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.

项目摘要 心肺逮捕（CA）是美国死亡/残疾的主要原因，预后不良和生存 费率。当前的CA治疗挑战在生理上是复杂的，因为它们涉及下灌注不足 [减少脑血流，（CBF）]，神经炎症和线粒体功能障碍。我们的长期目标 是确定最终控制神经元活力的这些复杂的调节元件。在我们的试点研究中，我们 发现新型血清/糖皮质激素调节的激酶1（SGK1）在脑神经元中高度表达 容易患缺血（例如海马和皮质）。通过GSK 650394抑制SGK1（特定 抑制剂）减轻了CA诱导的缺血，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在缺血期间可能起不利的作用。主要 该建议的目标是抑制SGK1并利用药理（特定SGK1抑制剂）和细胞类型 （神经元） - 在我们公认的CA的啮齿动物模型中，特定的遗传方法（例如，shRNA）回答 中央假设：CA后SGK1表达增强，这导致灌注不足， 神经炎症，线粒体功能障碍和神经功能缺陷。在AIM 1中，SGK1在 将研究CA诱导的灌注不足。如何确定SGK1引起CA诱导的灌注不足 通过两光子显微镜和激光斑点对比度成像（AIM 1A和1C）。此外，我们将确定 使用PCR，基于毛细管的潜在血管活性介质有助于SGK1介导的灌注不足 免疫测定和ELISA（AIM 1B）。在AIM 2中，我们将确定SGK是否负责神经炎症和 CA之后的线粒体功能障碍通过探索三个目标。首先，SGK1如何影响小胶质细胞 CA之后的激活/极化和星形胶质症，将通过大脑组织学和流动进行研究 细胞仪（AIM 2A）。其次，将通过通过抑制SGK1缓解CA诱导的神经炎症。 蛋白质芯片分析（AIM 2B）。第三，SGK1对线粒体离子稳态和能量学的有害影响 海马呼吸测定法和微光谱法（AIM 2C和2D）将研究。在AIM 3中， 我们将评估SGK1抑制剂针对CA诱导的神经元细胞死亡和 神经缺陷。利用脑组织学（甲甲基紫色和氟-jade C染色）（AIM 3A）和行为 试验（Y迷宫和新颖的对象识别测试）（AIM 3B），SGK1在神经系统缺陷中的作用将是 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 脑缺血相关疾病中的生存/死亡。由于FDA已批准超过46种激酶相关的药物 为了治疗各种疾病，我们的研究将立即转化为人类的临床试验 患有大约的患者