Role of RIPK2 in the neuroinflammatory response to ischemic stroke

摘要 脑卒中后神经炎症显著促进神经元损伤和神经功能缺损。 缺血半暗带中的延迟性细胞死亡与胶质细胞活化、募集和浸润相关， 外周免疫细胞进入大脑这是由促炎细胞因子的产生引发的， 趋化因子，有助于中风后的细胞死亡和血脑屏障（BBB）通透性。死亡的细胞 半暗带还释放促炎信号和损伤相关分子模式（DAMP）， 将驻留的小胶质细胞活化为促炎表型，从而进一步导致脑损伤。我们 总体目标是通过限制神经炎症来减少中风损害的扩散。 受体相互作用丝氨酸/苏氨酸蛋白激酶2（RIPK 2）是通过其介导的炎症的关键介质。 多种促炎和细胞死亡途径的激活。RIPK 2激酶活性的抑制消除 其信号传导以减轻外周中的炎症状况。RIPK 2在缺血性卒中中的作用仍然存在 然而，我们的初步数据显示，梗死面积大幅减少，卒中后改善， 与野生型相比，Ripk 2敲除（Ripk 2-/-）小鼠的急性和长期功能结局 （Ripk 2 +/+）小鼠。我们认为，RIPK 2是一个重要的启动和传播的促炎途径， 缺血性中风我们的主要目的是减弱其活性，并评估RIPK 2在体内的特定作用，因为RIPK 2在体内的作用可能是一种免疫抑制剂。 与中风病理学有关。我们假设RIPK 2信号在缺血性卒中中是有害的， 在骨髓细胞中的降解/抑制或选择性消融将改善结果。 目的1将使用高选择性的神经保护剂来确定缺血后RIPK 2阻断的神经保护作用。 RIPK 2抑制剂和在体内特异性降解RIPK 2的蛋白水解靶向嵌合体（PROTAC）。我们将 利用经受缺血性中风的两种性别的老年小鼠，并研究RIPK 2阻断对 梗死面积和长期行为结果。在目标2中，我们将确定RIPK 2封锁对 中风引起的神经炎症和研究神经保护机制。在目标3中，我们将剖析 RIPK 2在中风后神经炎症过程中的细胞特异性作用，使用RIPK 2 floxed小鼠与 在特定细胞类型中产生Cre重组酶的细胞系。我们将研究RIPK 2在骨髓中的作用- 谱系细胞和脑驻留小胶质细胞对中风损伤的影响。该项目将利用我们的专业知识和独特的 工具（Ripk 2 floxed小鼠，PROTAC和选择性抑制剂），以了解RIPK 2驱动的机制 缺血性中风中的炎症。这项研究可能会将RIPK 2确定为一种新的治疗方法。 靶向阻断神经炎症并促进缺血性卒中后神经元存活。

Abstract Neuroinflammation after stroke significantly contributes to neuronal damage and neurological impairment. Delayed cell death in the ischemic penumbra is associated with glial activation and recruitment and infiltration of peripheral immune cells to the brain. This is triggered by the production of pro-inflammatory cytokines and chemokines, contributing to cell death and blood-brain barrier (BBB) permeability after stroke. Dying cells in the penumbra also release pro-inflammatory signals and damage-associated molecular patterns (DAMPs) that activate resident microglia toward a pro-inflammatory phenotype, thus further contributing to brain injury. Our overall goal is to reduce the spread of stroke damage by limiting neuroinflammation. Receptor interacting serine/threonine protein kinase 2 (RIPK2) is a critical mediator of inflammation via its activation of multiple pro-inflammatory and cell death pathways. Inhibition of RIPK2’s kinase activity abolishes its signaling to alleviate inflammatory conditions in the periphery. The role of RIPK2 in ischemic stroke remains unexplored; however, our pilot data shows a substantial reduction in infarct size and improvement in post-stroke functional outcomes, both acutely and long-term, in Ripk2 knockout (Ripk2-/-) mice compared to wild-type (Ripk2+/+) mice. We propose that RIPK2 is an essential initiator and propagator of pro-inflammatory pathways in ischemic stroke. Our main objective is to attenuate its activity and assess the specific role of RIPK2 in vivo as it relates to stroke pathology. We hypothesize that RIPK2 signaling is detrimental in ischemic stroke, and RIPK2 degradation/inhibition or selective ablation in myeloid cells will improve outcomes. Aim 1 will determine the neuroprotective effect of RIPK2 blockade after ischemia using a highly selective RIPK2 inhibitor and a proteolysis-targeting chimera (PROTAC) that specifically degrades RIPK2 in vivo. We will utilize aged mice of both sexes subjected to ischemic stroke and investigate the effects of RIPK2 blockade on infarct size and long-term behavioral outcomes. In Aim 2, we will determine the impact of RIPK2 blockade on stroke-induced neuroinflammation and investigate neuroprotection mechanisms. In Aim 3, we will dissect the cell-specific role of RIPK2 in the neuroinflammatory process after stroke by using Ripk2 floxed mice crossed with lines producing Cre recombinase in specific cell types. We will study the contribution of RIPK2 from myeloid- lineage cells and brain-resident microglia to stroke injury. This project will leverage our expertise and unique tools (Ripk2 floxed mice, PROTAC, and selective inhibitors) to understand the mechanisms of RIPK2-driven inflammation in the context of ischemic stroke. This research may lead to identifying RIPK2 as a new therapeutic target to block neuroinflammation and promote neuronal survival in the aftermath of an ischemic stroke.