PINK1 overexpression protects against C2-ceramide-induced CAD cell death through the PI3K/AKT pathway.
The etiology of Parkinson's disease (PD) remains unknown. Mutations in several genes, including PINK1, have provided an understanding of the molecular mechanisms of this pathology. We analyzed the role of PINK1 overexpression (wild-type PINK1 or PINK1 with G309D or L347P mutations) on neurotoxicity associated with C2-ceramide exposure in CAD cells. CAD cells were transiently transfected with either PINK1 (wild type or mutated) or with empty vector and then treated with 25-μM C2-ceramide for 6 h. Cell viability and mitochondrial membrane potential were analyzed by flow cytometry, expression of Bax and Bcl-2 was determined by real-time PCR, and AKT phosphorylation was analyzed by western blot. CAD cells overexpressing wild-type PINK1 and treated with C2-ceramide showed lower percentages of depolarized mitochondria, lower expressions of Bax and higher expressions of Bcl-2 than non-transfected cells. In addition, wild-type PINK1 rescued C2-ceramide-induced inhibition of AKT phosphorylation. Overexpression of PINK1 G309D mutation caused an increase of depolarized mitochondria, a decrease of Bax and an increase in Bcl-2 expression levels. PINK1 L4347P mutation was associated with a higher drop in mitochondrial membrane potential and increased expression of Bax, with minimal variation in the expression of Bcl-2. PINK1 mutations did not result in variations of AKT phosphorylation. We suggest that by preventing mitochondrial dysfunction and reinforcing anti-apoptotic and neuronal survival pathways such as Bcl-2 and PI3K/AKT, PINK1 confers a neuroprotective effect against the neurotoxin C2-ceramide. These effects were abrogated by PINK1 mutations.
Universidad Colegio Mayor de Cundinamarca, Bogotá, Colombia.
Ruth Mélida
RM
Sánchez-Mora
Humberto
H
Arboleda
Gonzalo
G
Arboleda
D016923:Cell Death / Q000187:drug effects / Q000502:physiology; D002460:Cell Line; D002470:Cell Survival / Q000235:genetics; D006801:Humans; D051336:Mitochondrial Membranes / Q000187:drug effects / Q000473:pathology; D010300:Parkinson Disease / Q000201:enzymology / Q000235:genetics* / Q000473:pathology; D019869:Phosphatidylinositol 3-Kinases / Q000502:physiology*; D011494:Protein Kinases / Q000096:biosynthesis / Q000235:genetics*; D051057:Proto-Oncogene Proteins c-akt / Q000502:physiology*; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology*; D013110:Sphingosine / Q000031:analogs & derivatives* / Q000037:antagonists & inhibitors / Q000633:toxicity; D000099202:PTEN-Induced Putative Kinase
D019869:Phosphatidylinositol 3-Kinases; D011494:Protein Kinases; D051057:Proto-Oncogene Proteins c-akt; D013110:Sphingosine; D000099202:PTEN-Induced Putative Kinase; C064769:N-acetylsphingosine; C494918:AKT1 protein, human