Abstract

The emerging field of neuroimmunology, which examines the immune system’s role in nervous system development, homeostasis, injury and disease, has uncovered new landscapes for scientific exploration. Microglia, resident immune cells in the central nervous system (CNS), have been recognized as important for CNS function, yet fundamental questions about their molecular phenotypes and roles in CNS development and disease remain unanswered. Recent evidence of multi-dimensional heterogeneity, which underlies distinct microglial states, has only widened the knowledge gap, adding layers of complexity to our understanding of the biological nature of microglia in various CNS contexts. This dissertation, focuses on the following questions: (1) What is the role of specific microglial states in the CNS? – microglial function (2) How are microglial states governed? – microglial regulation (3) Do microglia change states over time and environmental context? – microglial plasticity For years, the microglia biology field has been unequipped to directly address these questions. This was due, in part, to the lack of tools available to study microglia on a state-specific level. My dissertation work overcomes this technical limitation, and offers new insights about the function, regulatory mechanisms, and plasticity of specific microglial states observed in development and disease. Proliferative-region associated microglia (PAM) and disease associated microglia (DAM) represent two microglial states observed in development and neurodegeneration, respectively. Previous studies have shown that PAM and DAM share similar gene signatures including the upregulation of the pattern recognition receptor, C-type lectin domain containing 7A, or Clec7a. In Chapter 2 of this dissertation, I detail the generation of a novel genetic tool, Clec7a-CreERT2 to study PAM and DAM in vivo. I demonstrate that this tool can be used as a reporter to faithfully label and visualize PAM in early developing white matter tracts, and DAM in pathologic brain regions of Alzheimer’s Disease and Multiple Sclerosis mouse models. By isolating, profiling and comparing PAM and DAM(s) using single-cell RNA sequencing, I show convergence and divergence of gene signatures that inform similar and unique functions of these microglial states. I propose that the unique characteristics we identified are influenced by context-specific environmental cues. Importantly, this new tool can be used for systematic comparison of microglial states in other models not discussed in this dissertation, including models of Parkinson’s Disease, Amyotrophic Lateral Sclerosis, as well as studies examining embryonic microglia. Unlike the homeostatic microglial state observed in steady state conditions, reactive microglial states like PAM and DAM have largely been resistant to traditional microglia depletion methods. Consequently, few advances have been made in deciphering the functional relevance of these states in their respective contexts. In Chapter 2, I also demonstrate how the Clec7a-CreERT2 mouse line can be used to elucidate the biological significance of microglial states like PAM and DAM. Using a dual transgenic mouse line, Clec7a-CreERT2;LSL-DTA, which enables Cre-mediated ablation of Clec7a expressing cells, I have established the first model to achieve state-specific depletion of DAM in vivo. I show that loss of DAM in a white matter injury model, resulted in an increase of aberrant myelination patterns and inefficient remyelination. These findings demonstrate that the signatures underlying DAM drive a protective microglial phenotype and are essential for CNS repair. Moreover, by validating this ablation strategy, the field is now able to employ this tool to elucidate the function of PAM in development, and further determine if the protective role of DAM, as observed in the white matter injury model, is conserved in other contexts of CNS injury and neurodegeneration. Over the last decade, there has been mounting evidence implicating microglia in numerous CNS diseases. For example, studies have shown that Alzheimer’s Disease risk genes are uniquely or highly expressed by microglia. Given this clear link to neurodegenerative diseases, microglia have been proposed to have significant therapeutic potential. Developing effective microglia-based therapies, however, requires knowing the factors that control microglial gene expression and function. While important regulators of microglia differentiation, proliferation and survival have been well characterized, it is less clear what governs the conversion of microglia to distinct reactive states. Finally, in Chapter 2, I discuss the use of an in vitro microglia model system to test bioinformatically predicted candidate regulators of PAM and DAM signatures. I demonstrate that these candidates alter microglial signatures, notably downregulating many of the genes associated with PAM and DAM. Identifying and validating these regulators provide useful insight for novel mechanistic targets to reprogram microglia into phenotypes that are beneficial for CNS health and recovery. As long-lived immune cells, microglia are exposed to a variety of CNS microenvironments within the developing, aged, and diseased brain. This, notably, has raised intriguing questions about if, when and how individual microglia change their transcriptomic, epigenetic, morphological, and metabolic phenotypes over time and contexts. In Chapter 3, I utilize the Clec7a-CreERT2 mouse tool, to directly address questions about microglia plasticity. Utilizing the reporter to label and track DAM in a white matter disease model, I show that microglia are highly plastic, transitioning between states in a context-dependent manner. Furthermore, in models of repetitive injury, I show that microglia plasticity and function is altered over time. The findings from this study pose the possibility that previously protective microglial states may, over time, directly or indirectly play roles in disease progression. Overall, this body of work contributes both technically and conceptually to the field of microglia biology and, more generally, the field of neuroimmunology. The conclusions drawn from these studies ultimately highlight that microglia are complex cells and justifies the need to study them in a state specific manner. Standardizing this approach will enable deeper understanding of the fundamental role of microglia in numerous biological contexts. Importantly, the value of the discoveries made in this dissertation extends beyond basic knowledge of microglia biology. The implications of microglia in neurodevelopmental, psychiatric and neurodegenerative disorders, many of which have no known cures, underscore these efforts as imperative for improving human health.

Committee Chair

Qingyun Li

Committee Members

Claudia Han; Erik Musiek; Gregory Wu; Marco Colonna

Degree

Doctor of Philosophy (PhD)

Author's Department

Biology & Biomedical Sciences (Neurosciences)

Author's School

Graduate School of Arts and Sciences

Document Type

Dissertation

Date of Award

4-21-2026

Language

English (en)

Author's ORCID

https://orcid.org/0000-0002-3531-2229

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Available for download on Thursday, April 20, 2028

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