GLUTAMATE NEUROTOXICITY IN NEURONAL NOS KNOCKOUTS

描述：（改编自申请人的摘要）在初级神经元中， 培养物、N-甲基-D-天冬氨酸（NMDA）神经毒性和体外模型 一氧化氮（NO）介导了脑缺血的发病机制。 尽管许多研究表明NO参与谷氨酸介导的 神经毒性，有证据表明，NO可能不发挥作用，在NMDA 神经毒性，实际上可能是神经保护性的。 NO可能 通过亚硝基化和失活NMDA受体来保护神经。 NO的双刃剑可能与其氧化还原状态有关， 自由基是有毒的，亚硝鎓离子是神经保护的。 在 为了更好地了解NO在各种生理过程中的作用， NO参与调节的过程，包括谷氨酸 神经毒性，申请人已经产生了携带选择性神经毒性的小鼠。 胚胎干细胞中神经元型NOS基因的定点缺失突变 细胞 利用NOS敲除小鼠及其野生型对照， 提出了一系列的实验来阐明潜在的机制 和NO参与神经毒性，并确定替代 毒性的途径。 脑缺血引起的神经元损伤可能 产生过量的NO。 联合氧糖剥夺 在神经元培养物中以及兴奋性氨基酸给药 将用作脑缺血的体外模型。 易感性 将在神经元NOS敲除中评估神经元损伤， 与野生型对照相比。 将进行实验， 确定神经元NOS的靶向缺失是否导致额外的 这些变化可能解释了对谷氨酸介导的 毒性 谷氨酸受体的分布和密度将是 评估。 45 Ca 2+的积累将被检查， 神经元NOS敲除中的兴奋性氨基酸，并与野生型相比 类型控件。 NADPH黄递酶或NOS神经元对NMDA有抗性 型神经毒性，对红藻氨酸盐和使君子酸盐高度敏感 神经毒性 NOS和NADPH黄递酶神经元共定位， 生长激素抑制素和神经肽Y。 NOS敲除者具有正常的NPY， 生长抑素神经元 因此，这些神经元对 NMDA、红藻氨酸和使君子酸将在NOS敲除与 野生型对照，以确定这些 神经元在NOS敲除中改变。 此外，分布 将在NOS神经元上检测谷氨酸受体，以评估是否 谷氨酸受体的差异表达解释了 对毒性的不同敏感性。 其他可能的途径 存在神经毒性，因为NOS抑制剂仅具有部分保护作用。 的 神经元NOS敲除提供了一个独特的机会，检查其他 谷氨酸神经毒性的潜在途径，而不受 因此，谷氨酸神经毒性和氧-葡萄糖剥夺， 将在神经元NOS敲除中检查神经元培养物， 与野生型对照相比， 其他潜在神经毒性途径的抑制剂。 这些试剂 将包括超氧阴离子和自由基的清除剂。 在 此外，磷脂酶A2抑制剂、环氧合酶 抑制剂和脂氧合酶抑制剂。

DESCRIPTION: (adapted from Applicant's Abstract) In primary neuronal cultures, N-methyl-D-aspartate (NMDA) neurotoxicity, and in vitro model of cerebral ischemia, is mediated, in part, by nitric oxide (NO). Despite numerous studies implicating NO in glutamate mediated neurotoxicity, there is evidence that NO may play no role in NMDA neurotoxicity and may in fact be neuroprotective. NO may be neuroprotective by nitrosylating and inactivating the NMDA receptor. NO s double edge sword may be related to it s redox state, with the NO radical being toxic and the nitrosonium ion being neuroprotective. In order to better understand the role of NO in a variety of physiologic processes in which NO has been implicated to regulate including glutamate neurotoxicity, the applicants have generated mice carrying a selective mutation in the neuronal NOS gene by targeted deletion in embryonic stem cells. Utilizing the NOS knockout mice and their wild type controls, a series of experiments are proposed to clarify the potential mechanisms and involvement of NO in neurotoxicity and to identify alternative pathways of toxicity. Neuronal damage due to cerebral ischemia may occur through excess NO production. Combined oxygen-glucose deprivation in neuronal cultures as well as excitatory amino acid administration will be used as in vitro model of cerebral ischemia. The susceptibility to neuronal injury will be evaluated in neuronal NOS knockouts and compared to wild type controls. Experiments will be performed to determine whether targeted deletion of neuronal NOS leads to additional changes that might account for the resistance to glutamate mediated toxicity. The distribution and density of glutamate receptors will be evaluated. 45Ca2+ accumulation will be examined in response to excitatory amino acids in neuronal NOS knockouts and compared to wild type controls. NADPH diaphorase or NOS neurons are resistant to NMDA type neurotoxicity and are highly susceptible to kainate and quisqualate neurotoxicity. NOS and NADPH diaphorase neuronal colocalize with somatostatin and NPY. The NOS knockouts possess normal NPY and somatostatin neurons. As such the susceptibility of these neurons to NMDA, kainate and quisqualate will be examined in NOS knockouts versus wild type controls to determine whether the susceptibility of these neurons is altered in NOS knockouts. Additionally, the distribution of glutamate receptors will be examined on NOS neurons to evaluate whether the differential expression of glutamate receptors accounts for differential susceptibility to toxicity. Other potential pathways of neurotoxicity exist as NOS inhibitors are only partially protective. The neuronal NOS knockouts provide a unique opportunity to examine other potential pathways of glutamate neurotoxicity without the influence of NO. As such, glutamate neurotoxicity and oxygen-glucose deprivation in neuronal cultures will be examined in the neuronal NOS knockouts and compared to wild type controls after the administration of a variety of inhibitors of other potential pathways of neurotoxicity. These agents will include scavengers of the superoxide anion, and free radicals. In addition, the effects of phospholipase A2 inhibitors, cyclooxygenase inhibitors, and lipoxygenase inhibitors will be examined.