Overexpression of VMAT-2 and DT-diaphorase protects substantia nigra-derived cells against aminochrome neurotoxicity

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 H-3-aminochrome 6.3 +/- 0.4 nmol/min/mg was similar to dopamine uptake 6.2 +/- 0.3 nmol/min/mg that which were dependent on ATP. Interestingly, aminochrome uptake was inhibited by 2 mu M lobeline but not reserpine (1 and 10 mu M). Incubation of cells overexpressing VMAT-2 and DT-diaphorase with 20 mu M 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 +/- 11 mu M) contrasting with a significant decrease in wild type cells (121 +/- 11 mu M, P<0.001); and (iv) a significant decrease in DNA laddering (21 +/- 8 pixels, P<0.001) cells in comparison with wild type cells treated with 20 mu M 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. (c) 2012 Elsevier B.V. All rights reserved.