CaMKII in global cerebral ischemia

描述（由申请人提供）：在美国，每年约有 600,000 人遭受心脏骤停（CA）并接受心肺复苏（CPR），导致大脑缺氧缺血（HI），从而导致大多数幸存者出现严重的神经功能缺损。没有药物治疗可以改善生存或长期神经系统结果。 CA引起的脑缺血引起的脑部缺血性损伤在很大程度上是由于通过NMDA型谷氨酸受体(NMDA)的病理性钙泛滥引发的谷氨酸兴奋性毒性，最终导致神经元细胞死亡。虽然脑缺血后导致损伤的病理学和级联事件很复杂，但 NMDAr 的过度刺激被认为是兴奋性毒性和缺血性神经元损伤的主要触发因素。 NMDAr 的兴奋毒性刺激会触发一系列 Ca2 依赖性信号通路，包括 Ca2 /钙调蛋白依赖性蛋白激酶 II (CaMKII) 的激活。我们最近的研究结果和初步数据表明，抑制 CaMKII 是一种最小化过度谷氨酸能刺激（兴奋性毒性）下游影响的新方法。 CaMKII 被认为是参与突触可塑性的生理谷氨酸信号传导的主要介质，特别是 CA1 神经元的突触增强，可能有助于海马学习。已经描述了 CaMKII 调节的两种主要形式：刺激的 CaMKII 活性和自主的 CaMKII 活性。 Ca2 /CaM 诱导 CaMKII 的刺激活性，并且通过残基 T286 的自磷酸化和/或直接结合到突触处的 NMDAr 亚基 NR2B（这也介导 CaMKII 在突触处的积累）诱导延长的自主（Ca2 独立）活性。此外，我们的初步研究揭示了 CaMKII 自主性和一氧化氮 (NO) 之间的新联系，一氧化氮是在脑缺血后的兴奋性毒性过程中产生的，会导致氧化应激和神经元损伤。我们的初步数据表明，NO 诱导的 CaMKII 亚硝基化/氧化可直接或通过保护 T286 免于去磷酸化促进自主 CaMKII 活性。我们最近证明，我们的新型 CaMKII 抑制剂 tatCN21（可阻断刺激活动和自主活动）在体外和实验性中风后提供强大的神经保护作用。相比之下，传统的 CaMKII 抑制剂（仅阻断刺激的活动）不提供攻击后神经保护。这表明抑制自主但不刺激的 CaMKII 活性是攻击后神经保护的相关药物靶点。 [重要的是，我们的初步结果表明，心脏骤停后施用 tatCN21 可产生显着的神经保护作用。] 目前的提案将利用我们的 新型小鼠 CA/CPR 模型利用几种缺乏每种形式的自主 CaMKII 活性的突变小鼠品系来揭示这些形式的 CaMKII 活性及其对缺血性神经元细胞死亡的相对贡献之间复杂的相互作用。我们假设（i）每种自主机制都有助于神经元细胞死亡，（ii）T286自磷酸化介导的CaMKII自主性具有最直接的重要性，（iii）新型亚硝基化机制通过延长T286磷酸化导致缺血性损伤，以及（iv）NR2B结合通过将CaMKII定位在nNOS附近实现有效的亚硝基化。

DESCRIPTION (provided by applicant): Each year in the United States ~600,000 people suffer from cardiac arrest (CA) and receive cardiopulmonary resuscitation (CPR), resulting in hypoxia-ischemia (HI) of the brain and consequently in severe neurological deficits in most of the survivors. No pharmacological treatment is available to improve survival or long-term neurological outcome. Ischemic damage in the brain following cerebral ischemia induced by CA is in large part due to glutamate excitotoxicity triggered by a pathological flood calcium through NMDA-type glutamate receptors (NMDAr), ultimately resulting in neuronal cell death. While the pathology and cascade of events leading to injury following cerebral ischemia is complex, overstimulation of NMDAr is considered the major triggering spark for excitotoxicity and ischemic neuronal damage. Excitotoxic stimulation of NMDAr triggers a series of Ca2+-dependent signaling pathways, including activation of Ca2+/calmodulin-dependent protein kinase II (CaMKII). Our recent findings, and preliminary data, demonstrate that inhibition of CaMKII is a novel approach to minimizing downstream effects of excessive glutamatergic stimulation (excitotoxicity). CaMKII is well established as a major mediator of physiological glutamate signaling involved in synaptic plasticity, particularly synaptic potentiation in CA1 neurons, likel contributing to hippocampal learning. Two major forms of CaMKII regulation have been described, stimulated and autonomous CaMKII activity. Stimulated activity is induced by Ca2+/CaM to CaMKII, and prolonged autonomous (Ca2+-independent) activity is induced by autophosphorylation of residue T286 and/or direct binding to NMDAr subunit NR2B at the synapse (which also mediates CaMKII accumulation at the synapse). Additionally, our preliminary studies reveal a novel link between CaMKII autonomy and nitric oxide (NO), which is produced during excitotoxicity following cerebral ischemia and contributes to oxidative stress and neuronal damage. Our preliminary data indicate that NO-induced nitrosylation/oxidation of CaMKII promotes autonomous CaMKII activity, directly and by protecting T286 from de-phosphorylation. We recently demonstrated that our new CaMKII inhibitor tatCN21 (which blocks both stimulated and autonomous activity) provides robust neuroprotection both in vitro and following experimental stroke. In contrast, traditional CaMKII inhibitors (which block only stimulated activity) do not provide post-insult neuroprotection. This indicates that inhibition of autonomous, but not stimulated CaMKII activity, is a relevant drug target for post-insult neuroprotection. [Importantly, our preliminary results demonstrate that administration of tatCN21 after cardiac arrest results in significant neuroprotection.] The current proposal will utilize our novel mouse CA/CPR model to take advantage of several mutant mouse strains deficient in each form of autonomous CaMKII activity to unravel the complex interactions between these forms of CaMKII activity and their relative contribution to ischemic neuronal cell death. We hypothesize that (i) each autonomy mechanism contributes to neuronal cell death, and that (ii) T286-autophosphorylation mediated CaMKII autonomy is of most direct importance, that (iii) the novel nitrosylation mechanisms contributes to ischemic damage by prolonging T286 phosphorylation, and that (iv) NR2B-binding enables efficient nitrosylation via localizing CaMKII near nNOS.