VPS35 mutation inhibits PINK1/parkin-mediated mitophagy via increased LRRK2 kinase activity

The p.D620N mutation in VPS35 causes an autosomal dominant form of Parkinson's disease via mechanisms that are poorly understood. PINK1 and parkin, two proteins whose loss-of-function underlies autosomal recessive Parkinson's disease, cooperate to mediate mitophagy, a quality control pathway for selective elimination of damaged mitochondria. PINK1/parkin-mediated mitophagy is disrupted by LRRK2 mutations, which are the most prevalent cause of autosomal dominant Parkinson's disease. Here, we investigated whether the p.D620N VPS35 mutation has an effect on PINK1/parkin-mediated mitophagy. We identified a novel family with autosomal dominant Parkinson's disease caused by a p.D620N VPS35 mutation. We cultured skin fibroblasts and induced pluripotent stem cell-derived dopaminergic neurons from the proband and from a second, unrelated Parkinson's disease patient with the p.D620N VPS35 mutation, and compared them with isogenic and non-isogenic control cells. PINK1/parkin-mediated mitophagy was severely impaired in VPS35 mutant fibroblasts and neurons, while non-selective, starvation-induced autophagy and lysosomal degradative capacity were preserved. siRNA-mediated VPS35 knockdown rescued the mitophagy defect in VPS35 mutant cells, whereas overexpression of wild-type VPS35 did not, suggesting a gain-of-function mechanism of the mutation. The VPS35 mutation did not interfere with activation of PINK1 or parkin after mitochondrial depolarization, but impaired mitochondrial recruitment of the autophagy receptor optineurin. LRRK2 kinase activity was increased in the VPS35 mutant cells, as shown by enhanced levels of the T73-phosphorylated form of the LRRK2 substrate RAB10. The enhanced level of phosphorylated RAB10 in VPS35 mutant cells was decreased by treatment with LRRK2 kinase inhibitors and by VPS35 knockdown. Importantly, the mitophagy defect of VPS35 mutant fibroblasts and neurons was fully rescued by LRRK2 kinase inhibitors as well as by overexpression of PPM1H, a phosphatase that dephosphorylates multiple RAB substrates of LRRK2. Finally, in situ proximity ligation experiments revealed that endogenous VPS35 and LRRK2 are proximity partners in human dopaminergic neurons and that this proximity relationship is enhanced by the VPS35 mutation. In conclusion, the VPS35 mutation impairs PINK1/parkin-mediated mitophagy via a gain-of-function mechanism that involves stimulation of LRRK2 kinase activity. Thus, a VPS35/LRRK2 axis linked to dominant Parkinson's disease intersects with a pathway mediated by proteins encoded by the recessive Parkinson's disease genes.