Mitochondrial Rho GTPase 1 (Miro1) protein is a well-known adaptor for mitochondrial transport and also regulates mitochondrial quality control and function. Furthermore, Miro1 was associated with mitochondrial-endoplasmic reticulum (ER) contact sites (MERCs), which are key regulators of cellular calcium homeostasis and the initiation of autophagy. Impairments of these mechanisms were linked to neurodegeneration in Parkinson’s disease (PD). We recently revealed that PD fibroblasts harboring Miro1 mutations displayed dysregulations in MERC organization and abundance, affecting mitochondrial homeostasis and clearance. We hypothesize that mutant Miro1 impairs the function of MERCs and mitochondrial dynamics, altering neuronal homeostasis and integrity in PD. PD skin fibroblasts harboring the Miro1-R272Q mutation were differentiated into patient-derived neurons. Live-cell imaging and immunocytochemistry were used to study mitophagy and the organization and function of MERCs. Markers of autophagy or mitochondrial function were assessed by western blotting. Quantification of organelle juxtapositions revealed an increased number of MERCs in patient-derived neurons. Live-cell imaging results showed alterations of mitochondrial dynamics and increased sensitivity to calcium stress, as well as reduced mitochondrial clearance. Finally, western blot analysis indicated a blockage of the autophagy flux in Miro1-mutant neurons. Miro1-mutant neurons display altered ER-mitochondrial tethering compared with control neurons. This alteration likely interferes with proper MERC function, contributing to a defective autophagic flux and cytosolic calcium handling capacity. Moreover, mutant Miro1 affects mitochondrial dynamics in neurons, which may result in disrupted mitochondrial turnover and altered mitochondrial movement.

中文翻译：

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帕金森病 Miro1 突变神经元中线粒体 - 内质网相互作用和线粒体自噬受损。

线粒体 Rho GTPase 1 (Miro1) 蛋白是众所周知的线粒体转运接头，还调节线粒体质量控制和功能。此外，Miro1 与线粒体-内质网 (ER) 接触位点 (MERC) 相关，后者是细胞钙稳态和自噬启动的关键调节剂。这些机制的损害与帕金森病 (PD) 的神经变性有关。我们最近发现携带 Miro1 突变的 PD 成纤维细胞显示出 MERC 组织和丰度的失调，影响线粒体稳态和清除。我们假设突变体 Miro1 会损害 MERC 和线粒体动力学的功能，改变 PD 中的神经元稳态和完整性。携带 Miro1-R272Q 突变的 PD 皮肤成纤维细胞分化为患者来源的神经元。活细胞成像和免疫细胞化学用于研究线粒体自噬以及 MERC 的组织和功能。通过蛋白质印迹评估自噬或线粒体功能的标志物。细胞器并置的量化显示患者源性神经元中的 MERC 数量增加。活细胞成像结果显示线粒体动力学的改变和对钙应激的敏感性增加，以及线粒体清除率降低。最后，蛋白质印迹分析表明 Miro1 突变神经元中的自噬通量被阻断。与对照神经元相比，Miro1 突变神经元显示出改变的 ER 线粒体束缚。这种改变可能会干扰正常的 MERC 功能，导致自噬通量和细胞溶质钙处理能力缺陷。此外，突变体 Miro1 影响神经元中的线粒体动力学，这可能导致线粒体更新中断和线粒体运动改变。