TY - JOUR
T1 - Withaferin A targets intermediate filaments glial fibrillary acidic protein and vimentin in a model of retinal gliosis
AU - Bargagna-Mohan, Paola
AU - Paranthan, Riya R.
AU - Hamza, Adel
AU - Dimova, Neviana
AU - Trucchi, Beatrice
AU - Srinivasan, Cidambi
AU - Elliott, Gregory I.
AU - Zhan, Chang Guo
AU - Lau, Daniel L.
AU - Zhu, Haiyan
AU - Kasahara, Kousuke
AU - Inagaki, Masaki
AU - Cambi, Franca
AU - Mohan, Royce
PY - 2010/3/5
Y1 - 2010/3/5
N2 - 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 G0/G1 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 with anolide as a potential treatment for diversegliosis dependent central nervous system traumatic injury conditions and diseases, and for orphan IF-dependent pathologies.
AB - 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 G0/G1 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 with anolide as a potential treatment for diversegliosis dependent central nervous system traumatic injury conditions and diseases, and for orphan IF-dependent pathologies.
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U2 - 10.1074/jbc.M109.093765
DO - 10.1074/jbc.M109.093765
M3 - Article
C2 - 20048155
AN - SCOPUS:77951234639
VL - 285
SP - 7657
EP - 7669
IS - 10
ER -