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P. Bargagna-Mohan, N. Dimova, R. Paranthan, F. Cambi, R. Mohan; Withaferin A, a Potential Regulator of Astrocyte Reactivity. Invest. Ophthalmol. Vis. Sci. 2008;49(13):4007. doi: https://doi.org/.
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Glial cells have an important role in both normal tissue functions and pathology of the brain and retina. During reactive gliosis, these cells undergo morphological changes and increase the production of two intermediate filaments (IFs), glial fibrillary acidic protein (GFAP) and vimentin. We previously showed that the angiogenesis inhibitor withaferin A (WFA) directly binds the IF protein vimentin in vascular endothelial cells and affects its function (Bargagna-Mohan et al., Chemistry & Biology 2007). We hypothesize that WFA may also modulate the expression and function of vimentin and GFAP in astrocytes.
Primary astrocytes were isolated from cerebral hemispheres of 2 day-old rats and maintained in D-MEM medium supplemented with 10% FBS. We performed dose-response and time-course studies to characterize the astrocytic reactions to WFA under normal and inflammatory conditions. Analysis of GFAP, vimentin and IΚB-α expression was determined by western blot analysis and immunohistochemistry.
GFAP and vimentin expression was down-regulated by WFA in a dose-dependent manner. Concentrations of WFA greater than 2µM caused cell-shape changes, reduction in process formation, and localization of GFAP was shown to be predominantly perinuclear. Upon longer treatment at high doses, astrocytes underwent apoptosis. In the presence of inflammatory cytokine tumor necrosis factor-α, WFA induced cytoskeletal alterations became further extenuated and the expression of IΚB-α was also potently inhibited.
GFAP and vimentin are overexpressed in a wide range of neurodegenerative pathologies and injury situations in the retina, making these IFs an excellent candidate as indicators of neural distress. We showed that WFA downregulates the expression of GFAP and vimentin in astrocytes, suggesting that WFA possibly attenuates astrocytic reactivity. These findings provide a molecular basis for testing WFA (and related withanolides) as a chemical genetic tool to study IF functions and as potential treatment for neurodegenerative and retinal diseases.
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