Mutations in the familial Parkinson's disease gene PINK1 enhance the sensitivity to apoptosis induced by proteasomal stress

Objectives: PINK1 is a putative mitochondrial protein kinase implicated in the pathophysiology of Parkinson's disease (PD). It has recently been shown that mutations in PINK1 are associated with early onset forms of PD. Since proteasome dysfunction and apoptotic cell death seem to play fundamental roles in PD and since mitochondria are crucially involved in apoptosis, we were interested whether 1) familial PINK1 mutations might sensitize cells to apoptosis induced by proteasome stress or 2) whether they might lead to a general sensitization to apoptotic stimuli.

Methods: Primary fibroblasts were obtained from all seven siblings of a family with three homozygous patients and three heterozygous carriers with the PINK1 mutant G309D. Cell death of fibroblasts or neuroblastoma cells was determined by 1) analysis of effector caspase activation (DEVDase assays) or by 2) uptake of propidium iodide and subsequent FACS analysis (FACScalibur, Becton-Dickinson, Heidelberg, Germany). Mitochondrial membrane potentials were measured by staining live cells with Tetramethylrhodamine methyl ester (TMRM) and subsequent FACS analysis (FACScalibur).

Results: Analysis of effector caspase activation and release of cytochrome C in primary fibroblast cultures revealed that homozygous expression of G309D was associated with potently enhanced susceptibility to apoptosis induced by the proteasome inhibitor MG132. Interestingly, cultures from heterozygous siblings also revealed enhanced cell death after treatment with MG132, although to a lesser extent. In contrast to MG132, sensitivity to cell death triggered by the unrelated death stimulus staurosporine was not affected by G309D. We have recently established neuroblastoma cell lines mitochondrially overexpressing GFP-tagged wild type and mutant PINK1 (G309D and mutant W437OPA) and are currently investigating the effect of both PINK1 mutations on apoptosis sensitivity and mitochondrial dysfunction in this neuronal model system.

Conclusions: We have shown that the PINK1 mutant G309D enhances susceptibility to apoptosis induced by proteasome dysfunction, but not by unrelated death stimuli activating the mitochondrial pathway of apoptosis. Since 1) wild type PINK1 was suggested to have a neuroprotective function and since 2) fibroblasts derived from heterozygous siblings carrying the G309D mutant showed an increased vulnerability to proteasome stress, this mutant might act in a dominant negative manner.