L-DOPA neurotoxicity is prevented by neuroprotective effects of erythropoietin

The neurotoxicity of L-3,4-dihydroxyphenylalanine (L-DOPA), one of the most important drugs for the treatment of Parkinson's disease, still remains controversial, although much more data on L-DOPA neurotoxicity have been presented. Considering the well known neuroprotective effects of erythropoietin (EPO), the inhibitory effects of EPO on L-DOPA neurotoxicity need to be evaluated. Neuronally differentiated PC12 (nPC12) cells were treated with different concentrations of L-DOPA and/or EPO for 24h. Cell viability was evaluated using trypan blue, 4',6-diamidino-2-phenylindole (DAPI) and TUNEL staining, and cell counting. Free radicals and intracellular signaling protein levels were measured with 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA) and Western blotting, respectively. L-DOPA reduced nPC12 cell viability at higher concentrations, but combined treatment with EPO and L-DOPA significantly restored cell viability. Free radicals and hydroxyl radical levels increased by L-DOPA were decreased after combined treatment of L-DOPA and EPO. Levels of survival-related intracellular signaling proteins decreased in nPC12 cells treated with 200 μM L-DOPA but increased significantly in cells treated with 200μM L-DOPA and 5 μM EPO. However, cleaved caspase-3, a death-related protein, increased in nPC12 cells treated with 200 μM L-DOPA but decreased significantly in cells treated with 200 μM L-DOPA and 5 μM EPO. Pretreatment with LY294002, a phosphatidylinositol 3-kinase inhibitor, prior to combined treatment with EPO and L-DOPA almost completely blocked the protective effects of EPO. These results indicate that EPO can prevent L-DOPA neurotoxicity by activating the PI3K pathway as well as reducing oxidative stress.