Chronic, low-grade inflammation (inflammaging) is a hallmark of aging and a major risk factor for both morbidity and mortality as well as being a major risk factor for a variety of age-related diseases, including neurodegenerative diseases. Despite the link between inflammation, aging, and age-associated diseases, the molecular mechanism(s) responsible for inflammaging is poorly understood. The objective of my current Merit VA grant was to determine if necroptosis, a cell death pathway that induces inflammation, played a role in inflammaging. We showed for the first time that necroptosis increased with age in various tissues including brain and was associated with an increase in various markers of inflammation. Importantly, we found that globally inhibiting/blocking necroptosis reduced markers of inflammation, which supported our original hypothesis. Interestingly, we found that the age-related increase in markers of necroptosis in the brain occurred primarily in neurons in the hippocampus and cortex even though there is little, if any loss of neurons. Thus, the activation of necroptosis in neurons does not appear to result in neuronal death. The overall objective of the current proposal is to determine the mechanism(s) by which neuronal activation of necroptosis can impact the aging brain without leading to neuronal death. We hypothesize that the activation of necroptosis in neurons leads to neuronal dysfunction and the release of necroptotic extracellular vesicles, which induce neuroinflammation when phagocytosed by microglia and/or astrocytes. We will test this hypothesis using mouse models in which necroptosis activation is blocked or induced specifically in neurons, e.g., Ripk3fl/fl and Mlklfl/fl mice crossed to transgenic Slick-H Cre transgenic mice to produce Ripk3NeuKO or MlklNeuKO mice in which necroptosis is blocked and crossing Ripk3- or Mlkl-knockin mice to SLICK-H Cre-transgenic mice to produce Ripk3NeuOE or MlklNeuOE mice, which overexpress RIPK3 or MLKL specifically in neurons. Using old (24- to 26-months) Ripk3NeuKO, MlklNeuKO, Ripk3NeuOE, and MlklNeuOE mice compared to adult (6-months) and old wild type (WT) mice, we will conduct the first experiments on the impact of necroptosis activation in a specific cell type (in this case neurons) on inflammation and the function of a cell in the absence of cell death. Aim 1. To determine the role of neuronal necroptosis activation in neuroinflammation. The impact of reducing or inducing the activation of necroptosis in neurons on neuroinflammation in old mice will be assessed by measuring proinflammatory cytokines/chemokines and markers of neuroinflammation in various regions of the brain. We predict that altering necroptosis specifically in neurons will in turn impact neuroinflammation. Aim 2. To determine the mechanism of how neuronal necroptosis activation leads to neuroinflammation. First, we will determine if blocking or inducing necroptosis activates specific populations of microglia...