Matthews C C, Figueiredo D M, Wollack J B, Fairweather N F, Dougan G, Hallewell R A, Cadet J L, Fishman P S
Department of Neurology, University of Maryland, School of Medicine, and Veteran's Affairs Medical Center, Baltimore, MD, 21201, USA.
J Mol Neurosci. 2000 Jun;14(3):155-66. doi: 10.1385/JMN:14:3:155.
There is evidence that raising cellular levels of Cu2+/Zn2+ superoxide dismutase (SOD1) can protect neurons from oxidative injury. We compared a novel method of elevating neuronal SOD activity using a recombinant hybrid protein composed of the atoxic neuronal binding domain of tetanus toxin (C fragment or TTC) and human SOD1 (hSOD1) with increasing cellular SOD levels through overexpression. Fetal murine cortical neurons or N18-RE-105 cells were incubated with the TTC-hSOD1 hybrid protein and compared to cells constitutively expressing hSOD1 for level of SOD activity, cellular localization of hSOD1, and capacity to survive glucose and pyruvate starvation. Cells incubated with TTC-hSOD1 showed a threefold increase in cellular SOD activity over control cells. This level of increase was comparable to fetal cortical neurons from transgenic mice constitutively expressing hSOD1 and transfected N18-RE-105 cells expressing a green fluorescent protein-hSOD1 fusion protein (GFP-hSOD1). Human SOD1 was distributed diffusely throughout the cytoplasm of the transgenic murine neurons and transfected N18-RE-105 cells. In contrast, cells incubated with TTC-hSOD1 showed hSOD1 localized to the cell surface and intra-cytoplasmic vesicles. The cells expressing hSOD1 showed enhanced survival in glucose- and pyruvate-free medium. Neither cortical neurons nor N18-RE-105 cells incubated in TTC-hSOD1 showed increased survival during starvation. Access to the site where toxic superoxides are generated or their targets may be necessary for the protective function of SOD1.
有证据表明提高细胞内铜/锌超氧化物歧化酶（SOD1）水平可保护神经元免受氧化损伤。我们将一种使用由破伤风毒素无毒神经元结合域（C片段或TTC）和人SOD1（hSOD1）组成的重组杂合蛋白提高神经元SOD活性的新方法，与通过过表达增加细胞SOD水平的方法进行了比较。将胎鼠皮质神经元或N18-RE-105细胞与TTC-hSOD1杂合蛋白一起孵育，并与组成性表达hSOD1的细胞比较SOD活性水平、hSOD1的细胞定位以及在葡萄糖和丙酮酸饥饿状态下的存活能力。与TTC-hSOD1一起孵育的细胞，其细胞SOD活性比对照细胞增加了三倍。这种增加水平与组成性表达hSOD1的转基因小鼠的胎鼠皮质神经元以及转染了绿色荧光蛋白-hSOD1融合蛋白（GFP-hSOD1）的N18-RE-105细胞相当。人SOD1弥漫性分布于转基因小鼠神经元和转染的N18-RE-105细胞的整个细胞质中。相比之下，与TTC-hSOD1一起孵育的细胞显示hSOD1定位于细胞表面和胞内小泡。表达hSOD1的细胞在无葡萄糖和丙酮酸的培养基中存活能力增强。在TTC-hSOD1中孵育的皮质神经元和N18-RE-105细胞在饥饿期间均未显示出存活能力增加。SOD1的保护功能可能需要作用于有毒超氧化物产生的部位或其靶点。
