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: 9922383
• 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/9922383
• 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/antagonist; insight; knock-down; male; nervous system disorder; neuroinflammation; neurological recovery; neuron loss; neuroprotection; novel therapeutics; 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.

摘要 中风后的神经炎症显著促进神经元细胞死亡。溴结构域和额外 末端结构域（BET）蛋白是炎症基因转录所必需的。有四种BET蛋白质： BRD 2、BRD 3、BRD 4和BRDT。BRD 2和BRD 4是丰富的并且普遍表达。BRD 3表达 在包括大脑在内的大多数组织中非常低，并且BRDT是睾丸特异性的。BET蛋白含有两个保守的 与乙酰化赖氨酸相关的溴结构域和末端外结构域。BET蛋白质已经改变 影响包括染色质重塑，组蛋白乙酰转移酶活性，并作为支架招募转录 因子;它们将染色质重塑与转录偶联。我们假设，BET封锁将提供一个 减少中风后细胞死亡的多管齐下的方法。BRD 2通常抑制过氧化物酶体增殖物 激活剂γ（PPARγ）活性，具有抗炎作用，因此我们预计抑制BRD 2将 增加抗炎基因转录。BRD 2敲低还降低核因子-κ B B（NF-κ B B） 激活，这是中风中促炎基因转录的主要调节因子。BRD 4充当NF-κ B B 因此，我们预测BRD 2和BRD 4抑制将减少促炎基因表达。 在缺血性脑中的转录。此外，由于BRD 2和BRD 4组成性抑制核因子， （红细胞衍生2）相关因子（Nrf 2）是抗氧化基因转录所必需的，我们预期BRD 2/4 抑制增加抗氧化基因的表达，减少氧化应激。关于这一点，我们知之甚少。 BET蛋白在中风中的作用，但我们的初步数据表明，BET抑制减少了啮齿动物的梗死 中风模型我们的长期目标是通过限制继发性损害的影响来减少中风损害的传播。 炎症我们的假设是，BET抑制剂通过限制脑缺血性卒中的神经保护作用， 继发性炎症的有害影响。我们的主要目标是确定BET 抑制对缺血性中风有保护作用。在目标1中，我们将确定BET阻断的神经保护作用。 在缺血后使用JQ 1（BET抑制剂）和dBET 1（降解BET的蛋白水解靶向嵌合体 蛋白质）。我们将利用雄性和雌性老年小鼠和大鼠缺血性中风，并将调查 BET阻断对梗死面积和长期行为结果的影响。在目标2中，我们将确定 BET阻断对中风诱导的神经炎症的影响。在目标3中，我们将剖析 通过使用BRD 4 floxed小鼠与产生BRD 4的品系杂交， 特定细胞类型中的Cre重组酶。我们将研究BRD 4在骨髓细胞中的作用， （BRD 4floxed/floxed x LysMCre/Cre）以及脑特异性内皮细胞（BRD 4floxed/floxed x Slco 1c 1-CreERT 2） 中风损伤。该项目将提供BET蛋白质如何有助于次级代谢的机制见解。 缺血性中风后的损伤。这些数据将产生积极的影响，因为它将为未来提供坚实的基础。 开发靶向BET蛋白以减少中风损伤的新治疗策略。