Vacuolar H-ATPase (v-ATPase) is a multi-subunit complex comprising two domains: the cytosolic V1 domain catalyzing ATP hydrolysis and the membranous V0 sector translocating protons across membranes. In addition to proton pumping, a direct function of the V0 proteolipid ring in membrane fusion has been proposed for yeast vacuolar fusion and synaptic vesicle exocytosis in Drosophila. Here, we show in cultured hippocampal neurons that in recycling synaptic vesicles, v-ATPases are only transiently assembled in a pH-dependent fashion during the tightly coupled cycle of exo-endocytosis. Upon locking v-ATPase in an assembled state by saliphenylhalamide, we observed use- and time-dependent release depression for stimuli exceeding release of primed vesicles but no abrogation of exocytosis. Thus, the membranous V0 sector is not part of the exocytotic fusion machinery. Instead, v-ATPase modulates release upstream of docking to favor fusion of fully filled synaptic vesicles.

In neurons, the continuous and dynamic endoplasmic reticulum (ER) network extends throughout the axon, and its dysfunction causes various axonopathies. However, it remains largely unknown how ER integrity and remodeling modulate presynaptic function in mammalian neurons. Here, we demonstrated that ER membrane receptors VAPA and VAPB are involved in modulating the synaptic vesicle (SV) cycle. VAP interacts with secernin‐1 (SCRN1) at the ER membrane via a single FFAT‐like motif. Similar to VAP, loss of SCRN1 or SCRN1‐VAP interactions resulted in impaired SV cycling. Consistently, SCRN1 or VAP depletion was accompanied by decreased action potential‐evoked Ca2+ responses. Additionally, we found that VAP‐SCRN1 interactions play an important role in maintaining ER continuity and dynamics, as well as presynaptic Ca2+ homeostasis. Based on these findings, we propose a model where the ER‐localized VAP‐SCRN1 interactions provide a novel control mechanism to tune ER remodeling and thereby modulate Ca2+ dynamics and SV cycling at presynaptic sites. These data provide new insights into the molecular mechanisms controlling ER structure and dynamics, and highlight the relevance of ER function for SV cycling.

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Changes in inhibitory connections are essential for experience-dependent circuit adaptations. Defects in inhibitory synapses are linked to neurodevelopmental disorders, but the molecular processes underlying inhibitory synapse formation are not well understood. Here we use high-resolution two-photon microscopy in organotypic hippocampal slices from GAD65-GFP mice of both sexes to examine the signaling pathways induced by the postsynaptic signaling molecule Semaphorin4D (Sema4D) during inhibitory synapse formation. By monitoring changes in individual GFP-labeled presynaptic boutons, we found that the primary action of Sema4D is to induce stabilization of presynaptic boutons within tens of minutes. Stabilized boutons rapidly recruited synaptic vesicles, followed by accumulation of postsynaptic gephyrin and were functional after 24 h, as determined by electrophysiology and immunohistochemistry. Inhibitory boutons are only sensitive to Sema4D at a specific stage during synapse formation and sensitivity to Sema4D is regulated by network activity. We further examined the intracellular signaling cascade triggered by Sema4D and found that bouton stabilization occurs through rapid remodeling of the actin cytoskeleton. This could be mimicked by the actin-depolymerizing drug latrunculin B or by reducing ROCK activity. We discovered that the intracellular signaling cascade requires activation of the receptor tyrosine kinase MET, which is a well known autism risk factor. By using a viral approach to reduce MET levels specifically in inhibitory neurons, we found that their axons are no longer sensitive to Sema4D signaling. Together, our data yield important insights into the molecular pathway underlying activitydependent Sema4D-induced synapse formation and reveal a novel role for presynaptic MET at inhibitory synapses.

Presynaptic metabotropic glutamate receptors (mGluRs) are essential for the control of synaptic transmission. However, a quantitative understanding of how the subsynaptic dynamics of these receptors is controlled and contributes to synaptic signaling remain poorly understood. Particularly, since the affinity of individual mGluR subtypes for glutamate differs considerably, the activation of mGluR subtypes critically depends on their precise subsynaptic distribution. Here, using super-resolution microscopy and single-molecule tracking, we unraveled novel molecular mechanisms that control the nanoscale distribution and mobility of presynaptic mGluRs in hippocampal neurons. We demonstrate that the high affinity group II receptor mGluR2 localizes diffusely along the axon, and is highly mobile, while the low affinity group III receptor mGluR7 is stably anchored at the active zone. We demonstrate that intracellular interactions modulate surface diffusion of mGluR2 while immobilization of mGluR7 at the active zone relies on its extracellular domain. Receptor activation or increases in synaptic activity did not alter the surface mobility of presynaptic mGluRs. Finally, computational modeling of presynaptic mGluR activity revealed that this particular nanoscale arrangement directly impacts their ability to modulate neurotransmitter release. Altogether, this study demonstrates that distinct mechanisms control surface mobility of presynaptic mGluRs to differentially contribute to glutamatergic synaptic transmission.