We tested the hypothesis that both VMAT-2 and DT-diaphorase are an important cellular defense against aminochrome-dependent neurotoxicity during dopamine oxidation. A cell line with VMAT-2 and DT-diaphorase over-expressed was created. The transfection of RCSN-3 cells with a bicistronic plasmid coding for VMAT-2 fused with GFP-IRES-DT-diaphorase cDNA induced a significant increase in protein expression of VMAT-2 (7-fold; P<0.001) and DT-diaphorase (9-fold; P<0.001), accompanied by a 4- and 5.5-fold significant increase in transport and enzyme activity, respectively. Studies with synaptic vesicles from rat substantia nigra revealed that VMAT-2 uptake of ³H-aminochrome 6.3 ± 0.4nmol/min/mg was similar to dopamine uptake 6.2 ± 0.3nmol/min/mg that which were dependent on ATP. Interestingly, aminochrome uptake was inhibited by 2μM lobeline but not reserpine (1 and 10μM). Incubation of cells overexpressing VMAT-2 and DT-diaphorase with 20μM aminochrome resulted in (i) a significant decrease in cell death (6-fold, P<0.001); (ii) normal ultra structure determined by transmission electron microscopy contrasting with a significant increase of autophagosome and a dramatic remodeling of the mitochondrial inner membrane in wild type cells; (iii) normal level of ATP (256 ± 11μM) contrasting with a significant decrease in wild type cells (121±11μM, P<0.001); and (iv) a significant decrease in DNA laddering (21 ± 8pixels, P<0.001) cells in comparison with wild type cells treated with 20μM aminochrome (269 ± 9). These results support our hypothesis that VMAT-2 and DT-diaphorase are an important defense system against aminochrome formed during dopamine oxidation.
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