Keller J N, Mark R J, Bruce A J, Blanc E, Rothstein J D, Uchida K, Waeg G, Mattson M P
Sanders-Brown Research Center on Aging, University of Kentucky, Lexington 40536, U.S.A.
Neuroscience. 1997 Oct;80(3):685-96. doi: 10.1016/s0306-4522(97)00065-1.
Removal of extracellular glutamate at synapses, by specific high-affinity glutamate transporters, is critical to prevent excitotoxic injury to neurons. Oxidative stress has been implicated in the pathogenesis of an array of prominent neurodegenerative conditions that involve degeneration of synapses and neurons in glutamatergic pathways including stroke, and Alzheimer's, Parkinson's and Huntington's diseases. Although cell culture data indicate that oxidative insults can impair key membrane regulatory systems including ion-motive ATPases and amino acid transport systems, the effects of oxidative stress on synapses, and the mechanisms that mediate such effects, are largely unknown. This study provides evidence that 4-hydroxynonenal, an aldehydic product of lipid peroxidation, mediates oxidation-induced impairment of glutamate transport and mitochondrial function in synapses. Exposure of rat cortical synaptosomes to 4-hydroxynonenal resulted in concentration- and time-dependent decreases in [3H]glutamate uptake, and mitochondrial function [assessed with the dye 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)]. Other related aldehydes including malondialdehyde and hexanal had little or no effect on glutamate uptake or mitochondrial function. Exposure of synaptosomes to insults known to induce lipid peroxidation (FeSO4 and amyloid beta-peptide) also impaired glutamate uptake and mitochondrial function. The antioxidants propyl gallate and glutathione prevented impairment of glutamate uptake and MTT reduction induced by FeSO4 and amyloid beta-peptide, but not that induced by 4-hydroxynonenal. Western blot analyses using an antibody to 4-hydroxynonenal-conjugated proteins showed that 4-hydroxynonenal bound to multiple cell proteins including GLT-1, a glial glutamate transporter present at high levels in synaptosomes. 4-Hydroxynonenal itself induced lipid peroxidation suggesting that, in addition to binding directly to membrane regulatory proteins, 4-hydroxynonenal potentiates oxidative cascades. Collectively, these findings suggest that 4-hydroxynonenal plays important roles in oxidative impairment of synaptic functions that would be expected to promote excitotoxic cascades.
通过特异性高亲和力谷氨酸转运体清除突触处的细胞外谷氨酸，对于防止神经元受到兴奋性毒性损伤至关重要。氧化应激与一系列突出的神经退行性疾病的发病机制有关，这些疾病涉及谷氨酸能通路中突触和神经元的退化，包括中风、阿尔茨海默病、帕金森病和亨廷顿病。尽管细胞培养数据表明氧化损伤会损害包括离子驱动ATP酶和氨基酸转运系统在内的关键膜调节系统，但氧化应激对突触的影响以及介导这种影响的机制在很大程度上尚不清楚。本研究提供了证据表明，4-羟基壬烯醛（脂质过氧化的醛类产物）介导了氧化诱导的突触中谷氨酸转运和线粒体功能的损伤。将大鼠皮质突触体暴露于4-羟基壬烯醛会导致[3H]谷氨酸摄取以及线粒体功能（用染料3-(4,5-二甲基噻唑-2-基)-2,5-二苯基四氮唑溴盐（MTT）评估）呈浓度和时间依赖性降低。其他相关醛类，包括丙二醛和己醛，对谷氨酸摄取或线粒体功能几乎没有影响。将突触体暴露于已知可诱导脂质过氧化的刺激物（硫酸亚铁和淀粉样β肽）也会损害谷氨酸摄取和线粒体功能。抗氧化剂没食子酸丙酯和谷胱甘肽可防止硫酸亚铁和淀粉样β肽诱导的谷氨酸摄取损伤和MTT还原，但不能防止4-羟基壬烯醛诱导的损伤。使用针对4-羟基壬烯醛结合蛋白的抗体进行的蛋白质印迹分析表明，4-羟基壬烯醛与多种细胞蛋白结合，包括GLT-1（一种在突触体中高水平存在的胶质谷氨酸转运体）。4-羟基壬烯醛本身会诱导脂质过氧化，这表明除了直接与膜调节蛋白结合外，4-羟基壬烯醛还会增强氧化级联反应。总的来说，这些发现表明4-羟基壬烯醛在突触功能的氧化损伤中起重要作用，而这有望促进兴奋性毒性级联反应。
