Overexpression of VMAT-2 and DT-diaphorase protects substantia nigra-derived cells against aminochrome neurotoxicity.
Overexpression of VMAT-2 and DT-diaphorase protects substantia nigra-derived cells against aminochrome neurotoxicity.
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DOI：
10.1016/j.bbadis.2012.03.010
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发表时间：
2012-07
影响因子：
6.2
通讯作者：
Segura-Aguilar, Juan
中科院分区：
文献类型：
--
作者：
Munoz, Patricia;Paris, Irmgard;Sanders, Laurie H.;Greenamyre, J. Timothy;Segura-Aguilar, Juan
我们验证了这一假设，即VMAT-2和DT-黄递酶在多巴胺氧化过程中都是对抗依赖于氨基色素的神经毒性的重要细胞防御。建立了高表达VMAT-2和DT-黄递酶的细胞系。将编码VMAT-2的双顺反子与GFP-IRES-dT-黄递酶基因融合后，RCSN-3细胞VMAT-2和DT-黄递酶的蛋白表达显著增加(7倍；P<；0.001)和DT-黄递酶(9倍；P<；0.001)，转运和酶活性分别增加4倍和5.5倍。对大鼠黑质突触小泡的研究表明，VMAT-2摄取~3H-氨基色素6.3±0.4nmol/min/mg与摄取多巴胺6.2±0.3nmol/min/mg相似，这是依赖于ATP的。有趣的是，2μM洛贝林对氨基色素摄取有抑制作用，而利血平(1和10μM)则无此作用。将高表达VMAT-2和DT-黄递酶的细胞与20μ的氨基铬孵育，结果显示：(1)细胞死亡显著减少(6倍，P<；0.001)；(2)透射电子显微镜观察到细胞超微结构正常，自噬小体显著增加，线粒体内膜显著重塑；(3)ATP256±11μM正常水平与野生型细胞显著减少(121±11μM，P<；0.001)；以及(Iv)DNA梯带显著减少(21±8像素，P<；0.001)细胞与20μM氨基铬处理的野生型细胞(269±9)比较。这些结果支持我们的假设，即VMAT-2和DT-黄递酶是对抗多巴胺氧化过程中形成的氨基色素的重要防御系统。
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 ± 0.3 nmol/min/mg that which were dependent on ATP. Interestingly, aminochrome uptake was inhibited by 2 μM lobeline but not reserpine (1 and 10 μM). Incubation of cells overexpressing VMAT-2 and DT-diaphorase with 20 μM 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 μM) contrasting with a significant decrease in wild type cells (121 ± 11 μM, 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 μM 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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影响因子：
16.2
作者：
Fon, EA;Pothos, EN;Edwards, RH
通讯作者：
Edwards, RH
影响因子：
6.1
作者：
Arriagada, C;Paris, I;Segura-Aguilar, J
通讯作者：
Segura-Aguilar, J
影响因子：
56.9
作者：
Conway, KA;Rochet, JC;Lansbury, PT
通讯作者：
Lansbury, PT
影响因子：
2.9
作者：
GIBB, WRG
通讯作者：
GIBB, WRG
影响因子：
2.5
作者：
Liang, CL;Nelson, O;German, DC
通讯作者：
German, DC