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John Wizeman, Paola Bargagna-Mohan, Sean O'Rourke, Royce Mohan; Novel GFAP Species in Retinal Gliosis. Invest. Ophthalmol. Vis. Sci. 2013;54(15):1953. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
Retinal gliosis occurs after injury and is an insidious process that also undermines several leading retinal diseases. A characteristic hallmark of gliosis is the upregulation of the type III intermediate filaments (IF) glial fibrillary acidic protein (GFAP) and vimentin. In injury models, the absence of GFAP and its binding partner vimentin has been shown to be protective. We have used the GFAP/vimentin targeting small molecule withaferin A (WFA; Bargagna-Mohan JBC 2010) as a chemical probe of GFAP expression in an injury model.
C57BL/6N, 129SvEV vimentin-deficient or wild type 129SvEV mice were subjected to a corneal alkali injury (1N NaOH) followed by epithelial debridement. Mice were allowed to heal (3 to 5 days), after which retinal cups were isolated and placed into explant organ culture in medium containing DMEM/F12 +10% FBS and antibiotics. Retinal cultures were treated with either WFA or the proteasome inhibitor epoxomicin for 3 days and soluble tissue extracts (200 mM NaCl, 1% NP-40) were subjected to western blot analysis.
As expected, injury increased 52kDa soluble GFAP expression in the retina. Unexpectedly, there was injury-related increase in novel high molecular weight (100-200 kDa) GFAP species in retinas from all mouse strains. This high molecular weight GFAP species was also susceptible to perturbation by WFA, with 50 nM WFA decreasing total GFAP levels by ~2.4 fold as compared to an injured, untreated retina. Higher concentrations of WFA had an impact on polyubiquitination levels. At 1 μM WFA, the total amount of ubiquitinated species was decreased ~3.8 fold compared to the injured, untreated retina. Treatment with epoxomicin caused accumulation of ubiquitinated GFAP species, a change that was also modulated with the addition of WFA.
Collectively, our results suggest that injury causes an induction of soluble GFAP expression in retinal Müller glia indicative of a novel post-translational modification. Importantly, these high molecular weight, modified GFAP species were targeted by WFA, mediated by a mechanism independent of proteasome inhibition. The identification of the causative modification to GFAP could lead to a deeper understanding of the mechanisms of gliosis caused by either injury or disease. The interaction of GFAP with the ubiquitin proteasome pathway remains a critical mechanism that needs to be characterized.
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