RESUMO
Mechanisms by which long corticospinal axons degenerate in hereditary spastic paraplegia (HSP) are largely unknown. Here, we have generated induced pluripotent stem cells (iPSCs) from patients with two autosomal recessive forms of HSP, SPG15 and SPG48, which are caused by mutations in the ZFYVE26 and AP5Z1 genes encoding proteins in the same complex, the spastizin and AP5Z1 proteins, respectively. In patient iPSC-derived telencephalic glutamatergic and midbrain dopaminergic neurons, neurite number, length and branching are significantly reduced, recapitulating disease-specific phenotypes. We analyzed mitochondrial morphology and noted a significant reduction in both mitochondrial length and their densities within axons of these HSP neurons. Mitochondrial membrane potential was also decreased, confirming functional mitochondrial defects. Notably, mdivi-1, an inhibitor of the mitochondrial fission GTPase DRP1, rescues mitochondrial morphology defects and suppresses the impairment in neurite outgrowth and late-onset apoptosis in HSP neurons. Furthermore, knockdown of these HSP genes causes similar axonal defects, also mitigated by treatment with mdivi-1. Finally, neurite outgrowth defects in SPG15 and SPG48 cortical neurons can be rescued by knocking down DRP1 directly. Thus, abnormal mitochondrial morphology caused by an imbalance of mitochondrial fission and fusion underlies specific axonal defects and serves as a potential therapeutic target for SPG15 and SPG48.
Assuntos
Proteínas Adaptadoras de Transporte Vesicular/genética , Proteínas de Transporte/genética , GTP Fosfo-Hidrolases/genética , Proteínas Associadas aos Microtúbulos/genética , Proteínas Mitocondriais/genética , Paraplegia Espástica Hereditária/genética , Axônios/efeitos dos fármacos , Axônios/patologia , Neurônios Dopaminérgicos/metabolismo , Neurônios Dopaminérgicos/patologia , Dinaminas , Humanos , Células-Tronco Pluripotentes Induzidas , Potencial da Membrana Mitocondrial/genética , Mesencéfalo/metabolismo , Mesencéfalo/patologia , Mitocôndrias/genética , Mitocôndrias/patologia , Dinâmica Mitocondrial/genética , Neurônios Motores/metabolismo , Neurônios Motores/patologia , Mutação , Crescimento Neuronal/efeitos dos fármacos , Crescimento Neuronal/genética , Quinazolinonas/farmacologia , Paraplegia Espástica Hereditária/tratamento farmacológico , Paraplegia Espástica Hereditária/fisiopatologiaRESUMO
Impaired axonal development and degeneration are implicated in many debilitating disorders, such as hereditary spastic paraplegia (HSP), amyotrophic lateral sclerosis (ALS), and periphery neuropathy. Human pluripotent stem cells (hPSCs) have provided researchers with an excellent resource for modeling human neuropathologic processes including axonal defects in vitro. There are a number of steps that are crucial when developing an hPSC-based model of a human disease, including generating induced pluripotent stem cells (iPSCs), differentiating those cells to affected cell types, and identifying disease-relevant phenotypes. Here, we describe these steps in detail, focusing on the neurodegenerative disorder HSP.
Assuntos
Axônios/ultraestrutura , Fibroblastos/citologia , Células-Tronco Pluripotentes Induzidas/citologia , Mitocôndrias/ultraestrutura , Neurônios/citologia , Paraplegia Espástica Hereditária/patologia , Animais , Axônios/metabolismo , Biomarcadores/metabolismo , Fator Neurotrófico Derivado do Encéfalo/farmacologia , Diferenciação Celular/efeitos dos fármacos , Reprogramação Celular/efeitos dos fármacos , Colágeno/química , Combinação de Medicamentos , Proteínas do Olho/genética , Proteínas do Olho/metabolismo , Células Alimentadoras/citologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Expressão Gênica , Fator Neurotrófico Derivado de Linhagem de Célula Glial/farmacologia , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Células-Tronco Pluripotentes Induzidas/metabolismo , Fator de Crescimento Insulin-Like I/farmacologia , Laminina/química , Camundongos , Mitocôndrias/metabolismo , Modelos Biológicos , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Fator de Transcrição PAX6 , Fatores de Transcrição Box Pareados/genética , Fatores de Transcrição Box Pareados/metabolismo , Fenótipo , Cultura Primária de Células , Proteoglicanas/química , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Paraplegia Espástica Hereditária/genética , Paraplegia Espástica Hereditária/metabolismoRESUMO
Spinal muscular atrophy (SMA), characterized by specific degeneration of spinal motor neurons, is caused by mutations in the survival of motor neuron 1, telomeric (SMN1) gene and subsequent decreased levels of functional SMN. How the deficiency of SMN, a ubiquitously expressed protein, leads to spinal motor neuron-specific degeneration in individuals affected by SMA remains unknown. In this study, we examined the role of SMN in mitochondrial axonal transport and morphology in human motor neurons by generating SMA type 1 patient-specific induced pluripotent stem cells (iPSCs) and differentiating these cells into spinal motor neurons. The initial specification of spinal motor neurons was not affected, but these SMA spinal motor neurons specifically degenerated following long-term culture. Moreover, at an early stage in SMA spinal motor neurons, but not in SMA forebrain neurons, the number of mitochondria, mitochondrial area and mitochondrial transport were significantly reduced in axons. Knocking down of SMN expression led to similar mitochondrial defects in spinal motor neurons derived from human embryonic stem cells, confirming that SMN deficiency results in impaired mitochondrial dynamics. Finally, the application of N-acetylcysteine (NAC) mitigated the impairment in mitochondrial transport and morphology and rescued motor neuron degeneration in SMA long-term cultures. Furthermore, NAC ameliorated the reduction in mitochondrial membrane potential in SMA spinal motor neurons, suggesting that NAC might rescue apoptosis and motor neuron degeneration by improving mitochondrial health. Overall, our data demonstrate that SMN deficiency results in abnormal mitochondrial transport and morphology and a subsequent reduction in mitochondrial health, which are implicated in the specific degeneration of spinal motor neurons in SMA.
Assuntos
Mitocôndrias/patologia , Neurônios Motores/patologia , Atrofia Muscular Espinal/patologia , Acetilcisteína/química , Animais , Transporte Biológico , Caspase 3/metabolismo , Caspase 7/metabolismo , Diferenciação Celular , Células Cultivadas , Modelos Animais de Doenças , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Cariotipagem , Potenciais da Membrana , Camundongos , Camundongos SCID , Reação em Cadeia da Polimerase , Prosencéfalo/fisiopatologiaRESUMO
Demyelination contributes to the functional impairment of irradiation injured spinal cord. One potential therapeutic strategy involves replacing the myelin-forming cells. Here, we asked whether transplantation of Olig2(+)-GFP(+)-oligodendrocyte precursor cells (OPCs), which are derived from Olig2-GFP-mouse embryonic stem cells (mESCs), could enhance remyelination and functional recovery after spinal cord irradiation injury. We differentiated Olig2-GFP-mESCs into purified Olig2(+)-GFP(+)-OPCs and transplanted them into the rats' cervical 4-5 dorsal spinal cord level at 4 months after irradiation injury. Eight weeks after transplantation, the Olig2(+)-GFP(+)-OPCs survived and integrated into the injured spinal cord. Immunofluorescence analysis showed that the grafted Olig2(+)-GFP(+)-OPCs primarily differentiated into adenomatous polyposis coli (APC(+)) oligodendrocytes (54.6±10.5%). The staining with luxol fast blue, hematoxylin & eosin (LFB/H&E) and electron microscopy demonstrated that the engrafted Olig2(+)-GFP(+)-OPCs attenuated the demyelination resulted from the irradiation. More importantly, the recovery of forelimb locomotor function was enhanced in animals receiving grafts of Olig2(+)-GFP(+)-OPCs. We concluded that OPC transplantation is a feasible therapy to repair the irradiated lesions in the central nervous system (CNS).