Clinically reported heterozygous mutations in the PINK1 kinase domain exert a gene dosage effect.
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 (DeltaPsi(m)) were assessed. Cell lines expressing kinase and C-terminus mutants exhibited a greater rate of apoptosis and decrease in DeltaPsi(m), 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 DeltaPsi(m) decrease than those expressing one copy of the mutant. In time-dependent 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.
Tan
Eng-King
EK
Department of Neurology, Singapore General Hospital, Singapore. gnrtek@sgh.com.sg
Refai
F Shaffra
FS
Siddique
Mobin
M
Yap
Karen
K
Ho
Patrick
P
Fook-Chong
Stephanie
S
Zhao
Yi
Y
D017209:Apoptosis / Q000235:genetics; D002460:Cell Line; D002470:Cell Survival; D018628:Gene Dosage; D006579:Heterozygote; D006801:Humans; D009154:Mutation; D018384:Oxidative Stress; D011494:Protein Kinases / Q000235:genetics*; D000099202:PTEN-Induced Putative Kinase
D011494:Protein Kinases; D000099202:PTEN-Induced Putative Kinase