Sheffield Institute for Translational Neuroscience, University of Sheffield, Sheffield, UK.
School of Chemistry, University of Sydney, Sydney, Australia.
Sci Rep. 2020 Sep 23;10(1):15485. doi: 10.1038/s41598-020-72345-4.
Mutations in PRKN 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 PRKN 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 PRKN mutant patient fibroblasts with a high dopaminergic neuron yield. We reveal changing mitochondrial phenotypes as neurons undergo a metabolic switch during differentiation. Fibroblasts from 4 controls and 4 PRKN 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. Direct conversion of control and PRKN 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. 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.
PRKN 基因突变是早发性帕金森病的最常见原因。Parkin 是一种 E3 泛素连接酶，在细胞自噬中发挥作用。PRKN 突变模型存在线粒体异常。患者来源的神经元是研究致病机制和治疗靶点的有前途的模型。在这里，我们从 PRKN 突变患者成纤维细胞中生成具有高多巴胺能神经元产量的诱导神经元祖细胞。我们揭示了神经元在分化过程中经历代谢转换时的线粒体表型变化。将 4 个对照和 4 个 PRKN 突变患者的成纤维细胞转化为诱导神经元祖细胞，随后分化为多巴胺能神经元。使用活细胞荧光成像、细胞 ATP 和活性氧产生定量来评估线粒体形态、功能和自噬。直接转化对照和 PRKN 突变患者的成纤维细胞会导致诱导神经元祖细胞及其分化产生高比例的多巴胺能神经元。我们能够观察到神经元在分化过程中经历代谢转换时线粒体表型的变化。我们的结果表明，当前神经元在分化早期糖酵解时，PRKN 缺乏不会损害自噬。然而，当神经元变得依赖氧化磷酸化时，PRKN 突变患者神经元中的自噬会严重受损。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。诱导神经元祖细胞转化可以产生高产量的多巴胺能神经元。线粒体表型，包括自噬状态，高度依赖于细胞的代谢状态。只有当神经元依赖氧化磷酸化时，才会发现线粒体异常的程度。这些数据提供了对 PRKN 突变细胞特异性影响的深入了解，特别是与自噬依赖性疾病表型有关，并为替代治疗方法提供了途径。
