RESUMEN
Previous investigations of the core gene regulatory circuitry that controls the pluripotency of embryonic stem (ES) cells have largely focused on the roles of transcription, chromatin and non-coding RNA regulators. Alternative splicing represents a widely acting mode of gene regulation, yet its role in regulating ES-cell pluripotency and differentiation is poorly understood. Here we identify the muscleblind-like RNA binding proteins, MBNL1 and MBNL2, as conserved and direct negative regulators of a large program of cassette exon alternative splicing events that are differentially regulated between ES cells and other cell types. Knockdown of MBNL proteins in differentiated cells causes switching to an ES-cell-like alternative splicing pattern for approximately half of these events, whereas overexpression of MBNL proteins in ES cells promotes differentiated-cell-like alternative splicing patterns. Among the MBNL-regulated events is an ES-cell-specific alternative splicing switch in the forkhead family transcription factor FOXP1 that controls pluripotency. Consistent with a central and negative regulatory role for MBNL proteins in pluripotency, their knockdown significantly enhances the expression of key pluripotency genes and the formation of induced pluripotent stem cells during somatic cell reprogramming.
Asunto(s)
Empalme Alternativo , Reprogramación Celular , Proteínas de Unión al ADN/metabolismo , Células Madre Embrionarias/citología , Células Madre Embrionarias/metabolismo , Proteínas de Unión al ARN/metabolismo , Empalme Alternativo/genética , Secuencias de Aminoácidos , Animales , Diferenciación Celular/genética , Línea Celular , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/deficiencia , Proteínas de Unión al ADN/genética , Fibroblastos/citología , Fibroblastos/metabolismo , Factores de Transcripción Forkhead/metabolismo , Técnicas de Silenciamiento del Gen , Células HEK293 , Células HeLa , Humanos , Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/metabolismo , Cinética , Ratones , Proteínas de Unión al ARN/química , Proteínas de Unión al ARN/genética , Proteínas Represoras/metabolismoRESUMEN
MECP2 mutations cause the X-linked neurodevelopmental disorder Rett Syndrome (RTT) by consistently altering the protein encoded by the MECP2e1 alternative transcript. While mutations that simultaneously affect both MECP2e1 and MECP2e2 isoforms have been widely studied, the consequence of MECP2e1 deficiency on human neurons remains unknown. Here we report the first isoform-specific patient induced pluripotent stem cell (iPSC) model of RTT. RTTe1 patient iPS cell-derived neurons retain an inactive X-chromosome and express only the mutant allele. Single-cell mRNA analysis demonstrated they have a molecular signature of cortical neurons. Mutant neurons exhibited a decrease in soma size, reduced dendritic complexity and decreased cell capacitance, consistent with impaired neuronal maturation. The soma size phenotype was rescued cell-autonomously by MECP2e1 transduction in a level-dependent manner but not by MECP2e2 gene transfer. Importantly, MECP2e1 mutant neurons showed a dysfunction in action potential generation, voltage-gated Na(+) currents, and miniature excitatory synaptic current frequency and amplitude. We conclude that MECP2e1 mutation affects soma size, information encoding properties and synaptic connectivity in human neurons that are defective in RTT.
Asunto(s)
Células Madre Pluripotentes Inducidas/patología , Células Madre Pluripotentes Inducidas/fisiología , Proteína 2 de Unión a Metil-CpG/genética , Neuronas/patología , Neuronas/fisiología , Síndrome de Rett/genética , Potenciales de Acción , Humanos , Mutación , Neuronas/metabolismo , Isoformas de Proteínas , Síndrome de Rett/patología , Síndrome de Rett/fisiopatologíaRESUMEN
The innate immune system recognizes nucleic acids during infection and tissue damage. Whereas viral RNA is detected by endosomal toll-like receptors (TLR3, TLR7, TLR8) and cytoplasmic RIG-I and MDA5, endosomal TLR9 and cytoplasmic DAI bind DNA, resulting in the activation of nuclear factor-kappaB and interferon regulatory factor transcription factors. However, viruses also trigger pro-inflammatory responses, which remain poorly defined. Here we show that internalized adenoviral DNA induces maturation of pro-interleukin-1beta in macrophages, which is dependent on NALP3 and ASC, components of the innate cytosolic molecular complex termed the inflammasome. Correspondingly, NALP3- and ASC-deficient mice display reduced innate inflammatory responses to adenovirus particles. Inflammasome activation also occurs as a result of transfected cytosolic bacterial, viral and mammalian (host) DNA, but in this case sensing is dependent on ASC but not NALP3. The DNA-sensing pro-inflammatory pathway functions independently of TLRs and interferon regulatory factors. Thus, in addition to viral and bacterial components or danger signals in general, inflammasomes sense potentially dangerous cytoplasmic DNA, strengthening their central role in innate immunity.
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Proteínas Portadoras/inmunología , Proteínas del Citoesqueleto/inmunología , Citosol/metabolismo , Citosol/virología , ADN/inmunología , Inmunidad Innata/inmunología , Inflamación/inmunología , Adenoviridae/genética , Adenoviridae/inmunología , Adenoviridae/fisiología , Animales , Proteínas Reguladoras de la Apoptosis , Proteínas Adaptadoras de Señalización CARD , Proteínas Portadoras/genética , Línea Celular , Proteínas del Citoesqueleto/deficiencia , Proteínas del Citoesqueleto/genética , Citosol/microbiología , ADN Viral/inmunología , Humanos , Inflamación/virología , Interleucina-1beta/inmunología , Interleucina-1beta/metabolismo , Macrófagos Peritoneales/inmunología , Macrófagos Peritoneales/metabolismo , Ratones , Ratones Endogámicos C57BL , Proteína con Dominio Pirina 3 de la Familia NLR , Procesamiento Proteico-PostraduccionalRESUMEN
Remodeling of cortical connectivity is thought to allow initially hippocampus-dependent memories to be expressed independently of the hippocampus at remote time points. Consistent with this, consolidation of a contextual fear memory is associated with dendritic spine growth in neurons of the anterior cingulate cortex (aCC). To directly test whether such cortical structural remodeling is necessary for memory consolidation, we disrupted spine growth in the aCC at different times following contextual fear conditioning in mice. We took advantage of previous studies showing that the transcription factor myocyte enhancer factor 2 (MEF2) negatively regulates spinogenesis both in vitro and in vivo. We found that increasing MEF2-dependent transcription in the aCC during a critical posttraining window (but not at later time points) blocked both the consolidation-associated dendritic spine growth and subsequent memory expression. Together, these data strengthen the causal link between cortical structural remodeling and memory consolidation and, further, identify MEF2 as a key regulator of these processes.
Asunto(s)
Espinas Dendríticas/fisiología , Giro del Cíngulo/fisiología , Factores Reguladores Miogénicos/fisiología , Animales , Condicionamiento Psicológico/fisiología , Hipocampo/fisiología , Factores de Transcripción MEF2 , Ratones , Neuronas/ultraestructura , Transcripción GenéticaRESUMEN
BACKGROUND: The X-linked PTCHD1 locus is strongly associated with autism spectrum disorder (ASD). Males who carry chromosome microdeletions of PTCHD1 antisense long non-coding RNA (PTCHD1-AS)/DEAD-box helicase 53 (DDX53) have ASD, or a sub-clinical form called Broader Autism Phenotype. If the deletion extends beyond PTCHD1-AS/DDX53 to the next gene, PTCHD1, which is protein-coding, the individuals typically have ASD and intellectual disability (ID). Three male siblings with a 90 kb deletion that affects only PTCHD1-AS (and not including DDX53) have ASD. We performed a functional analysis of DDX53 to examine its role in NGN2 neurons. METHODS: We used the clustered regularly interspaced short palindromic repeats (CRISPR) gene editing strategy to knock out DDX53 protein by inserting 3 termination codons (3TCs) into two different induced pluripotent stem cell (iPSC) lines. DDX53 CRISPR-edited iPSCs were differentiated into cortical excitatory neurons by Neurogenin 2 (NGN-2) directed differentiation. The functional differences of DDX53-3TC neurons compared to isogenic control neurons with molecular and electrophysiological approaches were assessed. RESULTS: Isogenic iPSC-derived control neurons exhibited low levels of DDX53 transcripts. Transcriptional analysis revealed the generation of excitatory cortical neurons and DDX53 protein was not detected in iPSC-derived control neurons by western blot. Control lines and DDX53-3TC neurons were active in the multi-electrode array, but no overt electrophysiological phenotype in either isogenic line was observed. CONCLUSION: DDX53-3TC mutation does not alter NGN2 neuronal function in these experiments, suggesting that synaptic deficits causing ASD are unlikely in this cell type.
Asunto(s)
Trastorno del Espectro Autista , ARN Helicasas DEAD-box , Células Madre Pluripotentes Inducidas , Humanos , Masculino , Trastorno del Espectro Autista/genética , ARN Helicasas DEAD-box/genética , Células Madre Pluripotentes Inducidas/metabolismo , Mutación , Neuronas/metabolismoRESUMEN
Helper-dependent adenovirus (hdAd) vectors have shown tremendous potential in animal models of human disease in numerous preclinical studies. Expression of a therapeutic transgene can be maintained for several years after a single administration of the hdAd vector. However, despite the long-term persistence of hdAd DNA in the transduced cell, little is known of the fate and structure of hdAd DNA within the host nucleus. In this study, we have characterized the assembly of hdAd DNA into chromatin in tissue culture. Eviction of the Ad DNA-packaging protein VII, histone deposition, and vector-associated gene expression all began within 2 to 6 h of host cell transduction. Inhibition of transcription elongation through the vector DNA template had no effect on the loss of VII, suggesting that transcription was not necessary for removal of the majority of protein VII. Vector DNA assembled into physiologically spaced nucleosomes within 6 h. hdAd vectors incorporated the histone H3 variant H3.3, which was dependent on the histone chaperone HIRA. Knockdown of HIRA reduced hdAd association with histones and reduced expression of the vector-carried transgene by 2- to 3-fold. Our study elucidates an essential role for hdAd DNA chromatinization for optimal vector gene expression.
Asunto(s)
Adenovirus Humanos/genética , Cromatina/metabolismo , ADN Viral/genética , ADN Viral/metabolismo , Expresión Génica , Vectores Genéticos , Línea Celular , Virus Helper/fisiología , HumanosRESUMEN
NMDA receptors (NMDARs), a prominent subtype of glutamatergic receptors, are implicated in the pathogenesis and development of neuropsychiatric disorders such as epilepsy, intellectual disability, autism spectrum disorder, and schizophrenia, and are therefore a potential therapeutic target in treating these disorders. Neurons derived from induced pluripotent stem cells (iPSCs) have provided the opportunity to investigate human NMDARs in their native environment. In this review, we describe the expression, function, and regulation of NMDARs in human iPSC-derived neurons and discuss approaches for utilizing human neurons for identifying potential drugs that target NMDARs in the treatment of neuropsychiatric disorders. A challenge in studying NMDARs in human iPSC-derived neurons is a predominance of those receptors containing the GluN2B subunit and low synaptic expression, suggesting a relatively immature phenotype of these neurons and delayed development of functional NMDARs. We outline potential approaches for improving neuronal maturation of human iPSC-derived neurons and accelerating the functional expression of NMDARs. Acceleration of functional expression of NMDARs in human iPSC-derived neurons will improve the modeling of neuropsychiatric disorders and facilitate the discovery and development of novel therapeutics targeting NMDARs for the treatment of these disorders.
Asunto(s)
Trastorno del Espectro Autista , Células Madre Pluripotentes Inducidas , Trastorno del Espectro Autista/tratamiento farmacológico , Trastorno del Espectro Autista/metabolismo , Evaluación Preclínica de Medicamentos , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Neuronas/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismoRESUMEN
Helper-dependent adenovirus (hdAd) vectors have shown great promise as therapeutic gene delivery vehicles in gene therapy applications. However, the level and duration of gene expression from hdAd can differ considerably depending on the nature of the noncoding stuffer DNA contained within the vector. For example, an hdAd containing 22 kb of prokaryotic DNA (hdAd-prok) expresses its transgene 60-fold less efficiently than a similar vector containing eukaryotic DNA (hdAd-euk). Here we have determined the mechanistic basis of this phenomenon. Although neither vector was subjected to CpG methylation and both genomes associated with cellular histones to similar degrees, hdAd-prok chromatin was actively deacetylated. Insertion of an insulator element between the transgene and the bacterial DNA derepressed hdAd-prok, suggesting that foreign DNA nucleates repressive chromatin structures that spread to the transgene. We found that Sp100B/Sp100HMG and Daxx play a role in repressing transgene expression from hdAd and act independently of PML bodies. Thus, we have identified nuclear factors involved in recognizing foreign DNA and have determined the mechanism by which associated genes are repressed.
Asunto(s)
Adenoviridae/genética , Expresión Génica , Vectores Genéticos , Virus Helper/genética , Transducción Genética , Transgenes , Cromatina/metabolismo , ADN , ADN Viral/metabolismo , Histona Desacetilasas/metabolismo , Histonas/metabolismo , Unión ProteicaRESUMEN
Genetic factors contribute to the development of autism spectrum disorder (ASD), and although non-protein-coding regions of the genome are being increasingly implicated in ASD, the functional consequences of these variants remain largely uncharacterized. Induced pluripotent stem cells (iPSCs) enable the production of personalized neurons that are genetically matched to people with ASD and can therefore be used to directly test the effects of genomic variation on neuronal gene expression, synapse function, and connectivity. The combined use of human pluripotent stem cells with genome editing to introduce or correct specific variants has proved to be a powerful approach for exploring the functional consequences of ASD-associated variants in protein-coding genes and, more recently, long non-coding RNAs (lncRNAs). Here, we review recent studies that implicate lncRNAs, other non-coding mutations, and regulatory variants in ASD susceptibility. We also discuss experimental design considerations for using iPSCs and genome editing to study the role of the non-protein-coding genome in ASD.
Asunto(s)
Trastorno del Espectro Autista/etiología , Trastorno del Espectro Autista/metabolismo , Estudios de Asociación Genética , Predisposición Genética a la Enfermedad , Células Madre Pluripotentes Inducidas/metabolismo , Modelos Biológicos , Neuronas/metabolismo , Alelos , Animales , Biomarcadores , Diferenciación Celular , Modelos Animales de Enfermedad , Regulación de la Expresión Génica , Estudios de Asociación Genética/métodos , Variación Genética , Humanos , Fenotipo , ARN no Traducido , SinapsisRESUMEN
Regulation of translation during human development is poorly understood, and its dysregulation is associated with Rett syndrome (RTT). To discover shifts in mRNA ribosomal engagement (RE) during human neurodevelopment, we use parallel translating ribosome affinity purification sequencing (TRAP-seq) and RNA sequencing (RNA-seq) on control and RTT human induced pluripotent stem cells, neural progenitor cells, and cortical neurons. We find that 30% of transcribed genes are translationally regulated, including key gene sets (neurodevelopment, transcription and translation factors, and glycolysis). Approximately 35% of abundant intergenic long noncoding RNAs (lncRNAs) are ribosome engaged. Neurons translate mRNAs more efficiently and have longer 3' UTRs, and RE correlates with elements for RNA-binding proteins. RTT neurons have reduced global translation and compromised mTOR signaling, and >2,100 genes are translationally dysregulated. NEDD4L E3-ubiquitin ligase is translationally impaired, ubiquitinated protein levels are reduced, and protein targets accumulate in RTT neurons. Overall, the dynamic translatome in neurodevelopment is disturbed in RTT and provides insight into altered ubiquitination that may have therapeutic implications.
Asunto(s)
Sistema Nervioso/crecimiento & desarrollo , Sistema Nervioso/patología , Síndrome de Rett/genética , Ribosomas/metabolismo , Ubiquitinación , Regiones no Traducidas 3'/genética , Animales , Secuencia de Bases , Femenino , Regulación del Desarrollo de la Expresión Génica , Glucólisis/genética , Células Madre Pluripotentes Inducidas/metabolismo , Proteína 2 de Unión a Metil-CpG/metabolismo , Ratones , Ubiquitina-Proteína Ligasas Nedd4/metabolismo , Neuronas/metabolismo , Unión Proteica , Biosíntesis de Proteínas , ARN no Traducido/genética , ARN no Traducido/metabolismo , Proteínas de Unión al ARN/metabolismo , Proteínas Ribosómicas/genética , Proteínas Ribosómicas/metabolismo , Factores de Transcripción/metabolismo , Ubiquitinación/genéticaRESUMEN
BACKGROUND: The Xp22.11 locus that encompasses PTCHD1, DDX53, and the long noncoding RNA PTCHD1-AS is frequently disrupted in male subjects with autism spectrum disorder (ASD), but the functional consequences of these genetic risk factors for ASD are unknown. METHODS: To evaluate the functional consequences of PTCHD1 locus deletions, we generated induced pluripotent stem cells (iPSCs) from unaffected control subjects and 3 subjects with ASD with microdeletions affecting PTCHD1-AS/PTCHD1, PTCHD1-AS/DDX53, or PTCHD1-AS alone. Function of iPSC-derived cortical neurons was assessed using molecular approaches and electrophysiology. We also compiled novel and known genetic variants of the PTCHD1 locus to explore the roles of PTCHD1 and PTCHD1-AS in genetic risk for ASD and other neurodevelopmental disorders. Finally, genome editing was used to explore the functional consequences of deleting a single conserved exon of PTCHD1-AS. RESULTS: iPSC-derived neurons from subjects with ASD exhibited reduced miniature excitatory postsynaptic current frequency and N-methyl-D-aspartate receptor hypofunction. We found that 35 ASD-associated deletions mapping to the PTCHD1 locus disrupted exons of PTCHD1-AS. We also found a novel ASD-associated deletion of PTCHD1-AS exon 3 and showed that exon 3 loss altered PTCHD1-AS splicing without affecting expression of the neighboring PTCHD1 coding gene. Finally, targeted disruption of PTCHD1-AS exon 3 recapitulated diminished miniature excitatory postsynaptic current frequency, supporting a role for the long noncoding RNA in the etiology of ASD. CONCLUSIONS: Our genetic findings provide strong evidence that PTCHD1-AS deletions are risk factors for ASD, and human iPSC-derived neurons implicate these deletions in the neurophysiology of excitatory synapses and in ASD-associated synaptic impairment.
Asunto(s)
Trastorno del Espectro Autista , Trastorno Autístico , Células Madre Pluripotentes Inducidas , Trastorno del Espectro Autista/genética , Trastorno Autístico/genética , Humanos , Masculino , Proteínas de la Membrana , Neuronas , SinapsisRESUMEN
NMDA receptors (NMDARs) are critical for physiological synaptic plasticity, learning, and memory and for pathological plasticity and neuronal death. The GluN1 subunit is encoded by a single gene, GRIN1, with 8 splice variants, but whether the diversity generated by this splicing has physiological consequences remains enigmatic. Here, we generate mice lacking from the GluN1 exon 5-encoded N1 cassette (GluN1a mice) or compulsorily expressing this exon (GluN1b mice). Despite no differences in basal synaptic transmission, long-term potentiation in the hippocampus is significantly enhanced in GluN1a mice compared with that in GluN1b mice. Furthermore, GluN1a mice learn more quickly and have significantly better spatial memory performance than do GluN1b mice. In addition, in human iPSC-derived neurons in autism spectrum disorder NMDARs show characteristics of N1-lacking GluN1. Our findings indicate that alternative splicing of GluN1 is a mechanism for controlling physiological long-lasting synaptic potentiation, learning, and memory.
Asunto(s)
Empalme Alternativo , Trastorno del Espectro Autista/genética , Potenciación a Largo Plazo/genética , Proteínas del Tejido Nervioso/genética , Neuronas/metabolismo , Receptores de N-Metil-D-Aspartato/genética , Animales , Trastorno del Espectro Autista/metabolismo , Trastorno del Espectro Autista/patología , Muerte Celular/genética , Diferenciación Celular , Potenciales Postsinápticos Excitadores/fisiología , Exones , Hipocampo/metabolismo , Hipocampo/patología , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Células Madre Pluripotentes Inducidas/patología , Ratones , Ratones Noqueados , Proteínas del Tejido Nervioso/deficiencia , Neuronas/patología , Cultivo Primario de Células , Subunidades de Proteína/deficiencia , Subunidades de Proteína/genética , Receptores de N-Metil-D-Aspartato/deficiencia , Memoria Espacial/fisiología , Sinapsis/metabolismo , Transmisión SinápticaRESUMEN
Heterozygous loss-of-function mutations in SHANK2 are associated with autism spectrum disorder (ASD). We generated cortical neurons from induced pluripotent stem cells derived from neurotypic and ASD-affected donors. We developed sparse coculture for connectivity assays where SHANK2 and control neurons were differentially labeled and sparsely seeded together on a lawn of unlabeled control neurons. We observed increases in dendrite length, dendrite complexity, synapse number, and frequency of spontaneous excitatory postsynaptic currents. These findings were phenocopied in gene-edited homozygous SHANK2 knockout cells and rescued by gene correction of an ASD SHANK2 mutation. Dendrite length increases were exacerbated by IGF1, TG003, or BDNF, and suppressed by DHPG treatment. The transcriptome in isogenic SHANK2 neurons was perturbed in synapse, plasticity, and neuronal morphogenesis gene sets and ASD gene modules, and activity-dependent dendrite extension was impaired. Our findings provide evidence for hyperconnectivity and altered transcriptome in SHANK2 neurons derived from ASD subjects.
Asunto(s)
Trastorno del Espectro Autista/genética , Trastorno del Espectro Autista/patología , Dendritas/patología , Proteínas del Tejido Nervioso/genética , Neuronas/patología , Trastorno del Espectro Autista/metabolismo , Técnicas de Cocultivo , Dendritas/metabolismo , Potenciales Postsinápticos Excitadores , Técnicas de Inactivación de Genes , Haploinsuficiencia , Humanos , Células Madre Pluripotentes Inducidas , Masculino , Plasticidad Neuronal , Neuronas/metabolismo , TranscriptomaRESUMEN
Autism spectrum disorder (ASD) is phenotypically and genetically heterogeneous. We present a CRISPR gene editing strategy to insert a protein tag and premature termination sites creating an induced pluripotent stem cell (iPSC) knockout resource for functional studies of ten ASD-relevant genes (AFF2/FMR2, ANOS1, ASTN2, ATRX, CACNA1C, CHD8, DLGAP2, KCNQ2, SCN2A, TENM1). Neurogenin 2 (NGN2)-directed induction of iPSCs allowed production of excitatory neurons, and mutant proteins were not detectable. RNA sequencing revealed convergence of several neuronal networks. Using both patch-clamp and multi-electrode array approaches, the electrophysiological deficits measured were distinct for different mutations. However, they culminated in a consistent reduction in synaptic activity, including reduced spontaneous excitatory postsynaptic current frequencies in AFF2/FMR2-, ASTN2-, ATRX-, KCNQ2-, and SCN2A-null neurons. Despite ASD susceptibility genes belonging to different gene ontologies, isogenic stem cell resources can reveal common functional phenotypes, such as reduced functional connectivity.
Asunto(s)
Trastorno Autístico/genética , Trastorno Autístico/fisiopatología , Edición Génica , Predisposición Genética a la Enfermedad , Neuronas/metabolismo , Neuronas/patología , Línea Celular , Electrodos , Técnicas de Inactivación de Genes , Células HEK293 , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Mutagénesis Insercional/genética , FenotipoRESUMEN
NMDA receptor (NMDAR)-mediated fast excitatory neurotransmission is implicated in a broad range of physiological and pathological processes in the mammalian central nervous system. The function and regulation of NMDARs have been extensively studied in neurons from rodents and other non-human species, and in recombinant expression systems. Here, we investigated human NMDARs in situ by using neurons produced by directed differentiation of human induced pluripotent stem cells (iPSCs). The resultant cells showed electrophysiological characteristics demonstrating that they are bona fide neurons. In particular, human iPSC-derived neurons expressed functional ligand-gated ion channels, including NMDARs, AMPA receptors, GABAA receptors, as well as glycine receptors. Pharmacological and electrophysiological properties of NMDAR-mediated currents indicated that these were dominated by receptors containing GluN2B subunits. The NMDAR currents were suppressed by genistein, a broad-spectrum tyrosine kinase inhibitor. The NMDAR currents were also inhibited by a Fyn-interfering peptide, Fyn(39-57), but not a Src-interfering peptide, Src(40-58). Together, these findings are the first evidence that tyrosine phosphorylation regulates the function of NMDARs in human iPSC-derived neurons. Our findings provide a basis for utilizing human iPSC-derived neurons in screening for drugs targeting NMDARs in neurological disorders.
Asunto(s)
Células Madre Pluripotentes Inducidas/citología , Neuronas/fisiología , Proteínas Proto-Oncogénicas c-fyn/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Diferenciación Celular , Células Cultivadas , Genisteína/farmacología , Humanos , Plasticidad Neuronal , Neuronas/metabolismo , Transmisión Sináptica/efectos de los fármacosRESUMEN
A progressive increase in MECP2 protein levels is a crucial and precisely regulated event during neurodevelopment, but the underlying mechanism is unclear. We report that MECP2 is regulated post-transcriptionally during in vitro differentiation of human embryonic stem cells (hESCs) into cortical neurons. Using reporters to identify functional RNA sequences in the MECP2 3' UTR and genetic manipulations to explore the role of interacting factors on endogenous MECP2, we discover combinatorial mechanisms that regulate RNA stability and translation. The RNA-binding protein PUM1 and pluripotent-specific microRNAs destabilize the long MECP2 3' UTR in hESCs. Hence, the 3' UTR appears to lengthen during differentiation as the long isoform becomes stable in neurons. Meanwhile, translation of MECP2 is repressed by TIA1 in hESCs until HuC predominates in neurons, resulting in a switch to translational enhancement. Ultimately, 3' UTR-directed translational fine-tuning differentially modulates MECP2 protein in the two cell types to levels appropriate for normal neurodevelopment.
Asunto(s)
Regulación de la Expresión Génica , Proteína 2 de Unión a Metil-CpG/genética , MicroARNs/metabolismo , Neuronas/metabolismo , Proteínas de Unión al ARN/metabolismo , Transcripción Genética , Regiones no Traducidas 3'/genética , Elementos Ricos en Adenilato y Uridilato/genética , Secuencia de Bases , Linaje de la Célula , Proliferación Celular , Secuencia Conservada/genética , Células Madre Embrionarias Humanas/citología , Células Madre Embrionarias Humanas/metabolismo , Humanos , Proteína 2 de Unión a Metil-CpG/metabolismo , Células-Madre Neurales/citología , Células-Madre Neurales/metabolismo , Prosencéfalo/embriología , Unión Proteica/genética , Biosíntesis de Proteínas , Estabilidad del ARN/genética , ARN Mensajero/genética , ARN Mensajero/metabolismoRESUMEN
BACKGROUND: Williams-Beuren Syndrome (WBS) is caused by the microdeletion of approximately 25 genes on chromosome 7q11.23, and is characterized by a spectrum of cognitive and behavioural features. RESULTS: We generated cortical neurons from a WBS individual and unaffected (WT) control by directed differentiation of induced pluripotent stem cells (iPSCs). Single cell mRNA analyses and immunostaining demonstrated very efficient production of differentiated cells expressing markers of mature neurons of mixed subtypes and from multiple cortical layers. We found that there was a profound alteration in action potentials, with significantly prolonged WBS repolarization times and a WBS deficit in voltage-activated K(+) currents. Miniature excitatory synaptic currents were normal, indicating that unitary excitatory synaptic transmission was not altered. Gene expression profiling identified 136 negatively enriched gene sets in WBS compared to WT neurons including gene sets involved in neurotransmitter receptor activity, synaptic assembly, and potassium channel complexes. CONCLUSIONS: Our findings provide insight into gene dysregulation and electrophysiological defects in WBS patient neurons.
Asunto(s)
Células Madre Pluripotentes Inducidas/citología , Modelos Biológicos , Neuronas/patología , Síndrome de Williams/patología , Potenciales de Acción , Diferenciación Celular , Forma de la Célula , Regulación hacia Abajo , Perfilación de la Expresión Génica , Hemicigoto , Humanos , Fenotipo , Canales de Potasio/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , Reproducibilidad de los Resultados , Síndrome de Williams/genética , Síndrome de Williams/fisiopatologíaRESUMEN
Autism spectrum disorder (ASD) is an early-onset neurodevelopmental disorder characterized by deficits in social communication, and restricted and repetitive patterns of behavior. Despite its high prevalence, discovery of pathophysiological mechanisms underlying ASD has lagged due to a lack of appropriate model systems. Recent advances in induced pluripotent stem cell (iPSC) technology and neural differentiation techniques allow for detailed functional analyses of neurons generated from living individuals with ASD. Refinement of cortical neuron differentiation methods from iPSCs will enable mechanistic studies of specific neuronal subpopulations that may be preferentially impaired in ASD. In this review, we summarize recent accomplishments in differentiation of cortical neurons from human pluripotent stems cells and efforts to establish in vitro model systems to study ASD using personalized neurons.
RESUMEN
Memory formation is thought to be mediated by dendritic-spine growth and restructuring. Myocyte enhancer factor 2 (MEF2) restricts spine growth in vitro, suggesting that this transcription factor negatively regulates the spine remodeling necessary for memory formation. Here we show that memory formation in adult mice was associated with changes in endogenous MEF2 levels and function. Locally and acutely increasing MEF2 function in the dentate gyrus blocked both learning-induced increases in spine density and spatial-memory formation. Increasing MEF2 function in amygdala disrupted fear-memory formation. We rescued MEF2-induced memory disruption by interfering with AMPA receptor endocytosis, suggesting that AMPA receptor trafficking is a key mechanism underlying the effects of MEF2. In contrast, decreasing MEF2 function in dentate gyrus and amygdala facilitated the formation of spatial and fear memory, respectively. These bidirectional effects indicate that MEF2 is a key regulator of plasticity and that relieving the suppressive effects of MEF2-mediated transcription permits memory formation.