Ischemic stroke remains a leading cause of death and serious long-term disability in the United States, with no effective therapy directly targeting neurons to increase post-ischemic neuronal viability and function. Brain calcium (Ca2+)-calmodulin (CaM)-dependent protein kinase II alpha (CaMKII?) is an essential neuronal kinase whose activity at the post-synaptic density (PSD) maintains neuronal health by regulating synaptic transmission and plasticity. CaMKII substrates involved in these processes at the PSD are N-methyl-D- aspartate receptor (NMDAR) and neuronal nitric oxide synthase (nNOS). Cerebral ischemia leads to sustained inactivation of CaMKII that critically affects post-ischemic neuronal death. Brain regions with the greatest suppression of CaMKII activity after ischemia are most susceptible to neuronal death, loss of CaMKII activity parallels post-ischemic neuronal damage, and CaMKII? deletion results in increased ischemic brain injury. Taken together, these findings support the view that CaMKII inactivation is involved in the molecular pathology of ischemic damage. However, the mechanisms that influence post-ischemic CaMKII activity are poorly understood, which restricts the exploration of its potential value as a therapeutic target. Post-translational modifications with ubiquitin have emerged as key regulator of protein stability and function. Our pilot studies suggest that cerebral ischemia induces CaMKII? subunit ubiquitination at the PSD, which results in deactivation of the enzyme. Given that CaMKII inactivation is involved in post-ischemic neuronal damage, we propose here to test the central hypothesis that ubiquitination of PSD-associated CaMKII? leads to post- ischemic CaMKII inactivation and contributes to ischemic brain injury. This hypothesis will be tested using in vitro (transient oxygen-glucose deprivation in mouse hippocampal slices; tOGD) and in vivo (transient middle cerebral artery occlusion in mice; tMCAO) approaches.
Aim 1 will address whether post-ischemic CaMKII? ubiquitination is responsible for its inactivation at the PSD, a site where CaMKII exerts pivotal functions to maintain neuronal health.
Aim 2 will elucidate the molecular mechanisms by which CaMKII inactivation by ubiquitination is achieved. In particular we will test whether post-ischemic CaMKII inactivation is the result of a reversible ubiquitin-dependent inhibition of CaM, ATP and substrate binding, major determinants of CaMKII activity. NMDAR and nNOS mediate post-ischemic neuronal death by producing toxic levels of reactive oxygen species (ROS) and nitric oxide (NO), and are key CaMKII phosphorylation targets at the PSD.
Aim 3 will determine whether CaMKII inactivation by ubiquitin leads to dysregulation of NMDAR and nNOS, which contributes to post-ischemic neuronal dysfunction and death. The proposed studies seek to identify the molecular mechanism responsible for post-ischemic CaMKII inactivation, and may provide new targets that may be harnessed for stroke therapy.
Cerebral ischemic stroke is a highly prevalent disease with no therapeutic options that directly target neurons to increase post-ischemic neuronal viability and function. Inactivation of the neuronal enzyme calcium- calmodulin-dependent kinase II (CaMKII) is implicated in neuronal death after cerebral ischemia, however it remains unknown what causes this dysfunction. The present application introduces ubiquitination, a post- translational protein modification, as reversible repressor of post-ischemic CaMKII activity, and, as such, may provide a new target that may be harnessed for ischemic stroke therapy.
