Autophagy dysfunction in Parkinson's Disease by VPS35 D620N

 描述（由申请人提供）：帕金森病（PD）是美国最常见的运动疾病。PD的主要临床运动症状是由于黑质中多巴胺能（DA）神经元的损失，自噬功能障碍与这种疾病密切相关。自噬是细胞内细胞器、大分子和蛋白质聚集体降解的过程。在PD中，主要是α-突触核蛋白的特征性毒性蛋白聚集体被认为是自噬去除的底物，并且通过自噬的清除改善了临床前模型结果。因此，调节自噬可能是对抗PD的有效策略。最近，据报道VPS 35中的一种导致PD的突变（D 620 N）可以阻止自噬。然而，其他小组对因果机制的初步研究仅限于HeLa细胞中的典型VPS 35蛋白相互作用物。为了克服这些限制，我们使用质谱和RNA测序（RNA seq）进行了无偏筛选，以确定广泛使用的PD细胞模型中的关键蛋白质相互作用物和途径。我们已经发现VPS 35蛋白相互作用物显示出RNA结合蛋白的高度富集，包括几种已知或怀疑是肌萎缩性侧索硬化症的病因的RNA结合蛋白。此外，D 620 N突变导致RNA结合蛋白相互作用的急剧减少。从我们的筛选中，融合肉瘤（FUS）和尤文肉瘤断点区1（EWSR 1）已成为介导VPS 35 D 620 N自噬功能障碍的主要候选者。基于RNA结合蛋白相互作用，我们检查了VPS 35 WT和D 620 N细胞的转录组，发现了指示RNA代谢和自噬改变的变化。我们假设VPS 35 D 620 N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VPS 35 D 620 N引起的自噬功能障碍和神经变性是否是由RNA结合蛋白活性改变引起的来测试我们的假设，并确定VPS 35 D 620 N引起促进自噬功能障碍的转录组变化。

 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.