RESUMEN
Expansion of the glutamine tract (poly-Q) in the protein huntingtin (HTT) causes the neurodegenerative disorder Huntington's disease (HD). Emerging evidence suggests that mutant HTT (mHTT) disrupts brain development. To gain mechanistic insights into the neurodevelopmental impact of human mHTT, we engineered male induced pluripotent stem cells to introduce a biallelic or monoallelic mutant 70Q expansion or to remove the poly-Q tract of HTT. The introduction of a 70Q mutation caused aberrant development of cerebral organoids with loss of neural progenitor organization. The early neurodevelopmental signature of mHTT highlighted the dysregulation of the protein coiled-coil-helix-coiled-coil-helix domain containing 2 (CHCHD2), a transcription factor involved in mitochondrial integrated stress response. CHCHD2 repression was associated with abnormal mitochondrial morpho-dynamics that was reverted upon overexpression of CHCHD2. Removing the poly-Q tract from HTT normalized CHCHD2 levels and corrected key mitochondrial defects. Hence, mHTT-mediated disruption of human neurodevelopment is paralleled by aberrant neurometabolic programming mediated by dysregulation of CHCHD2, which could then serve as an early interventional target for HD.
Asunto(s)
Encéfalo , Proteínas de Unión al ADN , Proteína Huntingtina , Enfermedad de Huntington , Células Madre Pluripotentes Inducidas , Mitocondrias , Proteínas Mitocondriales , Organoides , Factores de Transcripción , Humanos , Factores de Transcripción/metabolismo , Factores de Transcripción/genética , Proteínas de Unión al ADN/metabolismo , Proteínas de Unión al ADN/genética , Proteína Huntingtina/genética , Proteína Huntingtina/metabolismo , Organoides/metabolismo , Proteínas Mitocondriales/metabolismo , Proteínas Mitocondriales/genética , Encéfalo/metabolismo , Encéfalo/patología , Enfermedad de Huntington/metabolismo , Enfermedad de Huntington/genética , Enfermedad de Huntington/patología , Células Madre Pluripotentes Inducidas/metabolismo , Masculino , Mitocondrias/metabolismo , Mutación , Dinámicas Mitocondriales/genéticaRESUMEN
Huntington's disease (HD) is a neurodegenerative disorder caused by abnormal glutamine (Q) expansion in the huntingtin protein due to elongated CAG repeats in the gene HTT. We used non-integrative episomal plasmids to generate induced pluripotent stem cells (iPSCs) from three individuals affected by HD: CH1 (58Q), and two twin brothers CH3 (44Q) and CH4 (44Q). The iPSC lines exhibited one healthy HTT allele and one with elongated CAG repeats, as confirmed by PCR and sequencing. All iPSC lines expressed pluripotency markers, exhibited a normal karyotype, and generated cells of the three germ layers in vitro.
Asunto(s)
Proteína Huntingtina , Enfermedad de Huntington , Células Madre Pluripotentes Inducidas , Humanos , Enfermedad de Huntington/genética , Enfermedad de Huntington/patología , Células Madre Pluripotentes Inducidas/patología , Hermanos , Línea Celular , Proteína Huntingtina/genética , Alelos , MasculinoRESUMEN
We present a high-content analysis (HCA) protocol for monitoring the outgrowth capacity of human neurons derived from induced pluripotent stem cells (iPSCs). We describe steps to perform HCA imaging, followed by quantifying the morphology of dendrites and axons within a high-throughput system to evaluate neurons obtained through various differentiation approaches. This protocol can be used to screen for modulators of neuronal morphogenesis or neurotoxicity. The approach can be applied to patient-derived iPSCs to identify patient-specific defects and possible therapeutic strategies. For complete details on the use and execution of this protocol, please refer to Zink et al. (2020) and Inak et al. (2021). The protocol can be used in combination with Zink et al. (2022).
Asunto(s)
Células Madre Pluripotentes Inducidas , Síndromes de Neurotoxicidad , Diferenciación Celular/fisiología , Humanos , NeuronasRESUMEN
Despite the widespread use of human pluripotent stem cells (hPSCs), there is no general consensus with respect to monitoring the integrity of nuclear and mitochondrial genome of hPSCs. We argue that clear guidelines should be in place, and we have identified frequently asked questions that can be used to develop a practical guide for hPSCs users.