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

项目概要/摘要 好消息是，越来越多的缺血性中风患者存活下来。不幸的是幸存者 很少毫发无损-中风是美国长期残疾的主要原因。 部分原因是缺乏对中风后次级过程的了解。神经干细胞（NSC）已经证明 在中风恢复中的治疗益处。但关键问题仍然存在，如果得到回答，可能会提高临床使用。 神经干细胞通常迁移到病变处，分泌促修复因子，分化，并减少继发性损伤。 这些益处的核心是NSC发送和响应特定环境线索的能力。我们的数据 显示NSC功能受低密度脂蛋白受体相关蛋白（LRP 1）影响，LRP 1是一种多功能 通过多种机制调节细胞信号的受体。尽管在信号调制中具有广泛的作用， LRP 1在NSC生物学中的重要性实际上未被研究。我们在成年神经干细胞中敲除LRP 1， 小鼠大脑中动脉闭塞。我们发现LRP 1-KO在NSC消融迁移到缺血性病变。 我们还发现，功能障碍和病变大小减少。这一观察有些 通常认为NSC对损伤的反常物理定位对神经保护功效是重要的。 我们还发现LRP 1-KO消除了CXCR 4的表达，CXCR 4是一种趋化因子受体， 迁移至缺血性病变。因此，我们试图更好地了解LRP 1在神经干细胞中的表达受损是如何影响神经干细胞的功能的。 神经保护，并类似地了解CXCR 4的LRP 1调节的机制。我们的整体 假设LRP 1通过CXCR 4调节促进NSC向缺血性病变迁移， 还限制了内源性NSC神经保护反应。我们的数据表明，LRP 1是一个主要的驱动程序， 国家安全委员会对信号的反应该提案使用外科手术研究LRP 1，CXCR 4和NSC在卒中中的功能。 模型、体内和体外方法。我们利用Nestin-CreERt 2小鼠模型直接敲除LRP 1， 和/或CXCR 4，并追踪这些细胞的红色荧光蛋白表达。目标1测试 假设神经干细胞介导的神经保护是由于LRP 1的丢失，这促进了恢复 通过测量脑卒中后CXCR 4的表达， 在NSC中具有LRP 1-KO的小鼠中，或在NSC中具有CXCR 4-KO的小鼠中消融LRP 1。目标2检验假设 LRP 1（或CXCR 4）的缺失通过增强卒中后NSC的神经保护作用而具有神经保护作用， 通过询问对NSC卒中后反应和存活的影响，在体内和体外观察NSC的反应。目标3 测试LRP 1通过用结构域特异性的 构建体并阐明对CXCR 4的转录、信号传导、运输和降解的影响。数据 从这些研究中，将确定NSC-LRP 1作为治疗靶点的价值，确定 LRP 1介导的神经保护，并确定LRP 1调节CXCR 4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.