Autism spectrum disorders (ASD) are common, complex and heterogeneous neurodevelopmental disorders. Cellular and molecular mechanisms responsible for ASD pathogenesis have been proposed based on genetic studies, brain pathology, and imaging, but a major impediment to testing ASD hypotheses is the lack of human cell models. Here, we reprogrammed fibroblasts to generate induced pluripotent stem cells (iPSCs), neural progenitor cells (NPCs) and neurons from ASD individuals with early brain overgrowth and non-ASD controls with normal brain size. ASD-derived NPCs display increased cell proliferation due to dysregulation of a β-catenin/BRN2 transcriptional cascade. ASD-derived neurons display abnormal neurogenesis and reduced synaptogenesis leading to functional defects in neuronal networks. Interestingly, defects in neuronal networks could be rescued by IGF-1, a drug that is currently in clinical trials for ASD. This work demonstrates that selection of ASD subjects based on endophenotypes unraveled biologically relevant pathway disruption and revealed a potential cellular mechanism for the therapeutic effect of IGF-1.

Postsynaptic scaffolding proteins ensure efficient neurotransmission by anchoring receptors and signaling molecules in synapsespecific subcellular domains. In turn, posttranslational modifications of scaffolding proteins contribute to synaptic plasticity by remodeling the postsynaptic apparatus. Though these mechanisms are operant in glutamatergic synapses, little is known about regulation of GABAergic synapses, which mediate inhibitory transmission in the CNS. Here, we focused on gephyrin, the main scaffolding protein of GABAergic synapses. We identify a unique phosphorylation site in gephyrin, Ser270, targeted by glycogen synthase kinase 3β (GSK3β) to modulate GABAergic transmission. Abolishing Ser270 phosphorylation increased the density of gephyrin clusters and the frequency of miniature GABAergic postsynaptic currents in cultured hippocampal neurons. Enhanced, phosphorylation-dependent gephyrin clustering was also induced in vitro and in vivo with lithium chloride. Lithium is a GSK3β inhibitor used therapeutically as mood-stabilizing drug, which underscores the relevance of this posttranslational modification for synaptic plasticity. Conversely, we show that gephyrin availability for postsynaptic clustering is limited by Ca 2+ -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.GABA A receptors | lithium chloride | postsynaptic density | PSD95 | homeostatic plasticity P lasticity of chemical synapses endows neuronal networks with the capacity to store information by adjusting their functional connectivity. Hence, understanding the molecular underpinnings of synaptic plasticity is a fundamental quest of neuroscience. These mechanisms have been characterized most extensively at glutamatergic synapses, in which a core scaffolding protein, PSD95, forms a signaling complex assembled by proteins interacting via specific PDZ domains (1). In contrast, little is known about signals regulating GABAergic synapses, despite their ubiquitous presence throughout the CNS and their key role in the control of network activity and synchronization. In particular, the postsynaptic density (PSD) of GABAergic synapses, localized primarily on neuronal somata and dendritic shafts, remains ill characterized. Gephyrin, a 93-kDa cytoplasmic polypeptide, has emerged as a multifunctional protein mediating postsynaptic aggregation of GABA A receptors (GABA A R) and glycine receptors by forming a scaffold anchored to the cytoskeleton (2-4). However, the mechanisms of gephyrin and GABA A R clustering are poorly understood, although evidence for direct interaction between gephyrin and GABA A R is slowly emerging (5, 6). Though gephyrin is a phosphoprotein (7,8), the relevance of gephyrin phosphorylation for regulating GABAergic transmission has not been addressed.In the present work, we focused on gephyrin posttranslational modification for regulating its postsyna...

Background: Molecular mechanisms of plasticity at GABAergic synapses are presently unclear. Results: ERK phosphorylates gephyrin at Ser-268 to regulate size of gephyrin postsynaptic scaffold and strength of GABAergic transmission. Ser-268 phosphorylation by ERK is functionally coupled to Ser-270 phosphorylation by GSK3␤ to determine calpain action on gephyrin. Conclusion: Multiple signaling cascades regulate gephyrin postsynaptic clustering. Significance: Dynamic modulation of gephyrin clustering by phosphorylation regulates GABAergic synaptic transmission.

SummaryCollybistin (CB) is a guanine-nucleotide-exchange factor (GEF) selectively activating Cdc42. CB mutations cause X-linked mental retardation due to defective clustering of gephyrin, a postsynaptic protein associated with both glycine and GABA A receptors. Using a combination of biochemistry and cell biology we provide novel insights into the roles of the CB2 splice variants, CB2 SH3+ and CB2 SH3-, and their substrate, Cdc42, in regulating gephyrin clustering at GABAergic synapses. Transfection of Myc-tagged CB2 SH3+ and CB2 SH3-into cultured neurons revealed strong, but distinct, effects promoting postsynaptic gephyrin clustering, denoting mechanistic differences in their function. In addition, overexpression of constitutively active or dominant-negative Cdc42 mutants identified a new function of Cdc42 in regulating the shape and size of postsynaptic gephyrin clusters. Using biochemical assays and native brain tissue, we identify a direct interaction between gephyrin and Cdc42, independent of its activation state. Finally, our data show that CB2 SH3-, but not CB2 SH3+ , can form a ternary complex with gephyrin and Cdc42, providing a biochemical substrate for the distinct contribution of these CB isoforms in gephyrin clustering at GABAergic postsynaptic sites. Taken together, our results identify CB and Cdc42 as major regulators of GABAergic postsynaptic densities.

GABAA receptors (GABAARs) mediate the majority of fast inhibitory neurotransmission in the brain via synergistic association with the postsynaptic scaffolding protein gephyrin and its interaction partners. However, unlike their counterparts at glutamatergic synapses, gephyrin and its binding partners lack canonical protein interaction motifs; hence, the molecular basis for gephyrin scaffolding has remained unclear. In this study, we identify and characterize two new posttranslational modifications of gephyrin, SUMOylation and acetylation. We demonstrate that crosstalk between SUMOylation, acetylation and phosphorylation pathways regulates gephyrin scaffolding. Pharmacological intervention of SUMO pathway or transgenic expression of SUMOylation-deficient gephyrin variants rescued gephyrin clustering in CA1 or neocortical neurons of Gabra2-null mice, which otherwise lack gephyrin clusters, indicating that gephyrin SUMO modification is an essential determinant for scaffolding at GABAergic synapses. Together, our results demonstrate that concerted modifications on a protein scaffold by evolutionarily conserved yet functionally diverse signalling pathways facilitate GABAergic transmission.