Synaptic vesicle protein trafficking at the glutamate synapse

We investigated whether cortical glutamatergic and GABAergic release machineries can be differentiated on the basis of the nature and amount of proteins they express, by performing a quantitative analysis of the degree of co-localization of synaptotagmin (SYT) 1 and 2, synaptic vesicle protein 2 (SV2) A and B, and Rab3a and c in VGLUT1+, VGLUT2+, and VGAT+ terminals and synaptic vesicles (SVs) in rat cerebral cortex. Co-localization studies showed that VGLUT1 puncta had high levels of SV2A and B and of Rab3c, intermediate levels of SYT1, and low levels of SYT2 and Rab3c; VGLUT2 puncta exhibited intermediate levels of all presynaptic proteins studied; whereas vesicular GABA transporter (VGAT) puncta had high levels of SV2A and SYT2, intermediate levels of SYT1, Rab3a, and Rab3c, and low levels of SV2B. Since SV2B is reportedly expressed by glutamatergic neurons and we observed SV2B expression in VGAT puncta, we performed electron microscopic studies and found SV2B positive axon terminals forming symmetric synapses. Immunoisolation studies showed that the expression levels of the protein isoforms varied in the three populations of SVs. Expression of SYT1 was highest in VGLUT1–SVs, while SYT2 expression was similar in the three SV groups. Expression of SV2A was similarly high in all three SV populations, except for SV2B levels that were very low in VGAT SVs. Finally, Rab3a levels were similar in the three SV groups, while Rab3c levels were highest in VGLUT1–SVs. These quantitative results extend our previous studies on the differential expression of presynaptic proteins involved in neurotransmitter release in GABAergic and glutamatergic terminals and indicate that heterogeneity of the respective release machineries can be generated by the differential complement of SV proteins involved in distinct stages of the release process.

Background/Objectives: Glutamatergic neurotransmission is essential for normal functioning of the retina. Photoreceptor to bipolar and bipolar to ganglion cell signaling is mediated by L-glutamate, stored in, and released from vesicular glutamate transporter 1 (VGLUT1) containing synaptic vesicles. VGLUT1 is expressed postnatally, P2 onwards, and is required for glutamatergic retinal wave observed between P10 and P12 in the developing mouse retina. P9-P13 postnatal age is critical for retinal development as VGLUT1 expressing ribbon synapses activate in the outer and inner plexiform layers, and rod/cone mediated visual signaling commences in that period. Although it has been hypothesized that glutamatergic extrinsic signaling drives cell cycle exit and initiates cellular differentiation in the developing retina, it’s not clear whether intracellular, synaptic or extrasynaptic vesicular glutamate release contributes in this process. Recent studies attempted to decipher VGLUT’s role in retinal development. Here we investigate the potential effect of genetic loss of VGLUT1 on early postnatal histogenesis and development of retinal neural circuitry. Methods: We employed immunohistochemistry and electrophysiology to ascertain the density of glutamatergic, cholinergic and dopaminergic cells, spontaneous retinal activity and light responses in VGLUT1 null retina, and contrasted them to wildtype (WT) and melanopsin null retina. Results: We demonstrate here that VGLUT1 null retina shows signs of age dependent retinal degeneration, similar to other transgenic mice models with dysfunctional photoreceptor to bipolar cells synapses. Loss of VGLUT1 specifically alters glutamatergic cell density and morphological maturation of retinal ganglion cells. Moreover, VGLUT2 expression is lost in majority of VGLUT2 cones in absence of VGLUT1 co-expression, except when VGLUT2 co- expresses transiently with VGLUT3 in these cones, or when VGLUT1 null mice are dark reared. Conclusions: We present the first evidence that synaptic or extrasynaptic postnatal glutamate release from VGLUT1 containing vesicles impacts histogenesis of glutamatergic cells, pruning of retinal ganglion cell dendrites and VGLUT2 expression in cones.

Neuronal lysosomes and their biogenesis mechanisms are primarily thought to clear metabolites and proteins whose abnormal accumulation leads to neurodegenerative disease pathology. However, it remains unknown whether lysosomal sorting mechanisms regulate the levels of membrane proteins within synaptic vesicles. Using high-resolution deconvolution microscopy, we identified early endosomal compartments where both selected synaptic vesicle and lysosomal membrane proteins coexist with the adaptor protein complex 3 (AP-3) in neuronal cells. From these early endosomes, both synaptic vesicle membrane proteins and characteristic AP-3 lysosomal cargoes can be similarly sorted to brain synaptic vesicles and PC12 synaptic-like microvesicles. Mouse knockouts for two Hermansky-Pudlak complexes involved in lysosomal biogenesis from early endosomes, the ubiquitous isoform of AP-3 (Ap3b1(-/-)) and muted, defective in the biogenesis of lysosome-related organelles complex 1 (BLOC-1), increased the content of characteristic synaptic vesicle proteins and known AP-3 lysosomal proteins in isolated synaptic vesicle fractions. These phenotypes contrast with those of the mouse knockout for the neuronal AP-3 isoform involved in synaptic vesicle biogenesis (Ap3b2(-/-)), in which the content of select proteins was reduced in synaptic vesicles. Our results demonstrate that lysosomal and lysosome-related organelle biogenesis mechanisms regulate steady-state synaptic vesicle protein composition from shared early endosomes.

SV2 Renders Primed Synaptic Vesicles Competent for Ca 2+ -Induced Exocytosis. Chang WP, Südhof TC. J Neurosci 2009;29(4):883–897. Synaptic vesicle protein 2 (SV2), one of the first synaptic vesicle proteins identified, is characterized by multiple transmembrane regions that exhibit homology to sugar transporters, and by a highly glycosylated intravesicular sequence. Deletion of SV2 causes postnatal lethality in mice, primarily because of fulminant epilepsy. At the cellular level, deletion of SV2 impairs neurotransmitter release, but its function is unknown, and even the exact point at which release is affected in SV2-deleted synapses remains unclear. Using electrophysiological approaches, we now examine at what step in exocytosis the deletion of SV2 impairs release. Our data demonstrate that deletion of SV2 produces a decrease in evoked synaptic responses without causing changes in mini frequency, mini amplitude, the readily releasable pool of vesicles, or the apparent Ca 2+ sensitivity of vesicle fusion. These findings indicate that a previously unidentified step may couple priming of synaptic vesicles to Ca 2+ triggering of fusion, and that SV2 acts in this step to render primed synaptic vesicles fully Ca 2+ responsive. To investigate the structural requirements for this function of SV2, we used rescue experiments. We demonstrate that conserved charged residues within the transmembrane regions and the intravesicular glycosylation of SV2 are required for its normal folding and trafficking. In contrast, the conserved putative synaptotagmin-binding sequence of SV2 is fully dispensable. Viewed together, these observations suggest that SV2 functions in a maturation step of primed vesicles that converts the vesicles into a Ca 2+ - and synaptotagmin-responsive state.

Accumulation of glutamate, the primary excitatory neurotransmitter in the mammalian central nervous system, into presynaptic synaptic vesicles (SVs) depends upon three vesicular glutamate transporters (VGLUTs). Since VGLUTs are driven by a proton electrochemical gradient across the SV membrane generated by vacuolar-type H+-ATPases (V-ATPases), the rate of glutamate transport into SVs, as well as the amount of glutamate in SVs at equilibrium, are influenced by activities of both VGLUTs and V-ATPase. Despite emerging evidence that suggests various factors influencing glutamate transport by VGLUTs in vitro, little has been reported in physiological or pathological contexts to date. Historically, this was partially due to a lack of appropriate methods to monitor glutamate loading into SVs in living synapses. Furthermore, whether or not glutamate refilling of SVs can be rate-limiting for synaptic transmission is not well understood, primarily due to a lack of knowledge concerning the time required for vesicle reuse and refilling during repetitive stimulation. In this review, we first introduce a unique electrophysiological method to monitor glutamate refilling by VGLUTs in a giant model synapse from the calyx of Held in rodent brainstem slices, and we discuss the advantages and limitations of the method. We then introduce the current understanding of factors that potentially alter the amount and rate of glutamate refilling of SVs in this synapse, and discuss open questions from physiological viewpoints.

At conventional synapses of the central nervous system (CNS), fast synaptic transmission is mediated by the release of neurotransmitters (NTs) upon Ca2+-triggered synaptic vesicle (SV) exocytosis. Upon exocytosis SV proteins have to be resorted and retrieved from the surface by compensatory endocytosis in order to replenish the pool of NT-filled SVs. For my thesis I used pH-switchable dyes, both genetically encoded as well as new exogenous ones, for optically analysing SV protein molecules necessary for fusion as well as their retrieval. Exocytosis is mediated by the assembly of low-energy SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) protein complexes formed by the coil-coiling of three SNARE proteins: Synaptosomal associated protein - 25 (SNAP-25), Syntaxin 1A (Syx1A), and Synaptobrevin 2 (Syb2). However, it is unknown how many SNARE complexes are minimally needed for SV priming and fusion at CNS synapses. To resolve this issue, single vesicle fusion events were optically measured in real time using the genetically encoded probe SynaptopHluorin (SpH), a pH-sensitive green fluorescent protein (GFP), pHluorin (pHl) fused to the luminal domain of the SV SNARE Syb2. Fluorescence responses upon fusion displayed a quantal distribution of SpH molecules into SVs. Quantitative single molecule experiments revealed that the quantal size corresponds to single SpH molecule fluorescence. Surprisingly, when overexpressed on a genetic null background, SpH could fully rescue evoked SV fusion. However, SVs expressing only one copy of SpH were unable to rapidly fuse upon stimulation. Taken together, the first part of the study demonstrates that two copies of SpH and hence two SNARE complexes are necessary and sufficient for SV fusion during fast synaptic transmission.In order to maintain a steady-state rate of synaptic transmission the fused SV constituents are retrieved for further rounds of use by a compensatory process of endocytosis. Although clathrin-mediated endocytosis (CME) is thought to be the predominant mechanism of SV recycling, it seems too slow to account for fast recycling. Therefore, it has been suggested that a pre-sorted and pre-assembled pool of SV proteins on the presynaptic membrane might support the first phase of CME. In the second part of this study the spatial and temporal dynamics of such a readily retrievable pool (RRetP) of SV proteins at the presynaptic membrane of hippocampal neurons was monitored using a novel probe. By applying CypHer 5E, a new cyanine dye-based pH-sensitive exogenous marker, coupled to antibodies against luminal domains of SV proteins, the preferential retrieval of native SV constituents from the RRetP upon exocytosis was demonstrated. The functional size and capacity of this pool was found to closely resemble that of the readily releasable pool (RRP) of docked and primed SVs, suggesting that the RRetP can sustain SV recycling during moderate synaptic activity. Thus, the second part of the thesis demonstrates that small central synapses can avoid SV depletion in response to mild stimulation by having a preassembled pool of ready-to-go SV constituents (RRetP), which efficiently supports compensatory endocytosis to a significant degree without relying on freshly exocytosed SV constituents.

Specific transport proteins mediate the packaging of neurotransmitters into secretory vesicles and consequently require targeting to the appropriate intracellular compartment. To identify residues in the neuron-specific vesicular monoamine transporter (VMAT2) responsible for endocytosis, we examined the effect of amino (NH2-) and carboxyl (COOH-)-terminal mutations on steady state distribution and internalization. Deletion of a critical COOH-terminal domain sequence (AKEEKMAIL) results in accumulation of VMAT2 at the plasma membrane and a 50% reduction in endocytosis. Site-directed mutagenesis shows that replacement of the isoleucine-leucine pair within this sequence by alanine-alanine alone reduces endocytosis by 50% relative to wild type VMAT2. Furthermore, the KEEKMAIL sequence functions as an internalization signal when transferred to the plasma membrane protein Tac, and the mutation of the isoleucine-leucine pair also abolishes internalization of this protein. The closely related vesicular acetylcholine transporter (VAChT) contains a similar di-leucine sequence within the cytoplasmic COOH-terminal domain that when mutated results in accumulation of VAChT at the plasma membrane. The VAChT di-leucine sequence also confers internalization when appended to two other proteins and in one of these chimeras, conversion of the di-leucine sequence to di-alanine reduces the internalization rate by 50%. Both VMAT2 and VAChT thus use leucine-based signals for efficient endocytosis and as such are the first synaptic vesicle proteins known to use this motif for trafficking.

Effect of diazoxide on regulation of vesicular and plasma membrane GABA transporter genes and proteins in hippocampus of rats subjected to picrotoxin-induced kindling.

The regulated release of neurotransmitters at synapses is mediated by the fusion of neurotransmitter-filled synaptic vesicles with the plasma membrane. Continuous synaptic activity relies on the constant recycling of synaptic vesicle proteins into newly formed synaptic vesicles. At least two different mechanisms are presumed to mediate synaptic vesicle biogenesis at the synapse as follows: direct retrieval of synaptic vesicle proteins and lipids from the plasma membrane, and indirect passage of synaptic vesicle proteins through an endosomal intermediate. We have identified a vesicle population with the characteristics of a primary endocytic vesicle responsible for the recycling of synaptic vesicle proteins through the indirect pathway. We find that synaptic vesicle proteins colocalize in this vesicle with a variety of proteins known to recycle from the plasma membrane through the endocytic pathway, including three different glucose transporters, GLUT1, GLUT3, and GLUT4, and the transferrin receptor. These vesicles differ from "classical" synaptic vesicles in their size and their generic protein content, indicating that they do not discriminate between synaptic vesicle-specific proteins and other recycling proteins. We propose that these vesicles deliver synaptic vesicle proteins that have escaped internalization by the direct pathway to endosomes, where they are sorted from other recycling proteins and packaged into synaptic vesicles.

Botulinum neurotoxins (BoNTs) and tetanus neurotoxin (TeNT) are the most toxic proteins for humans. While BoNTs cause flaccid paralysis, TeNT causes spastic paralysis. Characterized BoNT serotypes enter neurons upon binding dual receptors, a ganglioside and a neuron-specific protein, either synaptic vesicle protein 2 (SV2) or synaptotagmin, while TeNT enters upon binding gangliosides as dual receptors. Recently, TeNT was reported to enter central nervous system (CNS) neurons upon synaptic vesicle cycling that was mediated by the direct binding to SV2, implying that TeNT and BoNT utilize common mechanisms to enter CNS neurons. This prompted an assessment of TeNT entry into CNS neurons, using the prototypic BoNT serotype A as a reference for SV2-mediated entry into synaptic vesicles, analyzing the heavy-chain receptor binding domain (HCR) of each toxin. Synaptic vesicle cycling stimulated the entry of HCR/A into neurons, while HCR/T entered neurons with similar levels of efficiency in depolarized and nondepolarized neurons. ImageJ analysis identified two populations of cell-associated HCR/T in synaptic vesicle cycling neurons, a major population which segregated from HCR/A and a minor population which colocalized with HCR/A. HCR/T did not inhibit HCR/A entry into neurons in competition experiments and did not bind SV2, the protein receptor for BoNT/A. Intoxication experiments showed that TeNT efficiently cleaved VAMP2 in depolarized neurons and neurons blocked for synaptic vesicle cycling. These experiments demonstrate that TeNT enters neurons by two pathways, one independent of stimulated synaptic vesicle cycling and one by synaptic vesicles independent of SV2, showing that TeNT and BoNT/A enter neurons by unique mechanisms.

The regulated release of neurotransmitter occurs via the fusion of synaptic vesicles (SVs) at specialized regions of the presynaptic membrane called active zones (AZs). These regions are defined by a cytoskeletal matrix assembled at AZs (CAZ), which functions to direct SVs toward docking and fusion sites and supports their maturation into the readily releasable pool. In addition, CAZ proteins localize voltage-gated Ca(2+) channels at SV release sites, bringing the fusion machinery in close proximity to the calcium source. Proteins of the CAZ therefore ensure that vesicle fusion is temporally and spatially organized, allowing for the precise and reliable release of neurotransmitter. Importantly, AZs are highly dynamic structures, supporting presynaptic remodeling, changes in neurotransmitter release efficacy, and thus presynaptic forms of plasticity. In this review, we discuss recent advances in the study of active zones, highlighting how the CAZ molecularly defines sites of neurotransmitter release, endocytic zones, and the integrity of synapses.

We studied the histogenesis of the lizard visual system (E30 to adulthood) by using a selection of immunohistochemical markers that had proved relevant for other vertebrates. By E30, the Pax6(+) pseudostratified retinal epithelium shows few newborn retinal ganglion cells (RGCs) in the centrodorsal region expressing neuron- and synaptic-specific markers such as betaIII-tubulin (Tuj1), synaptic vesicle protein-2 (SV2), and vesicular glutamate transporter-1 (VGLUT1). Concurrently, pioneer RGC axons run among the Pax2(+) astroglia in the optic nerve and reach the superficial optic tectum. Between E30 and E35, the optic chiasm and optic tract remain acellular, but the latter contains radial processes with subpial endfeet expressing vimentin (Vim). From E35, neuron- and synaptic-specific stainings spread in the retina and optic tectum, whereas retinal Pax6, and Tuj1/SV2 in RGC axons decrease. Müller glia and abundant optic nerve glia express a variety of glia-specific markers until adulthood. Subpopulations of optic nerve glia are also VGLUT1(+) and cluster differentiation-44 (CD44)-positive but cytokeratin-negative, unlike the case in other regeneration-competent species. Specifically, coexpression of CD44/Vim and glutamine synthetase (GS)/VGLUT1 reflects glial specialization, insofar as most CD44(+) glia are GS(-). In the adult optic tract and tectum, radial glia and free astroglia coexist. The latter show different immunocharacterization (Pax2(-)/CD44(-) /Vim(-)) compared with that in the optic nerve. We conclude that upregulation of Tuj1 and SV2 is required for axonal outgrowth and search for appropriate targets, whereas Pax2(+) optic nerve astroglia and Vim(+) radial glia may aid in early axonal guidance. Spontaneous axonal regrowth seems to succeed despite the heterogeneous mammalian-like glial environment in the lizard optic nerve.

Calcium-Dependent Synaptic Vesicle Trafficking Underlies Indefatigable Release at the Hair Cell Afferent Fiber Synapse

We describe a novel synaptic vesicle protein called SVOP that is distantly related to the synaptic vesicle proteins SV2A, SV2B, and SV2C (20-22% sequence identity). Both SVOP and SV2 contain 12 transmembrane regions. However, SV2 is highly glycosylated, whereas SVOP is not. Databank searches revealed that closely related homologs of SVOP are present in Caenorhabditis elegans and Drosophila (48% sequence identity), suggesting that SVOP is evolutionarily ancient. In contrast, no invertebrate orthologs of SV2 were detected. The sequences of SVOP and SV2 exhibit homology with transport proteins, in particular with mammalian organic cation and anion transporters. SVOP and SV2 are more distantly related to eukaryotic and bacterial phosphate, sugar, and organic acid transporters. SVOP is expressed at detectable levels only in brain and endocrine cells where it is primarily localized to synaptic vesicles and microvesicles. SVOP is present in all brain regions, with particularly high levels in large pyramidal neurons of the cerebral cortex. Immunocytochemical staining of adjacent rat brain sections for SVOP and SV2 demonstrated that SVOP and SV2 are probably coexpressed in most neurons. Although the functions of SV2 and SVOP remain obscure, the evolutionary conservation of SVOP, its hydrophobic nature, and its homology to transporters strongly support a role in the uptake of a novel, as yet unidentified component of synaptic vesicles. Thus synaptic vesicles contain two classes of abundant proteins with 12 transmembrane regions that are related to transporters, nonglycosylated SVOP and highly glycosylated SV2, suggesting that the transport functions of synaptic vesicles may be more complex than currently envisioned.

gamma-Aminobutyric acid (GABA) and glycine are stored into synaptic vesicles by a recently identified vesicular inhibitory amino acid transporter [VIAAT, also called vesicular GABA transporter (VGAT)]. Immunoblotting analysis revealed that rat brain VIAAT migrated as a doublet during sodium dodecyl sulfate-polyacrylamide gel electrophoresis, with a predominant slower band in all areas examined except olfactory bulb and retina. The slower band corresponded to a phosphorylated form of VIAAT as it was converted to the faster one by treating brain homogenates with alkaline phosphatase or with an endogenous phosphatase identified as type 2A protein-serine/threonine phosphatase using okadaic acid. In contrast, the recombinant protein expressed in COS-7 or PC12 cells co-migrated with the faster band of the brain doublet and was insensitive to alkaline phosphatase. To investigate the influence of VIAAT phosphorylation on vesicular neurotransmitter loading, purified synaptic vesicles were treated with alkaline phosphatase and assayed for amino acid uptake. However, neither GABA nor glycine uptake was affected by VIAAT phosphorylation. These results indicate that VIAAT is constitutively phosphorylated on cytosolic serine or threonine residues in most, but not all, regions of the rat brain. This phosphorylation does not regulate the vesicular loading of GABA or glycine, suggesting that it is involved at other stages of the synaptic vesicle life cycle.