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Functional genomic interrogation of inflammatory astrocyte reactivity


Astrocytes perform critical homeostatic functions in the central nervous system (CNS). In CNS injury or disease, astrocytes respond to pathophysiological perturbations by becoming “reactive”, which is defined as the adoption of context-specific cell states and associated alterations in morphology and function. Frequently, inflammatory processes play an important role in the pathophysiology of CNS injuries and diseases. As an immunocompetent CNS cell type, astrocytes actively participate in inflammatory signaling cascades involving interaction with both microglia as well as infiltrating peripheral immune cells. Proinflammatory cytokines, such as IL-1a, TNF, and C1q, induce inflammatory reactive astrocytes that lose homeostatic functions while concurrently releasing factors that are potentially harmful in specific contexts. Given that inflammatory astrocyte reactivity has been implicated in numerous neurodegenerative and neuroinflammatory diseases, in addition to being associated with normal aging, it is an attractive target for therapeutic development. However, the cellular pathways that control inflammatory astrocyte reactivity are still not fully understood.

In Chapter 1 of this dissertation, we broadly introduce astrocyte reactivity as a concept and the existing literature on inflammatory astrocyte reactivity.

In Chapter 2, we set out to characterize astrocyte reactivity in the context of Alzheimer’s disease (AD) using single-nucleus RNA-sequencing of post-mortem AD brain tissue. In addition to discovering that reactive astrocytes in AD downregulated homeostatic pathways, we also found that a specific subpopulation of excitatory neurons were selective vulnerable in AD, which we were able to validate using immunostaining in an independent cohort of cases and controls.

In Chapter 3, we developed a scalable method to generate hiPSC-derived astrocytes that allowed us to harness the power of pooled CRISPRi screening to systematically identify cellular pathways controlling inflammatory astrocyte reactivity. Following up on the top hits from CRISPRi screens with single-cell transcriptomics, we found that autocrine-paracrine IL-6 and interferon signaling drove two distinct inflammatory reactive states that were promoted by and inhibited by STAT3, respectively. Furthermore, we found that the inflammatory reactive states we identified corresponded to those observed in other experimental contexts, both in vitro and in vivo. Lastly, we also performed immunostaining supporting the existence of these inflammatory reactive states in human brains in the context of Alzheimer’s disease and hypoxic-ischemic encephalopathy.

In Chapter 4, we focused on how autophagic pathways are rewired in inflammatory reactive astrocytes and how this contributes to the neurotoxic activity of inflammatory reactive astrocytes. We found that lysosomes are remodeled and alkalinized in inflammatory reactive astrocytes, and that lysosome exocytosis drives astrocyte-mediated neurotoxicity. Through CRISPRi screening, we uncovered mTOR as a regulator of neurotoxic lysosome exocytosis. These results pinpoint lysosome remodeling and exocytosis in inflammatory reactive astrocytes as a potential therapeutic target.

We believe these studies contribute to our understanding of inflammatory astrocyte reactivity and may inform the development of therapeutics that modulate specific aspects of astrocyte reactivity for the treatment of neurodegenerative and neuroinflammatory diseases.

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