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.

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.

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.

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.

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.

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.