The functional genetic link of NLGN4X knockdown and neurodevelopment in neural stem cells.

Genetic mutations in NLGN4X (neuroligin 4), including point mutations and copy number variants (CNVs), have been associated with susceptibility to autism spectrum disorders (ASDs). However, it is unclear how mutations in NLGN4X result in neurodevelopmental defects. Here, we used neural stem cells (NSCs) as in vitro models to explore the impacts of NLGN4X knockdown on neurodevelopment. Using two shRNAmir-based vectors targeting NLGN4X and one control shRNAmir vector, we modulated NLGN4X expression and differentiated these NSCs into mature neurons. We monitored the neurodevelopmental process at Weeks 0, 0.5, 1, 2, 4 and 6, based on morphological analysis and whole-genome gene expression profiling. At the cellular level, in NSCs with NLGN4X knockdown, we observed increasingly delayed neuronal development and compromised neurite formation, starting from Week 2 through Week 6 post differentiation. At the molecular level, we identified multiple pathways, such as neurogenesis, neuron differentiation and muscle development, which are increasingly disturbed in cells with NLGN4X knockdown. Notably, several postsynaptic genes, including DLG4, NLGN1 and NLGN3, also have decreased expression. Based on in vitro models, NLGN4X knockdown directly impacts neurodevelopmental process during the formation of neurons and their connections. Our functional genomics study highlights the utility of NSCs models in understanding the functional roles of CNVs in affecting neurodevelopment and conferring susceptibility to neurodevelopmental diseases.

Mutations in the postsynaptic density signaling hub TNIK disrupt PSD signaling in human models of neurodevelopmental disorders.

A large number of synaptic proteins have been recurrently associated with complex brain disorders. One of these proteins, the Traf and Nck interacting kinase (TNIK), is a postsynaptic density (PSD) signaling hub, with many variants reported in neurodevelopmental disorder (NDD) and psychiatric disease. While rodent models of TNIK dysfunction have abnormal spontaneous synaptic activity and cognitive impairment, the role of mutations found in patients with TNIK protein deficiency and TNIK protein kinase activity during early stages of neuronal and synapse development has not been characterized. Here, using hiPSC-derived excitatory neurons, we show that TNIK mutations dysregulate neuronal activity in human immature synapses. Moreover, the lack of TNIK protein kinase activity impairs MAPK signaling and protein phosphorylation in structural components of the PSD. We show that the TNIK interactome is enriched in NDD risk factors and TNIK lack of function disrupts signaling networks and protein interactors associated with NDD that only partially overlap to mature mouse synapses, suggesting a differential role of TNIK in immature synapsis in NDD.

Human cerebellar organoids with functional Purkinje cells.

Research on human cerebellar development and disease has been hampered by the need for a human cell-based system that recapitulates the human cerebellum's cellular diversity and functional features. Here, we report a human organoid model (human cerebellar organoids [hCerOs]) capable of developing the complex cellular diversity of the fetal cerebellum, including a human-specific rhombic lip progenitor population that have never been generated in vitro prior to this study. 2-month-old hCerOs form distinct cytoarchitectural features, including laminar organized layering, and create functional connections between inhibitory and excitatory neurons that display coordinated network activity. Long-term culture of hCerOs allows healthy survival and maturation of Purkinje cells that display molecular and electrophysiological hallmarks of their in vivo counterparts, addressing a long-standing challenge in the field. This study therefore provides a physiologically relevant, all-human model system to elucidate the cell-type-specific mechanisms governing cerebellar development and disease.

TNiK is required for postsynaptic and nuclear signaling pathways and cognitive function.

Traf2 and NcK interacting kinase (TNiK) contains serine-threonine kinase and scaffold domains and has been implicated in cell proliferation and glutamate receptor regulation in vitro. Here we report its role in vivo using mice carrying a knock-out mutation. TNiK binds protein complexes in the synapse linking it to the NMDA receptor (NMDAR) via AKAP9. NMDAR and metabotropic receptors bidirectionally regulate TNiK phosphorylation and TNiK is required for AMPA expression and synaptic function. TNiK also organizes nuclear complexes and in the absence of TNiK, there was a marked elevation in GSK3ß and phosphorylation levels of its cognate phosphorylation sites on NeuroD1 with alterations in Wnt pathway signaling. We observed impairments in dentate gyrus neurogenesis in TNiK knock-out mice and cognitive testing using the touchscreen apparatus revealed impairments in pattern separation on a test of spatial discrimination. Object-location paired associate learning, which is dependent on glutamatergic signaling, was also impaired. Additionally, TNiK knock-out mice displayed hyperlocomotor behavior that could be rapidly reversed by GSK3ß inhibitors, indicating the potential for pharmacological rescue of a behavioral phenotype. These data establish TNiK as a critical regulator of cognitive functions and suggest it may play a regulatory role in diseases impacting on its interacting proteins and complexes.

Functional and phosphoproteomic analysis of ß-adrenergic receptor signaling at excitatory synapses in the CA1 region of the ventral hippocampus.

Activation of ß-adrenergic receptors (ß-ARs) not only enhances learning and memory but also facilitates the induction of long-term potentiation (LTP), a form of synaptic plasticity involved in memory formation. To identify the mechanisms underlying ß-AR-dependent forms of LTP we examined the effects of the ß-AR agonist isoproterenol on LTP induction at excitatory synapses onto CA1 pyramidal cells in the ventral hippocampus. LTP induction at these synapses is inhibited by activation of SK-type K+ channels, suggesting that ß-AR activation might facilitate LTP induction by inhibiting SK channels. However, although the SK channel blocker apamin enhanced LTP induction, it did not fully mimic the effects of isoproterenol. We therefore searched for potential alternative mechanisms using liquid chromatography-tandem mass spectrometry to determine how ß-AR activation regulates phosphorylation of postsynaptic density (PSD) proteins. Strikingly, ß-AR activation regulated hundreds of phosphorylation sites in PSD proteins that have diverse roles in dendritic spine structure and function. Moreover, within the core scaffold machinery of the PSD, ß-AR activation increased phosphorylation at several sites previously shown to be phosphorylated after LTP induction. Together, our results suggest that ß-AR activation recruits a diverse set of signaling pathways that likely act in a concerted fashion to regulate LTP induction.

Non-synaptic function of the autism spectrum disorder-associated gene SYNGAP1 in cortical neurogenesis.

Genes involved in synaptic function are enriched among those with autism spectrum disorder (ASD)-associated rare genetic variants. Dysregulated cortical neurogenesis has been implicated as a convergent mechanism in ASD pathophysiology, yet it remains unknown how 'synaptic' ASD risk genes contribute to these phenotypes, which arise before synaptogenesis. Here, we show that the synaptic Ras GTPase-activating (RASGAP) protein 1 (SYNGAP1, a top ASD risk gene) is expressed within the apical domain of human radial glia cells (hRGCs). In a human cortical organoid model of SYNGAP1 haploinsufficiency, we find dysregulated cytoskeletal dynamics that impair the scaffolding and division plane of hRGCs, resulting in disrupted lamination and accelerated maturation of cortical projection neurons. Additionally, we confirmed an imbalance in the ratio of progenitors to neurons in a mouse model of Syngap1 haploinsufficiency. Thus, SYNGAP1-related brain disorders may arise through non-synaptic mechanisms, highlighting the need to study genes associated with neurodevelopmental disorders (NDDs) in diverse human cell types and developmental stages.

The SH3 domain of postsynaptic density 95 mediates inflammatory pain through phosphatidylinositol-3-kinase recruitment.

Sensitization to inflammatory pain is a pathological form of neuronal plasticity that is poorly understood and treated. Here we examine the role of the SH3 domain of postsynaptic density 95 (PSD95) by using mice that carry a single amino-acid substitution in the polyproline-binding site. Testing multiple forms of plasticity we found sensitization to inflammation was specifically attenuated. The inflammatory response required recruitment of phosphatidylinositol-3-kinase-C2alpha to the SH3-binding site of PSD95. In wild-type mice, wortmannin or peptide competition attenuated the sensitization. These results show that different types of behavioural plasticity are mediated by specific domains of PSD95 and suggest novel therapeutic avenues for reducing inflammatory pain.

Knockdown of mental disorder susceptibility genes disrupts neuronal network physiology in vitro.

Schizophrenia and bipolar disorder are common diseases caused by multiple genes that disrupt brain circuits. While great progress has been made in identifying schizophrenia susceptibility genes, these studies have left two major unanswered mechanistic questions: is there a core biochemical mechanism that these genes regulate, and what are the electrophysiological consequences of the altered gene expression? Because clinical studies implicate abnormalities in neuronal networks, we developed a system for studying the neurophysiology of neuronal networks in vitro where the role of candidate disease genes can be rapidly assayed. Using this system we focused on three postsynaptic proteins DISC1, TNIK and PSD-93/DLG2 each of which is encoded by a schizophrenia susceptibility gene. We also examined the utility of this assay system in bipolar disorder (BD), which has a strong genetic overlap with schizophrenia, by examining the bipolar disorder susceptibility gene Dctn5. The global neuronal network firing behavior of primary cultures of mouse hippocampus neurons was examined on multi-electrode arrays (MEAs) and genes of interest were knocked down using RNAi interference. Measurement of multiple neural network parameters demonstrated phenotypes for these genes compared with controls. Moreover, the different genes disrupted network properties and showed distinct and overlapping effects. These data show multiple susceptibility genes for complex psychiatric disorders, regulate neural network physiology and demonstrate a new assay system with wide application.

A "multi-omics" analysis of blood-brain barrier and synaptic dysfunction in APOE4 mice.

Apolipoprotein E4 (APOE4), the main susceptibility gene for Alzheimer's disease, leads to blood-brain barrier (BBB) breakdown in humans and mice. Remarkably, BBB dysfunction predicts cognitive decline and precedes synaptic deficits in APOE4 human carriers. How APOE4 affects BBB and synaptic function at a molecular level, however, remains elusive. Using single-nucleus RNA-sequencing and phosphoproteome and proteome analysis, we show that APOE4 compared with APOE3 leads to an early disruption of the BBB transcriptome in 2-3-mo-old APOE4 knock-in mice, followed by dysregulation in protein signaling networks controlling cell junctions, cytoskeleton, clathrin-mediated transport, and translation in brain endothelium, as well as transcription and RNA splicing suggestive of DNA damage in pericytes. Changes in BBB signaling mechanisms paralleled an early, progressive BBB breakdown and loss of pericytes, which preceded postsynaptic interactome disruption and behavioral deficits that developed 2-5 mo later. Thus, dysregulated signaling mechanisms in endothelium and pericytes in APOE4 mice reflect a molecular signature of a progressive BBB failure preceding changes in synaptic function and behavior.

Endogenous Syngap1 alpha splice forms promote cognitive function and seizure protection.

Loss-of-function variants in SYNGAP1 cause a developmental encephalopathy defined by cognitive impairment, autistic features, and epilepsy. SYNGAP1 splicing leads to expression of distinct functional protein isoforms. Splicing imparts multiple cellular functions of SynGAP proteins through coding of distinct C-terminal motifs. However, it remains unknown how these different splice sequences function in vivo to regulate neuronal function and behavior. Reduced expression of SynGAP-α1/2 C-terminal splice variants in mice caused severe phenotypes, including reduced survival, impaired learning, and reduced seizure latency. In contrast, upregulation of α1/2 expression improved learning and increased seizure latency. Mice expressing α1-specific mutations, which disrupted SynGAP cellular functions without altering protein expression, promoted seizure, disrupted synapse plasticity, and impaired learning. These findings demonstrate that endogenous SynGAP isoforms with α1/2 spliced sequences promote cognitive function and impart seizure protection. Regulation of SynGAP-αexpression or function may be a viable therapeutic strategy to broadly improve cognitive function and mitigate seizure.

Genome Sequencing Variations in the Octodon degus, an Unconventional Natural Model of Aging and Alzheimer's Disease.

The degu (Octodon degus) is a diurnal long-lived rodent that can spontaneously develop molecular and behavioral changes that mirror those seen in human aging. With age some degu, but not all individuals, develop cognitive decline and brain pathology like that observed in Alzheimer's disease including neuroinflammation, hyperphosphorylated tau and amyloid plaques, together with other co-morbidities associated with aging such as macular degeneration, cataracts, alterations in circadian rhythm, diabetes and atherosclerosis. Here we report the whole-genome sequencing and analysis of the degu genome, which revealed unique features and molecular adaptations consistent with aging and Alzheimer's disease. We identified single nucleotide polymorphisms in genes associated with Alzheimer's disease including a novel apolipoprotein E (Apoe) gene variant that correlated with an increase in amyloid plaques in brain and modified the in silico predicted degu APOE protein structure and functionality. The reported genome of an unconventional long-lived animal model of aging and Alzheimer's disease offers the opportunity for understanding molecular pathways involved in aging and should help advance biomedical research into treatments for Alzheimer's disease.

A novel role for PSD-95 in mediating ethanol intoxication, drinking and place preference.

The synaptic signaling mechanisms mediating the behavioral effects of ethanol (EtOH) remain poorly understood. Post-synaptic density 95 (PSD-95, SAP-90, Dlg4) is a key orchestrator of N-methyl-D-aspartate receptors (NMDAR) and glutamatergic synapses, which are known to be major sites of EtOH's behavioral actions. However, the potential contribution of PSD-95 to EtOH-related behaviors has not been established. Here, we evaluated knockout (KO) mice lacking PSD-95 for multiple measures of sensitivity to the acute intoxicating effects of EtOH (ataxia, hypothermia, sedation/hypnosis), EtOH drinking under conditions of free access and following deprivation, acquisition and long-term retention of EtOH conditioned place preference (CPP) (and lithium chloride-induced conditioned taste aversion), and intoxication-potentiating responses to NMDAR antagonism. PSD-95 KO exhibited increased sensitivity to the sedative/hypnotic, but not ataxic or hypothermic, effects of acute EtOH relative to wild-type controls (WT). PSD-95 KO consumed less EtOH than WT, particularly at higher EtOH concentrations, although increases in KO drinking could be induced by concentration-fading and deprivation. PSD-95 KO showed normal EtOH CPP 1 day after conditioning, but showed significant aversion 2 weeks later. Lithium chloride-induced taste aversion was impaired in PSD-95 KO at both time points. Finally, the EtOH-potentiating effects of the NMDAR antagonist MK-801 were intact in PSD-95 KO at the dose tested. These data reveal a major, novel role for PSD-95 in mediating EtOH behaviors, and add to growing evidence that PSD-95 is a key mediator of the effects of multiple abused drugs.

Molecular architecture of postsynaptic Interactomes.

The postsynaptic density (PSD) plays an essential role in the organization of the synaptic signaling machinery. It contains a set of core scaffolding proteins that provide the backbone to PSD protein-protein interaction networks (PINs). These core scaffolding proteins can be seen as three principal layers classified by protein family, with DLG proteins being at the top, SHANKs along the bottom, and DLGAPs connecting the two layers. Early studies utilizing yeast two hybrid enabled the identification of direct protein-protein interactions (PPIs) within the multiple layers of scaffolding proteins. More recently, mass-spectrometry has allowed the characterization of whole interactomes within the PSD. This expansion of knowledge has further solidified the centrality of core scaffolding family members within synaptic PINs and provided context for their role in neuronal development and synaptic function. Here, we discuss the scaffolding machinery of the PSD, their essential functions in the organization of synaptic PINs, along with their relationship to neuronal processes found to be impaired in complex brain disorders.

Regulatory mechanisms in postsynaptic phosphorylation networks.

The modulation of the postsynaptic signaling machinery by protein phosphorylation has attracted much interest since it is key for the understanding of the regulation of a variety of synaptic functions. While advances in mass spectrometry have allowed us to begin performing large-scale analysis of protein phosphorylation in components of the PSD, the systematic collection of datasets and their functional significance within the context of regulatory signaling networks is in its infancy. Here, we will focus on the composition of the PSD phosphoproteome describing kinase, phosphatase, and protein domain modules involved in the regulation of phosphorylation signaling. We will discuss the impact of synaptic plasticity mechanisms such as long-term potentiation (LTP) in mammalian kinomes and describe the general rules of signaling organization in the PSD phosphoproteome.

Endogenous Cell Type-Specific Disrupted in Schizophrenia 1 Interactomes Reveal Protein Networks Associated With Neurodevelopmental Disorders.

BACKGROUND: Disrupted in schizophrenia 1 (DISC1) has been implicated in a number of psychiatric diseases along with neurodevelopmental phenotypes such as the proliferation and differentiation of neural progenitor cells. While there has been significant effort directed toward understanding the function of DISC1 through the determination of its protein-protein interactions within an in vitro setting, endogenous interactions involving DISC1 within a cell type-specific setting relevant to neural development remain unclear. METHODS: Using CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9) genome engineering technology, we inserted an endogenous 3X-FLAG tag at the C-terminus of the canonical DISC1 gene in human induced pluripotent stem cells (iPSCs). We further differentiated these cells and used affinity purification to determine protein-protein interactions involving DISC1 in iPSC-derived neural progenitor cells and astrocytes. RESULTS: We were able to determine 151 novel cell type-specific proteins present in DISC1 endogenous interactomes. The DISC1 interactomes can be clustered into several subcomplexes that suggest novel DISC1 cell-specific functions. In addition, the DISC1 interactome in iPSC-derived neural progenitor cells associates in a connected network containing proteins found to harbor de novo mutations in patients affected by schizophrenia and contains a subset of novel interactions that are known to harbor syndromic mutations in neurodevelopmental disorders. CONCLUSIONS: Endogenous DISC1 interactomes within iPSC-derived human neural progenitor cells and astrocytes are able to provide context to DISC1 function in a cell type-specific setting relevant to neural development and enables the integration of psychiatric disease risk factors within a set of defined molecular functions.

Gene-environment interactions in antisocial behavior are mediated by early-life 5-HT2A receptor activation.

The ontogeny of antisocial behavior (ASB) is rooted in complex gene-environment (G×E) interactions. The best-characterized of these interplays occurs between: a) low-activity alleles of the gene encoding monoamine oxidase A (MAOA), the main serotonin-degrading enzyme; and b) child maltreatment. The purpose of this study was to develop the first animal model of this G×E interaction, to help understand the neurobiological mechanisms of ASB and identify novel targets for its therapy. Maoa hypomorphic transgenic mice were exposed to an early-life stress regimen consisting of maternal separation and daily intraperitoneal saline injections and were then compared with their wild-type and non-stressed controls for ASB-related neurobehavioral phenotypes. Maoa hypomorphic mice subjected to stress from postnatal day (PND) 1 through 7 - but not during the second postnatal week - developed overt aggression, social deficits and abnormal stress responses from the fourth week onwards. On PND 8, these mice exhibited low resting heart rate - a well-established premorbid sign of ASB - and a significant and selective up-regulation of serotonin 5-HT2A receptors in the prefrontal cortex. Notably, both aggression and neonatal bradycardia were rescued by the 5-HT2 receptor antagonist ketanserin (1-3 mg kg-1, IP), as well as the selective 5-HT2A receptor blocker MDL-100,907 (volinanserin, 0.1-0.3 mg kg-1, IP) throughout the first postnatal week. These findings provide the first evidence of a molecular basis of G×E interactions in ASB and point to early-life 5-HT2A receptor activation as a key mechanism for the ontogeny of this condition. This article is part of the Special Issue entitled 'The neuropharmacology of social behavior: from bench to bedside'.

SynGO: An Evidence-Based, Expert-Curated Knowledge Base for the Synapse.

Synapses are fundamental information-processing units of the brain, and synaptic dysregulation is central to many brain disorders ("synaptopathies"). However, systematic annotation of synaptic genes and ontology of synaptic processes are currently lacking. We established SynGO, an interactive knowledge base that accumulates available research about synapse biology using Gene Ontology (GO) annotations to novel ontology terms: 87 synaptic locations and 179 synaptic processes. SynGO annotations are exclusively based on published, expert-curated evidence. Using 2,922 annotations for 1,112 genes, we show that synaptic genes are exceptionally well conserved and less tolerant to mutations than other genes. Many SynGO terms are significantly overrepresented among gene variations associated with intelligence, educational attainment, ADHD, autism, and bipolar disorder and among de novo variants associated with neurodevelopmental disorders, including schizophrenia. SynGO is a public, universal reference for synapse research and an online analysis platform for interpretation of large-scale -omics data (https://syngoportal.org and http://geneontology.org).

Synapse-associated protein 102/dlgh3 couples the NMDA receptor to specific plasticity pathways and learning strategies.

Understanding the mechanisms whereby information encoded within patterns of action potentials is deciphered by neurons is central to cognitive psychology. The multiprotein complexes formed by NMDA receptors linked to synaptic membrane-associated guanylate kinase (MAGUK) proteins including synapse-associated protein 102 (SAP102) and other associated proteins are instrumental in these processes. Although humans with mutations in SAP102 show mental retardation, the physiological and biochemical mechanisms involved are unknown. Using SAP102 knock-out mice, we found specific impairments in synaptic plasticity induced by selective frequencies of stimulation that also required extracellular signal-regulated kinase signaling. This was paralleled by inflexibility and impairment in spatial learning. Improvement in spatial learning performance occurred with extra training despite continued use of a suboptimal search strategy, and, in a separate nonspatial task, the mutants again deployed a different strategy. Double-mutant analysis of postsynaptic density-95 and SAP102 mutants indicate overlapping and specific functions of the two MAGUKs. These in vivo data support the model that specific MAGUK proteins couple the NMDA receptor to distinct downstream signaling pathways. This provides a mechanism for discriminating patterns of synaptic activity that lead to long-lasting changes in synaptic strength as well as distinct aspects of cognition in the mammalian nervous system.

NMDA receptor activation dephosphorylates AMPA receptor glutamate receptor 1 subunits at threonine 840.

Phosphorylation-dependent changes in AMPA receptor function have a crucial role in activity-dependent forms of synaptic plasticity such as long-term potentiation (LTP) and long-term depression (LTD). Although three previously identified phosphorylation sites in AMPA receptor glutamate receptor 1 (GluR1) subunits (S818, S831, and S845) appear to have important roles in LTP and LTD, little is known about the role of other putative phosphorylation sites in GluR1. Here, we describe the characterization of a recently identified phosphorylation site in GluR1 at threonine 840. The results of in vivo and in vitro phosphorylation assays suggest that T840 is not a substrate for protein kinases known to phosphorylate GluR1 at previously identified phosphorylation sites, such as protein kinase A, protein kinase C, and calcium/calmodulin-dependent kinase II. Instead, in vitro phosphorylation assays suggest that T840 is a substrate for p70S6 kinase. Although LTP-inducing patterns of synaptic stimulation had no effect on GluR1 phosphorylation at T840 in the hippocampal CA1 region, bath application of NMDA induced a strong, protein phosphatase 1- and/or 2A-mediated decrease in T840 phosphorylation. Moreover, GluR1 phosphorylation at T840 was transiently decreased by a chemical LTD induction protocol that induced a short-term depression of synaptic strength and persistently decreased by a chemical LTD induction protocol that induced a lasting depression of synaptic transmission. Together, our results show that GluR1 phosphorylation at T840 is regulated by NMDA receptor activation and suggest that decreases in GluR1 phosphorylation at T840 may have a role in LTD.

Haploinsufficiency leads to neurodegeneration in C9ORF72 ALS/FTD human induced motor neurons.

An intronic GGGGCC repeat expansion in C9ORF72 is the most common cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), but the pathogenic mechanism of this repeat remains unclear. Using human induced motor neurons (iMNs), we found that repeat-expanded C9ORF72 was haploinsufficient in ALS. We found that C9ORF72 interacted with endosomes and was required for normal vesicle trafficking and lysosomal biogenesis in motor neurons. Repeat expansion reduced C9ORF72 expression, triggering neurodegeneration through two mechanisms: accumulation of glutamate receptors, leading to excitotoxicity, and impaired clearance of neurotoxic dipeptide repeat proteins derived from the repeat expansion. Thus, cooperativity between gain- and loss-of-function mechanisms led to neurodegeneration. Restoring C9ORF72 levels or augmenting its function with constitutively active RAB5 or chemical modulators of RAB5 effectors rescued patient neuron survival and ameliorated neurodegenerative processes in both gain- and loss-of-function C9ORF72 mouse models. Thus, modulating vesicle trafficking was able to rescue neurodegeneration caused by the C9ORF72 repeat expansion. Coupled with rare mutations in ALS2, FIG4, CHMP2B, OPTN and SQSTM1, our results reveal mechanistic convergence on vesicle trafficking in ALS and FTD.