VPS35 D620N inhibits autophagy through disrupted hyaluronic acid-CD44 signaling

项目摘要-莫里森 帕金森病（PD）是美国最常见的运动疾病。主要临床运动症状 的PD是由于黑质多巴胺能（DA）神经元的丢失和自噬功能障碍引起的。 与这种疾病密切相关。自噬是一种负责细胞器降解的细胞过程， 大分子和蛋白质聚集体。在PD中，主要是α- 突触核蛋白被认为是自噬去除的底物， 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，初步 其他小组对因果机制的研究仅限于典型的VPS 35蛋白相互作用物， 宫颈癌细胞株为了克服这些限制，我们使用RNA进行了无偏筛选， 通过RNA测序（RNA seq）来鉴定在广泛使用的PD细胞模型中受影响的关键途径。我们有 鉴定的改变指示干扰的细胞外基质（ECM）-受体相互作用以及异常的细胞外基质（ECM）-受体相互作用。 AKT信号传导，已知调节自噬诱导的下游途径。透明质酸（HA）是 脑ECM的主要成分和通过CD 44的信号，CD 44是一种ECM受体，被我们的RNA识别为最高命中 Seq筛选，自噬调节AKT-mTOR通路，使该轴成为细胞凋亡的主要候选者。 介导VPS 35 D 620 N自噬阻断。此外，VPS 35在retromer中的良好作用 复合物，一种蛋白质复合物，指导质膜受体运输，表明改变运输 VPS 35突变体对CD 44的抑制作用可能是观察到的AKT途径活化改变的原因， 随后对自噬的抑制。本提案的中心假设是VPS 35 D 620 N块 自噬通过改变的CD 44运输通过失调的透明质酸-CD 44信号传导。我们提出 通过检测VPS 35突变表达细胞中HA-CD 44-AKT通路的激活来检验我们的假设; 通过突变表型的遗传和药理学拯救来验证该途径的重要性;以及 评估异常的CD 44活化是否导致增加的神经元损失。是否干扰CD 44 将确定VPS 35 D 620 N的运输是改变的信号传导的基础。

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.