Molecular Mechanisms Involved In The Generation Of Inhibitory Postsynaptic Structures

In the work presented here, a set of full-length size-selected and unamplified cDNA expression libraries [30.000 CFU in total; size-fractionated into 2-3, 3-4, and 4-5 kb pools of 10.000 CFU each] was screened for proteins that may function in gephyrin-dependent formation of inhibitory synapses similarly to CB or NL2. The assay we used is based on the previously described observation that in nonneuronal cells, coexpression of GFP-gephyrin together with CBSH3- alone or with CBSH3+ in the presence of NL2 leads to a redistribution of gephyrin into submembranous microclusters (Kins et al 2000, Poulopoulos et al 2009). In our assay, this phenotypic change was adopted by using Flp-In T-Rex-GFP-gephyrin HEK 293 cells inducibly expressing GFP-gephyrin (Papadopoulos et al 2017). Using this strategy, we were able to identify 7 clones from the cDNA library that induce redistribution (see Table 9). As a proof-of-principle for our screening assay, we were able to isolate previously known gephyrin-interacting proteins, such as GlyRβ and NL2 (Pfeiffer et al 1982, Poulopoulos et al 2009), thereby demonstrating the physiological relevance of the regulatory proteins identified by our approach. Furthermore, we identified novel candidate-proteins with potential roles in the formation of inhibitory synapses. These proteins are involved in the regulation of actin dynamics (WASF1/WAVE1, β-actin, HIP1R), phosphoinositide metabolism (PTPRN2, HIP1R) and transfer of membrane vesicles (HIP1R, NSF). We initially focused our studies in examining whether two of the candidate proteins, WAVE1 and GlyRβ, are relevant for the formation of inhibitory synapses in the mouse forebrain. First, we aimed to investigate the importance of WAVE1 for the formation and stability of GABAergic synapses. Our in vitro binding assays (see Figure 18) confirmed the interaction of the WRP with WAVE1, as previously described (Soderling et al 2002), but did not show a direct binding of WAVE1 to gephyrin (see Figure 18). In contrast, we indicated a specific interaction between the SH3-domain of CB and WAVE1 (see Figure 16), as untagged WAVE1 was found to consistently bind to the GST-tagged versions of the SH3-domain of CB (GST-SH3) and full-length, SH3-domain containing, CB (GST-CBSH3+), but not to GST alone or to CB lacking the SH3-domain (GST-CBSH3-). In order to use an untagged version of WAVE1 in the in vitro binding assay, GST-WAVE1 was cut with thrombin, resulting in fragmentation of WAVE1, which was also observed in the pulldown with GST-SH3, whereas the pulldown with GST-CB resulted mainly in a pulldown of the full-length WAVE1 (see Figure 16). This result, combined with a subsequent mass spectrometric analysis (see Figure 17) indicated that WAVE1 may have an additional, N-terminal, binding site for the full-length SH3 domain-containing CB. In addition to biochemical studies on possible interaction partners of WAVE1, this work investigated the effect of loss of WAVE1 on gephyrin clustering in the cerebellum, as well as hippocampus using immunohistochemical experiments in brain slices derived from 5-week-old WAVE1 KO mice. Our immunohistochemical analyses using WAVE1 KO mice (Soderling et al 2003) and their WT or +/- littermates, revealed no differences in the clustering of gephyrin in glutamatergic neurons, but a significant increase in the densities of dendritic gephyrin puncta in PV+ interneurons of the CA1 area of the hippocampus in brains of WAVE1 KO mice, as compared to controls (see Figure 21). This increase of gephyrin puncta in PV+ interneurons was accompanied by a decrease in the mean gephyrin puncta size (see Figure 22), as well as by a decrease in the percentages of gephyrin puncta apposed to the presynaptic marker VIAAT (see Figure 22), in slices derived from WAVE1 KOs, as compared to controls. The second candidate of the screen, which we examined more closely, was GlyRβ. In contrast to previous publications (Kirsch & Betz 1995) in our unbiased expression screen, the GlyR-β-induced formation of submembranous gephyrin microclusters was robust and consistent. In addition, we were able to demonstrate a localization of GlyRβ in the plasma membrane (see Figure 10). In order to study the expression pattern and the roles of the GlyRβ protein in selected regions and neuronal subtypes of the mouse forebrain, we generated two new mouse lines by using the CRISPR Cas9 technology and corresponding HDR fragments (see Figure 11 and Figure 12). The first line (see Figure 11; GLRB-CO) is a conditional KO line, carrying two loxP-sites in the intronic regions upstream of exons 8 and downstream of exon 9, respectively. Exons 8 and 9 encode half of the TMD2, the whole TMD3 and a part of the intracellular loop of GlyRβ, which we predict to result in the loss of the GlyRβ protein upon Cre-mediated recombination in mice. Mouse genotyping was carried out by PCR indicating the successful generation (see Figure 11). The HDR fragment for the second mouse line was designed to introduce loxP-sites in the regions upstream of exon 10 and downstream of the coding sequence, and to replace the Stop codon with the coding sequence of the HA-tag and a Stop codon. Initial PCR-based screening for homologous recombination in mice indicated the absence of the loxP-site upstream of exon 10 in the KI allele (see Figure 12). In contrast, the presence of the HA-tag and the second loxP-site in the KI allele was verified by PCR genotyping (see Figure 12). Furthermore, Western blot analysis of brain lysates derived from hom GLRB-HA mice and their WT-littermates using an HA-antibody verified the expression of a 58-60 kD protein in samples derived from GLRB-HA brains, but not in those from WT brains (see Figure 12). The size of this protein corresponds to a HA-tagged version of the full-length GlyRβ protein (Weltzien et al 2012). Finally, a comprehensive and unbiased screen of a cDNA library was successfully performed in this work, which identified both known and novel regulatory proteins involved in the gephyrin-dependent formation of inhibitory synapses. In the case of WAVE1, this work was able to indicate an important role of WAVE1 in the clustering of gephyrin at inhibitory synapses of PV+ interneurons. Our data provide evidence that all candidates identified in our screen have potentially important roles in the formation of gephyrin-dependent inhibitory synapses.2022-06-2