Clinically reported heterozygous mutations in the PINK1 kinase domain exert a gene dosage effect

Abstract

Mutations in the gene encoding phosphatase and tensin homolog (PTEN)-induced kinase 1 (PINK1) have been associated with the loss of dopaminergic neurons characteristic of familial and sporadic Parkinson disease. We developed an in vitro system of stable human dopaminergic neuronal cell lines coexpressing an equivalent copy of normal and mutant PINK1 to simulate "heterozygous" and "homozygous" states in patients. Mutants in the N-terminus, C-terminus, and kinase domain were generated and cloned into a two-gene mammalian expression vector to generate stable mammalian expression cell lines producing an equivalent copy number of wild-type/mutant PINK1. The cell lines were subjected to oxidative stress and the rate of apoptosis and change in mitochondrial membrane potential (Δψm) were assessed. Cell lines expressing kinase and C-terminus mutants exhibited a greater rate of apoptosis and decrease in Δψm, and increased time-dependent cell loss when subjected to oxidative stress compared to the wild-type. Cell lines expressing two copies of kinase mutants exhibited a greater apoptosis rate and Δψm decrease than those expressing one copy of the mutant. In timedependent experiments, there was a significant difference between "homozygous," "heterozygous," and wild-type cell lines, with decreasing cell survival in cell lines expressing mutant copies of PINK1 compared to the wild-type. We provided the first experimental evidence that clinically reported PINK1 heterozygous mutations exert a gene dosage effect, suggesting that haploinsufficiency of PINK1 is the most likely mechanism that increased the susceptibility to dopaminergic cellular loss. © 2009 Wiley-Liss, Inc.