Proper regulation of inflammatory responses is critical for effective microglia function in physiology and disease. While much emphasis is placed on mechanisms driving microglia activation, there remains a gap in knowledge on how microglia resolve or modulate inflammatory activation. Here we provide a comprehensive analysis of exosome regulation of microglia inflammatory response, highlighting a novel anti-inflammatory function of exosomes. First, microglia-derived exosomes contain immune molecules that are sufficient to propagate signals driving transcriptional changes associated with increased inflammatory cytokine production and phagocytic clearance in recipient microglia. Secondly, microglia with impaired exosome biogenesis display altered inflammatory kinetics, characterized by exaggerated and prolonged expression of pro-inflammatory molecules in response to acute and chronic aging-associated inflammation. This robust anti-inflammatory function of exosomes is manifested in part through the release of proinflammatory microRNAs, such miR-155. Taken together, our study identifies exosomes as critical mediators of inflammatory communication in the brain that play important roles in intracellular resolution of microglia inflammation.

Proteins of the major histocompatibility complex class I (MHC I), previously known mainly for antigen presentation to cells of the immune system, have recently been shown to play a crucial role in both developmental neural refinement and adult synaptic plasticity. However, their function in non-neuronal cell populations in the brain or the impact of cellular changes regulated by MHC I molecules on behavioral outputs have yet to be investigated. In this dissertation, I identify novel, non-immune roles for two specific classical MHC I molecules (out of as many as 50 encoded in the murine genome) in the brain. I demonstrate that H2-Kb is a negative regulator of proliferation in neural stem cells (NSCs), a unique cell type amenable to regenerative biology. Using genetic mouse models as well as primary NSC cultures, I show that deletion or knockdown of H2-Kb molecules causes increased proliferation and neurogenesis in the adult hippocampus in vivo as well as in a cell-type specific environment in vitro. Transcriptome analysis of NSCs lacking H2-Kb reveals that this enhanced proliferative potential is due to an induction of several growth factor receptor signaling pathways. I found functionally that one mechanism by which H2-Kb may exert its inhibition is through a physical interaction with fibroblast growth receptor 1, putatively dampening its signaling. Though also highly expressed in the hippocampus, I show that the other classical MHC I molecule, H2-Db, does not play a major role in NSC function. I thus probed other hippocampal processes and revealed a developmental necessity H2-Db in preserving normal locomotion, anxiety-related behavior, and spatial and associative memory. Furthermore, I show that its acute ablation in the adult hippocampus can remarkably improve hippocampus-dependent memory. The hippocampus, an extraordinary brain structure in its capacity for immense plasticity throughout life, is particularly vulnerable to deleterious conditions such as disease and aging. As MHC I molecules are known to increase in the aged and injured brain, understanding their precise functions in the hippocampus may provide opportunities to seize its neuroplastic and regenerative potential for therapeutic intervention.

Biologic sex matters in aging and neurodegeneration. A female advantage in lifespan is seen in humans and throughout the animal kingdom, and men and women suffer differing vulnerabilities to age-related neurodegenerative disorders, including Alzheimer’s disease (AD). More women suffer from AD, largely due to their longevity, as AD risk is highest in advanced age. In contrast, men with the disease die faster in populations worldwide and show greater cognitive deficits. We show the contribution of sex chromosome complement to these observations, in particular, the importance of the X chromosome. Using genetically modified mice we demonstrate (Chapter 2) that having XX sex chromosome complement increases survival during aging in male and female mice. We also show that XX sex chromosome complement in combination with ovaries extends lifespan. We go on to show (Chapter 3) that having two X chromosomes decreased mortality and conferred resilience to AD-related vulnerability in mice expressing the human amyloid precursor protein (hAPP). We then investigated how a second X chromosome can confer resilience against AD-related deficits. While XY and XX organisms express only one active X due to X chromosome inactivation (XCI), a few factors escape XCI and therefore are higher in females compared to males. One of these genes, Kdm6a, escapes in both mice and humans and is a histone demethylase linked to cognitive functions. We thus determined whether higher levels of Kdm6a – as found in the XX brain – could counter AD-related vulnerability in the XY brain. We first confirmed that the presence of a second X chromosome increased levels of Kdm6a in mouse hippocampus; it did so independent of gonadal phenotype or the presence of a Y chromosome. We then used lentiviral vectors in mice to modulate Kdm6a expression in vivo and in vitro. Overexpression of Kdm6a in the hippocampus of XY hAPP mice to higher levels observed in XX hAPP mice attenuated spatial learning and memory deficits in the Morris water maze. Similarly, overexpression of Kdm6a in XY primary neurons decreased Aβ toxicity in vitro. Furthermore, knockdown of Kdm6a in XX neurons to levels observed in XY neurons worsened Aβ toxicity in vitro. Finally, in humans, genetic variation in KDM6A was linked to higher brain expression and associated with less cognitive decline in aging and preclinical AD, suggesting relevance to human brain health. Our findings highlight the importance of sex chromosomes in aging and disease, as well as reveal a role for the baseline XCI escapee Kdm6a in countering AD-related deficits.

Traumatic brain injury (TBI) is the leading cause of neurological disability and the primary risk factor for development of neurodegenerative diseases and dementia in the United States. Over 2 million TBI-related incidents occur each year, and 3-5 million people currently suffer from chronic TBI-related disabilities. With the incidence of TBI on the rise, it is increasingly important to understand the underlying injury mechanisms and develop treatments for current and future patients. This dissertation investigates two such mechanisms contributing to TBI-induced cognitive decline, broadens the scope of TBI research to understand the effect of aging, and describes a new mouse model for frontal lobe TBI.

In chapter 1, I investigated the effect of aging on TBI-induced cognitive deficits and inflammatory response with emphasis on the contribution of peripheral, infiltrating monocytes. Our mouse model of TBI showed significantly greater chronic impairment of spatial memory in aging mice. Aging also exacerbated the infiltration of the peripheral monocytes while impairing expression of anti-inflammatory markers in peripheral monocytes and resident microglia. Furthermore, I observed that a subpopulation of the peripheral monocytes regained proliferative capabilities after infiltrating the injured brain, and the effect was significantly more pronounced in the old mice. In chapter 2, I targeted the integrated stress response (ISR) pathway using a novel small molecule at a chronic timepoint after injury. Inhibition of the ISR during behavioral testing fully restored spatial learning and memory in two different mouse models of TBI. The treatment additionally rescued deficits in long-term potentiation in the hippocampus at the chronic timepoint. In chapter 3, I applied the controlled cortical impact method to develop a frontal lobe injury mouse model to better mimic TBIs commonly observed among human patients. Markedly, mice that had received the frontal lobe TBI displayed deficits in prefrontal cortex-driven behavior similar to symptoms seen in human TBI patients. These chapters collectively add to our understanding of the biology of TBIs with the hope that we are a step closer to providing effective treatment for a major health crisis in the future.

Aging is a prominent risk factor for cognitive decline and neurodegenerative disease. Particularly sensitive to the effects of aging are functions governed by the hippocampus and indeed, impairments in hippocampal-dependent episodic and spatial memories have been observed in aged rodents, monkeys, and humans. While the mechanisms by which age-related cognitive impairments emerge are multifaceted, neuronal dysfunction and impaired plasticity are frequent hallmarks of brain aging thought to underlie cognitive decline. In the hippocampus, this is manifested as a loss of neurogenesis and decreased long-term potentiation, which is accompanied by corresponding decreases in synaptic density and synaptic protein expression. As the human population ages, understanding mechanisms governing hippocampal aging is increasingly important as means of identifying therapeutic targets to slow or even reverse aging in the brain. While significant work has characterized intrinsic cellular changes that contribute to age-related loss of neurogenesis, synaptic dysfunction, and cognitive decline, recent studies posit age-related changes in blood as a critical regulator of brain aging. Indeed, emerging studies utilizing the model of heterochronic parabiosis - in which the circulatory system of a young and old mouse is surgically joined - have revealed bidirectional pro- and anti- aging effects of old or young blood exposure on hippocampal neurogenesis, synaptic plasticity, and cognition. To date, studies have focused on age-related changes in blood-borne factors as regulators of hippocampal aging, and whether other components of blood contribute to hippocampal aging remains to be fully elucidated. Here, we assess the role of cellular aging of the hematopoietic system in driving hippocampal aging (Chapter 2) and test whether circulating exosomes in young blood contain rejuvenating capacity on the aged brain (Chapter 3). We find that cellular aging of the hematopoietic system, and downstream changes in blood-borne factors, drive age-related impairments in hippocampal cognition. Additionally we find that systemic administration of young plasma-derived exosomes fail to rejuvenate the aged hippocampus, but increase synaptic protein expression in the aged cortex.

Cognitive decline is emblematic of brain aging, ascribed in large part to age-related changes occurring at neuronal synapses. Dynamic post-translational protein modifications – in particular phosphorylation and O-GlcNAcylation – are known regulators of synaptic and cognitive function. However, how the landscape of synaptic PTMs changes with age has yet to be elucidated. In this study, age-dependent changes in phosphorylation and O-GlcNAcylation were characterized using a proteomics-based approach. Analyses implicate age-dependent changes in dynamic PTMs in age-related cognitive decline and vulnerability to neurodegenerative disorders, including Parkinson’s and Alzheimer’s disease. The functional relevance of age-related changes in PTMs, in particular of O-GlcNAcylation, was also investigated in vivo. Mimicking age-related decreased levels of neuronal O-GlcNAc recapitulated features of molecular, synaptic, and cognitive aging in mice, implicating that changes in O-GlcNAc promote brain dysfunction during aging. Moreover, increasing O-GlcNAcylation in the adult hippocampus enhanced cognition, proposing O-GlcNAc as a target for brain rejuvenation. Ultimately, this study proposes dynamic PTMs as novel molecular drivers of brain aging, and provides the foundation for future mechanistic studies to investigate site-specific molecular changes underlying synaptic and cognitive decline with age.