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.

Project Narrative Blood flow through the brain capillaries is impaired in Alzheimer’s disease, and preclinical research is needed to understand the bases for this impairment and to identify therapeutic avenues to improve capillary flow. This project will use cutting-edge microscopy and genetic approaches in live mice to study pericytes, a cell type that regulates capillary blood flow, and determine how contractile signals to these cells contributes to normal blood flow and reduced blood flow in models of Alzheimer’s disease-like pathology.