Project Summary/Abstract Focal cerebral ischemia (stroke) afflicts nearly 800,000 Americans each year and often results in permanent cognitive impairment or death. Efforts in developing a cerebroprotective stroke therapy have largely resulted in disappointment: the only approved pharmacological therapy is hemolytic treatment with tissue plasminogen activator (tPA; alteplase). In this project, we will further develop our optimized CaMKII inhibitor peptide tatCN19o as a cerebroprotective stroke treatment through comparative testing within the NINDS Stroke Preclinical Assessment Network (SPAN). The 30 amino-acid peptide is selective, stable, potent, water soluble, and has excellent chemistry, manufacturing, and control (CMC) properties. Importantly, tatCN19o was highly effective in vivo in global cerebral ischemia (GCI) models in both mouse and pig (the latter unpublished), even at extremely low doses of 0.01-0.02 mg/kg i.v.. The parent compound was also effective in vivo in a mouse model of acute ischemic stroke (transient middle cerebral artery occlusion; tMCAo). Neuroprotection was seen even at the latest time points tested so far after the various ischemic/excitotoxic insults (0.5h after global cerebral ischemia; 1h after stroke model; 6h in neuronal cultures). Here, SPAN will provide rigorous, unbiased testing of tatCN19o in rodent tMCAo models for direct comparison to other candidate interventions. Several tMCAo conditions are proposed for consideration by the network: compatibility with hemolytic tPA treatment (the current standard of care) and efficacy after varied insult duration. To most appropriately represent clinical populations, we propose parallel testing in adult and older animals of both sexes.