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

Abstract

Background:Mitochondrial dysfunction plays a prominent role in the pathogenesis of Parkinson’s disease (PD), and several genes linked to familial PD, including PINK1 and PARK2, are directly involved in processes such as mitophagy that maintain mitochondrial health. The dominant p.D620N variant in VPS35 has also been associated to 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 in VPS35. These cells were treated with the protonophore CCCP to induce PINK1/Parkin-mediated mitophagy, which was assessed using biochemical and microscopy approaches. Results:Mitochondria in 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 CCCP-induced 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 PINK1/Parkin-dependent mitophagy is impaired. Conclusion:Our findings extend the pool of evidence that the p.D620N mutant VPS35 causes mitochondrial dysfunction and suggest a converging pathogenic mechanism between VPS35, PINK1 and Parkin in PD.