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性痴呆（AD/ADRD）中， 周细胞导致脑灌注不足。然而，我们对血管活性信号的理解， 仍然缺乏周细胞收缩的细胞内机制。周细胞表达高水平的 内皮素-1A型受体（ETAR）和血栓素A2受体（TXA 2 R）。我们的核心假设是， ETAR和TXA 2 R信号传导对于优化通过脑毛细血管网络的血流是必不可少的， 在淀粉样蛋白β（A β）积累期间通过这些受体的活性有助于灌注不足。解决 根据这一假设，我们将使用具有创新成像方法的体内-体外管道和一种新的小鼠模型。 Cre-driver基因靶向CNS周细胞。这项工作将由调查人员进行， 在血流的体内双光子成像、离体脑切片实验、周细胞生物学和ETAR方面的专业知识 和TXA 2 R信号传导机制。目的1将检验周细胞中ETAR和TXA 2 R信号转导的假设， 提供基础毛细血管张力并协调通过脑毛细血管网络的流动的优化。ETAR（EDNRA 基因）和TXA 2 R（tbxa 2 r基因）在正常小鼠的毛细血管周细胞中将被条件性缺失。 在体内深双光子成像将用于研究毛细血管流动动力学的皮质层，并进入 清醒小鼠胼胝体的白色物质。一组血管指标，组织缺氧，神经炎症， 将评估行为结果。目的2将检验以下假设： ETAR和TXA 2 R需要毛细血管周细胞中的肌动球蛋白收缩机制，尽管α- 平滑肌肌动蛋白我们将使用化学遗传学来驱动G蛋白激活，如ETAR下游所见。 和TXA 2 R信号。周细胞将在离体脑切片中化学发生性收缩，并且 将给予肌动球蛋白和细胞骨架机制以剖析它们在周细胞收缩中的作用。此外，本发明还 在体内CNS毛细血管周细胞的化学发生活化将被表征为脑损伤的新模型。 低灌注目的3将检验异常ETAR和TXA 2 R信号传导驱动ETAR和TXA 2 R缺乏的假设。 毛细血管灌注和认知能力下降。ETAR的条件性遗传缺失或 将在两种AD模型中检查ETAR/TXA 2 R抑制剂的单独长期给药和ETAR/TXA 2 R抑制剂的单独长期给药。 如病理学（Tg-SwDi和5xFAD）。对脑血流量、大脑健康和行为指标的影响将在 考察本项目将通过以下几个方面来加深我们对AD/ADRD脑灌注不足的认识：（1）解密 正常和AD脑中周细胞收缩的机制;（2）利用新的毛细血管周细胞特异性 小鼠线解剖周细胞对血流的贡献;（3）表征毛细血管驱动的新模型 体内低灌注;（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中受影响最严重;（5）提供概念验证临床前研究，以测试是否调节 周细胞中ETAR和TXA 2 R信号通路可改善AD/ADRD患者毛细血管网络的流动和认知功能。

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.