Evidence for a role of the Bcl-2 family in the pathophysiology of Parkin-associated parkinsonism using a human fibroblast model

Parkinson's disease (PD) is a primarily sporadically occurring neurodegenerative disorder of advanced age. In the last few years several genes have been identified, including Parkin, coding for an E3 ligase involved in proteasomal protein degradation. Recent findings implicate oxidative damage and apoptosis as key molecular mechanisms of both sporadic and familial PD.

To characterize the possible role of molecular mechanisms in samples originating from patients with monogenic PD, we employed human skin fibroblasts from 8 Parkin mutation carriers (2 homozygous: delEx7, 1072Tdel and 1 compound heterozygous delEx7+c.1072Tdel; 5 heterozygous of either mutation) and 2 controls from the same family. After isolation of total proteins and subcellular fractionation, protein lysates were analyzed using Western blotting and immunocytochemistry to detect apoptosis-related changes. Oxy Dot blot and dihydorethidium staining served to monitor the status of oxidative stress. In order to evaluate the role of mitochondrial function, we challenged fibroblasts with paraquat, an oxidative stress inducer.

Western blotting revealed changes of Bax expression, with higher Bax levels in fibroblasts harbouring Parkin homozygous or compound heterozygous mutations, than in the control cells. On the contrary, levels of Bcl-2 were decreased in the fibroblasts with homozygous and compound heterozygous mutations. After paraquat treatment, changes on the level of Bax were more prominent: the level of mitochondrial Bax was increased over the cytosolic Bax. Compared to controls, the localization of cytochrome C was shifted from the mitochondrial towards the cytosolic fraction of all mutant cells. This finding became more obvious after paraquat treatment. An almost two-fold higher level of protein oxidation was detected in all mutant cells. Using immunostaining, we demonstrated an intensive localization of Bax in the mitochondrial network of mutant fibroblasts, whereas cytochrome C was diffusely localized throughout the cytosol. These changes were more prominent after paraquat treatment.

First, our results strongly indicate an involvement of apoptotic processes and oxidative cell damage leading to cell death and support involvement of mitochondrial dysfunction in the pathogenesis of Parkin-related PD. Second, we here present a novel approach to study the pathogenesis of cellular damage underlying genetic PD, establishing human skin fibroblasts from Parkin mutation carriers as a suitable model.