The molecular cell biology jigsaw of Parkinson's disease: bringing the pieces together

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PD is the second most common neurodegenerative disorder affecting 2-3% of the population over the age of 65. Seventeen genes have been associated with increased PD risk to date ("PARK genes"). The big challenge in the field is to order these genes into common pathways that may underpin the pathology. Common themes are gradually emerging, linking several of the best characterised PD associated genes to two overarching processes: endo-lysosomal trafficking and mitophagy. Both these processes are areas of intense research in the two collaborating host laboratories. Endo-lysosomal traffic is critical for the disposal of defective organelles and toxic protein aggregates by autophagy. PD is commonly associated with the accumulation of alpha-synuclein containing aggregates, called "Lewy bodies" in discrete regions of the brain. Mitophagy concerns the controlled disposal of defective mitochondria by selective autophagy. The resultant autophagosomes fuse with lysosomes where the degradation of enclosed material takes place. Failure to clear damaged mitochondria and aggregates in the dopaminergic neurons of the substantia nigra is thought to result in accumulation of reactive oxygen species and a calcium imbalance ultimately leading to cell death. We are interested in the functional links between the best characterised PD associated genes (LRRK2, VPS35, PINK1 and Parkin). Parkin is a ubiquitin E3 ligase that is activated by PINK1 and plays a critical role in mitophagy. Interestingly, we have shown that Parkin can ubiquitylate VPS35, a protein involved in vesicular trafficking. VPS35 mutants cause mis-sorting of newly sythesised lysosomal enzymes and defects in lysosome biogenesis. A PD associated mutant form of VPS35 has been found to activate the LRRK2 kinase, one of the most commonly mutated genes associated with familial PD. The most prominent substrates of LRRK2 belong to the Rab family of small GTPases, key regulators of vesicular trafficking and VPS35 function. The project will incorporate live and fixed cell imaging techniques and biochemical methods using model tissue culture cell systems. A proteomic component is anticipated and training in the preparation of samples for mass spectrometry as well as the analysis and interpretation of proteomic datasets will be provided. We will use CRISPR/Cas9 gene editing to generate isogenic cell lines that will allow for comparison between mutant and wild-type forms of PD associated genes. A component of the project will entail the use of dopaminergic neurons generated from neuronal precursor cells (NPCs) that can be derived from patient derived fibroblasts. This innovative system is advantageous compared with iPSC models as NPCs retain the epigenetic signatures associated with ageing.