Whole brain irradiation (WBI) leads to progressive dementia in ~50% of brain tumor patients who survive long- term after treatment, at least in part, due to dysregulation of CBF. Although the specific mechanisms for WBI- induced deceases in CBF and cognitive decline are not yet known, there is increasing evidence that alterations of the neurovascular unit play a crucial role. The objective of this proposal is to elucidate the mechanistic role of irradiation-induced astrocyte dysfunction in cognitive impairment. The central hypothesis is that irradiation causes astrocyte senescence and subsequent dysfunction, altering the production of vasodilator mediators and impairing neurovascular coupling (NVC) responses. The resulting neurovascular dysfunction contributes to decline in CBF and cognitive impairments. The proposed work is novel in that it will be the first to demonstrate that radiation-induced astrocyte senescence is a key driver of the effects of WBI on the brain. The results will likely identify specific mechanisms and reveal potential therapies that are capable of improving cerebral blood supply and restoring learning and memory. The following aims are proposed: 1) Elucidate the cellular mecha- nisms underlying WBI-induced impairment of NVC responses. The working hypothesis is that WBI impairs both eicosanoid-mediated and purinergic components of NVC responses. To test this hypothesis in a clinically rele- vant mouse model of WBI, pathways contributing to NVC responses will be assessed using laser speckle con- trast imaging, pharmacological tools and LC/MS/MS-based measurement of gliotransmitter release. The im- pact of pharmacological up-regulation of NVC responses on cognitive function of WBI-treated mice will be de- termined. 2) Determine how irradiation-induced senescence alters astrocyte function and phenotype. It is pre- dicted that irradiation induces senescence in astrocytes, which impairs cellular energy metabolism and the production/release of ATP and alters the cellular secretory profile, dysregulating the synthesis of vasoactive lipid mediators. To test these hypotheses senescent astrocytes will be isolated from WBI-treated mice and pri- mary human astrocyte cultures will be irradiated in vitro. We will combine advanced cellular imaging tech- niques and cutting-edge proteomics and biochemistry to investigate cellular energetics, gene expression and secretome signatures, the regulation of ATP release and the synthesis of lipid mediators. 3) Determine wheth- er elimination of senescent cells improves NVC and cognitive function in WBI-treated mice. It is hypothesized that activation of p16-dependent cellular senescence programs is responsible for WBI-induced neurovascular dysfunction and cognitive impairment. It is expected that elimination of senescent cells, either through genetic manipulation (p16-3MR mouse model) or by pharmacological means will restore neurovascular function and improve cognition in WBI-treated mice. Together, the proposed studies will identify a fundamental mechanism governing WBI-related neurovascular dysfunction eventually leading to cognitive impairment.