Withaferin A Targets Intermediate Filaments GFAP and Vimentin in A Model of Retinal Gliosis

Gliosis is a biological process that occurs during injury repair in the central nervous system (CNS) and is characterized by the overexpression of intermediate filaments (IFs) glial fibrillary acidic protein (GFAP) and vimentin. A common thread in many retinal diseases is reactive Müller cell gliosis, an untreatable condition that leads to tissue scarring and even blindness. Here, we demonstrate that the vimentin-targeting small molecule withaferin A (WFA) is a novel chemical probe of GFAP. Using molecular modeling studies that build on the x-ray crystal structure of tetrameric vimentin rod 2B domain we reveal that the WFA binding site is conserved in corresponding domain of tetrameric GFAP. Consequently, we demonstrate that WFA covalently binds soluble recombinant tetrameric human GFAP at cysteine 294. In cultured primary astrocytes, WFA binds to and downregulates soluble vimentin and GFAP expression to cause cell cycle G0/G1 arrest. Exploiting a chemical injury model that overexpresses vimentin and GFAP in retinal Müller glia, we demonstrate that systemic delivery of WFA downregulates soluble vimentin and GFAP expression in mouse retinas. This pharmacological knockdown of soluble IFs results in the impairment of GFAP filament assembly and inhibition of cell proliferative response in Müller glia. We further show that a more severe GFAP filament assembly-deficit manifests in vimentin-deficient mice, which is partly rescued by WFA. These findings illustrate WFA as a chemical probe of type III IFs and illuminate this class of withanolide as a potential treatment for diverse gliosisdependent CNS traumatic injury conditions and diseases, and for orphan IF-dependent pathologies. The overexpression of glial fibrillary acidic protein (GFAP) with vimentin is a hallmark of reactive gliosis in the central nervous system (CNS) (1,2). These IFs are expressed by reactive astrocytes and macro and microglia during traumatic and inflammatory injury and in a range of CNS degenerative diseases (2). In fact, an enigma of major retinal diseases, including agerelated macular degeneration, glaucoma, diabetic retinopathy and retinopathy of prematurity, is retinal gliosis, for which there is no available clinical treatment (3-5). Important fundamental insights on the structural and mechanical functions of IFs (6,7) have now been validated in mouse lines deficient in type III IFs (2). These studies have illuminated that, whereas overexpression of vimentin and GFAP during CNS stress response and injury repair contributes to scar formation (8), their deficiency can be protective of tissue functions in certain contexts. For instance, pathogenic angiogenesis is impaired in vimentin-deficient (Vim KO) mice due to decreased ability of newly formed blood vessels to cross the retinal inner limiting membrane in the model of hypoxiainduced retinal neovascularization (9). Interestingly, that study also identified in vimentin and GFAP double deficient (Vim GFAP dKO) http://www.jbc.org/cgi/doi/10.1074/jbc.M109.093765 The latest version is at JBC Papers in Press. Published on January 4, 2010 as Manuscript M109.093765 Copyright 2010 by The American Society for Biochemistry and Molecular Biology, Inc. by gest on O cber 0, 2017 hp://w w w .jb.org/ D ow nladed from