Abstract
Gliosis is a biological process that occurs during injury repair in the central nervous system and is characterized by the overexpression of the 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 the 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 down-regulates soluble vimentin and GFAP expression to cause cell cycle G(0)/G(1) arrest. Exploiting a chemical injury model that overexpresses vimentin and GFAP in retinal Müller glia, we demonstrate that systemic delivery of WFA down-regulates 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 gliosis-dependent central nervous system traumatic injury conditions and diseases, and for orphan IF-dependent pathologies.
Highlights
The overexpression of glial fibrillary acidic protein (GFAP)2 with vimentin is a hallmark of reactive gliosis in the central nervous system (CNS) [1, 2]
This is evident in primary amino acid sequences that make up the withaferin A (WFA) binding site having ϳ66% identity and 84% similarity between human GFAP and vimentin peptides, and as well, these sequences are evolutionarily conserved among vertebrate species, which suggests strong functional relevance [23]
Using chemical genetic approaches coupled with affinity purification strategies, we overcame this challenge with the discovery that WFA binds at a novel protein-protein interface of tetrameric vimentin [10]
Summary
General Methods—WFA was purchased from a commercial vendor (Chromadex, Santa Ana, CA) and WFA-Bt synthesis has been previously described [10]. Protein extracts of the retina or cell cultures were subjected to Western blotting as previously described [10] using primary antibodies (see supplemental data Table S2). Isolation of WFA-Bt-binding Proteins by Affinity Chromatography—The WFA-Bt ligand binding studies in mouse astrocytes were performed as previously reported for endothelial cells [10]. WFA-Bt Ligand Binding Studies with Recombinant Tetrameric GFAP—Purified soluble human recombinant GFAP [22] was incubated with vehicle or 5 M WFA for 30 min at 37 °C. Liquid Chromatography-Mass Spectrometric (LC-MS) Analysis of GFAP—Purified recombinant human tetrameric GFAP was treated with 5 M WFA or vehicle for 1 h and subjected to tryptic digestion. The hypothesis is strongly supported as the p values are larger than 0.1
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