John Jia En Chua

Quantitative comparison of glutamatergic and GABAergic synaptic vesicles unveils selectivity for few proteins including MAL2, a novel synaptic vesicle protein.

Synaptic vesicles (SVs) store neurotransmitters and release them by exocytosis. The vesicular neurotransmitter transporters discriminate which transmitter will be sequestered and stored by the vesicles. However, it is unclear whether the neurotransmitter phenotype of SVs is solely defined by the transporters or whether it is associated with additional proteins. Here we have compared the protein composition of SVs enriched in vesicular glutamate (VGLUT-1) and GABA transporters (VGAT), respectively, using quantitative proteomics. Of >450 quantified proteins, ∼50 were differentially distributed between the populations, with only few of them being specific for SVs. Of these, the most striking differences were observed for the zinc transporter ZnT3 and the vesicle proteins SV2B and SV31 that are associated preferentially with VGLUT-1 vesicles, and for SV2C that is associated mainly with VGAT vesicles. Several additional proteins displayed a preference for VGLUT-1 vesicles including, surprisingly, synaptophysin, synaptotagmins, and syntaxin 1a. Moreover, MAL2, a membrane protein of unknown function distantly related to synaptophysins and SCAMPs, cofractionated with VGLUT-1 vesicles. Both subcellular fractionation and immunolocalization at the light and electron microscopic level revealed that MAL2 is a bona-fide membrane constituent of SVs that is preferentially associated with VGLUT-1-containing nerve terminals. We conclude that SVs specific for different neurotransmitters share the majority of their protein constituents, with only few vesicle proteins showing preferences that, however, are nonexclusive, thus confirming that the vesicular transporters are the only components essential for defining the neurotransmitter phenotype of a SV.

Molecular Profiling of Synaptic Vesicle Docking Sites Reveals Novel Proteins but Few Differences between Glutamatergic and GABAergic Synapses

Neurotransmission involves calcium-triggered fusion of docked synaptic vesicles at specialized presynaptic release sites. While many of the participating proteins have been identified, the molecular composition of these sites has not been characterized comprehensively. Here, we report a procedure to biochemically isolate fractions highly enriched in docked synaptic vesicles. The fraction is largely free of postsynaptic proteins and most other organelles while containing most known synaptic vesicle and active zone proteins. Numerous presynaptic transmembrane proteins were also identified, together with over 30 uncharacterized proteins, many of which are evolutionarily conserved. Quantitative proteomic comparison of glutamate- and GABA-specific docking complexes revealed that, except of neurotransmitter-specific enzymes and transporters, only few proteins were selectively enriched in either fraction. We conclude that the core machinery involved in vesicle docking and exocytosis does not show compositional differences between the two types of synapses.

Quantitative analysis of synaptic vesicle Rabs uncovers distinct yet overlapping roles for Rab3a and Rab27b in Ca2+-triggered exocytosis.

Rab GTPases are molecular switches that orchestrate protein complexes before membrane fusion reactions. In synapses, Rab3 and Rab5 proteins have been implicated in the exo-endocytic cycling of synaptic vesicles (SVs), but an involvement of additional Rabs cannot be excluded. Here, combining high-resolution mass spectrometry and chemical labeling (iTRAQ) together with quantitative immunoblotting and fluorescence microscopy, we have determined the exocytotic (Rab3a, Rab3b, Rab3c, and Rab27b) and endocytic (Rab4b, Rab5a/b, Rab10, Rab11b, and Rab14) Rab machinery of SVs. Analysis of two closely related proteins, Rab3a and Rab27b, revealed colocalization in synaptic nerve terminals, where they reside on distinct but overlapping SV pools. Moreover, whereas Rab3a readily dissociates from SVs during Ca2+-triggered exocytosis, and is susceptible to membrane extraction by Rab-GDI, Rab27b persists on SV membranes upon stimulation and is resistant to GDI-coupled Rab retrieval. Finally, we demonstrate that selective modulation of the GTP/GDP switch mechanism of Rab27b impairs SV recycling, suggesting that Rab27b, probably in concert with Rab3s, is involved in SV exocytosis.

The GTPase Rab26 links synaptic vesicles to the autophagy pathway

Small GTPases of the Rab family not only regulate target recognition in membrane traffic but also control other cellular functions such as cytoskeletal transport and autophagy. Here we show that Rab26 is specifically associated with clusters of synaptic vesicles in neurites. Overexpression of active but not of GDP-preferring Rab26 enhances vesicle clustering, which is particularly conspicuous for the EGFP-tagged variant, resulting in a massive accumulation of synaptic vesicles in neuronal somata without altering the distribution of other organelles. Both endogenous and induced clusters co-localize with autophagy-related proteins such as Atg16L1, LC3B and Rab33B but not with other organelles. Furthermore, Atg16L1 appears to be a direct effector of Rab26 and binds Rab26 in its GTP-bound form, albeit only with low affinity. We propose that Rab26 selectively directs synaptic and secretory vesicles into preautophagosomal structures, suggesting the presence of a novel pathway for degradation of synaptic vesicles. DOI: http://dx.doi.org/10.7554/eLife.05597.001

The Architecture of an Excitatory Synapse

The functioning of excitatory synapses in the mammalian brain is governed by macromolecular complexes that are held together by protein-protein, protein-lipid and lipid-lipid interactions. On the presynaptic side, neurotransmitter (NT)-filled synaptic vesicles (SVs) are recruited to specialized

Phosphorylation-regulated axonal dependent transport of syntaxin 1 is mediated by a Kinesin-1 adapter

Presynaptic nerve terminals are formed from preassembled vesicles that are delivered to the prospective synapse by kinesin-mediated axonal transport. However, precisely how the various cargoes are linked to the motor proteins remains unclear. Here, we report a transport complex linking syntaxin 1a (Stx) and Munc18, two proteins functioning in synaptic vesicle exocytosis at the presynaptic plasma membrane, to the motor protein Kinesin-1 via the kinesin adaptor FEZ1. Mutation of the FEZ1 ortholog UNC-76 in Caenorhabditis elegans causes defects in the axonal transport of Stx. We also show that binding of FEZ1 to Kinesin-1 and Munc18 is regulated by phosphorylation, with a conserved site (serine 58) being essential for binding. When expressed in C. elegans, wild-type but not phosphorylation-deficient FEZ1 (S58A) restored axonal transport of Stx. We conclude that FEZ1 operates as a kinesin adaptor for the transport of Stx, with cargo loading and unloading being regulated by protein kinases.

Functions of Rab Proteins at Presynaptic Sites

Presynaptic neurotransmitter release is dominated by the synaptic vesicle (SV) cycle and entails the biogenesis, fusion, recycling, reformation or turnover of synaptic vesicles—a process involving bulk movement of membrane and proteins. As key mediators of membrane trafficking, small GTPases from the Rab family of proteins play critical roles in this process by acting as molecular switches that dynamically interact with and regulate the functions of different sets of macromolecular complexes involved in each stage of the cycle. Importantly, mutations affecting Rabs, and their regulators or effectors have now been identified that are implicated in severe neurological and neurodevelopmental disorders. Here, we summarize the roles and functions of presynaptic Rabs and discuss their involvement in the regulation of presynaptic function.

Synthesis of two SAPAP3 isoforms from a single mRNA is mediated via alternative translational initiation

In mammalian neurons, targeting and translation of specific mRNAs in dendrites contribute to synaptic plasticity. After nuclear export, mRNAs designated for dendritic transport are generally assumed to be translationally dormant and activity of individual synapses may locally trigger their extrasomatic translation. We show that the long, GC-rich 5′-untranslated region of dendritic SAPAP3 mRNA restricts translation initiation via a mechanism that involves an upstream open reading frame (uORF). In addition, the uORF enables the use of an alternative translation start site, permitting synthesis of two SAPAP3 isoforms from a single mRNA. While both isoforms progressively accumulate at postsynaptic densities during early rat brain development, their levels relative to each other vary in different adult rat brain areas. Thus, alternative translation initiation events appear to regulate relative expression of distinct SAPAP3 isoforms in different brain regions, which may function to influence synaptic plasticity.

Macromolecular complexes at active zones: integrated nano-machineries for neurotransmitter release

The release of neurotransmitters from synaptic vesicles exocytosing at presynaptic nerve terminals is a critical event in the initiation of synaptic transmission. This event occurs at specialized sites known as active zones. The task of faithfully executing various steps in the process is undertaken by careful orchestration of overlapping sets of molecular nano-machineries upon a core macromolecular scaffold situated at active zones. However, their composition remains incompletely elucidated. This review provides an overview of the role of the active zone in mediating neurotransmitter release and summarizes the recent progress using neuroproteomic approaches to decipher their composition. Key proteins of these nano-machineries are highlighted.

Phosphorylation of FEZ1 by Microtubule Affinity Regulating Kinases regulates its function in presynaptic protein trafficking.

Adapters bind motor proteins to cargoes and therefore play essential roles in Kinesin-1 mediated intracellular transport. The regulatory mechanisms governing adapter functions and the spectrum of cargoes recognized by individual adapters remain poorly defined. Here, we show that cargoes transported by the Kinesin-1 adapter FEZ1 are enriched for presynaptic components and identify that specific phosphorylation of FEZ1 at its serine 58 regulatory site is mediated by microtubule affinity-regulating kinases (MARK/PAR-1). Loss of MARK/PAR-1 impairs axonal transport, with adapter and cargo abnormally co-aggregating in neuronal cell bodies and axons. Presynaptic specializations are markedly reduced and distorted in FEZ1 and MARK/PAR-1 mutants. Strikingly, abnormal co-aggregates of unphosphorylated FEZ1, Kinesin-1 and its putative cargoes are present in brains of transgenic mice modelling aspects of Alzheimer’s disease, a neurodegenerative disorder exhibiting impaired axonal transport and altered MARK activity. Our findings suggest that perturbed FEZ1-mediated synaptic delivery of proteins arising from abnormal signalling potentially contributes to the process of neurodegeneration.