VPS35 D620N inhibits autophagy through disrupted hyaluronic acid-CD44 signaling

项目摘要--莫里森 帕金森氏病(PD)是美国最常见的运动疾病。主要临床运动症状 帕金森病的原因是黑质中多巴胺(DA)能神经元的丢失，自噬功能障碍 与这种疾病密切相关。自噬是细胞器降解的细胞过程， 大分子和蛋白质聚集体。在帕金森病患者中，特征毒性蛋白聚集体主要是α- 突触核蛋白被认为是自噬去除和自噬增强清除的底物 临床前模型结果。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，初步的 其他研究小组对致病机制的研究仅限于典型的VPS35蛋白相互作用 宫颈癌细胞系。为了克服这些限制，我们使用RNA进行了无偏筛选 测序(RNA序列)以确定在广泛使用的帕金森病细胞模型中受影响的关键通路。我们有 表明细胞外基质(ECM)-受体相互作用受扰的已识别变化以及异常 AKT信号，一条下游通路，调节自噬的诱导。透明质酸(HA)是 大脑ECM的主要成分和通过CD44的信号，CD44是一种ECM受体，被我们的RNA识别为最重要的受体 SEQ筛选，调节AKT-mTOR途径的自噬，使这个轴成为首选候选 调解VPS35 D620N自噬封锁。此外，VPS35的S在回溯中的作用已经确立 复合体，一种指导质膜受体运输的蛋白质复合体，表明改变的运输 VPS35突变体对CD44的激活可能与观察到的AKT途径激活和 随后对自噬的抑制。这一提议的中心假设是VPS35 D620N阻塞 通过调节失调的透明质酸-CD44信号通过改变CD44的运输实现自噬。我们建议 通过检测VPS35突变表达细胞中HA-CD44-AKT通路的激活来验证我们的假设； 通过对突变表型的遗传和药理学挽救来验证这一途径的重要性；以及 评估CD44异常激活是否导致神经元丢失增加。是否扰乱了CD44 VPS35 D620N的贩运是改变信号的基础，将被确定。

Project Summary - Morrison 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 a cervical cancer cell line. To overcome these limitations, we performed an unbiased screen using RNA sequencing (RNA seq) to identify key pathways affected in a widely used cellular model of PD. We have identified alterations indicative of perturbed extracellular matrix (ECM)-receptor interaction as well as aberrant AKT signaling, a downstream pathway known to regulate the induction of autophagy. Hyaluronic acid (HA) is the major component of brain ECM and signals via CD44, an ECM receptor identified as a top hit by our RNA Seq screen, to the autophagy regulating AKT-mTOR pathway, making this axis a prime candidate for mediating the VPS35 D620N autophagy blockade. Furthermore, VPS35’s well-established role in the retromer complex, a protein complex that directs plasma membrane receptor trafficking, suggests that altered trafficking of CD44 by the VPS35 mutant may be responsible for the observed alteration of AKT pathway activation and the subsequent repression of autophagy. The central hypothesis of this proposal is that VPS35 D620N blocks autophagy through dysregulated hyaluronic acid-CD44 signaling by altered trafficking of CD44. We propose testing our hypothesis by examining HA-CD44-AKT pathway activation in VPS35 mutant expressing cells; validating the importance of this pathway by genetic and pharmacological rescue of the mutant phenotype; and assessing whether aberrant CD44 activation leads to increased neuronal loss. Whether perturbed CD44 trafficking by VPS35 D620N underlies altered signaling will be determined.