RESUMO
Gamma-aminobutyric acid (GABA)-releasing interneurons play an important modulatory role in the cortex and have been implicated in multiple neurological disorders. Patient-derived interneurons could provide a foundation for studying the pathogenesis of these diseases as well as for identifying potential therapeutic targets. Here, we identified a set of genetic factors that could robustly induce human pluripotent stem cells (hPSCs) into GABAergic neurons (iGNs) with high efficiency. We demonstrated that the human iGNs express neurochemical markers and exhibit mature electrophysiological properties within 6-8 weeks. Furthermore, in vitro, iGNs could form functional synapses with other iGNs or with human-induced glutamatergic neurons (iENs). Upon transplantation into immunodeficient mice, human iGNs underwent synaptic maturation and integration into host neural circuits. Taken together, our rapid and highly efficient single-step protocol to generate iGNs may be useful to both mechanistic and translational studies of human interneurons.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Córtex Cerebral/metabolismo , Neurônios GABAérgicos/metabolismo , Células-Tronco Pluripotentes/metabolismo , Prosencéfalo/metabolismo , Ácido gama-Aminobutírico/metabolismo , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Biomarcadores/metabolismo , Diferenciação Celular , Linhagem Celular , Córtex Cerebral/citologia , Técnicas de Cocultura , Neurônios GABAérgicos/citologia , Expressão Gênica , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Humanos , Interneurônios/citologia , Interneurônios/metabolismo , Proteínas com Homeodomínio LIM/genética , Proteínas com Homeodomínio LIM/metabolismo , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Células-Tronco Neurais/citologia , Células-Tronco Neurais/metabolismo , Neuroglia/citologia , Neuroglia/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Técnicas de Patch-Clamp , Células-Tronco Pluripotentes/citologia , Cultura Primária de Células , Prosencéfalo/citologia , Sinapses/fisiologia , Transmissão Sináptica/fisiologia , Fator Nuclear 1 de Tireoide , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Spinocerebellar ataxia type 3 is caused by a polyglutamine expansion in the ataxin-3 protein, resulting in gain of toxic function of the mutant protein. The expanded glutamine stretch in the protein is the result of a CAG triplet repeat expansion in the penultimate exon of the ATXN3 gene. Several gene silencing approaches to reduce mutant ataxin-3 toxicity in this disease aim to lower ataxin-3 protein levels, but since this protein is involved in deubiquitination and proteasomal protein degradation, its long-term silencing might not be desirable. Here, we propose a novel protein modification approach to reduce mutant ataxin-3 toxicity by removing the toxic polyglutamine repeat from the ataxin-3 protein through antisense oligonucleotide-mediated exon skipping while maintaining important wild type functions of the protein. In vitro studies showed that exon skipping did not negatively impact the ubiquitin binding capacity of ataxin-3. Our in vivo studies showed no toxic properties of the novel truncated ataxin-3 protein. These results suggest that exon skipping may be a novel therapeutic approach to reduce polyglutamine-induced toxicity in spinocerebellar ataxia type 3.