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Principal Investigator: Han Peng
Organization: NORTHWESTERN UNIVERSITY AT CHICAGO
Fiscal Year: 2024
Award: $481,939
Funding agency: National Eye Institute
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 autophagy pathways for corneal diseases in
which extensive inflammation and aberrant differentiation are features (e.g., chemical burns).
Terms: <2019 novel corona virus><2019 novel coronavirus><2019-nCoV><20S Catalytic Proteasome><20S Core Proteasome><20S Proteasome><20S Proteosome><3-D><3-Dimensional><3D><ACE2><Alkalies><Anterior><Area><Arginine-Serpin><Atlases><Attenuated><Autophagocytosis><Autophagosome><Basal Cell><Basal Layer><Binding><Blindness><Body Tissues><COVID-19 virus><COVID19 virus><Cell Body><Cell Communication and Signaling><Cell Differentiation><Cell Differentiation process><Cell Line><Cell Signaling><CellLine><Cells><Chemical Burns><CoV-2><CoV2><Cornea><Corneal Diseases><Corneal Disorder><Data><Development><Differentation Markers><Differentiation Antigens><Differentiation Markers><Disease><Disorder><Down-Regulation><Epithelial Cells><Epithelium><Eye Burns><Eye diseases><Gene Expression><Genes><Genetic><Goals><Human><Immune infiltrates><Immunoglobulin Enhancer-Binding Protein><In Vitro><Inflammation><Inflammatory><Inflammatory Response><Intracellular Communication and Signaling><Invaded><KO mice><Knock-out Mice><Knockout Mice><Knowledge><Macropain><Macrophage><Macroxyproteinase><Marker Antigens><Mediating><Methodology><Mice><Mice Mammals><Modeling><Modern Man><Molecular><Molecular Interaction><Monocyte Arg-Serpin><Mucins><Mucus Glycoprotein><Multicatalytic Proteinase><Murine><Mus><Mφ><NF-kB><NF-kappa B><NF-kappaB><NFKB><Nuclear Factor kappa B><Nuclear Transcription Factor NF-kB><Null Mouse><PAI-2><PLANH2><Pathway interactions><Patients><Physiologic><Physiological><Plasminogen Activator Inhibitor 2><Plasminogen Activator Inhibitor Type II><Play><Population><Process><Proliferating><Prosome><Proteasome><Proteasome Endopeptidase Complex><Proteins><Proteosome><Receptor Protein><Regulation><Reporting><Rete Malpighii><Role><SARS corona virus 2><SARS-CO-V2><SARS-COVID-2><SARS-CoV-2><SARS-CoV2><SARS-associated corona virus 2><SARS-associated coronavirus 2><SARS-coronavirus-2><SARS-related corona virus 2><SARS-related coronavirus 2><SARSCoV2><Serine or Cysteine Proteinase Inhibitor Clade B Member 2><Severe Acute Respiratory Coronavirus 2><Severe Acute Respiratory Distress Syndrome CoV 2><Severe Acute Respiratory Distress Syndrome Corona Virus 2><Severe Acute Respiratory Distress Syndrome Coronavirus 2><Severe Acute Respiratory Syndrome CoV 2><Severe Acute Respiratory Syndrome-associated coronavirus 2><Severe Acute Respiratory Syndrome-related coronavirus 2><Severe acute respiratory syndrome associated corona virus 2><Severe acute respiratory syndrome coronavirus 2><Severe acute respiratory syndrome related corona virus 2><Signal Pathway><Signal Transduction><Signal Transduction Systems><Signaling><Strains Cell Lines><Stratum Basale><Stratum Germinativum><Stress><TRAF2><TRAF2 gene><TRAP3><Technology><Testing><Tissues><Transcription Factor NF-kB><Translating><Type 2 Plasminogen Activator Inhibitor><Wuhan coronavirus><angiotensin converting enzyme 2><angiotensin converting enzyme II><attenuate><attenuates><attenuation><autophagy><biological signal transduction><burn model><cellular differentiation><conditional knock-out><conditional knockout><cornea disorder><corneal><corneal epithelial><corneal epithelium><coronavirus disease 2019 virus><coronavirus disease-19 virus><cultured cell line><cytokine release syndrome><cytokine storm><developmental><dry eye><eye disorder><eye dryness><gain of function><global gene expression><global transcription profile><hCoV19><immune cell infiltrate><improved><in vivo><inhibition of autophagy><innovate><innovation><innovative><insight><kappa B Enhancer Binding Protein><knock-down><knockdown><limbal><loss of function><mouse model><multicatalytic endopeptidase complex><murine model><nCoV2><new drug target><new druggable target><new pharmacotherapy target><new therapeutic target><new therapy target><notch><notch protein><notch receptors><novel><novel drug target><novel druggable target><novel pharmacotherapy target><novel therapeutic target><novel therapy target><nuclear factor kappa beta><ocular disease><ocular disorder><ophthalmopathy><overexpress><overexpression><pathway><receptor><response><scRNA-seq><self-renew><self-renewal><single cell RNA-seq><single cell RNAseq><single cell expression profiling><single cell transcriptomic profiling><single-cell RNA sequencing><social role><stem><three dimensional><transcriptome><vision loss><visual loss>