Towards a unifying theory of Parkinson's disease: Investigation of the biochemical and genetic role of Rab GTPases

Mutations in the LRRK2 and PINK1 genes can be inherited in patients with familial forms of Parkinson's. LRRK2 and PINK1 function as a special class of enzymes known as protein kinases whose job is to label target proteins with a chemical phosphate group (in a process known as phosphorylation). Our laboratories have previously made significant advances in understanding the function of LRRK2 and PINK1 and recently identified that these enzymes target a different class of enzymes known as Rab GTPases. We now wish to better understand how LRRK2 and PINK1 controls Rabs and how mutations in these enzymes impact on Rab functioning in cells. Towards this goal we aim to identify the key Rabs controlled by LRRK2 and PINK1 and discover the molecules that Rabs bind to in order to execute downstream communications in the cell. To complement our analysis we will collaborate with genetics researchers to determine if Rabs themselves are mutated in families with Parkinson's. This project will lead to a fundamental understanding of the cellular pathways controlled by LRRK2 and PINK1 and improve our understanding of the critical pathways affected in Parkinson's.

This proposal builds on our recent exciting discovery that the LRRK2 and PINK1 kinases implicated in Parkinson's, regulate the phosphorylation and activity of Rab GTPases. LRRK2 directly phosphorylates numerous Rab isoforms on a conserved Thr/Ser residue
that lies at the centre of its effector binding motif (Thr72-Rab8A), inhibiting association with known effectors GDIs and Rabin-8 that are required for Rab8A activation. Activation of PINK1 induces phosphorylation of a distinct C-terminal residue of Rab isoforms (Ser111-Rab8A) through an as yet unknown intermediate kinase, which inhibits association with Rabin-8. Our findings therefore provide an opportunity to investigate novel biological roles for LRRK2 and PINK1 and the mechanisms implicated in Parkinson's. We will identify the key Rab isoforms that are phosphorylated by LRRK2 and PINK1 in brain and iPSC derived neurons (Alessi/Muqit/Gasser-labs). We will work on
delineating how Rab phosphorylation influences downstream signalling pathways (Alessi/Muqit-labs). Finally, we will investigate whether genetic variation in Rab GTPases and their effectors and interactors are linked to Parkinson's susceptibility (Gasser-lab). The results and reagents generated in this study together with the integration of biochemical, clinical and genetic analysis will make a major contribution to the assessment of Rab phosphorylation as central mediators of neurodegeneration.

Our proposal aims to determine fundamental new knowledge on the mechanisms that underlie neurodegeneration in Parkinson's disease (PD). This is of crucial importance since PD remains incurable and moreover, none of the currently available treatments influence disease progression. We wish to understand the role of a novel signal transduction pathway that we recently uncovered in which we discovered that Rab GTPases, are the downstream target of the protein kinases LRRK2 and PINK1. Both these kinases are encoded by genes that are mutated in Parkinson's highlighting the critical role of this pathway in neuronal survival. All significant discoveries in this proposal will be published in open access journals to ensure the greatest reach and impact of the work and should significantly advance knowledge in the Parkinson's field. Our proposal also employs state-of-the-art methodologies and high quality biochemical tools to study these pathways. These methodologies and tools will be extremely useful to many labs in the field for future work.

This proposal has significant potential in the development of a novel biomarker of PD. We are generating monoclonal antibodies against phosphorylated (activated) forms of Rab enzymes and will be testing their sensitivity and specificity in Parkinson's brains and patient derived cell lines. If successful these antibodies would be a significant boost for biotechnology companies developing biomarkers and could have immediate benefit to researchers conducting clinical trials of potential anti-PD therapies.

The proposal also has potential to advance new therapeutic strategies for PD, which will be of significant benefit to pharmaceutical companies. Our labs have a major advantage in engaging with the pharmaceutical industry since the MRC Protein Phosphorylation and Ubiquitylation Unit is integrated into the Division of Signal Transduction Therapy (DSTT), the largest UK academic-industry collaboration involving six of the world's largest pharmaceutical companies namely AstraZeneca, Boehringer Ingelheim, GlaxoSmithKline, Janssen Pharmaceutica, Pfizer and Merck KGaA. All new data arising from my work is made available to these companies prior to publication to enable them to consider the potential of the research for drug discovery. In addition we meet with company representatives three times per year, which gives my work significant exposure and maximises the possibilities for drug discovery and development by one of these companies.

Overall we strongly believe that this proposal under our direction will lead to significant progress in understanding Parkinson's and ultimately this will lead to improvements in human health.