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PINK1 Overexpression Protects Against C2-ceramide-Induced CAD Cell Death Through the PI3K/AKT Pathway

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Abstract

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.

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Acknowledgements

PINK1 constructs were a kind gift from Dr. Mark Cookson (NIH, USA). CAD cells were a kind gift from Dr. Dona M. Chikaraishi, Department of Neurobiology, Duke University Medical Center, Durham, NC, USA. This work was supported by grants from COLCIENCIAS (110145221189), DIB-Universidad Nacional de Colombia (20101007590, 20201009689) and the Facultad de Medicina, Universidad Nacional de Colombia.

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Authors and Affiliations

  1. Universidad Colegio Mayor de Cundinamarca, Bogotá, Colombia

    Ruth Mélida Sánchez-Mora

  2. Department of Pediatrics, Universidad Nacional de Colombia, Bogotá, Colombia

    Humberto Arboleda

  3. Department of Pathology, Universidad Nacional de Colombia, Bogotá, Colombia

    Gonzalo Arboleda

  4. Institute of Genetics, Universidad Nacional de Colombia, Bogotá, Colombia

    Ruth Mélida Sánchez-Mora, Humberto Arboleda & Gonzalo Arboleda

  5. Departamento de Patología, Facultad de Medicina, Universidad Nacional de Colombia, Bogotá, Colombia

    Gonzalo Arboleda

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  1. Ruth Mélida Sánchez-Mora
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  2. Humberto Arboleda
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  3. Gonzalo Arboleda
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Correspondence to Gonzalo Arboleda.

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Sánchez-Mora, R.M., Arboleda, H. & Arboleda, G. PINK1 Overexpression Protects Against C2-ceramide-Induced CAD Cell Death Through the PI3K/AKT Pathway. J Mol Neurosci 47, 582–594 (2012). https://doi.org/10.1007/s12031-011-9687-z

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