ORAI Channels and Neurovascular Coupling: Effects of Aging - PROJECT SUMMARY Neurovascular coupling (NVC) is an essential physiological process that rapidly directs blood flow to active brain regions, ensuring adequate delivery of oxygen and other vital nutrients. The cerebral capillary network is critical for NVC, acting as a highly specialized sensory system that detects regions of elevated neuronal activity and signals upstream arterioles to dilate, thereby directing blood flow to meet local metabolic needs. Ca2+ signaling mechanisms are essential for this capillary-to-arteriole dilation process, yet the mechanisms underlying Ca²⁺ influx and Ca2+ release from intracellular stores in brain capillary endothelial cells (ECs) remain poorly understood. ORAI1, ORAI2, and ORAI3 channels are critical mediators of store-operated Ca²⁺ entry (SOCE), a homeostatic response triggered by depletion of Ca²⁺ from the endoplasmic reticulum (ER). ER depletion Ca²⁺ induces a conformational change in stromal interaction molecule 1 (STIM1), an ER-resident transmembrane protein, allowing it to activate ORAI1 at the plasma membrane and initiate SOCE. Whereas ORAI1 is sufficient to mediate SOCE, ORAI2, and ORAI3 can modulate the process under specific physiological conditions by forming heteromultimeric channels with unique properties. ORAI channels also modify subcellular Ca²⁺ signals induced by phospholipase C-coupled receptors, indicating broad physiological significance. Our preliminary data reveal that ORAI channels are indispensable for sustaining capillary-to-arteriole dilation. Moreover, our initial findings suggest that the expression and functionality of ORAI channels in brain capillary ECs decline with age, a dynamic that we hypothesize contributes to impaired NVC, brain perfusion, and cognitive decline. This proposal aims to elucidate the role of ORAI channels and their associated Ca²⁺ signaling mechanisms in brain capillary ECs and how these processes are altered with aging. Preliminary findings show that brain capillary ECs express Stim1, Stim2, Orai1, and Orai3, but not Orai2. We developed new EC-specific Stim1-, Orai1-, and Orai3-knockout (ecKO) mice, as well as Orai1/Orai3 double-knockout mice and propose to use these models to investigate how ORAI1 and ORAI3 influence Ca²⁺ signaling in brain capillary ECs, how this signaling modulates capillary-to- arteriole dilation and cerebral blood flow (CBF) regulation, and how disruptions in these processes associated with aging contribute with cognitive decline. Proposed studies will investigate our ecKO mice and aged mice (up to 24 months) using an integrated, multimodal experimental approach that includes in vivo CBF measurements, behavioral analysis of learning and memory, patch-clamp electrophysiology of native brain capillary ECs, high- speed, high-resolution Ca2+ imaging, and ex vivo analysis of capillary-to-arteriole dilation using pressure myography. These insights will provide critical new understanding of NVC mechanisms at molecular and cellular levels and novel insight into neurovascular dysfunction during aging. More broadly, anticipated results may have implications for understanding brain perfusion deficits associated with hypertension, diabetes, cerebral small vessel diseases (cSVDs), Alzheimer’s and related disease (ADRD), and other cerebrovascular pathologies.
