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

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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.