Applying spatial transcriptomics and mouse molecular genetics to understand the mechanisms of fibrotic scarring for promoting neural repair after spinal cord injury

PROJECT SUMMARY Spinal cord injury (SCI) is a neurological condition that induces a wide array of transcriptional and functional changes to a variety of cell types in the central nervous system (CNS), including fibroblasts. After SCI, fibroblasts undergo a wound healing response called fibrotic scarring, in which they activate, proliferate, and migrate to the lesion site to produce extracellular matrix proteins. While acute fibrotic scarring is critical in orchestrating tissue repair and preserving tissue integrity; aberrant, chronic fibrotic scarring, has been implicated to contribute to the CNS limited regenerative potential after SCI. Chronic fibrotic scarring remains poorly understudied in elucidating the cellular mechanisms that govern its development, formation, extracellular influence. Transforming growth factor beta (TGF-β) has been extensively characterized as the central regulator of fibrotic scarring in the liver, heart, lung, and kidney while also being implicated in the CNS through sc-RNAseq and pharmacological evidence. However. there is no genetic, cell-specific evidence establishing the role of TGF-β and chronic fibrotic scarring after SCI and how the fibrotic scar shapes the injury site. This proposal aims to determine the contribution of TGF-β signaling in chronic fibrotic scarring and its role on the extracellular microenvironment using mice transgenic models and spatial transcriptomics. My overall hypothesis is that fibrotic scarring is modulated by TGF-β signaling and contributes to a nonpermissive microenvironment for neural repair and recovery after SCI. To test this hypothesis, I will conditionally delete TGF-β receptor 2 (TGFBR2) specifically in fibroblasts using a collagen type 1 alpha 2 promoter and assess fibrotic scarring in mouse models of SCI. Aim 1 will be focused on determining if fibrotic scarring is driven by TGF-β signaling in fibroblasts following SCI and its impact on hindlimb functional recovery. Recovery will be assessed in three locomotor behavioral assays. Aim 2 will leverage spatial transcriptomics to examine the impact of fibrotic scarring attenuation on various spatially resolved injury site cell populations, structures, and the overall microenvironment. Impacts on the genetic signatures of astrocytes, macrophages, and microglia will be of specific interest due to their role in neuroinflammation and neurotoxicity after SCI. Aim 3 will focus on manipulating fibrotic scarring to promote corticospinal tract axon regeneration after a dorsal hemisection SCI. This work will provide better insight into the cellular and molecular processes involved in fibrotic scarring after SCI, which will lead to more effective therapeutic interventions for spinal cord injury in the future.