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