Interrogating a white matter degeneration-specific astrocyte reactivity state and its role in governing repair-associated microglia specification and function.

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Project Summary Clearance of cellular debris from the degenerating central nervous system (CNS) white matter is inefficient. Debris accumulation, especially from myelin, precipitates maladaptive neuroinflammation which furthers disease progression and prevents repair. Chronic white matter degeneration that produces cellular debris and protracted innate immune inflammation is a hallmark of diverse disorders, including traumatic and ischemic CNS injury and Multiple Sclerosis (MS). The development of therapies aimed enhancing debris clearance to promote repair is thereby of broad translational relevance. Research aimed at uncovering central regulatory mechanisms underlying the inflammatory response that facilitates debris clearance after white matter insult is therefore prudent and necessary. The overarching objective of this research is to dissect molecular pathways of astrocyte- microglia interaction that govern pro-restorative CNS innate immune responses to white matter degeneration. The present research will investigate a newly identified mechanism of intercellular communication, through which a distinct subpopulation of white matter degeneration-reactive astrocytes regulate microglia specification and function required for the removal of inflammatory myelin debris and tissue repair. In Aim 1, we will use a transgenic, cell type-specific loss-of-function system, single-nucleus RNA-Sequencing (snRNA-Seq) and mouse models of acute and chronic white matter degeneration to assess the involvement of this unique astrocyte- mediated signaling pathway in regulating damage-responsive microglia specification required for myelin debris clearance and remyelination. In Aim 2, we will dissect the direct effects and receptor dependent mechanism of action for a novel astrocyte-derived molecular cue on microglia molecular profile, motility, and capacity for myelin debris phagocytosis using cultures of mouse primary and human iPSC-derived microglia. In Aim 3, we will determine cell-intrinsic regulatory pathways underlying molecularly and functionally distinct states of white matter astrocyte reactivity using cell-paired snRNA-Seq and single-nucleus Assay for Transposase-Accessible Chromatin using Sequencing (snATAC-Seq), over time, in a mouse model of chronic white matter degeneration. An integrative analysis pipeline will be used to generate gene regulatory programs of astrocyte reactivity, including dynamic changes in chromatin structure and context-specific combinations of transcriptional regulators (TR), which together govern genetic accessibility and distinct reactive changes in transcriptomic profile. Select TR of molecularly distinct states of white matter astrocyte reactivity will be functionally interrogated in vivo by loss-of-function studies. Together, this research will enhance the understanding of how astrocyte-microglia interactions shape CNS innate immune responses that enable white matter repair. These data may also inform new therapeutic avenues for harnessing restorative innate immune responses, involving astrocytes and microglia, to prevent or attenuate maladaptive white matter inflammation, as well as improve outcome and promote recovery after CNS injury and in disease.