Rabphilin-3A: A Multifunctional Regulator of Synaptic Vesicle Traffic

We have investigated the function of the synaptic vesicle protein Rabphilin-3A in neurotransmitter release at the squid giant synapse. Presynaptic microinjection of recombinant Rabphilin-3A reversibly inhibited the exocytotic release of neurotransmitter. Injection of fragments of Rabphilin-3A indicate that at least two distinct regions of the protein inhibit neurotransmitter release: the NH₂-terminal region that binds Rab3A and is phosphorylated by protein kinases and the two C2 domains that interact with calcium, phospholipid, and β-adducin. Each of the inhibitory fragments and the full-length protein had separate effects on presynaptic morphology, suggesting that individual domains were inhibiting a subset of the reactions in which the full-length protein participates. In addition to inhibiting exocytosis, constructs containing the NH₂ terminus of Rabphilin-3A also perturbed the endocytotic pathway, as indicated by changes in the membrane areas of endosomes, coated vesicles, and the plasma membrane. These results indicate that Rabphilin-3A regulates synaptic vesicle traffic and appears to do so at distinct stages of both the exocytotic and endocytotic pathways.     

Surprising complexity of the ancestral apoptosis network

Background

Apoptosis, one of the main types of programmed cell death, is regulated and performed by a complex protein network. Studies in model organisms, mostly in the nematode Caenorhabditis elegans, identified a relatively simple apoptotic network consisting of only a few proteins. However, analysis of several recently sequenced invertebrate genomes, ranging from the cnidarian sea anemone Nematostella vectensis, representing one of the morphologically simplest metazoans, to the deuterostomes sea urchin and amphioxus, contradicts the current paradigm of a simple ancestral network that expanded in vertebrates.

Results

Here we show that the apoptosome-forming CED-4/Apaf-1 protein, present in single copy in vertebrate, nematode, and insect genomes, had multiple paralogs in the cnidarian-bilaterian ancestor. Different members of this ancestral Apaf-1 family led to the extant proteins in nematodes/insects and in deuterostomes, explaining significant functional differences between proteins that until now were believed to be orthologous. Similarly, the evolution of the Bcl-2 and caspase protein families appears surprisingly complex and apparently included significant gene loss in nematodes and insects and expansions in deuterostomes.

Conclusion

The emerging picture of the evolution of the apoptosis network is one of a succession of lineage-specific expansions and losses, which combined with the limited number of 'apoptotic' protein families, resulted in apparent similarities between networks in different organisms that mask an underlying complex evolutionary history. Similar results are beginning to surface for other regulatory networks, contradicting the intuitive notion that regulatory networks evolved in a linear way, from simple to complex.     

Global gene expression analysis reveals reduced abundance of putative microRNA targets in human prostate tumours

Background

The availability of hundreds of bacterial genomes allowed a comparative genomic study of the Type VI Secretion System (T6SS), recently discovered as being involved in pathogenesis. By combining comparative and phylogenetic approaches using more than 500 prokaryotic genomes, we characterized the global T6SS genetic structure in terms of conservation, evolution and genomic organization.

Results

This genome wide analysis allowed the identification of a set of 13 proteins constituting the T6SS protein core and a set of conserved accessory proteins. 176 T6SS loci (encompassing 92 different bacteria) were identified and their comparison revealed that T6SS-encoded genes have a specific conserved genetic organization. Phylogenetic reconstruction based on the core genes showed that lateral transfer of the T6SS is probably its major way of dissemination among pathogenic and non-pathogenic bacteria. Furthermore, the sequence analysis of the VgrG proteins, proposed to be exported in a T6SS-dependent way, confirmed that some C-terminal regions possess domains showing similarities with adhesins or proteins with enzymatic functions.

Conclusion

The core of T6SS is composed of 13 proteins, conserved in both pathogenic and non-pathogenic bacteria. Subclasses of T6SS differ in regulatory and accessory protein content suggesting that T6SS has evolved to adapt to various microenvironments and specialized functions. Based on these results, new functional hypotheses concerning the assembly and function of T6SS proteins are proposed.     

The COG database: an updated version includes eukaryotes

Background

The availability of multiple, essentially complete genome sequences of prokaryotes and eukaryotes spurred both the demand and the opportunity for the construction of an evolutionary classification of genes from these genomes. Such a classification system based on orthologous relationships between genes appears to be a natural framework for comparative genomics and should facilitate both functional annotation of genomes and large-scale evolutionary studies.

Results

We describe here a major update of the previously developed system for delineation of Clusters of Orthologous Groups of proteins (COGs) from the sequenced genomes of prokaryotes and unicellular eukaryotes and the construction of clusters of predicted orthologs for 7 eukaryotic genomes, which we named KOGs after eukaryotic orthologous groups. The COG collection currently consists of 138,458 proteins, which form 4873 COGs and comprise 75% of the 185,505 (predicted) proteins encoded in 66 genomes of unicellular organisms. The eukaryotic orthologous groups (KOGs) include proteins from 7 eukaryotic genomes: three animals (the nematode Caenorhabditis elegans, the fruit fly Drosophila melanogaster and Homo sapiens), one plant, Arabidopsis thaliana, two fungi (Saccharomyces cerevisiae and Schizosaccharomyces pombe), and the intracellular microsporidian parasite Encephalitozoon cuniculi. The current KOG set consists of 4852 clusters of orthologs, which include 59,838 proteins, or ~54% of the analyzed eukaryotic 110,655 gene products. Compared to the coverage of the prokaryotic genomes with COGs, a considerably smaller fraction of eukaryotic genes could be included into the KOGs; addition of new eukaryotic genomes is expected to result in substantial increase in the coverage of eukaryotic genomes with KOGs. Examination of the phyletic patterns of KOGs reveals a conserved core represented in all analyzed species and consisting of ~20% of the KOG set. This conserved portion of the KOG set is much greater than the ubiquitous portion of the COG set (~1% of the COGs). In part, this difference is probably due to the small number of included eukaryotic genomes, but it could also reflect the relative compactness of eukaryotes as a clade and the greater evolutionary stability of eukaryotic genomes.

Conclusion

The updated collection of orthologous protein sets for prokaryotes and eukaryotes is expected to be a useful platform for functional annotation of newly sequenced genomes, including those of complex eukaryotes, and genome-wide evolutionary studies.     

Evolutionary history of bacteriophages with double-stranded DNA genomes

Background

Reconstruction of evolutionary history of bacteriophages is a difficult problem because of fast sequence drift and lack of omnipresent genes in phage genomes. Moreover, losses and recombinational exchanges of genes are so pervasive in phages that the plausibility of phylogenetic inference in phage kingdom has been questioned.

Results

We compiled the profiles of presence and absence of 803 orthologous genes in 158 completely sequenced phages with double-stranded DNA genomes and used these gene content vectors to infer the evolutionary history of phages. There were 18 well-supported clades, mostly corresponding to accepted genera, but in some cases appearing to define new taxonomic groups. Conflicts between this phylogeny and trees constructed from sequence alignments of phage proteins were exploited to infer 294 specific acts of intergenome gene transfer.

Conclusion

A notoriously reticulate evolutionary history of fast-evolving phages can be reconstructed in considerable detail by quantitative comparative genomics.

Open peer review

This article was reviewed by Eugene Koonin, Nicholas Galtier and Martijn Huynen.     

FPGA accelerator for protein secondary structure prediction based on the GOR algorithm

Background

Orthology analysis is an important part of data analysis in many areas of bioinformatics such as comparative genomics and molecular phylogenetics. The ever-increasing flood of sequence data, and hence the rapidly increasing number of genomes that can be compared simultaneously, calls for efficient software tools as brute-force approaches with quadratic memory requirements become infeasible in practise. The rapid pace at which new data become available, furthermore, makes it desirable to compute genome-wide orthology relations for a given dataset rather than relying on relations listed in databases.

Results

The program Proteinortho described here is a stand-alone tool that is geared towards large datasets and makes use of distributed computing techniques when run on multi-core hardware. It implements an extended version of the reciprocal best alignment heuristic. We apply Proteinortho to compute orthologous proteins in the complete set of all 717 eubacterial genomes available at NCBI at the beginning of 2009. We identified thirty proteins present in 99% of all bacterial proteomes.

Conclusions

Proteinortho significantly reduces the required amount of memory for orthology analysis compared to existing tools, allowing such computations to be performed on off-the-shelf hardware.     

Origins of amino acid transporter loci in trypanosomatid parasites

Background

In membrane trafficking, the mechanisms ensuring vesicle fusion specificity remain to be fully elucidated. Early models proposed that specificity was encoded entirely by SNARE proteins; more recent models include contributions from Rab proteins, Syntaxin-binding (SM) proteins and tethering factors. Most information on membrane trafficking derives from an evolutionarily narrow sampling of model organisms. However, considering factors from a wider diversity of eukaryotes can provide both functional information on core systems and insight into the evolutionary history of the trafficking machinery. For example, the major Qa/syntaxin SNARE families are present in most eukaryotic genomes and likely each evolved via gene duplication from a single ancestral syntaxin before the existing eukaryotic groups diversified. This pattern is also likely for Rabs and various other components of the membrane trafficking machinery.

Results

We performed comparative genomic and phylogenetic analyses, when relevant, on the SM proteins and components of the tethering complexes, both thought to contribute to vesicle fusion specificity. Despite evidence suggestive of secondary losses amongst many lineages, the tethering complexes are well represented across the eukaryotes, suggesting an origin predating the radiation of eukaryotic lineages. Further, whilst we detect distant sequence relations between GARP, COG, exocyst and DSL1 components, these similarities most likely reflect convergent evolution of similar secondary structural elements. No similarity is found between the TRAPP and HOPS complexes and the other tethering factors. Overall, our data favour independent origins for the various tethering complexes. The taxa examined possess at least one homologue of each of the four SM protein families; since the four monophyletic families each encompass a wide diversity of eukaryotes, the SM protein families very likely evolved before the last common eukaryotic ancestor (LCEA).

Conclusion

These data further support a highly complex LCEA and indicate that the basic architecture of the trafficking system is remarkably conserved and ancient, with the SM proteins and tethering factors having originated very early in eukaryotic evolution. However, the independent origin of the tethering complexes suggests a novel pattern for increasing complexity in the membrane trafficking system, in addition to the pattern of paralogous machinery elaboration seen thus far.     

Transsynaptic channelosomes

Recent findings demonstrate that synaptic channels are directly involved in the formation and maintenance of synapses by interacting with synapse organizers. The synaptic channels on the pre- and postsynaptic membranes possess non-conducting roles in addition to their functional roles as ion-conducting channels required for synaptic transmission. For example, presynaptic voltage-dependent calcium channels link the target-derived synapse organizer laminin β2 to cytomatrix of the active zone and function as scaffolding proteins to organize the presynaptic active zones. Furthermore, postsynaptic δ2-type glutamate receptors organize the synapses by forming transsynaptic protein complexes with presynaptic neurexins through synapse organizer cerebellin 1 precursor proteins. Interestingly, the synaptic clustering of AMPA receptors is regulated by neuronal activity-regulated pentraxins, while postsynaptic differentiation is induced by the interaction of postsynaptic calcium channels and thrombospondins. This review will focus on the non-conducting functions of ion-channels that contribute to the synapse formation in concert with synapse organizers and active-zone-specific proteins.     

Transsynaptic channelosomes: non-conducting roles of ion channels in synapse formation

Recent findings demonstrate that synaptic channels are directly involved in the formation and maintenance of synapses by interacting with synapse organizers. The synaptic channels on the pre- and postsynaptic membranes possess non-conducting roles in addition to their functional roles as ion-conducting channels required for synaptic transmission. For example, presynaptic voltage-dependent calcium channels link the target-derived synapse organizer laminin β2 to cytomatrix of the active zone and function as scaffolding proteins to organize the presynaptic active zones. Furthermore, postsynaptic δ2-type glutamate receptors organize the synapses by forming transsynaptic protein complexes with presynaptic neurexins through synapse organizer cerebellin 1 precursor proteins. Interestingly, the synaptic clustering of AMPA receptors is regulated by neuronal activity-regulated pentraxins, while postsynaptic differentiation is induced by the interaction of postsynaptic calcium channels and thrombospondins. This review will focus on the non-conducting functions of ion-channels that contribute to the synapse formation in concert with synapse organizers and active-zone-specific proteins.     

IRES-mediated translation of the carboxy-terminal domain of the horizontal cell specific connexin Cx55.5 in vivo and in vitro

Background

Changes of the interneuronal coupling mediated by electrical synapse proteins in response to light adaptation and receptive field shaping are a paramount feature in the photoreceptor/horizontal cell/bipolar cell (PRC/HC/BPC) complex of the outer retina. The regulation of these processes is not fully understood at the molecular level but they may require information transfer to the nucleus by locally generated messengers. Electrical synapse proteins may comprise a feasible molecular determinant in such an information-laden signalling pathway.

Results

Connexin55.5 (Cx55.5) is a connexin with horizontal cell-restricted expression in zebrafish accumulating at dendritic sites within the PRC/HC/BPC complex in form of hemichannels where light-dependent plasticity occurs. Here we provide evidence for the generation of a carboxy-terminal domain of Cx55.5. The protein product is translated from the Cx55.5 mRNA by internal translation initiation from an in-frame ATG codon involving a putative internal ribosome entry site (IRES) element localized in the coding region of Cx55.5. This protein product resembling an 11 kDa domain of Cx55.5 is partially located in the nucleus in vivo and in vitro.

Conclusion

Our results demonstrate the generation of a second protein from the coding region of Cx55.5 by an IRES mediated process. The nuclear occurrence of a fraction of this protein provides first evidence that this electrical synapse protein may participate in a putative cytoplasmic to nuclear signal transfer. This suggests that Cx55.5 could be involved in gene regulation making structural plasticity at the PRC/HC/BPC complex feasible.     

High throughput protein-protein interaction data: clues for the architecture of protein complexes

Background

High-throughput techniques are becoming widely used to study protein-protein interactions and protein complexes on a proteome-wide scale. Here we have explored the potential of these techniques to accurately determine the constituent proteins of complexes and their architecture within the complex.

Results

Two-dimensional representations of the 19S and 20S proteasome, mediator, and SAGA complexes were generated and overlaid with high quality pairwise interaction data, core-module-attachment classifications from affinity purifications of complexes and predicted domain-domain interactions. Pairwise interaction data could accurately determine the members of each complex, but was unexpectedly poor at deciphering the topology of proteins in complexes. Core and module data from affinity purification studies were less useful for accurately defining the member proteins of these complexes. However, these data gave strong information on the spatial proximity of many proteins. Predicted domain-domain interactions provided some insight into the topology of proteins within complexes, but was affected by a lack of available structural data for the co-activator complexes and the presence of shared domains in paralogous proteins.

Conclusion

The constituent proteins of complexes are likely to be determined with accuracy by combining data from high-throughput techniques. The topology of some proteins in the complexes will be able to be clearly inferred. We finally suggest strategies that can be employed to use high throughput interaction data to define the membership and understand the architecture of proteins in novel complexes.     

Predicting binding sites of hydrolase-inhibitor complexes by combining several methods

Background

Protein-protein interactions play a critical role in protein function. Completion of many genomes is being followed rapidly by major efforts to identify interacting protein pairs experimentally in order to decipher the networks of interacting, coordinated-in-action proteins. Identification of protein-protein interaction sites and detection of specific amino acids that contribute to the specificity and the strength of protein interactions is an important problem with broad applications ranging from rational drug design to the analysis of metabolic and signal transduction networks.

Results

In order to increase the power of predictive methods for protein-protein interaction sites, we have developed a consensus methodology for combining four different methods. These approaches include: data mining using Support Vector Machines, threading through protein structures, prediction of conserved residues on the protein surface by analysis of phylogenetic trees, and the Conservatism of Conservatism method of Mirny and Shakhnovich. Results obtained on a dataset of hydrolase-inhibitor complexes demonstrate that the combination of all four methods yield improved predictions over the individual methods.

Conclusions

We developed a consensus method for predicting protein-protein interface residues by combining sequence and structure-based methods. The success of our consensus approach suggests that similar methodologies can be developed to improve prediction accuracies for other bioinformatic problems.     

The presynaptic machinery at the synapse of <Emphasis Type="Italic">C. elegans</Emphasis>

Synapses are specialized contact sites that mediate information flow between neurons and their targets. Important physical interactions across the synapse are mediated by synaptic adhesion molecules. These adhesions regulate formation of synapses during development and play a role during mature synaptic function. Importantly, genes regulating synaptogenesis and axon regeneration are conserved across the animal phyla. Genetic screens in the nematode Caenorhabditis elegans have identified a number of molecules required for synapse patterning and assembly. C. elegans is able to survive even with its neuronal function severely compromised. This is in comparison with Drosophila and mice where increased complexity makes them less tolerant to impaired function. Although this fact may reflect differences in the function of the homologous proteins in the synapses between these organisms, the most likely interpretation is that many of these components are equally important, but not absolutely essential, for synaptic transmission to support the relatively undemanding life style of laboratory maintained C. elegans. Here, we review research on the major group of synaptic proteins, involved in the presynaptic machinery in C. elegans, showing a strong conservation between higher organisms and highlight how C. elegans can be used as an informative tool for dissecting synaptic components, based on a simple nervous system organization.     

Aneuploidy in health,disease, and aging

Synapse formation is a highly regulated process that requires the coordination of many cell biological events. Decades of research have identified a long list of molecular components involved in assembling a functioning synapse. Yet how the various steps, from transporting synaptic components to adhering synaptic partners and assembling the synaptic structure, are regulated and precisely executed during development and maintenance is still unclear. With the improvement of imaging and molecular tools, recent work in vertebrate and invertebrate systems has provided important insight into various aspects of presynaptic development, maintenance, and trans-synaptic signals, thereby increasing our understanding of how extrinsic organizers and intracellular mechanisms contribute to presynapse formation.Chemical synapses are highly specialized, asymmetric intercellular junction structures that are the basic units of neuronal communication. Proper development of synapses determines appropriate connectivity for the assembly of functional neuronal circuits. Synaptic circuits arise during development through a series of intricate steps (Waites et al., 2005; McAllister, 2007; Jin and Garner, 2008). First, spatiotemporal cues guide axons through complex cellular environments to contact their appropriate postsynaptic targets. At their destination, synapse formation is specified and initiated through adhesive interactions between synaptic partner cells or by local diffusible signaling molecules. Stabilization of intercellular contacts and assembly into functional synapses involves cytoskeletal rearrangements, aggregation, and insertion of pre- and postsynaptic components at nascent synaptic sites. Maturation and modulation of these newly formed synapses can then occur by altering the organization or composition of synaptic proteins and post-translational modifications to achieve its required physiological responsiveness (Budnik, 1996; Lee and Sheng, 2000). Conversely, retraction of contacts and elimination of inappropriate synaptic proteins help to refine the neuronal circuitry (Goda and Davis, 2003; Sanes and Yamagata, 2009).Over the last decade, new insights have furthered our understanding of synapse development through the identification of new molecular players and by advanced imaging technology that has allowed for high-resolution inspection of the dynamics and relative positions of synaptic proteins. This review will highlight recent results on the development of presynaptic specializations, and the roles of trans-synaptic organizers, intracellular synaptic proteins, and the cytoskeleton during the formation and maintenance of synapses.

Axonal transport of synaptic vesicle and active zone proteins

After cell fate determination and morphogenesis, neurons continue to differentiate by entering the phase of synapse formation. Most synaptic material required for this process is synthesized in the cell body of neurons and transported to synapses by microtubule (MT)-based molecular motors (Fig. 1). MTs are intrinsically polarized filaments with a plus and a minus end (Fig. 1 B). MT-based molecular motors use this polarity to transport cargoes to specific cellular locations. Examination of MTs by electron microscopy in dissociated cultured neurons showed that the organizations of MTs is different in axon and dendrite (Baas et al., 1988, 2006). In axons, all microtubules have their minus ends oriented toward the cell body and their plus ends extend distally. On the contrary, the MT polarity in dendrites is mixed. Recent studies tracking the movement of end-binding MT-capping proteins confirmed these results in vivo. Specifically, axonal MTs are uniformly organized with their plus ends pointing distally in all organisms. Dendrites of vertebrate neurons show more plus end–out MTs in vivo, whereas flies and worms have more minus end–out MTs in dendrites (Stepanova et al., 2003; Rolls et al., 2007; Stone et al., 2008).Open in a separate windowFigure 1.Regulatory steps during polarized motor-based transport of synaptic material. (A) At the Golgi apparatus, synaptic proteins have to be sorted into appropriate vesicles. These vesicles and other cargo such as mitochondria get loaded onto specific motor proteins. (B) Establishment of proper microtubule polarity along the axon determines anterograde and retrograde trafficking by plus end– and minus end–directed motor proteins such as kinesins and dynein. (C) At the appropriate destination, motor-cargo unloading occurs in a regulated fashion to achieve the appropriate distribution of synaptic boutons. At synapses, synaptic vesicle precursors give rise to mature synaptic vesicles. Proteins required for the SV cycle and trans-synaptic adhesion coalesce into the active zone (AZ) underneath the plasma membrane juxtaposed against the postsynaptic membrane.Does the difference in microtubule organization and polarity help to segregate synaptic cargoes between axons and dendrites? Recent studies have started to identify some molecules that create these differences in MT polarity in different neuronal subcellular compartments and show how disruption of their function affects synapse formation. For example, a recent paper showed that kinesin-1 is required to establish the predominantly minus end–out organization in the dendrites of Caenorhabditis elegans motor neurons (Yan et al., 2013). In kinesin-1/unc-116 mutants, dendrites adopt the axon-like MT polarity causing presynaptic cargoes to mislocalize into dendrites (Seeger and Rice, 2010; Yan et al., 2013). Similarly, loss of the MT-binding CRMP protein UNC-33 or the actin–spectrin adaptor protein ankyrin/UNC-44 in worms also results in MT polarity defects, which also results in ectopic localization of synaptic vesicles and active zone proteins into dendrites (Maniar et al., 2012). These results support the idea that MT polarity ensures the faithful targeting of presynaptic components to the axon. However, another way motors can distinguish between axons and dendrites is through MT-associated proteins (MAPs). In a recent study, Banker and colleagues showed that plus end–orienting kinesins can differentiate axon and dendrite, likely due to specific MT-binding proteins in these compartments (Huang and Banker, 2012).The direct regulation of motor activity by MTs or synaptic vesicle–associated proteins is likely to contribute to the trafficking of synaptic cargoes. Doublecortin, a MAP, binds to kinesin-3/KIF1A to affect the trafficking of the synaptic vesicle protein, synaptobrevin, in hippocampal neurons by altering the affinity of ADP-bound KIF1A to MTs (Liu et al., 2012). The Rab3 guanine nucleotide exchange factor, DENN/MADD, functions as an adaptor between kinesin-3 and GTP-Rab3–containing synaptic vesicles to promote the trafficking of synaptic vesicles in the axon (Niwa et al., 2008).Precise regulation of motor-based transport ensures that synaptic cargoes are delivered to and maintained at synapses. Several recent studies have provided evidence that two postmitotic cyclin-dependent kinases are important regulators of anterograde and retrograde trafficking of presynaptic cargoes. The kinase CDK-5 is required in many aspects of nervous system function. In the context of presynaptic development and function, CDK-5 has been shown to regulate the transport of synaptic vesicles and dense core vesicles, which contain neuropeptides, by inhibiting a dynein-mediated pathway that mobilizes presynaptic components to the somatodendritic compartments in C. elegans neurons (Ou et al., 2010; Goodwin et al., 2012). A paralogue of CDK-5, the PCT-1 kinase acts in a partially redundant pathway to prevent the mislocalization of presynaptic material to dendrites. In animals lacking both kinases or their activators, synaptic cargoes completely mislocalize to the dendrites, leaving an “empty” axon (Ou et al., 2010). Vertebrate CDK-5 also plays profound roles in the regulation of synaptic vesicle pools by modifying Ca²⁺ channels. Genetic ablation or pharmacological inhibition of CDK-5 increases the pool of synaptic vesicles that are docked at the active zone, termed the readily releasable pool, and potentiates synaptic function (Kim and Ryan, 2010, 2013). These results suggest that CDK-5 and its paralogue control local and global vesicle pools. Regulation of the exchange between these pools can affect membrane trafficking at presynaptic terminals as well as the overall polarity of neurons.To form synapses at defined locations, cargoes not only need to know how to “get on” the transport system but also need to know where to precisely “get off” at their destination (Fig. 1 C). Loss of a conserved small G-protein of the Arf-like family, ARL-8, in C. elegans, resulted in premature exit of synaptic cargoes during transport and showed ectopic aggregations of synaptic vesicles in the proximal axon. This causes a reduction in the number but an increase in the size of synapses (Klassen et al., 2010). ARL-8 localizes to both stable and trafficking synaptic vesicles and promotes trafficking by increasing kinesin-3 activity and suppressing aggregation-induced stoppage of synaptic cargoes along the axon (Wu et al., 2013). Hence, the balance between motor activity and aggregation propensity of trafficking cargoes may determine the number, size, and location of presynaptic terminals. Interestingly, the small GTPase Rab3, which normally associates with synaptic vesicles, has recently been shown to affect the distribution of active zone proteins at fly neuromuscular junction (NMJ) synapses, further suggesting that the trafficking of synaptic vesicles and formation of active zones are linked (Graf et al., 2009).Besides synaptic material, another major organelle cargo that is often present at the presynaptic terminal is mitochondria. The Milton–Miro complex functions as an adaptor between kinesin-1 and mitochondria to support axonal transport of mitochondria. Interestingly, the coupling of the Milton–Miro complex to kinesin is regulated by Ca²⁺ (Macaskill et al., 2009; Wang and Schwarz, 2009), providing a mechanism for neuronal activity controlling transport of mitochondria along the axon.Previous studies have suggested that components of the presynaptic active zone are transported in a preassembled form by Piccolo-Bassoon transport vesicles (PTVs) that may contain multiple components required to build a synapse (Zhai et al., 2001; Shapira et al., 2003). Recent studies found that Golgi-derived PTVs contain many active zone proteins including Piccolo, Bassoon, RIM1α, and ELKS2/CAST, but lack another active zone component, Munc-13, which may exit the Golgi on separate vesicles (Maas et al., 2012). Packing of various active zone components that have the propensity to self-assemble into separate vesicles may contribute a way to control synaptogenesis. This is interesting in light of the finding that Munc-13 can function as a protein scaffold for Bassoon and ELKS2 (Wang et al., 2009). The link between trafficking of synaptic vesicle and active zone components is not well understood. In vivo time-lapse imaging of synaptic vesicle and active zone trafficking showed that these components, possibly in the form of dense core vesicles, could be trafficked together in C. elegans neurons, suggestive of prepackaged presynaptic material during transport (Wu et al., 2013). Taken together, axonal transport of synaptic components is a necessary step for synapse formation and maintenance. The regulation of MTs, molecular motors, and synaptic cargoes ensure the targeting of appropriate proteins to synapses.

Role of the actin cytoskeleton in presynaptic assembly

Although MT-mediated transport is critical for long-range trafficking, actin-based mechanisms often organize local protein complexes in subcellular domains. A large body of work has described the role of the actin cytoskeleton in postsynaptic structure and function (Schubert and Dotti, 2007; Hotulainen and Hoogenraad, 2010). We will focus on more recent work that has highlighted the importance of the actin cytoskeleton in presynaptic formation.F-actin is required for presynaptic assembly during the early stages of synaptogenesis. Depolymerization of F-actin in young hippocampal neuronal cultures results in a reduction in the size and number of synapses. This effect was not seen with older cultures when synapses are more mature (Zhang and Benson, 2001). This observation correlates with an increase in both pre- and postsynaptic F-actin levels in newly formed synapses compared with mature synapses (Zhang and Benson, 2002).F-actin has been implicated in many steps of synapse assembly and function (Fig. 2; Cingolani and Goda, 2008). One of the roles that has been proposed for F-actin is to act as a scaffold for other presynaptic proteins (Sankaranarayanan et al., 2003). A recent study identified an F-actin–binding active zone molecule Neurabin/NAB-1 that is recruited by a presynaptic F-actin network (Chia et al., 2012). In addition, knockdown of Rac/Cdc42 GTPase exchange factor β-Pix resulted in a decrease in actin at synapses with a concomitant loss of synaptic vesicle clustering (Sun and Bamji, 2011). These studies demonstrate that F-actin at presynaptic sites can recruit and stabilize presynaptic components.Open in a separate windowFigure 2.Assembling the presynaptic active zone. Scaffolding proteins including Liprin, SYD-1, ELKS, Neurabin, Piccolo, and Bassoon form the dense protein network in the presynaptic cytomatrix that facilitates synaptic vesicle docking and fusion. The presynaptic F-actin networks are required for presynaptic assembly and maintenance.Studies of Drosophila NMJs have found that the presynaptic spectrin–actin cytoskeleton is important for synapse stability. Loss of presynaptic spectrin led to retraction of synapses (Pielage et al., 2005). Intriguingly, loss of postsynaptic spectrin increased the total number of the active zone specializations, termed T-bars, and affected the size and distribution of presynaptic sites. Thus, the spectrin cytoskeleton can impose a trans-synaptic influence on synapse development (Pielage et al., 2006).Given the importance of F-actin at synapses, it is crucial to understand the signaling pathways that instruct F-actin organization. Multiple studies have shown that signaling from synaptic cell adhesion molecules can lead to cytoskeletal rearrangements at synapses. Adhesion of hippocampal neurons to syndecan-2–coated beads is sufficient to induce F-actin clustering and downstream formation of presynaptic boutons (Lucido et al., 2009). In mice, the adhesion molecule L1CAM may bind to spectrin–actin adaptor ankyrin to mediate GABAergic synapse formation (Guan and Maness, 2010). Another adhesion molecule of the immunoglobulin superfamily SYG-1 in C. elegans has also been shown to be necessary and sufficient to recruit F-actin to synapses (Chia et al., 2012). In a recent study, secreted bone morphogenetic protein (BMP) can signal in a retrograde fashion to regulate Rac-GEF Trio expression in presynaptic neurons, which is important for controlling synaptic growth (Ball et al., 2010).Interestingly, presynaptic active zone proteins can also affect F-actin assembly (Fig. 2). Knockdown of Piccolo reduced activity-dependent assembly of F-actin at synapses and enhanced dispersion of Synapsin1a and synaptic vesicles in hippocampal neurons. Loss of Piccolo also resulted in a loss of Profilin 2, a regulator of actin polymerization (Waites et al., 2011).Various studies have begun to shed light on the actin regulators required for synaptic F-actin establishment and maintenance. Diaphanous, a formin-related gene that associates with barbed ends of F-actin, was found to function downstream of presynaptic receptor Dlar at fly NMJs. Spectrin–actin capping protein, Adducin, is enriched at presynaptic sites and is required to prevent synapse retraction and elimination (Bednarek and Caroni, 2011; Pielage et al., 2011). Activators of the Arp2/3 complex, WASP and WAVE, have also been implicated in the regulation of F-actin at synapses (Coyle et al., 2004; Stavoe et al., 2012; Zhao et al., 2013). This diversity of F-actin modulators suggests that there are probably different F-actin structures at different stages of development or even in subcellular domains within the synapse. This is supported by observations that F-actin can localize with synaptic vesicles, at the active zone and in the perisynaptic region (Bloom et al., 2003; Sankaranarayanan et al., 2003; Waites et al., 2011; Chia et al., 2012). Thus, much remains to be done in our understanding how distinct F-actin structures are formed and regulated to mediate various processes during synapse assembly and maintenance.

Assembly of the molecular network at presynaptic terminals

Although F-actin might help to initiate the presynaptic assembly process, many other ensuing molecular interactions are required to form the mature presynaptic apparatus (Fig. 2). The presynaptic active zone is comprised of a framework of scaffolding proteins that function as protein-binding hubs for other presynaptic components. Piccolo and Bassoon are important vertebrate multidomain proteins that traditionally have been widely used as active zone markers. Recent electrophysiology data on Piccolo mutant and Bassoon knockdown neurons showed that these molecules are dispensable for synaptic transmission but affect synaptic vesicle clustering (Mukherjee et al., 2010). Furthermore, Piccolo and Bassoon were found to be required for maintaining synapse integrity by regulating ubiquitination and degradation of presynaptic components (Waites et al., 2013).Forward genetic approaches in worms and flies have made important contributions to our understanding of the presynaptic cytomatrix. Studies have found that two active zone scaffolding molecules, SYD-1 and Liprin-α/SYD-2, are required for proper synapse formation (Zhen and Jin, 1999; Patel et al., 2006; Astigarraga et al., 2010; Owald et al., 2010; Stigloher et al., 2011). Interestingly, at fly NMJs, SYD-1 is necessary for clustering presynaptic neurexin that in turn clusters postsynaptic neuroligin (Owald et al., 2012). The presynaptic assembly function of SYD-1 and SYD-2 appears to be conserved because mutation analysis of mammalian SYD-1 and knockdown of Liprin-α both caused defects in presynaptic development and function (Spangler et al., 2013; Wentzel et al., 2013). In flies, the active zone T-bar structure is comprised of ERC/CAST family protein bruchpilot (brp) as the major active zone organizing protein (Fouquet et al., 2009). Brp is not only present at the active zone but also plays important scaffolding roles in localizing Ca²⁺ channels. In C. elegans, the Brp homologue ELKS-1 is also localized to the active zone; however, the importance of ELKS-1 during development of synapses was only revealed in sensitized genetic backgrounds (Dai et al., 2006; Patel and Shen, 2009), suggesting that there are likely redundant molecular pathways for presynaptic assembly. In the vertebrate system, loss of one of the three ELKS genes, surprisingly, caused an increase in the inhibitory synaptic transmission (Kaeser et al., 2009). Besides Brp, Rab3-interacting molecule (RIM) binding protein (RBP) was found to be important for active zone structural integrity in flies. Using super-resolution microscopy, RBP was found to surround Ca²⁺ channels at T-bars and loss of RBP resulted in defective Ca²⁺ channel clustering and reduced evoked neurotransmitter release (Liu et al., 2011).Assembly of the presynaptic active zone is subjected to several layers of regulation. The assembly process is balanced by inhibitory mechanisms that control the number and size of synapses. Loss of the E3 ubiquitin ligase Highwire/RPM-1 results in an increased number of synaptic boutons in flies and multiple active zones in worms (Wan et al., 2000; Zhen et al., 2000). Working together with F-box protein FSN-1, RPM-1 down-regulates the DLK MAP kinase signaling pathway (Liao et al., 2004; Nakata et al., 2005; Yan et al., 2009). Another E3 ubiquitin ligase, the SKP complex, has been shown to eliminate transient synapses during development in worms (Ding et al., 2007). Therefore, ubiquitin-mediated mechanisms play important roles in controlling the presynaptic assembly program.Other inhibitory mechanisms include SRPK79D, a serine–arginine protein kinase discovered in flies that represses T-bar formation (Johnson et al., 2009). In the mutant, the T-bar component Brp is ectopically accumulated in the axonal shaft. Regulator of synaptogenesis, RSY-1, limits the extent of presynaptic assembly by directly binding to active zone scaffold molecule Liprin-α/SYD-2 and SYD-1 (Patel and Shen, 2009). In addition, Liprin-α/SYD-2 may inhibit its own activity via intramolecular interactions (Taru and Jin, 2011; Chia et al., 2013).Taken together, the presynaptic assembly process driven by scaffolding molecules is controlled by complex inhibitory mechanisms to achieve the appropriate extent of aggregation in the process of synapse formation.

Trans-synaptic signals orchestrate pre- and postsynaptic formation

Coordinated pre- and postsynaptic development requires the precise apposition of presynaptic components to postsynaptic specializations. It is conceivable that signals from pre and postsynaptic sides functioning across the synaptic cleft coordinate synaptic differentiation reciprocally. Although a vast assortment of factors have been identified as synaptic organizers, the fact that genetic ablation of some synaptic organizers in vivo fails to elicit dramatic synaptic defects suggests the incomplete view of the trans-synaptic signaling. Moreover, the underlying mechanisms and the cross talk of these signaling pathways are still unclear. In recent years, an emerging body of literature has begun to shed light on trans-synaptic signaling and the importance of environmental cues in synapse formation.

Adhesion proteins instruct synaptic differentiation

A large body of literature suggests that trans-synaptic interactions between synaptic adhesion molecules function bi-directionally for synapse formation and maturation (Fig. 3). Neurexin–neuroligin is the first pair to be shown to induce pre- and postsynapse formation (Scheiffele et al., 2000; Graf et al., 2004; Chih et al., 2005; Nam and Chen, 2005; Chubykin et al., 2007). Recent in vitro studies have unveiled more components interacting with neurexin or neuroligin in specific synaptic differentiation events (Fig. 3, B and C). In early developmental stages, a secreted synaptic organizer, thrombospondin 1 (TSP1, see next section) increases the speed of synaptogenesis through neuroligin 1 (Xu et al., 2010). At excitatory synapses, a retrograde signaling controls synaptic vesicle clustering, neurotransmitter release, and presynaptic maturation by cooperation of neuroligin and N-cadherin (Wittenmayer et al., 2009; Stan et al., 2010; Aiga et al., 2011). A leucine-rich repeat transmembrane (LRRTM) protein family was also identified as an organizer of the function of excitatory synapses through interactions with neurexin (Linhoff et al., 2009). Further studies showed that binding of LRRTMs and neuroligins to neurexin acts redundantly to maintain excitatory synapses by preventing activity and Ca²⁺-dependent synapse elimination during early development, while performing divergent functions upon synapse maturation (de Wit et al., 2009; Ko et al., 2009, 2011; Soler-Llavina et al., 2011).Open in a separate windowFigure 3.Adhesive trans-synaptic signalings orchestrate excitatory and inhibitory synaptic assembly. Multiple pairs of trans-synaptic adhesion molecules organize synaptic differentiation and function on both pre- and postsynaptic sites. Note that different adhesion molecules are used at excitatory and inhibitory synapses. LPH1, latrophilin 1; α-DG, α-dystroglycan; β-DG, β-dystroglycan; S-SCAM, synaptic scaffolding molecule; Lasso, LPH1-associated synaptic surface organizer; IL-1RAcp, interleukin-1 receptor accessory protein.The function of neurexin and neuroligin in mediating synaptic differentiation has also been shown at Drosophila NMJs and mammalian CNS. In mammalian, although neither compound knockout of three neurexins nor two individual neuroligin knockout mice display severe defects in the number or morphology of synapses (Missler et al., 2003), the deletion of either neurexin or neuroligin affects the neurotransmitter release and in turn impairs the relevant behavior (Zhang et al., 2005; Blundell et al., 2009, 2010; Etherton et al., 2009; Jedlicka et al., 2011). Neurexin loss of function in fly leads to reduced number and defective morphology of synaptic boutons and active zones from early developmental stages (Li et al., 2007; Chen et al., 2010). In contrast, deletion of either neuroligin 1 or 2 causes NMJ defects and alternations of active zones only in the larval stage, indicating that they function mainly in the expansion of NMJs during development (Banovic et al., 2010; Sun et al., 2011). These abnormalities further impair synaptic transmission at the NMJs (Li et al., 2007; Banovic et al., 2010; Chen et al., 2010; Sun et al., 2011). Moreover, these phenotypes are enhanced when the Teneurin family of adhesion molecules is deleted, suggestive of functional redundancy between adhesion molecules (Mosca et al., 2012). Recently, it has been reported that an active zone protein, SYD-1, is required for the formation and function of the neurexin–neuroligin complex in flies (Fig. 2; Owald et al., 2012), providing an example of how trans-synaptic neurexin–neuroligin signaling orchestrates synaptic assembly bi-directionally. Interestingly, at postsynaptic sites, the NMDA receptor activity-triggered Ca²⁺-dependent cleavage of neuroligin 1 was found to destabilize presynaptic neurexin, reduce presynaptic release probability, and depress synaptic transmission (Peixoto et al., 2012). This observation raises a possibility that neurexin and neuroligin could fine-tune synaptogenesis both positively and negatively.Although Drosophila neuroligin and neurexin mutants share many phenotypes in synaptic differentiation, there are some unique features for each mutant, suggesting that they play distinct roles. For example, some aspects of synaptic specificity are achieved by different pairs of neurexin–neuroligin interactions. Neuroligin 1 promotes the growth and differentiation of excitatory synapses by binding to PSD-95, whose amount balances the ratio of excitatory-to-inhibitory synaptic specializations (Prange et al., 2004; Banovic et al., 2010). Neuroligin 2, on the contrary, binds to a scaffold protein gephyrin at inhibitory synapses, instructing inhibitory postsynaptic assembly (Fig. 3, B and C; Poulopoulos et al., 2009). Different isoforms of neurexin also contribute to the differentiation of excitatory and inhibitory synapses (Fig. 3, B and C; Chih et al., 2006; Graf et al., 2006; Kang et al., 2008).Other novel trans-synaptic interactions have also been identified to organize synaptic differentiation (Fig. 3, B and C). For example, Netrin-G ligand 3 (NGL-3), localized at postsynaptic region, induces excitatory synaptic differentiation by interacting with the receptor tyrosine phosphatase LAR family proteins, including PTPδ and PTPσ (Woo et al., 2009; Kwon et al., 2010). PTPδ can also trans-interact with Slitrk3 and IL-1 receptor accessory protein (IL-1RAcP) to promote presynaptic formation (Takahashi et al., 2012; Yoshida et al., 2012). Molecules that function in other neuronal developmental processes have also been shown to regulate synaptic differentiation. Farp1, essential for the dynamics of dendritic filopodia, regulates postsynaptic development and triggers a retrograde signal promoting active zone assembly by binding to SynCAM 1 (Cheadle and Biederer, 2012). Teneurins, instructing synaptic partner selection in fly olfactory system (Hong et al., 2012), act in synaptogenesis through trans-synaptic interaction at NMJs (Mosca et al., 2012). Another splice variant of a postsynaptic Teneurin-2 in rat, Lasso, binding with presynaptic Latrophilin 1 (LPH1), induces presynaptic Ca²⁺ signals and regulates synaptic function (Silva et al., 2011). Neural activity is also involved in controlling the growth of the presynapse. Conditioning or BDNF application induces presynaptic bouton development via an ephrin-B–dependent manner (Li et al., 2011), suggesting the role of EphB/ephrin-B signaling in activity-dependent synaptic modification.

Secreted molecules organize synapse differentiation

In addition to adhesion molecules, some secreted molecules also serve as synaptic organizers (Fig. 4). For example, the motor neuron–derived ligand agrin, which was the first identified secreted organizing molecule for postsynaptic differentiation, activates MuSK, a postsynaptic receptor tyrosine kinase, to regulate NMJ specialization (Glass et al., 1996; Zhou et al., 1999). Recently, a low-density lipoprotein receptor–related protein, LRP4, was identified as the co-receptor of agrin, forming a complex with MuSK and mediating MuSK signaling (Kim et al., 2008; Zhang et al., 2008). Several Wnts appears to act together with agrin to activate the LRP4–MuSK receptor complex to promote postsynaptic differentiation (Jing et al., 2009; Zhang et al., 2012). LRP4 also acts as a direct retrograde signal, functioning independently of MuSK for presynaptic differentiation (Yumoto et al., 2012), demonstrating that LRP4 acts as a bi-directional synaptic organizer (Fig. 4, left).Open in a separate windowFigure 4.Secreted trans-synaptic signaling at NMJs and CNS synapses. (Left) At Drosophila neuromuscular junctions (NMJs), Wnts are secreted from presynaptic terminals in association with Evi in the form of exosomes. In vertebrate NMJs, Wnt binds to the Agrin–LRP4–MuSK complex to regulate synapse formation. (Right) At CNS synapses, glia-derived thrombospondins (TSPs) and presynaptic neuron–derived cerebellin (Cbln) organize synapse differentiation and formation bi-directionally through binding to GluD2 and an isoform of neurexin (S4+) on the postsynaptic and presynaptic membranes, respectively. LTCC, L-type Ca²⁺ channel complex; AChR, acetyl choline receptor.Wnt is another well-characterized signaling molecule regulating many developmental processes including synaptic differentiation bi-directionally. Wnt regulates synaptic assembly both positively and negatively. For example, Wnt3 collaborates with agrin to promote the clustering of acetyl choline receptor (AChR) at the vertebrate NMJs (Henriquez et al., 2008), while Wnt3a inhibits AChR aggregation through β-catenin signaling (Wang et al., 2008). In the C. elegans NMJ, a Wnt molecule, CWN-2, stimulates the delivery and insertion of AchR to the postsynaptic membrane through the activation of a Frizzled–CAM-1 receptor complex (Jensen et al., 2012). Local Wnt gradient can suppress synapse formation in both C. elegans and Drosophila (Inaki et al., 2007; Klassen and Shen, 2007). Interestingly, in these contexts, Wnts are secreted from nonneuronal or nonsynaptic partner cells, suggesting that environmental factors can shape synaptic connections. Wnt can also be secreted from presynaptic neurons. A recent study demonstrated the trans-synaptic transmission of Wnt by exosome-like vesicles containing the Wnt-binding protein Evi at Drosophila NMJs (Fig. 4, left; Korkut et al., 2009; Koles et al., 2012). Presynaptic vesicular release of Evi is required for the secretion of Wnt. Intriguingly, different Wnt ligands regulate synapse formation in distinct cellular contexts. Wnt3a promotes excitatory synaptic assembly through CaMKII, whereas Wnt5a mediates inhibitory synapse formation by stabilizing GABA_A receptors (Cuitino et al., 2010; Ciani et al., 2011). This functional diversity indicates that different Wnts, receptors, and downstream pathways, as well as cell-specific contexts dictate the action of extracellular cues. Another conserved secreted molecule, netrin/UNC-6, can also pattern synapses by either promoting or inhibiting synapse formation (Colón-Ramos et al., 2007; Poon et al., 2008). Because Wnt and netrin often exist in gradients, these observations suggest that the localization of synapses can be specified by the gradient of extrinsic cues.In mammalian, several glia-derived cues have been shown to play important roles in regulating synapse formation or elimination. Thrombospondins (TSPs) are trans-synaptic organizers secreted from immature astrocytes (Christopherson et al., 2005). Both in vitro and in vivo data demonstrate the capacity of TSPs to increase synapse number, promote the localization of synaptic molecules, and refine the pre- and postsynaptic alignment (Christopherson et al., 2005; Eroglu et al., 2009). Recently, two transmembrane molecules were uncovered in mediating TSP-induced synaptogenesis (Fig. 4, right). Neuroligin 1 interacts with TSP1 with its extracellular domain mediating the acceleration of synaptogenesis in hippocampal neurons (Xu et al., 2010). α2δ-1, a subunit of the L-type Ca²⁺ channel complex (LTCC), was also identified as the postsynaptic receptor of TSP in excitatory CNS neurons (Eroglu et al., 2009). Interaction between TSP and α2δ–1 triggers the conformational changes and sequentially recruits synaptic scaffolding molecules and initiates synapse formation (Eroglu et al., 2009). Interestingly, TSP-induced synapses, although structurally normal and presynaptically active, are postsynaptically silent due to the lack of AMPA receptors (Christopherson et al., 2005), indicating the existence of other glia-derived signals involved in synapse formation. In fact, in cultured hippocampal neurons, a glia-derived neurotrophic factor GDNF enhances the pre- and postsynaptic adhesion by triggering the trans-homophilic interaction of its receptors GFRα1 localized at both pre- and postsynaptic sites (Ledda et al., 2007). Several other glia-derived factors have been shown to play critical roles in synaptogenesis. Astrocytes secrete extracellular molecules hevin and SPARC to regulate synapse formation in vitro and in vivo (Kucukdereli et al., 2011). Astrocytes also express a transmembrane adhesion protein, protocadherin-γ, serving as a local cue to promote synapse formation (Garrett and Weiner, 2009). TGF-β secreted from the NMJ glia acts together with the muscle-derived TGF-β to control synaptic growth (Fuentes-Medel et al., 2012). In a similar fashion, secretion of BDNF by vestibular supporting cells is required for synapse formation between hair cells and sensory organs (Gómez-Casati et al., 2010).Another important synaptic organizer is cerebellin (Cbln), a presynapse-derived complement protein, C1q-like family protein. In cbln1-null mice the number of parallel fibers (PF)–Purkinje synapses is dramatically reduced; the postsynaptic densities in the remaining synapses are larger than the apposite active zones (Hirai et al., 2005). Cbln was also found to regulate synaptic plasticity, as cbln1-null mice show impaired long-term depression in cerebellum (Hirai et al., 2005). These defects precisely resemble those in mice lacking a putative glutamate receptor, GluD2 (Kashiwabuchi et al., 1995; Kurihara et al., 1997), suggesting that Cbln1 and GluD2 function in synaptic differentiation through a common pathway. Interestingly, the C-terminal domain and N-terminal domains of GluD2 are indispensable for cerebella LTD and PF–Purkinje synaptic morphology, respectively (Kohda et al., 2007; Uemura et al., 2007; Kakegawa et al., 2008, 2009). Further studies suggested that Cbln1 directly binds to the N-terminal domain of GluD2 and recruits postsynaptic proteins by clustering GluD2 (Matsuda et al., 2010). Neurexin was recently reported as the presynaptic receptor of Cbln in promoting synaptogenesis (Uemura et al., 2010), which reinforces the understanding of Cbln-mediated trans-synaptic signaling: Cbln serves as a bi-directional synapse organizer by linking presynaptic neurexin and postsynaptic GluD2 (Fig. 4, right).Besides being required for synapse formation at early stages, genetic ablation of GluD2 in adult cerebellum leads to loss of PF–Purkinje synapses (Takeuchi et al., 2005), indicating that Cbln1–GluD2 signaling is also important for the maintenance of PF–Purkinje synapses. Chronic stimulation of neural activity decreases Cbln1 expression and diminishes the number of PF–Purkinje synapses (Iijima et al., 2009), suggesting the importance of Cbln1–GluD2 signaling for synaptic plasticity and homeostasis.Cbln subfamily proteins are widely expressed throughout the brain (Miura et al., 2006), suggesting that their synaptogenic roles may be wide spread in other regions of the brain. Cbln2 and 4 are also secreted proteins, whereas Cbln3 is retained in the cellular endomembrane system (Iijima et al., 2007). Cbln1 and 2, interacting with an isoform of presynaptic neurexin, induce synaptogenesis (Joo et al., 2011; Matsuda and Yuzaki, 2011). Notably, the cortical synapses induced by neurexin–Cbln signaling are preferentially inhibitory (Joo et al., 2011), distinguishing the effects of Cbln from neuroligin. GluD1 was recently found to be the postsynaptic receptor of Cbln1 and 2 in cortical neurons, mediating the differentiation of inhibitory presynapses (Yasumura et al., 2012). On the other side, Cbln4 selectively binds to the netrin receptor DCC in a netrin-displaceable manner (Fig. 4, right), suggesting a potential function of Cbln4 through DCC signaling pathway (Iijima et al., 2007). Intriguingly, C1q, although sharing similar structure with Cbln, serves an opposite role by regulating the synapse elimination: C1q released from retinal ganglion cells refines the retinogeniculate connections by eliminating unneeded synapses (Stevens et al., 2007).

Concluding remarks

Synapse development is regulated in multiple steps. Research over the last few years have uncovered many regulatory mechanisms on how trafficking of synaptic material is regulated and how scaffold proteins act with cytoskeleton networks and trans-synaptic signaling to instruct the synapse formation. Nevertheless, our understanding of the cellular and molecular mechanisms regulating synapse development is still incomplete. For example, how is the direction, speed, and amount of synaptic material being transported specified? How is a synapse’s size determined? How is synapse type and strength specified through adhesive and secreted trans-synaptic signaling? How do the redundant synapse-inducing pathways interact with each other? Given the rapidly emerging improvements of technologies, especially super-resolution microscopy and high-throughput genomics and proteomics, the synapse development field will likely rapidly evolve in the near future.     

A dual role of SNAP‐25 as carrier and guardian of synaptic transmission

In this issue, Matteoli and colleagues show that SNAP-25 levels regulate the efficacy of presynaptic glutamate release and thereby alter short-term plasticity, with potential relevance for psychiatric diseases.EMBO reports(2013) 14 7, 645–651 doi:10.1038/embor.2013.75Control of exocytotic neurotransmitter release is essential for communication in the nervous system and for preventing synaptic abnormalities. The function of synaptosomal-associated protein of 25 kDa (SNAP-25) as a crucial component of the core machinery required for synaptic vesicle fusion is well established, but evidence is growing to suggest an additional modulatory role in neurotransmission. In this issue of EMBO reports, Antonucci et al show that the efficacy of evoked glutamate release is modulated by the expression levels of SNAP-25—a function that might relate to the ability of SNAP-25 to modulate voltage-gated calcium channels and presynaptic calcium ion concentration [1]. Altered synaptic transmission and short-term plasticity due to changes in SNAP-25 expression might have direct consequences for brain function and for the development of neuropsychiatric disorders.Communication between neurons is essential for brain function and occurs through chemical neurotransmission at specialized cell–cell contacts termed ‘synapses''. Within the nerve terminal of the presynaptic neuron electrical stimuli cause the opening of voltage-gated calcium channels (VGCCs), which results in the influx of calcium ions. This triggers the exocytic release of neurotransmitter by fusion of synaptic vesicles with the presynaptic membrane. Released neurotransmitter molecules are detected by specific receptors expressed by the postsynaptic neuron.Calcium-induced synaptic vesicle fusion requires complex assembly between the soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein receptor (SNARE) synaptobrevin 2, located on the synaptic vesicle, and the abundant plasma membrane SNAREs SNAP-25 and syntaxin 1, on the opposing presynaptic plasma membrane. SNARE complex assembly is tightly regulated by Sec1/Munc18-like proteins [2]. Further regulatory factors such as the synaptic vesicle calcium-sensing protein synaptotagmin 1 couple the SNARE machinery to presynaptic calcium influx. SNARE-mediated neurotransmitter release occurs preferentially at the active zone—a presynaptic membrane domain specialized for exocytosis within which VGCCs are positioned close to docked synaptic vesicles through a proteinaceous cytomatrix and associated cell adhesion molecules [3,4].Altered short-term plasticity due to changes in SNAP-25 expression might have direct consequences for brain function and for the development of neuropsychiatric disordersAn unresolved conundrum in synaptic transmission remains—the observation that SNARE proteins, such as SNAP-25, are among the most highly expressed, in copy number, presynaptic proteins, whilst only a handful of SNARE complexes are needed to drive the fusion of a single synaptic vesicle [5]. Why, then, are SNAREs such as SNAP-25 so abundant? One possible explanation might be that SNARE proteins, in addition to forming trans-SNARE complexes, assemble with other proteins, and such partitioning might regulate neurotransmission. For example, SNAP-25 has been shown to negatively regulate VGCCs in glutamatergic but not in GABAergic neurons [6]. A secondary regulatory function of SNAP-25 is also supported by its genetic association with synaptic abnormalities such as schizophrenia and attention deficit hyperactivity disorder (ADHD) in humans [7]. SNAP-25 expression is reduced twofold in the hippocampus and frontal lobe from schizophrenic patients [8] and in animal models for ADHD [9]. Thus, SNAP-25 expression levels might crucially regulate normal synaptic function.A new study in this issue of EMBO reports by Antonucci and colleagues investigates the consequences of reduced SNAP-25 expression on synaptic function in SNAP-25^+/− heterozygous (Het) mutant mice. By using patch clamp electrophysiology, Antonucci et al revealed a selective enhancement of glutamatergic but not GABAergic neurotransmission as a result of reduced SNAP-25 expression. Several other parameters including the amplitude and frequency of miniature excitatory and inhibitory currents were unaffected. These data indicate that reduced levels of SNAP-25, an essential component of the fusion machinery, selectively enhance evoked release of glutamate whilst synaptic connectivity and postsynaptic glutamate receptor sensitivity remain unaltered. Further electrophysiological experiments in hippocampal neurons in culture showed that elevated glutamatergic transmission was probably due to increased release probability rather than changes in the number of fusion-prone, so-called ‘readily releasable synaptic vesicles''. This effect was occluded by pharmacologically induced calcium entry bypassing VGCCs, suggesting that altered calcium influx might underlie the differences in evoked glutamate release between wild-type and SNAP-25 Het neurons. As schizophrenia and ADHD are associated with changes in short-term plasticity, a paradigm reflecting presynaptic function, Antonucci et al analysed neurotransmission by paired-pulse stimulation—a protocol whereby two closely paired stimuli are applied within a 50 ms time interval. Wild-type neurons showed significant short-term facilitation, that is, a stronger response to the second stimulus as a result of increased calcium levels in the presynaptic compartment. By contrast, Het neurons had a reduced response to the second stimulus. Such paired-pulse depression is commonly viewed as a sign of increased release probability, which occurs when the first stimulus induces a partial depletion of release-ready synaptic vesicles during paired stimulation. As a consequence, the second stimulus evokes a comparably reduced response [3]. The switch from paired-pulse facilitation to depression was not fully reproduced in hippocampal slices from wild-type and Het mice, although facilitation seemed to be attenuated in SNAP-25 Het slices. One possible explanation for the apparent discrepancy between cultured neurons taken from newborn animals and acute slices from adult mice is the constant postnatal increase in SNAP-25 expression in SNAP-25 Het mice [10], which might partly counteract the defects caused by heterozygosity. Consistent with this explanation are data from rescue experiments by Antonucci et al, which showed that altered neurotransmission and defects in short-term plasticity in Het neurons can be gradually recovered in parallel with increased SNAP-25 expression. Moreover, cultured neurons show substantially higher levels of endogenous activity compared with acute slice preparations, leading to possible changes in the partitioning of SNAP-25 between SNARE complexes and association with VGCCs. Further experiments are clearly required to resolve these issues. Irrespective of these potential caveats, the combined data support the hypothesis that alterations in SNAP-25 expression underlie regulatory changes in neurotransmission, resulting in altered short-term plasticity and possibly disease.Many open questions remain. In particular, the precise mechanisms underlying elevated glutamatergic transmission and presynaptic plasticity under conditions of reduced SNAP-25 expression remain elusive. It has been shown before that free SNAP-25 inhibits Cav2.1-type VGCCs [6], an effect reversed by overexpression of synaptotagmin 1, which might associate with SNAP-25. Conversely, SNAP-25 occludes negative regulation of Cav2.2 VGCCs by free syntaxin 1 [3]. Hence, it is tempting to speculate that differential partitioning of SNAP-25 between free, SNARE-, synaptotagmin 1- and VGCC-complexed forms could regulate evoked neurotransmission (Fig 1). In this scenario, reduced SNAP-25 expression in Het animals and in schizophrenic and ADHD patients would be sufficient to sustain SNARE-mediated synaptic vesicle fusion but partially releases VGCCS from SNAP-25-mediated inhibition. This would result in elevated calcium influx and facilitated neurotransmission. Additional levels of regulation could be imposed by developmental switching between alternatively spliced ‘a'' and ‘b'' isoforms of SNAP-25 [11], age-dependent alterations in presynaptic protein turnover and post-translational modifications.Open in a separate windowFigure 1Effect of presynaptic SNAP-25 levels on calcium-induced glutamate release. Top: in wild-type (WT) neurons, SNARE-mediated calcium-triggered synaptic vesicle fusion is negatively regulated by complex formation between SNAP-25 and VGCCs. Bottom: reduced SNAP-25 expression in heterozygotes (Het;^+/−) partly releases VGCCs from SNAP-25-mediated clamping, resulting in elevated calcium influx through VGCCs and increased glutamate release through SNARE-mediated calcium-triggered synaptic vesicle fusion. Note that many key exocytotic proteins have been omitted for clarity. SNAP-25, synaptosomal-associated protein of 25 kDa; SNARE, soluble NSF attachment protein receptor; VGCC. voltage-gated calcium channel.Future studies need to address these possibilities, and their relationship to cognitive impairments and synaptic diseases, such as schizophrenia and ADHD.     

Presynaptic proteins involved in exocytosis inDrosophila melanogaster: A genetic analysis

Neuronal communication involves the fusion of neurotransmitter filled synaptic vesicles with the presynaptic terminal. This exocytotic event depends upon proteins present in three separate compartments: the synaptic vesicle, the synaptic cytosol, and the presynaptic membrane. Recent data indicate that the basic components of exocytotic pathways, including those used for neurotransmitter release, are conserved from yeast to human. Genetic dissection of the secretory pathway in yeast, identification of the target proteins cleaved by the clostridial neurotoxins and biochemical characterization of the interactions of synaptic proteins from vertebrates have converged to provide the SNARE (soluble NSF attachment protein receptor) hypothesis for vesicle trafficking. This model proposes that proteins present in the vesicle (v-SNAREs) interact with membrane receptors (t-SNAREs) to provide a molecular scaffold for cytosolic proteins involved in fusion. The hypothesis that these mechanisms function at the synapse relies largely uponin vitro evidence. Recently, genetic approaches in mice, C.elegans and the fruitfly,Drosophila melanagaster, have been used to dissect thein vivo function of numerous proteins involved in synaptic transmission. This review covers recent progress and insights provided by a genetic dissection of neurotransmitter release inDrosophila. In addition, we will provide evidence that the mechanisms for synaptic communication are highly conserved from invertebrates to vertebrates, makingDrosophila an ideal model system to further unravel the intricacies of synaptic transmission.     

Quercetin Targets Cysteine String Protein (CSPα) and Impairs Synaptic Transmission

Background

Cysteine string protein (CSPα) is a synaptic vesicle protein that displays unique anti-neurodegenerative properties. CSPα is a member of the conserved J protein family, also called the Hsp40 (heat shock protein of 40 kDa) protein family, whose importance in protein folding has been recognized for many years. Deletion of the CSPα in mice results in knockout mice that are normal for the first 2–3 weeks of life followed by an unexplained presynaptic neurodegeneration and premature death. How CSPα prevents neurodegeneration is currently not known. As a neuroprotective synaptic vesicle protein, CSPα represents a promising therapeutic target for the prevention of neurodegenerative disorders.

Methodology/Principal Findings

Here, we demonstrate that the flavonoid quercetin promotes formation of stable CSPα-CSPα dimers and that quercetin-induced dimerization is dependent on the unique cysteine string region. Furthermore, in primary cultures of Lymnaea neurons, quercetin induction of CSPα dimers correlates with an inhibition of synapse formation and synaptic transmission suggesting that quercetin interfers with CSPα function. Quercetin''s action on CSPα is concentration dependent and does not promote dimerization of other synaptic proteins or other J protein family members and reduces the assembly of CSPα:Hsc70 units (70kDa heat shock cognate protein).

Conclusions/Significance

Quercetin is a plant derived flavonoid and popular nutritional supplement proposed to prevent memory loss and altitude sickness among other ailments, although its precise mechanism(s) of action has been unclear. In view of the therapeutic promise of upregulation of CSPα and the undesired consequences of CSPα dysfunction, our data establish an essential proof of principle that pharmaceutical agents can selectively target the neuroprotective J protein CSPα.