Loss of PINK1 function decreases PP2A activity and promotes autophagy in dopaminergic cells and a murine model

Neurochem Int. 2011 Oct;59(5):572-81. doi: 10.1016/j.neuint.2011.03.023. Epub 2011 Jun 13.

Abstract

Parkinson's disease (PD) is the most common neurodegenerative movement disorder. Mutations in PTEN-induced kinase 1 (PINK1) are a frequent cause of recessive PD. Autophagy, a pathway for clearance of protein aggregates or impaired organelles, is a newly identified mechanism for PD development. However, it is still unclear what molecules regulate autophagy in PINK1-silenced cells. Here we report that autophagosome formation is promoted in the early phase in response to PINK1 gene silencing by lentivirus transfer vectors expressed in mouse striatum. Reduced PP2A activity and increased phosphorylation of PP2A at Y307 (inactive form of PP2A) were observed in PINK1-knockdown dopaminergic cells and striatum tissues. Treatment with C2-ceramide (an agonist of PP2A) reduced autophagy levels in PINK1-silenced MN9D cells, which suggests that PP2A plays an important role in the PINK1-knockdown-induced autophagic pathway. Furthermore, phosphorylation of Bcl-2 at S87 increased in PINK1-silenced cells and was negatively regulated by additional treatment with C2-ceramide, which indicates that Bcl-2 may be downstream of PP2A inactivation in response to PINK1 dysfunction. Immunoprecipitation also revealed dissociation of the Bcl-2/Beclin1 complex in PINK1-silenced cells, which was reversed by additional treatment with C2-ceramide, and correlated with changes in level of autophagy and S87 phosphorylation of Bcl-2. Finally, Western blots for cleaved caspase-9 and flow cytometry results for active caspase-3 revealed that PP2A inactivation is involved in the protective effect of autophagy on PINK1-silenced cells. Our findings show that downregulation of PP2A activity in PINK1-silenced cells promotes the protective effect of autophagy through phosphorylation of Bcl-2 at S87 and blockage of the caspase pathway. These results may have implications for identifying the mechanism of PD.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Apoptosis / genetics
  • Apoptosis / physiology
  • Autophagy / physiology*
  • Blotting, Western
  • Caspase 3 / metabolism
  • Caspase 9 / metabolism
  • Cell Death / physiology
  • Cell Line
  • Dopamine / physiology*
  • Fluorescent Antibody Technique, Direct
  • Gene Expression Regulation, Enzymologic / genetics
  • Gene Expression Regulation, Enzymologic / physiology
  • Genes, bcl-2 / genetics
  • Genetic Vectors
  • Immunoprecipitation
  • Lentivirus / genetics
  • Mice
  • Mice, Inbred C57BL
  • Phosphorylation
  • Plasmids / genetics
  • Protein Kinases / genetics*
  • Protein Kinases / physiology*
  • Protein Phosphatase 2 / metabolism*
  • RNA Interference
  • Transfection

Substances

  • Protein Kinases
  • PTEN-induced putative kinase
  • Protein Phosphatase 2
  • Caspase 3
  • Caspase 9
  • Dopamine