Establishment of Stably Transfected Rat Neuronal Cell Lines Expressing [alpha]-Synuclein GFP Fusion Proteins

Mutations in the α-synuclein gene have been linked to rare cases of familial Parkinson's disease (PD). α-Synuclein, a 140 amino acid polypeptide, is a major component of Lewy bodies (LB), a pathological hallmark of PD. Transgenic mice, Drosophila and marmosets (Challitrix jacchus) expressing either wild type (WT) or mutant human α-synuclein develop motor deficits, LB-like inclusions in some neurons and neuronal degeneration. The effects of human α-synuclein were investigated in a neuronal rat cell line (B103). Plasmids expressing WT and mutant human α-synuclein regulated by the cytomegalovirus (CMV) promoter were prepared and used for creating stably transfected neuronal rat cell lines. For localizing α-synuclein expression, stably transfected neuronal rat cell lines, expressing α-synuclein enhanced green fluorescent protein fusion proteins, regulated by either the CMV or the human platelet-derived growth factor ß promoter were generated. Over-expression of WT and A53T α-synuclein regulated by CMV promoter in stable transfectants resulted in formation of α-synuclein-immunopositive inclusion-like structures and mitochondrial alterations. Taken together, these results suggest that abnormal accumulation of α-synuclein could lead to mitochondrial alterations that might result in oxidative stress and eventually, cell death.[PUBLICATION ABSTRACT]