Project Summary – supplement application Studies over the past two decades have cast doubt on the amyloid seeding hypothesis of Alzheimer’s disease (AD), noting that early changes in microglia morphology and reactivity precede deposition of amyloid beta (Aβ) in affected brain regions. Moreover, these cellular changes occur prior to the appearance of Aβ plaques. In addition, the most significant risk factors for development of AD include genes variants associated with innate immunity in microglia, such as TREM2 and APOE4. These findings and others suggest an environmental link to early pathological changes in AD-sensitive brain regions associated with activation of innate immunity in glial cells. Environmental pesticides are one class of agents known to increase risk for AD. Many of these agents share a common mechanism in causing mitochondrial dysfunction and activation of inflammatory gene expression in microglia and astrocytes. However, the molecular signals regulating innate immune pathways in microglia in response to environmental pesticides are not well understood, particularly with respect to complex temporal changes in microglia phenotype associated with inflammation and autophagy in response to Aβ and Tau misfolding. The basis for this NOT- AG-21-018 Alzheimer’s-focused supplement application is the striking discovery from the parent research project in rotenone-treated mice of significant levels of alpha-synuclein protein aggregation in the hippocampus and entorhinal cortex, both regions affected in AD. Upon further interrogation of these brain regions, we found increased levels of both phosphorylated Tau (P-Tau) and Aβ, neither of which have been previously described in response to rotenone treatment in mice. Given that rotenone is a systemic inhibitor of mitochondrial complex I that is commonly used to model the effects of diverse pesticides affecting mitochondria, this raises the exciting possibility of a better model for the metabolic and neuroinflammatory derangements seen in AD that could be explored using powerful transgenic tools in mice. It is the central hypothesis of this supplement application that rotenone-dependent mitochondrial dysfunction causes activation of innate immune inflammatory signaling pathways in microglia that promote aggregation of Aβ and Tau in neurons, as well as amplifying astrocyte reactivity that contributes to neuroinflammation and cognitive decline. This hypothesis will be explored during the one-year supplement project period through the following two focused and complementary Specific Aims: Specific Aim 1 - Determine the spatio- temporal sequence of glial activation and aggregation of Aβ and Tau in mice exposed systemically to rotenone. Specific Aim 2 - Identify mechanisms by which rotenone enhances inflammatory activation of microglia and autophagy of Aβ+ and Tau+ protein aggregates. The rotenone mouse model developed in the parent research project and used in this supplement application shows chang...