Abstract/Summary

Mutation of Vacuolar Protein Sorting 35 (VPS35) [D620N] was first identified in a small number of families with late onset, autosomal dominant Parkinson’s disease (PD). VPS35 is an integral cargo component of the retromer complex, implicated in the retrograde transport of proteins from the endosomes to the trans-Golgi Network. The mechanism of mutation consequent neurodegeneration and resulting PD pathology is unclear, however recent studies have linked VPS35 mutation to increased kinase activity of Leucine Rich Repeat Kinase 2 (LRRK2), gain of function mutations of which are the most common cause of familial PD. The well characterised nematode model, Caenorhabditis elegans, shows great promise as a novel model for VPS35 function and potentially, the proposed LRRK2 interplay. Utilising a plethora of novel CRISPR/Cas9 modified C. elegans lines, encompassing orthologous PD relevant point mutations in VPS35 and LRRK2 orthologues, vps-35 and lrk-1 respectively, the mechanisms of vps-35 mutation and LRK-1 interplay have been examined. The novel vps-35[D638N] PD C. elegans model shows age-dependent impairments in dopaminergic behaviour, increased dopaminergic degeneration and cellular aberrations relevant to PD modelling, while studies of the genetics of this model suggest a more complex mechanism of mutation action than loss of function alone. Assessment of the functional conservation extent between human LRRK2 and C. elegans LRK-1, through characterisation of novel PD and catalytic point mutants suggest there are kinase activity dependent phenotypes and therefore it is suitable for dissecting the VPS35/LRRK2 interplay. Subsequent pharmacological and genetic models suggest that in C. elegans, LRK-1 kinase hyperactivation is implicated in vps-35[D638N] pathology, with proposed LRK-1 inhibition or genetic kinase ablation resulting in vps-35[D638N] phenotypic rescue. This study is the first to utilise CRISPR/Cas9 modified C. elegans for modelling PD gene function, has demonstrated this novel model could share sufficient conservation for this and has developed understanding of the VPS35/LRRK2 interplay.