Roy J, Minotti S, Dong L, Figlewicz D A, Durham H D
Montreal Neurological Institute and Department of Neurology and Neurosurgery, McGill University, Montreal, Quebec, Canada H3A 2B4.
J Neurosci. 1998 Dec 1;18(23):9673-84. doi: 10.1523/JNEUROSCI.18-23-09673.1998.
Mutations in the Cu/Zn-superoxide dismutase (SOD-1) gene are responsible for a subset of familial cases of amyotrophic lateral sclerosis. Using a primary culture model, we have demonstrated that normally nontoxic glutamatergic input, particularly via calcium-permeable AMPA/kainate receptors, is a major factor in the vulnerability of motor neurons to the toxicity of SOD-1 mutants. Wild-type and mutant (G41R, G93A, or N139K) human SOD-1 were expressed in motor neurons of dissociated cultures of murine spinal cord by intranuclear microinjection of plasmid expression vector. Both a general antagonist of AMPA/kainate receptors (CNQX) and a specific antagonist of calcium-permeable AMPA receptors (joro spider toxin) reduced formation of SOD-1 proteinaceous aggregates and prevented death of motor neurons expressing SOD-1 mutants. Partial protection was obtained by treatment with nifedipine, implicating Ca2+ entry through voltage-gated calcium channels as well as glutamate receptors in potentiating the toxicity of mutant SOD-1 in motor neurons. Dramatic neuroprotection was obtained by coexpressing the calcium-binding protein calbindin-D28k but not by increasing intracellular glutathione levels or treatment with the free radical spin trap agent, N-tert-butyl-alpha-phenylnitrone. Thus, generalized oxidative stress could have contributed in only a minor way to death of motor neurons expressing the mutant SOD-1. These studies demonstrated that the toxicity of these mutants is calcium-dependent and provide direct evidence that calcium entry during neurotransmission, coupled with deficiency of cytosolic calcium-binding proteins, is a major factor in the preferential vulnerability of motor neurons to disease.
铜/锌超氧化物歧化酶（SOD-1）基因突变是导致一部分家族性肌萎缩侧索硬化症病例的原因。利用原代培养模型，我们已经证明，正常情况下无毒的谷氨酸能输入，特别是通过钙通透性AMPA/海人藻酸受体的输入，是运动神经元对SOD-1突变体毒性易感性的一个主要因素。通过将质粒表达载体核内显微注射到小鼠脊髓解离培养物的运动神经元中，野生型和突变型（G41R、G93A或N139K）人类SOD-1得以表达。AMPA/海人藻酸受体的通用拮抗剂（CNQX）和钙通透性AMPA受体的特异性拮抗剂（乔罗蜘蛛毒素）都减少了SOD-1蛋白质聚集体的形成，并防止了表达SOD-1突变体的运动神经元死亡。硝苯地平治疗获得了部分保护作用，这表明通过电压门控钙通道以及谷氨酸受体的Ca2+内流在增强运动神经元中突变型SOD-1的毒性方面发挥了作用。通过共表达钙结合蛋白钙结合蛋白-D28k获得了显著的神经保护作用，但通过增加细胞内谷胱甘肽水平或用自由基自旋捕获剂N-叔丁基-α-苯基硝酮治疗则未获得这种作用。因此，全身性氧化应激对表达突变型SOD-1的运动神经元死亡的作用可能很小。这些研究表明，这些突变体的毒性是钙依赖性的，并提供了直接证据，即神经传递过程中的钙内流，加上胞质钙结合蛋白的缺乏，是运动神经元对疾病优先易感性的一个主要因素。
