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.
Heart attack and consequent cerebral ischemia is one of the leading causes of death and disability in the United States. Unfortunately, there are currently no drugs available to improve outcome and quality of life following severe heart attack. The proposed studies will demonstrate for the first time that autonomous CaMKII activity contributes to ischemia-induced neuronal cell death and suggest a novel interventional strategy to protect brain following heart attack.
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