RESUMEN
Visceral sensory pathways mediate homeostatic reflexes, the dysfunction of which leads to many neurological disorders1. The Bezold-Jarisch reflex (BJR), first described2,3 in 1867, is a cardioinhibitory reflex that is speculated to be mediated by vagal sensory neurons (VSNs) that also triggers syncope. However, the molecular identity, anatomical organization, physiological characteristics and behavioural influence of cardiac VSNs remain mostly unknown. Here we leveraged single-cell RNA-sequencing data and HYBRiD tissue clearing4 to show that VSNs that express neuropeptide Y receptor Y2 (NPY2R) predominately connect the heart ventricular wall to the area postrema. Optogenetic activation of NPY2R VSNs elicits the classic triad of BJR responses-hypotension, bradycardia and suppressed respiration-and causes an animal to faint. Photostimulation during high-resolution echocardiography and laser Doppler flowmetry with behavioural observation revealed a range of phenotypes reflected in clinical syncope, including reduced cardiac output, cerebral hypoperfusion, pupil dilation and eye-roll. Large-scale Neuropixels brain recordings and machine-learning-based modelling showed that this manipulation causes the suppression of activity across a large distributed neuronal population that is not explained by changes in spontaneous behavioural movements. Additionally, bidirectional manipulation of the periventricular zone had a push-pull effect, with inhibition leading to longer syncope periods and activation inducing arousal. Finally, ablating NPY2R VSNs specifically abolished the BJR. Combined, these results demonstrate a genetically defined cardiac reflex that recapitulates characteristics of human syncope at physiological, behavioural and neural network levels.
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Corazón , Reflejo , Células Receptoras Sensoriales , Síncope , Nervio Vago , Humanos , Área Postrema , Bradicardia/complicaciones , Bradicardia/fisiopatología , Gasto Cardíaco Bajo/complicaciones , Gasto Cardíaco Bajo/fisiopatología , Ecocardiografía , Corazón/fisiología , Frecuencia Cardíaca , Hipotensión/complicaciones , Hipotensión/fisiopatología , Flujometría por Láser-Doppler , Red Nerviosa , Reflejo/fisiología , Células Receptoras Sensoriales/fisiología , Análisis de Expresión Génica de una Sola Célula , Síncope/complicaciones , Síncope/etiología , Nervio Vago/citología , Nervio Vago/fisiologíaRESUMEN
A significant proportion of autism risk genes regulate synapse function, including plasticity, which is believed to contribute to behavioral abnormalities. However, it remains unclear how impaired synapse plasticity contributes to network-level processes linked to adaptive behaviors, such as experience-dependent ensemble plasticity. We found that Syngap1, a major autism risk gene, promoted measures of experience-dependent excitatory synapse strengthening in the mouse cortex, including spike-timing-dependent glutamatergic synaptic potentiation and presynaptic bouton formation. Synaptic depression and bouton elimination were normal in Syngap1 mice. Within cortical networks, Syngap1 promoted experience-dependent increases in somatic neural activity in weakly active neurons. In contrast, plastic changes to highly active neurons from the same ensemble that paradoxically weaken with experience were unaffected. Thus, experience-dependent excitatory synapse strengthening mediated by Syngap1 shapes neuron-specific plasticity within cortical ensembles. We propose that other genes regulate neuron-specific weakening within ensembles, and together, these processes function to redistribute activity within cortical networks during experience.
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Trastorno Autístico/genética , Plasticidad Neuronal/genética , Neuronas/metabolismo , Sinapsis/fisiología , Tacto , Proteínas Activadoras de ras GTPasa/metabolismo , Animales , Corteza Cerebral/fisiología , Epigénesis Genética , Femenino , Humanos , Masculino , Ratones , Técnicas de Placa-Clamp , Vibrisas , Proteínas Activadoras de ras GTPasa/genéticaRESUMEN
SYNGAP1 loss-of-function variants are causally associated with intellectual disability, severe epilepsy, autism spectrum disorder and schizophrenia. While there are hundreds of genetic risk factors for neurodevelopmental disorders (NDDs), this gene is somewhat unique because of the frequency and penetrance of loss-of-function variants found in patients combined with the range of brain disorders associated with SYNGAP1 pathogenicity. These clinical findings indicate that SYNGAP1 regulates fundamental neurodevelopmental processes that are necessary for brain development. Here, we describe four phenotypic domains that are controlled by Syngap1 expression across vertebrate species. Two domains, the maturation of cognitive functions and maintenance of excitatory-inhibitory balance, are defined exclusively through a review of the current literature. Two additional domains are defined by integrating the current literature with new data indicating that SYNGAP1/Syngap1 regulates innate survival behaviors and brain structure. These four phenotypic domains are commonly disrupted in NDDs, suggesting that a deeper understanding of developmental Syngap1 functions will be generalizable to other NDDs of known or unknown etiology. Therefore, we discuss the known molecular and cellular functions of Syngap1 and consider how these functions may contribute to the emergence of disease-relevant phenotypes. Finally, we identify major unexplored areas of Syngap1 neurobiology and discuss how a deeper understanding of this gene may uncover general principles of NDD pathobiology.
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Trastornos del Neurodesarrollo/genética , Fenotipo , Proteínas Activadoras de ras GTPasa/genética , Animales , Secuencia Conservada , Humanos , Mutación con Pérdida de Función , Ratones , Trastornos del Neurodesarrollo/metabolismo , Trastornos del Neurodesarrollo/patología , Proteínas Activadoras de ras GTPasa/química , Proteínas Activadoras de ras GTPasa/metabolismoRESUMEN
Chromatin states are believed to play a key role in distinct patterns of gene expression essential for self-renewal and pluripotency of embryonic stem cells (ESCs); however, the genes governing the establishment and propagation of the chromatin signature characteristic of pluripotent cells are poorly understood. Here, we show that conditional deletion of the histone acetyltransferase cofactor Trrap in mouse ESCs triggers unscheduled differentiation associated with loss of histone acetylation, condensation of chromatin into distinct foci (heterochromatization), and uncoupling of H3K4 dimethylation and H3K27 trimethylation. Trrap loss results in downregulation of stemness master genes Nanog, Oct4, and Sox2 and marked upregulation of specific differentiation markers from the three germ layers. Chromatin immunoprecipitation-sequencing analysis of genome-wide binding revealed a significant overlap between Oct4 and Trrap binding in ESCs but not in differentiated mouse embryonic fibroblasts, further supporting a functional interaction between Trrap and Oct4 in the maintenance of stemness. Remarkably, failure to downregulate Trrap prevents differentiation of ESCs, suggesting that downregulation of Trrap may be a critical step guiding transcriptional reprogramming and differentiation of ESCs. These findings establish Trrap as a critical part of the mechanism that restricts differentiation and promotes the maintenance of key features of ESCs.
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Proteínas Adaptadoras Transductoras de Señales/metabolismo , Células Madre Embrionarias/citología , Histona Acetiltransferasas/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Animales , Apoptosis/fisiología , Diferenciación Celular/fisiología , Cromatina/metabolismo , Inmunoprecipitación de Cromatina , Regulación hacia Abajo , Células Madre Embrionarias/enzimología , Células Madre Embrionarias/metabolismo , Regulación del Desarrollo de la Expresión Génica , Histona Acetiltransferasas/genética , Histonas/genética , Histonas/metabolismo , Ratones , Ratones Noqueados , Proteínas Nucleares/genética , Factor 3 de Transcripción de Unión a Octámeros/genética , Factor 3 de Transcripción de Unión a Octámeros/metabolismo , Regiones Promotoras GenéticasRESUMEN
Perception, a cognitive construct, emerges through sensorimotor integration (SMI). The molecular and cellular mechanisms that shape SMI within circuits that promote cognition are poorly understood. Here, we demonstrate that expression of the autism/intellectual disability gene, Syngap1, in mouse cortical excitatory neurons promotes touch sensitivity required to elicit perceptual behaviors. Cortical Syngap1 expression enabled touch-induced feedback signals within sensorimotor loops by assembling circuits that support tactile sensitivity. These circuits also encoded correlates of attention that promoted self-generated whisker movements underlying purposeful and sustained object exploration. As Syngap1 deficient animals explored objects with whiskers, relatively weak touch signals were integrated with relatively strong motor signals. This produced a signal-to-noise deficit consistent with impaired tactile sensitivity, reduced tactile exploration, and weak tactile learning. Thus, Syngap1 expression in cortex promotes tactile perception by assembling circuits that integrate touch and whisker motor signals. Deficient Syngap1 expression likely contributes to cognitive impairment through abnormal top-down SMI.
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The DOK1 gene is a putative tumour suppressor gene located on the human chromosome 2p13 which is frequently rearranged in leukaemia and other human tumours. We previously reported that the DOK1 gene can be mutated and its expression down-regulated in human malignancies. However, the mechanism underlying DOK1 silencing remains largely unknown. We show here that unscheduled silencing of DOK1 expression through aberrant hypermethylation is a frequent event in a variety of human malignancies. DOK1 was found to be silenced in nine head and neck cancer (HNC) cell lines studied and DOK1 CpG hypermethylation correlated with loss of gene expression in these cells. DOK1 expression could be restored via demethylating treatment using 5-aza-2'deoxycytidine. In addition, transduction of cancer cell lines with DOK1 impaired their proliferation, consistent with the critical role of epigenetic silencing of DOK1 in the development and maintenance of malignant cells. We further observed that DOK1 hypermethylation occurs frequently in a variety of primary human neoplasm including solid tumours (93% in HNC, 81% in lung cancer) and haematopoietic malignancy (64% in Burkitt's lymphoma). Control blood samples and exfoliated mouth epithelial cells from healthy individuals showed a low level of DOK1 methylation, suggesting that DOK1 hypermethylation is a tumour specific event. Finally, an inverse correlation was observed between the level of DOK1 gene methylation and its expression in tumour and adjacent non tumour tissues. Thus, hypermethylation of DOK1 is a potentially critical event in human carcinogenesis, and may be a potential cancer biomarker and an attractive target for epigenetic-based therapy.
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Metilación de ADN , Proteínas de Unión al ADN/genética , Neoplasias de Cabeza y Cuello/genética , Fosfoproteínas/genética , Regiones Promotoras Genéticas , Proteínas de Unión al ARN/genética , Adulto , Anciano , Azacitidina/análogos & derivados , Azacitidina/farmacología , Línea Celular Tumoral , Inhibidor p16 de la Quinasa Dependiente de Ciclina/genética , Proteínas de Unión al ADN/antagonistas & inhibidores , Decitabina , Femenino , Genes Supresores de Tumor , Neoplasias de Cabeza y Cuello/patología , Humanos , Masculino , Persona de Mediana Edad , Fosfoproteínas/antagonistas & inhibidores , Proteínas de Unión al ARN/antagonistas & inhibidores , Factores de Riesgo , Proteínas Supresoras de Tumor/genéticaRESUMEN
Constitutional epimutation is one of the causes for MLH1 gene inactivation associated with hereditary non-polyposis colon cancer (HNPCC) syndrome. Here we investigate MLH1 promoter hypermethylation in 110 sporadic early-onset colorectal cancer patients. Variable levels of hypermethylation were detected in 55 patients (50%). Importantly a reduced MLH1 gene expression was found in patients with high-level methylation, with the association of microsatellite instability (MSI) in their tumor cells. Such high-level methylation accounts for 7.4% of all patients included in this study. Furthermore, we found that in one case constitutional methylation affected both alleles, indicating a post-zygotic methylation dysregulation. Our findings suggest that constitutional epimutation is a mechanism underlying early-onset colorectal cancer, although it is involved in only a small proportion of patients, who require appropriate surveillance. Our findings provide further insight into the role of aberrant constitutional methylation in colon carcinogenesis and raise the question of whether prevalent low-level methylation constitutes a potential risk factor for cancer development.
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Proteínas Adaptadoras Transductoras de Señales/metabolismo , Alelos , Neoplasias Colorrectales/epidemiología , Neoplasias Colorrectales/genética , Metilación de ADN/genética , Proteínas Nucleares/metabolismo , Regiones Promotoras Genéticas/genética , Proteínas Adaptadoras Transductoras de Señales/genética , Adulto , Edad de Inicio , Secuencia de Bases , Estudios de Casos y Controles , Regulación Neoplásica de la Expresión Génica , Humanos , Inestabilidad de Microsatélites , Persona de Mediana Edad , Datos de Secuencia Molecular , Homólogo 1 de la Proteína MutL , Proteínas Nucleares/genéticaRESUMEN
BACKGROUND & AIMS: Hepatocellular carcinoma (HCC) is one of the most frequent human cancers and a major cause of cancer-related death worldwide. The major risk factors for developing HCC are infection by hepatitis B virus (HBV) and hepatitis C virus (HCV), chronic alcoholism, and aflatoxins; however, critical gene targets remain largely unknown. Herein, we sought to establish DNA methylation patterns in HCC and corresponding cirrhotic tissues and to identify DNA methylation changes associated with major risk factors. METHODS: We have established assays for quantitative analysis of DNA methylation levels in a panel of seven cancer-associated genes and repetitive elements, and combined these assays with a series of HCC tumors, associated with major risk factors, collected from two different geographical areas. RESULTS: We found a high frequency of aberrant hypermethylation of specific genes (RASSF1A, GSTP1, CHRNA3, and DOK1) in HCC tumors as compared to control cirrhotic or normal liver tissues, suggesting that aberrant hypermethylation exhibits non-random and tumor-specific patterns in HCC. Importantly, our analysis revealed an association between alcohol intake and the hypomethylation of MGMT and between hypermethylation of GSTP1 and HBV infection, indicating that hypermethylation of the genes analyzed in HCC tumors exhibits remarkably distinct patterns depending on associated risk factors. CONCLUSIONS: This study identifies aberrant DNA methylation of specific cellular genes in HCC and the major risk factors associated with these changes, providing information that could be exploited for biomarker discovery in clinics and molecular epidemiology.
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Carcinoma Hepatocelular/complicaciones , Carcinoma Hepatocelular/genética , Metilación de ADN , Hepatitis B/complicaciones , Hepatitis C/complicaciones , Neoplasias Hepáticas/complicaciones , Neoplasias Hepáticas/genética , Adulto , Anciano , Consumo de Bebidas Alcohólicas/efectos adversos , Secuencia de Bases , Cartilla de ADN/genética , ADN de Neoplasias/química , ADN de Neoplasias/genética , Proteínas de Unión al ADN/genética , Femenino , Silenciador del Gen , Gutatión-S-Transferasa pi/genética , Humanos , Masculino , Persona de Mediana Edad , Fosfoproteínas/genética , Proteínas de Unión al ARN/genética , Receptores Nicotínicos/genética , Factores de Riesgo , Proteínas Supresoras de Tumor/genéticaRESUMEN
Quantitative and reliable estimation of DNA methylation levels for multiple genomic regions pose a major challenge where starting DNA is available in very low quantity. Here we review major advances in the development of techniques for quantitative detection of DNA methylation in minute amount of DNA and describe a detailed protocol for quantitative Methylation Analysis of Minute DNA amounts after whole Bisulfitome Amplification (qMAMBA), a combination of techniques that allows quantitative and sensitive detection of DNA methylation at multiple CpG sites and for multiple gene assays. Recently we successfully used this technique to quantitatively detect DNA methylation for a set of cancer-related genes in lung cancer patient plasma samples [18]. This method involves genome-wide amplification of bisulfite-modified DNA template followed by quantitative methylation detection using pyrosequencing. This allows a precise assessment of DNA methylation at CpG sites and could be adapted for high-throughput settings. It can also be applied in conjunction with studies involving single-cells or laser capture microdissected samples. Thus, this method should facilitate DNA methylation studies aiming to discover epigenetic biomarkers, and should prove particularly valuable in profiling a large sample series of body fluids from molecular epidemiology studies.
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Líquidos Corporales/química , Metilación de ADN , ADN/metabolismo , Técnicas Genéticas , ADN/química , Humanos , Neoplasias/diagnóstico , Neoplasias/genéticaRESUMEN
Cytokinesis is the last step of mitotic cell division that separates the cytoplasm of dividing cells. Small molecule inhibitors targeting either the elements of the regulatory pathways controlling cytokinesis, or the terminal effectors have been of interest as potential drug candidates for the treatment of various diseases. Here we present a detailed protocol for a cell-based cytokinesis assay that can be used for the discovery of novel cytokinesis inhibitors. The assay is performed in a 96-well plate format in 48 h. Living cells, nuclei and nuclei of dead cells are identified by a single staining step using three fluorescent dyes, followed by rapid live cell imaging. The primary signal is the nuclei-to-cell ratio (NCR). In the presence of cytokinesis inhibitors, this ratio increases over time, as the ratio of multinucleated cells increases in the population. The ratio of dead nuclei to total nuclei provides a simultaneous measure of cytotoxicity. A screening window coefficient (Z`) of 0.65 indicates that the assay is suitable for screening purposes, as the positive and negative controls are well-separated. EC50 values can be reliably determined in a single 96-well plate by using only six different compound concentrations, enabling the testing of 4 compounds per plate. An excellent test-retest reliability (R 2 = 0.998) was found for EC50 values covering a ~1500-fold range of potencies. Established small molecule inhibitors of cytokinesis operating via direct action on actin dynamics or nonmuscle myosin II are used to demonstrate the robustness, simplicity and flexibility of the assay.
RESUMEN
It remains unclear to what extent neurodevelopmental disorder (NDD) risk genes retain functions into adulthood and how they may influence disease phenotypes. SYNGAP1 haploinsufficiency causes a severe NDD defined by autistic traits, cognitive impairment, and epilepsy. To determine if this gene retains therapeutically-relevant biological functions into adulthood, we performed a gene restoration technique in a mouse model for SYNGAP1 haploinsufficiency. Adult restoration of SynGAP protein improved behavioral and electrophysiological measures of memory and seizure. This included the elimination of interictal events that worsened during sleep. These events may be a biomarker for generalized cortical dysfunction in SYNGAP1 disorders because they also worsened during sleep in the human patient population. We conclude that SynGAP protein retains biological functions throughout adulthood and that non-developmental functions may contribute to disease phenotypes. Thus, treatments that target debilitating aspects of severe NDDs, such as medically-refractory seizures and cognitive impairment, may be effective in adult patients.
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Envejecimiento/metabolismo , Conducta , Encéfalo/metabolismo , Proteínas Activadoras de ras GTPasa/metabolismo , Potenciales de Acción , Animales , Conducta Animal , Electroencefalografía , Femenino , Humanos , Masculino , Memoria , Ratones , Ratones Mutantes , Convulsiones/metabolismo , Convulsiones/fisiopatología , Sueño , VigiliaRESUMEN
Cancers are traditionally viewed as a primarily genetic disorder, however this view has recently been modified by compelling evidence arguing that epigenetic events play important roles in most human cancers. Deregulation of epigenetic information (encoded in DNA methylation and histone modification patterns) in cells with pluripotent potential may alter defining properties of stem cells, self-renewal and differentiation potential, leading to cancer initiation and progression. The level of compaction of chromatin dictates accessibility to genomic DNA and therefore has a key role in establishing and maintaining distinct gene expression patterns and consequently pluripotent state and differentiation fates of stem cells. Unique properties of stem cells defined as "stemness" may be determined by acetylation and methylation of histones near gene promoters that regulate gene transcription, however these histone modifications elsewhere in the genome may also be important. In this review, we discuss new insights into possible mechanisms by which histone acetyltransferases (HATs) and histone acetylation in concert with other chromatin modifications may regulate pluripotency, and speculate how deregulation of histone marking may lead to tumourigenesis.
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Ensamble y Desensamble de Cromatina/fisiología , Cromatina/fisiología , Epigénesis Genética , Histonas/metabolismo , Neoplasias/genética , Células Madre Pluripotentes/fisiología , Células Madre/fisiología , Acetilación , Animales , Diferenciación Celular , Metilación de ADN , Histona Acetiltransferasas/metabolismo , Humanos , Modelos Biológicos , Neoplasias/etiología , Procesamiento Proteico-PostraduccionalRESUMEN
Knowledge on heritable changes in gene expression that result from epigenetic events is of increasing relevance in the development of strategies for prevention, early diagnosis and treatment of cancer. Histone acetylation and DNA methylation are epigenetic modifications whose patterns can be regarded as heritable marks that ensure accurate transmission of the chromatin states and gene expression profiles over many cell generations. Importantly, patterns and levels of DNA methylation and histone acetylation are profoundly altered in human cancers. Accumulating evidence suggests that an epigenetic cross-talk, i.e. interplay between DNA methylation and histone acetylation, may be involved in the process of gene transcription and aberrant gene silencing in tumours. Although the molecular mechanism of gene activation is relatively well understood, the hierarchical order of events and dependencies leading to gene silencing in the course of cancer development remain largely unknown. While some studies suggest that DNA methylation patterns guide histone modifications (including histone acetylation and methylation) during gene silencing, other studies argue that DNA methylation takes its cues primarily from histone modification states. In this review, we summarize current knowledge on the interplay between DNA methylation and histone modifications during gene silencing and its importance in the integration of environmental and intrinsic stimuli in the control of gene expression. We also discuss the importance of an epigenetic cross-talk in the protection against genetic changes in response to environmental genotoxins as well as the implication for cancer therapy and prevention.
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Metilación de ADN , Epigénesis Genética , Silenciador del Gen , Histonas/metabolismo , Acetilación , Ensamble y Desensamble de Cromatina , Contaminantes Ambientales/farmacología , Regulación de la Expresión Génica , Humanos , Mutágenos/farmacología , Transcripción Genética , Activación TranscripcionalRESUMEN
Cancer is traditionally viewed as a primarily genetic disorder, however it is now becoming accepted that cancer is also a consequence of abnormal epigenetic events. Genetic changes and aneuploidy are associated with alterations in DNA sequence, and they are a hallmark of the malignant process. Epigenetic alterations are universally present in human cancer and result in heritable changes in gene expression and chromatin structure over many cell generations without changes in DNA sequence, leading to functional consequences equivalent to those induced by genetic alterations. Importantly, intriguing evidence emerged suggesting that epigenetic changes may precede and provoke genetic changes. In this scenario, epigenetic events are primary events while genetic changes (such as mutations) may simply be a consequence of disrupted epigenetic states. This fact may explain why many genetic screens proved to be limited with regard to cancer causality and pathogenesis. Aberrant epigenetic events affect multiple genes and cellular pathways in a non-random fashion and this can predispose to induction and accumulation of genetic changes in the course of tumour initiation and progression. These considerations are critical for a better understanding of tumourigenesis and molecular events underlying the acquisition of drug resistance, as well as development of novel strategies for cancer therapy and prevention.
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Epigénesis Genética , Neoplasias/genética , Ensamble y Desensamble de Cromatina , Metilación de ADN , Silenciador del Gen , Histonas/metabolismo , Humanos , Modelos Genéticos , Mutación , Procesamiento Proteico-PostraduccionalRESUMEN
To deconstruct the architecture and function of brain circuits, it is necessary to generate maps of neuronal connectivity and activity on a whole-brain scale. New methods now enable large-scale mapping of the mouse brain at cellular and subcellular resolution. We developed a framework to automatically annotate, analyze, visualize and easily share whole-brain data at cellular resolution, based on a scale-invariant, interactive mouse brain atlas. This framework enables connectivity and mapping projects in individual laboratories and across imaging platforms, as well as multiplexed quantitative information on the molecular identity of single neurons. As a proof of concept, we generated a comparative connectivity map of five major neuron types in the corticostriatal circuit, as well as an activity-based map to identify hubs mediating the behavioral effects of cocaine. Thus, this computational framework provides the necessary tools to generate brain maps that integrate data from connectivity, neuron identity and function.
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Mapeo Encefálico , Encéfalo/citología , Red Nerviosa/fisiología , Vías Nerviosas/fisiología , Neuronas/fisiología , Animales , Regulación de la Expresión Génica/genética , Regulación de la Expresión Génica/fisiología , Genes Inmediatos-Precoces/fisiología , Glutamato Descarboxilasa/metabolismo , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Proteínas con Homeodominio LIM/genética , Proteínas con Homeodominio LIM/metabolismo , Masculino , Ratones Transgénicos , Actividad Motora , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Neuropéptido Y/metabolismo , Parvalbúminas/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
In the version of this article initially published online, Daniel Fürth was not listed as a corresponding author. The error has been corrected in the print, PDF and HTML versions of this article.
RESUMEN
In addition to cognitive impairments, neurodevelopmental disorders often result in sensory processing deficits. However, the biological mechanisms that underlie impaired sensory processing associated with neurodevelopmental disorders are generally understudied and poorly understood. We found that SYNGAP1 haploinsufficiency in humans, which causes a sporadic neurodevelopmental disorder defined by cognitive impairment, autistic features, and epilepsy, also leads to deficits in tactile-related sensory processing. In vivo neurophysiological analysis in Syngap1 mouse models revealed that upper-lamina neurons in somatosensory cortex weakly encode information related to touch. This was caused by reduced synaptic connectivity and impaired intrinsic excitability within upper-lamina somatosensory cortex neurons. These results were unexpected, given that Syngap1 heterozygosity is known to cause circuit hyperexcitability in brain areas more directly linked to cognitive functions. Thus, Syngap1 heterozygosity causes a range of circuit-specific pathologies, including reduced activity within cortical neurons required for touch processing, which may contribute to sensory phenotypes observed in patients.
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Red Nerviosa/fisiopatología , Trastornos de la Sensación/genética , Corteza Somatosensorial/fisiopatología , Percepción del Tacto/fisiología , Tacto/fisiología , Proteínas Activadoras de ras GTPasa/genética , Animales , Cognición/fisiología , Femenino , Haploinsuficiencia , Humanos , Masculino , Ratones , Neuronas/fisiología , Técnicas de Placa-Clamp , Sistema de Registros , Trastornos de la Sensación/fisiopatologíaRESUMEN
There is a pressing need to improve approaches for drug discovery related to neuropsychiatric disorders (NSDs). Therapeutic discovery in neuropsychiatric disorders would benefit from screening assays that can measure changes in complex phenotypes linked to disease mechanisms. However, traditional assays that track complex neuronal phenotypes, such as neuronal connectivity, exhibit poor scalability and are not compatible with high-throughput screening (HTS) procedures. Therefore, we created a neuronal phenotypic assay platform that focused on improving the scalability and affordability of neuron-based assays capable of tracking disease-relevant phenotypes. First, using inexpensive laboratory-level automation, we industrialized primary neuronal culture production, which enabled the creation of scalable assays within functioning neural networks. We then developed a panel of phenotypic assays based on culturing of primary neurons from genetically modified mice expressing HTS-compatible reporters that capture disease-relevant phenotypes. We demonstrated that a library of 1,280 compounds was quickly screened against both assays using only a few litters of mice in a typical academic laboratory setting. Finally, we implemented one assay in a fully automated high-throughput academic screening facility, illustrating the scalability of assays designed using this platform. These methodological improvements simplify the creation of highly scalable neuron-based phenotypic assays designed to improve drug discovery in CNS disorders.
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Our unique collection of memories determines our individuality and shapes our future interactions with the world. Remarkable advances into the neurobiological basis of memory have identified key epigenetic mechanisms that support the stability of memory. Various forms of epigenetic regulation at the levels of DNA methylation, histone modification, and non-coding RNAs (ncRNAs) can modulate transcriptional and translational events required for memory processes. By changing the cellular profile in the brain's emotional, reward, and memory circuits, these epigenetic modifications have also been linked to perseverant, pathogenic memories. In this review, we will delve into the relevance of epigenetic dysregulation to pathogenic memory mechanisms by focusing on two neuropsychiatric disorders perpetuated by aberrant memory associations: substance use disorder (SUD) and post-traumatic stress disorder (PTSD). As our understanding improves, neuroepigenetic mechanisms may someday be harnessed to develop novel therapeutic targets for the treatment of these chronic, relapsing disorders.
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BACKGROUND: Genetic haploinsufficiency of SYNGAP1/Syngap1 commonly occurs in developmental brain disorders, such as intellectual disability, epilepsy, schizophrenia, and autism spectrum disorder. Thus, studying mouse models of Syngap1 haploinsufficiency may uncover pathologic developmental processes common among distinct brain disorders. METHODS: A Syngap1 haploinsufficiency model was used to explore the relationship between critical period dendritic spine abnormalities, cortical circuit assembly, and the window for genetic rescue to understand how damaging mutations disrupt key substrates of mouse brain development. RESULTS: Syngap1 mutations broadly disrupted a developmentally sensitive period that corresponded to the period of heightened postnatal cortical synaptogenesis. Pathogenic Syngap1 mutations caused a coordinated acceleration of dendrite elongation and spine morphogenesis and pruning of these structures in neonatal cortical pyramidal neurons. These mutations also prevented a form of developmental structural plasticity associated with experience-dependent reorganization of brain circuits. Consistent with these findings, Syngap1 mutant mice displayed an altered pattern of long-distance synaptic inputs into a cortical area important for cognition. Interestingly, the ability to genetically improve the behavioral endophenotype of Syngap1 mice decreased slowly over postnatal development and mapped onto the developmental period of coordinated dendritic insults. CONCLUSIONS: Pathogenic Syngap1 mutations have a profound impact on the dynamics and structural integrity of pyramidal cell postsynaptic structures known to guide the de novo wiring of nascent cortical circuits. These findings support the idea that disrupted critical periods of dendritic growth and spine plasticity may be a common pathologic process in developmental brain disorders.