CaMKII in global cerebral ischemia

描述（由申请人提供）：在美国，每年约有60万人遭受心脏骤停（CA）并接受心肺复苏术（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障碍。没有药物治疗可以改善生存或长期神经预后。CA引起脑缺血后的脑缺血损伤在很大程度上是由于病理性钙通过nmda型谷氨酸受体（NMDAr）引发谷氨酸兴奋毒性，最终导致神经元细胞死亡。虽然脑缺血损伤的病理和级联反应是复杂的，但NMDAr的过度刺激被认为是兴奋毒性和缺血性神经元损伤的主要触发点。NMDAr的兴奋毒性刺激触发一系列Ca2+依赖性信号通路，包括Ca2+/钙调素依赖性蛋白激酶II （CaMKII）的激活。我们最近的发现和初步数据表明，抑制CaMKII是一种减少过度谷氨酸刺激（兴奋毒性）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。CaMKII调节的两种主要形式已经被描述，刺激和自主CaMKII活动。Ca2+/CaM诱导CaMKII激活活性，通过残基T286的自磷酸化和/或直接结合突触上的NMDAr亚基NR2B（这也介导了CaMKII在突触上的积累）诱导延长自主（Ca2+独立）活性。此外，我们的初步研究揭示了CaMKII自主性与一氧化氮（NO）之间的新联系，一氧化氮在脑缺血后的兴奋性毒性过程中产生，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CaMKII的亚硝基化/氧化直接促进了CaMKII的自主活性，并通过保护T286免于去磷酸化。我们最近证明，我们的新CaMKII抑制剂tatCN21（可阻断受刺激和自主活动）在体外和实验性中风后提供强大的神经保护。相比之下，传统的CaMKII抑制剂（仅阻断受刺激的活性）不能提供损伤后的神经保护。这表明抑制自主而非刺激的CaMKII活性是损伤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，在心脏骤停后给予tatCN21可产生显著的神经保护作用。当前的建议将利用我们的

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.