Pericyte control of capillary perfusion in the Alzheimer's disease brain

在阿尔茨海默病和阿尔茨海默病相关痴呆(AD/ADRD)中，毛细血管的异常收缩 周细胞导致脑低灌注量。然而，我们对血管活性信号和 周细胞收缩的细胞内机制仍然缺乏。周细胞表达高水平的 内皮素-1 A型受体(ETAR)和血栓素A2受体(TXA2R)。我们的中心假设是 ETAR和TXA2R信号是通过大脑毛细血管网络优化血液流动所必需的，而这种异常 在淀粉样β蛋白(A)积聚过程中，通过这些受体的活动导致了低灌注量。致信地址 这一假设，我们将使用体内-体外管道与创新的成像方法和一种新的小鼠 基因靶向CNS周细胞的CRE驱动程序。这项工作将由调查人员进行，并补充 擅长活体双光子血流成像、体外脑片实验、周细胞生物学和ETAR 以及TXA2R信号转导机制。目标1将验证这样的假设：周细胞中的ETAR和TXA2R信号 提供基本的毛细血管音调，并协调优化通过大脑毛细血管网络的流动。埃塔尔(Ednra) TXA2R(tbxa2r基因)和TXA2R(tbxa2r基因)将在正常小鼠的毛细血管周细胞中有条件地缺失。 在活体内，深度双光子成像将被用来研究穿过皮质层和进入 清醒小鼠的膝盖骨白质。一系列血管指标，组织缺氧，神经炎症，以及 将对行为结果进行评估。目标2将测试G蛋白信号下游的假说 ETAR和TXA2R需要毛细血管周细胞中肌动球蛋白的收缩机制，尽管α-1低表达。 平滑肌肌动蛋白。我们将使用化学遗传学来驱动G蛋白的激活，就像在Etar下游看到的那样 和TXA2R信号。周细胞将在体外脑片和阻滞剂中化学发生收缩 肌动球蛋白和细胞骨架机械将被用来分析它们在周细胞收缩中的作用。此外， 中枢神经系统毛细血管周细胞在体内的化学激活将是一种新的脑移植模型 低灌注率。目的3将测试假设，异常的ETAR和TXA2R信号驱动缺陷 A蓄积过程中的毛细血管灌流和认知功能下降。Etar或的条件性遗传缺失 TXA2R，以及分别在两种AD模型上长期给予ETAR/TXA2R抑制剂的情况。 类似病理(TG-Swdi和5xFAD)。对脑血流、脑健康和行为指标的影响 检查过了。这个项目将通过以下几个方面促进我们对AD/ADRD脑低灌注的理解：(1)破译 正常和AD脑中周细胞收缩的机制；(2)利用一种新的毛细血管周细胞特异性 小鼠品系解剖周细胞对血流的贡献；(3)表征一种新的毛细血管驱动模型 (4)采用先进的体内成像方法研究深部毛细血管网络 在AD/ADRD中受影响最大；(5)提供概念验证临床前研究以测试是否调节 周细胞内的ETAR和TXA2R信号通路可改善AD/ADRD的毛细血管网络血流和认知功能。

In Alzheimer's disease and Alzheimer's disease related dementias (AD/ADRD), aberrant contraction of capillary pericytes contributes to cerebral hypoperfusion. However, our understanding of the vasoactive signals and intracellular mechanisms underlying pericyte contractility remains lacking. Pericytes express high levels of endothelin-1 type A receptors (ETAR) and thromboxane A2 receptors (TXA2R). Our central hypothesis is that ETAR and TXA2R signaling is essential to optimize blood flow through brain capillary networks, and that aberrant activity through these receptors during amyloid beta (A) accumulation contributes to hypoperfusion. To address this hypothesis, we will use an in vivo-ex vivo pipeline with innovative imaging approaches and a novel murine Cre-driver to genetically target CNS pericytes. The work will be conducted by investigators with complementary expertise in in vivo two-photon imaging of blood flow, ex vivo brain slice experiments, pericyte biology, and ETAR and TXA2R signaling mechanisms. Aim 1 will test the hypothesis that ETAR and TXA2R signaling in pericytes provides basal capillary tone and orchestrates optimization of flow through brain capillary networks. ETAR (ednra gene), and separately, TXA2R (tbxa2r gene), will be conditionally deleted in capillary pericytes of normal mice. In vivo deep two-photon imaging will be used to study capillary flow dynamics across cortical layers and into callosal white matter of awake mice. A battery of vascular metrics, tissue hypoxia, neuroinflammation, and behavioral outcomes will be assessed. Aim 2 will test the hypothesis that G-protein signaling downstream of ETAR and TXA2R requires actomyosin contractile machinery in capillary pericytes, despite low expression of α- smooth muscle actin. We will use chemogenetics to drive G-protein activation, as seen downstream of ETAR and TXA2R signaling. Pericytes will be chemogenetically contracted in ex vivo brain slices and blockers of actomyosin and cytoskeletal machinery will be administered to dissect their roles in pericyte contraction. Further, chemogenetic activation of CNS capillary pericytes in vivo will be characterized as a new model of cerebral hypoperfusion. Aim 3 will test the hypothesis that aberrant ETAR and TXA2R signaling drives deficiency in capillary perfusion and cognitive decline during A accumulation. Conditional genetic deletion of ETAR or TXA2R, and separately, chronic administration of ETAR/TXA2R inhibitors will be examined in two models of AD- like pathology (Tg-SwDi and 5xFAD). Effects on cerebral blood flow, brain health, and behavioral metrics will be examined. This project will advance our understanding of brain hypoperfusion in AD/ADRD by: (1) Deciphering mechanisms of pericyte contractility in the normal and AD brain; (2) leveraging a novel capillary pericyte-specific mouse line to dissect pericyte contributions to blood flow; (3) characterizing a novel model of capillary-driven hypoperfusion in vivo; (4) employing advanced in vivo imaging approaches to study deep capillary networks most strongly affected in AD/ADRD; (5) providing proof of concept preclinical studies to test if modulation of ETAR and TXA2R signaling in pericytes can improve capillary network flow and cognition in AD/ADRD.