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），并因此在大多数幸存者中造成严重的神经功能缺损。没有药物治疗可用于改善生存率或长期神经系统结局。CA诱导的脑缺血后脑中的缺血性损伤在很大程度上是由于谷氨酸兴奋性毒性，其由通过NMDA型谷氨酸受体（NMDAr）的病理性钙洪水触发，最终导致神经元细胞死亡。虽然导致脑缺血后损伤的病理学和级联事件是复杂的，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火花。NMDAr的兴奋性毒性刺激触发一系列Ca 2+依赖性信号传导途径，包括Ca 2 +/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是一种新的方法，以尽量减少下游影响的过度兴奋性刺激（兴奋性毒性）。CaMKII被公认为参与突触可塑性的生理谷氨酸信号传导的主要介质，特别是在CA 1神经元中的突触增强，可能有助于海马学习。CaMKII调节的两种主要形式已被描述，刺激和自主CaMKII活性。刺激活性由Ca 2 +/CaM诱导为CaMKII，延长的自主（Ca 2+非依赖性）活性由残基T286的自磷酸化和/或在突触处直接结合至NMDAr亚基NR 2B（其也介导CaMKII在突触处的积累）诱导。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，这是在脑缺血后的兴奋性毒性过程中产生的，并有助于氧化应激和神经元损伤。我们的初步数据表明，NO诱导的亚硝基化/氧化的CaMKII促进自主CaMKII活性，直接和保护T286从去磷酸化。我们最近证明，我们的新的CaMKII抑制剂tatCN 21（阻断刺激和自主活动）在体外和实验性中风后都提供了强大的神经保护。相比之下，传统的CaMKII抑制剂（其仅阻断刺激活性）不提供损伤后神经保护。这表明，抑制自主的，但不是刺激的CaMKII活性，是一个相关的药物靶点损伤后的神经保护。[重要的是，我们的初步结果表明，心脏骤停后给予tatCN 21可产生显著的神经保护作用。目前的建议将利用我们的 新的小鼠CA/CPR模型，以利用几种突变小鼠品系的缺陷，在每一种形式的自主CaMKII活性，以解开这些形式的CaMKII活性和它们的相对贡献缺血性神经元细胞死亡之间的复杂的相互作用。我们假设：（i）每种自主机制都导致神经元细胞死亡，（ii）T286-自磷酸化介导的CaMKII自主性是最直接的重要性，（iii）新的亚硝基化机制通过延长T286磷酸化导致缺血性损伤，（iv）NR 2B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.