Protective effect of TAT-PARKIN on neuronal cells.

Parkinson’s disease is characterized by a progressive and selective loss of dopamine neurons in the substantia nigra pars compacta and decreased levels of striatal dopamine. Cytoplasmic inclusions (Lewy bodies) containing mainly aggregated alpha-synuclein (alpha-syn) and parkin are found in the surviving nigral dopaminergic neurons. Although largely sporadic, early-onset cases of Parkinson’s disease are associated with genetic mutations, including two missense mutations (alpha-synA53T and alpha-synA30P) in the alpha-syn gene. Recent studies suggest a protective role for parkin in dopaminergic neuron survival. Here we investigated a possible neuroprotective effect of parkin against alpha-synA30P-induced neurodegeneration, using rat pheochromocytoma PC12 cells. For this purpose, we have used a protein transduction domain derived from the human immunodeficiency TAT protein to engineer TAT-alpha-synA30P (Recchia et al. FASEB J. 2008) and TAT-parkin fusion proteins, to facilitate diffusion across the plasma membrane. Both TAT-parkin and TAT-alpha-synA30P were toxic to naive PC12 cells at concentrations above 100 uM. Incubation of PC12 cells with the dopaminergic neurotoxin 6-OHDA (5-300 uM) decreased cell viability in a time/concentration-dependent manner (40% at 0 hours, 70% at 6 hours). When PC12 cells were pre-incubated with 100 uM TAT-parkin for 24 hours, followed by challenge with 60 uM 6-OHDA, a neuroprotective effect was seen at time 0 only. However, when PC12 cells were pre-incubated with 100 uM TAT-parkin for 24 hours before treatment with 60 uM 6-OHDA for 2 hours, and then incubated a second time for 2-6 hours with 100 uM TAT-parkin, survival improved to 80% at all times, instead of 50% when treated only with 60 uM 6-OHDA. Our results show the utility of protein transduction domain technology in the study of neurodegenerative disease. The TAT-proteins described here represent useful tools for exploring the role of oxidative stress response and neuronal maintenance in Parkinson’s disease. This may lead to novel approaches in developing parkin as a therapeutic target for Parkinson’s disease, based on endogenous neuroprotective mechanisms.