Summary. We have shown that a pharmacological FAK inhibition and astrocyte FAK deletion are cerebroprotective after ischemic MCAO stroke in female, but not male, mice. A systemic small molecule FAK inhibitor treatment was efficacious when started 6 h after reperfusion following an MCAO, as shown by motor function and histological analyses of the injury site. We propose the FAK inhibitor or one in clinical trials for cancer as a candidate for preclinical assessment by the SPAN test sites, including replication in young adult mice, in aged mice, in rats and animals with comorbidities. This finding is based on work over more than ten years in our lab after we discovered a new integrin-FAK-STAT3 signaling pathway that regulates cytokine expression. In female mice, plasma vitronectin (VTN) that leaks into the brain injury activates integrins to exacerbate the progressive injury over the first two days which is mediated in large part by acute pro- inflammatory IL-6 expression in the brain. Stroke induced plasma VTN levels in females only and the levels correlate with worse tissue injury. The VTN-induced IL-6 mechanism was not affected by ovariectomy suggesting that sex hormones are not involved. Using cre-lox mice, we identified astroglial FAK as the major driver of the detrimental IL-6 peak in female, but not male, mice. Moreover, FAK14 was cerebroprotective in WT females but not in VTN-/- female littermates or in males. Thus, we have identified a pleiotropic mechanism that can be inhibited by a drug downstream of a detrimental blood protein and irrespective of its levels. In Aim 1, the Hagg lab will determine a dose-response curve in mice for the two FAK inhibitors to define the lowest dose that has maximal efficacy, and their potency. Outcome measures will be FAK phosphorylation and cytokine expression in brain tissue at 24 h after intraluminal filament MCAO with reperfusion and for reducing functional deficits and brain injury size at 7 d. The test sites would receive the most promising of the two inhibitors after quality control for the compounds we receive from suppliers. For Aim 2a, the test sites would replicate our finding that the FAK inhibitor is cerebroprotective after MCAO in young adult C57BL/6J females and not males. Outcome measures are injury size, as measured by repeated MRI, and sensorimotor function tests over 30 days, as defined by the current SPAN. Aim 2b would test it in aged mice and Aim 2c in young adult rats. Depending on the outcome, mouse or rat models of the most common risk factor comorbidities of human stroke, hypertension and diabetes, will be tested in Aim 2d. To broaden the potential clinical impact, Aim 2e will test the FAK inhibitor in a clot-tPA reperfusion model. In Aim 3, our lab will determine whether high VTN levels caused by comorbidities are a risk factor for worse stroke outcomes by analyzing plasma and brains from test site rodents. We also expect the risk to be reduced by the FAK inhibitor. Key milestones will be the selection of the FAK inhibitor and its dosing, replication and weighing its potential clinical promise in light of showing cerebroprotective effects in different models tested by the testing sites. If successful, we will contribute a new intervention which targets a specific mechanism and is well-tolerated, for clinical stroke trials.

Project Narrative This grant will preclinically assess, within a well-organized NINDS-supported network of leading stroke labs, whether FAK inhibitors can reduce neurological deficits and brain injury, measured by repeated MRI, after ischemic stroke. The lead inhibitor will be tested in young and aged female and male mice, in rats, and in animals mimicking human stroke comorbidities of hypertension and diabetes. We expect to identify a new treatment to maximize tissue protection and function as adjuvants of standard-of-care reperfusion treatments for subsequent testing in clinical stroke trials.