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

摘要 卒中后的神经炎症是神经细胞死亡的重要原因。溴域和Extra 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： BRD2、BRD3、BRD4和BRDT。Brd2和BRD4含量丰富，广泛表达。BRD3表达式 在包括大脑在内的大多数组织中含量很低，而BRDT是睾丸特有的。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一个端外结构域。BET蛋白有多种不同 影响包括染色质重塑，组蛋白乙酰转移酶活性，以及作为支架招募转录 因子；它们将染色质重塑与转录结合在一起。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。Brd2通常抑制过氧化物酶增殖物 激活剂γ(PPARγ)活性，具有抗炎作用，因此我们预计抑制BRD2将 增加抗炎基因转录。Brd2基因敲除也会降低核因子-B(NF-B) 激活，这是卒中促炎基因转录的主要调节因素。BRD4作为核因子-B 因此，我们预测BRD2和BRD4的抑制会降低促炎基因 脑缺血区的转录。此外，由于BRD2和BRD4结构性地抑制核因子 (红系衍生的2)相关因子(Nrf2)是抗氧化基因转录所必需的，我们预计BRD2/4 抑制增加抗氧化基因的表达，减少氧化应激。人们对此知之甚少 BET蛋白在卒中中的作用，但我们的初步数据显示，抑制BET可减少啮齿动物的脑梗塞 中风的模型。我们的长期目标是通过限制继发性中风的影响来减少中风损害的蔓延 发炎。我们的假设是，抑制BET对缺血性中风的神经保护作用是通过限制 继发性炎症的有害影响。我们的主要目标是确定BET的机制 抑制对缺血性卒中有保护作用。在目标1中，我们将确定BET受体阻滞剂的神经保护作用 缺血后使用JQ1(BET抑制剂)和dBET1(一种降解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遭受缺血性中风的大鼠，并将研究 BET阻断对心肌梗死面积和长期行为结局的影响。在目标2中，我们将确定 BET阻滞剂对卒中所致神经炎症的影响在目标3中，我们将剖析细胞特定的角色 BRD4在卒中后神经炎性过程中的作用 特定细胞类型中的Cre重组酶。我们将研究BRD4在髓系细胞中的作用 (BRD4 FLOXED/FLOXx LysMCre/Cre)以及脑特异性内皮细胞(BRD4FLOXED/FLOXx Slco1c1-CreerT2) 对中风的伤害。这个项目将提供关于BET蛋白如何对次要代谢做出贡献的机械性见解。 缺血性卒中后的损伤。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 开发针对BET蛋白的新治疗策略以减少中风损害。

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.