Autophagy dysfunction in Parkinson's Disease by VPS35 D620N

 描述(申请人提供)：帕金森氏病(PD)是美国最常见的运动疾病。帕金森病的主要临床运动症状是黑质多巴胺(DA)能神经元的丢失，自噬功能障碍与本病密切相关。自噬是负责细胞器、大分子和蛋白质聚集体降解的细胞过程。在帕金森病中，主要是α-突触核蛋白的特征毒性蛋白聚集体被认为是自噬去除和自噬清除的底物，改善了临床前模型的结果。因此，调控自噬可能是对抗帕金森病的有效策略。最近，VPS35(D620N)的一个导致帕金森病的突变被报道可以阻断自噬。然而，其他研究小组对病因机制的初步调查仅限于HeLa细胞中典型的VPS35蛋白相互作用。为了克服这些限制，我们使用质谱仪和RNA测序(RNA SEQ)进行了无偏筛选，以确定广泛使用的帕金森病细胞模型中的关键蛋白质相互作用因子和途径。我们已经发现，VPS35蛋白相互作用蛋白显示出对RNA结合蛋白的高度浓缩，包括几个已知或怀疑是肌萎缩侧索硬化症病因的蛋白。此外，D620N突变导致RNA结合蛋白相互作用显著减少。从我们的屏幕上看，融合肉瘤(FUS)和尤文肉瘤断点区1(EWSR1)已经成为介导VPS35 D620N自噬功能障碍的主要候选基因。基于RNA结合蛋白的相互作用，我们检测了VPS35 WT和D620N细胞的转录组，发现了RNA代谢和自噬的变化。我们假设VPS35 D620N通过RNA结合蛋白活性调节RNA代谢来抑制自噬并导致细胞死亡。我们建议通过确定VPS35 D620N的自噬功能障碍和神经退化是否是由RNA结合蛋白活性改变引起的来验证我们的假设，并建立VPS35 D620N引起的转录组变化，从而促进自噬功能障碍。

 DESCRIPTION (provided by applicant): Parkinson's disease (PD) is the most common motor disease in the USA. The primary clinical motor symptoms of PD result from loss of dopaminergic (DA) neurons in the substantia nigra with autophagy dysfunction being closely linked to this disease. Autophagy is a cellular process responsible for degradation of organelles, macromolecules, and protein aggregates. In PD, characteristic toxic protein aggregates of primarily alpha-synuclein are believed to be substrates for autophagic removal and clearance by autophagy improves preclinical model outcomes. Therefore, modulation of autophagy may be an effective strategy to combat PD. Recently, a PD-causing mutation in VPS35 (D620N) was reported to block autophagy. However, preliminary investigation by other groups into a causal mechanism was limited to canonical VPS35 protein interactors in HeLa cells. To overcome these limitations we have performed an unbiased screen using mass spectrometry and RNA sequencing (RNA seq) to identify key protein interactors and pathways in a widely-used cellular model of PD. We have discovered that VPS35 protein interactors show a high enrichment for RNA- binding proteins including several known or suspected to be causal for amyotrophic lateral sclerosis. Additionally, the D620N mutation resulted in a dramatic decrease in RNA-binding protein interaction. From our screen, Fused in Sarcoma (FUS) and Ewing sarcoma breakpoint region 1 (EWSR1) have emerged as lead candidates for mediating VPS35 D620N autophagy dysfunction. Based upon RNA-binding protein interaction, we examined the transcriptome of VPS35 WT and D620N cells and found changes indicative of alterations in RNA metabolism and autophagy. We hypothesize that VPS35 D620N inhibits autophagy and causes cell death by regulating RNA metabolism through RNA-binding protein activity. We propose testing our hypothesis by determining if autophagy dysfunction and neurodegeneration by VPS35 D620N is caused by altered RNA-binding protein activity and establish that VPS35 D620N causes transcriptome changes that facilitate autophagy dysfunction.