PROJECT SUMMARY Stroke is a leading cause of long-term disability in U.S. and worldwide. Post-stroke cognitive impairment (PSCI), a common sequela after stroke, is a decisive determinant of the quality of life for stroke survivors. Clinical studies have indicated that PSCI is common in both young and old stroke patients, even in cases of relative mild stroke and victims with successful thrombolysis and endovascular reperfusion therapies. However, the underlying mechanisms of PSCI remains poorly understood and no FDA approved treatment is available for PSCI. In this application, we propose to investigate the roles and therapeutic potential of DKK3 in PSCI and the underlying mechanisms using an experimental stroke model of transient middle cerebral artery occlusion (MCAO) followed by reperfusion. Previous studies (including ours) have demonstrated that mice subjected to transient MCAO developed long-term cognitive deficits that correlate with secondary damage to the hippocampus. Based on our promising pilot data, we hypothesize that DKK3 plays an important role not only in acute brain damage but also in secondary hippocampal damage and thereby represents a novel promising therapeutic target for treating cognitive impairment after ischemic stroke. First, we will determine the tempo-spatial regulation of DKK3 (and miR-125a) expression in the normal and ischemic brains at different time points after stroke, and evaluate the efficacy of early treatment versus delayed treatment by intranasal administration of recombinant DKK3 protein to ameliorate acute stroke injury and to improve long-term neurologic and cognitive outcomes after ischemic stroke (Aim1). Next, we will determine the mechanistic roles of DKK3 in neuropathology with the focus on the hippocampal mechanisms of PSCI after ischemic stroke (Aim 2). To test this hypothesis, the loss-of-function and gain- of-function experiments will be performed, in which conventional and conditional DKK3 knockout mice and functional reconstitution study with recombinant DKK3 via intranasal drug delivery will be utilized. Based on pilot data, we further hypothesize that ischemic stroke induces increase of miR-125a expression hence down-regulates DKK3 expression in the hippocampus, which contributes to PSCI induced by transient MCAO (Aim 3). We will determine the DKK3-dependent effects of miR-125a inhibition to improve PSCI after ischemic stroke. Both young adult and aged mice will be studied. The proposed studies may reveal previously unappreciated mechanisms underlying PSCI and provide a novel therapeutic approach to improve neurologic and cognitive outcomes following ischemic stroke.