Highlights d We establish a single-cell model to study synapses in iPSCderived neurons d This platform allows quantitative analysis of synaptic transmission and plasticity d The platform is validated for GABA-or glutamatergic iPSCderived human neurons d The platform is scalable and suitable for compound screening and disease modeling

Heterozygous mutations or deletions in the human Euchromatin histone methyltransferase 1 (EHMT1) gene cause Kleefstra syndrome, a neurodevelopmental disorder that is characterized by autistic-like features and severe intellectual disability (ID). Neurodevelopmental disorders including ID and autism may be related to deficits in activity-dependent wiring of brain circuits during development. Although Kleefstra syndrome has been associated with dendritic and synaptic defects in mice and Drosophila, little is known about the role of EHMT1 in the development of cortical neuronal networks. Here we used micro-electrode arrays and whole-cell patch-clamp recordings to investigate the impact of EHMT1 deficiency at the network and single cell level. We show that EHMT1 deficiency impaired neural network activity during the transition from uncorrelated background action potential firing to synchronized network bursting. Spontaneous bursting and excitatory synaptic currents were transiently reduced, whereas miniature excitatory postsynaptic currents were not affected. Finally, we show that loss of function of EHMT1 ultimately resulted in less regular network bursting patterns later in development. These data suggest that the developmental impairments observed in EHMT1-deficient networks may result in a temporal misalignment between activity-dependent developmental processes thereby contributing to the pathophysiology of Kleefstra syndrome.

The interaction of Munc18-1 helix 11 and 12 with the central region of the VAMP2 SNARE motif is essential for SNARE templating and synaptic transmission

Phosphorylation of Munc18 -1 (Stxbp1), a presynaptic organizer of synaptic vesicle fusion, is a powerful mechanism to regulate synaptic strength. Munc18-1 is a proposed substrate for the Down Syndromerelated kinase dual-specificity tyrosine phosphorylation-regulate kinase 1a (Dyrk1a) and mutations in both genes cause intellectual disability. However, the functional consequences of Dyrk1a-dependent phosphorylation of Munc18-1 for synapse function are unknown. Here, we show that the proposed Munc18-1 phosphorylation site, T479, is among the highly constrained phosphorylation sites in the coding regions of the gene and is also located within a larger constrained coding region. We confirm that Dyrk1a phosphorylates Munc18-1 at T479. Patch-clamp physiology in conditional null mutant hippocampal neurons expressing Cre and either wildtype, or mutants mimicking or preventing phosphorylation, revealed that synaptic transmission is similar among the three groups: frequency/ amplitude of mEPSCs, evoked EPSCs, paired pulse plasticity, rundown kinetics upon intense activity and the readily releasable pool. However, synapses expressing the phosphomimic mutant responded to intense activity with more pronounced facilitation. These data indicate that Dyrk1a-dependent Munc18-1 phosphorylation has a minor impact on synaptic transmission, only after intense activity, and that the role of genetic variation in both genes in intellectual disability may be through different mechanisms.Post-translational modification of synaptic proteins is a powerful way to regulate synaptic strength. Phosphorylation is probably the most well-studied mechanism of regulation of synaptic protein function. Many synaptic proteins have been identified in large-scale phospho-proteomics screens 1-4 , although the kinase and the function of the phosphorylation are often unknown. Presynaptic proteins (e.g. Synapsin, SNAP25, synaptotagmins, and Munc18) are phosphorylated by abundant (not neuro-specific) kinases like protein kinase C (PKC) and protein kinase A (PKA) and these modifications impact on synaptic transmission in a variety of ways (see review 5 ). However, many more (potential) phosphorylation sites in synaptic proteins are reported, for which the impact is unknown.Munc18-1 is a major regulator of synaptic transmission and among the best validated presynaptic phosphorylation targets. Loss of Munc18-1 in neurons leads to a loss of neurotransmitter release and cell death in vivo 6 and in vitro 7 . Munc18-1 null neurons survive for 4 days in culture but are subsequently unable to support viability 7 . The MUNC-18 gene in human is STXBP1, and exonic mutations in STXBP1 have been reported to cause STXBP1-encephalopathy, characterized by severe intellectual disability (ID), spasms, and epilepsy [8][9][10][11] . Many of these clinical symptoms are also observed in Stxbp1 mouse models of STXBP1-encephalopathy 12 .Several potential phosphorylation sites of Munc18-1 have been found 1 or predicted 13 . To date, all confirmed phosphorylation sites have major impact...