Synaptic vesicles in the brain harbor several SNARE proteins. With the exception of synaptobrevin2/VAMP2 (syb2) that is directly involved in vesicle fusion, the role of these SNAREs in neurotransmission is unclear. Here, we show that in mice while syb2 drives rapid Ca2+-dependent synchronous neurotransmission, the structurally homologous SNARE protein VAMP4 selectively maintains bulk Ca2+-dependent asynchronous release. At inhibitory nerve terminals, up- or down-regulation of VAMP4 causes a correlated change in asynchronous release. Biochemically, VAMP4 forms a stable complex with SNAREs syntaxin-1 and SNAP-25 that does not interact with complexins or synaptotagmin-1, proteins essential for synchronous neurotransmission. Optical imaging of individual synapses indicates that VAMP4 and syb2 trafficking show minimal overlap. Taken together, these findings suggest that VAMP4 and syb2 diverge functionally, traffic independently and support distinct forms of neurotransmission. These results provide molecular insight into how synapses diversify their release properties by taking advantage of distinct synaptic vesicle-associated SNAREs.

Summary Synaptic vesicle recycling is essential for maintaining normal synaptic function. The coupling of exocytosis and endocytosis is assumed to be Ca2+-dependent but the exact role of Ca2+ and its key effector synaptotagmin-1 (syt1) in regulation of endocytosis are poorly understood. Here, we probed the role of syt1 in single as well as multivesicle endocytic events using high resolution optical recordings. Our experiments showed that the slowed endocytosis phenotype previously reported after syt1 loss-of-function can also be triggered by other manipulations that promote asynchronous release such as Sr2+ substitution and complexin loss-of-function. The link between asynchronous release and slowed endocytosis was due to selective targeting of fused synaptic vesicles towards slow retrieval by the asynchronous release Ca2+ sensor synaptotagmin7. In contrast, after single synaptic vesicle fusion, syt1 acted as an essential determinant of synaptic vesicle endocytosis time course by delaying the kinetics of vesicle retrieval in response to increasing Ca2+ levels.

ALS (Amyotrophic Lateral Sclerosis) is a neurodegenerative disease characterized by the redistribution of the RNA binding protein TDP-43 in affected neurons: from predominantly nuclear to aggregated in the cytosol. However, the determinants of TDP-43 localization and the cellular insults that promote redistribution are incompletely understood. Here, we show that the putative Nuclear Export Signal (NES) is not required for nuclear egress of TDP-43. Moreover, when the TDP-43 domain which contains the putative NES is fused to a reporter protein, YFP, the presence of the NES is not sufficient to mediate nuclear exclusion of the fusion protein. We find that the previously studied “∆NES” mutant, in which conserved hydrophobic residues are mutated to alanines, disrupts both solubility and splicing function. We further show that nuclear export of TDP-43 is independent of the exportin XPO1. Finally, we provide evidence that nuclear egress of TDP-43 is size dependent; nuclear export of dTomato TDP-43 is significantly impaired compared to Flag TDP-43. Together, these results suggest nuclear export of TDP-43 is predominantly driven by passive diffusion.

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Recent studies suggest that spontaneous and action potential-evoked neurotransmitter release processes are independently regulated. However, the mechanisms that uncouple the two forms of neurotransmission remain unclear. In cultured mouse and rat neurons, we show that the two C2 domain-containing protein copine-6 is localized to presynaptic terminals and binds to synaptobrevin2 as well as other SNARE proteins in a Ca-dependent manner. Ca-dependent interaction of copine-6 with synaptobrevin2 selectively suppresses spontaneous neurotransmission in a reaction that requires the tandem tryptophan residues at the C-terminal region of synaptobrevin2. Accordingly, copine-6 loss of function augmented presynaptic Ca elevation-mediated neurotransmitter release. Intracellular Ca chelation, on the other hand, occluded copine-6-mediated suppression of release. We also evaluated the molecular specificity of the copine-6-dependent regulation of spontaneous release and found that overexpression of copine-6 did not suppress spontaneous release in synaptobrevin2-deficient neurons. Together, these results suggest that copine-6 acts as a specific Ca-dependent suppressor of spontaneous neurotransmission. Synaptic transmission occurs both in response to presynaptic action potentials and spontaneously, in the absence of stimulation. Currently, much more is understood about the mechanisms underlying action potential-evoked neurotransmission compared with spontaneous release. However, recent studies have shown selective modulation of spontaneous neurotransmission process by several neuromodulators, suggesting specific molecular regulation of spontaneous release. In this study, we identify copine-6 as a specific regulator of spontaneous neurotransmission. By both gain-of-function and loss-of-function experiments, we show that copine-6 functions as a Ca-dependent suppressor of spontaneous release. These results further elucidate the mechanisms underlying differential regulation of evoked and spontaneous neurotransmitter release.

Coupling between voltage-gated Ca(2+) influx and synaptic vesicle exocytosis is essential for rapid evoked neurotransmission. Acuna et al. show that the knockout of RIM-BPs, which are key structural components of this coupling, decreases the reliability of evoked neurotransmitter release.