Multi-pronged Approach to Recalibrating the Inflammatory Cascade in Ischemic Stroke with BET blockade

通过 BET 阻断重新校准缺血性中风炎症级联的多管齐下方法

• 批准号: 10456172
• 负责人: Eduardo Jesus Candelario-Jalil
• 金额: $ 38.52万
• 依托单位: UNIVERSITY OF FLORIDA
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-07-01 至 2024-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10456172
• 关键词: Acute; Address; Adult; Alteplase; Animal Model; Anti-Inflammatory Agents; Antiinflammatory Effect; Antioxidants; BRD2 gene; Basic Science; Behavior assessment; Binding; Blood - brain barrier anatomy; Brain; Brain Injuries; Bromodomain; Cause of Death; Cell Death; Cell Lineage; Cells; Cerebrovascular system; Cognitive; Data; Development; Dose; Endothelial Cells; Endothelium; Enterobacteria phage P1 Cre recombinase; Erythroid; Extravasation; FDA approved; Female; Flow Cytometry; Foundations; Future; Genes; Genetic Transcription; Goals; Hour; Immune; Infarction; Infiltration; Inflammation; Inflammatory; Injury; Ischemia; Ischemic Stroke; Knowledge; LoxP-flanked allele; Lysine; Magnetic Resonance Imaging; Measures; Mediator of activation protein; Microglia; Mission; Mus; Myelogenous; Myeloid Cells; National Institute of Neurological Disorders and Stroke; Neurologic Deficit; Nuclear; Nutrient; Oxidative Stress; Oxygen; Pathway interactions; Patients; Peroxisome Proliferators; Pharmaceutical Preparations; Plasma Proteins; Population; Process; Protac; Public Health; Rattus; Research; Rodent Model; Role; Secondary to; Signal Transduction; Stroke; Tertiary Protein Structure; Testing; Testis; Therapeutic; Time; Tissues; Treatment Protocols; Tumor-infiltrating immune cells; Work; aged; base; behavioral outcome; blood-brain barrier permeabilization; brain endothelial cell; cell type; chromatin remodeling; clinically relevant; disability; expectation; histone acetyltransferase; improved; inhibitor; insight; knock-down; male; nervous system disorder; neuroinflammation; neurological recovery; neuron loss; neuroprotection; novel therapeutic intervention; post stroke; pre-clinical; recruit; response; scaffold; sex; stroke model; stroke outcome; transcription factor

Abstract Neuroinflammation after stroke significantly contributes to neuronal cell death. Bromodomain and Extra Terminal Domain (BET) proteins are essential to inflammatory gene transcription. There are four BET proteins: BRD2, BRD3, BRD4, and BRDT. BRD2 and BRD4 are abundant and ubiquitously expressed. BRD3 expression is very low in most tissues including the brain, and BRDT is testis specific. BET proteins contain two conserved bromodomains that associate with acetylated lysines, and an extraterminal domain. BET proteins have varied effects including chromatin remodeling, histone acetyltransferase activity, and as scaffolds to recruit transcription factors; they couple chromatin remodeling with transcription. We hypothesize that BET blockade will provide a multipronged approach to reducing cell death after stroke. BRD2 normally represses peroxisome proliferator activator γ (PPARγ) activity, which has an anti-inflammatory effect, so we expect that inhibiting BRD2 will increase anti-inflammatory gene transcription. BRD2 knockdown also decreases nuclear factor-B (NF-B) activation, which is a major regulator of pro-inflammatory gene transcription in stroke. BRD4 acts as an NF-B co-activator, therefore we predict that BRD2 and BRD4 inhibition will decrease pro-inflammatory gene transcription in the ischemic brain. Furthermore, because BRD2 and BRD4 constitutively inhibit nuclear factor (erythroid-derived 2)-related factor (Nrf2) which is essential to antioxidant gene transcription, we expect BRD2/4 inhibition to increase expression of antioxidant genes, reducing oxidative stress. Little is known regarding the role of BET proteins in stroke, but our preliminary data shows that BET inhibition reduces infarct in a rodent model of stroke. Our long-term goal is to reduce the spread of stroke damage by limiting the effects of secondary inflammation. Our hypothesis is that BET inhibition is neuroprotective in ischemic stroke by limiting the deleterious effects of secondary inflammation. Our main objective is to determine the mechanism by which BET inhibition is protective in ischemic stroke. In Aim 1, we will determine the neuroprotective effect of BET blockade after ischemia using JQ1 (BET inhibitor) and dBET1 (a proteolysis-targeting chimera that degrades BET proteins). We will utilize male and female aged mice and rats subjected to ischemic stroke and will investigate the effects of BET blockade on infarct size and long-term behavioral outcomes. In Aim 2, we will determine the effects of BET blockade on stroke-induced neuroinflammation. In Aim 3, we will dissect the cell-specific role of BRD4 in the neuroinflammatory process after stroke by using BRD4 floxed mice crossed with lines producing Cre recombinase in specific cell types. We will study the contribution of BRD4 from myeloid-lineage cells (BRD4floxed/floxed x LysMCre/Cre) as well as from brain-specific endothelial cells (BRD4floxed/floxed x Slco1c1-CreERT2) to stroke injury. This project will provide mechanistic insights into how BET proteins contribute to secondary injury after ischemic stroke. These data will yield a positive impact as it will provide a strong foundation for future development of novel therapeutic strategies targeting BET proteins to reduce stroke damage.

抽象的 中风后的神经炎症显着导致神经元细胞死亡。溴化域和额外 末端结构域（BET）蛋白对于炎症基因转录至关重要。有四个BET蛋白： BRD2，BRD3，BRD4和BRDT。 BRD2和BRD4丰富且普遍表达。 BRD3表达 在包括大脑在内的大多数组织中，非常低，而BRDT是特定于睾丸的。 BET蛋白包含两个保守 与乙酰化的歌词和外交结构域相关的溴化群。 BET蛋白质有所不同 影响包括染色质重塑，组蛋白乙酰转移酶活性以及作为招募转录的脚手架的影响 因素；他们将染色质重塑与转录相结合。我们假设BET封锁将提供 多收益减少中风后细胞死亡的方法。 BRD2通常反映过氧化物组增殖物 激活剂γ（PPARγ）活性，具有抗炎作用，因此我们期望抑制BRD2会 增加抗炎基因转录。 BRD2敲低还降低了核因子-B（NF-B） 激活是中风中促炎基因转录的主要调节剂。 BRD4充当NF-B 因此，我们预测BRD2和BRD4抑制作用将降低促炎基因 缺血性大脑中的转录。此外，由于BRD2和BRD4组成型抑制核因子 （红细胞来源2）与抗氧化基因转录至关重要的相关因子（NRF2），我们期望BRD2/4 抑制增加抗氧化基因表达，减少氧化应激。关于 BET蛋白在中风中的作用，但是我们的初步数据表明，BET抑制会减少啮齿动物的梗塞 我们的长期目标是通过限制次级的影响来减少中风损伤的传播 炎。我们的假设是，BET抑制是通过限制的缺血性中风的神经保护作用 继发性炎症的有害作用。我们的主要目标是确定赌注的机制 在AIM 1中，我们将确定BET封锁的神经保护作用 缺血后使用JQ1（BET抑制剂）和DBET1（触发蛋白水解的嵌合体降解BET 蛋白质）。我们将利用雄性和雌性老年小鼠和遭受缺血性中风的大鼠，并将调查 BET封锁对梗塞大小和长期行为结果的影响。在AIM 2中，我们将确定 BET封锁对中风引起的神经炎症的影响。在AIM 3中，我们将剖析细胞特定的作用 中风后神经炎症过程中的BRD4使用BRD4 Floxed小鼠与生产线交叉 特定细胞类型中的CRE重组酶。我们将研究髓样细胞中BRD4的贡献 （Brd4floxed/Floxed X Lysmcre/Cre）以及脑特异性内皮细胞（BRD4Flox/Floxed X Slco1c1-Creert2） 中风受伤。该项目将提供有关BET蛋白如何促进次级的机械见解 缺血性中风后受伤。这些数据将产生积极的影响，因为它将为未来提供良好的基础 靶向BET蛋白的新型热策略的发展以减少中风损伤。