Abstract

BackgroundMitochondrial dysfunction has been implicated in the pathogenesis of Parkinson's disease (PD). Impairment of the mitochondrial electron transport chain (ETC) and an increased frequency in deletions of mitochondrial DNA (mtDNA), which encodes some of the subunits of the ETC, have been reported in the substantia nigra of PD brains. The identification of mutations in the PINK1 gene, which cause an autosomal recessive form of PD, has supported mitochondrial involvement in PD. The PINK1 protein is a serine/threonine kinase localized in mitochondria and the cytosol. Its precise function is unknown, but it is involved in neuroprotection against a variety of stress signalling pathways.Methodology/Principal FindingsIn this report we have investigated the effect of silencing PINK1 expression in human dopaminergic SH-SY5Y cells by siRNA on mtDNA synthesis and ETC function. Loss of PINK1 expression resulted in a decrease in mtDNA levels and mtDNA synthesis. We also report a concomitant loss of mitochondrial membrane potential and decreased mitochondrial ATP synthesis, with the activity of complex IV of the ETC most affected. This mitochondrial dysfunction resulted in increased markers of oxidative stress under basal conditions and increased cell death following treatment with the free radical generator paraquat.ConclusionsThis report highlights a novel function of PINK1 in mitochondrial biogenesis and a role in maintaining mitochondrial ETC activity. Dysfunction of both has been implicated in sporadic forms of PD suggesting that these may be key pathways in the development of the disease.

Highlights

  • Parkinson’s disease (PD) is characterized by loss of dopaminergic neurons in the substantia nigra of the brain, with surviving neurons typically containing intracytoplasmic protein inclusions known as Lewy bodies [1]

  • This report highlights a novel function of PINK1 in mitochondrial biogenesis and a role in maintaining mitochondrial electron transport chain (ETC) activity

  • Recombinant PINK1 mRNA levels were 10-fold higher than endogenous levels in this cell line. Treatment of these cells with the two different pairs of PINK1 siRNA reduced PINK1 mRNA levels by 60% when compared to scrambled control siRNA-treated cells. This resulted in a decrease in recombinant PINK1 protein expression, with the PINK1 #2 siRNA pair more effective (52% decrease compared to scrambled control siRNA; n = 3) than PINK1 #1 siRNA (39% decrease; n = 3) (Fig. 1B)

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Summary

Introduction

Parkinson’s disease (PD) is characterized by loss of dopaminergic neurons in the substantia nigra of the brain, with surviving neurons typically containing intracytoplasmic protein inclusions known as Lewy bodies [1]. The PINK1 protein is a serine/threonine kinase that has been localized to the cytosol and the inner membrane of mitochondria [8,9]. Impairment of the mitochondrial electron transport chain (ETC) and an increased frequency in deletions of mitochondrial DNA (mtDNA), which encodes some of the subunits of the ETC, have been reported in the substantia nigra of PD brains. The identification of mutations in the PINK1 gene, which cause an autosomal recessive form of PD, has supported mitochondrial involvement in PD. The PINK1 protein is a serine/threonine kinase localized in mitochondria and the cytosol. Its precise function is unknown, but it is involved in neuroprotection against a variety of stress signalling pathways

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