The influence of ApoE4 on signaling & poor outcome after traumatic brain injury

这个职业发展奖的建议旨在定义潜在的机制，预测结果后， 创伤性脑损伤（TBI），同时为申请人提供培训机会， 弗吉尼亚TBI影响约15%的军人。退伍军人受到反复TBI是在增加 发展长期神经退行性疾病的风险。治疗选择有限，主要是因为 对TBI引起的神经功能缺损背后的病理学知之甚少。 载脂蛋白E（ApoE）有几个等位基因，表达E4等位基因的患者预后较差， 特别是在TBI后的长期内。尽管围绕ApoE 4等位基因进行了广泛的研究，但没有一项研究能够证明 研究主要的ApoE受体，低密度脂蛋白相关蛋白1（LRP 1）， TBI后的结果。然而，有证据表明LRP 1可以在调节TBI结果中发挥作用- - LRP 1从质膜和胞质溶胶中去除多种细胞蛋白质， 在决定质膜上蛋白质表达中的重要作用。 ApoE 4通过与LRP 1结合并经历受体介导的内吞作用进入细胞。相对于其他 ApoE等位基因（E2、E3）、ApoE 4内吞作用和受体再循环受损。在LRP 1的情况下， 再循环被认为极大地改变了LRP 1作为清除受体的能力，因此改变了环境。 蛋白质在质膜上的表达，从而改变细胞对损伤的反应。对于这一提议， 我专注于了解LRP 1在单个细胞类型星形胶质细胞中对TBI病理学的贡献。不 星形胶质细胞不仅是大脑中ApoE的主要生产者，它们还在决定ApoE的表达方面发挥着巨大的作用。 通过限制TBI后损伤的二次扩散来获得结果。我的工作假设是关键LRP 1 功能被星形胶质细胞中的ApoE 4结合破坏，从而调节正常的细胞反应， 使脑对创伤后炎症和细胞死亡敏感。我的研究策略将检验这个假设 同时提供明确的培训目标和职业机会，以培养独立性。 目的1检验星形胶质细胞LRP 1缺失影响TBI预后的假设。星形胶质细胞特异性LRP 1 已经产生了小鼠，小鼠TBI模型将测试急性和慢性结果。目的2 将检验ApoE 4对TBI结果的影响取决于LRP 1的假设。TBI样细胞培养模型 将机械地测试ApoE 4和LRP 1对细胞存活的影响。将生成一条新的鼠标线 通过将ApoE 4靶向替代小鼠与星形胶质细胞-LRP 1敲除模型杂交，然后观察 TBI后的结果将与目标1类似地进行测试。目的3检验ApoE 4通过以下方式改变结果的假设： 通过炎症性肿瘤坏死α受体1（TNFR 1）增加信号传导， 刺激细胞死亡。这一目标将使用细胞培养模型进行机械测试，以测试以下因素的影响： ApoE 4和LRP 1对TNFR 1质膜表达、再循环和TNFR 1介导的细胞死亡的影响 我的职业目标是成为VA系统内成功的独立调查员，最终获得 MERIT资助并为TBI退伍军人的更好结果做出贡献。我组建了一个团队 LRP 1生物学、神经科学，最重要的是TBI领域的专家。科学目标将 准备我的职业生涯研究TBI在小鼠模型的影响，而职业目标将提高 科学的批判性思维，并提供机会，网络和介绍我的结果。这些将是 通过咨询委员会一年两次的评价、出席国家会议、出席 期刊俱乐部，提高granitarian，并出版手稿产生的拟议工作。我的同事 导师还将提供与成功的独立研究实验室的正确方向有关的培训。总之， 我希望本提案中详述的活动将提供一个例外的环境， 独立的职业生涯。

This career development award proposal aims to define potential mechanisms that predict outcome after traumatic brain injury (TBI), while at the same time providing the applicant with training opportunities within the VA. TBI affects approximately 15% military population. Veterans were subjected to repeated TBI are at increased risk of developing long-term neurodegenerative disorders. Treatment options are limited, mainly because the pathology behind neurological deficits due to TBI is poorly understood. Apolipoprotein E (ApoE) has several alleles, and patients that express the E4 allele have worse outcomes, particularly in the long term after TBI. Despite a wide range of studies centered on the ApoE4 allele, none can be found that investigate the influence of a major ApoE receptor, low density lipoprotein related protein 1 (LRP1), on outcomes after TBI. Nevertheless, evidence suggests that LRP1 can play a role in modulating TBI outcome- -LRP1 removes a large variety of cellular proteins, both from the plasma membrane and in the cytosol, and plays an important role in determining protein expression at the plasma membrane. ApoE4 enters cells by binding to LRP1 and undergoing receptor mediated endocytosis. In contrast to other ApoE alleles (E2, E3), ApoE4 endocytosis and receptor recycling are impaired. In the case of LRP1, impaired recycling is postulated to greatly alter ability of LRP1 to act as a clearance receptor, therefore changing the milieu of proteins expressed on the plasma membrane and so altering cellular response to damage. For this proposal, I am focused on understanding the contribution of LRP1 to TBI pathology in a single cell type, astrocytes. Not only are astrocytes the primary producers of ApoE in the brain, they also play a tremendous role in determining outcome by limiting secondary spread of damage after TBI. My working hypothesis is that critical LRP1 function is disrupted by ApoE4 binding in astrocytes, thereby modulating normal cellular responses and sensitizing the brain to inflammation and cell death after TBI. My research strategy will test this hypothesis while also providing clear training objectives and career opportunities in order to develop independence. Aim 1 tests the hypothesis that loss of astrocyte LRP1 worsens outcome in TBI. Astrocyte-specific LRP1 mice have been generated, and mouse TBI models will test outcome in the acute and chronic outcome. Aim 2 will test the hypothesis that the impact of ApoE4 on TBI outcome depends on LRP1. TBI-like cell culture models will mechanistically test the influence of ApoE4 and LRP1 on cell survival. One new mouse line will be generated by crossing ApoE4 targeted replacement mice into the astrocyte-LRP1 knockout model, and then the effect on outcome after TBI will be tested similar to aim 1. Aim 3 tests the hypothesis that ApoE4 worsens outcome by increasing signaling through the inflammatory tumor necrosis alpha receptor 1 (TNFR1), elevating cytokine stimulated cell death. This aim will be tested mechanistically using cell culture models to test the influence of ApoE4 and LRP1 on TNFR1 plasma membrane expression, recycling, and TNFR1-mediated cell death. My career goal is to become a successful independent investigator within the VA system, eventually obtaining MERIT funding and contributing to better outcomes in Veterans with TBI. I have assembled a team that includes experts in the field of LRP1 biology, neuroscience, and most importantly, TBI. The scientific objectives will prepare me for a career studying the effects of TBI in mouse models, while the career objectives will improve scientific critical thinking and provide opportunities for networking and presentation of my results. These will be achieved via biannual evaluation by the advisory committee, attendance at national conferences, attending journal club, improving grantsmanship, and publishing manuscripts resulting from the proposed work. My co- mentors will also provide training related to proper direction of a successful independent research lab. Altogether, I expect that the activities detailed in this proposal will provide an exceptional environment from which to start an independent career.