Aging is the primary risk factor for most neurodegenerative disorders including Alzheimer's disease (AD). The dysfunction seen in organisms because of aging has a multifaceted origin at the molecular level. Understanding how altered molecular signaling in key cellular pathways generates age-related decline is essential to develop better, more targeted therapies that ameliorate dysfunction. AD and related diseases are the most crippling cognitive threat to our aging population. There is no cure for AD, and this is partly due to a poor understanding of how aging and inflammation impact pathogenesis and neural plasticity. It is imperative that we gain a more comprehensive understanding of the biological basis for brain aging. We hypothesize that the neuroimmune system plays a critical role in aging pathology. Microglia are the brain's resident macrophage and the major immune effector in the CNS. ?Activated? microglia assume a pro-inflammatory phenotype, secreting cytotoxic factors. An additional process mediated by anti-inflammatory molecules, inhibits the pro- inflammatory phenotype and shifts microglial toward a restorative phenotype that promotes debris clearance and tissue repair. Aging perturbs microglial biology such that aging microglia become hyper-responsive to pro- inflammatory stimuli, generate excess cytotoxic factors, and become insensitive to anti-inflammatory molecules. Aged microglia are also increasingly unable to assume an anti-inflammatory phenotype. In this way, dysfunctional microglia become neurotoxic and increase CNS vulnerability. We have previously described age- related changes in rodent microglia using mass-spectrometry-based proteomics. We identified RICTOR, a subunit of mTORC2, as an upstream regulator predicted to be inhibited with age. This prediction coincided with upregulated pro-inflammatory signaling, downregulated anti-inflammatory signaling, and impaired cellular metabolism and energy regulation. We validated this finding by demonstrating reduced RICTOR expression in aged primary mouse microglia when compared with their younger counterparts. We also conducted targeted knockdown of RICTOR in BV2 cells and observed a phenotype resembling aged microglia, validating a primary role for RICTOR in the aging phenotype. The present study will determine the consequences of RICTOR knockdown in vivo on microglial phenotype and cognitive behavior in young mice. We hypothesize that in vivo microglial RICTOR deletion will induce an aging microglial phenotype that will decrease brain resilience and have widespread impact on cognition and neural plasticity. We predict that knockout animals will manifest a phenotype characterized by dysregulated neuroinflammation, impaired cognitive and behavioral performance, and reduced brain plasticity. Our goal is to identify the molecular substrates of in vivo microglial dysfunction via mTORC2 signaling with the experiments detailed in the proposal.
Understanding the molecular basis for age-related neurodegeneration is key in developing therapies that prevent or cure diseases like Alzheimer?s. Our proposed experiments bridge the knowledge gap in how the neuroimmune system exacerbates age-related neurodegeneration with targeted, in vivo, RICTOR knockdown in microglia. Our goal is to identify the molecular substrates of in vivo microglial dysfunction via RICTOR-mTORC2 signaling and determine how dysfunction in this pathway worsens neurodegenerative pathology.
