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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.
Assuntos
Síndrome de Rett , Humanos , Camundongos , Animais , Síndrome de Rett/genética , RNA Mensageiro , Meia-Vida , Proteína 2 de Ligação a Metil-CpG/metabolismo , Regulação da Expressão GênicaRESUMO
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.
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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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Transtorno do Espectro Autista , RNA Helicases DEAD-box , Células-Tronco Pluripotentes Induzidas , Humanos , Masculino , Transtorno do Espectro Autista/genética , RNA Helicases DEAD-box/genética , Células-Tronco Pluripotentes Induzidas/metabolismo , Mutação , Neurônios/metabolismoRESUMO
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.
Assuntos
Proteína 2 de Ligação a Metil-CpG , Síndrome de Rett , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Proteína 2 de Ligação a Metil-CpG/genética , Mutação , Neurônios/metabolismo , Fenótipo , Síndrome de Rett/genéticaRESUMO
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.
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Neurogênese/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transdução de Sinais/genética , Antígeno-1 Intracelular de Células T/genética , Antígeno-1 Intracelular de Células T/metabolismo , Transtorno do Espectro Autista/genética , Sítios de Ligação , Diferenciação Celular/genética , Linhagem Celular , Técnicas de Silenciamento de Genes , Células-Tronco Embrionárias Humanas/metabolismo , Humanos , Imunoprecipitação/métodos , Células-Tronco Neurais/metabolismo , Neurônios/metabolismo , Ligação Proteica , RNA Interferente Pequeno/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Ribossomos/metabolismo , Análise de Sequência de RNA/métodos , TransfecçãoRESUMO
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.
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Transtorno do Espectro Autista/etiologia , Transtorno do Espectro Autista/metabolismo , Estudos de Associação Genética , Predisposição Genética para Doença , Células-Tronco Pluripotentes Induzidas/metabolismo , Modelos Biológicos , Neurônios/metabolismo , Alelos , Animais , Biomarcadores , Diferenciação Celular , Modelos Animais de Doenças , Regulação da Expressão Gênica , Estudos de Associação Genética/métodos , Variação Genética , Humanos , Fenótipo , RNA não Traduzido , SinapsesRESUMO
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 Nervoso/crescimento & desenvolvimento , Sistema Nervoso/patologia , Síndrome de Rett/genética , Ribossomos/metabolismo , Ubiquitinação , Regiões 3' não Traduzidas/genética , Animais , Sequência de Bases , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Glicólise/genética , Células-Tronco Pluripotentes Induzidas/metabolismo , Proteína 2 de Ligação a Metil-CpG/metabolismo , Camundongos , Ubiquitina-Proteína Ligases Nedd4/metabolismo , Neurônios/metabolismo , Ligação Proteica , Biossíntese de Proteínas , RNA não Traduzido/genética , RNA não Traduzido/metabolismo , Proteínas de Ligação a RNA/metabolismo , Proteínas Ribossômicas/genética , Proteínas Ribossômicas/metabolismo , Fatores de Transcrição/metabolismo , Ubiquitinação/genéticaRESUMO
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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Transtorno do Espectro Autista , Transtorno Autístico , Células-Tronco Pluripotentes Induzidas , Transtorno do Espectro Autista/genética , Transtorno Autístico/genética , Humanos , Masculino , Proteínas de Membrana , Neurônios , SinapsesRESUMO
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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Diferenciação Celular , Linhagem da Célula , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Sistemas CRISPR-Cas , Autorrenovação Celular , Separação Celular , Ectoderma/citologia , Ectoderma/metabolismo , Edição de Genes , Humanos , Miócitos Cardíacos/citologia , Miócitos Cardíacos/metabolismo , Neurônios/citologia , Neurônios/metabolismo , Organoides , Fenótipo , Linfócitos T/metabolismo , Sequenciamento Completo do GenomaRESUMO
S17 is a clonogenic bone marrow stromal (BMS) cell line derived from mouse that has been extensively used to assess both human and murine hematopoiesis support capacity. However, very little is known about the expression of potassium ion channels and their function in cell survival and migration in these cells. Thus, the present study was designed to characterize potassium ion channels using electrophysiological and molecular biological approaches in S17 BMS cells. The whole-cell configuration of the patch clamp technique has been applied to identify potassium ion currents and reverse transcription polymerase chain reaction (RT-PCR) used to determine their molecular identities. Based on gating kinetics and pharmacological modulation of the macroscopic currents we found the presence of four functional potassium ion channels in S17 BMS cells. These include a current rapidly activated and inactivated, tetraethylammonium-sensitive, (IKV ) in most (50%) cells; a fast activated and rapidly inactivating A-type K + current (IK A -like); a delayed rectifier K + current (IK DR ) and an inward rectifier potassium current (IK IR ), found in, respectively 4.5%, 26% and 24% of these cells. RT-PCR confirmed the presence of mRNA transcripts for the alpha subunit of the corresponding functional ion channels. Additionally, functional assays were performed to investigate the importance of potassium currents in cell survival and migration. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide analyses revealed a reduction in cell viability, while wound healing assays revealed reduced migration potential in cells incubated with different potassium channel blockers. In conclusion, our data suggested that potassium currents might play a role in the maintenance of overall S17 cell ionic homeostasis directly affecting cell survival and migration.
Assuntos
Movimento Celular , Células-Tronco Mesenquimais/metabolismo , Canais de Potássio/metabolismo , Potássio/metabolismo , Animais , Linhagem Celular , Movimento Celular/efeitos dos fármacos , Sobrevivência Celular , Ativação do Canal Iônico , Cinética , Potenciais da Membrana , Células-Tronco Mesenquimais/efeitos dos fármacos , Camundongos , Bloqueadores dos Canais de Potássio/farmacologia , Canais de Potássio/efeitos dos fármacos , Canais de Potássio/genética , Transdução de SinaisRESUMO
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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Transtorno do Espectro Autista/genética , Transtorno do Espectro Autista/patologia , Dendritos/patologia , Proteínas do Tecido Nervoso/genética , Neurônios/patologia , Transtorno do Espectro Autista/metabolismo , Técnicas de Cocultura , Dendritos/metabolismo , Potenciais Pós-Sinápticos Excitadores , Técnicas de Inativação de Genes , Haploinsuficiência , Humanos , Células-Tronco Pluripotentes Induzidas , Masculino , Plasticidade Neuronal , Neurônios/metabolismo , TranscriptomaRESUMO
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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Transtorno Autístico/fisiopatologia , Contactinas/metabolismo , Antígenos de Histocompatibilidade/metabolismo , Histona-Lisina N-Metiltransferase/metabolismo , Células-Tronco Pluripotentes Induzidas/fisiologia , Rede Nervosa/fisiologia , Neurônios/fisiologia , Adolescente , Adulto , Células Cultivadas , Criança , Contactinas/deficiência , Contactinas/genética , Fenômenos Eletrofisiológicos , Feminino , Heterozigoto , Antígenos de Histocompatibilidade/genética , Histona-Lisina N-Metiltransferase/deficiência , Histona-Lisina N-Metiltransferase/genética , Humanos , Cinesinas/genética , Cinesinas/metabolismo , Masculino , Pessoa de Meia-Idade , Modelos Teóricos , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Técnicas de Patch-Clamp , Enzimas de Conjugação de Ubiquitina/genética , Enzimas de Conjugação de Ubiquitina/metabolismo , Adulto Jovem , Proteína Gli3 com Dedos de Zinco/genética , Proteína Gli3 com Dedos de Zinco/metabolismoRESUMO
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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Transtorno Autístico/genética , Transtorno Autístico/fisiopatologia , Edição de Genes , Predisposição Genética para Doença , Neurônios/metabolismo , Neurônios/patologia , Linhagem Celular , Eletrodos , Técnicas de Inativação de Genes , Células HEK293 , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Mutagênese Insercional/genética , FenótipoRESUMO
Mass spectrometry (MS) analysis of human post-mortem central nervous system (CNS) tissue and induced pluripotent stem cell (iPSC)-based directed differentiations offer complementary avenues to define protein signatures of neurodevelopment. Methodological improvements of formalin-fixed, paraffin-embedded (FFPE) protein isolation now enable widespread proteomic analysis of well-annotated archival tissue samples in the context of development and disease. Here, we utilize a shotgun label-free quantification (LFQ) MS method to profile magnetically enriched human cortical neurons and neural progenitor cells (NPCs) derived from iPSCs. We use these signatures to help define spatiotemporal protein dynamics of developing human FFPE cerebral regions. We show that the use of high resolution Q Exactive mass spectrometers now allow simultaneous quantification of >2700 proteins in a single LFQ experiment and provide sufficient coverage to define novel biomarkers and signatures of NPC maintenance and differentiation. Importantly, we show that this abbreviated strategy allows efficient recovery of novel cytoplasmic, membrane-specific and synaptic proteins that are shared between both in vivo and in vitro neuronal differentiation. This study highlights the discovery potential of non-comprehensive high-throughput proteomic profiling of unfractionated clinically well-annotated FFPE human tissue from a diverse array of development and diseased states.
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Cérebro/embriologia , Cérebro/metabolismo , Proteômica/métodos , Diferenciação Celular , Linhagem Celular , Feto/embriologia , Formaldeído , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Espectrometria de Massas , Modelos Biológicos , Células-Tronco Neurais/metabolismo , Neurônios/metabolismo , Inclusão em Parafina , Proteoma/metabolismo , Fixação de TecidosRESUMO
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.
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Regulação da Expressão Gênica , Proteína 2 de Ligação a Metil-CpG/genética , MicroRNAs/metabolismo , Neurônios/metabolismo , Proteínas de Ligação a RNA/metabolismo , Transcrição Gênica , Regiões 3' não Traduzidas/genética , Elementos Ricos em Adenilato e Uridilato/genética , Sequência de Bases , Linhagem da Célula , Proliferação de Células , Sequência Conservada/genética , Células-Tronco Embrionárias Humanas/citologia , Células-Tronco Embrionárias Humanas/metabolismo , Humanos , Proteína 2 de Ligação a Metil-CpG/metabolismo , Células-Tronco Neurais/citologia , Células-Tronco Neurais/metabolismo , Prosencéfalo/embriologia , Ligação Proteica/genética , Biossíntese de Proteínas , Estabilidade de RNA/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
Pluripotent mouse embryonic stem cells (mESC) are cell lines derived from the inner cell mass of blastocyst-stage early mammalian embryos. Since ion channel modulation has been reported to interfere with both growth and differentiation process in mouse and human ESC it is important to characterize the electrophysiological properties of newly generated mESC and compare them to other lines. In this work, we studied the intercellular communication by way of gap junctions in a Brazilian derived mESC (USP-1, generated by Dr. Lygia Pereira's group) and characterized its electrophysiological properties. We used immunofluorescence and RT-PCR to reveal the presence of connexin 43 (Cx43), pluripotency markers and ion channels. Using a co-culture of neonatal mouse cardiomyocytes with mESC, where the heart cells expressed the enhanced Green Fluorescent Protein, we performed dye injections to assess functional coupling between the two cell types observing dye diffusion. The patch-clamp study showed outward currents identified as two types of potassium currents, transient outward potassium current (Ito) and delayed rectifier outward potassium current (Iks), by use of specific drug blockage. Calcium or sodium currents in undifferentiated mESC were not identified. We conclude that USP-1 mESC has functional Cx43 channels establishing intercellular communication among themselves and with cardiomyocytes and has a similar electrophysiological profile compared to other mESC cell lines.
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Diferenciação Celular/fisiologia , Células-Tronco Embrionárias/fisiologia , Miócitos Cardíacos/fisiologia , Animais , Animais Recém-Nascidos , Brasil , Comunicação Celular , Corantes , Células-Tronco Embrionárias/citologia , Humanos , Imuno-Histoquímica , Camundongos , Miócitos Cardíacos/citologia , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
INTRODUCTION: The objective of this work was to evaluate the efficacy of placenta-derived mesenchymal stem cell (MSC) therapy in a mouse model of myocardial infarction (MI). Since MSCs can be obtained from two different regions of the human term placenta (chorionic plate or villi), cells obtained from both these regions were compared so that the best candidate for cell therapy could be selected. METHODS: For the in vitro studies, chorionic plate MSCs (cp-MSCs) and chorionic villi MSCs (cv-MSCs) were extensively characterized for their genetic stability, clonogenic and differentiation potential, gene expression, and immunophenotype. For the in vivo studies, C57Bl/6 mice were submitted to MI and, after 21 days, received weekly intramyocardial injections of cp-MSCs for 3 weeks. Cells were also stably transduced with a viral construct expressing luciferase, under the control of the murine stem cell virus (MSCV) promoter, and were used in a bioluminescence assay. The expression of genes associated with the insulin signaling pathway was analyzed in the cardiac tissue from cp-MSCs and placebo groups. RESULTS: Morphology, differentiation, immunophenotype, and proliferation were quite similar between these cells. However, cp-MSCs had a greater clonogenic potential and higher expression of genes related to cell cycle progression and genome stability. Therefore, we considered that the chorionic plate was preferable to the chorionic villi for the isolation of MSCs. Sixty days after MI, cell-treated mice had a significant increase in ejection fraction and a reduction in end-systolic volume. This improvement was not caused by a reduction in infarct size. In addition, tracking of cp-MSCs transduced with luciferase revealed that cells remained in the heart for 4 days after the first injection but that the survival period was reduced after the second and third injections. Quantitative reverse transcription-polymerase chain reaction revealed similar expression of genes involved in the insulin signaling pathway when comparing cell-treated and placebo groups. CONCLUSIONS: Improvement of cardiac function by cp-MSCs did not require permanent engraftment and was not mediated by the insulin signaling pathway.
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Insulina/fisiologia , Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais/fisiologia , Infarto do Miocárdio/terapia , Animais , Volume Cardíaco , Diferenciação Celular , Forma Celular , Células Cultivadas , Feminino , Humanos , Camundongos Endogâmicos C57BL , Infarto do Miocárdio/fisiopatologia , Miocárdio/patologia , Fenótipo , Transdução de Sinais , Volume SistólicoRESUMO
DNA damage repair (DDR) is an orchestrated process encompassing the injury detection to its complete resolution. DNA double-strand break lesions are repaired mainly by two distinct mechanisms: the error-free homologous recombination (HR) and the error-prone non-homologous end-joining. Galectin-3 (GAL3) is the unique member of the chimeric galectins subfamily and is reported to be involved in several cancer development and progression related events. Recently our group described a putative protein interaction between GAL3 and BARD1, the main partner of breast and ovarian cancer susceptibility gene product BRCA1, both involved in HR pathway. In this report we characterized GAL3/BARD1 protein interaction and evaluated the role of GAL3 in DDR pathways using GAL3 silenced human cells exposed to different DNA damage agents. In the absence of GAL3 we observed a delayed DDR response activation, as well as a decrease in the G 2/M cell cycle checkpoint arrest associated with HR pathway. Moreover, using a TAP-MS approach we also determined the protein interaction network of GAL3.
Assuntos
Dano ao DNA , Reparo do DNA , Galectina 3/metabolismo , Antineoplásicos/farmacologia , Proteína BRCA1/metabolismo , Proteínas Sanguíneas , Carboplatina/farmacologia , Etoposídeo/farmacologia , Pontos de Checagem da Fase G2 do Ciclo Celular , Galectina 3/genética , Galectinas , Inativação Gênica , Células HEK293 , Células HeLa , Humanos , Mitomicina/farmacologia , Mapas de Interação de Proteínas , Transdução de Sinais , Proteínas Supressoras de Tumor/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , UbiquitinaçãoRESUMO
Properties of induced pluripotent stem cells (iPSC) have been extensively studied since their first derivation in 2006. However, the modification in reactive oxygen species (ROS) production and detoxification caused by reprogramming still needs to be further elucidated. The objective of this study was to compare the response of iPSC generated from menstrual blood-derived mesenchymal stem cells (mb-iPSC), embryonic stem cells (H9) and adult menstrual blood-derived mesenchymal stem cells (mbMSC) to ROS exposure and investigate the effects of reprogramming on cellular oxidative stress (OS). mbMSC were extremely resistant to ROS exposure, however, mb-iPSC were 10-fold less resistant to H(2)O(2), which was very similar to embryonic stem cell sensitivity. Extracellular production of ROS was also similar in mb-iPSC and H9 and almost threefold lower than in mbMSC. Furthermore, intracellular amounts of ROS were higher in mb-iPSC and H9 when compared with mbMSC. As the ability to metabolize ROS is related to antioxidant enzymes, we analysed enzyme activities in these cell types. Catalase and superoxide dismutase activities were reduced in mb-iPSC and H9 when compared with mbMSC. Finally, cell adhesion under OS conditions was impaired in mb-iPSC when compared with mbMSC, albeit similar to H9. Thus, reprogramming leads to profound modifications in extracellular ROS production accompanied by loss of the ability to handle OS.