AMPA/Kainate Receptors, Free Radicals, And Motor Neuron Injury

描述(申请人提供)：观察到肌萎缩侧索硬化症患者脑脊液中谷氨酸的增加，以及运动神经元(MN)对谷氨酸受体介导的(“兴奋性毒性”)损伤的选择性易感性的发现，支持兴奋性毒性在疾病中MN丢失中的作用。过去的研究已经强调了可能导致这种脆弱性的因素；与大多数其他神经元相比，MNS的兴奋毒性激活导致更大的线粒体钙超载和异常强烈的活性氧物种(ROS)的产生。然而，虽然星形胶质细胞谷氨酸转运体是CMS摄取谷氨酸的主要原因，而且它们的损伤似乎是ALS细胞外谷氨酸升高的基础，但它们功能障碍的原因尚不清楚。最近的研究提供了一个可能的线索，表明MNS内产生的这种ROS响应兴奋性毒性激活本身可能会导致周围星形胶质细胞对谷氨酸的摄取中断。这些观察提示了一种机制，即兴奋性MN损伤与谷氨酸运输的氧化中断有关，并为ALS的前馈模型提供了基础，在该模型中，一系列激发因素可能导致一种常见的疾病途径。这项建议的主要目的是使用培养和切片模型来扩展这些研究，并进一步检验MNS内ROS的产生有助于体内局部谷氨酸运输丧失的假设。将在野生型小鼠和携带与ALS家族性形式相关的超氧化物歧化酶(SOD)突变的小鼠的分离培养模型和切片培养模型中检验MNS中激发毒性ROS的生成及其穿透周围组织和扰乱谷氨酸运输的能力(这提供了该疾病的最佳动物模型)。超氧化物歧化酶突变小鼠将被用来检测在紧邻大腹角MN的区域发生氧化变化和运输中断的程度，如果MN是ROS的来源，就会预测到这一点。最后，将在切片和动物模型中测试选定的药物干预措施减少这些病理过程的能力。希望这些研究将进一步阐明导致ALS选择性MN丢失的事件序列，从而促进新治疗策略的开发。

DESCRIPTION (provided by applicant): Observations of increased CSF glutamate in ALS, together with findings that motor neurons (MNs) are selectively vulnerable to glutamate receptor mediated ("excitotoxic") injury support an excitotoxic contribution to MN loss in the disease. Past studies have highlighted factors that may underlie this vulnerability; in comparison to most other neurons, excitotoxic activation of MNs induces greater mitochondrial Ca2+ overload and exceptionally strong reactive oxygen species (ROS) generation. However, while astroglial glutamate transporters account for most glutamate uptake in the CMS, and their damage appears to underlie extracellular glutamate elevations in ALS, the reason for their dysfunction has been unclear. Providing a possible clue, recent studies suggest that this ROS generated within MNs in response to excitotoxic activation may in itself cause disruption of glutamate uptake in surrounding astrocytes. These observations suggest a mechanism that causatively links excitotoxic MN damage with oxidative disruption of glutamate transport, and provide the basis the for a feed forward model of ALS, in which a range of inciting factors could lead into a common disease pathway. The broad aim of this proposal is to use culture and slice models to extend these studies and further examine the hypothesis that ROS generation within MNs contributes to the loss of regional glutamate transport in vivo. Excitotoxic ROS generation within MNs and its ability to penetrate surrounding tissue and disrupt glutamate transport will be examined in dissociated and slice culture models from wild type mice as well as mice harboring superoxide dismutase (SOD) mutations associated with familial forms of ALS (which provide the best animal models of the disease). SOD mutant mice will be used to examine the degree to which oxidative changes and disruption of transport occurs in regions immediately surrounding large ventral horn MNs, as would be predicted if the MNs are the source of the ROS. Finally, the ability of selected pharmacological interventions to decrease these pathological processes will be tested in slice and animal models. It is hoped that these studies will further clarify sequences of events culminating in selective MN loss in ALS, and thereby facilitate development of new treatment strategies.