Wild-type PINK1 Prevents Basal and Induced Neuronal Apoptosis, a Protective Effect Abrogated by Parkinson Disease-related Mutations

Agnes Petit,Toshitaka Kawarai,Erwan Paitel,Nobuo Sanjo,Mary Maj,Michael Scheid,Fusheng Chen,Yongjun Gu,Hiroshi Hasegawa,Shabnam Salehi-Rad,Linda Wang,Ekaterina Rogaeva,Paul Fraser,Brian Robinson,Peter St George-Hyslop,Anurag Tandon

doi:10.1074/jbc.m505143200

Agnes Petit, Toshitaka Kawarai + Show 14 more

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https://doi.org/10.1074/jbc.m505143200

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Abstract

Mutations in the PTEN-induced kinase 1 (PINK1) gene have recently been implicated in autosomal recessive early onset Parkinson Disease (1, 2). To investigate the role of PINK1 in neurodegeneration, we designed human and murine neuronal cell lines expressing either wild-type PINK1 or PINK1 bearing a mutation associated with Parkinson Disease. We show that under basal and staurosporine-induced conditions, the number of terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling (TUNEL)-positive cells was lower in wild-type PINK1 expressing SH-SY5Y cells than in mock-transfected cells. This phenotype was due to a PINK1-mediated reduction in cytochrome c release from mitochondria, which prevents subsequent caspase-3 activation. We show that overexpression of wild-type PINK1 strongly reduced both basal and staurosporine-induced caspase 3 activity. Overexpression of wild-type PINK1 also reduced the levels of cleaved caspase-9, caspase-3, caspase-7, and activated poly(ADP-ribose) polymerase under both basal and staurosporine-induced conditions. In contrast, Parkinson disease-related mutations and a kinase-inactive mutation in PINK1 abrogated the protective effect of PINK1. Together, these results suggest that PINK1 reduces the basal neuronal pro-apoptotic activity and protects neurons from staurosporine-induced apoptosis. Loss of this protective function may therefore underlie the degeneration of nigral dopaminergic neurons in patients with PINK1 mutations.

Highlights

Responsible for inherited forms of PD have been identified
We demonstrate that PTEN-induced kinase 1 (PINK1) reduces neuronal apoptosis by reducing the release of cytochrome c, thereby limiting the subsequent activation of caspases-9, -7, and -3 and PARP
Human SH-SY5Y neuroblastoma cells stably transfected with wildtype human PINK1 express a ϳ63-kDa protein recognized by our rabbit polyclonal antibody that is directed against the first 24 amino acids at the N terminus of human PINK1 including the mitochondrial targeting motif (Fig. 2A)

Summary

Introduction

Responsible for inherited forms of PD have been identified. Mutations in the ␣-synuclein [5], LRRK2 (leucine-rich repeat kinase 2) and UCH-L1 (ubiquitin C-terminal esterase L1) genes cause dominant forms of familial PD. Mutations in parkin [6], DJ-1 [7, 8], and the newly identified PTEN (phosphatase and tensin homologue on chromosome 10)-induced kinase 1 (PINK1) [1, 2] are responsible for recessive forms of familial PD. To further investigate the role of PINK1 in neuronal apoptosis, we overexpressed this protein in SH-SY5Y human neuroblastoma cells and N2a murine neurons. We demonstrate that PINK1 reduces neuronal apoptosis by reducing the release of cytochrome c, thereby limiting the subsequent activation of caspases-9, -7, and -3 and PARP. We show that this protective effect is abrogated by PD-related mutations and by rendering the kinase inactive through removal of a key lysine residue in the catalytic domain (Lys-219)

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