The role of the synaptic vesicle priming factors Munc13-1 and Munc13-2 in synaptic vesicle docking

Munc13 proteins are key regulators of synaptic vesicle release, as evidenced by the complete loss of evoked and spontaneous neurotransmitter release in the absence of Munc13 priming factors. Munc13s reside in the dense protein network at the presynaptic active zone. Their mode of action is believed to involve the opening of the t-SNARE Syntaxin-1 by displacing Munc18-1, allowing t-SNARE acceptor complex formation (Ma et al., 2011). A previous study has shown that KO of Munc13 proteins results in a massive decrease in the number of “docked” or “membrane-attached” synaptic vesicles in neurons (Siksou et al., 2009). Therefore I decided to start with the reanalysis of synaptic vesicle docking in Munc13-1/2 DKO neurons in order to validate my experimental system (Augustin et al., 1999a; Varoqueaux et al., 2002). Homozygous Munc13-2 KO neurons are phenotypically indistinguishable from wild-type neurons (Varoqueaux et al., 2002). In this study, I therefore compared four Munc13-1/2 DKO animals (N=4) with four littermate control animals (N=4) from two cultures. The control animals were either wild-type or heterozygous for the Munc13-1 KO allele in a homozygous Munc13-2 KO background, or heterozygous for Munc13-1 and Munc13-2 KO alleles.

3.1.3.1.1. 2D-EM analysis of synaptic morphology in Munc13-1/2 DKO neurons

In a first experiment, ultrastructural parameters of Munc13-1/2 DKO synapses were analyzed in electron micrographs from 60 nm ultrathin EPON sections after high-pressure freezing fixation, freeze-substitution and EPON embedding in order to rule out any major changes of presynaptic morphology that could influence the results obtained from a high-resolution electron tomographic analysis (Fig. 3.3). I did not observe any major morphological changes between control and Munc13-1/2 DKO synaptic profiles, although a potential increase in the size of synaptic vesicles was apparent in Munc13-1/2 DKO

terminals (Fig. 3.3 A, B). I found that the total number of synaptic vesicles per synaptic profiles in Munc13-1/2 DKO synapses was unchanged in comparison to control synaptic profiles (control: 61.44 ± 3.195, n=106; Munc13-1/2 DKO: 70.53 ± 4.203, n=103 / P=0.2565, n.s.; Fig. 3.3 C). Moreover, I did not detect any significant changes in the number of synaptic vesicles normalized to the presynaptic terminal size (synaptic vesicles per 0.01 µm² terminal area or synaptic vesicle terminal density) (control: 1.633 ± 0.059, n=106; Munc13-1/2 DKO: 1.489 ± 0.057, n=103 / P=0.0558, n.s.) nor in the measured length of the PSD (control: 371.1 ± 11.95, n=106; Munc13-1/2 DKO: 393.7 ± 12.97, n=103 / P =0.1895, n.s.; Fig.3.3 D, F). However, I did observe a small decrease in the number of synaptic vesicles normalized to the size of the synaptic vesicle cluster (synaptic vesicles per 0.01 µm² cluster area or synaptic vesicle cluster density) (control: 3.149 ± 0.056, n=106; Munc13-1/2 DKO: 2.883 ± 0.055, n=103 / P=0.0018, **; Fig. 3.3 E). I also noticed an increase in the number of vesicular structures with a larger diameter than synaptic vesicles, possibly representing components of the endocytotic recycling pathway in the presynaptic terminal (control: 1.000 ± 0.127, n=106; Munc13-1/2 DKO: 1.650 ± 0.198, n=103 / P =0.0462, *; Fig. 3.3 G). A recent study indicated a role of proteins from the Munc13 family in LDCV release in neurons, without detecting any changes in the number of LDCVs in presynaptic terminals from Munc13-1/2 DKO and control neurons (van de Bospoort et al., 2012). I could confirm these results in our experimental setting as I failed to observe a difference in the number of LDCVs per synaptic profile (control: 0.236 ± 0.056, n=106; Munc13-1/2 DKO: 0.233 ± 0.054, n=103 / P=0.8375, n.s.; Fig. 3.3 H).

3.1.3.1.2. 3D-ET analysis of synaptic vesicle docking in Munc13-1/2 DKO neurons

I then moved on to analyze synaptic vesicle docking in presynaptic terminals from electron tomographic reconstructions of 200 nm-thick semithin sections through glutamatergic, spine synapses of control and Munc13-1/2 DKO samples (Fig. 3.4 A-F). For the three-dimensional analysis of electron tomograms, I compared samples from three Munc13-1/2 DKO animals (N=3) with samples from two littermate controls (N=2). Control animals were either wild-type for the Munc13-1 KO allele in a homozygous Munc13-2 KO background, or heterozygous for both Munc13-1 and Munc13-2 KO alleles. In agreement with previous publications, I detected an almost complete loss of membrane-attached vesicles (0-2 nm)

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Figure 3.3. Two-dimensional ultrastructural analysis of synaptic morphology in Munc13-1/2 DKO neurons

Electron micrographs of control (A) and Munc13-1/2 DKO (B) synaptic profiles acquired from 60 nm-thick ultrathin sections. Mean number of synaptic vesicles (SVs) per synaptic profile (C). Mean number of SVs normalized to synaptic terminal area (SV terminal density; D). Mean number of SVs normalized to SV cluster area (SV cluster density; E). Mean postsynaptic density (PSD) length (F) Mean number of endosomes per synaptic profile (G). Mean number of large dense-core vesicles (LDCVs) per synaptic profile (H). C-H: Control: N=4, n=106; Munc13-1/2 DKO: N=4, n=103 (Mean + SEM), P<0.001: ***; P<0.01: **; P<0.05: *. Scale bar: B, 500 nm.

normalized to 0.01 µm² active zone area in Munc13-1/2 DKO in comparison to control synaptic profiles (control: 0.979 ± 0.134, n=16; Munc13-1/2 DKO: 0.042 ± 0.004, n=15 / P<0.001, ***; Fig. 3.4 I), which was still statistically significant after including vesicles that are closely tethered (0-4 nm) normalized to 0.01 µm² active zone area (control: 1.651 ± 0.201, n=16; Munc13-1/2 DKO: 0.145 ± 0.089, n=15 / P<0.001, ***; Fig. 3.4 J). 14 out of 15 synaptic profiles (93%) harbored no membrane-attached (0-2 nm) vesicles, and 12 synaptic profiles (80%) lacked docked and closely tethered vesicles (0-4 nm). The number of membrane-attached (0-2 nm) and closely tethered (0-4 nm) synaptic vesicles was reduced to 4% and 11% of control profiles, respectively. However, the number of vesicles that were close to the active zone (within 40 nm) normalized to 0.01 µm² active zone area was unchanged (control: 2.487 ± 0.188, n=16; Munc13-1/2 DKO: 2.113 ± 0.179, n=15 / P=0.1626, n.s.; Fig. 3.4 H). Consistent with this finding, the synaptic vesicles in Munc13-1/2 DKO synapses seem to accumulate at a close distance to the active zone membrane, with a peak in the vesicle distribution around 8-10 nm (Fig. 3.4 M). Those

membrane-proximal, but not docked vesicles, were often linked to the presynaptic plasma membrane by thin, long filaments similar to previous findings (Siksou et al., 2009). I also detected a decrease in the number of vesicles within 100 nm distance from the active zone membrane, normalized to 0.01 µm² active zone area (control: 6.547 ± 0.395, n=16;

Munc13-1/2 DKO: 5.023 ± 0.384, n=15 / P=0.0099, **; Fig. 3.4 G). A likely explanation for this is a dramatic increase in the mean outer synaptic vesicle diameter, first observed in two-dimensional electron micrographs from Munc13-1/2 DKO synapses, and subsequently confirmed by three-dimensional electron tomographic analysis (control:

46.25 ± 0.485, n=16; Munc13-1/2 DKO: 50.15 ± 0.715, n=15; P<0.001, ***; Fig. 3.4 K, N).

This increase in the outer vesicle diameter in Munc13-1/2 DKO presynaptic terminals leads to a corresponding 28% increase in the synaptic vesicle volume (control: 52473 ± 1656, n=16; Munc13-1/2 DKO: 67158 ± 2853, n=15; P<0.001, ***; Fig. 3.4 L). The increased number of larger vesicular structures, possibly components of the endocytotic recycling pathway or precursors of vesicular biosynthesis, in the presynaptic terminals in Munc13-1/2 DKO synaptic profiles raises the possibility that synaptic trafficking or vesicle recycling is disrupted in the absence of Munc13 proteins.

In summary, the ultrastructural findings presented here are in agreement with previously published data, showing an almost complete loss of membrane-associated synaptic vesicles in synapses lacking synaptic vesicle priming molecules of the Munc13 family (Siksou et al., 2009). Moreover, the inability of synaptic vesicles to dock to the presynaptic active zone membrane strongly supports the hypothesis that the terms morphological synaptic vesicle “docking” and physiological synaptic vesicle “priming” may describe the same molecular process.

3.1.3.2. The role of the synaptic vesicle priming factors CAPS-1 and CAPS-2