Kinase regulation in cerebral ischemia

脑缺血中的激酶调节

• 批准号: 10416366
• 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/10416366
• 关键词: Affect; Behavioral trial; Bioenergetics; Biological; Blood Circulation; Blood capillaries; Brain; Brain Injuries; Cardiopulmonary Arrest; Cause of Death; Cell Survival; Cells; Cerebral Ischemia; Cerebrovascular Circulation; Cessation of life; 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 (Brain); Histology; Homeostasis; Human; Immunoassay; Immunohistochemistry; Inflammation; Inflammatory; Injury; Ions; Ischemia; Kidney; Laser Scanning Microscopy; Laser Speckle Imaging; Lead; Learning; Life Style; Link; Liver; Measures; Mediating; Mediator of activation protein; Memory; Memory impairment; Messenger RNA; Microcirculation; Microglia; Mitochondria; Monitor; Nerve Degeneration; Neurologic Deficit; Neurons; Norepinephrine; Organ; Patients; Persons; Pharmaceutical Preparations; Pharmacology; Pharmacotherapy; Phosphotransferases; Physiological; Pilot Projects; Play; Prognosis; Protein Microchips; Protein-Serine-Threonine Kinases; Proteins; Rattus; Regulation; Regulatory Element; Reporting; Resuscitation; Rodent Model; Role; Secondary to; Sgk protein; Stains; Survival Rate; Sympathetic Nervous System; Techniques; Testing; Therapeutic; Translating; United States; Up-Regulation; Vasoconstrictor Agents; astrogliosis; base; 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; 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 在美国，心脏骤停（CA）是死亡/残疾的主要原因，预后和生存率较差 rates.目前CA治疗的挑战是生理复杂的，因为它们涉及低灌注 [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 就是识别这些复杂的调控元件，它们最终控制着神经元的活力。在试点研究中，我们 发现新型血清/糖皮质激素调节激酶1（SGK 1）在脑神经元中高度表达， 易受缺血影响的组织（例如，海马和皮质）。通过GSK 650394（特异性）抑制SGK 1 抑制剂）减轻CA诱导的低灌注、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SGK 1可能在缺血期间起有害作用。主 本提案的目的是抑制SGK 1并利用药理学（特异性SGK 1抑制剂）和细胞类型 （神经元）特异性遗传方法（例如，shRNA）在我们成熟的CA啮齿动物模型中的表达，以回答 中心假设：CA后SGK 1表达增强，导致低灌注， 神经炎症、线粒体功能障碍和神经缺陷。在目标1中，SGK 1在以下方面的作用： 将研究CA诱导的低灌注。将确定SGK 1如何引起CA诱导的低灌注 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，导致SGK 1介导的低灌注的潜在血管活性介质 免疫测定和ELISA（目的1b）。在目标2中，我们将确定SGK是否负责神经炎症， 线粒体功能障碍后CA探索三个目标。首先，SGK 1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动力学研究 流式细胞术（Aim 2a）。其次，将通过以下分析SGK 1减轻CA诱导的神经炎症的抑制： 蛋白芯片测定（Aim 2b）。第三，SGK 1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分光光度法进行研究（目标2c和2d）。在目标3中， 我们将评估SGK 1抑制剂对CA诱导的神经元细胞死亡的治疗潜力， 神经缺陷利用脑组织学（甲酚紫和Fluoro-Jade C染色）（Aim 3a）和行为学 试验（Y-迷宫和新物体识别测试）（目的3b），SGK 1在神经功能缺损中的作用将被 测定这项研究的成功完成将揭示SGK 1在神经元细胞中的基本作用。 脑缺血相关疾病的生存/死亡。自FDA批准超过46种激酶相关药物以来 对于治疗各种疾病，我们的研究将迅速转化为人类临床试验， 患有CA的患者。