Kinase regulation in cerebral ischemia

脑缺血中的激酶调节

• 批准号: 10589927
• 负责人: Reggie Hui-Chao Lee
• 金额: $ 36.75万
• 依托单位: LOUISIANA STATE UNIV HSC SHREVEPORT
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-03-15 至 2027-02-28
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10589927
• 关键词: Affect; Behavioral trial; Bioenergetics; Biological; Blood capillaries; Brain; Brain Injuries; Cardiopulmonary Arrest; Cause of Death; Cell Survival; Cerebral Ischemia; Cerebrovascular Circulation; Cessation of life; Circulation; Clinical Trials; Complex; Data; Disease; Emergency treatment; Enzyme-Linked Immunosorbent Assay; Enzymes; Eukaryotic Cell; Family; Flow Cytometry; Foundations; Genes; Genus Hippocampus; Goals; Heart; Heart Arrest; Hippocampus; Histology; Homeostasis; Human; Immunoassay; Immunohistochemistry; Inflammation; Inflammatory; Injury; Ions; Ischemia; Ischemic Brain Injury; Kidney; Laser Scanning Microscopy; Laser Speckle Imaging; Learning; Life Style; Link; Liver; Measures; Mediating; Mediator; Memory; Memory impairment; Messenger RNA; Microcirculation; Microglia; Mitochondria; Monitor; Nerve Degeneration; Neurologic Deficit; Neurons; Norepinephrine; Organ; Pathology; Patients; Persons; Pharmaceutical Preparations; Pharmacotherapy; Phosphotransferases; Physiological; Pilot Projects; Play; Predisposition; Prognosis; Protein Microchips; Protein-Serine-Threonine Kinases; Proteins; Rattus; Regulation; Regulatory Element; Reporting; Resuscitation; Rodent Model; Role; Secondary to; Sgk protein; Spirometry; Stains; Survival Rate; Sympathetic Nervous System; Techniques; Testing; Therapeutic; Translating; United States; Up-Regulation; Vasoconstrictor Agents; astrogliosis; cell type; chromatin immunoprecipitation; cresyl violet; cytokine; disability; fluoro jade; genetic approach; hypoperfusion; inhibitor; member; mitochondrial dysfunction; mouse model; neoplastic; neuroinflammation; neuron loss; neuronal survival; neuropeptide Y; novel; novel therapeutic intervention; object recognition; overexpression; pharmacologic; small hairpin RNA; therapeutic target; two photon microscopy; two-photon

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.

项目概要 心肺骤停 (CA) 是美国死亡/残疾的主要原因，预后和生存率较差 费率。当前的 CA 治疗挑战在生理上很复杂，因为它们涉及灌注不足 [脑血流量减少（CBF）]、神经炎症和线粒体功能障碍。我们的长期目标 的目的是识别这些最终控制神经元活力的复杂调节元件。在我们的试点研究中，我们 发现新型血清/糖皮质激素调节激酶 1 (SGK1) 在大脑神经元中高表达 容易缺血的部位（例如海马体和皮质）。通过 GSK 650394 抑制 SGK1（特定 抑制剂）减轻CA引起的低灌注、神经炎症、线粒体缺陷、神经元细胞死亡， 和学习/记忆缺陷；这表明 SGK1 可能在缺血期间发挥有害作用。初级 该提案的目标是抑制 SGK1 并利用药理学（特定 SGK1 抑制剂）和细胞类型 在我们完善的 CA 啮齿动物模型中使用（神经元）特异性遗传方法（例如 shRNA）来回答 中心假设：CA后SGK1表达增强，导致灌注不足， 神经炎症、线粒体功能障碍和神经功能缺陷。在目标 1 中，SGK1 的作用 将研究 CA 引起的低灌注。 SGK1 如何导致 CA 诱导的低灌注将被确定 通过双光子显微镜和激光散斑对比成像（目标 1a 和 1c）。此外，我们将确定 使用 PCR、基于毛细血管的方法研究导致 SGK1 介导的低灌注的潜在血管活性介质 免疫测定和 ELISA（目标 1b）。在目标 2 中，我们将确定 SGK 是否负责神经炎症以及 通过探索三个目标来治疗 CA 后的线粒体功能障碍。一、SGK1如何影响小胶质细胞 CA 后的激活/极化和星形胶质细胞增生，将通过脑组织学和血流进行研究 细胞计数（目标 2a）。其次，抑制 SGK1 可减轻 CA 诱导的神经炎症 蛋白质芯片测定（目标 2b）。三、SGK1对线粒体离子稳态和能量学的有害影响 将分别通过 Seahorse 呼吸测定法和显微荧光光谱法进行研究（目标 2c 和 2d）。在目标 3 中， 我们将评估 SGK1 抑制剂对 CA 诱导的神经元细胞死亡的治疗潜力， 神经功能缺陷。利用脑组织学（甲酚紫和 Fluoro-Jade C 染色）（目标 3a）和行为学 试验（Y 迷宫和新物体识别测试）（目标 3b），SGK1 在神经缺陷中的作用将是 决定。成功完成拟议的研究将揭示 SGK1 在神经元中的基本作用 脑缺血相关疾病的生存/死亡。自 FDA 已批准超过 46 种激酶相关药物以来 针对各种疾病的治疗，我们的研究将及时转化为人体临床试验 患有CA的患者。