Molecular basis and cellular effects of Parkinson’s disease causing mutations in the retromer complex.

这一建议旨在研究解释为什么反转录复合体亚基突变导致遗传性帕金森病的基本分子缺陷。Retromer是一种进化保守的内体多蛋白复合物，它协调内化整体膜蛋白的内吞循环。逆转录子亚基内的氨基酸交换突变最近被证明以常染色体显性方式引起晚发性遗传性帕金森病。了解这些突变的致病机制的大部分努力都集中在逆转录物在整体膜蛋白循环中的长期确立和规范作用上。我们最近发现了逆转录酶的一个主要新功能，即作为内吞小GTPase RAB7活性和定位的主要调节剂。我们的初步数据表明，反转录亚基VPS35 （VPS35- d620n）中最普遍的帕金森相关突变改变了反转录体介导的RAB7活性控制，因为它导致RAB7过度失活，并伴随溶酶体中这种小GTPase的丢失。因此，VPS35-D620N突变在控制RAB7活性方面起到了“功能获得”突变的作用，这可能解释了这种遗传畸变的常染色体显性性质。此外，我们还利用定量蛋白质组学鉴定了一系列由反转录物和反转录物相关蛋白调控的其他小gtpase。这些发现有可能为后转录酶在帕金森病中的作用建立一个新的范式，值得深入研究。通过基因编辑、定量蛋白质组学和先进成像技术的创新组合，我们试图研究逆转录酶在RAB gtpase控制中的生理作用，以及致病突变对RAB7和其他几种不受调控的RAB gtpase调节的各种细胞途径的影响。本研究将阐明逆转录酶介导的对RAB GTPase活性的控制如何维持晚期内吞功能、自噬、mTOR信号传导和线粒体动力学，这些都可能在逆转录酶突变时被解除调控。我们有信心，我们提出的工作将改变我们对逆转录酶的看法，从而为其在生理学和疾病中的作用开辟新的研究途径。

This proposal seeks to investigate the fundamental molecular defects that explain why mutations in subunits of the retromer complex cause hereditary Parkinson’s disease. Retromer is an evolutionary conserved, endosomal multi-protein complex that orchestrates the endocytic recycling of internalized integral membrane proteins. Amino acid exchanging mutations within retromer subunits have recently been demonstrated to cause late onset, hereditary Parkinson’s disease in an autosomal dominant manner. Most of the efforts to understand the pathogenic mechanism of these mutations have focused on retromer’s long established and canonical role in the recycling of integral membrane proteins. We have very recently identified a major new function for retromer as a master regulator of the activity and localization of the late endocytic small GTPase RAB7. Our preliminary data indicate that the most prevalent Parkinson’s associated mutation within the retromer subunit VPS35 (VPS35-D620N) alters retromer mediated control of RAB7 activity, as it leads to excessive deactivation of RAB7 and a concomitant loss of this small GTPase from lysosomes. Thus, the VPS35-D620N mutation acts a “gain of function” mutation in the control of RAB7 activity, which may explain the autosomal dominant nature of this genetic aberration. In addition, we have utilized quantitative proteomics to identify a whole host of other small GTPases that are regulated by retromer and retromer asociated proteins. These findings have the potential to establish a new paradigm for the role of retromer in Parkinsonism, which warrants in depth investigation. Through an innovative combination of gene editing, quantitative proteomics and advanced imaging techniques, we seek to investigate the physiological role of retromer in the control of RAB GTPases as well as the effects of the pathogenic mutation on various cellular pathways that are regulated by RAB7 and several other deregulated RAB GTPases. The proposed research will elucidate how retromer mediated control of RAB GTPase activity maintains late endocytic function, autophagy, mTOR signaling and mitochondrial dynamics, all of which are likely deregulated upon mutation of retromer. We are confident, that our proposed work will change the way we think abour retromer, thereby opening up new avenues of research in its role in physiology and in disease.