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

这项建议试图调查基本的分子缺陷，以解释为什么逆转录复合体亚单位的突变会导致遗传性帕金森氏症。逆转聚体是一种进化保守的内体多蛋白复合体，它协调内化的完整膜蛋白在细胞内的循环。最近发现，逆转录子亚基内的氨基酸交换突变可导致常染色体显性遗传的晚发遗传性帕金森氏病。了解这些突变的致病机制的大部分努力都集中在逆转录分子在完整膜蛋白循环中的长期确立和规范作用上。最近，我们发现了逆转聚体的一个重要新功能，它是晚期内吞小GTP酶RAB7活性和定位的主要调节因子。我们的初步数据表明，最常见的帕金森病相关突变VPS35(VPS35-D620N)改变了逆转录分子介导的RAB7活性控制，因为它导致RAB7过度失活，并伴随着溶酶体中这个小GTP酶的丢失。因此，VPS35-D620N突变在RAB7活性的控制中起着“功能获得”突变的作用，这可能解释了这种遗传异常的常染色体显性本质。此外，我们还利用定量蛋白质组学方法鉴定了一系列受逆转录和逆转录相关蛋白调控的小分子GTP酶。这些发现有可能为逆转聚体在帕金森病中的作用建立一个新的范式，这是值得深入研究的。通过基因编辑、定量蛋白质组学和先进的成像技术的创新组合，我们试图研究逆转聚体在RAB GTP酶控制中的生理作用，以及致病突变对RAB7和其他几种非调控的RAB GTP酶调控的各种细胞通路的影响。这项研究将阐明逆转录病毒介导的RAB GTPase活性控制如何维持晚期的内吞功能、自噬、mTOR信号和线粒体动力学，所有这些都可能在逆转录病毒突变时被解除调控。我们相信，我们提议的工作将改变我们对逆转录病毒的看法，从而为研究其在生理学和疾病中的作用开辟新的途径。

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.