CaMKII in global cerebral ischemia

描述(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(CPR)，导致脑缺氧缺血(HI)，从而导致大多数幸存者严重的神经功能障碍。目前还没有可用于改善存活率或长期神经学结果的药物治疗。CA诱导的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NMDAR引起的谷氨酸兴奋性毒性，最终导致神经细胞死亡。虽然脑缺血后损伤的病理和级联事件是复杂的，但过度刺激NMDAR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的兴奋毒性刺激触发了一系列钙依赖的信号通路，包括激活钙/钙调蛋白依赖的蛋白激酶II(CaMKII)。我们最近的发现和初步数据表明，抑制CaMKII是将过度谷氨酸能刺激(兴奋毒性)的下游影响降至最低的一种新方法。CaMKII是生理谷氨酸信号转导的主要介质，参与突触可塑性，特别是CA1神经元的突触增强，可能与海马区的学习有关。已经描述了两种主要的CaMKII调节形式，刺激的和自主的CaMKII活性。钙/钙调素对CaMKII的刺激活性是由CaMKII诱导的，而T286残基的自动磷酸化和/或直接与突触上的NMDAR亚单位NR2B结合(这也介导了CaMKII在突触上的积聚)诱导了长时间的自主(非钙依赖)活性。此外，我们的初步研究揭示了CaMKII自主与一氧化氮(NO)之间的新联系，一氧化氮(NO)是在脑缺血后的兴奋性毒性过程中产生的，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的亚硝化/氧化直接促进CaMKII的自主活性，并通过保护T286免于去磷酸化。我们最近证明了我们的新的CaMKII抑制剂talCN21(它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(只阻断刺激的活动)不能提供侮辱后的神经保护。这表明，抑制自主的但不被刺激的CaMKII活性是侮辱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，在心脏骤停后使用talCN21可以显著地保护神经。]目前的提案将利用我们的 新的小鼠CA/CPR模型，利用几个缺乏每种形式自主CaMKII活性的突变小鼠品系来揭示这些形式的CaMKII活性与它们对缺血性神经细胞死亡的相对贡献之间的复杂相互作用。我们假设(I)每种自主机制都有助于神经细胞死亡，(Ii)T286-自动磷酸化介导的CaMKII自主是最直接的，(Iii)新的亚硝化机制通过延长T286磷酸化而促进缺血损伤，(Iv)NR2B结合通过将CaMKII定位在nNOS附近而使有效的亚硝化。

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.