(Scale bars: 2 m.) To assess possible in vivo effects of lithium on GABAergic synapses, we examined the effects of chronic LiCl administration in adult mice over 72 h on gephyrin cluster and GABAergic presynaptic terminal density in four brain regions (Fig. underscores the relevance of this posttranslational modification for AMG 208 synaptic plasticity. Conversely, we show that gephyrin availability for postsynaptic clustering is limited by Ca2+-dependent gephyrin cleavage by the cysteine protease calpain-1. Together, these findings identify gephyrin as synaptogenic molecule regulating GABAergic synaptic plasticity, likely contributing to the therapeutic action of lithium. and and Table S1), unchanged in size (Fig. 1and and and 3UTR and mCherry-homer (green; to visualize the transfected dendrites). Presynaptic terminals were stained with synapsin-1 (blue). The residual gephyrin clusters are AMG 208 present in nontransfected cells. (and and mRNA 3UTR to deplete endogenous gephyrin without affecting expression of eGFP-constructs (which lack the 3UTR), as reported earlier (12). To demonstrate its specificity, we used the shRNA with three point-mutations in its sequence (3UTR-3m). Cells were analyzed after 11 + 7 DIV by triple-fluorescence with a presynaptic marker (Fig. 1 and and and and Table S1). Functional Analysis of Gephyrin Ser270 Phosphorylation Mutants. Next, to assess the functional relevance of Ser270 phosphorylation, whole-cell patch-clamp recordings of miniature inhibitory postsynaptic currents (mIPSCs) were performed. Overexpression of AMG 208 WT eGFP-gephyrin did not influence mIPSC amplitudes or interevent intervals compared with mock-transfected cells present on the same coverslip (Fig. 2 and Table S2), indicating that recombinant gephyrin did not cause measurable overexpression artifacts. In contrast, the average amplitude of mIPSCs recorded in neurons expressing S270A was 10% larger than control (Fig. 2and Table S2), suggesting increased density of functional GABAergic synapses. In cells transfected with S270E mutant, mIPSCs were much like WT or mock-transfected cells, reflecting the results of Fig. 1. Finally, the rise and decay time constants of mIPSCs did not switch appreciably (Fig. S1), suggesting no differences in localization or functional properties of GABAAR in transfected neurons. Thus, constitutive blockade of gephyrin Ser270 phosphorylation allows formation of supernumerary, functional GABAergic synapses in cultured neurons. Open in a separate windows Fig. 2. Effects of WT AMG 208 and mutant eGFP-gephyrin expression on GABAergic mIPSCs. ( 0.001 compared with WT eGFP-gephyrin. Identifying the Kinase Pathway Regulating Ser270 Site in Gephyrin. We tested different protein kinase inhibitors for their effect on gephyrin clustering, expecting proline-directed serine/threonine kinases to be effective, as a proline residue flanks Ser270 (DTASLSTTPSEsPR). Accordingly, overnight treatment of neuronal cultures with inhibitors of the nonCproline-directed kinases PKA (H-89, 5 M; KT5720; 1 M) and PKC (calphostin C; 2 M) experienced no observable effect on eGFP-gephyrin clustering. However, overnight exposure to the GSK3 inhibitor GSK3-IX (5 M) induced a marked increase in postsynaptic eGFP-gephyrin cluster AMG 208 density (Fig. 3 and and Table S1), mimicking the phenotype of the S270A mutant. In control experiments, GSK3-IX exposure did not change the phenotype of eGFP-S270E mutant (Fig. 3 and Table S1), confirming the selectivity of GSK3 action on Ser270. As seen in Table S1, the size of WT and S270E gephyrin clusters was significantly reduced by GSK3-IX, suggesting that GSK3 inhibition limits gephyrin availability. Open in a separate windows Fig. 3. GSK3 phosphorylates gephyrin at Ser270. (and and and and and Table S1). Next, we tested whether Ser270 phosphorylation Rabbit Polyclonal to Pim-1 (phospho-Tyr309) underlies these effects of lithium. In neurons transfected with S270E mutant, no effect on gephyrin cluster density was observed after 12-h exposure to 20-mM LiCl (Fig. 4 and and Table S1), confirming the specificity of lithium inhibition of GSK3. However, as observed above with GSK3-IX, the size of clusters was reduced (Table S1), indicating that enlargement of eGFP-gephyrin clusters after LiCl exposure involves an additional mechanism. Open in a separate windows Fig. 4. LiCl affects postsynaptic gephyrin clusters in cultured hippocampus neurons. (and and = 5 cells; 98 clusters; ** 0.0001), PSD95-eGFP cotransfected.