REGULATION OF NEURONAL MITOCHONDRIAL MRNA METABOLISM

尽管有证据表明线粒体能量代谢的改变可能参与神经退行性疾病的病理生理过程，但对氧化磷酸化系统酶复合体的线粒体DNA编码的信使核糖核酸(MtmRNA)代谢的调节机制尚不清楚。我们观察到，细胞内钠([Na+]i)的升高显著降低了培养神经元中mtmRNA的水平。这是出乎意料的，因为高[Na+]i会增加能量需求，而能量需求通常会上调线粒体的新陈代谢。长期目标：了解钠离子依赖调控线粒体基因表达的机制(S)，并确定这可能如何有助于中风和神经退行性疾病中选择性神经元延迟死亡的易感性。具体目的：验证高[Na+]i对线粒体基因表达的抑制是由兴奋性毒性刺激引起的，并与迟发性神经元死亡有关的假说。理论基础：原代培养神经元暴露于兴奋性谷氨酸水平后的高[Na+]i抑制线粒体基因表达，损害细胞能量代谢，影响依赖电子传递的线粒体活动(例如，ATP合成，[Ca2+]i缓冲自由基的产生和对钙离子诱导的细胞色素c：释放的敏感性)，并增加神经元对坏死性和凋亡性细胞死亡的易感性。方法：原代培养的神经元暴露于兴奋性谷氨酸后，将监测线粒体mRNA水平、相应蛋白亚基、相关酶活性(即细胞色素氧化酶活性)和线粒体呼吸，并在没有和存在延迟的二次谷氨酸暴露的情况下，将其与细胞ATP水平和神经元死亡标志物进行24小时的比较。意义：本项目研究了[Na+]i升高导致延迟性细胞死亡的新机制。对这一机制的理解可能会导致新的神经保护干预措施的发展。

Mechanisms regulating metabolism of mitochondrial DNA-encoded mRNA (mtmRNA) for enzyme complexes of oxidative phosphorylation system are not well understood although evidence indicates that alterations in mitochondrial energy metabolism likely to contribute to the pathophysiology of neurodegenerative diseases. We have observed that elevation of intracellular sodium ([Na+]i) markedly decreases levels of mtmRNA in cultured neurons. This was unexpected since high [Na+]i increases energy demand which normally up-regulates mitochondrial metabolism. Long-term Goal: To understand the mechanism(s) of Na+-dependent regulation of mitochondrial gene expression and to determine how this may contribute to selective neuronal vulnerability to delayed neuronal death in stroke and neurodegenerative disorders. Specific Aim: Test the hypothesis that inhibition of mitochondrial gene expression by high [Na+]i is elicited by an excitotoxic insult and that it contributes to delayed neuronal death. Rationale: High [Na+]i following exposure of primary neuronal cultures to excitotoxic levels of glutamate inhibits mitochondrial gene expression, compromises cellular energy metabolism, affects electron transport-dependent mitochondrial activities (e.g., ATP synthesis, [Ca2+]i buffering free radical generation and sensitivity to Ca2+-induced release of cytochrome c: and increases neuronal vulnerability to both necrotic and apoptotic cell death. Approach: Levels of mitochondrial mRNA, respective protein subunits, associated enzyme activity (i.e., cytochrome oxidase activity) and mitochondrial respiration will be monitored in primary neuronal cultures after exposure to excitotoxic levels of glutamate and compared to cellular ATP levels and markers of neuronal death over 24 hours in the absence and presence of a delayed, secondary glutamate exposure. Significance: This project examines a new mechanism by which elevated [Na+]i contribute to delayed cell death. An understanding of this mechanism could lead to the development of novel neuroprotective interventions.