Abstract . Alzheimer's disease (AD) is a devastating neurodegenerative disorder affecting millions of Americans. Despite decades of research to understand memory and cognitive deficits and numerous clinical trials to treat the disease, mechanisms underlying AD development and progression remain unclear and outcomes of clinical trials uncertain. Intriguingly, an early event in AD is a decrease of cerebral blood flow (CBF) that has been associated with oligomeric amyloid β (Aβ) accumulation and changes in the diameter of cerebral blood vessels in both human with AD and animal models of AD. Efforts to explored whether the vasculature contributes to AD have mainly centered on mechanisms of endothelial dysfunction. However, how vascular smooth muscle (VSM), which contain the contractile apparatus to modulate arterial/arteriole diameter and CBF, are affected by Aβ leading to AD development and progression are poorly understood. The overall objective of this proposal is to address these fundamental knowledge gaps by providing a comprehensive evaluation of a link between cerebral VSM dysfunction and Aβ accumulation/exposure. We will address the novel central hypothesis that Aβ exposure alters VSM function and vascular reactivity by modifying the clustering and activity of the ion channel CaV1.2, which is essential for VSM contraction. We further hypothesize that the phosphorylation state of a single CaV1.2 amino acid - S1928 – mediates Aβ-dependent effects on CaV1.2 spatiotemporal properties. This innovative hypotheses are formulated on the basis of strong preliminary data indicating an unanticipated and remarkable effect of Aβ exposure in increasing S1928 phosphorylation. This was correlated with increased CaV1.2 activity and the induction of coupled CaV1.2 events upon Aβ exposure. Increased CaV1.2 coupling results in a net amplification of Ca2+ influx leading to enhanced vasoconstriction and altered CBF in response to Aβ, thus underscoring the significance of this supplement. Beyond the unforeseen role for S1928 in control of arterial CaV1.2 and vascular function in response to Aβ, an emerging and innovative concept is that pS1928 is a major risk factor in AD. Our multiscale contemporary approach that includes innovative microscopy techniques, sophisticated biochemistry, electrophysiology, in silico analysis and unique animal models will be implemented to explore the following aims. Aim 1 will test the hypothesis that Aβ exposure increases CaV1.2 clustering, activity and coupled gating. Aim 2 will test the hypothesis that pS1928 is essential for Aβ-induced CaV1.2 clustering and coupled gating. The impact of the application lies in uncovering fundamental new mechanistic insight of a link between cerebral VSM dysfunction and AD that could be exploited for the rational development of treatment strategies that reduce the risk of vascular and neuronal complications.