Blood-brain barrier P-glycoprotein: a new target for Alzheimer's disease

描述（由申请人提供）：我们在阿尔茨海默病（AD）中降低p -糖蛋白（P-gp）表达和血脑屏障运输活性的机制方面缺乏基本的理解。缺乏这方面的知识是一个重要的临床问题，因为它阻碍了有效治疗的发展，以增强大脑中AB的清除，降低AB的大脑水平，从而防止AD患者的认知能力下降。研究者的长期目标是更好地了解神经退行性疾病中调节血脑屏障功能的分子机制，这一目标可能会导致治疗AD的新治疗策略。这一特殊应用的目的是确定AD中P-gp减少的机制，验证这一机制作为保护P-gp的靶标，并测试一种恢复P-gp的新治疗策略。实现这些目标有望降低AD患者的A¿脑水平并改善认知。基于初步数据，核心假设是A¿介导P-gp的蛋白酶体降解，阻断蛋白酶体降解可保护P-gp，通过PXR激活恢复P-gp水平可减轻A¿脑负荷并改善AD小鼠的认知能力。该研究的基本原理是确定减少脑毛细血管P-gp的机制，并保护和/或恢复P-gp以改善A¿脑清除，这将潜在地为降低AD患者A¿脑水平提供新的治疗靶点。为了实现这一应用的目标，我们将通过追求以下三个具体目标来验证我们的中心假设：1)确定A¿介导的血脑屏障P-gp减少的机制。2)在AD小鼠模型中验证泛素-蛋白酶体系统作为保护P-gp的靶标。3)开发一种治疗策略来减少AD小鼠模型的认知能力下降。在Aim 1中，我们将抑制泛素-蛋白酶体系统，以确定A¿介导的P-gp还原所涉及的步骤，并确定P-gp的表达、运输活性和泛素化。在Aim 2中，我们将用泛素-蛋白酶体系统抑制剂治疗hAPP小鼠，监测P-gp表达、运输活性和泛素化，并测量A¿脑水平。我们将通过脑灌注来评估P-gp在体内的活性，并进行摇尾试验来确定P-gp变化的后果。在Aim 3中，我们将对hAPP小鼠进行为期2年的ppn喂养研究，以评估pxr介导的P-gp恢复对AB脑水平的长期治疗效果。定期测定P-gp表达和转运活性、A -脑负荷和认知能力。这项拟议中的研究具有创新性，因为它专注于两种独立的策略，专门用于增强AD患者大脑对A¿的清除。这项提议的研究意义重大，因为它有望提供两种新的治疗策略，以降低大脑负担和减缓阿尔茨海默病的进展。拟议的研究是转译性的，因为这两种策略的药物，抑制泛素-蛋白酶体系统和PXR激活，目前都在市场上，这两种治疗策略都有可能转化为临床治疗AD患者。

DESCRIPTION (provided by applicant): We have a fundamental lack in understanding the mechanism that reduces P-glycoprotein (P-gp) expression and transport activity at the blood-brain barrier in Alzheimer's disease (AD). Lack of this knowledge is a significant clinical problem since it prevents development of an effective therapy to enhance AB clearance from the brain, lower AB brain levels, and thus prevent cognitive decline in AD. The long-term goal of the investigator is to better understand the molecular mechanisms that regulate blood-brain barrier function in neurodegenerative disorders, a goal which may lead to new therapeutic strategies to treat AD. The objectives of this particular application are to identify the mechanism responsible for P-gp reduction in AD, to validate this mechanism as a target to protect P-gp, and to test a novel therapeutic strategy for restoring P-gp. Accomplishing these objectives is expected to reduce A¿ brain levels and improve cognition in AD. Based on preliminary data, the central hypothesis is that A¿ mediates proteasomal degradation of P-gp, that blocking proteasomal degradation protects P-gp, and that restoring P-gp levels through PXR activation reduces A¿ brain burden and improves cognition in mice with AD. The rationale for the proposed research is that identifying the mechanism that reduces brain capillary P-gp and protecting and/or restoring P-gp to improve A¿ brain clearance will potentially provide novel therapeutic targets to lower A¿ brain levels in AD. To accomplish the objectives of this application, we will test our central hypothesis by pursuing the following three specific aims: 1) Identify the mechanism of A¿-mediated P-gp reduction at the blood-brain barrier. 2) Validate the ubiquitin-proteasome system as a target to protect P-gp in an AD mouse model. 3) Develop a therapeutic strategy to reduce cognitive decline in an AD mouse model. In Aim 1, we will inhibit the ubiquitin-proteasome system to identify the steps involved in A¿-mediated P-gp reduction, and determine expression, transport activity, and ubiquitination of P-gp. In Aim 2, we will treat hAPP mice with inhibitors of the ubiquitin-proteasome system, monitor P-gp expression, transport activity, and ubiquitination, and measure A¿ brain levels. We will conduct brain perfusion to assess P-gp activity in vivo and perform tail-flick assays to determine the consequence of changes in P-gp. In Aim 3, we will con- duct a 2-year PCN-feeding study with hAPP mice to assess the long-term therapeutic effect of PXR-mediated P-gp restoration on AB brain levels. P-gp expression and transport activity, A¿ brain load, and cognition will be periodically determined. The proposed research is innovative because it focuses on two independent strategies designed specifically to enhance A¿ clearance from the brain in AD. The proposed research is significant because it holds the promise of two new therapeutic strategies to lower A¿ brain burden and slow progression of AD. The proposed research is translational because drugs for either strategy, inhibition of the ubiquitin- proteasome system and PXR activation, are currently on the market, and both therapeutic strategies could potentially be translated into the clinic for the treatment of AD patients.