A Munc13-1 conditional KO mouse line

The aim of the second part of the present study was to generate a Munc13-1 conditional KO mouse line. Munc13-1 conventional KO mice exhibit severe impairments in synaptic transmission in glutamatergic synapses, causing death of the animals at birth (Augustin et al., 1999a). Within the last years it became evident that Munc13s are not only essential for priming synaptic vesicles under basal conditions, but also serve as important modulators of synaptic vesicle release through a wide range of molecular interactions with second messengers (Ca²⁺, DAG) in the presynaptic terminal (Lipstein et al., 2013; Rhee et al., 2002). Moreover, it has been shown that Munc13s play a crucial role in presynaptic short- and long-term plasticity processes in a variety of excitatory neuron types (e.g. Calyx of Held, hippocampal mossy fiber synapse) (Breustedt et al., 2010; Chen et al., 2013;

Lipstein et al., 2013; Yang and Calakos, 2011; Zhao et al., 2012a, 2012b). The majority of these studies revealed a dominant role of Munc13-1 in the investigated neuron types, a problem that required elaborated approaches to study the role of Munc13-1 in

physiological networks, e.g. in organotypic slice culture systems or by viral overexpression of dominant-negative Munc13-1 constructs in vivo (Chen et al., 2013; Yang and Calakos, 2011; Zhao et al., 2012a, 2012b). With the help of a Munc13-1 CKO mouse line, it will not only be possible to assess the consequences of Munc13-1 loss in vivo, but also to study the functions of Munc13-2 and -3 in the absence of Munc13-1. Mice carrying a genetic deletion for Munc13-2, -3 or for both of these isoforms are viable and fertile indicating that Munc13-1 can compensate for their loss and to a large extent maintain adequate synaptic transmission in the affected synapses (Augustin et al., 2001; Chen et al., 2013;

Varoqueaux et al., 2002).

In the present study, I was able to generate Munc13-1 CKO mice and demonstrate that homozygously floxed animals from that line are viable and fertile. Moreover, viral expression of the Cre-recombinase protein in dissociated neurons successfully induces Cre-mediated deletion of exon 21 in the Munc13-1 gene, tested by PCR. In addition, Western blot analysis demonstrated the absence of Munc13-1 protein in Cre-recombinase expressing cultures, and of truncation products capable of exerting dominant-negative effects. Preliminary electrophysiological experiments in Cre-recombinase expressing autaptic cultures revealed significant reductions in EPSC amplitudes and RRP sizes in response to hypertonic sucrose solution by ~80%. To test whether Cre-mediated excision can take place in vivo, I started to cross the floxed Munc13-1 allele into a mouse line expressing Cre-recombinase under the adenoviral Ella promotor, which is expected to excise exon 21 of Munc13-1 in all neurons at an early developmental time-point (Lakso et al., 1996). Genotyping of the first offspring from these breeding pairs by PCR revealed the presence of heterozygous mice, carrying one Munc13-1 WT and one Munc13-1 Cre allele.

Litters from double heterozygous breeding pairs are expected to contain mice which will carry two Munc13-1 Cre alleles and that will hopefully phenotypically resemble full Munc13-1 KO mice (Augustin et al., 1999a). Additionally, the floxed Munc13-1 allele will be crossed into a Munc13-2/3 DKO genetic background. This mouse line can be used to completely shut down synaptic transmission in a subset of neurons or at different developmental time-points, depending on the choice of promoter controlling Cre-recombinase expression in specific mouse lines (Kaartinen and Nagy, 2001). Moreover, in these genetic backgrounds viral overexpression of Munc13 constructs carrying mutations in crucial regulatory domains (e.g. C2B, C1) in vivo can help to understand their respective functions in plasticity processes in defined, mature neuronal networks that have been difficult to study in culture (e.g. Calyx of Held).

148

5. Summary

In the present study, a combination of organotypic slice culture, cryo-fixation and three-dimensional electron tomographic microscopy was employed to analyse synaptic vesicle docking in the absence of key proteins mediating neurotransmitter release. In this experimental setting, loss of priming proteins from the Munc13 and CAPS family caused an almost complete absence of docked synaptic vesicles. In both cases, reductions in the number of membrane-attached synaptic vesicles correlated well with previously observed physiological deficits in synaptic vesicle priming.

These findings indicate that morphological synaptic vesicle docking and functional priming are correlates of the same molecular process. Genetic deletion, or reduced expression, of the individual SNARE proteins Synaptobrevin-2, SNAP25 and Syntaxin-1 resulted in a decreased number of membrane-attached synaptic vesicles, indicating that (partial) SNARE complex assembly underlies the molecular mechanism of synaptic vesicle docking and priming. Moreover, my data indicate that upregulation of Synaptobrevin-1 may partially compensate for the loss of Synaptobrevin-2 function in docking/priming in a subset of glutamatergic synapses. This finding supports the hypothesis that residual release observed in neurons after genetic deletion of SNARE proteins is mediated by alternative SNARE homologues. Genetic deletion of Munc13s results in an almost complete depletion of release-competent (docked/primed) synaptic vesicles, consistent with the view that Munc13s initiate synaptic vesicle docking/priming, possibly via their interaction with the t-SNARE Syntaxin-1. However, the precise stage at which SNARE-complex assembly is arrested in Munc13-deficient synapses could not be identified in this study.

Loss of the Ca²⁺-sensor Synaptotagmin-1 caused a decrease in total and membrane-attached synaptic vesicles. However, the reductions were not as dramatic as it would have been expected for a molecule that was formerly proposed to be the vesicular partner in vesicle docking. Based on our findings, Synaptotagmin-1 might have a role (1) in tethering synaptic vesicles to the plasma membrane prior to final membrane-attachment or (2) in clamping synaptic vesicles in a fusion-competent state until the arrival of the Ca²⁺-signal for triggering fusion. Genetic deletion of Complexin caused no significant changes in the number of docked synaptic vesicles, a result that provides support for a facilitatory, rather than inhibitory, role of Complexins prior to synaptic vesicle fusion.

Synapses lacking Munc13 priming proteins, or the SNARE proteins Synaptobrevin-2 or SNAP25, exhibited increased synaptic vesicle sizes, indicating that the key components of

the synaptic release machinery might also play an important role in presynaptic membrane or protein recycling.

In a second project, I generated a conditional Munc13-1 knock-out mouse line, which will be used to study the role of Munc13-1 in defined neuronal networks in vivo. Moreover, it will allow the function of other Munc13 isoforms (bMunc13-2, ubMunc13-2 and Munc13-3) to be studied in synapses which are otherwise dominated by Munc13-1. In this study, I demonstrate that homozygously floxed mice are viable and fertile and that after lentiviral Cre-recombinase expression in dissociated hippocampal neuron cultures, Munc13-1 protein levels are undetectable by Western blot analysis. Cre-recombinase expressing autaptic neurons exhibit a massive reduction in the EPSC and RRP sizes as described for the conventional Munc13-1 knock-out mouse line (Augustin et al., 1999a).

150