Project Summary/Abstract: Poor wound healing is a major health issue in insulin-resistant diabetes. Degeneration of nerves in diabetes contributes to the delay in healing and is associated with reduction in basal keratinocyte migration across the wound bed. Improved understanding of the communication between neurons and keratinocytes, which is critical for wound repair, may lead to new interventions. Cutaneous sensory nerves are now recognized to comprise several subtypes characterized by different markers and functions. Identifying the neuron subtype(s) involved in wound healing may provide clues to new therapeutic directions. To explore the impact of specific neuron-keratinocyte communication on wound healing, we will initially ablate specific neuron subsets in healthy mouse skin using genetic expression of diphtheria toxin receptors and will evaluate the impact on healing of splinted wounds. We will confirm subgroup neurons involvement by chemogenetically introducing and activating stimulatory designer receptors (DREADDs), which we expect to accelerate healing if activated in a nerve subset that is important for normal healing. We have shown that nerve degeneration results from neuronal hyperexcitability and that introducing inhibitory DREADDs into the majority of sensory nerves in a mouse model of diabetes both suppresses this excitation and reverses the nerve degeneration, although the impact on healing is unexplored. Building on this observation, we will introduce these inhibitory DREADDs into specific neuronal subsets in diabetic mice to delineate the impact on healing and whether one or more subtype of neurons is key to the degeneration and healing impairment. These studies in healthy and diabetic mice will allow us to capture unwounded and wound edge skin for conducting transcriptomic analysis. In particular, we will evaluate changes in expression of G-protein coupled receptors (GPCRs). Activation of these GPCRs with selective agonists should replicate the observed effects of DREADDs. We anticipate that these studies will implicate targets in keratinocytes for small molecule drug discovery using high throughput technology to assess calcium signals, migration, proliferation, and toxicity. Best candidates will be tested topically in cultured 3D human diabetic wound models and subsequently in our type 2 diabetic mouse models towards finding new interventions to promote wound healing. These proposed studies will increase our understanding of the role that nerve afferent subsets play in diabetic vs. normal wound healing. Furthermore, by identifying responsible subsets of nerves and gene expression patterns that are altered during diabetic wound healing, we can screen and advance preclinical trials of new small molecules that can be applied topically to promote healing of diabetic wounds.

Project Narrative: Impaired wound healing in individuals with diabetes remains a major health issue that demands better treatment. We propose that specific subsets of nerves that extend into skin are critical for wound healing but their communication with skin cells is defective in diabetes. Using various genetic techniques, we will study the role that nerve subsets play in healing and, based on alterations we discover or induce in diabetic skin cells and nerves, will find new small drugs that can be applied topically to promote wound healing.