The role of retromer in Parkinson's disease

Abstract

Parkinson’s disease (PD) is an irreversible neurological disorder characterized by protein aggregate accumulation, endo-lysosomal dysfunction and neuronal cell death. Recently, several novel point mutations linked to late onset PD were identified in the retromer subunit; Vps35. Retromer is a highly conserved protein sorting complex that governs the endosome-to-Trans Golgi Network (TGN) trafficking pathway (hereafter referred to as retrograde sorting). While a great deal of evidence currently exists detailing the mechanisms of retromer in the retrograde sorting process, very little information conclusively highlights how sorting is misguided by retromer in PD. Firstly, this thesis investigated the importance of retromer in regulating overall lysosomal function and clearance of α-synuclein. This study demonstrated the consistent accumulation of aggregated α-synuclein in Rab7A positive compartments following stable depletion of the retromer complex. Upon use of exogenous stimuli, a large percentage of retromer depleted cells displayed aggregates positive for α-synuclein, when compared to non-silenced cells. Further, this accumulation in the late endocytic network negatively regulated the ability for material derived from the TGN to be appropriately processed in the late endosomal network, further perturbing the ability for aggregated α-synuclein protein to be cleared and likely contributing to the death of the cell. Secondly, I identified several novel shortfalls of retromer in the various point mutations that are linked to PD and additionally, expanded on how retromer plays a fundamental role in preservation of the endo-lysosomal network. This thesis demonstrates that Vps35 D620N, Vps35 P316S or Vps35 R524W do not disrupt the overall formation of the retromer complex, but functionally demonstrates incorrect sorting of specific cargo to the TGN. The failed sorting observed by Vps35 D620N containing retromer results in vacuolization of the late endosome, and large disruptions to the overall localization of endocytic compartments. As a result, the lysosomal hydrolase, Cathepsin D, an enzyme needed for the degradation of α-synuclein, is not transported to the late endocytic network and can be detected in the extracellular medium. Subsequent investigation into additional point mutations (Vps35 P316S and Vps35 R524W) highlighted the inability for Vps35 R524W containing retromer to correctly associate with the endosome compartment. While Vps35 P316S containing retromer appears to function identical to that of the wild type retromer, Vps35 R524W negatively regulates retromer-dependent machinery at the endosome surface, leads to the mislocalization of receptors and delays the ability of α-synuclein to be cleared from the cell. Collectively, it is clear that the findings presented here strongly implicate the PD-linked Vps35 variants as loss-of-function mutations and lead to the presence of cellular phenotypes witnessed in PD. Lastly, I investigated the use of a pharmacological agent to enhance retromer’s function that consequently, aided in significant clearance of α-synuclein inclusions that were observed following the depletion of Vps35 or expression of PD linked mutations Vps35 P316S and Vps35 R524W. These findings support the notion that therapeutic intervention against retromer may aid in delaying the cellular phenotypes observed in PD tissue and subsequently, bridge the gap between the mammalian retromer complex, the lysosome and human disease.