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Heterozygous loss-of-function mutations in SHANK2 are associated with autism spectrum disorder (ASD). We generated cortical neurons from induced pluripotent stem cells derived from neurotypic and ASD-affected donors. We developed sparse coculture for connectivity assays where SHANK2 and control neurons were differentially labeled and sparsely seeded together on a lawn of unlabeled control neurons. We observed increases in dendrite length, dendrite complexity, synapse number, and frequency of spontaneous excitatory postsynaptic currents. These findings were phenocopied in gene-edited homozygous SHANK2 knockout cells and rescued by gene correction of an ASD SHANK2 mutation. Dendrite length increases were exacerbated by IGF1, TG003, or BDNF, and suppressed by DHPG treatment. The transcriptome in isogenic SHANK2 neurons was perturbed in synapse, plasticity, and neuronal morphogenesis gene sets and ASD gene modules, and activity-dependent dendrite extension was impaired. Our findings provide evidence for hyperconnectivity and altered transcriptome in SHANK2 neurons derived from ASD subjects.Using a novel assay, Ellis et al. show that stem cell-derived neurons from individuals with autism carrying SHANK2 mutations are hyperconnected, have impaired activity-dependent dendrite extension, and have perturbed transcription of ASD gene modules.

Activation of inflammasomes leads to maturation of the pro-inflammatory cytokine IL-1β; this study adds DNA to the growing list of exogenous and endogenous inflammasome activators. The innate immune system recognizes nucleic acids during infection and tissue damage. Whereas viral RNA is detected by endosomal toll-like receptors (TLR3, TLR7, TLR8) and cytoplasmic RIG-I and MDA5, endosomal TLR9 and cytoplasmic DAI bind DNA 1 , resulting in the activation of nuclear factor-κB and interferon regulatory factor transcription factors. However, viruses also trigger pro-inflammatory responses 2 , which remain poorly defined. Here we show that internalized adenoviral DNA induces maturation of pro-interleukin-1β in macrophages, which is dependent on NALP3 and ASC, components of the innate cytosolic molecular complex termed the inflammasome. Correspondingly, NALP3- and ASC-deficient mice display reduced innate inflammatory responses to adenovirus particles. Inflammasome activation also occurs as a result of transfected cytosolic bacterial, viral and mammalian (host) DNA, but in this case sensing is dependent on ASC but not NALP3. The DNA-sensing pro-inflammatory pathway functions independently of TLRs and interferon regulatory factors. Thus, in addition to viral and bacterial components or danger signals in general, inflammasomes sense potentially dangerous cytoplasmic DNA, strengthening their central role in innate immunity.

NMDA receptors (NMDARs), a prominent subtype of glutamatergic receptors, are implicated in the pathogenesis and development of neuropsychiatric disorders such as epilepsy, intellectual disability, autism spectrum disorder, and schizophrenia, and are therefore a potential therapeutic target in treating these disorders. Neurons derived from induced pluripotent stem cells (iPSCs) have provided the opportunity to investigate human NMDARs in their native environment. In this review, we describe the expression, function, and regulation of NMDARs in human iPSC-derived neurons and discuss approaches for utilizing human neurons for identifying potential drugs that target NMDARs in the treatment of neuropsychiatric disorders. A challenge in studying NMDARs in human iPSC-derived neurons is a predominance of those receptors containing the GluN2B subunit and low synaptic expression, suggesting a relatively immature phenotype of these neurons and delayed development of functional NMDARs. We outline potential approaches for improving neuronal maturation of human iPSC-derived neurons and accelerating the functional expression of NMDARs. Acceleration of functional expression of NMDARs in human iPSC-derived neurons will improve the modeling of neuropsychiatric disorders and facilitate the discovery and development of novel therapeutics targeting NMDARs for the treatment of these disorders.

Autism spectrum disorder (ASD) is an early-onset neurodevelopmental disorder characterized by deficits in social communication, and restricted and repetitive patterns of behavior. Despite its high prevalence, discovery of pathophysiological mechanisms underlying ASD has lagged due to a lack of appropriate model systems. Recent advances in induced pluripotent stem cell (iPSC) technology and neural differentiation techniques allow for detailed functional analyses of neurons generated from living individuals with ASD. Refinement of cortical neuron differentiation methods from iPSCs will enable mechanistic studies of specific neuronal subpopulations that may be preferentially impaired in ASD. In this review, we summarize recent accomplishments in differentiation of cortical neurons from human pluripotent stems cells and efforts to establish in vitro model systems to study ASD using personalized neurons.

Remodeling of cortical connectivity is thought to allow initially hippocampus-dependent memories to be expressed independently of the hippocampus at remote time points. Consistent with this, consolidation of a contextual fear memory is associated with dendritic spine growth in neurons of the anterior cingulate cortex (aCC). To directly test whether such cortical structural remodeling is necessary for memory consolidation, we disrupted spine growth in the aCC at different times following contextual fear conditioning in mice. We took advantage of previous studies showing that the transcription factor myocyte enhancer factor 2 (MEF2) negatively regulates spinogenesis both in vitro and in vivo. We found that increasing MEF2-dependent transcription in the aCC during a critical posttraining window (but not at later time points) blocked both the consolidation-associated dendritic spine growth and subsequent memory expression. Together, these data strengthen the causal link between cortical structural remodeling and memory consolidation and, further, identify MEF2 as a key regulator of these processes.

The authors report that endogenous myocyte enhance factor 2 (MEF2) levels affect spatial and fear memory formation in adult mice. MEF2-induced memory disruption was rescued by interfering with AMPA receptor endocytosis. Memory formation is thought to be mediated by dendritic-spine growth and restructuring. Myocyte enhancer factor 2 (MEF2) restricts spine growth in vitro , suggesting that this transcription factor negatively regulates the spine remodeling necessary for memory formation. Here we show that memory formation in adult mice was associated with changes in endogenous MEF2 levels and function. Locally and acutely increasing MEF2 function in the dentate gyrus blocked both learning-induced increases in spine density and spatial-memory formation. Increasing MEF2 function in amygdala disrupted fear-memory formation. We rescued MEF2-induced memory disruption by interfering with AMPA receptor endocytosis, suggesting that AMPA receptor trafficking is a key mechanism underlying the effects of MEF2. In contrast, decreasing MEF2 function in dentate gyrus and amygdala facilitated the formation of spatial and fear memory, respectively. These bidirectional effects indicate that MEF2 is a key regulator of plasticity and that relieving the suppressive effects of MEF2-mediated transcription permits memory formation.

Background The X-linked PTCHD1 locus is strongly associated with autism spectrum disorder (ASD). Males who carry chromosome microdeletions of PTCHD1 antisense long non-coding RNA ( PTCHD1-AS )/DEAD-box helicase 53 ( DDX53 ) have ASD, or a sub-clinical form called Broader Autism Phenotype. If the deletion extends beyond PTCHD1-AS / DDX53 to the next gene, PTCHD1 , which is protein-coding, the individuals typically have ASD and intellectual disability (ID). Three male siblings with a 90 kb deletion that affects only PTCHD1-AS (and not including DDX53 ) have ASD. We performed a functional analysis of DDX53 to examine its role in NGN2 neurons. Methods We used the clustered regularly interspaced short palindromic repeats (CRISPR) gene editing strategy to knock out DDX53 protein by inserting 3 termination codons (3TCs) into two different induced pluripotent stem cell (iPSC) lines. DDX53 CRISPR-edited iPSCs were differentiated into cortical excitatory neurons by Neurogenin 2 (NGN-2) directed differentiation. The functional differences of DDX53 -3TC neurons compared to isogenic control neurons with molecular and electrophysiological approaches were assessed. Results Isogenic iPSC-derived control neurons exhibited low levels of DDX53 transcripts. Transcriptional analysis revealed the generation of excitatory cortical neurons and DDX53 protein was not detected in iPSC-derived control neurons by western blot. Control lines and DDX53 -3TC neurons were active in the multi-electrode array, but no overt electrophysiological phenotype in either isogenic line was observed. Conclusion DDX53 -3TC mutation does not alter NGN2 neuronal function in these experiments, suggesting that synaptic deficits causing ASD are unlikely in this cell type.

Genetic factors contribute to the development of autism spectrum disorder (ASD), and although non-protein-coding regions of the genome are being increasingly implicated in ASD, the functional consequences of these variants remain largely uncharacterized. Induced pluripotent stem cells (iPSCs) enable the production of personalized neurons that are genetically matched to people with ASD and can therefore be used to directly test the effects of genomic variation on neuronal gene expression, synapse function, and connectivity. The combined use of human pluripotent stem cells with genome editing to introduce or correct specific variants has proved to be a powerful approach for exploring the functional consequences of ASD-associated variants in protein-coding genes and, more recently, long non-coding RNAs (lncRNAs). Here, we review recent studies that implicate lncRNAs, other non-coding mutations, and regulatory variants in ASD susceptibility. We also discuss experimental design considerations for using iPSCs and genome editing to study the role of the non-protein-coding genome in ASD.

This study identifies MBNL proteins as negative regulators of alternative splicing events that are differentially regulated between ES cells and other cell types; several lines of evidence show that these proteins repress an ES cell alternative splicing program and the reprogramming of somatic cells to induced pluripotent stem cells. MBNL proteins and pluripotency Ben Blencowe and colleagues identify the muscleblind-like RNA binding proteins MBNL1 and MBNL2 as negative regulators of alternative splicing events that are differentially regulated between embryonic stem cells and other cell types. Several lines of evidence show that they are involved in the regulation of embryonic-stem-cell-like alternative splicing patterns. The authors also identify a regulatory role during the reprogramming of fibroblasts to induced pluripotent stem (iPS) cells. Previous investigations of the core gene regulatory circuitry that controls the pluripotency of embryonic stem (ES) cells have largely focused on the roles of transcription, chromatin and non-coding RNA regulators 1 , 2 , 3 . Alternative splicing represents a widely acting mode of gene regulation 4 , 5 , 6 , 7 , 8 , yet its role in regulating ES-cell pluripotency and differentiation is poorly understood. Here we identify the muscleblind-like RNA binding proteins, MBNL1 and MBNL2, as conserved and direct negative regulators of a large program of cassette exon alternative splicing events that are differentially regulated between ES cells and other cell types. Knockdown of MBNL proteins in differentiated cells causes switching to an ES-cell-like alternative splicing pattern for approximately half of these events, whereas overexpression of MBNL proteins in ES cells promotes differentiated-cell-like alternative splicing patterns. Among the MBNL-regulated events is an ES-cell-specific alternative splicing switch in the forkhead family transcription factor FOXP1 that controls pluripotency 9 . Consistent with a central and negative regulatory role for MBNL proteins in pluripotency, their knockdown significantly enhances the expression of key pluripotency genes and the formation of induced pluripotent stem cells during somatic cell reprogramming.

NMDA receptor (NMDAR)-mediated fast excitatory neurotransmission is implicated in a broad range of physiological and pathological processes in the mammalian central nervous system. The function and regulation of NMDARs have been extensively studied in neurons from rodents and other non-human species and in recombinant expression systems. Here, we investigated human NMDARs in situ by using neurons produced by directed differentiation of human induced pluripotent stem cells (iPSCs). The resultant cells showed electrophysiological characteristics demonstrating that they are bona fide neurons. In particular, human iPSC-derived neurons expressed functional ligand-gated ion channels, including NMDARs, AMPA receptors, GABA A receptors, as well as glycine receptors. Pharmacological and electrophysiological properties of NMDAR-mediated currents indicated that these were dominated by receptors containing GluN2B subunits. The NMDAR currents were suppressed by genistein, a broad-spectrum tyrosine kinase inhibitor. The NMDAR currents were also inhibited by a Fyn-interfering peptide, Fyn(39–57), but not a Src-interfering peptide, Src(40–58). Together, these findings are the first evidence that tyrosine phosphorylation regulates the function of NMDARs in human iPSC-derived neurons. Our findings provide a basis for utilizing human iPSC-derived neurons in screening for drugs targeting NMDARs in neurological disorders.