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R. R. Paranthan, P. Bargagna-Mohan, C. Srinivasan, D. L. Lau, R. Mohan; Probing Biological Functions of Type III Intermediate Filament Nanotubes in vivo. Invest. Ophthalmol. Vis. Sci. 2009;50(13):2427.
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Vimentin and glial fibrillar acidic protein (GFAP) are type III intermediate filaments (IFs) that co-assemble into ‘biological nanotubes" by lateral elongation. This process is dynamically elaborated in retinal Muller glia, which is an elegant system for studying IF pathophysiology. We exploited the small molecule vimentin-targeting agent, withaferin A (WFA) (Bargagna-Mohan et al., Chemistry & Biology 2007), to investigate how GFAP IFs are structurally regulated under stress in genetic and drug-induced deficiency of vimentin.
We employed the alkali burn injury model (brief application of 0.15 N NaOH with removal of corneal epithelium) to cause retinal gliosis in wild-type 129 Svev or vimentin-deficient (vim-/-) mice. Mice were systemically treated with 2 mg/kg withaferin A for seven days to cause conditional downregulation of vimentin expression in vivo. Mouse eyes were cryo-embedded and processed for immunohistochemical analysis using epifluorescence detection. Soluble tetrameric and polymeric forms of GFAP and vimentin were fractionated by their differential solubility and analyzed by western blotting. Tissue sections stained for GFAP (at least 16 per sample group) were characterized by computer-assisted measurement of IFs and validated by statistical analysis.
We show that GFAP displays characteristic laminar staining of Muller glia, showing expression across the entire retina. In vim-/- mice, formation of GFAP-homotypic filaments is impaired, and characterized by reduction in polymer length and isoform diversity. Greater abundance of GFAP isoforms is confined to soluble tetrameric pools in vimentin deficiency, as apposed to the abundance of GFAP isoforms being found in insoluble pools of wild-type mice. WFA treatment in wild-type mice causes GFAP-stained IFs to also show such a reduction of filament length and isoform diversity. In vim-/- mice, WFA treatment stimulates GFAP IF formation, which is characterized by longer filaments and denser filament structures. By comparison of staining data and western blot analyses, we identified an unique GFAP isoform that is produced in Muller glia of vim-/- mice by WFA treatment. We speculate that this GFAP isoform is targeted by WFA and partially restores a vimentin-like function to assist in homotypic GFAP polymer growth in vivo.
Our findings identify novel features of the type III IFs that are elaborated under different genetic, environmental and pharmacological conditions. We plan to exploit these characteristics for tissue regeneration applications in the future.
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