Parkinson’s disease-associated VPS35 mutant reduces mitochondrial membrane potential and impairs PINK1/Parkinmediated mitophagy

Background:Mitochondrial dysfunction plays a prominent role in the pathogenesis of Parkinson’s disease(PD),and several genes linked to familial PD,including PINK1(encoding PTEN-induced putative kinase 1[PINK1])and PARK2(encoding the E3 ubiquitin ligase Parkin),are directly involved in processes such as mitophagy that maintain mitochondrial health.The dominant p.D620N variant of vacuolar protein sorting 35 ortholog(VPS35)gene is also associated with familial PD but has not been functionally connected to PINK1 and PARK2.Methods:To better mimic and study the patient situation,we used CRISPR-Cas9 to generate heterozygous human SH-SY5Y cells carrying the PD-associated D620N variant of VPS35.These cells were treated with a protonophore carbonyl cyanide m-chlorophenylhydrazone(CCCP)to induce the PINK1/Parkin-mediated mitophagy,which was assessed using biochemical and microscopy approaches.Results:Mitochondria in the VPS35-D620N cells exhibited reduced mitochondrial membrane potential and appeared to already be damaged at steady state.As a result,the mitochondria of these cells were desensitized to the CCCPinduced collapse in mitochondrial potential,as they displayed altered fragmentation and were unable to accumulate PINK1 at their surface upon this insult.Consequently,Parkin recruitment to the cell surface was inhibited and initiation of the PINK1/Parkin-dependent mitophagy was impaired.Conclusion:Our findings extend the pool of evidence that the p.D620N mutation of VPS35 causes mitochondrial dysfunction and suggest a converging pathogenic mechanism among VPS35,PINK1 and Parkin in PD.