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

描述（由申请人提供）：ALS患者脑脊液谷氨酸升高的观察，以及运动神经元（MNs）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，支持了该疾病中兴奋性毒性对MN损失的贡献。过去的研究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的兴奋毒性激活诱导线粒体Ca2+过载和异常强的活性氧（ROS）产生。然而，虽然星形胶质细胞谷氨酸转运体在CMS中占大多数谷氨酸摄取，并且它们的损伤似乎是ALS细胞外谷氨酸升高的基础，但其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内响应兴奋毒性激活产生的ROS本身可能导致周围星形胶质细胞谷氨酸摄取的破坏。这些观察结果表明，兴奋毒性MN损伤与谷氨酸运输的氧化破坏之间存在因果关系，并为ALS的前馈模型提供了基础，在该模型中，一系列刺激因素可能导致共同的疾病途径。本提案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的产生导致体内区域谷氨酸运输丧失的假设。MNs内兴奋毒性ROS的产生及其穿透周围组织和破坏谷氨酸运输的能力将在野生型小鼠以及携带与家族型ALS相关的超氧化物歧化酶（SOD）突变的小鼠的分离和切片培养模型中进行研究（这提供了该疾病的最佳动物模型）。SOD突变小鼠将被用来检测在大腹角MNs周围区域发生的氧化变化和运输中断的程度，如果MNs是ROS的来源，则可以预测。最后，选择药物干预减少这些病理过程的能力将在切片和动物模型中进行测试。希望这些研究能够进一步阐明ALS患者选择性MN丧失的事件序列，从而促进新的治疗策略的发展。

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.