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P. Bargagna-Mohan, R. R. Paranthan, V. Neamtu, G. I. Elliott, R. Mohan; Vimentin and Glial Fibrillar Acidic Protein are Targeted by Withaferin A in Retinal Gliosis. Invest. Ophthalmol. Vis. Sci. 2009;50(13):69.
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© ARVO (1962-2015); The Authors (2016-present)
Reactive gliosis is an insidious pathogenic process resulting from trauma injury to the central nervous system (CNS) and it also presents in retinal diseases such as age-related macular degeneration, diabetic retinopathy and glaucoma. A hallmark feature of this process is the overexpression of type III intermediate filaments (IFs), glial fibrillar acidic protein (GFAP) and vimentin. We have exploited withaferin A (WFA) to cause vimentin-target deficiency (Bargagna-Mohan et al., Chemistry & Biology 2007) in the mouse retina. Here we validate our hypothesis that chemical genetic perturbation of vimentin function(s) can phenocopy the genetic deficiency of this IF target in vivo.
We employed the alkali burn injury model (brief application of 0.15 N NaOH with removal of corneal epithelium) to cause retinal gliosis. Wild-type 129 Svev or vimentin-deficient mice were systemically treated with injections of withaferin A for seven days and killed. Mouse eyes were processed for cryo-embedded immunohistochemical analysis with epifluorescence detection and western blot analysis. IF expression in Muller cells was characterized from digital images by computer-assisted measurement of glial fibrillar acidic protein (GFAP)-stained structures and data validated by statistical analysis.
We previously identified that vimentin and GFAP expression is targeted by WFA in cultured astrocytes (Bargagna-Mohan P. et al. IOVS 2008; ARVO E-abstract 4007). Here we show in the alkali injury model of retinal gliosis that the overexpression of vimentin and GFAP in ganglion astrocytes and Muller cells is also pharmacologically targeted by WFA in vivo. Wild-type mice treated with WFA show downregulated vimentin expression, with GFAP-stained IFs showing reduction in filament lengths and isoform distribution caused by drug treatment. Under stress, this WFA-induced phenotype mimics the functional perturbation of GFAP IFs produced by genetic deficiency of vimentin. Expression levels of cellular retinaldehyde binding protein and glutamine synthase were not affected by WFA or vimentin genetic deficiency, but their localization paralleled that of GFAP in each treatment group.
Our findings identify WFA as an important chemical genetic probe of IF functions in retinal Muller cells. The use of WFA to probe the pathobiology of IFs in other CNS stress conditions warrant further investigation considering that previous studies have defined their pathological role in gliosis and glial scar formation in trauma models of injury.
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