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

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• 作者：Patricia Muñ ; oz^a ; Irmgard Paris^a ; ^b ; Laurie H. Sanders^c ; J. Timothy Greenamyre^c ; Juan Segura-Aguilar^a ; ^{jsegura@med.uchile.cl}
• 关键词：Dopamine ; Neuromelanin ; VMAT-2 ; DT-diaphorase ; Aminochrome ; Parkinson's disease
• 刊名：Biochimica et Biophysica Acta (BBA)/Molecular Basis of Disease
• 出版年：2012
• 期刊代码：19_09254439
• 类别：bio
• 出版时间：July, 2012
• 卷：1822
• 期：7
• 页码：1125-1136
• 文件大小：2028 K

摘要

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 卤 0.4 nmol/min/mg was similar to dopamine uptake 6.2 卤 0.3 nmol/min/mg that which were dependent on ATP. Interestingly, aminochrome uptake was inhibited by 2 渭M lobeline but not reserpine (1 and 10 渭M). Incubation of cells overexpressing VMAT-2 and DT-diaphorase with 20 渭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 卤 11 渭M) contrasting with a significant decrease in wild type cells (121 卤 11 渭M, P < 0.001); and (iv) a significant decrease in DNA laddering (21 卤 8 pixels, P < 0.001) cells in comparison with wild type cells treated with 20 渭M aminochrome (269 卤 9). These results support our hypothesis that VMAT-2 and DT-diaphorase are an important defense system against aminochrome formed during dopamine oxidation.