The role of Wnt signaling in treating glucocorticoid-induced glaucoma

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PROJECT SUMMARY/ABSTRACT Glucocorticoid (GC)-induced ocular hypertension (OHT) and glaucoma (GIG) occur in ~40% of the general population and ~90% primary open angle glaucoma (POAG) patients. Our published studies and preliminary data show that canonical Wnt signaling activation inhibits glucocorticoid receptor signaling and GC-induced OHT. We hypothesize that activation of canonical Wnt signaling inhibits GIG, and this inhibition requires nucle- ar β-catenin, glucocorticoid receptor (GR), and epigenetic modification enzymes including HDAC1 and MeCP2. Our goal is to elucidate the mechanism of GIG. Our objective is to inhibit GC-induced OHT without compromis- ing GC’s anti-inflammatory effects. Our rationale is that Wnt activation is a potential approach to prevent and treat OHT/GIG, and studying GIG will help us to better understand POAG. Here, we propose three specific aims to test our central hypothesis. SA1. Explore how Wnt signaling modulates GC-induced OHT and anti- inflammatory effects in mouse eyes. We will determine SA1.1) If canonical Wnt signaling activation inhibits GC-induced OHT using canonical Wnt signaling reporter mice; SA1.2) If β-catenin is necessary for inhibiting GC-induced OHT using conditional knockout mice; and SA1.3) If activated canonical Wnt signaling compro- mises GC’s anti-inflammatory effects using mouse uveitis models. SA2. Determine the role of canonical Wnt signaling in glucocorticoid responsiveness in human eyes. SA2.1) Determine if Dkk1 (a Wnt signaling in- hibitor) is elevated while canonical Wnt signaling is inhibited. We will collect aqueous humor and trabecular meshwork tissues from GIG eyes as well as from non-GIG eyes to study Dkk1, β-catenin and axin2 levels. We expect to see higher Dkk1 levels but lower β-catenin and axin2 in GIG eyes. SA2.2) Determine if canonical Wnt signaling affects GC responsiveness in human donor eyes. We will perfuse paired donor eyes with DEX to identify responsiveness. For responder eyes, we will co-treat one eye with CHIR (a small molecule Wnt signal- ing activator) and expect it will relieve OHT. For non-responder eyes, we will co-treat one eye with Dkk1 and expect it will induce OHT. SA3. Determine the molecular mechanism of canonical Wnt signaling and β- catenin in GC responsiveness and TM homeostasis. SA3.1) Determine Wnt signaling induced gene ex- pression and associated chromatin accessibility in GC response. We will determine whether Wnt signaling has differential regulation of GC-induced genes using RNA-seq and Assay for Transposase-Accessible Chromatin with high-throughput Sequencing (ATAC-seq). SA3.2) Determine the role of β-catenin-GR/HDAC1/MeCP2 nu- clear complex in regulating GR signaling. The components and binding pattern of this complex will be deter- mined using Co-IP, Mass-Spec, FLIM-FRET, and ChIP-qPCR. In summary, we propose a novel approach to remove GC side effects without compromising its therapeutic effects. The compound that we test may serve as a lead compound in future drug development for GIG, and it would enable long-term, undisrupted use of GCs for various eye diseases upon the success of this study.