Loss of individual SNARE proteins can be partially compensated

In the present study, I observed docking deficits for neurons lacking the individual SNARE proteins. The greatest reduction in synaptic vesicle membrane-attachment was observed for SNAP25 KO neurons. 13 out of 25 tomograms from SNAP25 KO synapses (52%) were completely devoid of membrane-attached vesicles (0-2 nm) with 7 out of 25 tomograms (28%) lacking both, physically docked and closely tethered synaptic vesicles (0-4 nm). Synapses lacking SNAP25 showed a reduction in the numbers of membrane-attached and closely tethered vesicles to 21% and 35% of controls, respectively. In low density cultures from SNAP25 KO neurons, cells degenerated during culturing, but survival was prolonged by plating cells at high densities. In such a continental culture setting, it has been described that 20-30% of SNAP25 KO neurons did not exhibit action potential- evoked release. In the remaining cells only very small EPSC amplitudes could be measured and spontaneous release and the RRP size probed by hypertonic sucrose solution was found to be reduced to 14% and 10% of controls, respectively (Bronk et al., 2007).

The fact that the pool of readily releasable fusion-competent and docked vesicles as described here are not completely absent, but rather reduced, raises the possibility that alternative SNARE proteins compensate for the loss of SNAP25 (Bronk et al., 2007). One such candidate SNARE molecule is SNAP23, a neuronally expressed SNAP25 homologue capable of rescuing sucrose-evoked, but not synchronous action potential-evoked, release in cultured hippocampal neurons (Delgado-Martínez et al., 2007).

Recently, SNAP23 has been described to reside specifically in postsynaptic terminals, where it has a functional role in N-methyl-D-aspartate (NMDA) receptor exocytosis (Suh et al., 2010). Consistent with the findings of Suh et al., no specific co-localization was

detected between the glutamatergic vesicle marker VGLUT1 and SNAP23, which appeared rather in apposition to excitatory terminals in organotypic hippocampal slices.

Based on its localization, it is unlikely that SNAP23 can compensate for SNAP25 and account for the remaining docked vesicles observed in SNAP25 KO synapses. Other possible candidates and SNAP25 homologues that are expressed in the brain are SNAP29 and SNAP47 (Holt et al., 2006; Steegmaier, 1998). SNAP29 lacks the membrane anchor present in SNAP25 and SNAP23, but is present on multiple membranes and can bind to a broad range of Syntaxins, including Syntaxin-1A (Steegmaier, 1998). SNAP29 has, however, been proposed to function as an inhibitor of synaptic transmission (Pan et al., 2005). SNAP47 also lacks a membrane anchor, but is enriched on intracellular membranes and can form SNARE complexes with Synaptobrevin-2 and Syntaxin-1 to mediate proteoliposome fusion in vitro (Holt et al., 2006). More recently, SNAP-47 has been described to function in postsynaptic AMPA (α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid) receptor exocytosis during long-term potentiation (Jurado et al., 2013). It would be interesting to investigate whether changes in the expression level or localization of these alternative SNAP25 homologues might explain the residual synaptic vesicle docking and neurotransmitter release in SNAP25 deficient cells.

In comparison to controls, Syntaxin-1A / Syntaxin-1B^YFP neurons exhibited a reduction in the numbers of membrane-attached (0-2 nm) and closely tethered (0-4 nm) vesicles by 38% and 35%, respectively. Previous electrophysiological analyses in hippocampal autaptic neurons of these mutant had revealed a 65% reduction of the RRP size and an 80% reduction of EPSC size compared to controls (Arancillo et al., 2013). In comparison to the severe electrophysiological deficits in terms of the number of primed synaptic vesicles, I only detected milder effects on the number of membrane-attached synaptic vesicles. This discrepancy can most likely be attributed to the differences in the culture system, since organotypic slices were cultured for up to five weeks in a dense neuronal network, which is considerably longer than is possible with autaptic cell cultures. It is likely that during this extended culture period, compensatory, likely activity-dependent effects result in stabilization of the remaining Syntaxin-1B^YFP molecules, potentially via cell homeostatic mechanisms functioning to protect Syntaxin-1B^YFP from degradation. A recent study suggested that expression of Syntaxin-2, -3 and -4, which are BoNT/C resistant, can rescue neuronal cell death induced by proteolytic cleavage of Syntaxin-1 (Peng et al., 2013). However, attempts to culture Syntaxin-1A/B DKO neurons in an organotypic slice

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couple of days, indicating that most likely no other Syntaxin isoform can compensate for the loss of Syntaxin-1 in neurons in culture.

The analysis of synaptic vesicle docking in Synaptobrevin-2 KO neurons revealed reductions in the numbers of membrane-attached (0-2) and closely tethered (0-4 nm) vesicles by 33% and 43%, respectively, compared to control samples. In a mass culture system, all Synaptobrevin-2 KO neurons were described to be able to release. However, evoked and spontaneous release were strongly impaired and the RRP size measured after hypertonic sucrose application was reduced to 10% of control levels (Schoch et al., 2001). Here, it was noticed that 33% of Synaptobrevin-2 KO synapses seemed to be less affected by the loss of the v-SNARE, since they exhibited comparable numbers of membrane-attached synaptic vesicles as control synapses, whereas the numbers of membrane-attached and closely-tethered vesicles in the remaining KO synapses were reduced by 95%. By immunohistochemical stainings, I demonstrated that Synaptobrevin-1, another Synaptobrevin isoform expressed in the brain (Takamori et al., 2006), has been upregulated in glutamatergic synapses in comparison to control slices. In a previous study, Synaptobrevin-1 was not detected by Western blot analysis in P0 Synaptobrevin-2 KO mouse brain homogenates (Schoch et al., 2001). However, in situ hybridization experiments mapping the developmental pattern of Synaptobrevin-1 expression in the hippocampus have demonstrated that this Synaptobrevin isoform is first expressed at around P14 (Allen Developing Mouse Brain Atlas). In the present study, a striking increase in the number of glutamatergic synapses that exhibited Synaptobrevin-1 immunoreactivity from 8% in control to 36% in Synaptobrevin-2 KO organotypicic slices was demonstrated. This finding was compelling, since control samples expressed Synaptobrevin-1 preferentially at non-glutamatergic synapses, with the majority of Synaptobrevin-1 puncta not colocalizing with VGLUT1. The presence of Synaptobrevin-1 in 36% of all glutamatergic synapses correlated well with the observation that 33% of the analyzed tomograms exhibited normal synaptic vesicle docking. It can therefore be postulated that loss of Synaptobrevin-2 is at least partially compensated for by an upregulation of Synaptobrevin-1. The question as to whether Synaptobrevin-1 can rescue the physiological deficits observed in Synaptobrevin-2 KO neurons has not previously been addressed to my knowledge. However, it was not possible to detect Synaptobrevin-1 in all glutamatergic terminals, explaining the presence of Synaptobrevin-2 KO synapses that harbored only very low numbers of docked synaptic vesicles. It cannot be excluded that additional Synaptobrevin homologues may also compensate for the loss of Synaptobrevin-2. A promising candidate is VAMP7, a tetanus toxin-insensitive Synaptobrevin-2 homologue that has been shown to reside in presynaptic terminals in the

brain, e.g. in the hippocampal mossy fiber synapses, and has been implicated in asynchronous synaptic vesicle release (Hua et al., 2011b; Muzerelle et al., 2003;

Scheuber et al., 2006).

In conclusion, the presence of a small pool of docked synaptic vesicles in the absence of the major synaptic SNARE molecules could be attributed to a compensatory effect by other SNARE isoforms or homologues. Above all, the majority of SNAP25 KO neurons have been shown to degenerate in culture, indicating that survival of the remaining neurons depends on an alternative SNARE isoform. Indeed, such a compensatory effect appears to operate in Synaptobrevin-2 deficient samples, in which an upregulation of Synaptobrevin-1 can rescue normal vesicle docking in a subset of glutamatergic synapses.

4.6. Increased vesicle sizes do not cause the deficiency in synaptic