LRP1 as a novel regulator of CXCR4 in adult neural stem cells and post-stroke response

项目摘要/摘要 好消息是，经历缺血性中风的患者越来越多地存活下来。不幸的是，幸存者 中风是美国长期残疾的主要原因。干预措施仅限于 部分原因是对中风后的继发过程缺乏洞察力。神经干细胞已经证明 对中风康复的治疗益处。但关键问题仍然存在，如果得到解决，可能会加强临床应用。 神经干细胞通常迁移到病变处，分泌修复因子，分化，减少继发性损害。 这些好处的核心是NSC发送和响应特定环境线索的能力。我们的数据 显示神经干细胞功能受低密度脂蛋白受体相关蛋白(LRP1)的影响，LRP1是一种多功能蛋白 通过多种机制调节细胞信号的受体。尽管在信号调制方面发挥了广泛的作用， LRP1在神经干细胞生物学中的重要性几乎没有被研究过。我们在成年NSCs中敲除了LRP1，并对 小鼠至大脑中动脉闭塞。我们发现LRP1-KO可促进神经干细胞向缺血灶的迁移。 我们还发现，功能损害和病变大小都有所减少。这一观察结果有点 神经干细胞对损伤的矛盾物理定位通常被认为是神经保护效果的重要因素。 我们还发现，LRP1-KO抑制了趋化因子受体CXCR4的表达，CXCR4是一种趋化因子受体，在 转移到缺血灶。因此，我们试图更好地了解神经干细胞中LRP1的表达是如何受损的 神经保护，以及类似地理解LRP1调控CXCR4的机制。我们的整体 假设LRP1通过调节CXCR4促进NSC向缺血损伤迁移， 但也限制了内源性神经干细胞的神经保护反应。我们的数据表明，LRP1是一个主要的驱动因素 NSC对信号的反应。本研究通过外科手术研究卒中患者LRP1、CXCR4和NSC的功能 模型、体内和体外方法。我们利用Nestin-CreERt2小鼠模型直接敲除LRP1 和/或CXCR4在神经干细胞中的表达，并用红色荧光蛋白的表达来追踪这些细胞。AIM 1测试 假设NSC介导的神经保护是由于LRP1的丢失，LRP1促进了恢复 通过挽救CXCR4的表达来评估卒中后预后独立于CXCR4的影响 在NSCs中用LRP1-KO或在NSCs中CXCR4-KO消融LRP1。《目标2》检验了这一假设 LRP1(或CXCR4)的丢失通过增强中风后NSC的神经保护作用而起到神经保护作用 通过询问在体内和体外对NSC卒中后反应和存活率的影响来作出反应。目标3 检测LRP1通过区域特异性拯救LRP1来调节CXCR4表达的机制 构建并阐明CXCR4对转录、信号、运输和降解的影响。数据 这些研究将确定NSC-LRP1作为治疗靶点的价值，确定不同的机制 LRP1介导的神经保护，并确定LRP1调节CXCR4表达的机制。

Project Summary/Abstract The good news is that increasingly, patients who experience an ischemic stroke survive. Unfortunately, survivors are rarely unscathed — stroke is the leading cause of long-term disability in the U.S. Interventions are limited in part due to a lack of insight into secondary processes post-stroke. Neural stem cells (NSCs) have demonstrated therapeutic benefit in stroke recovery. But key questions remain that if answered, could enhance clinical use. NSCs normally migrate to the lesion, secrete pro-reparative factors, differentiate, and reduce secondary damage. Central to these benefits is the ability of NSCs to send and respond to specific environmental cues. Our data show NSC function is influenced by low-density lipoprotein receptor-related protein (LRP1), a multifunctional receptor that modulates cell signaling via multiple mechanisms. Despite an expansive role in signal modulation, the importance of LRP1 in NSC biology is virtually unstudied. We knocked-out LRP1 in adult NSCs and subjected mice to middle-cerebral artery occlusion. We found LRP1-KO in NSCs ablated migration to ischemic lesions. We also discovered that functional impairment and lesion size were reduced. This observation is somewhat paradoxical-physical localization of NSCs to lesions is often considered important to neuroprotective efficacy. We also found that LRP1-KO ablates expression of CXCR4, a chemokine receptor which is essential for migration to ischemic lesions. Thus, we seek to better understand how impaired LRP1 expression in NSCs is neuroprotective, and similarly to understand the mechanisms underlying LRP1 regulation of CXCR4. Our overall hypothesis is that LRP1 promotes NSC migration toward ischemic lesions through CXCR4 regulation, yet also limits the endogenous NSC-neuroprotective response. Our data suggest that LRP1 is a major driver of NSC response to signals. This proposal investigates LRP1, CXCR4, and NSC function in stroke using surgical models, in vivo, and in vitro approaches. We utilize a Nestin-CreERt2 mouse model to direct knock-out of LRP1 and/or CXCR4 in NSCs and to track these cells with expression of red fluorescent protein. Aim 1 tests the hypothesis that NSC-mediated neuroprotection is due to loss of LRP1, which promotes recovery independent of effects on CXCR4 by measuring post-stroke outcomes after rescuing expression of CXCR4 in mice with LRP1-KO in NSCs, or ablating LRP1 in mice with CXCR4-KO in NSCs. Aim 2 tests the hypothesis that loss of LRP1 (or CXCR4) is neuroprotective by enhancing post-stroke NSC neuroprotective response by interrogating the effect on NSC post-stroke response and survival, both in vivo and in vitro. Aim 3 tests the mechanism by which LRP1 regulates CXCR4 expression by rescuing LRP1 with domain-specific constructs and elucidating the effect on transcription, signaling, trafficking, and degradation of CXCR4. Data from these studies will define the value of NSC-LRP1 as a therapeutic target, identify discrete mechanisms of LRP1-mediated neuroprotection, and define the mechanism by which LRP1 regulates CXCR4 expression.