Kinase regulation in cerebral ischemia

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Project Summary Cardiopulmonary arrest (CA) is a major cause of death/disability in the U.S. with poor prognosis and survival rates. The current CA therapeutic challenges are physiologically complex because they involved hypoperfusion [decreased cerebral blood flow, (CBF)], neuroinflammation, and mitochondrial dysfunction. Our long-term goal is to identify these complex regulatory elements that ultimately control neuronal viability. In our pilot study, we discovered that novel serum/glucocorticoid-regulated kinase 1 (SGK1) is highly expressed in brain NEURONS that are susceptible to ischemia (e.g., hippocampus and cortex). Inhibition of SGK1 via GSK 650394 (specific inhibitor) alleviated CA-induced hypoperfusion, neuroinflammation, mitochondrial deficits, neuronal cell death, and learning/memory deficits; this suggests SGK1 may play a detrimental role during ischemia. The primary goal of this proposal is to inhibit SGK1 and utilize pharmacological (specific SGK1 inhibitor) and cell type (neuron)-specific genetic approaches (e.g., shRNA) in our well-established rodent models of CA to answer the central hypothesis: SGK1 expression is enhanced after CA, which leads to hypoperfusion, neuroinflammation, mitochondrial dysfunctional, and neurological deficits. In Aim 1, the role of SGK1 in CA-induced hypoperfusion will be investigated. How SGK1 causes CA-induced hypoperfusion will be determined via two-photon microscopy and laser speckle contrast imaging (Aim 1a and 1c). Furthermore, we will identify potential vasoactive mediators that contribute to SGK1-mediated hypoperfusion using PCR, capillary-based immunoassay, and ELISA (Aim 1b). In Aim 2, we will determine if SGK is responsible for neuroinflammation and mitochondrial dysfunction after CA by exploring three objectives. First, how SGK1 affects microglia activation/polarization and astrogliosis following CA, which will be investigated via brain histology and flow cytometry (Aim 2a). Second, inhibition of SGK1 alleviated CA-induced neuroinflammation will be analyzed via protein chip assay (Aim 2b). Third, the harmful effects of SGK1 on mitochondrial ion homeostasis and energetics will be studied by Seahorse respirometry and microspectrofluorometry, respectively (Aim 2c and 2d). In Aim 3, we will evaluate the therapeutic potential of the SGK1 inhibitor against CA-induced neuronal cell death and neurological deficits. Utilizing brain histology (Cresyl violet and Fluoro-Jade C staining) (Aim 3a) and behavioral trials (Y-maze and novel object recognition test) (Aim 3b), the role of SGK1 in neurological deficits will be determined. Successful completion of the proposed study will reveal the fundamental roles of SGK1 in neuronal survival/death in cerebral ischemia-related diseases. Since the FDA has approved over 46 kinase-related drugs for the treatment of various diseases, our study will be promptly translated into human clinical trials for the patients suffering from CA.