Analysis in vitro of the neuroprotective action of recombinant human TAT-parkin and parkin over-expression in the 6-hydroxydopamine model of Parkinson's disease (2010)

Parkinson's disease is a chronic progressive neurodegenerative movement disorder characterized by a profound and selective loss of nigrostriatal dopaminergic neurons. A major hurdle in the development of neuroprotective therapies is due to limited understanding of disease processes leading to the death of neurons. The etiology of dopaminergic neuronal demise in Parkinson's disease is elusive, but a combination of genetic and environmental factors seems to play a critical role. Mutations in parkin are known to be the predominant cause of autosomal recessive, early-onset parkinsonism. Parkin is an E3 ubiquitin ligase, an essential component of the ubiquitin-proteasomal system, and its over-expression is neuroprotective in several cellular models of apoptosis. Cell-penetrating peptides, e.g. linked to the transactivator of transcription (TAT) provide an attractive delivery system across biological membranes. In the first part of the study, we describe the construction, purification, and characterization of a TAT-parkin fusion protein, and its neuroprotective activity in an in vitro model of Parkinson's disease. Western blot and immunocytochemical analyses showed TAT-parkin to transduce PC12 pheocromocytoma cells and Chinese hamster ovary cells and to localize in the nucleus, cytoplasm, and mitochondria. Moreover, TAT-parkin exhibited ubiquitination activity in vitro. Importantly, TAT-parkin at nanomolar concentrations protected nerve growth factor-differentiated PC12 cells against the dopaminergic neurotoxin 6-hydroxydopamine (6-OHDA), but only if present in the culture medium before, during, and after treatment with 6-OHDA. Based on these results, the second part of the study was designed to assess the pro-survival action of parkin using a system in which parkin is over-expressed. To accomplish this, stable PC12 cells transfectants were established with plasmids carrying either human wild-type (WT) parkin, or the human R42P mutation (parkinR42P). Non-transfected PC12 cells were used as control. Clones were screened for the expression of human parkin gene by Reverse Transcription-Polymerase Chain Reaction and Western blotting. The selected clones and un-transfected PC12 cells were treated with 50 and 75 μM 6-OHDA for different times. Cell-viability decreased in a time-dependent manner in both clonal and control cells, but the PC12 parkinR42P expressing clone and un-trasfected cells showed a statistically significant reduction in cell viability already from 2 h. In contrast, in the PC12 parkin (WT) expressing clone cell viability was significantly reduced only from 12 h. As abnormalities in the ubiquitin-proteasome system and in the autophagy-lysosome pathway are thought to be involved in the etiopathogenesis of Parkinson's disease, we treated the above cells with either a specific proteasome inhibitor (MG132) or the macroautophagy inhibitor 3-methyladenine (3-MA) for further 16 h. Improved survival was again evident in PC12 parkin (WT) expressing cells. This latter also showed an increase in the amount of ubiquitinated proteins and in the basal level of autophagy activation. Nevertheless, co-administration of the proteasome and autophagy inhibitors, neutralized the protective effect of PC12 parkin (WT) expression. Over-expression of WT parkin thus appears able to protect cells from oxidative stress by 6-OHDA and from the neurotoxicity of proteasome or macroautophagy inhibitors, and confirmed that the R42P amino-acid substitution affects parkin physiological action. Taken together, our data suggest that parkin, either as a recombinant TAT-fusion protein or when over-expressed is strongly neuroprotective, preventing dopaminergic PC12 cell death under various stress conditions. Parkin may thus represent a potential new therapeutic target in Parkinson's disease. Moreover, these findings support also the view that protein transduction strategies are well suited for intracellular translocation of proteins and, in particular, point to the application of designing fusion proteins with a protein transduction domain for potential therapeutic benefit, including Parkinson's disease.