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Significance Mammalian central synapses of diverse functions contribute to highly complex brain organization, but the molecular basis of synaptic diversity remains open. This is because current synapse proteomics are restricted to the “average” composition of abundant synaptic proteins. Here, we demonstrate a subcellular proteomic workflow that can identify and quantify the deep proteome of synaptic vesicles, including previously missing proteins present in a small percentage of central synapses. This synaptic vesicle proteome revealed many proteins of physiological and pathological relevance, particularly in the low-abundance range, thus providing a resource for future investigations on diversified synaptic functions and neuronal dysfunctions., Current proteomic studies clarified canonical synaptic proteins that are common to many types of synapses. However, proteins of diversified functions in a subset of synapses are largely hidden because of their low abundance or structural similarities to abundant proteins. To overcome this limitation, we have developed an “ultra-definition” (UD) subcellular proteomic workflow. Using purified synaptic vesicle (SV) fraction from rat brain, we identified 1,466 proteins, three times more than reported previously. This refined proteome includes all canonical SV proteins, as well as numerous proteins of low abundance, many of which were hitherto undetected. Comparison of UD quantifications between SV and synaptosomal fractions has enabled us to distinguish SV-resident proteins from potential SV-visitor proteins. We found 134 SV residents, of which 86 are present in an average copy number per SV of less than one, including vesicular transporters of nonubiquitous neurotransmitters in the brain. We provide a fully annotated resource of all categorized SV-resident and potential SV-visitor proteins, which can be utilized to drive novel functional studies, as we characterized here Aak1 as a regulator of synaptic transmission. Moreover, proteins in the SV fraction are associated with more than 200 distinct brain diseases. Remarkably, a majority of these proteins was found in the low-abundance proteome range, highlighting its pathological significance. Our deep SV proteome will provide a fundamental resource for a variety of future investigations on the function of synapses in health and disease.

Newly generated synaptic vesicles (SVs) are re-acidified by the activity of the vacuolar-type H

Neurotransmission is based on the exocytic fusion of synaptic vesicles (SVs) followed by endocytic membrane retrieval and the reformation of SVs. Conflicting models have been proposed regarding the mechanisms of SV endocytosis, most notably clathrin/ AP-2-mediated endocytosis and clathrin-independent ultrafast endocytosis. Partitioning between these pathways has been suggested to be controlled by temperature and stimulus paradigm. We report on the comprehensive survey of six major SV proteins to show that SV endocytosis in hippocampal neurons at physiological temperature occurs independent of clathrin while the endocytic retrieval of a subset of SV proteins including the vesicular transporters for glutamate and GABA depend on sorting by the clathrin adaptor AP-2. Our findings highlight a clathrin-independent role of the clathrin adaptor AP-2 in the endocytic retrieval of select SV cargos from the presynaptic cell surface and suggest a unified model for the endocytosis of SV membranes at mammalian central synapses.

Replenishment of readily releasable synaptic vesicles (SVs) with vesicles in the recycling pool is important for sustained transmitter release during repetitive stimulation. Kinetics of replenishment and available pool size define synaptic performance. However, whether all SVs in the recycling pool are recruited for release with equal probability is unknown. Here, using comprehensive optical imaging for various presynaptic endosomal SNARE proteins in cultured hippocampal neurons, we demonstrate that part of the recycling pool bearing the endosomal Q–SNARE Syntaxin 7 (Stx7) is preferentially mobilized for release during high–frequency repetitive stimulation. Recruitment of the SV pool marked with the Stx7–reporter requires high intra–terminal Ca2+ concentrations and actin polymerization. Furthermore, disruption of Stx7 function by overexpressing the N–terminal domain selectively abolished this pool. Thus, our data indicate that endosomal membrane fusion involving Stx7 is essential for adaptation of synapses to respond high-frequency repetitive stimulation.

Ca2+ transport into synaptic vesicles (SVs) at the presynaptic terminals has been proposed to be an important process for regulating presynaptic [Ca2+] during stimulation as well as at rest. However, the molecular identity of the transport system remains elusive. Previous studies have demonstrated that isolated SVs exhibit two distinct Ca2+ transport systems depending on extra-vesicular (cytosolic) pH; one is mediated by a high affinity Ca2+ transporter which is active at neutral pH and the other is mediated by a low affinity Ca2+/H+ antiporter which is maximally active at alkaline pH of 8.5. In addition, synaptic vesicle glycoprotein 2 s (SV2s), a major SV component, have been proposed to contribute to Ca2+ clearance from the presynaptic cytoplasm. Here, we show that at physiological pH, the plasma membrane Ca2+ ATPases (PMCAs) are responsible for both the Ca2+/H+ exchange activity and Ca2+ uptake into SVs. The Ca2+/H+ exchange activity monitored by acidification assay exhibited high affinity for Ca2+ (Km ~ 400 nM) and characteristic divalent cation selectivity for the PMCAs. Both activities were remarkably reduced by PMCA blockers, but not by a blocker of the ATPase that transfers Ca2+ from the cytosol to the lumen of sarcoplasmic endoplasmic reticulum (SERCA) at physiological pH. Furthermore, we rule out the contribution of SV2s, putative Ca2+ transporters on SVs, since both Ca2+/H+ exchange activity and Ca2+ transport were unaffected in isolated vesicles derived from SV2-deficient brains. Finally, using a PMCA1-pHluorin construct that enabled us to monitor cellular distribution and recycling properties in living neurons, we demonstrated that PMCA1-pHluorin localized to intracellular acidic compartments and recycled at presynaptic terminals in an activity-dependent manner. Collectively, our results imply that vesicular PMCAs may play pivotal roles in both presynaptic Ca2+ homeostasis and the modulation of H+ gradient in SVs.

GABA acts as the major inhibitory neurotransmitter in the mammalian brain, shaping neuronal and circuit activity. For sustained synaptic transmission, synaptic vesicles (SVs) are required to be recycled and refilled with neurotransmitters using an H(+) electrochemical gradient. However, neither the mechanism underlying vesicular GABA uptake nor the kinetics of GABA loading in living neurons have been fully elucidated. To characterize the process of GABA uptake into SVs in functional synapses, we monitored luminal pH of GABAergic SVs separately from that of excitatory glutamatergic SVs in cultured hippocampal neurons. By using a pH sensor optimal for the SV lumen, we found that GABAergic SVs exhibited an unexpectedly higher resting pH (∼6.4) than glutamatergic SVs (pH ∼5.8). Moreover, unlike glutamatergic SVs, GABAergic SVs displayed unique pH dynamics after endocytosis that involved initial overacidification and subsequent alkalization that restored their resting pH. GABAergic SVs that lacked the vesicular GABA transporter (VGAT) did not show the pH overshoot and acidified further to ∼6.0. Comparison of luminal pH dynamics in the presence or absence of VGAT showed that VGAT operates as a GABA/H(+) exchanger, which is continuously required to offset GABA leakage. Furthermore, the kinetics of GABA transport was slower (τ > 20 s at physiological temperature) than that of glutamate uptake and may exceed the time required for reuse of exocytosed SVs, allowing reuse of incompletely filled vesicles in the presence of high demand for inhibitory transmission.

Summary Recycling of synaptic vesicles (SVs) at presynaptic terminals is required for sustained neurotransmitter release. Although SV endocytosis is a rate-limiting step for synaptic transmission, it is unclear whether the rate of the subsequent SV refilling with neurotransmitter also influences synaptic transmission. By analyzing vesicular glutamate transporter 1 (VGLUT1)-deficient calyx of Held synapses, in which both VGLUT1 and VGLUT2 are co-expressed in wild-type situation, we found that VGLUT1 loss causes a drastic reduction in SV refilling rate down to ∼25% of wild-type values, with only subtle changes in basic synaptic parameters. Strikingly, VGLUT1-deficient synapses exhibited abnormal synaptic failures within a few seconds during high-frequency repetitive firing, which was recapitulated by manipulating presynaptic Cl− concentrations to retard SV refilling. Our data show that the speed of SV refilling can be rate limiting for synaptic transmission under certain conditions that entail reduced VGLUT levels during development as well as various neuropathological processes.

Targeting gene expression to a particular subset of neurons helps study the cellular function of the nervous system. Although neuron-specific promoters, such as the synapsin I promoter and the α-CaMKII promoter, are known to exhibit selectivity for excitatory glutamatergic neurons in vivo, the cell type-specificity of these promoters has not been thoroughly tested in culture preparations. Here, by using hippocampal culture preparation from the VGAT-Venus transgenic mice, we examined the ability of five putative promoter sequences of glutamatergic-selective markers including synapsin I, α-CaMKII, the vesicular glutamate transporter 1 (VGLUT1), Dock10 and Prox1. Among these, a genomic fragment containing a 2.1 kb segment upstream of the translation start site (TSS) of the VGLUT1 implemented in a lentiviral vector with the Tet-Off inducible system achieved the highest preferential gene expression in glutamatergic neurons. Analysis of various lengths of the VGLUT1 promoter regions identified a segment between −2.1 kb and −1.4 kb from the TSS as a responsible element for the glutamatergic selectivity. Consistently, expression of channelrhodopsin under this promoter sequence allowed for selective light-evoked activation of excitatory neurons. Thus, the lentiviral system carrying the VGLUT1 promoter fragment can be used to effectively target exogenous gene expression to excitatory glutamatergic neurons in cultures.

Since the discovery of a putative Cl(-) channel on synaptic vesicle (SV) membranes, attempts to establish its molecular identity have proven surprisingly difficult. Recent evidence has emerged to support the idea that the vesicular glutamate transporter (VGLUT), whose main function is to concentrate the excitatory neurotransmitter glutamate into SVs, can also act as the Cl(-) permeation pathway. Here, I summarize studies investigating the putative Cl(-) channel on SVs and discuss the possible roles of VGLUT-mediated Cl(-) transport on glutamate loading.

SummaryRett syndrome (RTT) is a neurodevelopmental disorder caused by MECP2 mutations. Although emerging evidence suggests that MeCP2 deficiency is associated with dysregulation of mechanistic target of rapamycin (mTOR), which functions as a hub for various signaling pathways, the mechanism underlying this association and the molecular pathophysiology of RTT remain elusive. We show here that MeCP2 promotes the posttranscriptional processing of particular microRNAs (miRNAs) as a component of the microprocessor Drosha complex. Among the MeCP2-regulated miRNAs, we found that miR-199a positively controls mTOR signaling by targeting inhibitors for mTOR signaling. miR-199a and its targets have opposite effects on mTOR activity, ameliorating and inducing RTT neuronal phenotypes, respectively. Furthermore, genetic deletion of miR-199a-2 led to a reduction of mTOR activity in the brain and recapitulated numerous RTT phenotypes in mice. Together, these findings establish miR-199a as a critical downstream target of MeCP2 in RTT pathogenesis by linking MeCP2 with mTOR signaling.

During synaptic vesicle (SV) recycling, the vacuolar-type H+ATPase creates a proton electrochemical gradient (ΔμH+) that drives neurotransmitter loading into SVs. Given the low estimates of free luminal protons, it has been envisioned that the influx of a limited number of protons suffices to establish ΔμH+. Consistent with this, the time constant of SV re-acidification was reported to be m/pH), corresponding to an accumulation of ∼1200 protons during re-acidification. Together, our results uncover hitherto unrecognized robust proton influx and storage in SVs that can restrict the rate of neurotransmitter refilling.

Efficient retrieval of the synaptic vesicle (SV) membrane from the presynaptic plasma membrane, a process called endocytosis, is crucial for the fidelity of neurotransmission, particularly during sustained neural activity. Although multiple modes of endocytosis have been identified, it is clear that the efficient retrieval of the major SV cargos into newly formed SVs during any of these modes is fundamental for synaptic transmission. It is currently believed that SVs are eventually reformed via a clathrin-dependent pathway. Various adaptor proteins recognize SV cargos and link them to clathrin, ensuring the efficient retrieval of the cargos into newly formed SVs. Here, we summarize our current knowledge of the molecular signatures within individual SV cargos that underlie efficient retrieval into SV membranes, as well as discuss possible contributions of the mechanisms under physiological conditions.

The quantal hypothesis for the release of neurotransmitters at the chemical synapse has gained wide acceptance since it was first worked out at the motor endplate in frog skeletal muscle in the 1950s. Considering the morphological identification of synaptic vesicles at the nerve terminals that appeared to be homogeneous in size, the hypothesis proposed that signal transduction at synapses is mediated by the release of neurotransmitters packed in synaptic vesicles that are individually uniform in size; the amount of transmitter in a synaptic vesicle is called a quantum. Although quantal size – the amplitude of the postsynaptic response elicited by the release of neurotransmitters from a single vesicle – clearly depends on the number and sensitivity of the postsynaptic receptors, accumulating evidence has also indicated that the amount of neurotransmitters stored in synaptic vesicles can be altered by various presynaptic factors. Here, I provide an overview of the concepts and underlying presynaptic molecular underpinnings that may regulate quantal size.

Uptake of glutamate into synaptic vesicles is mediated by vesicular glutamate transporters (VGLUTs). Although glutamate uptake has been shown to depend critically on Cl(-), the precise contribution of this ion to the transport process is unclear. We found that VGLUT1, and not ClC-3 as proposed previously, represents the major Cl(-) permeation pathway in synaptic vesicles. Using reconstituted VGLUT1, we found that the biphasic dependence of glutamate transport on extravesicular Cl(-) is a result of the permeation of this anion through VGLUT1 itself. Moreover, we observed that high luminal Cl(-) concentrations markedly enhanced loading of glutamate by facilitation of membrane potential-driven uptake and discovered a hitherto unrecognized transport mode of VGLUT1. Because a steep Cl(-) gradient across the synaptic vesicle membrane exists in endocytosed synaptic vesicles, our results imply that the transport velocity and the final glutamate content are highly influenced, if not determined, by the extracellular Cl(-) concentration.

Neurotransmitter uptake into synaptic vesicles is mediated by vesicular neurotransmitter transporters. Although these transporters belong to different families, they all are thought to share a common overall topology with an even number of transmembrane domains. Using epitope-specific antibodies and mass spectrometry we show that the vesicular GABA transporter (VGAT) possesses an uneven number of transmembrane domains, with the N terminus facing the cytoplasm and the C terminus residing in the synaptic vesicle lumen. Antibodies recognizing the C terminus of VGAT (anti-VGAT-C) selectively label GABAergic nerve terminals of live cultured hippocampal and striatal neurons as confirmed by immunocytochemistry and patch-clamp electrophysiology. Injection of fluorochromated anti-VGAT-C into the hippocampus of mice results in specific labeling of GABAergic synapsesin vivo. Overall, our data open the possibility of studying novel GABA release sites, characterizing inhibitory vesicle trafficking, and establishing their contribution to inhibitory neurotransmission at identified GABAergic synapses.

Vesicular glutamate transporters (VGLUTs) are essential to glutamatergic synapses and determine the glutamatergic phenotype of neurones. The three known VGLUT isoforms display nearly identical uptake characteristics, but the associated expression domains in the adult rodent brain are largely segregated. Indeed, indirect evidence obtained in young VGLUT1-deficient mice indicated that in cells that co-express VGLUT1 and VGLUT2, the transporters may be targeted to different synaptic vesicles, which may populate different types of synapses formed by the same neurone. Direct evidence for a systematic segregation of VGLUT1 and VGLUT2 to distinct synapses and vesicles is lacking, and the mechanisms that may convey this segregation are not known. We show here that VGLUT1 and VGLUT2 are co-localized in many layers of the young hippocampus. Strikingly, VGLUT2 co-localizes with VGLUT1 in the mossy fibers at early stages. Furthermore, we show that a fraction of VGLUT1 and VGLUT2 is carried by the same vesicles at these stages. Hence, hippocampal neurones co-expressing VGLUT1 and VGLUT2 do not appear to sort them to separate vesicle pools. As the number of transporter molecules per vesicle affects quantal size, the developmental window where VGLUT1 and VGLUT2 are co-expressed may allow for greater plasticity in the control of quantal release.

Membrane traffic in eukaryotic cells involves transport of vesicles that bud from a donor compartment and fuse with an acceptor compartment. Common principles of budding and fusion have emerged, and many of the proteins involved in these events are now known. However, a detailed picture of an entire traffickingorganelle is not yet available. Using synaptic vesicles as a model, we have now determined the protein and lipid composition; measured vesicle size, density, and mass; calculated the average protein and lipid mass per vesicle; and determined the copy number of more than a dozen major constituents. A model has been constructed that integrates all quantitative data and includes structural models of abundant proteins. Synaptic vesicles are dominated by proteins, possess a surprising diversity of trafficking proteins, and, with the exception of the V-ATPase that is present in only one to two copies, contain numerous copies of proteins essential for membrane traffic and neurotransmitter uptake. ispartof: Cell vol:127 issue:4 pages:671-673 ispartof: location:United States status: published

Glutamate is the principal excitatory neurotransmitter in the mammalian central nervous system (CNS). Glutamate is first synthesized in the cytoplasm of presynaptic terminals before being loaded into synaptic vesicles, which fuse with the plasma membrane, releasing their contents, in response to neuronal activity. The important process of synaptic vesicle loading is mediated by a transport protein, collectively known as vesicular glutamate transporter (VGLUT). Controlling the activity of these transporters could potentially modulate the efficacy of glutamatergic neurotransmission. In recent years, three isoforms of mammalian VGLUTs have been cloned and molecularly characterized in detail. Probing these three VGLUTs has been proven to be the most reliable way of visualizing sites of glutamate release in the mammalian CNS. Immunohistochemical studies on VGLUTs suggest that glutamatergic neurons are categorized into subgroups depending on which VGLUT isoform they contain. Recent studies on VGLUT1-deficient mice have led various models to be postulated concerning the possible roles of VGLUTs in synaptic physiology, such as presynaptic regulation of quantal size and activity-dependent short-term plasticity.

The SNARE proteins are essential components of the intracellular fusion machinery. It is thought that they form a tight four-helix complex between membranes, in effect initiating fusion. Most SNAREs contain a single coiled-coil region, referred to as the SNARE motif, directly adjacent to a single transmembrane domain. The neuronal SNARE SNAP-25 defines a subfamily of SNARE proteins with two SNARE helices connected by a longer linker, comprising also the proteins SNAP-23 and SNAP-29. We now report the initial characterization of a novel vertebrate homologue termed SNAP-47. Northern blot and immunoblot analysis revealed ubiquitous tissue distribution, with particularly high levels in nervous tissue. In neurons, SNAP-47 shows a widespread distribution on intracellular membranes and is also enriched in synaptic vesicle fractions. In vitro, SNAP-47 substituted for SNAP-25 in SNARE complex formation with the neuronal SNAREs syntaxin 1a and synaptobrevin 2, and it also substituted for SNAP-25 in proteoliposome fusion. However, neither complex assembly nor fusion was as efficient as with SNAP-25. ispartof: Journal of Biological Chemistry vol:281 issue:25 pages:17076-17083 ispartof: location:United States status: published

Fast chemical neurotransmission at the synapse is mediated by the release of neurotransmitters from synaptic vesicles (SVs) by exocytosis. In the mammalian central nervous system, the majority of neurons utilize amino acids such as glutamate, γ-aminobutyric acid (GABA), and glycine. Glutamate is the major excitatory neurotransmitter, whereas GABA and glycine are inhibitory. These amino acids are present at relatively high levels in the cytoplasm of presynaptic terminals and are accumulated into SVs for their exocytotic release. Over the past several decades, this essential process has been biochemically characterized and proteins responsible for neurotransmitter loading have been molecularly identified. Analysis of knockout animals has elucidated physiological significance of this process and moreover has deepened our understanding of glutamatergic and GABAergic neural circuits. However, the precise mechanism of the transport system remains largely unknown. In this chapter, I overview advances in the vesicular loading process and discuss some controversial concepts that may have important consequences for synaptic transmission.

SNARE proteins (soluble NSF-attachment protein receptors) are thought to be central components of the exocytotic mechanism in neurosecretory cells, but their precise function remained unclear. Here, we show that each of the vesicle-associated SNARE proteins (v-SNARE) of a chromaffin granule, synaptobrevin II or cellubrevin, is sufficient to support Ca(2+)-dependent exocytosis and to establish a pool of primed, readily releasable vesicles. In the absence of both proteins, secretion is abolished, without affecting biogenesis or docking of granules indicating that v-SNAREs are absolutely required for granule exocytosis. We find that synaptobrevin II and cellubrevin differentially control the pool of readily releasable vesicles and show that the v-SNARE's amino terminus regulates the vesicle's primed state. We demonstrate that dynamics of fusion pore dilation are regulated by v-SNAREs, indicating their action throughout exocytosis from priming to fusion of vesicles.

Classical neurotransmitters, including monoamines, acetylcholine, glutamate, GABA, and glycine, are loaded into synaptic vesicles by means of specific transporters. Vesicular monoamine transporters are under negative regulation by α subunits of trimeric G-proteins, including Gαo2and Gαq. Furthermore, glutamate uptake, mediated by vesicular glutamate transporters (VGLUTs), is decreased by the nonhydrolysable GTP-analog guanylylimidodiphosphate. Using mutant mice lacking various Gα subunits, including Gαo1, Gαo2, Gαq, and Gα11, and a Gαo2-specific monoclonal antibody, we now show that VGLUTs are exclusively regulated by Gαo2. G-protein activation does not affect the electrochemical proton gradient serving as driving force for neurotransmitter uptake; rather, Gαo2exerts its action by specifically affecting the chloride dependence of VGLUTs. All VGLUTs show maximal activity at ∼5 mmchloride. Activated Gαo2shifts this maximum to lower chloride concentrations. In contrast, glutamate uptake by vesicles isolated from Gαo2-/-mice have completely lost chloride activation. Thus, Gαo2acts on a putative regulatory chloride binding domain that appears to modulate transport activity of vesicular glutamate transporters.

Quantal neurotransmitter release at excitatory synapses depends on glutamate import into synaptic vesicles by vesicular glutamate transporters (VGLUTs). Of the three known transporters, VGLUT1 and VGLUT2 are expressed prominently in the adult brain, but during the first two weeks of postnatal development, VGLUT2 expression predominates. Targeted deletion of VGLUT1 in mice causes lethality in the third postnatal week. Glutamatergic neurotransmission is drastically reduced in neurons from VGLUT1-deficient mice, with a specific reduction in quantal size. The remaining activity correlates with the expression of VGLUT2. This reduction in glutamatergic neurotransmission can be rescued and enhanced with overexpression of VGLUT1. These results show that the expression level of VGLUTs determines the amount of glutamate that is loaded into vesicles and released and thereby regulates the efficacy of neurotransmission.

Hippocampal interneurons comprise a heterogeneous group of locally acting GABAergic neurons. In addition to their variability in cotransmitter content and receptor profile, they express a variety of potassium channels that specify their individual properties. Here we describe a new type of large GABA-containing neuron in rodent hippocampus that is characterized by an axonal sorting of the potassium channel Kir3.3. The parent cell bodies of the Kir3.3-positive axons are located in CA3, as assessed by primary cultures derived from hippocampal subareas. At postnatal day 14 these neurons appear at the border between stratum oriens and stratum pyramidale of CA3, from where their axons pass through stratum pyramidale to join the mossy fiber tract. In adult hippocampus, high levels of Kir3.3 channel protein exist in axons that run with the mossy fiber tract. Kir3.3 and the vesicular GABA transporter could be identified in subpopulations of large synaptic terminals that probably derive from Kir3.3 neurons. Axonal sorting of Kir3.3 appears to be typical of a group of large inhibitory neurons, including Purkinje cells and a novel type of interneuron in CA3. Kir3.3 neurons might modulate the activity of CA3 circuitries and consequently memory processing in the hippocampus.

Changes in the vesicular transmitter content modulate synaptic strength and may contribute to synaptic plasticity. Several transporters mediating transmitter uptake into small synaptic vesicles (SSVs) have been identified but their regulation is largely unknown. Here we show by quantitative immunoelectron microscopy that the heterotrimeric G-protein subunits Galphao(2), Galpha(q/11), Gbeta(2), and Ggamma(7) are associated with vesicle-containing areas in terminals of cerebellar parallel fibers. These terminals also contain the vesicular glutamate transporter 1 (VGLUT1). In contrast, SSVs of climbing fiber terminals that contain VGLUT2 express one of the Gbeta-subunits Gbeta(1), Gbeta(3), or Gbeta(4), Ggamma(7), and one Galpha-subunit, probably Galphao(2). Glutamate uptake into cerebellar SSVs was inhibited by more than 50% by GMppNp, an activator of G proteins. Thus, vesicle populations with different subtypes of vesicular glutamate transporters contain functional G proteins with distinct subunit profiles. Heterotrimeric G proteins may play an important role in the control of vesicular filling.

In humans, mutations in the alpha-synuclein gene or exposure to the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) produce Parkinson's disease with loss of dopaminergic neurons and depletion of nigrostriatal dopamine. alpha-Synuclein is a vertebrate-specific component of presynaptic nerve terminals that may function in modulating synaptic transmission. To test whether MPTP toxicity involves alpha-synuclein, we generated alpha-synuclein-deficient mice by homologous recombination, and analyzed the effect of deleting alpha-synuclein on MPTP toxicity using these knockout mice. In addition, we examined commercially available mice that contain a spontaneous loss of the alpha-synuclein gene. As described previously, deletion of alpha-synuclein had no significant effects on brain structure or composition. In particular, the levels of synaptic proteins were not altered, and the concentrations of dopamine, dopamine metabolites, and dopaminergic proteins were unchanged. Upon acute MPTP challenge, alpha-synuclein knockout mice were partly protected from chronic depletion of nigrostriatal dopamine when compared with littermates of the same genetic background, whereas mice carrying the spontaneous deletion of the alpha-synuclein gene exhibited no protection. Furthermore, alpha-synuclein knockout mice but not the mice with the alpha-synuclein gene deletion were slightly more sensitive to methamphetamine than littermate control mice. These results demonstrate that alpha-synuclein is not obligatorily coupled to MPTP sensitivity, but can influence MPTP toxicity on some genetic backgrounds, and illustrate the need for extensive controls in studies aimed at describing the effects of mouse knockouts on MPTP sensitivity.

Glutamate is the major excitatory neurotransmitter in the mammalian CNS. It is loaded into synaptic vesicles by a proton gradient-dependent uptake system and is released by exocytosis upon stimulation. Recently, two mammalian isoforms of a vesicular glutamate transporter, VGLUT1 and VGLUT2, have been identified, the expression of which enables quantal release of glutamate from glutamatergic neurons. Here, we report a novel isoform of a human vesicular glutamate transporter (hVGLUT3). The predicted amino acid sequence of hVGLUT3 shows 72% identity to both hVGLUT1 and hVGLUT2. hVGLUT3 functions as a vesicular glutamate transporter with similar properties to the other isoforms when it is heterologously expressed in a neuroendocrine cell line. Although mammalian VGLUT1 and VGLUT2 exhibit a complementary expression pattern covering all glutamatergic pathways in the CNS, expression of hVGLUT3 overlaps with them in some brain areas, suggesting molecular diversity that may account for physiological heterogeneity in glutamatergic synapses.

Glutamate is the major excitatory neurotransmitter in the mammalian central nervous system. Synaptic vesicles are loaded with neurotransmitter by means of specific vesicular transporters. Here we show that expression of BNPI, a vesicle-bound transporter associated with sodium-dependent phosphate transport, results in glutamate uptake by intracellular vesicles. Substrate specificity and energy dependence are very similar to glutamate uptake by synaptic vesicles. Stimulation of exocytosis--fusion of the vesicles with the cell membrane and release of their contents--resulted in quantal release of glutamate from BNPI-expressing cells. Furthermore, we expressed BNPI in neurons containing GABA (gamma-aminobutyric acid) and maintained them as cultures of single, isolated neurons that form synapses to themselves. After stimulation of these neurons, a component of the postsynaptic current is mediated by glutamate as it is blocked by a combination of the glutamate receptor antagonists, but is insensitive to a GABA(A) receptor antagonist. We conclude that BNPI functions as vesicular glutamate transporter and that expression of BNPI suffices to define a glutamatergic phenotype in neurons.

Background: The vesicular GABA transporter (VGAT) loads GABA and glycine from the neuronal cytoplasm into synaptic vesicles. To address functional importance of VGAT during embryonic development, we generated global VGAT knockout mice and analyzed them., Results: VGAT knockouts at embryonic day (E) 18.5 exhibited substantial increases in overall GABA and glycine, but not glutamate, contents in the forebrain. Electrophysiological recordings from E17.5-18.5 spinal cord motoneurons demonstrated that VGAT knockouts presented no spontaneous inhibitory postsynaptic currents mediated by GABA and glycine. Histological examination of E18.5 knockout fetuses revealed reductions in the trapezius muscle, hepatic congestion and little alveolar spaces in the lung, indicating that the development of skeletal muscle, liver and lung in these mice was severely affected., Conclusion: VGAT is fundamental for the GABA- and/or glycine-mediated transmission that supports embryonic development. VGAT knockout mice will be useful for further investigating the roles of VGAT in normal physiology and pathophysiologic processes.

Using PC12 cells that express transfected human growth hormone (hGH) as a secreted reporter protein, we have searched for Rab proteins that function in exocytosis. Among the Rab proteins tested, we found that besides the previously described Rab3 proteins, only members of the Rab11 family (Rab11a, 11b, and 25) impaired Ca2+-induced exocytosis. Rab11b, which is enriched in brain, had the strongest effect. Consistent with a role in exocytosis, Rab11 and Rab3 proteins were colocalized with other vesicle proteins on secretory vesicles in PC12 cells and on mature synaptic vesicles in brain. Rab11b mutants that fix Rab11b in the GTP- or GDP-bound state both effectively inhibited Ca2+-induced exocytosis but seemed to act by distinct mechanisms: whereas GDP-bound Rab11b greatly stimulated constitutive secretion of hGH and depleted hGH stores in secretory vesicles, GTP-bound Rab11b only had a moderate effect on constitutive secretion and no effect on vesicular hGH stores. These results suggest that, consistent with a GTP-dependent regulation of Rab function, GDP-bound Rab11b indirectly inhibits Ca2+-triggered exocytosis by causing the loss of hGH from the PC12 cells, whereas GTP-bound Rab11b directly impairs Ca2+-triggered exocytosis. In contrast to neuroendocrine PC12 cells in which GTP- and GDP-bound Rab11b inhibited Ca2+-induced, but not constitutive, exocytosis, in non-neuronal cells GTP- and GDP-bound Rab11b inhibited constitutive exocytosis and caused an accumulation of cellular hGH. Viewed together, our data suggest that, in addition to other functions, Rab11 has a specific role in neuronal and neuroendocrine but not in non-neuronal cells as a GTP-dependent switch between regulated and constitutive secretory pathways.

a Institut fur Anatomie/Neurowissenschaftliches Zentrum der Charite, Humboldt-Universitat zu Berlin, Philippstrasse 12, D-10115, Berlin, Germany b Centre for Molecular Biology and Neuroscience and Department of Anatomy, Institute of Basic Medical Sciences, University of Oslo, P.O. Box 1105-Blindern, N-0317 Oslo, Norway c Max-Planck-Institut fur Biophysikalische Chemie, Am Fassberg 11, D-37077 Gottingen, Germany d Departments of Neurology and Physiology, Graduate Programs in Neuroscience, Cell Biology, and Biomedical Sciences, 513 Parnassus Avenue, UCSF School of Medicine, San Francisco, CA 9414, USA e Institut fur Pharmakologie, Universitatsklinikum Benjamin Franklin, Freie Universitat zu Berlin, Thielallee 67-73, D-14195 Berlin, Germany f Section of Medical Statistics, Institute of Basic Medical Sciences, University of Oslo, P.O. Box 1105-Blindern, N-0317 Oslo, Norway g Institut fur Physiologische Chemie II, Heinrich-Heine-Universitat, Dusseldorf, Universitatstrasse 1, D-40225 Dusseldorf, Germany

Synaptic vesicles from mammalian brain are among the best characterized trafficking organelles. However, so far it has not been possible to characterize vesicle subpopulations that are specific for a given neurotransmitter. Taking advantage of the recent molecular characterization of vesicular neurotransmitter transporters, we have used an antibody specific for the vesicular GABA transporter (VGAT) to isolate GABA-specific synaptic vesicles. The isolated vesicles are of exceptional purity as judged by electron microscopy. Immunoblotting revealed that isolated vesicles contain most of the major synaptic vesicle proteins in addition to VGAT and are devoid of vesicular monoamine and acetylcholine transporters. The vesicles are 10-fold enriched in GABA uptake activity when compared with the starting vesicle fraction. Furthermore, glutamate uptake activity and glutamate-induced but not chloride-induced acidification are selectively lost during immunoisolation. We conclude that the population of GABA-containing synaptic vesicles is separable and distinct from vesicle populations transporting other neurotransmitters.