RESUMEN
BACKGROUND: Neuronal development is a tightly controlled process involving multi-layered regulatory mechanisms. While transcriptional pathways regulating neurodevelopment are well characterized, post-transcriptional programs are still poorly understood. TIA1 is an RNA-binding protein that can regulate splicing, stability, or translation of target mRNAs, and has been shown to play critical roles in stress response and neurodevelopment. However, the identity of mRNAs regulated by TIA1 during neurodevelopment under unstressed conditions is still unknown. METHODS AND RESULTS: To identify the mRNAs targeted by TIA1 during the first stages of human neurodevelopment, we performed RNA immunoprecipitation-sequencing (RIP-seq) on human embryonic stem cells (hESCs) and derived neural progenitor cells (NPCs), and cortical neurons under unstressed conditions. While there was no change in TIA1 protein levels, the number of TIA1 targeted mRNAs decreased from pluripotent cells to neurons. We identified 2400, 845, and 330 TIA1 mRNA targets in hESCs, NPC, and neurons, respectively. The vast majority of mRNA targets in hESC were genes associated with neurodevelopment and included autism spectrum disorder-risk genes that were not bound in neurons. Additionally, we found that most TIA1 mRNA targets have reduced ribosomal engagement levels. CONCLUSION: Our results reveal TIA1 mRNA targets in hESCs and during human neurodevelopment, indicate that translation repression is a key process targeted by TIA1 binding and implicate TIA1 function in neuronal differentiation.
Asunto(s)
Neurogénesis/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Transducción de Señal/genética , Antígeno Intracelular 1 de las Células T/genética , Antígeno Intracelular 1 de las Células T/metabolismo , Trastorno del Espectro Autista/genética , Sitios de Unión , Diferenciación Celular/genética , Línea Celular , Técnicas de Silenciamiento del Gen , Células Madre Embrionarias Humanas/metabolismo , Humanos , Inmunoprecipitación/métodos , Células-Madre Neurales/metabolismo , Neuronas/metabolismo , Unión Proteica , ARN Interferente Pequeño/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Ribosomas/metabolismo , Análisis de Secuencia de ARN/métodos , TransfecciónRESUMEN
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.
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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 contribution of mRNA half-life is commonly overlooked when examining changes in mRNA abundance during development. mRNA levels of some genes are regulated by transcription rate only, but others may be regulated by mRNA half-life only shifts. Furthermore, transcriptional buffering is predicted when changes in transcription rates have compensating shifts in mRNA half-life resulting in no change to steady-state levels. Likewise, transcriptional boosting should result when changes in transcription rate are accompanied by amplifying half-life shifts. During neurodevelopment there is widespread 3'UTR lengthening that could be shaped by differential shifts in the stability of existing short or long 3'UTR transcript isoforms. We measured transcription rate and mRNA half-life changes during induced human Pluripotent Stem Cell (iPSC)-derived neuronal development using RATE-seq. During transitions to progenitor and neuron stages, transcriptional buffering occurred in up to 50%, and transcriptional boosting in up to 15%, of genes with changed transcription rates. The remaining changes occurred by transcription rate only or mRNA half-life only shifts. Average mRNA half-life decreased two-fold in neurons relative to iPSCs. Short gene isoforms were more destabilized in neurons and thereby increased the average 3'UTR length. Small RNA sequencing captured an increase in microRNA copy number per cell during neurodevelopment. We propose that mRNA destabilization and 3'UTR lengthening are driven in part by an increase in microRNA load in neurons. Our findings identify mRNA stability mechanisms in human neurodevelopment that regulate gene and isoform level abundance and provide a precedent for similar post-transcriptional regulatory events as other tissues develop.
RESUMEN
Transcriptional changes in Rett syndrome (RTT) are assumed to directly correlate with steady-state mRNA levels, but limited evidence in mice suggests that changes in transcription can be compensated by post-transcriptional regulation. We measure transcription rate and mRNA half-life changes in RTT patient neurons using RATEseq, and re-interpret nuclear and whole-cell RNAseq from Mecp2 mice. Genes are dysregulated by changing transcription rate or half-life and are buffered when both change. We utilized classifier models to predict the direction of transcription rate changes and find that combined frequencies of three dinucleotides are better predictors than CA and CG. MicroRNA and RNA-binding Protein (RBP) motifs are enriched in 3'UTRs of genes with half-life changes. Nuclear RBP motifs are enriched on buffered genes with increased transcription rate. We identify post-transcriptional mechanisms in humans and mice that alter half-life or buffer transcription rate changes when a transcriptional modulator gene is mutated in a neurodevelopmental disorder.
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Síndrome de Rett , Humanos , Ratones , Animales , Síndrome de Rett/genética , ARN Mensajero , Semivida , Proteína 2 de Unión a Metil-CpG/metabolismo , Regulación de la Expresión GénicaRESUMEN
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.
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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
Rett syndrome (RTT) is a severe neurodevelopmental disorder primarily caused by heterozygous loss-of-function mutations in the X-linked gene MECP2 that is a global transcriptional regulator. Mutations in the methyl-CpG binding domain (MBD) of MECP2 disrupt its interaction with methylated DNA. Here, we investigate the effect of a novel MECP2 L124W missense mutation in the MBD of an atypical RTT patient with preserved speech in comparison to severe MECP2 null mutations. L124W protein had a limited ability to disrupt heterochromatic chromocenters due to decreased binding dynamics. We isolated two pairs of isogenic WT and L124W induced pluripotent stem cells. L124W induced excitatory neurons expressed stable protein, exhibited increased input resistance and decreased voltage-gated Na+ and K+ currents, and their neuronal dysmorphology was limited to decreased dendritic complexity. Three isogenic pairs of MECP2 null neurons had the expected more extreme morphological and electrophysiological phenotypes. We examined development and maturation of L124W and MECP2 null excitatory neural network activity using micro-electrode arrays. Relative to isogenic controls, L124W neurons had an increase in synchronous network burst frequency, in contrast to MECP2 null neurons that suffered a significant decrease in synchronous network burst frequency and a transient extension of network burst duration. A biologically motivated computational neural network model shows the observed changes in network dynamics are explained by changes in intrinsic Na+ and K+ currents in individual neurons. Our multilevel results demonstrate that RTT excitatory neurons show a wide spectrum of morphological, electrophysiological and circuitry phenotypes that are dependent on the severity of the MECP2 mutation.
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Proteína 2 de Unión a Metil-CpG , Síndrome de Rett , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Proteína 2 de Unión a Metil-CpG/genética , Mutación , Neuronas/metabolismo , Fenotipo , Síndrome de Rett/genéticaRESUMEN
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.
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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.
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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
Induced pluripotent stem cell (iPSC)-derived neurons are increasingly used to model Autism Spectrum Disorder (ASD), which is clinically and genetically heterogeneous. To study the complex relationship of penetrant and weaker polygenic risk variants to ASD, 'isogenic' iPSC-derived neurons are critical. We developed a set of procedures to control for heterogeneity in reprogramming and differentiation, and generated 53 different iPSC-derived glutamatergic neuronal lines from 25 participants from 12 unrelated families with ASD. Heterozygous de novo and rare-inherited presumed-damaging variants were characterized in ASD risk genes/loci. Combinations of putative etiologic variants (GLI3/KIF21A or EHMT2/UBE2I) in separate families were modeled. We used a multi-electrode array, with patch-clamp recordings, to determine a reproducible synaptic phenotype in 25% of the individuals with ASD (other relevant data on the remaining lines was collected). Our most compelling new results revealed a consistent spontaneous network hyperactivity in neurons deficient for CNTN5 or EHMT2. The biobank of iPSC-derived neurons and accompanying genomic data are available to accelerate ASD research. Editorial note: This article has been through an editorial process in which authors decide how to respond to the issues raised during peer review. The Reviewing Editor's assessment is that all the issues have been addressed (see decision letter).
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Trastorno Autístico/fisiopatología , Contactinas/metabolismo , Antígenos de Histocompatibilidad/metabolismo , N-Metiltransferasa de Histona-Lisina/metabolismo , Células Madre Pluripotentes Inducidas/fisiología , Red Nerviosa/fisiología , Neuronas/fisiología , Adolescente , Adulto , Células Cultivadas , Niño , Contactinas/deficiencia , Contactinas/genética , Fenómenos Electrofisiológicos , Femenino , Heterocigoto , Antígenos de Histocompatibilidad/genética , N-Metiltransferasa de Histona-Lisina/deficiencia , N-Metiltransferasa de Histona-Lisina/genética , Humanos , Cinesinas/genética , Cinesinas/metabolismo , Masculino , Persona de Mediana Edad , Modelos Teóricos , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Técnicas de Placa-Clamp , Enzimas Ubiquitina-Conjugadoras/genética , Enzimas Ubiquitina-Conjugadoras/metabolismo , Adulto Joven , Proteína Gli3 con Dedos de Zinc/genética , Proteína Gli3 con Dedos de Zinc/metabolismoRESUMEN
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.
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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
Induced pluripotent stem cells (iPSC) derived from healthy individuals are important controls for disease-modeling studies. Here we apply precision health to create a high-quality resource of control iPSCs. Footprint-free lines were reprogrammed from four volunteers of the Personal Genome Project Canada (PGPC). Multilineage-directed differentiation efficiently produced functional cortical neurons, cardiomyocytes and hepatocytes. Pilot users demonstrated versatility by generating kidney organoids, T lymphocytes, and sensory neurons. A frameshift knockout was introduced into MYBPC3 and these cardiomyocytes exhibited the expected hypertrophic phenotype. Whole-genome sequencing-based annotation of PGPC lines revealed on average 20 coding variants. Importantly, nearly all annotated PGPC and HipSci lines harbored at least one pre-existing or acquired variant with cardiac, neurological, or other disease associations. Overall, PGPC lines were efficiently differentiated by multiple users into cells from six tissues for disease modeling, and variant-preferred healthy control lines were identified for specific disease settings.
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Diferenciación Celular , Linaje de la Célula , Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/metabolismo , Sistemas CRISPR-Cas , Autorrenovación de las Células , Separación Celular , Ectodermo/citología , Ectodermo/metabolismo , Edición Génica , Humanos , Miocitos Cardíacos/citología , Miocitos Cardíacos/metabolismo , Neuronas/citología , Neuronas/metabolismo , Organoides , Fenotipo , Linfocitos T/metabolismo , Secuenciación Completa del GenomaRESUMEN
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.
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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
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.