Luxembourg Centre for Systems Biomedicine (LCSB), University of Luxembourg, Esch-sur-Alzette, Luxembourg.
Department of Pathology and Cell Biology, Columbia University Medical Center, New York, New York, USA.
Mov Disord. 2021 Mar;36(3):704-715. doi: 10.1002/mds.28365. Epub 2020 Nov 3.
VPS35 is part of the retromer complex and is responsible for the trafficking and recycling of proteins implicated in autophagy and lysosomal degradation, but also takes part in the degradation of mitochondrial proteins via mitochondria-derived vesicles. The p.D620N mutation of VPS35 causes an autosomal-dominant form of Parkinson's disease (PD), clinically representing typical PD.
Most of the studies on p.D620N VPS35 were performed on human tumor cell lines, rodent models overexpressing mutant VPS35, or in patient-derived fibroblasts. Here, based on identified target proteins, we investigated the implication of mutant VPS35 in autophagy, lysosomal degradation, and mitochondrial function in induced pluripotent stem cell-derived neurons from a patient harboring the p.D620N mutation.
We reprogrammed fibroblasts from a PD patient carrying the p.D620N mutation in the VPS35 gene and from two healthy donors in induced pluripotent stem cells. These were subsequently differentiated into neuronal precursor cells to finally generate midbrain dopaminergic neurons.
We observed a decreased autophagic flux and lysosomal mass associated with an accumulation of α-synuclein in patient-derived neurons compared to controls. Moreover, patient-derived neurons presented a mitochondrial dysfunction with decreased membrane potential, impaired mitochondrial respiration, and increased production of reactive oxygen species associated with a defect in mitochondrial quality control via mitophagy.
We describe for the first time the impact of the p.D620N VPS35 mutation on autophago-lysosome pathway and mitochondrial function in stem cell-derived neurons from an affected p.D620N carrier and define neuronal phenotypes for future pharmacological interventions. © 2020 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.
VPS35 是 retromer 复合物的一部分，负责与自噬和溶酶体降解相关的蛋白质的运输和回收，但也参与通过线粒体衍生小泡降解线粒体蛋白质。VPS35 的 p.D620N 突变导致常染色体显性形式的帕金森病（PD），临床上代表典型的 PD。
大多数关于 p.D620N VPS35 的研究都是在人肿瘤细胞系、过表达突变 VPS35 的啮齿动物模型或在患者来源的成纤维细胞中进行的。在这里，我们基于鉴定的靶蛋白，研究了携带 p.D620N 突变的患者来源的诱导多能干细胞衍生神经元中的突变 VPS35 在自噬、溶酶体降解和线粒体功能中的作用。
我们将来自携带 VPS35 基因 p.D620N 突变的 PD 患者和两名健康供体的成纤维细胞重编程为诱导多能干细胞，然后将其分化为神经前体细胞，最终生成中脑多巴胺能神经元。
与对照组相比，我们观察到患者来源的神经元中自噬通量和溶酶体质量降低，α-突触核蛋白积累。此外，与线粒体质量控制缺陷相关的自噬缺陷，患者来源的神经元表现出线粒体功能障碍，表现为膜电位降低、线粒体呼吸受损、活性氧产生增加。
我们首次描述了 p.D620N VPS35 突变对受影响的 p.D620N 携带者来源的干细胞衍生神经元中自噬溶酶体途径和线粒体功能的影响，并定义了未来药物干预的神经元表型。
