RESUMO
Fused in sarcoma (FUS) is a pathogenic RNA-binding protein in amyotrophic lateral sclerosis (ALS). We previously reported that FUS stabilizes Synaptic Ras-GTPase activating protein 1 (Syngap1) mRNA at its 3' untranslated region (UTR) and maintains spine maturation. To elucidate the pathologic roles of this mechanism in ALS patients, we identified the SYNGAP1 3'UTR variant rs149438267 in seven (four males and three females) out of 807 ALS patients at the FUS binding site from a multicenter cohort in Japan. Human-induced pluripotent stem cell (hiPSC)-derived motor neurons with the SYNGAP1 variant showed aberrant splicing, increased isoform α1 levels, and decreased isoform γ levels, which caused dendritic spine loss. Moreover, the SYNGAP1 variant excessively recruited FUS and heterogeneous nuclear ribonucleoprotein K (HNRNPK), and antisense oligonucleotides (ASOs) blocking HNRNPK altered aberrant splicing and ameliorated dendritic spine loss. These data suggest that excessive recruitment of RNA-binding proteins, especially HNRNPK, as well as changes in SYNGAP1 isoforms, are crucial for spine formation in motor neurons.SIGNIFICANCE STATEMENT It is not yet known which RNAs cause the pathogenesis of amyotrophic lateral sclerosis (ALS). We previously reported that Fused in sarcoma (FUS), a pathogenic RNA-binding protein in ALS, stabilizes synaptic Ras-GTPase activating protein 1 (Syngap1) mRNA at its 3' untranslated region (UTR) and maintains dendritic spine maturation. To elucidate whether this mechanism is crucial for ALS, we identified the SYNGAP1 3'UTR variant rs149438267 at the FUS binding site. Human-induced pluripotent stem cell (hiPSC)-derived motor neurons with the SYNGAP1 variant showed aberrant splicing, which caused dendritic spine loss along with excessive recruitment of FUS and heterogeneous nuclear ribonucleoprotein K (HNRNPK). Our findings that dendritic spine loss is because of excess recruitment of RNA-binding proteins provide a basis for the future exploration of ALS-related RNA-binding proteins.
Assuntos
Esclerose Lateral Amiotrófica , Sarcoma , Masculino , Feminino , Humanos , Esclerose Lateral Amiotrófica/metabolismo , Regiões 3' não Traduzidas/genética , Proteína FUS de Ligação a RNA/genética , Proteína FUS de Ligação a RNA/metabolismo , Ribonucleoproteínas Nucleares Heterogêneas Grupo K/genética , Espinhas Dendríticas/metabolismo , Mutação , Proteínas de Ligação a RNA/genética , RNA Mensageiro/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteínas Ativadoras de GTPase/genética , Proteínas Ativadoras de GTPase/metabolismo , Sarcoma/genética , Proteínas Ativadoras de ras GTPase/genéticaRESUMO
One of the key areas in stem cell research is the identification of factors capable of promoting the expansion of Neural Stem Cell/Progenitor Cells (NSPCs) and understanding their molecular mechanisms for future use in clinical settings. We previously identified Macrophage Migration Inhibitory Factor (MIF) as a novel factor that can support the proliferation and/or survival of NSPCs based on in vitro functional cloning strategy and revealed that MIF can support the proliferation of human brain tumor-initiating cells (BTICs). However, the detailed downstream signaling for the functions has largely remained unknown. Thus, in the present study, we newly identified translationally-controlled tumor protein-1 (TPT1), which is expressed in the ventricular zone of mouse embryonic brain, as a downstream target of MIF signaling in mouse and human NSPCs and human BTICs. Using gene manipulation (over or downregulation of TPT1) techniques including CRISPR/Cas9-mediated heterozygous gene disruption showed that TPT1 contributed to the regulation of cell proliferation/survival in mouse NSPCs, human embryonic stem cell (hESC) derived-NSPCs, human-induced pluripotent stem cells (hiPSCs) derived-NSPCs and BTICs. Furthermore, gene silencing of TPT1 caused defects in neuronal differentiation in the NSPCs in vitro. We also identified the MIF-CHD7-TPT1-SMO signaling axis in regulating hESC-NSPCs and BTICs proliferation. Intriguingly, TPT1suppressed the miR-338 gene, which targets SMO in hESC-NSPCs and BTICs. Finally, mice with implanted BTICs infected with lentivirus-TPT1 shRNA showed a longer overall survival than control. These results also open up new avenues for the development of glioma therapies based on the TPT1 signaling pathway.
Assuntos
Fatores Inibidores da Migração de Macrófagos , Células-Tronco Neoplásicas , Células-Tronco Neurais , Proteína Tumoral 1 Controlada por Tradução , Animais , Encéfalo/metabolismo , Proliferação de Células/fisiologia , Humanos , Oxirredutases Intramoleculares , Fatores Inibidores da Migração de Macrófagos/genética , Fatores Inibidores da Migração de Macrófagos/metabolismo , Camundongos , MicroRNAs/metabolismo , Proteínas de Neoplasias/metabolismo , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neurais/metabolismo , Proteína Tumoral 1 Controlada por Tradução/genéticaRESUMO
Amyotrophic lateral sclerosis (ALS) is an adult-onset incurable motor neuron (MN) disease. The reasons for selective MN vulnerability in ALS are unknown. Axonal pathology is among the earliest signs of ALS. We searched for novel modulatory genes in human MN axon shortening affected by TARDBP mutations. In transcriptome analysis of RNA present in the axon compartment of human-derived induced pluripotent stem cell (iPSC)-derived MNs, PHOX2B (paired-like homeobox protein 2B) showed lower expression in TARDBP mutant axons, which was consistent with axon qPCR and in situ hybridization. PHOX2B mRNA stability was reduced in TARDBP mutant MNs. Furthermore, PHOX2B knockdown reduced neurite length in human MNs. Finally, phox2b knockdown in zebrafish induced short spinal axons and impaired escape response. PHOX2B is known to be highly express in other types of neurons maintained after ALS progression. Collectively, TARDBP mutations induced loss of axonal resilience, which is an important ALS-related phenotype mediated by PHOX2B downregulation.
Assuntos
Esclerose Lateral Amiotrófica/metabolismo , Axônios/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas de Homeodomínio/metabolismo , Células-Tronco Pluripotentes Induzidas/metabolismo , Neurônios Motores/metabolismo , Fatores de Transcrição/metabolismo , Peixe-Zebra/metabolismo , Esclerose Lateral Amiotrófica/genética , Animais , Proteínas de Ligação a DNA/genética , Regulação da Expressão Gênica , Técnicas de Silenciamento de Genes/métodos , Proteínas de Homeodomínio/genética , Humanos , Mutação , Fenótipo , Fatores de Transcrição/genética , TranscriptomaRESUMO
Fused in sarcoma/translated in liposarcoma (FUS) is a causative gene of amyotrophic lateral sclerosis (ALS). Mutated FUS causes accumulation of DNA damage and cytosolic stress granule (SG) formation, thereby motor neuron (MN) death. However, key molecular aetiology remains unclear. Here, we applied a novel platform technology, iBRN, "Non- biased" Bayesian gene regulatory network analysis based on induced pluripotent stem cell (iPSC)-derived cell model, to elucidate the molecular aetiology using transcriptome of iPSC-derived MNs harboring FUSH517D. iBRN revealed "hub molecules", which strongly influenced transcriptome network, such as miR-125b-5p-TIMELESS axis and PRKDC for the molecular aetiology. Next, we confirmed miR-125b-5p-TIMELESS axis in FUSH517D MNs such that miR-125b-5p regulated several DNA repair-related genes including TIMELESS. In addition, we validated both introduction of miR-125b-5p and knocking down of TIMELESS caused DNA damage in the cell culture model. Furthermore, PRKDC was strongly associated with FUS mis-localization into SGs by DNA damage under impaired DNA-PK activity. Collectively, our iBRN strategy provides the first compelling evidence to elucidate molecular aetiology in neurodegenerative diseases.
Assuntos
Esclerose Lateral Amiotrófica/genética , Redes Reguladoras de Genes/fisiologia , Células-Tronco Pluripotentes Induzidas/fisiologia , MicroRNAs/genética , Proteína FUS de Ligação a RNA/genética , Esclerose Lateral Amiotrófica/metabolismo , Teorema de Bayes , Linhagem Celular Tumoral , Dano ao DNA/fisiologia , Técnicas de Inativação de Genes/métodos , Humanos , MicroRNAs/biossíntese , Proteína FUS de Ligação a RNA/biossínteseRESUMO
Leucine-rich repeat kinase 2 (LRRK2) is the causal gene of the autosomal dominant hereditary form of Parkinson's disease (PD), PARK8. We have previously reported that induced pluripotent stem cells (iPSCs) from a PARK8 patient with I2020T LRRK2 mutation replicated to some extent the pathologic phenotype evident in the brain of PD patients. In the present study, we generated gene-corrected iPSCs line, KEIUi001-A, using TALEN-mediated genome editing. KEIUi001-A retained a normal karyotype and pluripotency, i.e. the capacity to differentiate into cell types of the three germ layers. This iPSCs will be valuable for clarifying various aspects of LRRK2-related pathology.
Assuntos
Células-Tronco Pluripotentes Induzidas , Doença de Parkinson , Humanos , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/genética , Mutação , Doença de Parkinson/genética , FenótipoRESUMO
Obtaining differentiated cells with high physiological functions by an efficient, but simple and rapid differentiation method is crucial for modeling neuronal diseases in vitro using human pluripotent stem cells (hPSCs). Currently, methods involving the transient expression of one or a couple of transcription factors have been established as techniques for inducing neuronal differentiation in a rapid, single step. It has also been reported that microRNAs can function as reprogramming effectors for directly reprogramming human dermal fibroblasts to neurons. In this study, we tested the effect of adding neuronal microRNAs, miRNA-9/9*, and miR-124 (miR-9/9*-124), for the neuronal induction method of hPSCs using Tet-On-driven expression of the Neurogenin2 gene (Ngn2), a proneural factor. While it has been established that Ngn2 can facilitate differentiation from pluripotent stem cells into neurons with high purity due to its neurogenic effect, a long or indefinite time is required for neuronal maturation with Ngn2 misexpression alone. With the present method, the cells maintained a high neuronal differentiation rate while exhibiting increased gene expression of neuronal maturation markers, spontaneous calcium oscillation, and high electrical activity with network bursts as assessed by a multipoint electrode system. Moreover, when applying this method to iPSCs from Alzheimer's disease (AD) patients with presenilin-1 (PS1) or presenilin-2 (PS2) mutations, cellular phenotypes such as increased amount of extracellular secretion of amyloid ß42, abnormal oxygen consumption, and increased reactive oxygen species in the cells were observed in a shorter culture period than those previously reported. Therefore, it is strongly anticipated that the induction method combining Ngn2 and miR-9/9*-124 will enable more rapid and simple screening for various types of neuronal disease phenotypes and promote drug discovery.
Assuntos
MicroRNAs/metabolismo , Doenças do Sistema Nervoso/genética , Neurogênese/fisiologia , Neurônios/metabolismo , Células-Tronco Pluripotentes/metabolismo , Diferenciação Celular , Humanos , Neurônios/citologia , Fenótipo , TransfecçãoRESUMO
Knock-in (KI) gene targeting can be employed for a wide range of applications in stem cell research. However, vectors for KI require multiple complicated processes for construction, including multiple times of digestion/ligation steps and extensive restriction mapping, which has imposed limitations for the robust applicability of KI gene targeting. To circumvent this issue, here we introduce versatile and systematic methods for generating KI vectors by molecular cloning. In this approach, we employed the Multisite Gateway technology, an efficient in vitro DNA recombination system using proprietary sequences and enzymes. KI vector construction exploiting these methods requires only efficient steps, such as PCR and recombination, enabling robust KI gene targeting. We show that combinatorial usage of the KI vectors generated using this method and site-specific nucleases enabled the precise integration of fluorescent protein genes in multiple loci of human and common marmoset (marmoset; Callithrix jacchus) pluripotent stem cells. The methods described here will facilitate the usage of KI technology and ultimately help to accelerate stem cell research.
Assuntos
DNA Recombinante/genética , Técnicas de Introdução de Genes/métodos , Marcação de Genes/métodos , Vetores Genéticos/genética , Animais , Callithrix/genética , Clonagem Molecular/métodos , Desoxirribonucleases/genética , Genes Reporter/genética , Proteínas de Fluorescência Verde/genética , Humanos , Pesquisa com Células-TroncoRESUMO
We developed a reporter system that can be used in a dual manner in visualizing mature osteoblast formation. The system is based on a helper-dependent adenoviral vector (HDAdV), in which a fluorescent protein, Venus, is expressed under the control of the 19-kb human osteocalcin (OC) genomic locus. By infecting human and murine primary osteoblast (POB) cultures with this reporter vector, the cells forming bone-like nodules were specifically visualized by the reporter. In addition, the same vector was utilized to efficiently knock-in the reporter into the endogenous OC gene of human induced pluripotent stem cells (iPSCs), by homologous recombination. Neural crest-like cells (NCLCs) derived from the knock-in reporter iPSCs were differentiated into osteoblasts forming bone-like nodules and could be visualized by the expression of the fluorescent reporter. Living mature osteoblasts were then isolated from the murine mixed POB culture by fluorescence-activated cell sorting (FACS), and their mRNA expression profile was analyzed. Our study presents unique utility of reporter HDAdVs in stem cell biology and related applications.
Assuntos
Adenoviridae/genética , Diferenciação Celular , Células-Tronco Embrionárias/citologia , Células-Tronco Pluripotentes Induzidas/citologia , Osteoblastos/citologia , Osteogênese , Osteossarcoma/patologia , Animais , Neoplasias Ósseas/metabolismo , Neoplasias Ósseas/patologia , Células-Tronco Embrionárias/metabolismo , Genes Reporter , Vetores Genéticos/administração & dosagem , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Osteoblastos/metabolismo , Osteossarcoma/metabolismoRESUMO
BACKGROUND: The characteristic structure of motor neurons (MNs), particularly of the long axons, becomes damaged in the early stages of amyotrophic lateral sclerosis (ALS). However, the molecular pathophysiology of axonal degeneration remains to be fully elucidated. METHOD: Two sets of isogenic human-induced pluripotent stem cell (hiPSCs)-derived MNs possessing the single amino acid difference (p.H517D) in the fused in sarcoma (FUS) were constructed. By combining MN reporter lentivirus, MN specific phenotype was analyzed. Moreover, RNA profiling of isolated axons were conducted by applying the microfluidic devices that enable axon bundles to be produced for omics analysis. The relationship between the target gene, which was identified as a pathological candidate in ALS with RNA-sequencing, and the MN phenotype was confirmed by intervention with si-RNA or overexpression to hiPSCs-derived MNs and even in vivo. The commonality was further confirmed with other ALS-causative mutant hiPSCs-derived MNs and human pathology. FINDINGS: We identified aberrant increasing of axon branchings in FUS-mutant hiPSCs-derived MN axons compared with isogenic controls as a novel phenotype. We identified increased level of Fos-B mRNA, the binding target of FUS, in FUS-mutant MNs. While Fos-B reduction using si-RNA or an inhibitor ameliorated the observed aberrant axon branching, Fos-B overexpression resulted in aberrant axon branching even in vivo. The commonality of those phenotypes was further confirmed with other ALS causative mutation than FUS. INTERPRETATION: Analyzing the axonal fraction of hiPSC-derived MNs using microfluidic devices revealed that Fos-B is a key regulator of FUS-mutant axon branching. FUND: Japan Agency for Medical Research and development; Japanese Ministry of Education, Culture, Sports, Science and Technology Clinical Research, Innovation and Education Center, Tohoku University Hospital; Japan Intractable Diseases (Nanbyo) Research Foundation; the Kanae Foundation for the Promotion of Medical Science; and "Inochi-no-Iro" ALS research grant.
Assuntos
Esclerose Lateral Amiotrófica/genética , Proteínas Proto-Oncogênicas c-fos/genética , Proteína FUS de Ligação a RNA/genética , Esclerose Lateral Amiotrófica/patologia , Animais , Axônios/metabolismo , Axônios/patologia , Diferenciação Celular/genética , Linhagem Celular , Edição de Genes/métodos , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Lentivirus/genética , Neurônios Motores/metabolismo , Mutação , Neurogênese/genética , Fenótipo , RNA Interferente Pequeno/genéticaRESUMO
The mechanism by which dopaminergic neurons are selectively affected in Parkinson's disease is not fully understood. In this study, we found a dramatic increase in the expression of catechol-O-methyltransferase (COMT), along with a lower level of DNA methylation, in induced pluripotent stem cell-derived dopaminergic neurons from patients with parkin (PARK2) gene mutations compared to those from healthy controls. In addition, a significant increase in the expression of COMT was found in dopaminergic neurons of isogenic PARK2 induced pluripotent stem cell lines that mimicked loss of function of PARK2 by CRISPR Cas9 technology. In dopamine transporter (DAT)-Cre mice, overexpression of COMT, specifically in dopaminergic neurons of the substantia nigra, produced cataleptic behaviours associated with impaired motor coordination. These findings suggest that upregulation of COMT, likely resulting from DNA hypomethylation, in dopaminergic neurons may contribute to the initial stage of neuronal dysfunction in Parkinson's disease.
Assuntos
Catecol O-Metiltransferase/genética , Neurônios Dopaminérgicos/metabolismo , Doença de Parkinson/metabolismo , Ubiquitina-Proteína Ligases/genética , Animais , Proteínas da Membrana Plasmática de Transporte de Dopamina/genética , Proteínas da Membrana Plasmática de Transporte de Dopamina/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Camundongos Transgênicos , Doença de Parkinson/genética , Doença de Parkinson/fisiopatologia , Substância Negra/metabolismoRESUMO
Genome editing technology greatly facilitates the genetic modification of various cells and animals. The common marmoset (Callithrix jacchus), a small non-human primate which exhibits high reproductive efficiency, is a widely used animal model in biomedical research. Developing genome editing techniques in the common marmoset will further enhance its utility. Here, we report the successful establishment of a knock-in (KI) method for marmoset embryonic stem cells (ESCs), which is based on the CRISPR-Cas9 system. The use of CRISPR-Cas9, mediated by homologous recombination (HR), enhanced the KI efficiency in marmoset ESCs. Furthermore, we succeeded in performing KI in early-stage marmoset embryos. In the course of the experiments, we found that HR in the marmoset ESCs is innately highly efficient. This suggested that the marmoset possesses a repair mechanism for DNA double-strand breaks. The current study will facilitate the generation of genetically modified marmosets and gene function analysis in the marmoset.
Assuntos
Sistemas CRISPR-Cas , Quebras de DNA de Cadeia Dupla , Embrião de Mamíferos/citologia , Células-Tronco Embrionárias/citologia , Edição de Genes , Técnicas de Introdução de Genes/métodos , Células-Tronco Neurais/citologia , Animais , Callithrix , Embrião de Mamíferos/metabolismo , Células-Tronco Embrionárias/metabolismo , Feminino , Fatores de Transcrição Forkhead/antagonistas & inibidores , Fatores de Transcrição Forkhead/genética , Marcação de Genes , Recombinação Homóloga , Humanos , Masculino , Modelos Animais , Proteína Proteolipídica de Mielina/antagonistas & inibidores , Proteína Proteolipídica de Mielina/genética , Células-Tronco Neurais/metabolismoRESUMO
Parkinson disease (PD) is a progressive neurological disease caused by selective degeneration of dopaminergic (DA) neurons in the substantia nigra. Although most cases of PD are sporadic cases, familial PD provides a versatile research model for basic mechanistic insights into the pathogenesis of PD. In this study, we generated DA neurons from PARK2 patient-specific, isogenic PARK2 null and PARK6 patient-specific induced pluripotent stem cells and found that these neurons exhibited more apoptosis and greater susceptibility to rotenone-induced mitochondrial stress. From phenotypic screening with an FDA-approved drug library, one voltage-gated calcium channel antagonist, benidipine, was found to suppress rotenone-induced apoptosis. Furthermore, we demonstrated the dysregulation of calcium homeostasis and increased susceptibility to rotenone-induced stress in PD, which is prevented by T-type calcium channel knockdown or antagonists. These findings suggest that calcium homeostasis in DA neurons might be a useful target for developing new drugs for PD patients.
Assuntos
Canais de Cálcio Tipo T/metabolismo , Neurônios Dopaminérgicos/patologia , Mitocôndrias/metabolismo , Estresse Oxidativo , Doença de Parkinson/metabolismo , Doença de Parkinson/patologia , Apoptose/efeitos dos fármacos , Cálcio/metabolismo , Bloqueadores dos Canais de Cálcio/química , Bloqueadores dos Canais de Cálcio/farmacologia , Linhagem Celular , Neurônios Dopaminérgicos/metabolismo , Homeostase/efeitos dos fármacos , Humanos , Células-Tronco Pluripotentes Induzidas , Mitocôndrias/efeitos dos fármacos , Modelos Biológicos , Crescimento Neuronal/efeitos dos fármacos , Fármacos Neuroprotetores/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Proteínas Quinases/metabolismo , Rotenona/toxicidade , Ubiquitina-Proteína Ligases/metabolismoRESUMO
Multiple-system atrophy (MSA) is a neurodegenerative disease characterized by autonomic failure with various combinations of parkinsonism, cerebellar ataxia, and pyramidal dysfunction. We previously reported that functionally impaired variants of COQ2, which encodes an essential enzyme in the biosynthetic pathway of coenzyme Q10, are associated with MSA. Here, we report functional deficiencies in mitochondrial respiration and the antioxidative system in induced pluripotent stem cell (iPSC)-derived neurons from an MSA patient with compound heterozygous COQ2 mutations. The functional deficiencies were rescued by site-specific CRISPR/Cas9-mediated gene corrections. We also report an increase in apoptosis of iPSC-derived neurons from MSA patients. Coenzyme Q10 reduced apoptosis of neurons from the MSA patient with compound heterozygous COQ2 mutations. Our results reveal that cellular dysfunctions attributable to decreased coenzyme Q10 levels are related to neuronal death in MSA, particularly in patients with COQ2 variants, and may contribute to the development of therapy using coenzyme Q10 supplementation.
Assuntos
Células-Tronco Pluripotentes Induzidas/metabolismo , Atrofia de Múltiplos Sistemas/metabolismo , Atrofia de Múltiplos Sistemas/patologia , Neurônios/metabolismo , Ubiquinona/análogos & derivados , Adulto , Alquil e Aril Transferases/metabolismo , Sequência de Aminoácidos , Atrofia/metabolismo , Atrofia/patologia , Sequência de Bases , Feminino , Humanos , Células-Tronco Pluripotentes Induzidas/patologia , Masculino , Pessoa de Meia-Idade , Mitocôndrias/metabolismo , Mutação/genética , Neurônios/patologia , Ubiquinona/metabolismoRESUMO
Ghrelin exerts a wide range of physiological actions throughout the body and appears to be a promising target for disease therapy. Endogenous ghrelin receptors (GHSRs) are present in extrahypothalamic sites including the substantia nigra pars compacta (SNc), which is related to phenotypic dysregulation or frank degeneration in Parkinson's disease (PD). Here we found a dramatic decrease in the expression of GHSR in PD-specific induced pluripotent stem cell (iPSC)-derived dopaminergic (DAnergic) neurons generated from patients carrying parkin gene (PARK2) mutations compared to those from healthy controls. Consistently, a significant decrease in the expression of GHSR was found in DAnergic neurons of isogenic PARK2-iPSC lines that mimicked loss of function of the PARK2 gene through CRISPR Cas9 technology. Furthermore, either intracerebroventricular injection or microinjection into the SNc of the selective GHSR1a antagonist [D-Lys3]-GHRP6 in normal mice produced cataleptic behaviors related to dysfunction of motor coordination. These findings suggest that the down-regulation of GHSRs in SNc-DA neurons induced the initial dysfunction of DA neurons, leading to extrapyramidal disorder under PD.
Assuntos
Neurônios Dopaminérgicos/metabolismo , Regulação para Baixo , Atividade Motora , Doença de Parkinson/patologia , Doença de Parkinson/fisiopatologia , Receptores de Grelina/genética , Substância Negra/metabolismo , Substância Negra/patologia , Animais , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Neurônios Dopaminérgicos/efeitos dos fármacos , Regulação para Baixo/efeitos dos fármacos , Técnicas de Introdução de Genes , Técnicas de Inativação de Genes , Humanos , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Células-Tronco Pluripotentes Induzidas/metabolismo , Injeções Intraventriculares , Masculino , Camundongos Endogâmicos C57BL , Atividade Motora/efeitos dos fármacos , Oligopeptídeos/farmacologia , Doença de Parkinson/metabolismo , Receptores de Grelina/antagonistas & inibidores , Receptores de Grelina/metabolismo , Ubiquitina-Proteína Ligases/metabolismoRESUMO
Dravet syndrome (DS) is an infantile epileptic encephalopathy mainly caused by de novo mutations in the SCN1A gene encoding the α1 subunit of the voltage-gated sodium channel Nav1.1. As an in vitro model of this disease, we previously generated an induced pluripotent stem cell (iPSC) line from a patient with DS carrying a c.4933C>T (p.R1645*) substitution in SCN1A. Here, we describe developing a genome-edited control cell line from this DS iPSC line by substituting the point mutation with the wild-type residue. This artificial control iPSC line will be a powerful tool for research into the pathology of DS.
Assuntos
Técnicas de Cultura de Células/métodos , Epilepsias Mioclônicas/patologia , Edição de Genes , Canal de Sódio Disparado por Voltagem NAV1.1/genética , Nucleases dos Efetores Semelhantes a Ativadores de Transcrição/metabolismo , Adulto , Sequência de Bases , Linhagem Celular , Feminino , Humanos , Células-Tronco Pluripotentes Induzidas , Repetições de Microssatélites/genética , Mycoplasma/isolamento & purificaçãoRESUMO
Patient-specific induced pluripotent stem cells (iPSCs) show promise for use as tools for in vitro modeling of Parkinson's disease. We sought to improve the efficiency of dopaminergic (DA) neuron induction from iPSCs by the using surface markers expressed in DA progenitors to increase the significance of the phenotypic analysis. By sorting for a CD184high/CD44- fraction during neural differentiation, we obtained a population of cells that were enriched in DA neuron precursor cells and achieved higher differentiation efficiencies than those obtained through the same protocol without sorting. This high efficiency method of DA neuronal induction enabled reliable detection of reactive oxygen species (ROS) accumulation and vulnerable phenotypes in PARK2 iPSCs-derived DA neurons. We additionally established a quantitative system using the mt-mKeima reporter system to monitor mitophagy in which mitochondria fuse with lysosomes and, by combining this system with the method of DA neuronal induction described above, determined that mitophagy is impaired in PARK2 neurons. These findings suggest that the efficiency of DA neuron induction is important for the precise detection of cellular phenotypes in modeling Parkinson's disease.
Assuntos
Neurônios Dopaminérgicos/citologia , Neurônios Dopaminérgicos/metabolismo , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Apoptose , Diferenciação Celular/fisiologia , Linhagem Celular , Humanos , Receptores de Hialuronatos/metabolismo , Mitofagia/fisiologia , Modelos Neurológicos , Células-Tronco Neurais/citologia , Células-Tronco Neurais/metabolismo , Doença de Parkinson/etiologia , Doença de Parkinson/metabolismo , Doença de Parkinson/patologia , Espécies Reativas de Oxigênio/metabolismoRESUMO
Hearing impairments are the most common symptom of congenital defects, and they generally remain intractable to treatment. Pendred syndrome, the most frequent syndromic form of hereditary hearing loss, is associated with mutations in the anion exchanger pendrin. Loss of pendrin function as an anion exchanger is thought to be causative, but rodent models do not exhibit progressive deafness. Here, we report a degenerative phenotype exhibiting mutant pendrin aggregates and increased susceptibility to cellular stresses in cochlear epithelial cells induced from patient-derived induced pluripotent stem cells (iPSCs). These degenerative phenotypes were rescued by site-specific gene corrections. Moreover, low-dose rapamycin and metformin reduced aggregation and cell death. Our results provide an unexpected, comprehensive understanding of deafness due to "degenerative cochlear disease" and may contribute to rational therapeutic development. This iPSC-based disease model provides an approach to the study of pathogenesis and therapeutic development for hereditary hearing loss.
Assuntos
Cóclea/patologia , Perda Auditiva/congênito , Perda Auditiva/terapia , Células-Tronco Pluripotentes Induzidas/metabolismo , Adolescente , Adulto , Linhagem Celular , Criança , Pré-Escolar , Feminino , Bócio Nodular/genética , Bócio Nodular/patologia , Bócio Nodular/terapia , Perda Auditiva/patologia , Perda Auditiva Neurossensorial/genética , Perda Auditiva Neurossensorial/patologia , Perda Auditiva Neurossensorial/terapia , Células-Tronco Embrionárias Humanas/efeitos dos fármacos , Células-Tronco Embrionárias Humanas/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Recém-Nascido , Troca Iônica , Proteínas de Membrana Transportadoras/química , Proteínas de Membrana Transportadoras/metabolismo , Metformina/farmacologia , Fenótipo , Agregados Proteicos/efeitos dos fármacos , Sirolimo/farmacologia , Transportadores de Sulfato , Aqueduto Vestibular/anormalidades , Aqueduto Vestibular/patologiaRESUMO
Amyotrophic lateral sclerosis (ALS) is a late-onset motor neuron disorder. Although its neuropathology is well understood, the cellular and molecular mechanisms are yet to be elucidated due to limitations in the currently available human genetic data. In this study, we generated induced pluripotent stem cells (iPSC) from two familial ALS (FALS) patients with a missense mutation in the fused-in sarcoma (FUS) gene carrying the heterozygous FUS H517D mutation, and isogenic iPSCs with the homozygous FUS H517D mutation by genome editing technology. These cell-derived motor neurons mimicked several neurodegenerative phenotypes including mis-localization of FUS into cytosolic and stress granules under stress conditions, and cellular vulnerability. Moreover, exon array analysis using motor neuron precursor cells (MPCs) combined with CLIP-seq datasets revealed aberrant gene expression and/or splicing pattern in FALS MPCs. These results suggest that iPSC-derived motor neurons are a useful tool for analyzing the pathogenesis of human motor neuron disorders.
Assuntos
Diferenciação Celular/genética , Células-Tronco Pluripotentes Induzidas/metabolismo , Neurônios Motores/metabolismo , Mutação de Sentido Incorreto , Proteína FUS de Ligação a RNA/genética , Adulto , Esclerose Lateral Amiotrófica/genética , Esclerose Lateral Amiotrófica/metabolismo , Esclerose Lateral Amiotrófica/patologia , Sequência de Bases , Células Cultivadas , Citosol/metabolismo , Saúde da Família , Feminino , Edição de Genes , Perfilação da Expressão Gênica/métodos , Heterozigoto , Homozigoto , Humanos , Células-Tronco Pluripotentes Induzidas/patologia , Masculino , Microscopia de Fluorescência , Modelos Genéticos , Neurônios Motores/patologia , Linhagem , Proteína FUS de Ligação a RNA/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Adulto JovemRESUMO
The CNS contains many diverse neuronal subtypes, and most neurological diseases target specific subtypes. However, the mechanism of neuronal subtype specificity of disease phenotypes remains elusive. Although in vitro disease models employing human pluripotent stem cells (PSCs) have great potential to clarify the association of neuronal subtypes with disease, it is currently difficult to compare various PSC-derived subtypes. This is due to the limited number of subtypes whose induction is established, and different cultivation protocols for each subtype. Here, we report a culture system to control the regional identity of PSC-derived neurons along the anteroposterior (A-P) and dorsoventral (D-V) axes. This system was successfully used to obtain various neuronal subtypes based on the same protocol. Furthermore, we reproduced subtype-specific phenotypes of amyotrophic lateral sclerosis (ALS) and Alzheimer's disease (AD) by comparing the obtained subtypes. Therefore, our culture system provides new opportunities for modeling neurological diseases with PSCs.
Assuntos
Doença de Alzheimer/patologia , Esclerose Lateral Amiotrófica/patologia , Células-Tronco Neurais/patologia , Neurônios/patologia , Células-Tronco Pluripotentes/patologia , Doença de Alzheimer/metabolismo , Esclerose Lateral Amiotrófica/metabolismo , Técnicas de Cultura de Células , Linhagem Celular , Proteínas Hedgehog/metabolismo , Humanos , Células-Tronco Neurais/citologia , Células-Tronco Neurais/metabolismo , Neurogênese , Neurônios/citologia , Neurônios/metabolismo , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/metabolismo , Transdução de Sinais , Via de Sinalização WntRESUMO
Multisite Gateway technology is a DNA cloning method based on in vitro site-specific recombination that is becoming increasingly popular because it allows quick and highly efficient assembly of multiple DNA fragments into a vector backbone. In the conventional Gateway Multisite strategy, cloning of multiple DNA fragments requires recombination of multiple entry clones with a single destination vector. The -limitation of this approach is that as the number of entry clones increases, the efficiency of the assembly reactions decreases due to difficulty in successfully recognizing multiple pairs of matched att signals simultaneously. To address this problem, we have devised methods to generate modular expression clones, modular entry clones, and modular destination vectors. These allow many DNA fragments to be -assembled stepwise into complex expression clones. We describe here how to construct these intermediate clones and vectors, and how to use these modules to construct expression clones comprising ten or more DNA -segments. These principles can be applied to make multicomponent DNAs for many applications.