Mechanisms of reticulophagy and ER stress mitigation in epidermis

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Project Summary/Abstract The epidermis forms a multi-layered epithelium that serves as a protective shield for the body, preventing dehydration and pathogen invasion. Its principal cellular constituents, keratinocytes, continually regenerate the cutaneous barrier via a specialized form of differentiation as they move outward in the tissue. At the end of their life in the skin’s outermost layers, keratinocytes initiate a cellular remodeling program called cornification in which they eliminate their organelles to form flattened, keratinized cells. The importance of this process is underscored by many disorders of cornification linked to defective keratinocyte maturation. Despite the advent of biologic treatments for inflammatory skin diseases, development of similar targeted therapies for epidermal barrier dysfunction has been limited by an incomplete understanding of the pathways driving keratinocyte differentiation. The proposed aims address this knowledge gap by applying advanced microscopy, gene editing, and optogenetics to define the mechanisms mediating organelle degradation in human epidermis. This proposal builds on the PI’s K08 project, which found that cornifying keratinocytes induce autophagy, a lysosomal degradation pathway, to break down organelles. The K08-funded work showed that differentiating keratinocytes upregulate an autophagy receptor, NIX, which marked mitochondria and instructed cells in the upper tissue layers to break down these organelles, a step that was essential for epidermal maturation. Our planned experiments will expand the K08 project scope to include the endoplasmic reticulum (ER), testing the hypothesis that keratinocytes utilize distinct autophagy receptors to orchestrate breakdown of the ER (called reticulophagy). Aim 1 will determine how reticulophagy drives programmed ER degradation during cornification and Aim 2 will assess if reticulophagy mitigates damage from ER stress in keratinocytes. Preliminary studies identified candidate receptors that initiate reticulophagy in either a constitutive manner during cornification or upon organelle injury due to ER stress. We will use gain- and loss-of-function approaches in organotypic skin to determine the role of these receptors in directing epidermal morphogenesis and mitigating ER stress. As well, we will leverage live biosensor imaging and optogenetic tools to define how reticulophagy alters signaling mediators that control keratinocyte differentiation, including calcium and reactive oxygen species. Results from the planned work promise to identify novel strategies for normalizing epidermal differentiation in disorders of cornification but also for repairing barrier dysfunction in common diseases like atopic dermatitis and psoriasis. Augmenting the investment by the University of Washington to ensure his success as a physician-scientist, R03 funding will advance the PI’s career development and position him to compete for R01-level funding as he transitions to an independent investigator. The proposed project enhances the PI’s expertise at the intersection of cell biology and dermatology, building an innovative research program that complements his clinical specialty and provides a platform for future translational work to find novel treatments for skin barrier disorders.