Withaferin A targets intermediate filaments glial fibrillary acidic protein and vimentin in a model of retinal gliosis.
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.
Bargagna-Mohan
Paola
P
Departmentsof Ophthalmology & Visual Sciences, University of Kentucky, Lexington, Kentucky 40536, USA.
Paranthan
Riya R
RR
Hamza
Adel
A
Dimova
Neviana
N
Trucchi
Beatrice
B
Srinivasan
Cidambi
C
Elliott
Gregory I
GI
Zhan
Chang-Guo
CG
Lau
Daniel L
DL
Zhu
Haiyan
H
Kasahara
Kousuke
K
Inagaki
Masaki
M
Cambi
Franca
F
Mohan
Royce
R
D000818:Animals; D001253:Astrocytes / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D002453:Cell Cycle / Q000187:drug effects; D002478:Cells, Cultured; D056743:Cyclin D3 / Q000378:metabolism; D050760:Cyclin-Dependent Kinase Inhibitor p27 / Q000378:metabolism; D004875:Ergosterol / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D005904:Glial Fibrillary Acidic Protein / Q000235:genetics / Q000378:metabolism*; D005911:Gliosis / Q000378:metabolism / Q000473:pathology; D006801:Humans; D051379:Mice; D018345:Mice, Knockout; D008958:Models, Molecular; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012160:Retina / Q000187:drug effects / Q000378:metabolism / Q000473:pathology; D012162:Retinal Degeneration / Q000378:metabolism / Q000473:pathology; D014746:Vimentin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D054358:Withanolides
D056743:Cyclin D3; D005904:Glial Fibrillary Acidic Protein; D011994:Recombinant Proteins; D014746:Vimentin; D054358:Withanolides; D050760:Cyclin-Dependent Kinase Inhibitor p27; C009684:withaferin A; D004875:Ergosterol