ZINC NEUROTOXICITY

说明（来自申请人的摘要）：谷氨酸受体和谷氨酸受体 钙离子介导的神经毒性是过去研究的重点。 最近，我们也开始研究一种相关的神经毒性， 增强谷氨酸受体激活，但介导的锌2+，而不是 Ca2+。Zn 2+介导的神经毒性可能导致中枢神经元死亡 在某些损伤后，例如短暂的全脑缺血。我们的中央 假设细胞外Zn 2+可以通过以下方式杀死神经元：1）进入跨膜细胞， 质膜，主要是通过电压门控钙通道（VGCC）， 去极化神经元; 2）增加细胞内游离Zn 2+（[Zn 2 +]i）; 3） 干扰糖酵解，导致ATP水平下降; 4）触发 细胞凋亡（在较低的Zn 2+水平）。拟议的实验将测试以下方面： 在培养的小鼠皮层神经元中， Zn 2+诱导的神经元死亡的潜在机制，以促进开发 治疗对策，可用于减少脑损伤后， 心脏骤停 培养的神经元将暴露于不同浓度的细胞外 锌短期（“快速毒性”）或长期（“缓慢毒性”）的时间段。 我们计划定义跨膜Zn 2+流入（测量） 用膜片钳和放射性同位素通量技术），[Zn 2 +]I（用染料 视频显微镜），细胞Zn 2+含量（用原子吸收法测量 光谱学或电感耦合等离子体光谱学），以及细胞 细胞凋亡（v.s.坏死）。我们还将测量由此产生的神经元水平 ATP、NAD+、NADH和糖酵解中间产物，线粒体跨膜 电位和细胞质活性氧（用 二氢乙锭染料）。最后，我们将测试细胞的遗传扰动， 锌2+稳态，特别是增加或减少表达的关键 质膜Zn 2+转运蛋白，ZnT-1，或主要的神经细胞内 Zn ~（2+）结合蛋白-金属硫蛋白-Ⅲ（metallothionein-III）会引起 脆弱性锌+神经毒性预测的中心假说。

DESCRIPTION (From the Applicant's Abstract): Glutamate receptor- and Ca2+-mediated neurotoxicity was the focus of study during past grant periods. Recently, we have begun to examine a related form of neurotoxicity, also enhanced by glutamate receptor activation but mediated by Zn2+ rather than Ca2+. Zn2+-mediated neurotoxicity likely contributes to central neuronal death after certain insults, such as transient global ischemia. Our Central Hypothesis is that extracellular Zn2+ can kill neurons by: 1) entering across the plasma membrane, largely through voltage-gated Ca2+ channels (VGCCs) in depolarized neurons; 2) increasing intracellular free Zn2+ ([Zn2+]i); 3) interfering with glycolysis, causing ATP levels to fall; 4) triggering apoptosis (at lower Zn2+ levels). The proposed experiments will test aspects of this central hypothesis in cultured murine cortical neurons, delineating mechanisms underlying Zn2+-induced neuronal death to advance efforts to develop therapeutic countermeasures that might be used to reduce brain damage after cardiac arrest. Cultured neurons will be exposed to varying concentrations of extracellular zinc for brief ("fast toxicity") or prolonged ("slow toxicity") time periods. We plan to define the relationships linking transmembrane Zn2+ influx (measured with patch-clamp and radio-isotope flux techniques), [Zn2+]I (measured with dye videomacroscopy), cellular Zn2+ content (measured with atomic absorption spectroscopy or inductively-coupled plasma spectroscopy), and cellular apoptosis (v.s. necrosis). We will also measure resultant neuronal levels of ATP, NAD+, NADH and glycolytic intermediates, mitochondrial transmembrane potential, and cytoplasmic reactive oxygen species (measured with dihydroethidium dye). Finally, we will test genetic perturbations of cellular Zn2+ homeostasis, specifically increased or decreased expression of the key plasma membrane Zn2+ transporter, ZnT-1, or the major neuronal intracellular Zn2+ binding protein, metallothionein-III, will produce the changes in vulnerability to Zn2+ neurotoxicity predicted by the central hypothesis.