Hidden proteome of synaptic vesicles in the mammalian brain.

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.

Unique pH dynamics in GABAergic synaptic vesicles illuminates the mechanism and kinetics of GABA loading.

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.

Monitoring of vacuolar-type H+ ATPase-mediated proton influx into synaptic vesicles.

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 <5 s, much faster than glutamate loading (τ of ∼ 15 s) and thus unlikely to be rate limiting for neurotransmitter loading. However, such estimates have relied on pHluorin-based probes that lack sensitivity in the lower luminal pH range. Here, we reexamined re-acidification kinetics using the mOrange2-based probe that should report the SV pH more accurately. In recordings from cultured mouse hippocampal neurons, we found that re-acidification took substantially longer (τ of ∼ 15 s) than estimated previously. In addition, we found that the SV lumen exhibited a large buffering capacity (∼ 57 mm/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.

Vesicular glutamate transporters as anion channels?

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.

Compartmentalization of soluble endocytic proteins in synaptic vesicle clusters by phase separation.

Synaptic vesicle (SV) clusters, which reportedly result from synapsin's capacity to undergo liquid-liquid phase separation (LLPS), constitute the structural basis for neurotransmission. Although these clusters contain various endocytic accessory proteins, how endocytic proteins accumulate in SV clusters remains unknown. Here, we report that endophilin A1 (EndoA1), the endocytic scaffold protein, undergoes LLPS under physiologically relevant concentrations at presynaptic terminals. On heterologous expression, EndoA1 facilitates the formation of synapsin condensates and accumulates in SV-like vesicle clusters via synapsin. Moreover, EndoA1 condensates recruit endocytic proteins such as dynamin 1, amphiphysin, and intersectin 1, none of which are recruited in vesicle clusters by synapsin. In cultured neurons, like synapsin, EndoA1 is compartmentalized in SV clusters through LLPS, exhibiting activity-dependent dispersion/reassembly cycles. Thus, beyond its essential function in SV endocytosis, EndoA1 serves an additional structural function by undergoing LLPS, thereby accumulating various endocytic proteins in dynamic SV clusters in concert with synapsin.

Quantitative Analysis of Presynaptic Vesicle Luminal pH in Cultured Neurons.

Newly generated synaptic vesicles (SVs) are re-acidified by the activity of the vacuolar-type H+-ATPases. Since H+ gradient across SV membrane drives neurotransmitter uptake into SVs, precise measurements of steady-state vesicular pH and dynamics of re-acidification process will provide important information concerning the H+-driven neurotransmitter uptake. Indeed, we recently demonstrated distinct features of steady state and dynamics of vesicular pH between glutamatergic vesicles and GABAergic vesicles in cultured hippocampal neurons. In this article, we focus on an experimental protocol and setup required to determine steady-state luminal pH of SVs in living neurons. This protocol is composed of efficient expression of a pH-sensitive fluorescent protein in the lumen of SVs in cultured neurons, and recordings of its fluorescence changes under a conventional fluorescent microscope during local applications of acidic buffer and ionophores-containing solution at a given pH. The method described here can be easily applied for measuring luminal pH of different types of secretory organelles and other acidic organelles such as lysosomes and endosomes in cultured cell preparations.

Clathrin-independent endocytic retrieval of SV proteins mediated by the clathrin adaptor AP-2 at mammalian central synapses.

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/adaptor protein complex 2 (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 mouse 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 revised model for the endocytosis of SV membranes at mammalian central synapses.

Physiological Perspectives on Molecular Mechanisms and Regulation of Vesicular Glutamate Transport: Lessons From Calyx of Held Synapses.

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.

The endosomal Q-SNARE, Syntaxin 7, defines a rapidly replenishing synaptic vesicle recycling pool in hippocampal neurons.

Upon the arrival of repetitive stimulation at the presynaptic terminals of neurons, replenishment of readily releasable synaptic vesicles (SVs) with vesicles in the recycling pool is important for sustained neurotransmitter release. Kinetics of replenishment and the available pool size define synaptic performance. However, whether all SVs in the recycling pool are recruited for release with equal probability and speed is unknown. Here, based on comprehensive optical imaging of various presynaptic endosomal SNARE proteins in cultured hippocampal neurons, all of which are implicated in organellar membrane fusion in non-neuronal cells, we show 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 an Stx7-reporter requires actin polymerization, as well as activation of the Ca2+/calmodulin signaling pathway, reminiscent of rapidly replenishing SVs characterized previously in calyx of Held synapses. Furthermore, disruption of Stx7 function by overexpressing its N-terminal domain selectively abolished this pool. Thus, our data indicate that endosomal membrane fusion involving Stx7 forms rapidly replenishing vesicles essential for synaptic responses to high-frequency repetitive stimulation, and also highlight functional diversities of endosomal SNAREs in generating distinct exocytic vesicles in the presynaptic terminals.

Vesicular Glutamate Transporter Expression Ensures High-Fidelity Synaptic Transmission at the Calyx of Held Synapses.

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.

Unique luminal localization of VGAT-C terminus allows for selective labeling of active cortical GABAergic synapses.

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 synapses in 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.

Expression of plasma membrane calcium ATPases confers Ca2+/H+ exchange in rodent synaptic vesicles.

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.

Development of lentiviral vectors for efficient glutamatergic-selective gene expression in cultured hippocampal neurons.

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.

Molecular Signatures Underlying Synaptic Vesicle Cargo Retrieval.

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.

Galphao2 regulates vesicular glutamate transporter activity by changing its chloride dependence.

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 alpha subunits of trimeric G-proteins, including Galpha(o2) and Galpha(q). Furthermore, glutamate uptake, mediated by vesicular glutamate transporters (VGLUTs), is decreased by the nonhydrolysable GTP-analog guanylylimidodiphosphate. Using mutant mice lacking various Galpha subunits, including Galpha(o1), Galpha(o2), Galpha(q), and Galpha11, and a Galpha(o2)-specific monoclonal antibody, we now show that VGLUTs are exclusively regulated by Galpha(o2). G-protein activation does not affect the electrochemical proton gradient serving as driving force for neurotransmitter uptake; rather, Galpha(o2) exerts its action by specifically affecting the chloride dependence of VGLUTs. All VGLUTs show maximal activity at approximately 5 mm chloride. Activated Galpha(o2) shifts this maximum to lower chloride concentrations. In contrast, glutamate uptake by vesicles isolated from Galpha(o2-/-) mice have completely lost chloride activation. Thus, Galpha(o2) acts on a putative regulatory chloride binding domain that appears to modulate transport activity of vesicular glutamate transporters.

VGLUTs: 'exciting' times for glutamatergic research?

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.

Presynaptic Molecular Determinants of Quantal Size.

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 1950's. Considering the morphological identification of synaptic vesicles (SVs) 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 SVs 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 SVs 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.

Divergent functions of neuronal Rab11b in Ca2+-regulated versus constitutive exocytosis.

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.

miR-199a Links MeCP2 with mTOR Signaling and Its Dysregulation Leads to Rett Syndrome Phenotypes.

Rett 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.

Valproic acid selectively suppresses the formation of inhibitory synapses in cultured cortical neurons.

Valproic acid (VPA) has been used to treat epileptic patients because of its ability to potentiate GABA signaling in the brain. Despite its clinical significance, VPA administration during pregnancy increases the risk of congenital abnormalities, such as neural tube defects and neurodevelopmental disorders including autism. Furthermore, recent studies revealed that early postnatal administration of VPA also leads to neurodevelopmental deficits in rodents. Here, using cultured cortical neurons derived from postnatal day 1 rats, we examined whether exposure to VPA would affect synapse formation. When neurons were exposed to 1mM VPA during early development, expression of the vesicular GABA transporter (VGAT) was selectively reduced, whereas other synaptic markers, including the vesicular glutamate transporters 1 and 2 (VGLUT1 and 2), were not affected. This VPA effect was mediated through inhibition of histone deacetylases (HDACs), since the effects were mostly recapitulated by an HDAC inhibitor, trichostatin A, but not by a VPA derivative, valpromide, which lacks HDAC inhibitor activity. Immunocytochemical analysis demonstrated that VPA exposure resulted in a retardation of axonal growth specific to GABAergic neurons and a decrease in VGAT-positive synapses. Since disturbance of the excitatory and inhibitory (E-I) balance has been implicated as a potential cause of multiple psychiatric disorders, our results may account for one of the cellular mechanisms underlying the pathogenesis of VPA-induced neurodevelopmental impairments.