Enlarged synaptic vesicles in Synaptobrevin-1, SNAP25 and Munc13 KO synapses

Throughout the study, I noticed a significant increase in the vesicle diameter in synapses lacking either Munc13 proteins, or the SNARE proteins Synaptobrevin-2 or SNAP25. This increase in the vesicle size assessed by three-dimensional analysis of electron tomograms was accompanied by an accumulation of large vacuolar endosomal structures in two-dimensional electron micrographs in the aforementioned genotypes. Moreover, synapses from Syntaxin-1A / Syntaxin-1B^YFP samples exhibited more endosomes per synaptic profile, despite normal synaptic vesicle sizes. The latter discrepancy is likely due

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KO slices, I detected an increase in the size of VGLUT1 positive puncta, which might be associated to the respective increase in the measured synaptic vesicle volumes for these genotypes. The increase in vesicle size is unlikely to result from an increase in vesicular neurotransmitter content, since the mEPSC amplitudes were unchanged, or even slightly decreased, in Synaptobrevin-2 and SNAP25 KO mice, respectively (Bronk et al., 2007;

Schoch et al., 2001). However, recent data implicating SNARE proteins in postsynaptic receptor trafficking/exocytosis emphasize the need for caution when estimating vesicular quantal sizes by analysis of mEPSCs in SNARE-deletion mutants (Jurado et al., 2013).

Indeed, it is likely that the observed increases in vesicle size are correlated to altered presynaptic membrane recycling in the absence of synaptic vesicle fusion. This coupling of exo-and endocytotic processes is thought to be largely Ca²⁺ and SNARE protein dependent (Deák et al., 2004; Hosoi et al., 2009; Koo et al., 2011; Peng et al., 2013; Wu et al., 2009; Xu et al., 2013). Early studies proposed that Synaptobrevin-2 could be involved in a fast endocytosis process in hippocampal synapses, since genetic deletion of Synaptobrevin-2 causes an increase in synaptic vesicle sizes (Deák et al., 2004). The observed defects in endocytosis are most likely caused by an inability of Synaptobrevin-2 to interact normally with its endocytic adaptors AP180 and clathrin assembly lymphoid myeloid leukemia (CALM). AP180 KO mice exhibited retention of Synaptobrevin-2, but not VGLUT1, in the plasma membranes and an increase in synaptic vesicle sizes comparable with that observed in Synaptobrevin-2 KO synapses in the present study (Koo et al., 2011).

SNAP25 and Syntaxin-1 were shown to interact with intersectin and dynamin, respectively, both molecules involved in synaptic membrane recycling (Galas et al., 2000;

Okamoto et al., 1999). A recent study proposed a mechanism according to which the observed cell death after genetic deletion or proteolytic cleavage of Syntaxin-1 and SNAP25 is most likely caused by a defect in t-SNARE dependent membrane recycling processes rather than by the defects observed in exocytosis (Peng et al., 2013).

Interestingly, a SNAP25 knock-in mouse line carrying a mutation that abolishes protein kinase C-dependent phosphorylation of SNAP25 (SNAP25^S187A) results in a reduction of presynaptic SNAP25 levels accompanied by a significant increase in the size of glutamatergic terminals in the striatum as assessed by VGLUT1 immunolabeling and electron microscopy (Nakata et al., 2012). Moreover, these mice commonly exhibited seizures and neurons with the knock-in mutation presented an accumulation of α-Synuclein in presynaptic terminals, a feature associated with a range of neurodegenerative diseases (α-Synucleinopathies), including Parkinson's disease.

Interestingly, these α-Synuclein accumulations seem to reside at the periactive zone, a

canonical site of endocytosis, indicating a relationship between altered endocytosis and SNAP25 expression levels in the synapse (Nakata et al. 2012). Taken together, the data presented here support the hypothesis that SNAP25 plays a role in presynaptic membrane recycling.

In the Calyx of Held synapse, a large, highly specialized synapse in the auditory pathway, exo- and endocytosis can be monitored by membrane capacitance measurements made at the presynaptic terminals (reviewed in Schneggenburger and Forsythe 2006). During high synaptic activity, e.g. upon by high-frequency stimulation, the pool of fusion-competent vesicles has to be constantly refilled while action potential-triggered fusion remains ongoing. The rate of this constant refilling of the RRP has been shown to be diminished when endocytosis is pharmacologically blocked or when the SNARE proteins Synaptobrevin, SNAP25 and Syntaxin were proteolytically cleaved (Hosoi et al., 2009; Xu et al., 2013). One interpretation of these findings was that the rate of RRP refilling is most likely not limited by the number of presynaptic vesicles present, but rather by the speed by which key players of the molecular release machinery (i.e. SNAREs, Synaptotagmins, and vesicular neurotransmitter transporter) can be cleared from active zone release sites and/or recycled into release-competent synaptic vesicles (Hosoi et al., 2009).

The 28% increase in vesicle volume observed in Munc13-1/2 DKO samples is more difficult to explain, since so far no defects in endocytosis have been observed in these mice (Varoqueaux et al., 2002). Synapses lacking all Munc13 isoforms are completely silent, therefore exo-endocytosis coupling cannot be measured by capacitance measurements or by stimulation of FM dye uptake into vesicles. One possible explanation for the observed increase in synaptic vesicle volume could be that a constant block of neurotransmitter release prevents important vesicular proteins (i.e. Synaptotagmin-1, Synaptobrevin-2) needed for endocytosis from ever reaching the plasma membrane, a hypothesis which would be difficult to test experimentally in Munc13-1/2 DKO mice that die at birth and whose neurons are completely silent (Diril et al., 2006; Koo et al., 2011;

Varoqueaux et al., 2002). However, it has been proposed that in hippocampal and Calyx of Held synapses, an increase in Ca²⁺-concentrations not only triggers release, but also endocytosis of a "readily-retrievable pool" of vesicles (Hua et al., 2011a; Wu et al., 2009).

In the Calyx of Held, the Ca²⁺-dependence of endocytosis has been linked to Calmodulin, a Ca²⁺-dependent regulator of Munc13s (Wu et al., 2009). Indeed, Calmodulin inhibitors induce a defect in RRP refilling, which may be attributable to a slower rate of endocytosis

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the Calyx of Held, indicating that Munc13-1 might be the major target in Ca²⁺-Calmodulin dependent presynaptic plasticity processes (Lipstein et al., 2013).

The results of the present study support the possibility that Munc13s function in both synaptic vesicle priming and in coupling exo- and endocytosis at the active zone. Ca²⁺ -Calmodulin/Munc13 interactions during high synaptic activity might then potentiate the rate of pool refilling by boosting fast endocytosis and therefore fast release machinery and membrane recycling during release. Indeed, Munc13s may be interesting candidates for such a function due to their central position at the active zone and their interactions with RIM proteins, which link Munc13s and vesicles in close proximity of Ca²⁺-channels (Han et al., 2011; Kaeser et al., 2011). Moreover, it has recently been shown that synaptosomes lacking RIM1α, a major Munc13-1 and ubMunc13-2 binding partner at the active zone, exhibited a 26% increase in synaptic vesicle volumes, which closely correlates with the 28% increase observed in Munc13-1/2 DKO synaptic profiles in the present study (Fernández-Busnadiego et al., 2013). RIM1α KO mice are viable, despite exhibiting defects in synaptic vesicle priming/docking, and a 60% decrease in Munc13 levels (Andrews-Zwilling et al., 2006; Deng et al., 2011; Han et al., 2011; Schoch et al., 2002).

These findings indicate that the interaction between Munc13s, RIM proteins and potentially Ca²⁺- channels at the active zone might have a direct role in mediating exo- and endocytosis coupling.