InteractiveFly: GeneBrief

Synapsin: Biological Overview | References

BIOLOGICAL OVERVIEW

Synapsin is a phosphoprotein reversibly associated with synaptic vesicles. Synapsin function in mediating synaptic activity was investigated during intense stimulation at Drosophila motor boutons. Electron microscopy analysis of synapsin⁻ boutons demonstrated that synapsin maintains vesicle clustering over the periphery of the bouton. Cyclosporin A pretreatment disrupted peripheral vesicle clustering, presumably due to increasing synapsin phosphorylated state. Labeling recycling vesicles with a fluorescent dye FM1-43 followed by photoconversion of the dye into electron dense product demonstrated that synapsin deficiency does not affect mixing of the reserve and recycling vesicle pools but selectively reduces the size of the reserve pool. Intense stimulation produced a significant increase in vesicle abundance and vesicle redistribution toward the central core of synapsin ⁺ boutons, while in synapsin⁻ boutons the area occupied by vesicles did not change and the increase in vesicle numbers was not as prominent. However, intense stimulation produced an increase in basal release at synapsin⁻ but not in synapsin⁺ boutons, suggesting that synapsin may direct vesicles to the reserve pool. Finally, synapsin deficiency inhibited an increase in quantal size and formation of endosome-like cisternae, which was activated either by intense electrical stimulation or by high K⁺ application. Taken together, these results elucidate a novel synapsin function, specifically, promoting vesicle reuptake and reserve pool formation upon intense stimulation (Akbergenova, 2010).

The question of how neuronal terminals maintain synaptic transmission during intense neuronal stimulation remains fascinating and controversial. It is now understood that synaptic vesicles undergo a series of preparatory steps to be properly activated for neurotransmitter release, and, according to their functional state, vesicles can be subdivided into several functional pools (Schneggenburger, 2002 for review). The recycling pool maintains exo/endocytosis at moderate stimulation paradigms, and it is refilled by newly recycled vesicles, while the reserve pool is a depot of synaptic vesicles which contribute to release only during intense stimulation (Rizzoli, 2005 for review). Multiple pathways have been proposed for recycling of synaptic vesicles upon exocytosis, but the relationship between these pathways and functional vesicle pools is not fully understood (Akbergenova, 2010).

At the majority of synapses, the recycling and reserve pools are spatially intermixed (Rizzoli, 2005 for review). However, at Drosophila Ib type motor boutons, optical studies relying on vesicle staining with FM1-43 dye lead to a view that the recycling pool occupies the periphery of the bouton and the reserve pool is spread towards its central core (Kidokoro, 2004; Kuromi, 2005; Verstreken, 2005). This view has been recently revised by two ultrastructural studies (Akbergenova, 2009; Denker, 2009) demonstrating that at this synapse vesicles generally cluster over the periphery of the bouton, but the reserve and recycling pools are spatially intermixed. Furthermore, it was demonstrated that intense stimulation produces an increase in vesicle abundance, as well as vesicle redistribution towards the central core of the bouton (Akbergenova, 2009a). This form of plasticity was associated with an increased synaptic enhancement during a subsequent stimulation (Akbergenova, 2010).

Vesicle recycling may follow different routes or pathways, including active zone recycling, clathrin-mediated endocytosis, endosomal intermediates, or bulk membrane uptake. An intense synaptic activity is often associated with the recycling pathway that involves a formation of endosome-like structures or cisternae. Recently, it was recognized that endosome-like structures may be capable of exocytosis, and that this process may produce enlarged neurosecretory quanta. Thus, formation of endosome-like cisternae and enlarged vesicles is likely to represent a pathway that enhances synaptic efficacy (Akbergenova, 2010).

In summary, a neuronal terminal may sustain an intense stimulation by mobilizing reserve vesicles into the recycling pathway, by formation of extra vesicles, or by formation of enlarged vesicles and quanta. The goal of this study was to understand how these mechanisms are regulated by a synaptic vesicle protein synapsin (Akbergenova, 2010).

Synapsins, abundant and highly conserved family of phoshoproteins reversibly associated with vesicles, have been implicated in maintaining the reserve pool (Bloom, 2003; Akbergenova, 2007; Gitler, 2008) and regulating mobilization of vesicles into the recycling pool (Chi, 2003; Cousin, 2003; Menegon, 2006; Baldelli, 2007). In their dephosphorylated form, synapsins attach to synaptic vesicles and trigger actin polymerization (Bloom, 2003), while synapsin phosphorylation causes its dissociation from vesicles (Hosaka, 1999). Synapsin-dependent regulation of the reserve and recycling vesicle pools was found to be critical during sustained stimulation (Humeau, 2001). Consistently, synapsin deficiency produces enhanced synaptic depression (Rosahl, 1995; Gitler, 2004; Samigullin, 2004; Akbergenova, 2010 and references therein).

To investigate the role of synapsin in activity dependent plasticity at the Drosophila motor boutons, advantage was taken of the synapsin knockout [synapsin⁻] fly (Godenschwege, 2004) that was shown to have defects in complex behavior, learning and memory (Michels, 2005). This study has investigated how synaptic ultrastructure and activity is regulated upon intense stimulation in synapsin⁻ boutons (Akbergenova, 2010).

Synapsin functions in enabling a nerve terminal to maintain and enhance its activity during sustained stimulation. This synapsin function is likely to be mediated by several mechanisms. First, synapsin maintains vesicle organization that leaves void and, possibly, reserve spaces; these reserve spaces may be filled with extra vesicles upon intense stimulation. Second, synapsin is likely to participate in the process of vesicle recycling more directly, by directing the extra vesicles formed upon intense stimulation into the reserve pool. Finally, synapsin is required for an activation of the recycling pathway that produces enlarged vesicles/quanta upon maintained depolarization (Akbergenova, 2010).

Synapsin functioning is responsible for peripheral vesicle organization in Drosophila Ib motor boutons. Normally, vesicles in these boutons cluster over the periphery of the bouton leaving void spaces. This study has demonstrated that this vesicle organization is disrupted in synapsin null mutants. Furthermore, synapsin, similar to vesicles, has a peripheral distribution. This result suggests that synapsin may perform its function by binding to vesicles and tethering them at a peripheral cluster. In support of this idea, it was also demonstrated that a treatment by a calcineurin inhibitor CycA disrupts both peripheral vesicle clustering and the peripheral distributiuon of synapsin (Akbergenova, 2010).

The latter findings compel revisiting an earlier hypothesis that CycA treatment mobilizes reserve vesicles into the recycling pathway. The above interpretation was largely based upon the observation that FM1-43 fluorescence spread towards the center of the bouton when a mild stimulation paradigm was employed in the presence of CycA. The data suggests a different interpretation for this CycA action. It is known that CycA application completely blocks synapsin dephosphorylation at sites 4,5,6 (Jovanovic, 2001), which are controlled by MAP kinase/calcineurin activity. Interestingly, one of these sites (S62) is conserved between mammals and Drosophila (Genbank, COBALT alignment). Thus, it is likely that one of the consequences of CycA treatment is an increase in synapsin phosphorylated forms, and, consequentially, synapsin dispersion from vesicles followed by a dispersion of vesicles over the entire bouton (Akbergenova, 2010).

The synapsin function in maintaining vesicle clustering appears to be rather general among different types of organisms and synapses. Indeed, the studies performed at the giant lamprey synapse showed that synapsin neutralization disrupts vesicle clustering (Bloom, 2003; Hilfiker, 1999). Vesicle clustering is widely observed at mammalian CNS synapses even though regularities in vesicle distribution are not immediately evident. In synapsin null mutants vesicles are generally depleted (Rosahl, 1995; Li, 1995; Gitler, 2004; this study), and the degree of depletion may depend on the terminal region in relation to the plasma membrane. A tomography study (Siksou, 2007) demonstrated that vesicles are linked into clusters by a filamentous matrix, and that synapsin belongs to these clusters. Thus, the general synapsin function in vesicle clustering is evident. This study suggests an important physiological function of this clustering. Specifically, it suggests that synapsin may enable nerve terminals to maintain reserve spaces that can be filled by newly formed vesicles upon intense stimulation (Akbergenova, 2010).

This study has demonstrated that synapsin is a critical player in the process of structural potentiation, a form of plasticity which produces formation of extra vesicles upon intense stimulation (Akbergenova, 2009). It is thought that the major underlying mechanism of this structural potentiation is excess endocytosis (Engisch, 1998) that may be accelerated by intense synaptic activity (Wu, 2005). The possibility that a part of the newly formed vesicle pool is produced by anterograde axonal vesicle trafficking cannot be completely excluded (Akbergenova, 2010).

Hypothetically, actin/synapsin interactions could contribute to the latter mechanism by reducing vesicle mobility and promoting vesicle capturing in the terminals, thus enhancing vesicle supply via axonal transport. However, recent studies indicate that this possibility is unlikely. There is, in contrast emerging evidence that synapsin may contribute to endocytosis upon intense stimulation. In particular, nerve stimulation has been shown to produce synapsin immunoreactivity in the region of endocytic zones (Bloom, 2003). Furthermore, sustained stimulation of synapsin null terminals produced enhanced synaptic depression, as was observed in several preparations (Rosahl, 1995; Gitler, 2004; Samigullin, 2004), including Drosophila NMJ (this study). It is therefore suggested that a synapsin-dependent mechanism may enhance endocytosis upon sustained intense activity and direct the newly formed vesicles into the reserve pool (Akbergenova, 2010).

This study clearly demonstrates a critical role of synapsin in the latter mechanism, namely directing newly formed vesicles into the reserve pool, as well as maintaining the reserve pool. First, FM1-43 photoconversion experiments demonstrated that the reserve pool is diminished in synapsin⁻ terminals, while the recycling pool is unaffected. Second, recordings of synaptic activity from potentiated boutons demonstrated that the vesicles newly formed upon potentiation are directed into the reserve pool in WT but not in synapsin⁻ terminals. These results directly demonstrate synapsin functioning in forming and maintaining the reserve pool and suggest that this function is carried out via mediating compensatory endocytosis during sustained activity (Akbergenova, 2010).

Synapses may respond to maintained depolarization by activation of a recycling pathway that produces numerous endosome-like cisternae. This endosome-like structures are likely to represent a mixture of intermediates from three pathways, including bulk membrane uptake. Some of the endosome-like structures may fuse with the presynaptic membrane (Akbergenova, 2010).

Recent rigorous study at the calyx synapse demonstrated that intense activity initiates compound vesicle fusion, which is followed by formation of enlarged vesicles, subsequent release of enlarged neurosecretory quanta, and a compensatory bulk membrane reuptake. This study at the Drosophila NMJ produced similar conclusions, showing no FM1-43 staining at some of the enlarged vesicles (presumably formed by compound vesicle fusion), a strong and uniform FM1-43 uptake at other endosome-like structures (presumably produced by bulk endocytosis), and a non-uniform staining pattern (presumably, a fusion of stained and non-stained vesicles). The formation of enlarged vesicles produces an increase in quantal size that is likely to represent a mechanism for enhancing synaptic activity in response to a sustained stimulation. Indeed, enlarged vesicles/quanta appear as a result of enhanced motor activity in vivo, implying a physiological significance of this mechanism (Akbergenova, 2010).

This study has demonstrated that both formation of enlarged vesicles/cisternae and release of enlarged quanta critically depend on the presence of synapsin. Synapsin⁻ terminals lack the ability to increase the quantal size in response to sustained depolarization, since the formation of enlarged vesicles/cisternae is inhibited in synapsin⁻ terminals. It is important to note that synapsin deficiency inhibits activity-dependent formation of both endosome-like structures and vesicles. This finding, rules out the possibility that extra vesicles produced upon intense stimulation could be formed from endosome-like cisternae (Akbergenova, 2010).

Thus this study reveals a novel synapsin function, specifically, enhancing synaptic transmission via an increase in the size of a neurosecretory quantum. Together with earlier studies demonstrating that the formation of enlarged vesicles/cisternae depends on actin polymerization, these results may elucidate a specific molecular pathway of the compound vesicle fusion, suggesting that some of the observed endosome-like structures may be formed via an actin/synapsin-dependent association of synaptic vesicles (Akbergenova, 2010).

In summary, this study has demonstrated that the ability of a synapse to respond to a sustained stimulation is compromised in synapsin⁻ terminals in several ways. First, synapsin⁻ terminals have a diminished reserve pool and a limited ability to sustain synaptic activity in response to continuous stimulation. Second, synapsin⁻ terminals have a reduced ability to increase vesicle abundance in response to intense stimulation. Third, the vesicles newly formed in response to an intense stimulation are not directed to the reserve pool in the absence of synapsin. Finally, synapsin is required for an activation of a recycling pathway that produces enlarged vesicles/quanta in response to a maintained depolarization (Akbergenova, 2010).

A liquid phase of synapsin and lipid vesicles

Neurotransmitter-containing synaptic vesicles (SVs) form tight clusters at synapses. These clusters act as a reservoir from which SVs are drawn for exocytosis during sustained activity. Several components associated with SVs that are likely to help form such clusters have been reported, including synapsin. This study found that synapsin can form a distinct liquid phase in an aqueous environment. Other scaffolding proteins could coassemble into this condensate but were not necessary for its formation. Importantly, the synapsin phase could capture small lipid vesicles. The synapsin phase rapidly disassembled upon phosphorylation by calcium/calmodulin-dependent protein kinase II, mimicking the dispersion of synapsin 1 that occurs at presynaptic sites upon stimulation. Thus, principles of liquid-liquid phase separation may apply to the clustering of SVs at synapses (Milovanovic, 2018).

The presence of synaptic vesicle (SV) clusters is a defining feature of nerve terminals. SVs are tightly packed in these structures, which are well distinguished from the surrounding cytoplasm, although there is no evidence for a restraining boundary. Vesicles intermix within the clusters and can be exchanged between them. Although SV clusters present at synapses are anchored to active zones of secretion, active-zone proteins are not required for their formation and small clusters also occur in developing axons before synapse formation. How the motility of SVs within clusters is compatible with their spatial confinement remains unknown. Recently, liquid-liquid phase separation has been shown to be a mechanism through which components of the cytoplasm (proteins and RNAs) can assemble into distinct compartments (biomolecular condensates) not delimited by a membrane. A key feature of proteins that can undergo liquid-liquid phase separation is their ability to engage in multivalent, low-affinity interactions, either through intrinsically disordered regions (IDRs) or through association with binding partners. A major constituent of the matrix that connects SVs is synapsin, whose abundance in nerve terminals is severalfold higher than the abundance of any other protein specifically localized in this matrix. Synapsin comprises an adenosine triphosphate (ATP)- binding module of unclear physiological function, flanked by an N-terminal short region that partially penetrates membranes and a C-terminal IDR with multiple SRC homology 3 (SH3) domain-binding motifs. This prompted a hypothesis that synapsin may be a key constituent of a biomolecular condensate that includes SVs (Milovanovic, 2018).

To assess whether synapsin, which forms homo and heterodimers, can phase-separate through interactions of its IDR, enhanced green fluorescent protein (EGFP)-tagged synapsin 1 was incubated in a buffer of physiological salt concentration and pH on a glass-bottom dish at room temperature. After a lag time of tens of minutes, synapsin 1 alone formed micrometer-sized droplets, with the size of the droplets correlating with its concentration (0.5 to 20 &mi;M concentrations tested). The coalescence of synapsin 1 into droplets was confirmed by performing the incubation in suspension and measuring turbidity. These concentrations were not above the physiological range, as synapsin 1 is estimated to reach concentrations above 100 &mi;M in nerve terminals. Droplets of synapsin 1 had the expected properties of a liquid phase: They fused with each other, and bleaching of several droplets revealed that synapsin 1 molecules swiftly exchanged into and out of synapsin 1 droplets [half-time (t1/2) = 65 s]. Additionally, fluorescence recovery after photobleaching (FRAP) of a small area within the droplet was followed by rapid recovery (t1/2 = 40 s) of fluorescence, reflecting local rearrangement of synapsin 1 molecules. Analysis of two purified fragments of synapsin 1 confirmed that its IDR (amino acids 421 to 706), but not its folded central ATP-binding module (amino acids 113 to 420), which is known to dimerize, formed droplets. Droplet formation by the IDR alone was as efficient as droplet formation by the full-length protein. Synapsin 2, a paralog of synapsin 1 that can heterodimerize with synapsin 1, also contains a C-terminal IDR, albeit shorter than the IDR of synapsin 1. Accordingly, synapsin 2 also phase-separated. Increasing the salt concentration above the physiological range impaired droplet formation, implicating charge-dependent interactions in their formation (Milovanovic, 2018).

Polyvalent interactions between SH3 domain-containing proteins and proteins harboring cognate proline-rich motifs can also generate distinct liquid phases. Synapsin 1 interacts with several SH3 domain-containing proteins via its IDR. One such protein, intersectin (Drosophila homolog: Dap160), is a component of a network of protein-protein interactions that facilitates the clustering of SVs in conjunction with synapsin. Thus, this study examined whether, upon incubation with SH3 domain-containing binding partners such as intersectin and growth factor receptor-bound 2 (GRB2), synapsin 1 phase-separated together with them. GRB2 and intersectin contain two and five SH3 domains, respectively (Milovanovic, 2018).

Synapsin 1 was mixed with either GRB2 or a fragment from human intersectin comprising its five SH3 domains [(SH3)5-intersectin] and incubated at room temperature in physiological salt concentration. After some delay, droplets appeared containing both synapsin and its binding partners. Droplet growth, after the initial nucleation, was faster than with synapsin 1 alone. As before, droplets grew progressively or by fusing with each other, revealing a liquid state. The same concentration (10 μM) was used for synapsin 1 and its partners, although synapsin is thought to be the most abundant matrix protein within SV clusters. This was meant to reflect the presence at synapses of multiple SH3 domain-containing synapsin 1 ligands, and thus a higher collective concentration of these proteins than the concentration of any one of them. The synapsin 1:(SH3)5-intersectin droplets were larger, which could be explained by the higher valence of the intersectin fragment (five SH3 domains, although with different affinities for synapsin) relative to GRB2 (two SH3 domains) (Milovanovic, 2018).

The cytoplasm of a synaptic bouton is a crowded environment filled with organelles and macromolecules. To mimic this environment in subsequent experiments, polyethylene glycol (PEG), a crowding reagent, was added to the buffer. In the presence of 3% PEG 8000, droplets of synapsin 1 alone or of synapsin 1 and its binding partners formed immediately, with no lag phase. FRAP confirmed that, even under these conditions, proteins were mobile within the droplets, with a faster recovery time for synapsin 1 in droplets generated with GRB2 (t1/2 at 1.5 min) than in those generated with (SH3)5-intersectin (t1/2 at 3.2 min). This, again, possibly reflects the higher valence of this protein relative to GRB2. Recovery of fluorescence was observed both when a region within a droplet and when the entire droplet was bleached, indicating that recovery results from both molecular rearrangements within the droplet and the exchange of molecules with the dilute phase. Presence of a synapsin 1 binding partner, (SH3)5-intersectin, had a biphasic effect on droplet formation. It enhanced this process at low-to-moderate excess stoichiometric ratio but inhibited it when added in large excess. Thus, (SH3)5-intersectin is not only a 'client' of synapsin but also an active player in the formation of the liquid condensate. The negative effect at high stoichiometric ratio may be due to the masking of sites within the synapsin IDR that interact with each other. Recruitment of synapsin interactors into the droplets was specific and did not occur with proteins that do not bind synapsin (Milovanovic, 2018).

Synapsin binds SVs. If its phase-separating properties are involved in SV cluster formation, synapsin 1 should be capable of capturing vesicles into such a phase. Thus synapsin 1 was incubated with small lipid vesicles (~50 to 150 nm in diameter) mimicking SVs in lipid composition supplemented with a fluorescently labeled lipid, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(cyanine 5) (DOPE-Cy5). Formation of synapsin droplets correlated with the appearance of droplets positive for the labeled lipid, whereas no droplets positive for the lipid were observed in the absence of synapsin 1. Synapsin 1 condensates did not recruit vesicles lacking negatively charged phospholipids, which is expected, given that negatively charged lipids are necessary for synapsin binding to vesicles. Other well-characterized protein liquid condensates that do not bind lipid membranes, such as droplets composed of cytoplasmic protein noncatalytic region of tyrosine kinase adaptor protein 1 (NCK) and a fragment of neural Wiskott-Aldrich syndrome protein (N-WASP) that does not include the phospholipid-binding region, did not sequester lipid vesicles. Lipid vesicles were mobile within the synapsin phase, as indicated by FRAP. Furthermore, lipid vesicles and SH3-domain synapsin 1 interactors coassembled with synapsin. Electron microscopy (EM) analysis showed that these droplets were represented by clusters of small vesicles, whereas in the absence of synapsin 1, vesicles remained dispersed (Milovanovic, 2018).

Synapsin 1 is a major presynaptic phosphoprotein that undergoes multisite phosphorylation. Sustained nerve-terminal stimulation to trigger massive neurotransmitter release also induces the calcium-dependent phosphorylation of synapsin 1. This results in its dissociation from SVs and dispersion within the nerve-terminal cytosol, as SVs are consumed by exocytosis. If the formation of a biomolecular condensate by synapsin has a physiological importance in its coassembly with SVs, one would expect synapsin 1 droplets to disassemble upon calcium-dependent phosphorylation. Two prominent phosphorylation sites for calcium/calmodulin-dependent protein kinase II (CaMKII), called sites 2 and 3, are present in its IDR. Addition of CaMKII, calcium, and calmodulin to synapsin 1-containing samples did not disperse droplets. Indeed, CaMKII, which binds synapsin 1, was recruited into the droplets. However, further addition of ATP (200 μM) to induce synapsin 1 phosphorylation caused rapid dispersal of both synapsin 1 and CaMKII [mean lifetime of 5.9 s]. Importantly, CaMKII also disassembled synapsin 1-liposome droplets. As a control, protein kinase C was added to the droplets, for which synapsin 1 is not a substrate, but neither addition of the kinase nor the subsequent addition of ATP (200 μM) affected the droplets. The lack of effect of ATP in the latter experiment also rules out that droplet dispersion may be explained by a hydrotrope action of this nucleotide. Such an action, as reported for liquid droplets generated by other proteins, occurs only at much higher ATP concentrations than the one used in the phosphorylation assay (Milovanovic, 2018).

Strong evidence points to a physiological master role of synapsin in the clustering of SVs at living synapses. In neuronal cultures from mice that lack all three synapsins, the number of SVs at synapses is lower than that in wild-type mice, and this decrease is selective for SVs away from active zones. These results were extended. Even at synapses in situ-both excitatory (cerebellar mossy fibers) and inhibitory (deep cerebellar nuclei) nerve terminals-not only the total number but also the packing of SVs was notably lower in synapsin triple-knockout mice than in wild-type mice (Milovanovic, 2018).

Collectively, these findings demonstrate that synapsin can form a separate liquid biomolecular condensate either alone or together with binding partners for its IDR, with lipid vesicles, or with both. Interactions occurring at the presynapse in situ are expected to be more complex than the interactions of synapsin with the two SH3 domain-containing proteins and artificial lipid membranes described here. Because SVs are membranous organelles selectively recruited into clusters, there must be additional factors that help provide specificity. However, the minimal systems used here provide some insight into the mechanisms responsible for the properties of SV clusters. Clusters of other membranous organelles may self-organize according to similar principles without the need for a surrounding membrane or protein-based structure to confine them (Milovanovic, 2018).

Genetic dissection of aversive associative olfactory learning and memory in Drosophila larvae

Memory formation is a highly complex and dynamic process. It consists of different phases, which depend on various neuronal and molecular mechanisms. In adult Drosophila it was shown that memory formation after aversive Pavlovian conditioning includes-besides other forms-a labile short-term component that consolidates within hours to a longer-lasting memory. Accordingly, memory formation requires the timely controlled action of different neuronal circuits, neurotransmitters, neuromodulators and molecules that were initially identified by classical forward genetic approaches. Compared to adult Drosophila, memory formation was only sporadically analyzed at its larval stage. This study deconstructed the larval mnemonic organization after aversive olfactory conditioning. After odor-high salt conditioning (establishing an aversive olfactory memory) larvae form two parallel memory phases; a short lasting component that depends on cyclic adenosine 3'5'-monophosphate (cAMP) signaling and synapsin gene function. In addition, this study shows for the first time for Drosophila larvae an anesthesia resistant component, which relies on radish and bruchpilot gene function, protein kinase C (PKC) activity, requires presynaptic output of mushroom body Kenyon cells and dopamine function. Given the numerical simplicity of the larval nervous system this work offers a unique prospect for studying memory formation of defined specifications, at full-brain scope with single-cell, and single-synapse resolution (Widmann, 2016).

Memory formation and consolidation usually describes a chronological order, parallel existence or completion of distinct short-, intermediate- and/or long-lasting memory phases. For example, in honeybees, in Aplysia, and also in mammals two longer-lasting memory phases can be distinguished based on their dependence on de novo protein synthesis. In adult Drosophila classical odor-electric shock conditioning establishes two co-existing and interacting forms of memory--ARM and LTM--that are encoded by separate molecular pathways (Widmann, 2016).

Seen in this light, memory formation in Drosophila larvae established via classical odor-high salt conditioning seems to follow a similar logic. It consist of LSTM (larval short lasting component) and LARM (anesthesia resistant memory). Aversive olfactory LSTM was already described in two larval studies using different negative reinforcers (electric shock and quinine) and different training protocols (differential and absolute conditioning). The current results introduce for the first time LARM that was also evident directly after conditioning but lasts longer than LSTM. LARM was established following different training protocols that varied in the number of applied training cycles and the type of negative or appetitive reinforcer. Thus, LSTM and LARM likely constitute general aspects of memory formation in Drosophila larvae that are separated on the molecular level (Widmann, 2016).

Memory formation depends on the action of distinct molecular pathways that strengthen or weaken synaptic contacts of defined sets of neurons. The cAMP/PKA pathway is conserved throughout the animal kingdom and plays a key role in regulating synaptic plasticity. Amongst other examples it was shown to be crucial for sensitization and synaptic facilitation in Aplysia, associative olfactory learning in adult Drosophila and honeybees, long-term associative memory and long-term potentiation in mammals (Widmann, 2016).

For Drosophila larvae two studies by Honjo (2005) and Khurana (2009) suggest that aversive LSTM depends on intact cAMP signaling. In detail, they showed an impaired memory for rut and dnc mutants following absolute odor-bitter quinine conditioning and following differential odor-electric shock conditioning. Thus, both studies support the interpretation of the current results. It is argued that odor-high salt training established a cAMP dependent LSTM due to the observed phenotypes of rut, dnc and syn mutant larvae. The current molecular model is summarized in A molecular working hypothesis for LARM formation. Yet, it has to be mentioned that all studies on aversive LSTM in Drosophila larvae did not clearly distinguish between the acquisition, consolidation and retrieval of memory. Thus, future work has to relate the observed genetic functions to these specific processes (Widmann, 2016).

In contrast, LARM formation utilizes a different molecular pathway. Based on different experiments, it was ascertained, that LARM formation, consolidation and retrieval is independent of cAMP signaling itself, PKA function, upstream and downstream targets of PKA, and de-novo protein synthesis. Instead it was found that LARM formation, consolidation and/or retrieval depends on radish (rsh) gene function, brp gene function, dopaminergic signaling and requires presynaptic signaling of MB KCs (Widmann, 2016).

Interestingly, studies on adult Drosophila show that rsh and brp gene function, as well as dopaminergic signaling and presynaptic MB KC output are also necessary for adult ARM formation. Thus, although a direct comparison of larval and adult ARM is somehow limited due to several variables (differences in CS, US, training protocols, test intervals, developmental stages, and coexisting memories), both forms share some genetic aspects. This is remarkable as adult ARM and LARM use different neuronal substrates. The larval MB is completely reconstructed during metamorphosis and the initial formation of adult ARM requires a set of MB α/β KCs that is born after larval life during puparium formation (Widmann, 2016).

In addition, this study has demonstrated the necessity of PKC signaling for LARM formation in MB KCs. The involvement of the PKC pathway for memory formation is also conserved throughout the animal kingdom. For example, it has been shown that PKC signaling is an integral component in memory formation in Aplysia, long-term potentiation and contextual fear conditioning in mammals and associative learning in honeybees. In Drosophila it was shown that PKC induced phosphorylation cascade is involved in LTM as well as in ARM formation. Although the exact signaling cascade involved in ARM formation in Drosophila still remains unclear, this study has established a working hypothesis for the underlying genetic pathway forming LARM based on the current findings and on prior studies in different model organisms. Thereby this study does not take into account findings in adult Drosophila. These studies showed that PKA mutants have increased ARM and that dnc sensitive cAMP signaling supports ARM. Thus both studies directly link PKA signaling with ARM formation. (Widmann, 2016).

KCs have been shown to act on MB output neurons to trigger a conditioned response after training. Work from different insects suggests that the presynaptic output of an odor activated KCs is strengthened if it receives at the same time a dopaminergic, punishment representing signal. The current results support these models as they show that LARM formation requires accurate dopaminergic signaling and presynaptic output of MB KCs. Yet, for LARM formation dopamine receptor function seems to be linked with PKC pathway activation. Indeed, in honeybees, adult Drosophila and vertebrates it was shown that dopamine receptors can be coupled to Gαq proteins and activate the PKC pathway via PLC and IP3/DAG signaling. As potential downstream targets of PKC radish and bruchpilot are suggested. Interference with the function of both genes impairs LARM. The radish gene encodes a functionally unknown protein that has many potential phosphorylation sites for PKA and PKC. Thus considerable intersection between the proteins Rsh and PKC signaling pathway can be forecasted. Whether this is also the case for the bruchpilot gene that encodes for a member of the active zone complex remains unknown. The detailed analysis of the molecular interactions has to be a focus of future approaches. Therefore, the current working hypothesis can be used to define educated guesses. For instance, it is not clear how the coincidence of the odor stimulus and the punishing stimulus are encoded molecularly. The same is true for ARM formation in adult Drosophila. Based on the working hypothesis it can be speculated that PKC may directly serve as a coincidence detector via a US dependent DAG signal and CS dependent Ca²⁺ activation (Widmann, 2016).

Do the current findings in general apply to learning and memory in Drosophila larvae? To this the most comprehensive set of data can be found on sugar reward learning. Drosophila larva are able to form positive associations between an odor and a number of sugars that differ in their nutritional value. Using high concentrations of fructose as a reinforcer in a three cycle differential training paradigm (comparable to the one used in this study for high salt learning and fructose learning) other studies found that learning and/or memory in syn⁹⁷ mutant larvae is reduced to ~50% of wild type levels. Thus, half of the memory seen directly after conditioning seems to depend on the cAMP-PKA-synapsin pathway. The current results in turn suggest that the residual memory seen in syn⁹⁷ mutant larvae is likely LARM. Thus, aversive and appetitive olfactory learning and memory share general molecular aspects. Yet, the precise ratio of the cAMP-dependent and independent components rely on the specificities of the used odor-reinforcer pairings. Two additional findings support this conclusion. First, a recent study has shown that memory scores in syn⁹⁷ mutant larvae are only lower than in wild type animals when more salient, higher concentrations of odor or fructose reward are used. Usage of low odor or sugar concentrations does not give rise to a cAMP-PKA-synapsin dependent learning and memory phenotype. Second, another study showed that learning and/or memory following absolute one cycle conditioning using sucrose sugar reward is completely impaired in rut¹, rut²⁰⁸⁰ and dnc¹ mutants. Thus, for this particular odor-reinforcer pairing only the cAMP pathway seems to be important. Therefore, a basic understanding of the molecular pathways involved in larval memory formation is emerging. Further studies, however, will be necessary in order to understand how Drosophila larvae make use of the different molecular pathways with respect to a specific CS/US pairing (Widmann, 2016).

cindr, the Drosophila homolog of the CD2AP Alzheimer's disease risk gene, is required for synaptic transmission and proteostasis

The Alzheimer's disease (AD) susceptibility gene, CD2-associated protein (CD2AP), encodes an actin binding adaptor protein, but its function in the nervous system is largely unknown. Loss of the Drosophila ortholog cindr enhances neurotoxicity of human Tau, which forms neurofibrillary tangle pathology in AD. Cindr is expressed in neurons and present at synaptic terminals. cindr mutants show impairments in synapse maturation and both synaptic vesicle recycling and release. Cindr associates and genetically interacts with 14-3-3zeta, regulates the ubiquitin-proteasome system, and affects turnover of Synapsin and the plasma membrane calcium ATPase (PMCA). Loss of cindr elevates PMCA levels and reduces cytosolic calcium. Studies of Cd2ap null mice support a conserved role in synaptic proteostasis, and CD2AP protein levels are inversely related to Synapsin abundance in human postmortem brains. These results reveal CD2AP neuronal requirements with relevance to AD susceptibility, including for proteostasis, calcium handling, and synaptic structure and function (Ojelade, 2019).

Alzheimer's disease (AD) is a progressive and incurable neurodegenerative disorder that is estimated to affect 13 million people in the United States by 2050 (Alzheimer's Association). At autopsy, AD is characterized by extracellular neuritic plaques and intraneuronal neurofibrillary tangles, predominantly composed of misfolded and aggregated β-amyloid (Aβ) peptide and the microtubule-associated protein tau (MAPT or Tau), respectively. Substantial evidence indicates that AD pathology disrupts synaptic function at an early stage, and synapse loss is strongly associated with clinical progression. Human genome-wide association studies (GWASs) have discovered nearly 30 common variants associated with AD risk; however, most of these newly implicated genes remain poorly studied, especially in the nervous system. Prior work leveraged a human MAPT transgenic model in the fruit fly, Drosophila melanogaster, to identify AD susceptibility gene candidates that interact with Tau-mediated mechanisms. Among the findings, knockdown of cindr, the ortholog of the AD risk gene CD2-associated protein (CD2AP), enhanced Tau-induced neurodegeneration (Ojelade, 2019).

Before its identification from AD GWASs, loss-of-function mutations in CD2AP were identified as a rare cause of familial, autosomal dominant, focal segmental glomerulosclerosis, and progressive renal dysfunction has been recapitulated in mice hetero- or homozygous for a Cd2ap knockout allele. CD2AP encodes an adaptor protein that contains three SH3 motifs, a proline-rich region, and an actin binding domain. In the kidney, CD2AP is required to maintain structural integrity of the slit diaphragm, the specialized junctions between the foot processes of glomerular podocytes. Additional studies support a role for CD2AP in cytoskeletal anchoring of adhesion complexes and regulation of membrane localization for cell surface receptors via endocytosis. Several other AD susceptibility loci harbor genes with links to cell adhesion (CASS4, FERMT2, and PTK2B) and endocytosis (BIN1, PICALM, and RIN3). Nevertheless, the function of CD2AP in the adult nervous system, or its mechanism in neurological disorders, remains largely unknown. Studies in mice have connected CD2AP to both axonal growth (Harrison, 2016) and maintenance of blood-brain barrier integrity (Cochran, 2015). CD2AP has also been implicated in amyloid precursor protein trafficking and processing in cultured neurons (Ubelmann, 2017), although Cd2ap loss of function did not affect total Aβ levels or amyloid plaque burden in AD mouse models (Ojelade, 2019).

Besides suggesting potential interactions with Tau (Shulman, 2014), investigations in the fruit fly have implicated cindr with conserved roles in nephrocytes, retinal development, and oogenesis. This study has used Drosophila to elucidate the role of cindr in the nervous system, confirming a requirement for synaptic vesicle endocytosis and revealing requirements for ubiquitin-proteasome-mediated protein turnover and presynaptic Ca2+ homeostasis. Moreover, evidence is presented that the role of CD2AP in synaptic proteostasis is conserved in the mammalian brain and may contribute to vulnerability for Tau-mediated neurotoxic mechanisms in AD (Ojelade, 2019).

CD2AP is an AD susceptibility gene, but to date, its function in the adult nervous system is poorly defined. A cross-species experimental strategy implicates Cindr in synaptic biology and defines potential links to AD pathogenic mechanisms. First, Cindr was shown to be present at the synapse and interacts with other presynaptic proteins. Second, cindr loss of function causes impairments in synapse maturation and synaptic vesicle recycling and release. Third, the synaptic structural and functional defects are directly linked to requirements for Cindr in UPS-mediated protein turnover and Ca2+ homeostasis. Lastly, this study shows that Cindr functions with 14-3-3ζ to regulate proteasome activity, and this role is likely conserved in mouse and human brains. The results therefore enhance understanding of cindr/CD2AP and suggest potential mechanisms for its contribution to AD susceptibility (Ojelade, 2019).

Synaptic loss is strongly associated with cognitive decline in AD, and both Aβ and Tau have been linked to synaptic dysfunction. In Drosophila, loss of cindr causes a combination of endocytic and exocytic synaptic defects. Prior studies have implicated cindr/CD2AP in endocytosis in other contexts. Consistent with this, loss of cindr caused a reduced synaptic vesicle density at the NMJ and depression of synaptic responses during high-frequency stimulation, reminiscent of other genes implicated in endocytosis. Notable among these is like-AP180, which is homologous to another AD susceptibility gene PICALM. Loss of cindr caused reduced evoked potentials and enhanced facilitation, which both result from diminished presynaptic Ca2+, possibly because of increased PMCA levels. Altered Ca2+ handling has also been linked to AD pathogenesis, in which PMCA has been implicated. PMCA is a Ca2+ efflux pump with an established requirement for neuronal Ca2+ homeostasis. It was possible to rescue cindr phenotypes either directly through increasing extracellular [Ca2+] or indirectly by manipulating buffer pH to suppress PMCA activity; however, the possibility cannot be excluded that other Ca2+ channels or transporters may participate. Overall, this study confirms a role for Cindr in synaptic vesicle endocytosis and recycling and highlight an unexpected requirement for presynaptic Ca2+ homeostasis and resulting vesicle exocytosis and release (Ojelade, 2019).

Actin cytoskeletal dynamics have been implicated in synaptic vesicle recycling and plasticity, including at the Drosophila NMJ. Cindr is an actin binding protein, and this study found that cindr loss of function stabilizes F-actin in the Drosophila nervous system, consistent with prior studies in both flies and human cells. Changes in actin could potentially influence many functions and phenotypes addressed in this study. Pharmacological manipulations that disrupt actin have been reported to attenuate ghost bouton formation and impair synaptic vesicle endocytosis. Based on biochemical studies, actin may also bind and regulate human PMCA to modulate Ca2+ homeostasis. In mammals, Synapsins have been shown to tether synaptic vesicles to the actin cytoskeleton, thereby regulating the vesicle reserve pool and activity-dependent, presynaptic plasticity. In Drosophila, mutations in synapsin attenuate the formation of NMJ ghost boutons following stimulation. While loss of cindr leads to an increase in Synapsin, this is unlikely to explain the accompanying ghost bouton formation, because Synapsin overexpression does not cause this phenotype. Although elevations were identified in Synapsin and other presynaptic proteins in Cd2ap null mice, it will be important in future work to directly confirm a conserved role for Cd2ap in the function of mammalian central synapses. In the rat peripheral nervous system, Cd2ap was implicated to participate in signal transduction during axonal outgrowth accompanying collateral sprouting, an important contributor to neuronal plasticity (Ojelade, 2019).

dhat reduced CD2AP expression is protective for AD; however, this interpretation contradicts experimental findings that cindr/CD2AP (1) is required for neuronal homeostasis and synaptic function and (2) knockdown enhances Tau-induced neurodegeneration. Moreover, the true causal variant or variants at the CD2AP locus remain unknown. A sequencing study identified several AD cases harboring a rare, CD2AP coding variant (K633R) predicted to be damaging. Although this finding requires replication, it underscores the importance of fine mapping to definitively identify the responsible allelic variants and their mechanisms. Furthermore, because available brain RNA sequencing data are from bulk tissue, it was not possible to differentiate the impact of CD2AP risk alleles on gene expression in neurons versus other cell types (e.g., astrocytes, endothelial cells, or microglia). CD2AP is broadly expressed, and although the studies define important neuronal requirements, prior work highlights complementary functions in other cell types and tissues. Finally, it is possible that susceptibility genes and the implicated biological pathways have dynamic and pleiotropic relationships with AD phenotypes, even flipping from protection to risk during distinct temporal or cell-type-specific disease phases. For example, while reduced neuronal [Ca2+] may attenuate synaptic strength and thereby increase vulnerability to early AD synaptic dysfunction, the identical perturbation may protect against late excitotoxic mechanisms. Thus, it is possible, if not likely, that CD2AP impinges on multiple targets and pathways to affect AD pathogenesis. In conclusion, although substantial challenges remain, functional dissection of susceptibility genes like CD2AP holds enormous promise for refinement of AD mechanistic models and development of improved therapeutics (Ojelade, 2019).

MAPK/Erk-dependent phosphorylation of synapsin mediates formation of functional synapses and short-term homosynaptic plasticity

MAPK/Erk is a protein kinase activated by neurotrophic factors involved in synapse formation and plasticity, which acts at both the nuclear and cytoplasmic level. Synapsin proteins are synaptic-vesicle-associated proteins that are well known to be MAPK/Erk substrates at phylogenetically conserved sites. However, the physiological role of MAPK/Erk-dependent synapsin phosphorylation in regulating synaptic formation and function is poorly understood. This study examined whether synapsin acts as a physiological effector of MAPK/Erk in synaptogenesis and plasticity. To this aim, an in vitro model was developed of soma-to-soma paired Helix B2 neurons, which establish bidirectional excitatory synapses. It was found that the formation and activity-dependent short-term plasticity of these synapses is dependent on the MAPK/Erk pathway. To address the role of synapsin in this pathway, non-phosphorylatable and pseudo-phosphorylated Helix synapsin mutants were generated at the MAPK/Erk sites. Overexpression experiments revealed that both mutants interfere with presynaptic differentiation, synapsin clustering, and severely impair post-tetanic potentiation, a form of short-term homosynaptic plasticity. These findings show that MAPK/Erk-dependent synapsin phosphorylation has a dual role both in the establishment of functional synaptic connections and their short-term plasticity, indicating that some of the multiple extranuclear functions of MAPK/Erk in neurons can be mediated by the same multifunctional presynaptic target (Giachello, 2010).

Age-associated increase of the active zone protein Bruchpilot within the honeybee mushroom body

In honeybees, age-associated structural modifications can be observed in the mushroom bodies. Prominent examples are the synaptic complexes (microglomeruli, MG) in the mushroom body calyces, which were shown to alter their size and density with age. It is not known whether the amount of intracellular synaptic proteins in the MG is altered as well. The presynaptic protein Bruchpilot (BRP) is localized at active zones and is involved in regulating the probability of neurotransmitter release in the fruit fly, Drosophila melanogaster. This study explored the localization of the honeybee BRP (Apis mellifera BRP, AmBRP) in the bee brain and examined age-related changes in the AmBRP abundance in the central bee brain and in microglomeruli of the mushroom body calyces. Predominant AmBRP localization is reported near the membrane of presynaptic boutons within the mushroom body MG. The relative amount of AmBRP was increased in the central brain of two-week old bees whereas the amount of Synapsin, another presynaptic protein involved in the regulation of neurotransmitter release, shows an increase during the first two weeks followed by a decrease. In addition, an age-associated modulation was demonstrated of AmBRP located near the membrane of presynaptic boutons within MG located in mushroom body calyces where sensory input is conveyed to mushroom body intrinsic neurons. The observed age-associated AmBRP modulation might be related to maturation processes or to homeostatic mechanisms that might help to maintain synaptic functionality in old animals (Gehring, 2017).

In Drosophila, it has been demonstrated that Bruchpilot (DmBRP) in Kenyon cells plays a critical role in the formation of an anesthesia-resistant memory (Knapek, 2011): a 70% reduction of DmBRP in the Kenyon cells reduces this type of memory significantly. Accordingly, an age-associated increase of BRP, as observed in this experiment, might facilitate memory formation in fruit fly and possibly also in honeybees. However, Gupta (2016) demonstrated that an age-induced increase of DmBRP, which could be mimicked by an increase of the BRP copy number, did not facilitate anesthesia-resistant memory but instead blocked a cold-sensitive, anesthesia-sensitive memory. Based on these results, it was proposed that, in the Drosophila nervous system, aging synapses might steer towards the upper limit of their operational range by increasing BRP levels. This age-dependent process might limit synaptic plasticity and contribute to impairment of memory formation with age (Gehring, 2017).

Previous studies demonstrated that the packing density of boutons in lip and dense collar decreases with age resulting in fewer boutons in a defined area, i.e. a region of interest (ROI), of these neuropils. Thus, one would predict that presynaptic proteins in lip and dense collar are decreasing with age due to the decreased packing density of boutons resulting in fewer boutons per ROI that were analyzed. Indeed, this prediction proves true for Synapsin in the dense collar in this study, since an age-associated reduction was observed of the number of anti-SYNORF1-positive pixels. However, this is not the case for Synapsin in the lip where the number of anti-SYNORF1-positive pixels does not change with age. What might be the reason for this finding? It was shown that, in addition to the decrease in density, the mean volume of individual boutons increases with age in the lip and the dense collar. This increase is stronger in the lip than in the collar. Thus, the decrease in bouton density and the increase in bouton volume most likely counteract each other in the lip and this might be the reason why no change is seen in the amount of Synapsin in the lip (Gehring, 2017).

As it is the case with Synapsin, age-associated alterations in the structural organization of lip and collar boutons might influence the detection of anti-BRP^last200-positive pixels. Thus, the ratio between the median number of anti-BRP^last200-positive pixels to the median number of anti-SYNORF1-positive pixels per ROI was calculated, thereby factoring out the influence of morphological changes in the density and volume of the boutons on the detection of anti-BRP^last200-positive pixels. The ratios, i.e. the relative area, and thus probably the amount, of AmBRP increased in an age-associated manner in both, lip and collar: In the dense collar and the lip, the relative amount of AmBRP is significantly increased in 43-day-old bees. In addition, an increase was observed in the relative amount of AmBRP in the first week after emergence in the lip (Gehring, 2017).

AmBRP is a protein predominately located at presynapses. Due to the age-associated increase in bouton volume, boutons with a larger surface might also have more active zones. Increased numbers of active zones per bouton would lead to increased AmBRP levels which would provide an explanation for the observed age-associated increase in the relative amount of AmBRP. Indeed, this hypothesis could hold true for the collar as it was shown that the number of active zones per bouton is increased in 35-day-old bees compared with 1-day-old bees and that the proportion of ribbon vs. non-ribbon type active zones is increased in 35-day-old bees compared to 1-day-old bees. The latter is interesting, because ribbon-synapses in bees resemble T-bar-shaped synapses in fruit flies, that contain BRP, whereas non-ribbon synapses do not resemble this synapse type. Thus, these data are in line with findings of an increase of AmBRP from day 1, day 8 and day 15 to day 43 (Gehring, 2017).

In contrast to the collar, the number of active zones per bouton remains unchanged between 1- and 35-day-old bees in the lip. However, the same study showed that also in lip boutons the proportion of ribbon vs. non-ribbon type active zones increases. Thus, the AmBRP increase in the lip might not be indicative for the formation of new active zones and thus new synapses. Rather, it is suggested that, in the lip, it is the amount of AmBRP at existing active zones that is altered in an age-associated manner. As mentioned above, this alteration seems to take place twice: Early after emergence and late in the bees' lifetime between day 29 and 43. It might well be that an alteration of the amount of AmBRP at existing synapses shifts the proportion of ribbon vs non-ribbon active zones such that ribbon-active zones are increasing in an age-dependent manner (Gehring, 2017).

What might be the cause of the observed age-associated alterations of AmBRP in the lip and collar? Based on the existing literature, the first increase of AmBRP in the lip could be due to maturation processes in the olfactory system. The lip can be regarded as part of this olfactory system as projection neurons from the antennal lobes convey odor information onto MB Kenyon cells in this region. Neuropils belonging to the olfactory system such as the antennal lobes are not yet fully developed in newly emerged bees and mature during the first days after emergence. These maturation processes occurring in the antennal lobe might also influence synaptic connections, and thereby probably the amount of AmBRP, in upstream odor processing centers such as the lip (Gehring, 2017).

In addition to an AmBRP increase during the first week of a bee's life, increased AmBRP levels were found in very old bees (43-day-old) in the lip, but also in the collar. Similar results were observed at neuromuscular junctions of aged fruit flies (Gupta, 2016). The authors found that, with progression of age, the number of BRP-labeled spots, which indicate active zones, per bouton increased up to an age of 42 days and that this increase is accompanied by an increase in bouton volume. It is known from studies on endocytosis mutants, that an increase in number of boutons and active zones compensates a decrease of synaptic vesicle exocytosis. Thus, increased AmBRP levels at boutons in older insects might represent compensatory mechanisms for age-associated lower synaptic transmission. This hypothesis is in line with the view that age-associated synaptic alterations might be the consequence of adaptive processes due to neuronal plasticity that compensate for age-dependent cognitive impairments. Indeed, it has been demonstrated that a drop in postsynaptic excitability drives an increase of presynaptic scaffolds. According to the authors, this increase of presynaptic scaffolds might lead to an increase of synaptic vesicle release, which has been shown to be age-dependent (Gupta, 2016). In line, in a fruit fly model of Alzheimer's disease, an age-dependent reduction of the amount of BRP and the synaptic vesicle release probability has been observed suggesting that presynaptic β-amyloid plaques in the fruit fly brain might hinder a compensation of age-dependent processes that could be related to the amount of BRP (Gehring, 2017).

A striking feature of honeybee workers is their age-related division of labor. Individual workers perform different tasks within and outside the hive in an age-dependent manner: For the first 2-3 weeks after adult emergence, workers perform in-hive duties such as brood care and food processing, and start to forage for nectar and pollen outside the hive thereafter. This behavioral plasticity has been suggested to have both age- and experience-related determinants. Therefore, it should be taken into account that age-associated processes observed in honeybees are not only due to their chronological age but also due to the task they fulfill because of their age and because of the state of the colony. Thus, the age-associated effects observed in this study could be due to the (unknown) age-dependent signal that triggers the switch between the two tasks, due to experiences made when fulfilling the age-associated task, or due to the internal state of the colony. In the latter case, the observed effects would not be due to the bees' age but to the state of the colony. Since bees of defined ages were observed in a colony that was not manipulated, it is proposed that this study observed 'normally' aging bees and that the effects that were observed are directly or indirectly associated with the bees' age (Gehring, 2017).

This study reports that the level of the presynaptic proteins, Synapsin and AmBRP, are modified in an age-associated manner in the honeybee brain. An early increase was found in the relative amount of AmBRP during the first week after emergence in the MB lip, which was hypothesized to be due to maturation processes in the olfactory system. This study has shown that both MB regions, lip and collar, have increased amounts of AmBRP in 43-day-old bees. Given that BRP is homologous to the vertebrate ELKS/CAST/ERC protein, which is part of the presynaptic active zone, it will be interesting if these proteins are altered in an age-associated manner in vertebrates as well and if an AmBRP increase compensates for age-dependent cognitive impairments (Gehring, 2017).

A small pool of vesicles maintains synaptic activity in vivo

Chemical synapses contain substantial numbers of neurotransmitter-filled synaptic vesicles, ranging from approximately 100 to many thousands. The vesicles fuse with the plasma membrane to release neurotransmitter and are subsequently reformed and recycled. Stimulation of synapses in vitro generally causes the majority of the synaptic vesicles to release neurotransmitter, leading to the assumption that synapses contain numerous vesicles to sustain transmission during high activity. This assumption was tested by an approach termed cellular ethology, monitoring vesicle function in behaving animals (10 animal models, nematodes to mammals). Using FM dye photooxidation, pHluorin imaging, and HRP uptake it was found that only approximately 1%-5% of the vesicles recycled over several hours, in both CNS synapses and neuromuscular junctions. These vesicles recycle repeatedly, intermixing slowly (over hours) with the reserve vesicles. The latter can eventually release when recycling is inhibited in vivo but do not seem to participate under normal activity. Vesicle recycling increased only to ~5% in animals subjected to an extreme stress situation (frog predation on locusts). Synapsin, a molecule binding both vesicles and the cytoskeleton, may be a marker for the reserve vesicles: the proportion of vesicles recycling in vivo increased to 30% in synapsin-null Drosophila. It is concluded that synapses do not require numerous reserve vesicles to sustain neurotransmitter release and thus may use them for other purposes, examined in an accompanying paper (Denker, 2011a).

It is hypothesized that a physical barrier may inhibit the release of the majority of the vesicles, perhaps by lowering their mobility. Synapsin has been proposed to bind to synaptic vesicles and/or to the cytoskeleton, thus acting as a 'glue' keeping vesicles in a clustered (possibly inactive) state (see Cesca, 2011). To test whether vesicle clustering may affect their release in vivo, synapsin-null Drosophila larvae (Godenschwege, 2004) were studied. Indeed, fluorescence recovery after photobleaching (FRAP) experiments revealed that the vesicles were significantly more mobile in synapsin-null animals. The vesicle labeling was also much stronger in vivo, with approximately 30% of the vesicles labeled at 2-4 h after injection, allowing the conclusion that synapsin is one potential marker for the inactive vesicles (Denker, 2011a).

Because synapsin is not the only molecule cross-linking vesicles, it is not unexpected that a substantial nonreleasing pool of vesicles persists in the synapsin-null animals. Importantly, synapsin is a soluble molecule, rather than an integral component of the vesicles, and therefore it is not likely to constitute a permanent vesicle tag. Thus, an intermixing of the active and inactive vesicles should take place over time, although it seems to be on a very slow scale, of a few hours (Denker, 2011a)

It is suggested that most vesicles do not participate directly in neurotransmitter release and vesicle recycling. The number of vesicles functioning in vitro has been much discussed in the last decades, with many conflicting studies. Generally, NMJ works conclude that many or most vesicles can be used under stimulation, although some vesicle pools are used preferentially, whereas the remaining vesicles constitute a reserve pool. One group of studies has concluded that a substantial proportion of the vesicles in hippocampal neurons remain unused even during strong nonphysiological stimulation, albeit other investigations suggested that all vesicles may be releasable (Denker, 2011a and references therein)

The classic reserve pool of the literature typically comprises 50%-80% of all vesicles. This concept can hardly apply to an in vivo situation in which this study found more than 90% to be 'in reserve' or else to serve another purpose. It is difficult to imagine physiological situations that would require synapses to release substantially more vesicles at any one time. For example, the ventral muscles of the Drosophila larva would certainly be used more frequently if the larva attempted to avoid a predator, but with Drosophila movement being inherently slow, no great rates of activity could ever be attained. Similarly, stress increases the use of vesicles in locusts, but even in this life-or-death situation the jumping behavior is infrequent. Importantly, because predation eventually results in the death of all locusts analyzed, no higher stimulation could take place in vivo -- no life situation could have induced a stronger need for movement (and hence vesicle recycling). Finally, the rates of activity cannot increase endlessly/continually, at least for the NMJs, because the muscles would stop responding (Denker, 2011a).

The reserve vesicles seem to be clustered and are therefore rather immobile. They would simply stay linked to other vesicles and/or to the cytoskeleton (perhaps via synapsin), at tens or hundreds of nanometers from the active zones. This hypothesis is in agreement with two recent findings on vesicle mobility and recycling: first, only a small pool of vesicles is mobile in cultured hippocampal synapses, with all other vesicles immobile ('fixed'). The fixed vesicles did not become more mobile upon physiological stimulation, suggesting that they may not be involved in neurotransmitter release and recycling under normal circumstances. Second, in the same preparation only few active (mobile) vesicles have their membranes optimally sorted via endosomes. All other vesicles were found to be reluctant to release and did not seem to use endosomes when forced to release via prolonged stimulation in vitro. It is tempting to hypothesize that the reason why most synaptic vesicles do not sort their membranes optimally (via endosomes) is that these vesicles are not normally intended for recycling (in vivo) (Denker, 2011a).

The current data do not exclude the existence of synapses that use most of their vesicles. They also clearly indicate that the pool tags are not permanent, with the active and inactive vesicles intermixing slowly over time, so that in a strict sense all vesicles may be eventually used in neurotransmitter release. Additionally, the intermixing might be faster at higher temperatures (at least for invertebrates, which were maintained at 21°C in these experiments). However, at any point in time only a small proportion of the vesicles recycle. It is therefore unlikely that physiological activity would deplete all vesicles at one time, which is the only event that would justify the need for a large reserve pool of vesicles. In a related study (Denker, 2011b), the hypothesis is presented that these vesicles support release indirectly, functioning primarily as a molecular buffer for proteins involved in vesicle recycling. Thus, all vesicles seem to have some role in synaptic physiology, although only a minority is actually involved in neurotransmitter release at any one time (Denker, 2011a).

Cellular site and molecular mode of synapsin action in associative learning

Synapsin is an evolutionarily conserved, presynaptic vesicular phosphoprotein. This study asked where and how synapsin functions in associative behavioral plasticity. Upon loss or reduction of synapsin in a deletion mutant or via RNAi, respectively, Drosophila larvae are impaired in odor-sugar associative learning. Acute global expression of synapsin and local expression in only the mushroom body, a third-order 'cortical' brain region, fully restores associative ability in the mutant. No rescue is found by synapsin expression in mushroom body input neurons or by expression excluding the mushroom bodies. On the molecular level, it was found that a transgenically expressed synapsin with dysfunctional PKA-consensus sites cannot rescue the defect of the mutant in associative function, thus assigning synapsin as a behaviorally relevant effector of the AC-cAMP-PKA cascade. It is therefore suggested that synapsin acts in associative memory trace formation in the mushroom bodies, as a downstream element of AC-cAMP-PKA signaling. These analyses provide a comprehensive chain of explanation from the molecular level to an associative behavioral change (Michels, 2011).

The associative defect in the syn⁹⁷-mutant (Michels, 2005) can be phenocopied by an RNAi-mediated knock-down of synapsin, and can be rescued by acutely restoring synapsin. In terms of site of action, locally restoring synapsin in the mushroom bodies fully restores associative ability, whereas restoring synapsin in the projection neurons does not. If synapsin is restored in wide areas of the brain excluding the mushroom bodies, learning ability is not restored either. Therefore, it was concluded that a synapsin-dependent memory trace is located in the mushroom bodies, and suggest that this likely is the only site where such a trace is established regarding odor-sugar short-term memory in larval Drosophila. In terms of mode of action, it was found that a synapsin protein that carries dysfunctional PKA sites cannot rescue the syn⁹⁷-mutant learning defect. It is therefore suggested that synapsin functions as a downstream element of AC-cAMP-PKA signaling in associative function (Michels, 2011).

Arguably, the Rutabaga type I adenylyl cyclase acts as a detector of the coincidence between an aminergic reinforcement signal (appetitive learning: octopamine; aversive learning: dopamine) and the odor-specific activation of the mushroom body neurons. Initially, this notion had been based on mutant and biochemical analyses in Drosophila and physiology in Aplysia. Indeed, activation of mushroom body neurons in temporal coincidence with dopamine application increases cAMP levels in wild-type, but not AC-deficient flies (rut²⁰⁸⁰), and a corresponding AC-dependence of PKA activation has been shown by mushroom body costimulation with octopamine. However, the downstream effects of the AC-cAMP-PKA cascade has remained clouded. This study suggests that, similar to the situation in snails, one of these PKA effectors is synapsin, such that synapsin phosphorylation allows a transient recruitment of synaptic vesicles from the reserve pool to the readily releasable pool. A subsequent presentation of the learned odor could then draw upon these newly recruited vesicles. This scenario also captures the lack of additivity of the syn⁹⁷ and rut²⁰⁸⁰ mutations in adult odor-shock associative function, and the selective defect of the syn⁹⁷-mutation in short- rather than longer-term memory (Michels, 2011).

Given that the memory trace established in the current paradigm likely is localized to few cells relative to the brain as a whole, given that these are transient, short-term memory traces, and given the possibility of dephosphorylation, it is not unexpected that Nuwal (2011) did not uncovered either predicted PKA site of synapsin as being phosphorylated in a biochemical approach, using whole-brain homogenates from untrained adult Drosophila (for similar results in Drosophila embryos see Zhai, 2008). Given the likely spatial and temporal restriction of these events in vivo, immunohistological approaches are warranted to see whether, where, and under which experimental conditions synapsin phosphorylated at either of its PKA sites indeed can be detected (Michels, 2011).

Interestingly, the evolutionarily conserved N-terminal PKA-1 site undergoes ADAR-dependent mRNA editing, which despite the genomically coded RRFS motif yields a protein carrying RGFS. This editing event, as judged from whole-brain homogenates, occurs for most but not all synapsin and, as suggested by in vitro assays of an undecapeptide with bovine PKA, may reduce phosphorylation rates by PKA. Given that the successfully rescuing UAS-syn construct codes the edited RGFS sequence, it should be interesting to see whether this rescue is conferred by residual phosphorylation at PKA-1, and/or by phosphorylation of the evolutionarily nonconserved PKA-2 site. Last, one may ask whether an otherwise wild-type synapsin protein featuring a nonedited RRFS motif is also rescuing associative function (Michels, 2011).

In any event, the finding that the PKA consensus sites of synapsin are required to restore learning in the syn⁹⁷-mutant is the first functional argument to date, in any experimental system, to suggest synapsin as an effector of the AC-cAMP-PKA cascade in associative function (Michels, 2011).

Synapsin regulates activity-dependent outgrowth of synaptic boutons at the Drosophila neuromuscular junction

Patterned depolarization of Drosophila motor neurons can rapidly induce the outgrowth of new synaptic boutons at the larval neuromuscular junction (NMJ), providing a model system to investigate mechanisms underlying acute structural plasticity. Correlative light and electron microscopy analysis revealed that new boutons typically form near the edge of postsynaptic reticulums of presynaptic boutons. Unlike mature boutons, new varicosities have synaptic vesicles which are distributed uniformly throughout the bouton and undeveloped postsynaptic specializations. To characterize the presynaptic mechanisms mediating new synaptic growth induced by patterned activity, the formation of new boutons was investigated in NMJs lacking synapsin [Syn^-], a synaptic protein important for vesicle clustering, neurodevelopment, and plasticity. Budding of new boutons at Syn^- NMJs was significantly diminished, and new boutons in Syn^- preparations were smaller and had reduced synaptic vesicle density. Since synapsin is a target of protein kinase A (PKA), whether activity-dependent synaptic growth is regulated via a cAMP/PKA/synapsin pathway was assayed. Preparations were pretreated with forskolin to raise cAMP levels; this manipulation significantly enhanced activity-dependent synaptic growth in control but not Syn^- preparations. To examine the trafficking of synapsin during synaptic growth, transgenic animals were generated expressing fluorescently tagged synapsin. Fluorescence recovery after photobleaching analysis revealed that patterned depolarization promoted synapsin movement between boutons. During new synaptic bouton formation, synapsin redistributed upon stimulation toward the sites of varicosity outgrowth. These findings support a model whereby synapsin accumulates at sites of synaptic growth and facilitates budding of new boutons via a cAMP/PKA-dependent pathway (Vasin, 2014).

Bruchpilot, a synaptic active zone protein for anesthesia-resistant memory

In Drosophila, aversive associative memory of an odor consists of heterogeneous components with different stabilities. This study reports that Bruchpilot (Brp), a ubiquitous presynaptic active zone protein, is required for olfactory memory. Brp was shown before to facilitate efficient vesicle release, particularly at low stimulation frequencies. Transgenic knockdown in the Kenyon cells of the mushroom body, the second-order olfactory interneurons, revealed that Brp is required for olfactory memory. Brp in the Kenyon cells preferentially functions for anesthesia-resistant memory. Another presynaptic protein, Synapsin, has been shown previously to be required selectively for the labile anesthesia-sensitive memory, which is less affected in brp knockdown. Thus, consolidated and labile components of aversive olfactory memory can be dissociated by the function of different presynaptic proteins (Knapek, 2011).

In flies, middle-term olfactory memory after a single training cycle comprises functionally dissociable forms of memory: the labile ASM and the stable ARM. In contrast to ASM, the molecular basis of ARM formation is poorly understood. Only a few molecules have been shown to be important for ARM so far. This study demonstrated that Brp in the Kenyon cells of the mushroom body is preferentially required for ARM. Although there is no apparent developmental defect in the downregulation of Brp (Wagh, 2006), that study does not specify the requirement for ARM in the adult (Knapek, 2011).

The Brp protein is specifically localized to the active zone at the presynaptic terminals, in which it forms electron dense projections. Interestingly, the Radish protein that is also required for ARM is highly enriched in the lobes of the mushroom body. Thus, Brp and Radish might interact at the active zones to regulate neurotransmission underlying ARM (Knapek, 2011).

Several memory mutants have been shown to have a selective phenotype in ASM. Consistent with the parallel memory formation of ASM and ARM, the brp knockdown and a rutabaga mutation caused an additive memory deficit. Interestingly, Synapsin is required for ASM preferentially, and the null mutation caused no augmentation of the memory phenotype in the rutabaga single mutant (Knapek, 2010). Thus, the complementary forms of memory might recruit differential signaling mechanisms that rely on distinct presynaptic machineries (Knapek, 2011).

In a current model of memory dynamics, ARM gradually develops after training, whereas ASM occupies the largest part of early memory and decays more quickly. Although Radish and Brp are selectively required for ARM measured at 3 h after training, flies lacking either of the proteins are impaired also in immediate memory. By applying cold anesthesia for STM, this study found that STM does contain a significant ARM component. The consistent requirement of Brp for short-term and 3 h ARM may contribute to a synaptic mechanism of memory that is stable against amnesic treatment (Knapek, 2011).

If ARM and ASM were formed at the same synapses of Kenyon cells, how could the two synaptic proteins Brp and Synapsin dissociate these different forms of memory? Notably, Brp and Synapsin are meant to be required for distinct components of action potential-evoked vesicle release. In vertebrates, Synapsin has been shown to be particularly important for recruitment of synaptic vesicles from reserve pools at high stimulation frequencies (Pieribone, 1995; Rosahl, 1995; Gitler, 2004; Sun, 2006). Consistently, synapsin mutants show normal quantal content (number of synaptic vesicles released per action potential) at moderate action potential frequencies. Similarly, Drosophila Synapsin maintains the reserve pool of vesicles and mediates mobilization of the reserve pool during intense stimulation (Akbergenova, 2007). The brp null mutant in contrast shows decreased quantal contents particularly in response to the first arrival of an action potential. Vesicle release after subsequently following high-frequency spikes however is less affected, suggesting the importance in vesicle release at low-frequency stimulation. The two different modes of neurotransmission (e.g., different release probabilities during high- vs low-frequency stimulation) could therefore differentiate ASM and ARM, even if the traces of these different forms of memory resided in the same synapses of the Kenyon cells (Knapek, 2011).

Alternatively, the memory traces of ASM and ARM could be spatially separated within the same neurons, i.e., localized at different synapse populations. In the lobes, Kenyon cell axons have multiple compartments that are intersected by transverse extrinsic neurons. This study found that brp knockdown in the α/β neurons affected ARM. This is consistent with a previous report, in which inhibition of the output of the α/β neurons impaired ARM. Interestingly, inhibition of a specific type of dopaminergic neurons that synapse onto another restricted compartment of the β lobe selectively affected ASM (Aso, 2010). Thus, associative plasticity underlying ASM and ARM could be formed by stimulating different synapses of the same neurons. This hypothesis may be tested in future by the identification of extrinsic neurons that are specifically required for ARM and corresponding functional imaging of memory traces (Knapek, 2011).

Synapsin function in GABA-ergic interneurons is required for short-term olfactory habituation

In Drosophila, short-term (STH) and long-term habituation (LTH) of olfactory avoidance behavior are believed to arise from the selective potentiation of GABAergic synapses between multiglomerular local circuit interneurons (LNs) and projection neurons in the antennal lobe. However, the underlying mechanisms remain poorly understood. This study shows that synapsin (syn) function is necessary for STH and that syn⁹⁷-null mutant defects in STH can be rescued by syn⁺ cDNA expression solely in the LN1 subset of GABAergic local interneurons. As synapsin is a synaptic vesicle-clustering phosphoprotein, these observations identify a presynaptic mechanism for STH as well as the inhibitory interneurons in which this mechanism is deployed. Serine residues 6 and/or 533, potential kinase target sites of synapsin, are necessary for synapsin function suggesting that synapsin phosphorylation is essential for STH. Consistently, biochemical analyses using a phospho-synapsin-specific antiserum show that synapsin is a target of Ca²⁺ calmodulin-dependent kinase II (CaMKII) phosphorylation in vivo. Additional behavioral and genetic observations demonstrate that CaMKII function is necessary in LNs for STH. Together, these data support a model in which CaMKII-mediated synapsin phosphorylation in LNs induces synaptic vesicle mobilization and thereby presynaptic facilitation of GABA release that underlies olfactory STH. Finally, the striking observation that LTH occurs normally in syn⁹⁷ mutants indicates that signaling pathways for STH and LTH diverge upstream of synapsin function in GABAergic interneurons (Sadanandappa, 2013).

Mechanisms for synaptic plasticity have been intensively analyzed in reduced preparations, e.g., in hippocampal or cortical-slice preparations and in neuromuscular synapses, e.g., of Aplysia, crayfish, frog, lamprey, and Drosophila. In contrast, synaptic mechanisms underlying specific forms of behavioral learning are less well understood in vivo, in terms of the signaling pathways engaged by experience as well as the cell types in which these mechanisms operate. The analysis of such in vivo mechanisms of plasticity requires an accessible neural circuit, whose properties are measurably altered by experience (Sadanandappa, 2013).

The olfactory response in Drosophila is initiated by the activation of odorant receptors expressed in the olfactory sensory neurons (OSNs), which project into the antennal lobe (AL) and form synapses with glomerulus-specific projection neurons (PNs) that wire to both, the mushroom bodies (MB) and the lateral protocerebrum. OSNs and PNs also activate multiglomerular excitatory and inhibitory local interneurons (LNs), which mediate lateral and intraglomerular inhibition in the AL. Strong evidence has been proved for a model in which Drosophila olfactory habituation, i.e., reduced olfactory avoidance caused by previous exposure to an odorant arises through potentiation of inhibitory transmission between GABAergic LNs and PNs of the AL (Das, 2011). This study analyzes the molecular basis of this potentiation and propose a mechanism that could lead to increased presynaptic GABA release after odorant exposure (Sadanandappa, 2013).

Synapsins are a conserved family of synaptic vesicle-associated proteins. They are predominantly associated with the reserve pool of synaptic vesicles and their phosphorylation by kinases such as calcium-dependent protein kinases (CaMKs), protein kinase A (PKA), and MAPK/Erk, result in the mobilization of these vesicles and thereby induces presynaptic facilitation. While synapsin may play key roles in behavioral plasticity in mammals, its functions in learning and memory remain mysterious in part because mammals have three synapsins (I, II, and III) encoded by three different genes (Sadanandappa, 2013).

Recent studies of the single synapsin gene in Drosophila show that it is required for adult anesthesia-sensitive memory of odor-shock association (Knapek, 2010). Larval associative memory requires synapsin with intact phosphorylation target sites (Ser6 and Ser533) likely operating in a subset of MB neuron (Sadanandappa, 2013).

This study asked whether, where, and how synapsin functions in olfactory habituation. The observations indicate that short-term habituation (STH) requires synapsin function as well as its CaMKII-dependent phosphorylation in the LN1 subset of inhibitory interneurons in the AL. These point to presynaptic facilitation of GABA release as being a crucial mechanism for STH. The observation that long-term habituation (LTH) forms normally in syn⁹⁷ mutants indicates that this form of long-term memory can be encoded without transition through a short-term memory stage (Sadanandappa, 2013).

Previous studies have established the important roles for vertebrate and invertebrate synapsins in short-term synaptic plasticity. The conclusion that increased GABA release underlies olfactory STH is built on previous studies that have established the role of synapsin in synaptic vesicle mobilization and presynaptic facilitation. Several in vivo and in vitro studies have provided evidence that synapsin phosphorylation and dephosphorylation regulate the effective size of different vesicle pools and thereby control neurotransmitter release. In the mollusks Aplysia californica and Helix pomatia/aspersa, the phosphorylation and redistribution of synapsin induced by the PKA or MAPK/Erk-pathways mediates short-term facilitation of transmitter release (Byrne, 1996; Angers, 2002; Giachello, 2010). In Drosophila larval neuromuscular junctions, post-tetanic potentiation of transmitter release requires synapsin function and is accompanied by mobilization of a reserve pool of synaptic vesicles. And in the mouse CNS (Cui, 2008), enhanced ERK signaling in the inhibitory neurons results in increased levels of synapsin-1 phosphorylation and enhanced GABA release from hippocampal interneurons (Sadanandappa, 2013).

In context of these prior studies of synapsin, the current observation that synapsin and its phosphorylation are necessary in GABAergic local interneurons for STH indicates that the facilitation of GABA release from LNs attenuates excitatory signals in the AL and thereby results in the reduced behavioral response, characteristic of habituation. The molecular reversibility of phosphorylation could potentially account for the spontaneous recovery of the olfactory response after STH. These in vivo observations on synapsin function constitute a substantial advance as they show that synapsin-mediated plasticity, usually observed in response to experimentally enforced electrophysiological stimulations, can underlie behavioral learning and memory induced by sensory experience. In addition, by placing these changes in an identified subpopulation of local inhibitory interneurons in the AL, they implicate presynaptic plasticity of LNs as a mechanism necessary for habituation and thereby substantially clarify a neural circuit mechanism for this form of nonassociative memory (Sadanandappa, 2013).

A significant question is how synapsin phosphorylation is regulated in vivo for synaptic plasticity and how this contributes to altered circuit function that underlies behavioral learning. This has been difficult to address for two reasons: first, due to the complexity and potential promiscuity of kinase signaling pathways, and second, this requires not only the identification of neurons that show synapsin-dependent plasticity, but also concurrent understanding of the circuit functions of these neurons and their postsynaptic target(s) in vivo. The latter has been a particular challenge in neural networks for behavioral memory. For instance, although synapsin- and S6/S533-dependent odor-reward memory trace localized to the MBs (Michels, 2011), the downstream targets of the MB to mediate learned behavior are only beginning to be unraveled. This makes it difficult to comprehensively interpret the complex role of synapsin in associative memory illustrated by the finding that 3 min memory and anesthesia-sensitive 2 h memory require synapsin, whereas 5 h memory and anesthesia-resistant 2 h memory do not (Knapek, 2010). In the Drosophila neural circuit that underlies olfactory habituation, this study presents evidence that is most parsimoniously explained by a model in which CaMKII regulates synapsin phosphorylation in GABAergic LNs of the AL, neurons that are known to inhibit projection neurons that transmit olfactory input to higher brain centers (Sadanandappa, 2013).

in vivo CaMKII studies not only show the requirement of its function in olfactory habituation, but also demonstrate that the predominant form of synapsin, which arises from pre-mRNA editing, is a potent substrate for CaMKII. Thus, expression of an inhibitory CaMKII peptide in neurons not only reduces (yet does not abolish) the S6-phosphorylated form of synapsin detected using S6 phospho-specific antibody, it also blocks olfactory habituation, thereby providing experimental evidence for presynaptic function of CaMKII. These findings provide in vivo support for a model that proposed the primacy of CaMKII regulation of synapsin function; however, the structural basis for this regulation may be slightly different as invertebrate synapsins lack the D domain found on mammalian homologs that appear to be the major targets of CaMKII-dependent synapsin phosphorylation (Sadanandappa, 2013).

These conclusions on the role of CaMKII must be qualified in two ways. First, though tight correlations were shown between CaMKII phosphorylation of synapsin and the protein's function in STH, the data fall short of establishing causality. Second, it was not possible to directly demonstrate that odorant exposure that induces STH results in CaMKII-dependent synapsin phosphorylation in LNs. Attempts to address the latter issue failed because of technical difficulties in using phospho-specific antibodies for in vivo immunohistochemistry. Finally, although it is proposed that CaMKII phosphorylation is necessary for synapsin function during STH, it remains possible that other additional kinases, e.g., PKA and CaMKI, also contribute to synapsin regulation in vivo required for STH (Sadanandappa, 2013).

Several studies have discriminated between mechanisms of short-term and long-term memory (STM and LTM) formation. Most have focused on the distinctive requirement for protein synthesis in LTM and have not established whether STM is a necessary step in the formation of LTM or whether STM and LTM arise via distinctive, if partially overlapping molecular pathways. However, a few reports describe experimental perturbations that greatly reduce STM, without altering LTM (Sadanandappa, 2013).

In cultured sensorimotor synapses, which provide a surrogate model for behavioral sensitization in Aplysia, it has been shown that synaptic application of a selective 5-HT receptor antagonist blocked short-term facilitation while leaving long-term facilitation unaffected. In contrast, somatic application of the same antagonist selectively blocked long-term facilitation. This showed that long-term synaptic plasticity, though triggered by similar inputs (5-HT in this case), can progress through a pathway that does not require short-term plasticity (Sadanandappa, 2013).

In mammalian systems, it has been shown that a variety of pharmacological infusions into the hippocampal CA1 region or in entorhinal/ parietal cortex inhibited STM without affecting LTM of a shock avoidance memory task. In Drosophila, different isoforms of A-kinase anchor protein (AKAP) interacts with cAMP-PKA and play a distinct role in the formation of STM and LTM (Lu, 2007; Zhao, 2013). While these studies indicate that LTM is not built on STM, they do not rule out the possibility that they share an early common synaptic mechanism, but differ in subsequent mechanisms of consolidation, which occur in anatomically distinct brain regions (Sadanandappa, 2013).

The current observations on the absolute requirement for synapsin in STH but not LTH extend a model in which LTM can be encoded without transition through STM, particularly because STH and LTH involve different timescales of plasticity in the very same olfactory neurons. In the simple learning circuit for STH and LTH, the current observations are interpreted in a biochemical model. While both STH and LTH occur through potentiation of iLN-PN synapses, it is proposed that the signaling mechanisms for short- and long-term synaptic plasticity diverge before the stage of synapsin phosphorylation in GABAergic local interneurons. Thus, odorant exposure results in the activation of key signaling molecules such as PKA and CaMKII in LNs required for both forms of olfactory habituation. However, the kinases affect STH through synapsin phosphorylation, which results in a rapid but transient increase in evoked GABA release. Meanwhile, the same signaling molecules also participate in the activation of translational and/or transcriptional control machinery, which act, on a slower timescale, to cause the formation of additional GABAergic synapses that persist stably for much longer periods of time (Sadanandappa, 2013).

The unusual behavioral state, in which an animal is unable to form an STM but capable of longer term memory may have some clinical significance. Recently, a comprehensive mouse model for Down syndrome, which is trisomic for ~92% of the human chromosome 21, was shown to have defective STM of novel object recognition, while still showing normal LTM in this task, which is conceptually similar to behavioral habituation. Thus, it is suggested that further studies may eventually identify several other molecules which, like synapsin, are selectively necessary for short-term but not for long-term memory (Sadanandappa, 2013).

The neuro-ecology of Drosophila pupation behavior

Many species of Drosophila form conspecific pupa aggregations across the breeding sites. These aggregations could result from species-specific larval odor recognition. To test this hypothesis larval odors of D. melanogaster and D. pavani, two species that coexist in the nature, were tested. When stimulated by those odors, wild type and vestigial (vg) third-instar larvae of D. melanogaster pupated on conspecific larval odors, but individuals deficient in the expression of the odor co-receptor Orco randomly pupated across the substrate, indicating that in this species, olfaction plays a role in pupation site selection. Larvae are unable to learn but can smell, the Synapsin (Syn^97CS) and rut strains of D. melanogaster, did not respond to conspecific odors or D. pavani larval cues, and they randomly pupated across the substrate, suggesting that larval odor-based learning could influence the pupation site selection. Thus, Orco, Syn97CS and rut loci participated in the pupation site selection. When stimulated by conspecific and D. melanogaster larval cues, D. pavani larvae also pupated on conspecific odors. The larvae of D. gaucha, a sibling species of D. pavani, did not respond to D. melanogaster larval cues, pupating randomly across the substrate. In nature, D. gaucha is isolated from D. melanogaster. Interspecific hybrids, which result from crossing pavani female with gaucha males clumped their pupae similarly to D. pavani, but the behavior of gaucha female x pavani male hybrids was similar to D. gaucha parent. The two sibling species show substantial evolutionary divergence in organization and functioning of larval nervous system. D. melanogaster and D. pavani larvae extracted information about odor identities and the spatial location of congener and alien larvae to select pupation sites. It is hypothesized that larval recognition contributes to the cohabitation of species with similar ecologies, thus aiding the organization and persistence of Drosophila species guilds in the wild (Del Pino, 2014).

Drosophila evaluates and learns the nutritional value of sugars

Living organisms need to search for and ingest nutritional chemicals, and gustation plays a major role in detecting and discriminating between chemicals present in the environment. Using Drosophila as a model organism, it was asked whether animals have the ability to evaluate the nutritional value of sugars. In flies, chemosensilla on the tarsi and labellum are the gustatory organs used to discriminate between edible and nonedible compounds. It was noticed that Drosophila do not assign nutritional values to all sweet chemicals. D-arabinose is sweet to flies, but it provides them with no nutrition. By contrast, the sugar alcohol D-sorbitol is not sensed as sweet, but flies can live on it. Behavioral and electrophysiological measurements were performed to confirm these gustatory and feeding responses. It was found that Drosophila can learn the nutritional value of nonsweet D-sorbitol when it is associated with an odor cue. The learning process involved the synapsin molecule, suggesting that a neuronal mechanism is involved. It is proposed that Drosophila uses neural machinery to detect, evaluate, and learn the nutritional value of foods after ingestion (Fujita, 2011).

In an insect body, D-sorbitol is synthesized from D-glucose. It is then stored in the body during the diapause period and used for energy conservation and cold stability. Because D-sorbitol provides energy and supports other functions in flies, it is clear that flies need to recognize and ingest D-sorbitol. The feeding behavior of Drosophila is induced by sequential stimulation of taste cells in the tarsus on the legs, labellum on the proboscis, and interpseudotracheal papilla located inside the labellum. D-sorbitol was found to invoked feeding without stimulating external taste cells (Fujita, 2011).

Tests were performed to see whether a neural system was involved in learning. To this end, the syn⁹⁷ mutant, which is a null mutant of the synapsin gene that encodes a protein necessary for synaptic function, was used. The gene is associated with reduced learning ability in adult flies and larvae. syn⁹⁷ mutant flies showed significantly reduced learning ability, demonstrating that a neural system is involved in nutritional value learning (Fujita, 2011).

How do flies detect D-sorbitol and start feeding behavior? Is it possible that pharyngeal taste cells are involved? If these taste cells play a major role in regulating ingestion and in sensing D-sorbitol, flies would consume a large amount of D-sorbitol once feeding starts. But no evidence for this was found in the feeding assay. As food deprivation increases, spontaneous extension of the proboscis may initiate feeding on D-sorbitol-containing agar medium, and after ingestion the internal sensor might send a positive signal to initiate feeding behavior, despite providing no initial external gustatory input. Evidence is available to support the hypothesis that flies control feeding behavior by a putative internal sensor. poxn flies with no external taste cells also survive on standard medium. These flies may recognize the nutritional value of substances or other factors in sugar without sensing sugar sweetness. In fact, Gr64 null mutant flies have no glucose receptors but can survive on glucose-containing medium. It has been postulated that an internal sensor must exist to monitor the hemolymph sugar level, and a putative receptor molecule has been identified. Such a nutrient body sensor mechanism might inhibit feeding behavior if the ingested sugar provides no nutrition. A similar phenomenon was reported in mutant mice with deficient taste reception that regulate feeding behavior by providing feedback on the nutritional state in the body after ingestion. It has been proposed that a dopaminergic neural reward system is involved (Fujita, 2011).

This study reveals that flies learn the nutritional value of sugar, but the process by which they learn this is still unclear. Gustatory receptor neurons send axons to the subesophageal ganglion. It remains to be elucidated how information from the subesophageal ganglion is transferred to the central brain. A detailed functional anatomy of the subesophageal ganglion may help to elucidate the neural network of taste learning. Future studies could elucidate how the nutritional state is internally sensed and how that the information is processed in the fly brain (Fujita, 2011).

Distinct presynaptic and postsynaptic dismantling processes of Drosophila neuromuscular junctions during metamorphosis

Synapse remodeling is a widespread and fundamental process that underlies the formation of neuronal circuitry during development and in adaptation to physiological and/or environmental changes. However, the mechanisms of synapse remodeling are poorly understood. Synapses at the neuromuscular junction (NMJ) in Drosophila larvae undergo dramatic and extensive remodeling during metamorphosis to generate adult-specific synapses. To explore the molecular and cellular processes of synapse elimination, confocal microscopy, live imaging, and electron microscopy (EM) of NMJ synapses were performed during the early stages of metamorphosis in Drosophila in which the expressions of selected genes were genetically altered. It is reported that the localization of the postsynaptic scaffold protein Disc large (Dlg) becomes diffuse first and then undetectable, as larval muscles undergo histolysis, whereas presynaptic vesicles aggregate and are retrogradely transported along axons in synchrony with the formation of filopodia-like structures along NMJ elaborations and retraction of the presynaptic plasma membrane. EM revealed that the postsynaptic subsynaptic reticulum vacuolizes in the early stages of synapse dismantling concomitant with diffuse localization of Dlg. Ecdysone is the major hormone that drives metamorphosis. Blockade of the ecdysone signaling specifically in presynaptic neurons by expression of a dominant-negative form of ecdysone receptors delayed presynaptic but not postsynaptic dismantling. However, inhibition of ecdysone signaling, as well as ubiquitination pathway or apoptosis specifically in postsynaptic muscles, arrested both presynaptic and postsynaptic dismantling. These results demonstrate that presynaptic and postsynaptic dismantling takes place through different mechanisms and that the postsynaptic side plays an instructive role in synapse dismantling (Liu, 2010).

The Drosophila NMJ is an attractive model system for studying synaptogenesis but has only rarely been exploited to study synapse elimination. The present study used the Drosophila NMJ to study synapse elimination in the early stages of metamorphosis during which extensive synapse elimination occurs. This study unveiled distinct presynaptic and postsynaptic dismantling processes. Presynaptic elimination is characterized by the formation of prominent filopodial structures. The presynaptic membrane then retracts toward the nerve-muscle contact site with decreased bouton number and enlarged bouton size, accompanied by SV aggregation and retrograde axonal transport of SVs. It is worth pointing out that the precise timing of synapse dismantling revealed by immunostaining and live imaging is different, and this is probably attributable to the fact that the samples were analyzed under different conditions. For example, animals were kept at 25°C for immunostaining but were maintained at 20°C during live imaging. It is well known that filopodia are present in growth cones and play an important role in neurite outgrowth. The filopodia-like structures observed during synapse elimination presumably sense and explore the environment. The data also demonstrate that retrograde axonal transport plays an important role in presynaptic elimination. It is expected that the retrogradely transported synaptic constituents are reused to form adult-specific synaptic connections, although the final fate of the motor neuron MN4a innervating muscle 4 has not been determined. During the metamorphic period from 4 to 11 h APF we examined for the complete synapse elimination, neither disrupted synaptic microtubules were observed as reported for the local synapse disassembly in Drosophila larvae nor was an axonal 'retraction bulb' as seen during mammalian NMJ synapse elimination; a retraction bulb appears when a presynaptic terminal is detached mostly or completely from the postsynaptic specialization (Liu, 2010).

The first signs of postsynaptic elimination were the blurred and diffuse localization of postsynaptic markers Dlg and CD8-GFP-Shaker at 4 h APF, followed by a more expanded distribution of GluRs and vacuolization of SSR in 6 h APF. The postsynaptic components of Dlg and CD8-GFP-Shaker were almost completely eliminated at 9 h APF. Completion of the synapse dismantling process starting from the diffusion of postsynaptic Dlg at 4 h APF takes ~7 h. It is remarkable to note that the patterns of elimination of postsynaptic Dlg and GluRs are different; Dlg is eliminated by a diffusion-degradation process, whereas no diffusion of GluRs was observed before degradation, indicating that they are eliminated by different mechanisms. The differential elimination of Dlg and GluRs is consistent with the previous finding that the synaptic localization of GluR IIA is independent of Dlg. Interestingly, as for Drosophila GluRs, mammalian NMJ postsynaptic acetylcholine receptors are eliminated without the intermediate process of diffusion (Liu, 2010).

Synapse elimination is distinct from axonal and dendritic pruning. In Drosophila, the pruning of axonal and dendritic processes during metamorphosis closely resembles the pathological process of Wallerian degeneration, a process in which part of the axon separated from the nucleus of the neuron degenerates. In both axonal pruning of central mushroom body neurons and dendritic elimination of peripheral sensory neurons, severing of neuronal processes is preceded by microtubule depolymerization and followed by cytoplasmic blebbing and degeneration. It is conceivable that synapse elimination and pruning of neuronal processes are closely interconnected, but the time course of the two discrete processes has yet to be determined. It has been shown that specific E2/E3 ubiquinating enzymes and caspases (i.e., UbcD1-Diap1-Dronc) are involved in dendritic pruning. But it is unknown whether those molecules also participate in NMJ synapse elimination. However, this study has demonstrated that disruption of ubiquitination and apoptosis pathways on the postsynaptic side arrests synaptic elimination. It will be of great interest to identify the specific ubiquinating enzymes and caspases that participate in synapse dismantling during metamorphosis. It is worth noting that glial cells play an important role in axonal pruning of mushroom body neurons and olfactory receptor neurons. This study found no evidence to suggest that glial cells play a role in NMJ synapse elimination (Liu, 2010).

Synapse disassembly or instability in Drosophila NMJ terminals have been described. However, this process is fundamentally different from that reported in this paper in several aspects. First, local synapse disassembly, the disassembly of distal synaptic boutons or a branch of the whole synaptic terminals of a motor neuron, a process that occurs during synapse growth in larval development was examined in previous papers, whereas this paper studied the elimination of complete NMJ 4 synapses in synchrony with muscle histolysis during metamorphosis. Also, the synapse elimination reported in this study is different from that of mammalian NMJ synapses; the former involves muscle destruction, whereas the latter does not. Second, the processes of local versus general synapse disassembly are different. In local synapse disassembly, presynaptic dismantling precedes postsynaptic dismantling: the presynaptic microtubule cytoskeleton retracts first, followed by the elimination of synaptic release machinery (i.e., the vesicle-associated protein synapsin) and ultimately the disassembly of the postsynaptic apparatus including the postsynaptic GluRs and the scaffold Dlg. However, in the elimination of complete synaptic terminals during metamorphosis, postsynaptic dismantling starts first, followed by presynaptic dismantling. Third, disrupting the dynactin complex destabilizes local synapses, leading to more synaptic 'footprints' that are defined as the withdrawal of presynaptic components from clearly defined postsynaptic specialization containing Dlg was studied previously. It was argued that the dynactin complex functions locally within presynaptic terminals to maintain synapse stability. Although the current study not examined synaptic footprints during metamorphosis, this study reports that disrupting the dynactin complex in presynaptic neurons delays presynaptic dismantling specifically, whereas the postsynaptic components disassemble normally, indicating that dynactin-mediated retrograde axonal transport is required for presynaptic elimination. These results demonstrate that the dynactin complex functions differently in distinct cellular contexts (Liu, 2010).

Two independent lines of evidence indicate that the postsynaptic rather than presynaptic side plays an instructive role in synapse elimination during metamorphosis. First, immunostaining showed that postsynaptic dismantling precedes presynaptic elimination by ~1 h. Second, blockade of retrograde axonal transport and ecdysone signaling specifically in presynaptic neurons delayed presynaptic dismantling only but postsynaptic dismantling proceeded normally. However, inactivation of ecdysone signaling, ubiquitination, or apoptosis pathways in postsynaptic muscles arrested both presynaptic and postsynaptic dismantling. It is noted that synapse elimination is closely correlated with muscle destruction. Indeed, muscle histolysis might be the primary cause of NMJ synapse elimination during metamorphosis. These results together indicate that postsynaptic elimination is independent of presynaptic elimination, but presynaptic elimination depends on postsynaptic elimination; in other words, postsynaptic elimination triggers presynaptic elimination. This hypothesis is supported by a previous report that local ecdysone treatment of the hawkmoth, Manduca sexta, to induce local muscle degeneration results in loss of synaptic contacts in the treated region, whereas neighboring NMJ synapses remain intact. The current data are also consistent with mounting evidence from mammalian studies supporting a major role for the postsynaptic side in synapse elimination. However, the downstream targets of the ubiquitination, apoptosis, or ecdysone pathways in the postsynaptic muscles that are crucial for initiating presynaptic dismantling are currently unknown. It will be of great interest to identify these targets (Liu, 2010).

External and circadian inputs modulate synaptic protein expression in the visual system of Drosophila melanogaster

In the visual system of Drosophila the retina photoreceptors form tetrad synapses with the first order interneurons, amacrine cells and glial cells in the first optic neuropil (lamina), in order to transmit photic and visual information to the brain. Using the specific antibodies against synaptic proteins; Bruchpilot (BRP), Synapsin (SYN), and Disc Large (DLG), the synapses in the distal lamina were specifically labeled. Then their abundance was measured as immunofluorescence intensity in flies held in light/dark (LD 12:12), constant darkness (DD), and after locomotor and light stimulation. Moreover, the levels of proteins (SYN and DLG), and mRNAs of the brp, syn, and dlg genes, were measured in the fly's head and brain, respectively. In the head, SYN and DLG oscillations were not detected. It was found, however, that in the lamina, DLG oscillates in LD 12:12 and DD but SYN cycles only in DD. The abundance of all synaptic proteins was also changed in the lamina after locomotor and light stimulation. One hour locomotor stimulations at different time points in LD 12:12 affected the pattern of the daily rhythm of synaptic proteins. In turn, light stimulations in DD increased the level of all proteins studied. In the case of SYN, however, this effect was observed only after a short light pulse (15 min). In contrast to proteins studied in the lamina, the mRNA of brp, syn, and dlg genes in the brain was not cycling in LD 12:12 and DD, except the mRNA of dlg in LD 12:12. The abundance of BRP, SYN and DLG in the distal lamina, at the tetrad synapses, is regulated by light and a circadian clock while locomotor stimulation affects their daily pattern of expression. The observed changes in the level of synaptic markers reflect the circadian plasticity of tetrad synapses regulated by the circadian clock and external inputs, both specific and unspecific for the visual system.

Synapsin is selectively required for anesthesia-sensitive memory

Odor-shock memory in Drosophila consists of heterogeneous components each with different dynamics. A null mutant for the evolutionarily conserved synaptic protein Synapsin entails a memory deficit selectively in early memory, leaving later memory as well as sensory motor function unaffected. Notably, a consolidated memory component remaining after cold-anesthesia is not impaired, suggesting that only anesthesia-sensitive memory (ASM) depends on Synapsin. The lack of Synapsin does not further impair the memory deficit of mutants for the rutabaga gene encoding the type I adenylyl cyclase. This suggests that cAMP signaling, through a Synapsin-dependent mechanism, may underlie the formation of a labile memory component (Knapek, 2010).

The inclusion of the memory phenotype of the synapsin mutant in rutabaga may suggest Synapsin as a part of the cAMP signaling cascade and, if they would function in the same cells, as a potential downstream target of PKA. This is consistent with physiological studies demonstrating that cAMP signaling is selectively required for the recruitment of the reserve pool vesicles (Kuromi, 2000; Kuromi, 2002), as well as with the selective requirement of rutabaga for ASM, but not for ARM (Isabel, 2004). Both results fit very well with the physiology and memory of the synapsin mutant (Akbergenova, 2007). These lines of argument suggest that cAMP/PKA signaling might regulate the reserve-pool vesicle recruitment through Synapsin and that this process might underlie ASM (Knapek, 2010).

Synapsin maintains the reserve vesicle pool and spatial segregation of the recycling pool in Drosophila presynaptic boutons

Optical detection of the lipophylic dye FM1-43 and focal recordings of quantal release were employed to investigate how synapsin affects vesicle cycling at the neuromuscular junction of synapsin knockout (Syn KO) Drosophila. Loading the dye employing high K+ stimulation, which presumably involves the recycling pool of vesicles in exo/endocytosis, stained the periphery of wild type (WT) boutons, while in Syn KO the dye was redistributed towards the center of the bouton. When endocytosis was promoted by cyclosporin A pretreatment, the dye uptake was significantly enhanced in WT boutons, and entire boutons were stained, suggesting staining of the reserve vesicle pool. In Syn KO boutons, the same loading paradigm produced fainter staining and significantly faster destaining. When the axon was stimulated electrically, a distinct difference in dye loading patterns was observed in WT boutons at different stimulation frequencies: a low stimulation frequency (3 Hz) produced a ring-shaped staining pattern, while at a higher frequency (10 Hz) the dye was redistributed towards the center of the bouton and the fluorescence intensity was significantly increased. This difference in staining patterns was essentially disrupted in Syn KO boutons, although synapsin did not affect the rate of quantal release. Stimulation of the nerve in the presence of bafilomycin, the blocker of the transmitter uptake, produced significantly stronger depression in Syn KO boutons. These results suggest that synapsin maintains the reserve pool of vesicles and segregation between the recycling and reserve pools, and that it mediates mobilization of the reserve pool during intense stimulation (Akbergenova, 2007).

The conserved protein kinase-A target motif in synapsin of Drosophila is effectively modified by pre-mRNA editing

Synapsins are abundant synaptic vesicle associated phosphoproteins that are involved in the fine regulation of neurotransmitter release. The Drosophila member of this protein family contains three conserved domains (A, C, and E) and is expressed in most or all synaptic terminals. Similar to mouse mutants, synapsin knock-out flies show no obvious structural defects but are disturbed in complex behaviour, notably learning and memory. This study demonstrates that the N-terminal phosphorylation consensus motif RRxS that is conserved in all synapsins investigated so far, is modified in Drosophila by pre-mRNA editing. In mammals this motif represents the target site P1 of protein kinase A (PKA) and calcium/calmodulin dependent protein kinase I/IV. The result of this editing, by which RRFS is modified to RGFS, can be observed in cDNAs of larvae and adults and in both isolated heads and bodies. It is also seen in several newly collected wild-type strains and thus does not represent an adaptation to laboratory culture conditions. A likely editing site complementary sequence is found in a downstream intron indicating that the synapsin pre-mRNA can form a double-stranded RNA structure that is required for editing by the adenosine deaminase acting on RNA (ADAR) enzyme. A deletion in the Drosophila Adar gene generated by transposon remobilization prevents this modification, proving that the ADAR enzyme is responsible for the pre-mRNA editing described in this study. Evidence is provided for a likely function of synapsin editing in Drosophila. The N-terminal synapsin undeca-peptide containing the genomic motif (RRFS) represents an excellent substrate for in-vitro phosphorylation by bovine PKA while the edited peptide (RGFS) is not significantly phosphorylated. Thus pre-mRNA editing by ADAR could modulate the function of ubiquitously expressed synapsin in a cell-specific manner during development and adulthood. It is concluded that, similar to several other neuronal proteins of Drosophila, synapsin is modified by ADAR-mediated recoding at the pre-mRNA level. This editing likely reduces or abolishes synapsin phosphorylation by PKA. Since synapsin in Drosophila is required for various forms of behavioural plasticity, it will be fascinating to investigate the effect of this recoding on learning and memory (Diegelmann, 2006; full text of article).

A role for Synapsin in associative learning: the Drosophila larva as a study case

Synapsins are evolutionarily conserved, highly abundant vesicular phosphoproteins in presynaptic terminals. They are thought to regulate the recruitment of synaptic vesicles from the reserve pool to the readily-releasable pool, in particular when vesicle release is to be maintained at high spiking rates. As regulation of transmitter release is a prerequisite for synaptic plasticity, the fruit fly was used to ask whether Synapsin has a role in behavioral plasticity as well; in fruit flies, Synapsin is encoded by a single gene (syn). This question was tackled for associative olfactory learning in larval Drosophila by using the deletion mutant syn^97CS, which had been backcrossed to the Canton-S wild-type strain (CS) for 13 generations. A molecular account is provided of the genomic status of syn^97CS by PCR; the absence of gene product was shown on Western blots and nerve-muscle preparations. Olfactory associative learning in syn^97CS larvae was found to be reduced to approximately 50% of wild-type CS levels; however, responsiveness to the to-be-associated stimuli and motor performance in untrained animals are normal. In addition, two novel behavioral control procedures were introduced to test stimulus responsiveness and motor performance after 'sham training.' Wild-type CS and syn^97CS were fpimd perform indistinguishably also in these tests. Thus, larval Drosophila can be used as a case study for a role of Synapsin in associative learning (Michels, 2005; full text of article).

Flies lacking all synapsins are unexpectedly healthy but are impaired in complex behaviour

Vertebrate synapsins are abundant synaptic vesicle phosphoproteins that have been proposed to fine-regulate neurotransmitter release by phosphorylation-dependent control of synaptic vesicle motility. However, the consequences of a total lack of all synapsin isoforms due to a knock-out of all three mouse synapsin genes have not yet been investigated. In Drosophila a single synapsin gene encodes several isoforms and is expressed in most synaptic terminals. Thus the targeted deletion of the synapsin gene of Drosophila eliminates the possibility of functional knock-out complementation by other isoforms. Unexpectedly, synapsin null mutant flies show no obvious defects in brain morphology, and no striking qualitative changes in behaviour are observed. Ultrastructural analysis of an identified 'model' synapse of the larval nerve muscle preparation revealed no difference between wild-type and mutant, and spontaneous or evoked excitatory junction potentials at this synapse were normal up to a stimulus frequency of 5 Hz. However, when several behavioural responses were analysed quantitatively, specific differences between mutant and wild-type flies are noted. Adult locomotor activity, optomotor responses at high pattern velocities, wing beat frequency, and visual pattern preference are modified. Synapsin mutant flies show faster habituation of an olfactory jump response, enhanced ethanol tolerance, and significant defects in learning and memory as measured using three different paradigms. Larval behavioural defects are described in a separate paper. It is concluded that Drosophila synapsins play a significant role in nervous system function, which is subtle at the cellular level but manifests itself in complex behaviour (Godenschwege, 2004).

Invertebrate synapsins: A single gene codes for several isoforms in Drosophila

Vertebrate synapsins constitute a family of synaptic proteins that participate in the regulation of neurotransmitter release. Information on the presence of synapsin homologs in invertebrates has been inconclusive. A Drosophila gene coding for at least two inferred proteins was cloned that both contain a region with 50% amino acid identity to the highly conserved vesicle- and actin-binding 'C' domain of vertebrate synapsins. Within the C domain coding sequence, the positions of two introns have been conserved exactly from fly to human. The positions of three additional introns within this domain are similar. The Drosophila synapsin gene (Syn) is widely expressed in the nervous system of the fly. The gene products are detected in all or nearly all conventional synaptic terminals. A single amber (UAG) stop codon terminates the open reading frame (ORF1) of the most abundant transcript of the Syn gene 140 amino acid codons downstream of the homology domain. Unexpectedly, the stop codon is followed by another 443 in-frame amino acid codons (ORF2). Using different antibodies directed against ORF1 or ORF2, it was demonstrated that in the adult fly small and large synapsin isoforms are generated. The small isoforms are only recognized by antibodies against ORF1; the large isoforms bind both kinds of antibodies. It is suggested that the large synapsin isoform in Drosophila may be generated by UAG read-through. Implications of such an unconventional mechanism for the generation of protein diversity from a single gene are discussed (Klagges, 1996; full text of article).

A comparative analysis of the mobility of 45 proteins in the synaptic bouton

Many proteins involved in synaptic transmission are well known, and their features, as their abundance or spatial distribution, have been analyzed in systematic studies. This has not been the case, however, for their mobility. To solve this, the motion of 45 GFP-tagged synaptic proteins expressed in cultured hippocampal neurons, was analyzed using fluorescence recovery after photobleaching, particle tracking, and modeling. Synaptic vesicle proteins, endo- and exocytosis cofactors, cytoskeleton components, and trafficking proteins were compared. Movement was influenced by the protein association with synaptic vesicles, especially for membrane proteins. Surprisingly, protein mobility also correlated significantly with parameters as the protein lifetimes, or the nucleotide composition of their mRNAs. Protein movement was analyzed thoroughly, taking into account the spatial characteristics of the system. This resulted in a first visualization of overall protein motion in the synapse, which should enable future modeling studies of synaptic physiology (Reshetniak, 2020).

This study provides a first comparative study of protein mobility in the synaptic bouton, encompassing 45 different proteins, from different types and classes. The results confirm several expectations, including the lower mobility of membrane proteins when compared to soluble proteins, or the lower mobility of virtually all proteins in the synapse, when compared to the axon. Other expected observations were that the movement rates of the same proteins in the axon and in the synapse correlated little, presumably due to the different conditions encountered there, or that the mobility of soluble proteins was only little controlled by their molecular size. Several other observations could be made, including relations between protein mobility and structural parameters, mRNA composition, or protein lifetimes (Reshetniak, 2020).

The measurements, as indicated above, were performed with the caveat that the proteins that were measured were more abundant than under normal (wild-type) conditions. The overexpression levels that were observed were mild, but they may nevertheless contribute to artifacts, namely to an over-estimation of the mobility of individual proteins. If one protein is expressed too highly, its copy numbers saturate all binding to interacting partners, and the un-bound molecules end up moving randomly in the synapse, presumably at the highest possible speeds. The various validations that performed suggest that this is not a major problem. At the same time, the reported values should be taken as maximal mobility estimates, due to this issue. Native (non-tagged) proteins have not been investigated often, with only a handful of studies available. Several such studies were reproduced well by the current data. At the same time, a FRAP measurement of knock-in Munc13 provided a substantially lower mobility, with the shortest time constant measured in cultured mouse cortical neurons being around ~3 min, as opposed to a few seconds in the current measurements. This difference probably has both technical and biological grounds. The previous work imaged the synapses at intervals of a few minutes, to avoid photobleaching. Analyzing the current Munc13 data at 20-30 s of intervals (as opposed to two times per second, as in original data) raised the time constant from ~4 to ~30 s. Analyzing such data every few minutes would presumably result in an even longer time constant. Also, the previous work bleached multiple boutons in the same area, which probably resulted in bleaching a considerable proportion of the fast-moving molecules in the respective axons, which will reduce the fluorescence recovery. Nevertheless, it is still possible that Munc13 mobility is particularly sensitive to overexpression (Reshetniak, 2020).

In spite of these caveats, several conclusions could nevertheless be drawn. First, synaptic protein mobility seems to be influenced by the interaction of the proteins with the vesicles. For soluble proteins, it has been hypothesized that strong interactions to the vesicle cluster cause their enrichment in synapses. This was observed especially for synapsin, whose slow movement in synapses was paralleled by strong binding to vesicles. This effect was even more strongly visible for membrane proteins and is mostly explained by the fact that molecules that are more highly enriched in synaptic vesicles are present at lower levels on the plasma membrane. This implies that large fractions of these proteins will recover slowly during FRAP, through the infrequent active transport of synaptic vesicles. This will result in large time constants for the respective proteins. However, this is not the only explanation for this observation. The time constants of bona fide synaptic vesicle proteins are also higher in the axon, when compared to non-vesicular proteins. As all of these molecules are found in axons mainly as molecules fused to the plasma membrane, an explanation based on the transport of synaptic vesicles seems unlikely. A potential solution to this question is that synaptic vesicle proteins may diffuse in the axon in the form of assemblies composed of multiple molecules. This issue has been discussed for several decades, and it is still open for further interpretation. However, a series of recent observations, made mainly through super-resolution imaging of fused synaptic vesicles, suggested that such assemblies are indeed present in the axon, and may even be the dominant form in which vesicle proteins are found in the axonal compartment (Reshetniak, 2020).

Second, soluble unstructured proteins also appeared to move more slowly in synapses. This observation is especially interesting in the context of a recently proposed mechanism of synaptic vesicle cluster segregation. It has been suggested that synaptic vesicles, together with synaptic vesicle binding proteins, form a distinct liquid phase via liquid-liquid phase separation within the synapses. By definition, material exchange between liquid phases is slower than free diffusion; therefore, it is expected that soluble proteins of synaptic vesicle cluster would have slower recovery rates. Since the presence of multiple disordered coils is one of the main structural characteristics of proteins known to take part in liquid phase separation, the current observations fit very well with this model (Reshetniak, 2020).

Third, several correlations could be found to the presence of different amino acids in the protein sequence, or to the presence of particular nucleotides in the mRNA sequence. While the correlations to specific amino acids were relatively easy to interpret, the links to mRNA composition are less obvious. Different parameters of protein homeostasis have been linked to the mRNA composition in mammalian cells, and especially to the mRNA secondary structure or to the GC contents. At the same time, the mRNA composition has been suggested to control the folding conformation of specific proteins. It is still unclear whether the relations between mRNA composition and cell biology parameters are causative in nature, but they are sufficiently strong to enable reasonable predictions of protein abundance, lifetime, and translation rate. Overall, it is therefore not entirely surprising that protein mobility also correlates with mRNA composition, albeit it is difficult to explain why a high percentage of adenine correlates with higher mobility. One hypothesis could be based on the observation that proteins related to specialized function, including synapse formation, are encoded by GC-rich genes. In contrast, proteins involved in cell proliferation and in general cellular metabolism are encoded by AU-rich genes. This implies that “less synaptic” proteins would have mRNAs containing higher adenine percentages than bona fide synaptic proteins. The former would interact less with synaptic vesicles or other synaptic components, and would therefore be more mobile than true synaptic proteins. This hypothesis is supported by the fact that the top adenine-containing targets, which influence mostly these correlations, are SNAP23, Rab5, VAMP4, and SNAP29, trafficking molecules that are not specific to synapses in any fashion. At the opposite end, the top adenine-lacking proteins are the synaptic vesicle markers synaptophysin, synaptogyrin, and vGlut (glutamate transporter), along with the endocytosis cofactor epsin, further confirming this hypothesis (Reshetniak, 2020).

Finally, the FRAP data was analyzed thoroughly, to provide diffusion coefficients for the different proteins. These coefficients were validated by several types of measurements, as described above, and should provide a good starting point for models of synaptic physiology. Laboratories specialized in neuronal and synaptic modeling could exploit the entire dataset by introducing the different protein amounts and mobilities in multi-reaction synaptic models. Importantly, the data could be compared and combined with any dataset on hippocampal cultured synapses, which are a commonly used experimental model, for which large numbers of functional datasets are available (Reshetniak, 2020).

It is concluded that the current work provides a novel resource for the analysis of synaptic function, which should enable synaptic modeling with substantially higher precision than in the past (Reshetniak, 2020).

Vesicle clustering in a living synapse depends on a synapsin region that mediates phase separation

Liquid-liquid phase separation is an increasingly recognized mechanism for compartmentalization in cells. Recent in vitro studies suggest that this organizational principle may apply to synaptic vesicle clusters. This study tested this possibility by performing microinjections at the living lamprey giant reticulospinal synapse. Axons are maintained at rest to examine whether reagents introduced into the cytosol enter a putative liquid phase to disrupt critical protein-protein interactions. Compounds that perturb the intrinsically disordered region of synapsin (see Drosophila Synapsin), which is critical for liquid phase organization in vitro, cause dispersion of synaptic vesicles from resting clusters. Reagents that perturb SH3 domain interactions with synapsin are ineffective at rest. These results indicate that synaptic vesicles at a living central synapse are organized as a distinct liquid phase maintained by interactions via the intrinsically disordered region of synapsin (Pechstein, 2020).

Chemical synapses can sustain neurotransmitter release at high rates by mobilizing synaptic vesicles (SVs) from a pool clustered at the release sites. Despite the critical role of SV clusters in neural signaling, the mechanisms underlying their organization remain unclear. Two principal models have been proposed, both of which are centered at synapsins that are essential for the maintenance of the major, distal part of SV clusters. In the first model, called the scaffold model here, the clustering of SVs is suggested to depend on their anchoring via synapsins to the cytoskeleton, because synapsins can bind both SVs and cytoskeletal components like actin, spectrin, and microtubules. Key to the scaffold model is the observation that Ca2+-dependent phosphorylation of synapsin by Ca2+/calmodulin-dependent protein kinase II (CaMKII) causes its dissociation from SVs and actin, thereby enabling vesicle mobilization. A variant of the scaffold model implies that SVs are kept in place by intervesicular filaments, or tethers, that are partly composed of synapsin but also contain unidentified components. In the second model, called the liquid phase model here, SV clustering is suggested to result from phase separation (Milovanovic, 2018). Liquid-liquid phase separation is an increasingly recognized mechanism for membraneless subcellular compartmentalization, in which a distinct liquid phase forms in the cytosol because of weak multimeric interactions between proteins or between RNA and proteins (Banani, 2017; Gomes, 2019). In the case of protein-based liquid phase organization, the interactions typically involve intrinsically disordered regions (IDRs) and/or modular binding domains such as SH2 and SH3 domains. In support of the liquid phase model for SV clustering, in vitro studies have shown that recombinant synapsin, or its isolated IDR (also termed region D), can form droplets in aqueous solution (Milovanovic, 2018). Droplet formation was enhanced by adding synapsin-binding SH3 domains and disrupted by CaMKII phosphorylation. Moreover, small liposomes could be captured in synapsin droplets (Milovanovic, 2018). At present, neither of the two models of SV clustering has been rigorously tested in vivo. Moreover, with regard to liquid-liquid phase separation, in vitro results are not easily transferred to a cellular context (Pechstein, 2020).

This study examined whether the requirements for synapsin droplet formation in vitro pertain to SV clusters at the lamprey giant reticulospinal synapse. Earlier studies in this model have demonstrated that SV clusters in stimulated synapses depend on synapsin. In the present experiments, synapses were maintained at rest to test whether reagents microinjected into the axonal cytosol would enter a putative vesicular liquid phase to disrupt critical protein-protein interactions. This study found that compounds that perturb the synapsin IDR cause dispersion of SVs from resting clusters. However, reagents that perturb SH3 domain interactions with synapsin are ineffective (Pechstein, 2020).

Recent studies with purified components suggest that presynaptic compartments, including the SV cluster and the active zone, may be organized by liquid-liquid phase separation (Milovanovic, 2018, Wu, 2019). However, the precise biological relevance of these data has been unclear. This study used a living giant synapse to examine the conditions underlying organization of the SV cluster. The results fulfill two main criteria predicted by the liquid phase model. First, fusion proteins and antibodies microinjected into the axonal cytosol readily entered, and affected, key interaction sites within resting SV clusters containing thousands of densely packed vesicles. This finding is in line with the property of a liquid phase compartment. The low level of spontaneous vesicle turnover in the lamprey synapse underscores that the SV cluster can be regarded as near a complete resting state. If the cluster had been organized as a rigid scaffold structure, it would have been expected that the reagents penetrated less effectively, thus not giving rise to complete dispersion of clustered vesicles. Second, and most importantly, the intrinsically disordered regions (IDR) defined as critical for synapsin droplet formation in vitro was found to be equally critical for maintaining SV clusters in living synapses. Disturbance of the IDR, by binding of antibodies or the SH3A domain, led to complete dissociation of the distal part of SV clusters. In vitro experiments confirmed the efficacy of the reagents used (Pechstein, 2020).

The SH3A domain potently inhibited droplet formation of the IDR. Its efficacy may suggest that the region within the lamprey IDR that contains the SH3A binding site plays an important role to support IDR-IDR interactions. In mammalian synapsin I, at least two SH3A binding sites have been identifiedr. Thus, it cannot be excluded that occlusion of several SH3A domain binding sites may be required to disrupt liquid droplet formation. Higher than stoichiometric amounts of SH3A domains are more efficient to disperse synapsin IDR phase separation in vitro, suggesting that SH3A acts on other proline-rich regions in synapsin IDR at high concentrations. However, the antibodies stimulated droplet formation in vitro, suggesting that the divalent antibodies may drive aberrant droplet formation with the IDR. In the living synapse, the antibodies may thus have acted in a more complex way by perturbing endogenous IDR-IDR interactions and by forcing formation of antibody-IDR condensates that disrupt recruitment of vesicles to liquid droplets. This interpretation agrees with in vivo observations of electron-dense condensates associated with SVs (Pechstein, 2020).

Although the present results do not formally disprove the scaffold model, several arguments can be raised against it. The original version of the scaffold model implies that SVs are anchored via actin filaments. This possibility appears unlikely for two reasons. First, it would suggest that the C and E regions, which mediate actin binding, would be important, rather than the IDR. Second, actin filaments are mainly localized around SV clusters, with few filaments penetrating their interior. The version of the scaffold model suggesting a role for intervesicular tethers is more difficult to rule out, because their molecular identity is unclear, with only part consisting of synapsin. However, the estimated number of synapsin molecules per vesicle does not correlate well with the number of tethers. Moreover, intervesicular tethers can still be observed after knockout of synapsin, which argues against their primary role in SV clustering (Pechstein, 2020).

It is important to note that the present results only concern the resting synapse, and the situation may be somewhat different during activity. Stimulation causes a fraction of the vesicular synapsin pool to disperse into the axon, at least partly because of its phosphorylation by CaMKII. It is conceivable, and consistent with in vitro data (Milovanovic, 2018), that the mobile synapsin fraction temporarily leaves the liquid phase to reenter it during rest (Pechstein, 2020).

This study found that the antibodies directed to region E of synapsin did not effectively disrupt vesicle clusters at rest. In earlier experiments, these antibodies were shown to deplete the distal vesicle pool when the microinjection was followed by action potential stimulation. This stimulus-dependent effect can be explained by the stimulation-induced dispersal of synapsin discussed earlier. Following its dispersion in the cytosol, synapsin may have been captured by the E region-directed antibodies and thus prevented from reassociating with SVs to maintain a liquid phase. It is possible that the marked stimulus-dependent effect is enhanced by exposure of a normally hidden E region. The E region-directed antibodies produced a minor, non-significant loss of vesicles. The lack of a consistent effect suggests that the E region is not involved in maintaining the liquid phase. It is speculated that the modest effect results from mobility of some vesicles even at rest that may be linked with minute dispersion of synapsin. It is also possible that the E region-directed antibodies exerted a small influence on the adjacent IDR within a liquid phase (Pechstein, 2020).

Another notable observation was the occurrence of numerous scattered SVs and SV aggregates in the axonal cytosol following IDR perturbation, which sheds new light on a long-standing problem. Knockout of synapsins is known to cause a general decrease in the number of SVs, which has led to the proposition that synapsins play a role in SV maintenance, possibly via its ATP-binding C region. In an alternative hypothesis, the loss of SVs was proposed to be secondary to a clustering defect, eventually resulting in SV degradation. The dispersion of SVs following acute IDR perturbation supports a primary role of synapsin in vesicle clustering in central synapses (Pechstein, 2020).

A proximal pool of SVs remained tethered at the active zone after IDR perturbation. The mechanisms underlying the organization of this membrane-proximal pool are presently unclear. It may be organized by scaffolding, with the vesicles tethered to filaments extending from the active zone. Alternatively, these vesicles may compose a distinct liquid phase organized by other proteins than synapsin. The active zone has been proposed to form a distinct liquid phase involving RIM, RIM-BP, and calcium channels, but it is unclear how far into the cytosol this phase may extend (Pechstein, 2020).

Intersectin, amphiphysin, endophilin, and syndapin are all localized in SV clusters at rest. Upon stimulation, they partly redistribute to the periactive zone, where they participate in different aspects of SV endocytosis. Although the endocytic functions of these proteins are well established, their possible roles within the SV cluster remain unclear. No effect on resting SV clusters was observed by disrupting intersectin or amphiphysin SH3 domain interactions with synapsin, which is consistent with results on intersectin- and amphiphysin-deficient mice. In previous studies, no effect on clusters was observed after perturbing endophilin or syndapin SH3 domain interactions. Thus, individual interactions with any one of these four presynaptic SH3 domain-containing proteins are dispensable for the maintenance of resting SV clusters in vivo. However, this study does not rule out the possibility that multivalent interactions simultaneously involving these proteins may promote liquid phase organization of the SV cluster (Pechstein, 2020).

The accumulation of SH3 domain-containing proteins in SV clusters may merely reflect passive buffering, but they may also play active roles at this location. In the case of intersectin, a role in SV mobilization has been proposed, because deletion of intersectins I and II in mice causes enhanced synaptic depression during high-frequency stimulation. Such enhanced depression is consistent with impaired SV mobilization, but it could also be secondary to an endocytosis defect compromising the clearance of release sites. The roles of SH3 domain-containing proteins in SV clusters thus remain an important topic for future studies (Pechstein, 2020).

Vesicle tethering on the surface of phase-separated active zone condensates from rat brains

Tethering of synaptic vesicles (SVs) to the active zone determines synaptic strength, although the molecular basis governing SV tethering is elusive. This study discovered that small unilamellar vesicles (SUVs) and SVs from rat brains coat on the surface of condensed liquid droplets formed by active zone proteins RIM, RIM-BP, and ELKS via phase separation. Remarkably, SUV-coated RIM/RIM-BP condensates are encapsulated by synapsin/SUV condensates, forming two distinct SUV pools reminiscent of the reserve and tethered SV pools that exist in presynaptic boutons. The SUV-coated RIM/RIM-BP condensates can further cluster Ca(2+) channels anchored on membranes. Thus, this study has reconstituted a presynaptic bouton-like structure mimicking the SV-tethered active zone with its one side attached to the presynaptic membrane and the other side connected to the synapsin-clustered SV condensates (see Drosophila Synapsin). The distinct interaction modes between membraneless protein condensates and membrane-based organelles revealed here have general implications in cellular processes, including vesicular formation and trafficking, organelle biogenesis, and autophagy (Wu, 2020).

Active zones in presynaptic terminal boutons of synapses are intimately involved in neurotransmitter release, because active zones are responsible for tethering and docking synaptic vesicles (SVs) to be near release sites at plasma membranes, priming SVs for action potential-evoked fusion and clustering voltage-gated Ca2+channels (VGCCs) for precise spatiotemporal control of Ca2+level at the release sites. Electron microscopy and super-resolution optical imaging experiments have shown that active zone proteins, including RIM, RIM-BP, ELKS, Munc13, and Liprin, are located within a narrow space of <100 nm from the presynaptic terminal membranes. Biochemically, these active zone proteins are all multi-domain scaffold proteins without intrinsic enzymatic activities, and these proteins can form an intricate molecular network by specific protein-protein interactions, suggesting that active zones are large and dense protein-based molecular assemblies. Such active zone assemblies were proposed to be anchored to presynaptic plasma membranes by directly binding to lipids or transmembrane proteins, such as LAR and VGCCs, but none of them alone has been proved to be essential. Genetic and cell biology studies have shown that these proteins function together and in redundant ways in organizing active zones and in modulating neurotransmitter releases. For example, removal of each active zone component in rodents individually showed little impact on the active zone structures and vesicle tethering to active zones, except that removal of RIM led to 50% reduction of tethered vesicles. Combined removals of RIM with RIM-BP or with ELKS led to disintegration of active zone molecular assemblies, near-total loss of vesicle tethering and docking, and dramatic impairments of synaptic release. It had been recently shown that RIM and RIM-BP, when mixed in vitro, can autonomously form condensed molecular assembly via phase separation. The RIM/RIM-BP condensates can cluster the cytosolic tail of VGCC (VGCC-CT) tethered to lipid membranes, forming highly concentrated RIM/RIM-BP/VGCC-CT assembly on the surface of membranes. The formation of condensed RIM/RIM-BP/VGCC-CT on the surface of lipid membranes via phase separation provides an explanation to how condensed active zone protein assemblies, which are not enclosed by any physical barriers, can form at presynaptic boutons. The highly dynamic nature of the RIM/RIM-BP/VGCC-CT condensates may also explain why the dense projection structures in active zones can be observed by electron microscopy (EM) only when synapses are chemically fixed. The presence of dense RIM clusters within the active zone has also been observed in living neurons. Phase separation-mediated formation of condensed molecular assemblies has also been suggested to form the postsynaptic density and for AMPA receptor synaptic clustering and transmission (Zeng, 2019). Formation of condensed molecular assemblies via phase separation is likely to be advantageous for synapse formation and plasticity, although such research is in its early stage (Wu, 2020).

Although tethering of SVs to release sites of presynaptic membranes requires the active zone, how SVs are physically tethered to active zones is not known. Recently, Milovanovic (2018) showed that lipid vesicles can be clustered by coacervation with the synapsin condensates, providing a mechanism for maintaining large reserve pool SVs away from active zones. However, numerous EM studies have shown that tethered SVs are discretely distributed within active zone sheets and the number of tethered SVs is linearly proportional to the areas of active zone sheets. In EM images of chemically fixed synapses, SVs are evenly separated by the dense projections, which are grid-like structures on active zone sheets. In addition, perturbation of synapsin affects only the reserve pool SVs, and the tethered pool remains unchanged. Conversely, disruption of active zone scaffolds impairs only the tethered pool SVs without changing the reserve pool. Thus, tethering of SVs to the active zones is likely mediated by mechanisms that are very different from that seen in the reserve pool SV clustering by synapsin. This study demonstrates that negatively charged liposomes, as well as SVs purified from rat brains, coat on the surface of the phase-separated RIM/RIM-BP condensates. By tethering VGCC-CT to the giant unilamellar vesicle (GUV) membranes, this study shows that the RIM/RIM-BP condensates can bind to and cluster VGCC-CT on GUV membranes on one hand and link SUVs on the other hand, resembling active zone-mediated tethering of SVs to presynaptic membrane release sites. Remarkably, the synapsin-clustered SUV condensates and the SUV-coated RIM/RIM-BP condensates form phase-in-phase assemblies, which recapitulate coexistence of reserved and tethered pools of SVs in presynaptic boutons. The finding that the RIM/RIM-BP condensates can simultaneously link two totally different types of membrane-based cellular organelles suggests a wide range of unanticipated cellular roles of membraneless biological condensates formed by phase separation (Wu, 2020).

Tethered SVs are typically defined by their physical distance(30 nm or less) to the presynaptic plasma membranes. Decades of past research have identified molecules, including RIM, RIM-BP,ELKS, and Munc13, that are critical for SVs to tether and dock to presynaptic membranes. However, how SVs are retained at the active zone remains elusive. This study discovered that SVs coat on the surface of condensed droplets formed by the active zone proteins, including RIM, RIM-BP, and ELKS, via phase separation. This finding may explain several observations made in past decades of studies. First, the number of tethered SVs is proportional to the surface area of the active zone. Second, SVs coated on the surface of RIM/RIM-BP condensates are highly dynamic but are very close to both fusion machineries and presynaptic plasma membranes; thus, such active zone tethered SVs are primed for rapid fusion. Third, the RIM/RIM-BP condensates can cluster Ca2+ channels on the membranes. The direct coating of SVs on the surface of the RIM/RIM-BP droplets can physically link clustered Ca2+ channels with SVs for Ca2+-regulated fusion reactions. Fourth, formation of the RIM/RIM-BP condensates can greatly enhance the weak interaction between RIM/RIM-BP and negatively charged lipids on SVs, and thus allow stable interaction between the active zone molecular assembly and SVs. Fifth, the binding of SVs to the liquid-like active zone may shape the active zone into grid-like structures observed by EM under chemical fixing conditions. The condensed droplets formed by proteins, including RIM, RIM-BP, and ELKS, are highly dense but still very dynamic. Chemical fixation may capture such dynamic but dense assemblies referred to as dense projections. The coating of SVs on the surfaces of active zones likely involves proteins in addition to RIM, RIM-BP, and ELKS examined in this study. Proteins such as Munc13, Munc18, Liprin, Piccolo, Bassoon,SNAREs, the Rab family small GTPases, synaptotagmins, and so on can modulate SV docking and priming. These proteins may also participate in the regulation of active zone assembly and SVs coating. It is noted that the SV-coated RIM/RIM-BP condensates are not physically insulated by SVs. Proteins can readily enter in or escape from the SV-coated active zone protein condensates. Thus, such SV-coated active zone protein condensates can connect with molecular components both near and distal to the presynaptic membranes (Wu, 2020).

Another major finding of this study is that interactions of SVs with different protein condensates formed via phase separation can be radically different. SVs co-phase separate with synapsin, leading to clustering and concentration of SVs. Given that synapsin is massively concentrated in presynaptic boutons, the protein is ideally suited for maintaining the vast majority of SVs as the reserve pool. Strikingly, SVs do not enter the condensed phase formed by the active zone proteins, but instead coat on the surface of the condensates. Remarkably, the synapsin/SUV condensates encapsulate the SUV-coated RIM/RIM-BP droplets when the two phase-separated condensates are mixed, suggesting that the synapsin-clustered reserve SV pool can co-exist with the active zone-tethered SVs, and the two pools of SVs can exchange with each other. Finally, this study has been able to reconstitute a minimalistic presynaptic bouton-like structure, in which the active zone-coated vesicles are attached to the presynaptic plasma membrane and the synapsin-clustered reserve pool SV condensates are situated more distal to the presynaptic plasma membrane but directly interact with the active zone condensates. This reconstituted system recapitulates the basic features of the SV clustering and tethering in presynaptic boutons. In real synapses, the presynaptic plasma membranes together with various membrane proteins likely serve as the starting layer structure to organize the subsequent active zone layer, tethered SVs, and synapsin-clustered reserve pool SVs, forming the elaborate and polarized multiphase organizations. It should be noted that the scale/size of the reconstituted, presynaptic bouton-like assemblies here is much larger than the sizes of real presynaptic boutons. The awkward size of pre-synaptic boutons has presented challenges to investigate whether the layered structures observed in EM studies are indeed formed via the multiphase organization seen in this in vitro study. New technologies will need to be developed to answer this question in the future (Wu, 2020).

In a broader cell biology context, distinct mode of interactions between membraneless organelles and various membrane demarcated organelles, similar to what is observed in this study, may be broadly adopted by cells to modulate processes such as organelle biogenesis, vesicle formation and trafficking, autophagy, and membrane-associated signaling assembly formation (Wu, 2020).

PRICKLE1 interaction with SYNAPSIN I reveals a role in autism spectrum disorders

The frequent comorbidity of Autism Spectrum Disorders (ASDs) with epilepsy suggests a shared underlying genetic susceptibility; several genes, when mutated, can contribute to both disorders. Recently, PRICKLE1 missense mutations were found to segregate with ASD. However, the mechanism by which mutations in this gene might contribute to ASD is unknown. To elucidate the role of PRICKLE1 in ASDs, studies were carried out in Prickle1(+/-) mice and Drosophila, yeast, and neuronal cell lines. Mice with Prickle1 mutations were shown to exhibit ASD-like behaviors. To find proteins that interact with PRICKLE1 in the central nervous system, a yeast two-hybrid screen was performed with a human brain cDNA library and a peptide was isolated with homology to SYNAPSIN I (SYN1), a protein involved in synaptogenesis, synaptic vesicle formation, and regulation of neurotransmitter release. Endogenous Prickle1 and Syn1 co-localize in neurons and physically interact via the SYN1 region mutated in ASD and epilepsy. Finally, a mutation in PRICKLE1 disrupts its ability to increase the size of dense-core vesicles in PC12 cells. Taken together, these findings suggest PRICKLE1 mutations contribute to ASD by disrupting the interaction with SYN1 and regulation of synaptic vesicles (Paemka, 2013).

Search PubMed for articles about Drosophila Synapsin

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Banani, S. F., Lee, H. O., Hyman, A. A. and Rosen, M. K. (2017). Biomolecular condensates: organizers of cellular biochemistry. Nat Rev Mol Cell Biol 18(5): 285-298. PubMed ID: 28225081

Bloom, O., et al. (2003). Colocalization of synapsin and actin during synaptic vesicle recycling. J. Cell Biol. 161: 737-747. PubMed ID: 12756235

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date revised: 3 January 2020

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