RESUMEN
The engineering of autologous patient T cells for adoptive cell therapies has revolutionized the treatment of several types of cancer1. However, further improvements are needed to increase response and cure rates. CRISPR-based loss-of-function screens have been limited to negative regulators of T cell functions2-4 and raise safety concerns owing to the permanent modification of the genome. Here we identify positive regulators of T cell functions through overexpression of around 12,000 barcoded human open reading frames (ORFs). The top-ranked genes increased the proliferation and activation of primary human CD4+ and CD8+ T cells and their secretion of key cytokines such as interleukin-2 and interferon-γ. In addition, we developed the single-cell genomics method OverCITE-seq for high-throughput quantification of the transcriptome and surface antigens in ORF-engineered T cells. The top-ranked ORF-lymphotoxin-ß receptor (LTBR)-is typically expressed in myeloid cells but absent in lymphocytes. When overexpressed in T cells, LTBR induced profound transcriptional and epigenomic remodelling, leading to increased T cell effector functions and resistance to exhaustion in chronic stimulation settings through constitutive activation of the canonical NF-κB pathway. LTBR and other highly ranked genes improved the antigen-specific responses of chimeric antigen receptor T cells and γδ T cells, highlighting their potential for future cancer-agnostic therapies5. Our results provide several strategies for improving next-generation T cell therapies by the induction of synthetic cell programmes.
Asunto(s)
Linfocitos T CD8-positivos , Neoplasias , Linfocitos T CD4-Positivos , Proliferación Celular , Humanos , Inmunoterapia Adoptiva , Activación de Linfocitos/genéticaRESUMEN
Whole-exome sequencing of autism spectrum disorder (ASD) probands and unaffected family members has identified many genes harboring de novo variants suspected to play a causal role in the disorder. Of these, chromodomain helicase DNA-binding protein 8 (CHD8) is the most recurrently mutated. Despite the prevalence of CHD8 mutations, we have little insight into how CHD8 loss affects genome organization or the functional consequences of these molecular alterations in neurons. Here, we engineered two isogenic human embryonic stem cell lines with CHD8 loss-of-function mutations and characterized differences in differentiated human cortical neurons. We identified hundreds of genes with altered expression, including many involved in neural development and excitatory synaptic transmission. Field recordings and single-cell electrophysiology revealed a 3-fold decrease in firing rates and synaptic activity in CHD8+/- neurons, as well as a similar firing-rate deficit in primary cortical neurons from Chd8+/- mice. These alterations in neuron and synapse function can be reversed by CHD8 overexpression. Moreover, CHD8+/- neurons displayed a large increase in open chromatin across the genome, where the greatest change in compaction was near autism susceptibility candidate 2 (AUTS2), which encodes a transcriptional regulator implicated in ASD. Genes with changes in chromatin accessibility and expression in CHD8+/- neurons have significant overlap with genes mutated in probands for ASD, intellectual disability, and schizophrenia but not with genes mutated in healthy controls or other disease cohorts. Overall, this study characterizes key molecular alterations in genome structure and expression in CHD8+/- neurons and links these changes to impaired neuronal and synaptic function.
Asunto(s)
Trastorno del Espectro Autista , Trastorno Autístico , Humanos , Animales , Ratones , Trastorno Autístico/genética , Trastorno del Espectro Autista/genética , Cromatina/genética , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Expresión Génica , Factores de Transcripción/genéticaRESUMEN
Overexpression of mouse neurogenin ( Neurog) 2 alone or in combination with mouse Neurog2/1 in human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) can rapidly produce high-yield excitatory neurons. Here, we report a detailed characterization of human neuronal networks induced by the expression of human NEUROG2 together with human NEUROG2/1 in hESCs using molecular, cellular, and electrophysiological measurements over 60 d after induction. Both excitatory synaptic transmission and network firing activity increased over time. Strikingly, inhibitory synaptic transmission and GABAergic cells were identified from NEUROG2/1 induced neurons (iNs). To illustrate the application of such iNs, we demonstrated that the heterozygous knock out of SCN2A, whose loss-of-function mutation is strongly implicated in autism risk, led to a dramatic reduction in network activity in the NEUROG2/1 iNs. Our findings not only extend our understanding of the NEUROG2/1-induced human neuronal network but also substantiate NEUROG2/1 iNs as an in vitro system for modeling neuronal and functional deficits on a human genetic background.-Lu, C., Shi, X., Allen, A., Baez-Nieto, D., Nikish, A., Sanjana, N. E., Pan, J. Q. Overexpression of NEUROG2 and NEUROG1 in human embryonic stem cells produces a network of excitatory and inhibitory neurons.
Asunto(s)
Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Neuronas/citología , Neuronas/metabolismo , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Diferenciación Celular/genética , Diferenciación Celular/fisiología , Línea Celular , Humanos , Inmunohistoquímica , Proteínas del Tejido Nervioso/genética , Técnicas de Placa-Clamp , Transmisión Sináptica/genética , Transmisión Sináptica/fisiologíaRESUMEN
BACKGROUND: Ewing sarcoma (ES) is driven by fusion of the Ewing sarcoma breakpoint region 1 gene (EWSR1) with an E26 transformation-specific (ETS) transcription factor (EWS-ETS), most often the Friend leukemia integration 1 transcription factor (FLI1). Neuropeptide Y (NPY) is an EWS-FLI1 transcriptional target; it is highly expressed in ES and exerts opposing effects, ranging from ES cell death to angiogenesis and cancer stem cell propagation. The functions of NPY are regulated by dipeptidyl peptidase IV (DPPIV), a hypoxia-inducible enzyme that cleaves the peptide and activates its growth-promoting actions. The objective of this study was to determine the clinically relevant functions of NPY by identifying the associations between patients' ES phenotype and their NPY concentrations and DPP activity. METHODS: NPY concentrations and DPP activity were measured in serum samples from 223 patients with localized ES and 9 patients with metastatic ES provided by the Children's Oncology Group. RESULTS: Serum NPY levels were elevated in ES patients compared with the levels in a healthy control group and an osteosarcoma patient population, and the elevated levels were independent of EWS-ETS translocation type. Significantly higher NPY concentrations were detected in patients with ES who had tumors of pelvic and bone origin. A similar trend was observed in patients with metastatic ES. There was no effect of NPY on survival in patients with localized ES. DPP activity in sera from patients with ES did not differ significantly from that in healthy controls and patients with osteosarcoma. However, high DPP levels were associated with improved survival. CONCLUSIONS: Systemic NPY levels are elevated in patients with ES, and these high levels are associated with unfavorable disease features. DPPIV in serum samples from patients with ES is derived from nontumor sources, and its high activity is correlated with improved survival.
Asunto(s)
Neoplasias Óseas/sangre , Dipeptidil Peptidasa 4/sangre , Neuropéptido Y/sangre , Proteínas de Fusión Oncogénica/genética , Proteína Proto-Oncogénica c-fli-1/genética , Proteína EWS de Unión a ARN/genética , Sarcoma de Ewing/sangre , Adolescente , Animales , Neoplasias Óseas/genética , Neoplasias Óseas/mortalidad , Línea Celular Tumoral , Niño , Dipeptidil Peptidasa 4/genética , Regulación Neoplásica de la Expresión Génica , Humanos , Ratones , Ratones SCID , Trasplante de Neoplasias , Neuropéptido Y/genética , Neuropéptido Y/metabolismo , Osteosarcoma/sangre , Osteosarcoma/genética , ARN Mensajero/genética , Receptores de Neuropéptido Y/genética , Sarcoma de Ewing/genética , Sarcoma de Ewing/mortalidad , Trasplante HeterólogoRESUMEN
Autism Spectrum Disorder (ASD) is a highly heritable condition with diverse clinical presentations. Approximately 20% of ASD's genetic susceptibility is imparted by de novo mutations of major effect, most of which cause haploinsufficiency. We mapped enhancers of two high confidence autism genes - CHD8 and SCN2A and used CRISPR-based gene activation (CRISPR-A) in hPSC-derived excitatory neurons and cerebral forebrain organoids to correct the effects of haploinsufficiency, taking advantage of the presence of a wildtype allele of each gene and endogenous gene regulation. We found that CRISPR-A induced a sustained increase in CHD8 and SCN2A expression in treated neurons and organoids, with rescue of gene expression levels and mutation-associated phenotypes, including gene expression and physiology. These data support gene activation via targeting enhancers of haploinsufficient genes, as a therapeutic intervention in ASD and other neurodevelopmental disorders.
RESUMEN
Neurogenins are proneural transcription factors required to specify neuronal identity. Their overexpression in human pluripotent stem cells rapidly produces cortical-like neurons with spiking activity and, because of this, they have been widely adopted for human neuron disease models. However, we do not fully understand the key downstream regulatory effectors responsible for driving neural differentiation. Here, using inducible expression of NEUROG1 and NEUROG2, we identify transcription factors (TFs) required for directed neuronal differentiation by combining expression and chromatin accessibility analyses with a pooled in vitro CRISPR-Cas9 screen targeting all ~1900 TFs in the human genome. The loss of one of these essential TFs (ZBTB18) yields few MAP2-positive neurons. Differentiated ZBTB18-null cells have radically altered gene expression, leading to cytoskeletal defects and stunted neurites and spines. In addition to identifying key downstream TFs for neuronal differentiation, our work develops an integrative multi-omics and TFome-wide perturbation platform to rapidly characterize essential TFs for the differentiation of any human cell type.
Asunto(s)
Células Madre Pluripotentes , Factores de Transcripción , Humanos , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Neurogénesis/genética , Neuronas/metabolismo , Diferenciación Celular/genética , Células Madre Pluripotentes/metabolismoRESUMEN
Ewing sarcoma family of tumors (ESFT) is a group of aggressive pediatric malignancies driven by the EWS-FLI1 fusion protein, an aberrant transcription factor up-regulating specific target genes, such as neuropeptide Y (NPY) and its Y1 and Y5 receptors (Y5Rs). Previously, we have shown that both exogenous NPY and endogenous NPY stimulate ESFT cell death via its Y1 and Y5Rs. Here, we demonstrate that this effect is prevented by dipeptidyl peptidases (DPPs), which cleave NPY to its shorter form, NPY(3-36), not active at Y1Rs. We have shown that NPY-induced cell death can be abolished by overexpression of DPPs and enhanced by their down-regulation. Both NPY treatment and DPP blockade activated the same cell death pathway mediated by poly(ADP-ribose) polymerase (PARP-1) and apoptosis-inducing factor (AIF). Moreover, the decrease in cell survival induced by DPP inhibition was blocked by Y1 and Y5R antagonists, confirming its dependence on endogenous NPY. Interestingly, similar levels of NPY-driven cell death were achieved by blocking membrane DPPIV and cytosolic DPP8 and DPP9. Thus, this is the first evidence of these intracellular DPPs cleaving releasable peptides, such as NPY, in live cells. In contrast, another membrane DPP, fibroblast activation protein (FAP), did not affect NPY actions. In conclusion, DPPs act as survival factors for ESFT cells and protect them from cell death induced by endogenous NPY. This is the first demonstration that intracellular DPPs are involved in regulation of ESFT growth and may become potential therapeutic targets for these tumors.
Asunto(s)
Dipeptidil-Peptidasas y Tripeptidil-Peptidasas/metabolismo , Sarcoma de Ewing/metabolismo , Línea Celular Tumoral , Humanos , ARN Interferente Pequeño , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Sarcoma de Ewing/enzimología , Sarcoma de Ewing/patologíaRESUMEN
BACKGROUND: Prebiotics are food ingredients, usually non-digestible oligosaccharides, that are selectively fermented by populations of beneficial gut bacteria. Endoxylanases, altering the naturally present cereal arabinoxylans, are commonly used in the bread industry to improve dough and bread characteristics. Recently, an in situ method has been developed to produce arabinoxylan-oligosaccharides (AXOS) at high levels in breads through the use of a thermophilic endoxylanase. AXOS have demonstrated potentially prebiotic properties in that they have been observed to lead to beneficial shifts in the microbiota in vitro and in murine, poultry and human studies. METHODS: A double-blind, placebo controlled human intervention study was undertaken with 40 healthy adult volunteers to assess the impact of consumption of breads with in situ produced AXOS (containing 2.2 g AXOS) compared to non-endoxylanase treated breads. Volatile fatty acid concentrations in faeces were assessed and fluorescence in situ hybridisation was used to assess changes in gut microbial groups. Secretory immunoglobulin A (sIgA) levels in saliva were also measured. RESULTS: Consumption of AXOS-enriched breads led to increased faecal butyrate and a trend for reduced iso-valerate and fatty acids associated with protein fermentation. Faecal levels of bifidobacteria increased following initial control breads and remained elevated throughout the study. Lactobacilli levels were elevated following both placebo and AXOS-breads. No changes in salivary secretory IgA levels were observed during the study. Furthermore, no adverse effects on gastrointestinal symptoms were reported during AXOS-bread intake. CONCLUSIONS: AXOS-breads led to a potentially beneficial shift in fermentation end products and are well tolerated.
Asunto(s)
Pan , Endo-1,4-beta Xilanasas/metabolismo , Aditivos Alimentarios/metabolismo , Mucosa Intestinal/microbiología , Oligosacáridos/metabolismo , Prebióticos , Xilanos/metabolismo , Adolescente , Adulto , Bifidobacterium/crecimiento & desarrollo , Bifidobacterium/aislamiento & purificación , Bifidobacterium/metabolismo , Pan/efectos adversos , Pan/análisis , Estudios Cruzados , Método Doble Ciego , Ácidos Grasos Volátiles/análisis , Ácidos Grasos Volátiles/metabolismo , Heces/química , Heces/microbiología , Femenino , Fermentación , Humanos , Masculino , Persona de Mediana Edad , Oligosacáridos/efectos adversos , Oligosacáridos/análisis , Prebióticos/efectos adversos , Prebióticos/análisis , Xilanos/efectos adversos , Xilanos/análisis , Adulto JovenRESUMEN
In pluripotent cells, a delicate activation-repression balance maintains pro-differentiation genes ready for rapid activation. The identity of transcription factors (TFs) that specifically repress pro-differentiation genes remains obscure. By targeting â¼1,700 TFs with CRISPR loss-of-function screen, we found that ZBTB11 and ZFP131 are required for embryonic stem cell (ESC) pluripotency. ESCs without ZBTB11 or ZFP131 lose colony morphology, reduce proliferation rate, and upregulate transcription of genes associated with three germ layers. ZBTB11 and ZFP131 bind proximally to pro-differentiation genes. ZBTB11 or ZFP131 loss leads to an increase in H3K4me3, negative elongation factor (NELF) complex release, and concomitant transcription at associated genes. Together, our results suggest that ZBTB11 and ZFP131 maintain pluripotency by preventing premature expression of pro-differentiation genes and present a generalizable framework to maintain cellular potency.
Asunto(s)
Células Madre Embrionarias , Células Madre Pluripotentes , Animales , Humanos , Ratones , Diferenciación Celular/genética , Sistemas CRISPR-Cas , Células Madre Embrionarias/metabolismo , Estratos Germinativos/metabolismo , Células Madre Pluripotentes/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
Adverse prognosis in Ewing sarcoma (ES) is associated with the presence of metastases, particularly in bone, tumor hypoxia and chromosomal instability (CIN). Yet, a mechanistic link between these factors remains unknown. We demonstrate that in ES, tumor hypoxia selectively exacerbates bone metastasis. This process is triggered by hypoxia-induced stimulation of the neuropeptide Y (NPY)/Y5 receptor (Y5R) pathway, which leads to RhoA over-activation and cytokinesis failure. These mitotic defects result in the formation of polyploid ES cells, the progeny of which exhibit high CIN, an ability to invade and colonize bone, and a resistance to chemotherapy. Blocking Y5R in hypoxic ES tumors prevents polyploidization and bone metastasis. Our findings provide evidence for the role of the hypoxia-inducible NPY/Y5R/RhoA axis in promoting genomic changes and subsequent osseous dissemination in ES, and suggest that targeting this pathway may prevent CIN and disease progression in ES and other cancers rich in NPY and Y5R.
Asunto(s)
Neoplasias Óseas , Sarcoma de Ewing , Neoplasias Óseas/genética , Inestabilidad Cromosómica , Humanos , Hipoxia , Neuropéptido Y/genética , Neuropéptido Y/metabolismo , Receptores de Neuropéptido Y/genética , Receptores de Neuropéptido Y/metabolismo , Sarcoma de Ewing/patología , Proteína de Unión al GTP rhoA/genética , Proteína de Unión al GTP rhoA/metabolismoRESUMEN
BACKGROUND: Many neurodegenerative diseases develop only later in life, when cells in the nervous system lose their structure or function. In many forms of neurodegenerative diseases, this late-onset phenomenon remains largely unexplained. RESULTS: Analyzing single-cell RNA sequencing from Alzheimer's disease (AD) and Huntington's disease (HD) patients, we find increased transcriptional heterogeneity in disease-state neurons. We hypothesize that transcriptional heterogeneity precedes neurodegenerative disease pathologies. To test this idea experimentally, we use juvenile forms (72Q; 180Q) of HD iPSCs, differentiate them into committed neuronal progenitors, and obtain single-cell expression profiles. We show a global increase in gene expression variability in HD. Autophagy genes become more stable, while energy and actin-related genes become more variable in the mutant cells. Knocking down several differentially variable genes results in increased aggregate formation, a pathology associated with HD. We further validate the increased transcriptional heterogeneity in CHD8+/- cells, a model for autism spectrum disorder. CONCLUSIONS: Overall, our results suggest that although neurodegenerative diseases develop over time, transcriptional regulation imbalance is present already at very early developmental stages. Therefore, an intervention aimed at this early phenotype may be of high diagnostic value.
Asunto(s)
Regulación de la Expresión Génica , Heterogeneidad Genética , Predisposición Genética a la Enfermedad , Modelos Biológicos , Enfermedades Neurodegenerativas/etiología , Células Madre Pluripotentes/metabolismo , Adulto , Perfilación de la Expresión Génica , Redes Reguladoras de Genes , Antecedentes Genéticos , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Mutación , RNA-Seq , Análisis de la Célula Individual/métodosRESUMEN
Overexpression of NEUROG2 and NEUROG1 (NEUROG2/1) in human embryonic stem cells (hESCs) rapidly produces functional networks of excitatory and inhibitory neurons. To facilitate the use of this efficient inducible human neuron model in neuroscience research, we generated hESCs with doxycycline-inducible NEUROG2/1 via lentivirus and a tdTomato fluorescent reporter knock-in at the MAP2 locus using the CRISPR nuclease Cas9. Upon doxycycline-driven induction of NEUROG2/1, these hESCs differentiate within days into cells that are uniformly MAP2 immunoreactive and tdTomato fluorescent.
Asunto(s)
Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Técnicas de Cultivo de Célula/métodos , Línea Celular/citología , Técnicas de Sustitución del Gen , Genes Reporteros , Células Madre Embrionarias Humanas/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Secuencia de Bases , Femenino , Humanos , Reproducibilidad de los ResultadosRESUMEN
Neuropeptide Y (NPY) is a multifunctional neurotransmitter acting via G protein-coupled receptors - Y1R, Y2R and Y5R. NPY activities, such as its proliferative effects, are mediated by multiple receptors, which have the ability to dimerize. However, the role of this receptor interplay in NPY functions remains unclear. The goal of the current study was to identify NPY receptor interactions, focusing on the ligand-binding fraction, and determine their impact on the mitogenic activity of the peptide. Y1R, Y2R and Y5R expressed in CHO-K1 cells formed homodimers detectable on the cell surface by cross-linking. Moreover, Y1R and Y5R heterodimerized, while no Y2R/Y5R heterodimers were detected. Nevertheless, Y5R failed to block internalization of its cognate receptor in both Y1R/Y5R and Y2R/Y5R transfectants, indicating Y5R transactivation upon stimulation of the co-expressed receptor. These receptor interactions correlated with an augmented mitogenic response to NPY. In Y1R/Y5R and Y2R/Y5R transfectants, the proliferative response started at picomolar NPY concentrations, while nanomolar concentrations were needed to trigger proliferation in cells transfected with single receptors. Thus, our data identify direct and indirect heterotypic NPY receptor interactions as the mechanism amplifying its activity. Understanding these processes is crucial for the design of treatments targeting the NPY system.
Asunto(s)
Proliferación Celular/fisiología , Neuropéptido Y/metabolismo , Receptores de Neuropéptido Y/metabolismo , Animales , Células CHO , Cricetulus , Multimerización de Proteína/fisiologíaRESUMEN
Understanding neurological diseases requires tractable genetic systems. Engineered 3D neural tissues are an attractive choice, but how the cellular transcriptomic profiles in these tissues are affected by the encapsulating materials and are related to the human-brain transcriptome is not well understood. Here, we report the characterization of the effects of culturing conditions on the transcriptomic profiles of induced neuronal cells, as well as a method for the rapid generation of 3D co-cultures of neuronal and astrocytic cells from the same pool of human embryonic stem cells. By comparing the gene-expression profiles of neuronal cells in culture conditions relevant to the developing human brain, we found that modifying the degree of crosslinking of composite hydrogels can tune expression patterns so they correlate with those of specific brain regions and developmental stages. Moreover, by using single-cell sequencing, we show that our engineered tissues recapitulate transcriptional patterns of cell types in the human brain. The analysis of culturing conditions will inform the development of 3D neural tissues for use as tractable models of brain diseases.
RESUMEN
The postsynaptic scaffolding protein SH3 and multiple ankyrin repeat domains 3 (SHANK3) is critical for the development and function of glutamatergic synapses. Disruption of the SHANK3-encoding gene has been strongly implicated as a monogenic cause of autism, and Shank3 mutant mice show repetitive grooming and social interaction deficits. Although basal ganglia dysfunction has been proposed to underlie repetitive behaviors, few studies have provided direct evidence to support this notion and the exact cellular mechanisms remain largely unknown. Here, we utilized the Shank3B mutant mouse model of autism to investigate how Shank3 mutation may differentially affect striatonigral (direct pathway) and striatopallidal (indirect pathway) medium spiny neurons (MSNs) and its relevance to repetitive grooming behavior in Shank3B mutant mice. We found that Shank3 deletion preferentially affects synapses onto striatopallidal MSNs. Striatopallidal MSNs showed profound defects, including alterations in synaptic transmission, synaptic plasticity, and spine density. Importantly, the repetitive grooming behavior was rescued by selectively enhancing the striatopallidal MSN activity via a Gq-coupled human M3 muscarinic receptor (hM3Dq), a type of designer receptors exclusively activated by designer drugs (DREADD). Our findings directly demonstrate the existence of distinct changes between 2 striatal pathways in a mouse model of autism and indicate that the indirect striatal pathway disruption might play a causative role in repetitive behavior of Shank3B mutant mice.
Asunto(s)
Trastorno Autístico , Cuerpo Estriado , Proteínas del Tejido Nervioso , Plasticidad Neuronal/genética , Sustancia Negra , Transmisión Sináptica/genética , Animales , Trastorno Autístico/genética , Trastorno Autístico/metabolismo , Trastorno Autístico/fisiopatología , Cuerpo Estriado/metabolismo , Cuerpo Estriado/fisiopatología , Modelos Animales de Enfermedad , Humanos , Ratones , Ratones Noqueados , Proteínas de Microfilamentos , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Sustancia Negra/metabolismo , Sustancia Negra/fisiopatologíaRESUMEN
In developing cerebellar interneurons, NMDA increases spontaneous GABA release by activating presynaptic NMDA receptors. We investigated the role of these receptors on differentiating basket/stellate cells in cerebellar cultures grown under conditions allowing functional synaptic transmission. Presynaptic GABAergic boutons were visualized either by GAD65 immunostaining or by using cells derived from GAD65-enhanced green fluorescent protein (eGFP) transgenic mice, in which cerebellar basket/stellate cells express eGFP. After the first week in culture, whole-cell recordings from granule cells reveal that acute application of NMDA increases miniature IPSC (mIPSC) frequency. Interestingly, after 2 weeks, the mIPSC frequency increases compared with the first week but is not modulated by NMDA. Furthermore, in cultures chronically treated with NMDA for 1 week, the size of the GABAergic boutons increases. This growth is paralleled by increased mIPSC frequency and the loss of NMDA sensitivity. Direct patch-clamp recording from these presynaptic terminals reveals single NMDA-activated channels, showing multiple conductance levels, and electronic propagation from the somatodendritic compartment. Our results demonstrate that NMDA receptors alter GABAergic synapses in developing cerebellar cultures by increasing the size of the terminal and spontaneous GABA release. These findings parallel changes in inhibitory synaptic efficacy seen in vivo in developing GABAergic interneurons of the molecular layer of the cerebellum.
Asunto(s)
Cerebelo/citología , Interneuronas/fisiología , N-Metilaspartato/farmacología , Terminales Presinápticos/metabolismo , Receptores de N-Metil-D-Aspartato/fisiología , Ácido gamma-Aminobutírico/metabolismo , Animales , Axones/química , Factor Neurotrófico Derivado del Encéfalo/farmacología , Células Cultivadas/efectos de los fármacos , Células Cultivadas/fisiología , Dendritas/química , Maleato de Dizocilpina/farmacología , Agonistas de Aminoácidos Excitadores/farmacología , Antagonistas de Aminoácidos Excitadores/farmacología , Genes Reporteros , Interneuronas/ultraestructura , Ratones , Ratones Transgénicos , Técnicas de Placa-Clamp , Terminales Presinápticos/efectos de los fármacos , Receptores de N-Metil-D-Aspartato/efectos de los fármacosRESUMEN
Genetic studies have revealed significant overlaps of risk genes among psychiatric disorders. However, it is not clear how different mutations of the same gene contribute to different disorders. We characterized two lines of mutant mice with Shank3 mutations linked to ASD and schizophrenia. We found both shared and distinct synaptic and behavioral phenotypes. Mice with the ASD-linked InsG3680 mutation manifest striatal synaptic transmission defects before weaning age and impaired juvenile social interaction, coinciding with the early onset of ASD symptoms. On the other hand, adult mice carrying the schizophrenia-linked R1117X mutation show profound synaptic defects in prefrontal cortex and social dominance behavior. Furthermore, we found differential Shank3 mRNA stability and SHANK1/2 upregulation in these two lines. These data demonstrate that different alleles of the same gene may have distinct phenotypes at molecular, synaptic, and circuit levels in mice, which may inform exploration of these relationships in human patients.
Asunto(s)
Trastorno del Espectro Autista/genética , Mutación/genética , Proteínas del Tejido Nervioso/genética , Neuronas/metabolismo , Esquizofrenia/genética , Conducta Social , Transmisión Sináptica/genética , Envejecimiento , Animales , Trastorno del Espectro Autista/metabolismo , Conducta Animal , Modelos Animales de Enfermedad , Hipocampo/metabolismo , Hipocampo/fisiopatología , Ratones Transgénicos , Proteínas de Microfilamentos , Proteínas del Tejido Nervioso/metabolismo , Esquizofrenia/metabolismoRESUMEN
A fundamental impediment to understanding the brain is the availability of inexpensive and robust methods for targeting and manipulating specific neuronal populations. The need to overcome this barrier is pressing because there are considerable anatomical, physiological, cognitive and behavioral differences between mice and higher mammalian species in which it is difficult to specifically target and manipulate genetically defined functional cell types. In particular, it is unclear the degree to which insights from mouse models can shed light on the neural mechanisms that mediate cognitive functions in higher species, including humans. Here we describe a novel recombinant adeno-associated virus that restricts gene expression to GABAergic interneurons within the telencephalon. We demonstrate that the viral expression is specific and robust, allowing for morphological visualization, activity monitoring and functional manipulation of interneurons in both mice and non-genetically tractable species, thus opening the possibility to study GABAergic function in virtually any vertebrate species.
Asunto(s)
Encéfalo/virología , Dependovirus/aislamiento & purificación , Neuronas GABAérgicas/virología , Interneuronas/fisiología , Vertebrados/virología , Animales , Conducta Animal , Encéfalo/metabolismo , Células Cultivadas , Dependovirus/genética , Femenino , Neuronas GABAérgicas/patología , Vectores Genéticos/genética , Ratones Endogámicos C57BLRESUMEN
Ewing sarcoma (ES) develops in bones or soft tissues of children and adolescents. The presence of bone metastases is one of the most adverse prognostic factors, yet the mechanisms governing their formation remain unclear. As a transcriptional target of EWS-FLI1, the fusion protein driving ES transformation, neuropeptide Y (NPY) is highly expressed and released from ES tumors. Hypoxia up-regulates NPY and activates its pro-metastatic functions. To test the impact of NPY on ES metastatic pattern, ES cell lines, SK-ES1 and TC71, with high and low peptide release, respectively, were used in an orthotopic xenograft model. ES cells were injected into gastrocnemius muscles of SCID/beige mice, the primary tumors excised, and mice monitored for the presence of metastases. SK-ES1 xenografts resulted in thoracic extra-osseous metastases (67%) and dissemination to bone (50%) and brain (25%), while TC71 tumors metastasized to the lungs (70%). Bone dissemination in SK-ES1 xenografts associated with increased NPY expression in bone metastases and its accumulation in bone invasion areas. The genetic silencing of NPY in SK-ES1 cells reduced bone degradation. Our study supports the role for NPY in ES bone invasion and provides new models for identifying pathways driving ES metastases to specific niches and testing anti-metastatic therapeutics.