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Royce Mohan, Riya R. Paranthan, Paola Bargagna-Mohan; A Chemical Genetic Approach for Targeting Intermediate Filaments: Towards Rational Drug Design for Retinal Gliosis. Invest. Ophthalmol. Vis. Sci. 2011;52(14):5660.
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Gliosis is an insidius process that underlies retinal and choroidal scarring. It is a common pathology from trauma and widely prevalent in many leading neurodegenerative diseases. The two critical targets of gliosis, vimentin and glial fibrillary acidic protein (GFAP), are newly discovered binding receptors of the potent inhibitor withaferin A (WFA) (Bargagna-Mohan et al., JBC 14: 7657-7669 ; 2010). The goal of this study is to rationally develop WFA for treatment of retinal gliosis by investigating its extended efficacy on GFAP targeting.
Wild type and vimentin-deficient (Vim KO) 129Svev mice were subjected to alkali burn with epithelial scrape injury to elicit retinal gliosis. Withaferin A (2 mg/kg/day; intraperitoneal injection) was provided daily for 7 and 14 days. Proteins were separated into soluble (low salt extracted) and insoluble (high salt extracted) fractions for western blot analysis. Cryosections of mouse eyes and western blots were probed with antibodies against vimentin, GFAP and cyclin dependent kinase inhibtor p27Kip1.
In injured wild-type mice, insoluble polymeric GFAP was induced 12-fold over control, with vimentin expression being induced 1.5-fold over its basal high levels in Muller glia at day 7. WFA potently downregulated soluble GFAP at days 7 and 14 resulting in filament fragmentation and also downregulated expression of polymeric GFAP. To validate WFA directly targets GFAP independent of vimentin, we exploited Vim KO mice for target validation studies. The in vivo expression of soluble GFAP is induced 4-fold at 7 days reaching 16-fold higher levels at day 14. WFA treatment reduces partly (2-fold) expression of GFAP at 7 days and compleletly abrogates all GFAP isoform expression at 14 days in Vim KO retinas. Thus, polymeric GFAP staining in Muller glia was observed only at the end-feet at day 14 in Vim KO mice, as observed in wild type WFA-treated mice. Confirming that these effects of WFA result in Muller glial cell cycle arrest is shown by restoration of injury-induced downregulation of p27Kip1 expression at days 7 and 14 to levels found in uninjured mice.
We provide the first significant milestone in intermediate filament drug discovery showing the efficacy of WFA to dually target vimentin and GFAP exression in vivo during retinal gliosis. This study thus illustrates use of the chemical genetic approach for drug and target discovery, which we will discuss as a new paradigm for complex diseases.
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