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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.
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Moléculas de Adhesión Celular Neuronal/genética , Moléculas de Adhesión Celular Neuronal/metabolismo , Células-Madre Neurales/fisiología , Neurogénesis , Neuronas/fisiología , Diferenciación Celular/genética , Células Cultivadas , Trastornos Generalizados del Desarrollo Infantil/genética , Variaciones en el Número de Copia de ADN , Perfilación de la Expresión Génica , Regulación del Desarrollo de la Expresión Génica , Técnicas de Silenciamiento del Gen , Humanos , Desarrollo de Músculos , Neuritas/fisiología , Mutación PuntualRESUMEN
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.
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Cerebelo , Células de Purkinje , Humanos , Lactante , Metencéfalo , OrganoidesRESUMEN
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.
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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.
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Aprendizaje por Asociación/fisiología , Trastornos del Conocimiento/enzimología , Giro Dentado/enzimología , Aprendizaje Discriminativo/fisiología , Proteínas del Tejido Nervioso/fisiología , Densidad Postsináptica/enzimología , Proteínas Serina-Treonina Quinasas/fisiología , Detección de Señal Psicológica/fisiología , Percepción Espacial/fisiología , Animales , Núcleo Celular/enzimología , Trastornos del Conocimiento/fisiopatología , Giro Dentado/patología , Ácido Glutámico/fisiología , Glucógeno Sintasa Quinasa 3/antagonistas & inhibidores , Glucógeno Sintasa Quinasa 3/fisiología , Glucógeno Sintasa Quinasa 3 beta , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Potenciales Postsinápticos Miniatura/fisiología , Proteínas del Tejido Nervioso/deficiencia , Neurogénesis/fisiología , Fenotipo , Fosforilación , Densidad Postsináptica/fisiología , Procesamiento Proteico-Postraduccional , Proteínas Serina-Treonina Quinasas/biosíntesis , Proteínas Serina-Treonina Quinasas/deficiencia , Proteínas Serina-Treonina Quinasas/genética , Proteínas Recombinantes de Fusión/fisiologíaRESUMEN
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.
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Receptores Adrenérgicos beta , Transducción de Señal , Isoproterenol/farmacología , Hipocampo , Potenciación a Largo PlazoRESUMEN
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.
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Trastorno del Espectro Autista , Trastornos del Neurodesarrollo , Animales , Ratones , Humanos , Trastorno del Espectro Autista/genética , Proteínas Activadoras de ras GTPasa/genética , Trastornos del Neurodesarrollo/genética , Fenotipo , Neurogénesis/genéticaRESUMEN
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.
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Trastorno Bipolar/genética , Trastornos Mentales/genética , Red Nerviosa/fisiología , Neuronas/fisiología , Esquizofrenia/genética , Animales , Células Cultivadas , Técnicas de Silenciamiento del Gen , Predisposición Genética a la Enfermedad , Quinasas del Centro Germinal , Guanilato-Quinasas/genética , Hipocampo/citología , Hipocampo/fisiología , Humanos , Ratones , Proteínas del Tejido Nervioso/genética , Neuronas/citología , Proteínas Serina-Treonina Quinasas/genética , Interferencia de ARN , Proteínas Supresoras de Tumor/genéticaRESUMEN
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.
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Enfermedad de Alzheimer , Apolipoproteína E4 , Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/metabolismo , Animales , Apolipoproteína E3/genética , Apolipoproteína E3/metabolismo , Apolipoproteína E4/genética , Apolipoproteína E4/metabolismo , Barrera Hematoencefálica/metabolismo , Humanos , Ratones , Ratones Transgénicos , PericitosRESUMEN
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.
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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.
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Consumo de Bebidas Alcohólicas/genética , Intoxicación Alcohólica/genética , Intoxicación Alcohólica/psicología , Aprendizaje por Asociación/efectos de los fármacos , Conducta de Elección/efectos de los fármacos , Condicionamiento Clásico/efectos de los fármacos , Guanilato-Quinasas/genética , Proteínas de la Membrana/genética , Medio Social , Animales , Antimaníacos/farmacología , Homólogo 4 de la Proteína Discs Large , Maleato de Dizocilpina/farmacología , Antagonistas de Aminoácidos Excitadores/farmacología , Femenino , Inyecciones Intraperitoneales , Cloruro de Litio/toxicidad , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Transducción de Señal/genética , Gusto/efectos de los fármacos , Gusto/genéticaRESUMEN
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.
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Proteínas de la Membrana/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Densidad Postsináptica/metabolismo , Sinapsis/metabolismo , Animales , Línea Celular , Humanos , Unión Proteica , Dominios y Motivos de Interacción de ProteínasRESUMEN
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.
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Fosfoproteínas/metabolismo , Sinapsis/metabolismo , Animales , Humanos , FosforilaciónRESUMEN
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.
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Diferenciación Celular , Proteínas del Tejido Nervioso/genética , Células-Madre Neurales/fisiología , Trastornos del Neurodesarrollo/genética , Esquizofrenia/genética , Astrocitos/metabolismo , Proteína 9 Asociada a CRISPR/genética , Células Cultivadas , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas/genética , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Mutación , Células-Madre Neurales/metabolismo , Mapas de Interacción de Proteínas/genéticaRESUMEN
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 (1-3â¯mgâ¯kg-1, IP), as well as the selective 5-HT2A receptor blocker MDL-100,907 (volinanserin, 0.1-0.3â¯mgâ¯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'.
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Trastorno de Personalidad Antisocial/metabolismo , Interacción Gen-Ambiente , Privación Materna , Receptor de Serotonina 5-HT2A/metabolismo , Estrés Psicológico/metabolismo , Factores de Edad , Animales , Animales Recién Nacidos , Trastorno de Personalidad Antisocial/psicología , Relación Dosis-Respuesta a Droga , Femenino , Locomoción/efectos de los fármacos , Locomoción/fisiología , Masculino , Ratones , Ratones Transgénicos , Corteza Prefrontal/efectos de los fármacos , Corteza Prefrontal/metabolismo , Ratas , Antagonistas del Receptor de Serotonina 5-HT2/farmacología , Estrés Psicológico/psicologíaRESUMEN
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).
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Encéfalo/citología , Ontología de Genes , Proteómica , Programas Informáticos , Sinapsis/fisiología , Animales , Encéfalo/fisiología , Bases de Datos Genéticas , Humanos , Bases del Conocimiento , Potenciales Sinápticos/fisiología , SinaptosomasRESUMEN
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.
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Aprendizaje por Laberinto/fisiología , Plasticidad Neuronal/fisiología , Neuropéptidos/fisiología , Receptores de N-Metil-D-Aspartato/fisiología , Potenciales de Acción , Animales , Homólogo 4 de la Proteína Discs Large , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Guanilato-Quinasas , Técnicas In Vitro , Péptidos y Proteínas de Señalización Intracelular/fisiología , Potenciación a Largo Plazo/fisiología , Masculino , Proteínas de la Membrana/fisiología , Ratones , Ratones Noqueados , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Neuropéptidos/deficiencia , Tiempo de Reacción/fisiología , Receptores de N-Metil-D-Aspartato/metabolismo , Natación , Transmisión Sináptica/fisiologíaRESUMEN
GTPase-activating proteins (GAPs) and guanine exchange factors (GEFs) play essential roles in regulating the activity of small GTPases. Several GAPs and GEFs have been shown to be present at the postsynaptic density (PSD) within excitatory glutamatergic neurons and regulate the activity of glutamate receptors. However, it is not known how synaptic GAP and GEF proteins are organized within the PSD signaling machinery, if they have overlapping interaction networks, or if they associate with proteins implicated in contributing to psychiatric disease. Here, we determine the interactomes of three interacting GAP/GEF proteins at the PSD, including the RasGAP Syngap1, the ArfGAP Agap2, and the RhoGEF Kalirin, which includes a total of 280 interactions. We describe the functional properties of each interactome and show that these GAP/GEF proteins are highly associated with and cluster other proteins directly involved in GTPase signaling mechanisms. We also utilize Agap2 as an example of GAP/GEFs localized within multiple neuronal compartments and determine an additional 110 interactions involving Agap2 outside of the PSD. Functional analysis of PSD and non-PSD interactomes illustrates both common and unique functions of Agap2 determined by its subcellular location. Furthermore, we also show that these GAPs/GEFs associate with several proteins involved in psychiatric disease.
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Proteínas Activadoras de GTPasa/metabolismo , Factores de Intercambio de Guanina Nucleótido/metabolismo , Guanosina Trifosfato/metabolismo , Trastornos Mentales/metabolismo , Densidad Postsináptica/metabolismo , Proteínas Activadoras de ras GTPasa/metabolismo , Animales , Trastornos Mentales/patología , Ratones , Dominios y Motivos de Interacción de Proteínas , Transducción de SeñalRESUMEN
The postsynaptic density (PSD) contains a collection of scaffold proteins used for assembling synaptic signaling complexes. However, it is not known how the core-scaffold machinery associates in protein-interaction networks or how proteins encoded by genes involved in complex brain disorders are distributed through spatiotemporal protein complexes. Here using immunopurification, proteomics and bioinformatics, we isolated 2,876 proteins across 41 in vivo interactomes and determined their protein domain composition, correlation to gene expression levels and developmental integration to the PSD. We defined clusters for enrichment of schizophrenia, autism spectrum disorders, developmental delay and intellectual disability risk factors at embryonic day 14 and adult PSD in mice. Mutations in highly connected nodes alter protein-protein interactions modulating macromolecular complexes enriched in disease risk candidates. These results were integrated into a software platform, Synaptic Protein/Pathways Resource (SyPPRes), enabling the prioritization of disease risk factors and their placement within synaptic protein interaction networks.
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Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas del Tejido Nervioso/genética , Densidad Postsináptica/genética , Sinapsis/metabolismo , Animales , Encefalopatías/genética , Encefalopatías/metabolismo , Modelos Animales de Enfermedad , Guanilato-Quinasas/genética , Proteínas de la Membrana/genética , Ratones Transgénicos , Esquizofrenia/genética , Esquizofrenia/metabolismo , Transducción de Señal/genética , Sinapsis/genéticaRESUMEN
BACKGROUND: Protein domains can be viewed as portable units of biological function that defines the functional properties of proteins. Therefore, if a protein is associated with a disease, protein domains might also be associated and define disease endophenotypes. However, knowledge about such domain-disease relationships is rarely available. Thus, identification of domains associated with human diseases would greatly improve our understanding of the mechanism of human complex diseases and further improve the prevention, diagnosis and treatment of these diseases. METHODS: Based on phenotypic similarities among diseases, we first group diseases into overlapping modules. We then develop a framework to infer associations between domains and diseases through known relationships between diseases and modules, domains and proteins, as well as proteins and disease modules. Different methods including Association, Maximum likelihood estimation (MLE), Domain-disease pair exclusion analysis (DPEA), Bayesian, and Parsimonious explanation (PE) approaches are developed to predict domain-disease associations. RESULTS: We demonstrate the effectiveness of all the five approaches via a series of validation experiments, and show the robustness of the MLE, Bayesian and PE approaches to the involved parameters. We also study the effects of disease modularization in inferring novel domain-disease associations. Through validation, the AUC (Area Under the operating characteristic Curve) scores for Bayesian, MLE, DPEA, PE, and Association approaches are 0.86, 0.84, 0.83, 0.83 and 0.79, respectively, indicating the usefulness of these approaches for predicting domain-disease relationships. Finally, we choose the Bayesian approach to infer domains associated with two common diseases, Crohn's disease and type 2 diabetes. CONCLUSIONS: The Bayesian approach has the best performance for the inference of domain-disease relationships. The predicted landscape between domains and diseases provides a more detailed view about the disease mechanisms.
Asunto(s)
Enfermedad de Crohn/genética , Diabetes Mellitus Tipo 2/genética , Dominios Proteicos , Área Bajo la Curva , Teorema de Bayes , Enfermedad/genética , Estudios de Asociación Genética , Funciones de Verosimilitud , FenotipoRESUMEN
BACKGROUND: Atypical synapse development and plasticity are implicated in many neurodevelopmental disorders (NDDs). NDD-associated, high-confidence risk genes have been identified, yet little is known about functional relationships at the level of protein-protein interactions, which are the dominant molecular bases responsible for mediating circuit development. METHODS: Proteomics in three independent developing neocortical synaptosomal preparations identified putative interacting proteins of the ligand-activated MET receptor tyrosine kinase, an autism risk gene that mediates synapse development. The candidates were translated into interactome networks and analyzed bioinformatically. Additionally, three independent quantitative proximity ligation assays in cultured neurons and four independent immunoprecipitation analyses of synaptosomes validated protein interactions. RESULTS: Approximately 11% (8/72) of MET-interacting proteins, including SHANK3, SYNGAP1, and GRIN2B, are associated with NDDs. Proteins in the MET interactome were translated into a novel MET interactome network based on human protein-protein interaction databases. High-confidence genes from different NDD datasets that encode synaptosomal proteins were analyzed for being enriched in MET interactome proteins. This was found for autism but not schizophrenia, bipolar disorder, major depressive disorder, or attention-deficit/hyperactivity disorder. There is correlated gene expression between MET and its interactive partners in developing human temporal and visual neocortices but not with highly expressed genes that are not in the interactome. Proximity ligation assays and biochemical analyses demonstrate that MET-protein partner interactions are dynamically regulated by receptor activation. CONCLUSIONS: The results provide a novel molecular framework for deciphering the functional relations of key regulators of synaptogenesis that contribute to both typical cortical development and to NDDs.