Investigating Oatp-Mediated Delivery of Statins to the Brain in Males and Females: Relevance to Neuroprotective Treatment for Ischemic Stroke

项目总结 在美国，中风是第五大死因。已批准的中风疗法受限于狭义 治疗窗口期、出血性转化的风险和再灌注损伤。因此，有一个关键的 需要能够改善中风后神经功能的神经保护性药物。目前，3-羟基-3- 甲基戊二酰辅酶A(HMG CoA)还原酶抑制剂(即他汀类药物)可用于中风患者 已被证明在改善认知和运动结果方面的效用。我们实验室的研究发现了一种特定的 使他汀类药物成为治疗中风有效药物的生物学机制：通过血液转运- 脑屏障(BBB)通过内源性摄取转运体有机阴离子转运多肽1A4(Oatp1a4)。 我们第一次证明，阿托伐他汀需要Oatp介导的转运来减少大脑 短暂性大脑中动脉损伤后24小时脑梗塞体积和感觉运动功能的改善 闭塞(TMCAO)。我们还观察到tMCAO AS雌性大鼠对阿托伐他汀摄取的增加。 与年龄匹配的男性同龄人相比；然而，目前尚不清楚这些差异是否在燕麦中- 血脑屏障介导的转运导致阿托伐他汀预防中风进展和/或的能力发生变化 急性期/亚急性期神经认知功能障碍加重。我们的目标是评估性作为一种 他汀类药物在缺血脑中转运的生物学变量以及这些差异如何影响他汀类药物 作为中风疗法的疗效。此F31应用程序的中心假设是：i)功能 TMCAO后Oatp1a4在血脑屏障的表达在男性和女性之间存在差异； 他汀类药物的神经保护特性和/或它们对中风后神经预后的影响 受BBB Oatp1a4活性性别差异的影响。两个目标将检验这些假设： 目的1：研究Oatp1a4介导的他汀类药物在血脑屏障转运的性别差异。 卒中。我们将使用tMCAO模型在雄性和雌性SD大鼠身上进行研究。Oatp1a4本地化 蛋白质表达将分别使用共聚焦显微镜和Western blotting进行评估。从血液到- 他汀类药物的脑转运将使用原位脑灌注进行测量，这是一种最先进的研究方法 在BBB的毒品运输。 目的2：评估他汀类药物相关神经保护和功能的性别差异 卒中患者的神经功能恢复。在tMCAO手术的雄性和雌性SD大鼠中，我们将使用共聚焦显微镜 和蛋白质印迹分析，以检查与神经保护相关的分子生物标记物。我们还将评估 使用稳健行为测试(即旋转杆分析，Morris)的tMCAO动物的运动和认知表现 水迷宫，新颖的物体识别测试)。 总体而言，该项目将提供有关他汀类药物作为中风神经保护药物疗效的关键机制数据。 此外，该项目与美国国立卫生研究院将性别作为中风生物学变量的研究目标直接一致。

PROJECT SUMMARY Stroke is the fifth leading cause of death in the United States. Approved stroke therapies are limited by narrow treatment windows, the risk of hemorrhagic transformation, and reperfusion injury. Therefore, there is a critical need for neuroprotective drugs that can improve post-stroke neurological performance. Currently, 3-hydroxy-3- methylglutaryl coenzyme A (HMG CoA) reductase inhibitors (i.e., statins) are given to stroke patients due to their proven utility in improving cognitive and motor outcomes. Studies in our laboratory have uncovered a specific biological mechanism that enables statins to be effective drugs for stroke treatment: transport across the blood- brain barrier (BBB) via the endogenous uptake transporter organic anion transporting polypeptide 1a4 (Oatp1a4). We have shown, for the first time, that Oatp-mediated transport is required for atorvastatin to reduce cerebral infarction volume and improve sensorimotor performance at 24 h following transient middle cerebral artery occlusion (tMCAO). We also observed increased atorvastatin uptake in female rats subjected to tMCAO as compared with their age-matched male counterparts; however, it is unknown if these differences in Oatp- mediated transport at the BBB cause variations in atorvastatin’s ability to prevent stroke progression and/or worsening of neurocognitive deficits in the acute/subacute period. Our goals are to assess the role of sex as a biological variable on statin transport in the ischemic brain and to determine how these differences affect statin efficacy as stroke therapeutics. The central hypotheses of this F31 application are i) that functional expression of Oatp1a4 at the BBB is different in males as compared to females following tMCAO; and ii) that statin neuroprotective properties and/or their effects on post-stroke neurological outcomes are influenced by sex-dependent differences in BBB Oatp1a4 activity. Two aims will test these hypotheses: Aim 1: Investigate sex-dependent differences in Oatp1a4-mediated transport of statins at the BBB in stroke. We will perform our studies in male and female SD rats using the tMCAO model. Oatp1a4 localization and protein expression will be assessed using confocal microscopy and western blotting, respectively. Blood-to- brain transport of statins will be measured using in situ brain perfusion, a state-of-the-art methodology to study drug transport at the BBB. Aim 2: Evaluate sex-dependent differences in statin-associated neuroprotection and functional neurological recovery in stroke. In tMCAO operated male and female SD rats, we will use confocal microscopy and western blot analysis to examine molecular biomarkers associated with neuroprotection. We will also assess motor and cognitive performance in tMCAO-animals using robust behavioral tests (i.e., rotarod analysis, Morris Water Maze, Novel Object Recognition test). Overall, this project will provide critical mechanistic data on efficacy of statins as neuroprotective drugs for stroke. Furthermore, this project directly aligns with NIH goals in studying sex as a biological variable in stroke.