Mitochondrial Impairment Increases FL-PINK1 Levels by Calcium-Dependent Gene Expression

Neurobiol Dis. 2014 Feb;62(100):426-40. doi: 10.1016/j.nbd.2013.10.021. Epub 2013 Oct 29.

Abstract

Mutations of the PTEN-induced kinase 1 (PINK1) gene are a cause of autosomal recessive Parkinson's disease (PD). This gene encodes a mitochondrial serine/threonine kinase, which is partly localized to mitochondria, and has been shown to play a role in protecting neuronal cells from oxidative stress and cell death, perhaps related to its role in mitochondrial dynamics and mitophagy. In this study, we report that increased mitochondrial PINK1 levels observed in human neuroblastoma SH-SY5Y cells after carbonyl cyanide m-chlorophelyhydrazone (CCCP) treatment were due to de novo protein synthesis, and not just increased stabilization of full length PINK1 (FL-PINK1). PINK1 mRNA levels were significantly increased by 4-fold after 24h. FL-PINK1 protein levels at this time point were significantly higher than vehicle-treated, or cells treated with CCCP for 3h, despite mitochondrial content being decreased by 29%. We have also shown that CCCP dissipated the mitochondrial membrane potential (Δψm) and induced entry of extracellular calcium through L/N-type calcium channels. The calcium chelating agent BAPTA-AM impaired the CCCP-induced PINK1 mRNA and protein expression. Furthermore, CCCP treatment activated the transcription factor c-Fos in a calcium-dependent manner. These data indicate that PINK1 expression is significantly increased upon CCCP-induced mitophagy in a calcium-dependent manner. This increase in expression continues after peak Parkin mitochondrial translocation, suggesting a role for PINK1 in mitophagy that is downstream of ubiquitination of mitochondrial substrates. This sensitivity to intracellular calcium levels supports the hypothesis that PINK1 may also play a role in cellular calcium homeostasis and neuroprotection.

Keywords: CCCP; Calcium; Mitophagy; PINK1; Parkinson's disease; SH-SY5Y.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Autophagy / drug effects
  • Calcium / metabolism*
  • Carbonyl Cyanide m-Chlorophenyl Hydrazone / toxicity
  • Cell Line, Tumor
  • Gene Expression*
  • Humans
  • Mitochondria / drug effects
  • Mitochondria / enzymology*
  • Mitochondria / metabolism*
  • Mitophagy / drug effects
  • Mitophagy / physiology
  • Neuroblastoma / enzymology
  • Neuroblastoma / metabolism
  • Protein Kinases / genetics
  • Protein Kinases / metabolism*
  • Proto-Oncogene Proteins c-fos / metabolism
  • Proton Ionophores / toxicity

Substances

  • Proto-Oncogene Proteins c-fos
  • Proton Ionophores
  • Carbonyl Cyanide m-Chlorophenyl Hydrazone
  • Protein Kinases
  • PTEN-induced putative kinase
  • Calcium