PROJECT SUMMARY/ABSTRACT The long-term goal of this project is to understand the functions of selective autophagy in corneal inflammation as well as corneal/limbal epithelial differentiation. Selective autophagy is a specialized form of autophagy and can control signaling pathways in cells by specifically degrading key components of these pathways. Even though our studies have advanced our understanding on the roles of non selective autophagy in corneal/limbal epithelia, the roles of selective autophagy in corneal inflammation and corneal/limbal epithelial differentiation remain unclear. Single-cell RNA-sequencing (scRNA-seq) methodologies enable the interrogation of relatively rare cell populations (e.g., stem and early transit amplifying (TA) cells). This technology also allows the comparison of different physiological and/or disease states. Thus, we took advantage of Beclin1+/- (Beclin1 het) mice, a well-established mouse model with compromised autophagy and conducted a scRNA-seq analysis in the ocular anterior segmental tissue from wild-type (WT) and beclin1 het mice. scRNA-seq data suggest that autophagy plays a significant role in response to corneal inflammation, epithelial differentiation, and the regulation of Notch signaling. Our preliminary data show that genetic attenuation of autophagy enhances corneal inflammation. Interestingly, TRAF2, a positive regulator of inflammation, is regulated by NDP52- mediated selective autophagy. Furthermore, we have demonstrated that attenuation of p62-mediated selective autophagy markedly increased: (i) stem/TA cell enriched human limbal epithelial cell (HLEC) differentiation; and (ii) Notch intracellular domain (Notch ICD, an active form of Notch) in HLECs in a proteosome independent way. The scRNA-seq data also suggested that Wdfy1, which is preferentially expressed in the mouse limbal epithelial basal layer, was positively regulated by autophagy. Knockdown of Wdfy1 enhanced HLEC differentiation. Therefore, we will question if: (i) NDP52-mediated selective autophagy degrades TRAF2 and consequently has an inhibitory role in the inflammatory response in both corneal epithelial cells and macrophages (Aim1); and (ii) downregulation of p62-mediated selective autophagy allows for limbal epithelial differentiation via increasing Notch ICD (active form) and thus decreasing Wdfy1 expression (Aim2). To accomplish these goals, we will capitalize on our ability to conduct gain- and loss-of-function studies of selective autophagy-related genes in submerged and 3D organotypic raft cultures of primary limbal/corneal epithelial cells as well as mice. Knowledge gained from this study will provide molecular insights into how selective autophagy regulates corneal inflammation and limbal epithelial differentiation. A better understanding of the physiological importance of selective autophagy in corneal inflammation and differentiation will translate into the development of novel therapies by targeting selective autopha...