Background Mutations in parkin are the most common cause of early onset Parkinson's disease. Parkin is an E3 ubiquitin ligase, functioning in mitophagy. Mitochondrial abnormalities are present in parkin mutant models. Patient derived neurons are a promising model in which to study pathogenic mechanisms and therapeutic targets. Here we generate induced neuronal progenitor cells from parkin mutant patient fibroblasts with a high dopaminergic neuron yield. We reveal changing mitochondrial phenotypes as neurons undergo a metabolic switch during differentiation. Methods Fibroblasts from 4 controls and 4 parkin mutant patients were transformed into induced neuronal progenitor cells and subsequently differentiated into dopaminergic neurons. Mitochondrial morphology, function and mitophagy were evaluated using live cell fluorescent imaging, cellular ATP and reactive oxygen species production quantification. Results Direct conversion of control and parkin mutant patient fibroblasts results in induced neuronal progenitor and their differentiation yields high percentage of dopaminergic neurons. We were able to observe changing mitochondrial phenotypes as neurons undergo a metabolic switch during differentiation. Our results show that when pre-neurons are glycolytic early in differentiation mitophagy is unimpaired by PRKN deficiency. However as neurons become oxidative phosphorylation dependent, mitophagy is severely impaired in the PRKN mutant patient neurons. These changes correlate with changes in mitochondrial function and morphology; resulting in lower neuron yield and altered neuronal morphology. Conclusions Induced neuronal progenitor cell conversion can produce a high yield of dopaminergic neurons. The mitochondrial phenotype, including mitophagy status, is highly dependent on the metabolic status of the cell. Only when neurons are oxidative phosphorylation reliant the extent of mitochondrial abnormalities are identified. These data provide insight into cell specific effects of PRKN mutations, in particular in relation to mitophagy dependent disease phenotypes and provide avenues for alternative therapeutic approaches.

中文翻译：

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氧化开关驱动多巴胺能帕金突变患者神经元的线粒体缺陷。

背景帕金森突变是帕金森氏病早期发作的最常见原因。Parkin是一种E3泛素连接酶，在线粒体中起作用。帕金突变体模型中存在线粒体异常。患者来源的神经元是一种有前途的模型，可用于研究其致病机制和治疗靶标。在这里，我们从具有高多巴胺能神经元产量的帕金突变患者成纤维细胞中生成诱导的神经元祖细胞。我们揭示了变化的线粒体表型，因为神经元在分化过程中经历了代谢转换。方法将4例对照和4例parkin突变患者的成纤维细胞转化为诱导的神经元祖细胞，然后分化为多巴胺能神经元。使用活细胞荧光成像评估线粒体的形态，功能和线粒体，细胞ATP和活性氧的产生定量。结果对照和帕金突变型患者成纤维细胞的直接转化产生了诱导的神经元祖细胞，它们的分化产生了高百分比的多巴胺能神经元。当神经元在分化过程中经历代谢转换时，我们能够观察到线粒体表型的变化。我们的结果表明，当前神经元在分化早期被糖酵解时，噬菌体不会因PRKN缺乏而受损。但是，由于神经元变得依赖于氧化磷酸化，因此PRKN突变患者神经元的线粒体功能受到严重损害。这些变化与线粒体功能和形态的变化相关。导致神经元产量降低和神经元形态改变。结论诱导的神经元祖细胞转化可以产生高产的多巴胺能神经元。线粒体表型，包括线粒体状态，高度依赖于细胞的代谢状态。只有在神经元的氧化磷酸化依赖线粒体异常的程度时，才能确定。这些数据提供了对PRKN突变的细胞特异性作用的洞察力，特别是与线粒体依赖性疾病表型有关的洞察力，并为替代治疗方法提供了途径。