Universidad Colegio Mayor de Cundinamarca, Bogotá, Colombia.
J Mol Neurosci. 2012 Jul;47(3):582-94. doi: 10.1007/s12031-011-9687-z. Epub 2012 Jan 3.
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.
帕金森病（PD）的病因仍然未知。几个基因的突变，包括 PINK1，为了解这种病理学的分子机制提供了依据。我们分析了 PINK1 过表达（野生型 PINK1 或具有 G309D 或 L347P 突变的 PINK1）对 CAD 细胞中 C2-神经酰胺暴露相关神经毒性的作用。CAD 细胞瞬时转染 PINK1（野生型或突变型）或空载体，然后用 25-μM C2-神经酰胺处理 6 小时。通过流式细胞术分析细胞活力和线粒体膜电位，实时 PCR 测定 Bax 和 Bcl-2 的表达，Western blot 分析 AKT 磷酸化。与未转染细胞相比，过表达野生型 PINK1 并用 C2-神经酰胺处理的 CAD 细胞显示出更低比例的去极化线粒体、更低的 Bax 表达和更高的 Bcl-2 表达。此外，野生型 PINK1 挽救了 C2-神经酰胺诱导的 AKT 磷酸化抑制。PINK1 G309D 突变的过表达导致去极化线粒体增加、Bax 减少和 Bcl-2 表达水平增加。PINK1 L4347P 突变与线粒体膜电位下降幅度更大和 Bax 表达增加有关，Bcl-2 表达变化最小。PINK1 突变不会导致 AKT 磷酸化的变化。我们认为，通过防止线粒体功能障碍和加强抗凋亡和神经元存活途径，如 Bcl-2 和 PI3K/AKT，PINK1 对 C2-神经酰胺的神经毒素具有神经保护作用。这些作用被 PINK1 突变所阻断。
