. (1991). A small GTP-binding protein dissociates from synaptic vesicles during exocytosis. Nature 349: 79-81. PubMed ID: 1845915

Frank, C. A. et al. (2006). Mechanisms underlying the rapid induction and sustained expression of synaptic homeostasis. Neuron 52: 663-677. PubMed ID: 17114050

Frank, C. A., Pielage, J. and Davis, G. W. (2009). A presynaptic homeostatic signaling system composed of the Eph receptor, ephexin, Cdc42, and CaV2.1 calcium channels. Neuron 61: 556-569. PubMed ID: 19249276

Graf, E. R., et al. (2009). Rab3 dynamically controls protein composition at active zones. Neuron 64(5): 663-77. PubMed ID: 20005823

Grosshans, B. L. Ortiz, D. and Novick, P. (2006). Rabs and their effectors: Achieving specificity in membrane traffic. Proc. Natl. Acad. Sci. 103: 11821-11827. PubMed ID: 16882731

Hibino, H., et al. (2002). RIM binding proteins (RBPs) couple Rab3-interacting molecules (RIMs) to voltage-gated Ca2+ channels. Neuron 34: 411-423. PubMed ID: 11988172

Iwasaki, K., et al. (1997). aex-3 encodes a novel regulator of presynaptic activity in C. elegans. Neuron 18: 613-622. PubMed ID: 9136770

Johnston, P. R., et al. (1991). rab3A attachment to the synaptic vesicle membrane mediated by a conserved polyisoprenylated carboxy-terminal sequence. Neuron 7: 101-109. PubMed ID: 1648935

Kanno, E., et al. (2010). Comprehensive screening for novel rab-binding proteins by GST pull-down assay using 60 different mammalian Rabs. Traffic 11: 491-507. PubMed ID: 20070612

Kiyonaka, S., et al. (2007). RIM1 confers sustained activity and neurotransmitter vesicle anchoring to presynaptic Ca2+ channels. Nat. Neurosci. 10: 691-701. PubMed ID: 17496890

Mahoney, T. R., et al. (2006). Regulation of synaptic transmission by RAB-3 and RAB-27 in Caenorhabditis elegans. Mol. Biol. Cell 6: 2617-2625. PubMed ID: 16571673

Müller, M., Pym, E. C., Tong, A. and Davis, G. W. (2011). Rab3-GAP controls the progression of synaptic homeostasis at a late stage of vesicle release. Neuron 69(4): 749-62. PubMed ID: 21338884

Nottingham, R. M. and Pfeffer, S. R. (2009). Defining the boundaries: Rab GEFs and GAPs. Proc. Natl. Acad. Sci. 106: 14185-14186. PubMed ID: 19706500

Peled, E. S. and Isacoff, E. Y. (2011). Optical quantal analysis of synaptic transmission in wild-type and rab3-mutant Drosophila motor axons. Nat. Neurosci. 14(4): 519-26. PubMed ID: 21378971

Pelkey, K. A. and McBain, C. J. (2007). Differential regulation at functionally divergent release sites along a common axon. Curr. Opin. Neurobiol. 17: 366-373. PubMed ID: 17493799

Patel, M. R. and Shen, K. (2009). RSY-1 is a local inhibitor of presynaptic assembly in C. elegans. Science 323: 1500-1503. PubMed ID: 19286562

Schoch, S., et al (2002). RIM1alpha forms a protein scaffold for regulating neurotransmitter release at the active zone. Nature 415: 321-326. PubMed ID: 11797009

Schluter, O. M.. Basu, J., Sudhof, T. C. and Rosenmund, C. (2006). Rab3 superprimes synaptic vesicles for release: implications for short-term synaptic plasticity. J. Neurosci. 26: 1239-1246. PubMed ID: 16436611

Simon, D. J. et al. (2008). The microRNA miR-1 regulates a MEF-2-dependent retrograde signal at neuromuscular junctions. Cell 133: 903-915.

Sudhof, T. C. (2004). The synaptic vesicle cycle. Annu. Rev. Neurosci. 27: 509-547. PubMed ID: 15217342

Takamori, S., et al. (2006). Molecular anatomy of a trafficking organelle. Cell 127: 831-846. PubMed ID: 17110340

Viquez, N. M., et al. (2009). PP2A and GSK-3beta act antagonistically to regulate active zone development. J. Neurosci. 29: 11484-11494. PubMed ID: 19759297

Wairkar, Y. P., et al. (2009). Unc-51 controls active zone density and protein composition by downregulating ERK signaling. J. Neurosci. 29: 517-528. PubMed ID: 19144852

Wang, Y., et al (1997). Rim is a putative Rab3 effector in regulating synaptic-vesicle fusion. Nature 38: 593-598. PubMed ID: 13197706

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date revised: 2 January 2023

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