AbstractParkinson’s disease (PD) is the second most common progressive neurodegenerative disorder. As PD remains incurable, only symptomatic treatment is available for patients. To identify targets for therapeutic intervention and also recognise early stage disease biomarkers, current research revolves heavily around the known genetic causes of PD. These gene encoded proteins are a strong starting point for understanding downstream signalling cascades that may contribute to PD pathology. PINK1 is mutated in early-onset forms of PD, and its downstream signalling cascade is the main focus of this thesis.
PINK1 was previously demonstrated to phosphorylate downstream substrates, Parkin and ubiquitin at Ser65, whilst also mediating the indirect phosphorylation of a subset of Rab GTPases. I first identified four Rabs including Rab1B, Rab8A, Rab8B and Rab13, to be indirectly phosphorylated by PINK1 at a Ser111 residue whilst a further four Rabs including Rab10, Rab38, Rab33b and Rab35 were also indirectly phosphorylated across various sites. Large-scale kinase assay screens were employed to identify the direct upstream Rab kinase. Although the Ser111 kinase remained elusive, further novel Thr72 Rab kinases were identified.
In order to elucidate the function of Rab Ser111 phosphorylation, I went on to fully characterise a novel p-Ser111 Rab8A antibody and attempt to determine p-Ser111 Rab8A cellular localisation. Rab8A total and p-Ser111 co-localised to Rab5 and additional known interactor proteins, however future work is required confirm these interactions occur endogenously and potentially distinguish differential localisation between phosphorylated and unphosphorylated Rab.
I finally went on to determine the role of PINK1-mediated Rab phosphorylation within additional PD-associated pathways, including LRRK2 and VPS35. With the presence of p-Ser111 Rab8A, the ability of LRRK2 to phosphorylate Rab at Thr72 was significantly reduced both in vitro and in cell-based experiments. This demonstrated the first evidence of cross-talk between PINK1 and LRRK2 pathways, demonstrating a possible protective mechanism to reduce p-Thr72 Rabs. After extensive investigation into the pathway crosstalk of VPS35 D620N (PD mutant) and PINK1, no robust link was observed.
This thesis has led to the development Rab8A p-Ser111 antibody as a key tool for the field in addition to further understanding into the role of PINK1-mediated Rab Ser111 phosphorylation, both independently and in the context of further PD-pathways.
|Date of Award||2019|
|Supervisor||Miratul Muqit (Supervisor) & Dario Alessi (Supervisor)|