Abstract
Purpose: :
During gliosis, Muller glial cells increase expression of type III intermediate filaments (IFs), glial fibrillary acidic protein (GFAP) and vimentin and their reorganization and disassembly is required for cells to re-enter the cell cycle. Therefore, controlling IF assembly is a desired mechanism to limit Muller cell proliferation during retinal injury. We previously reported that Withaferin A (WFA) targets vimentin to downregulate its expression in vitro and in vivo. In this report, we investigated WFA’s control of Muller cell cycle in a model of retinal gliosis.
Methods: :
Retinal gliosis was induced in wild-type (WT) or vimentin-deficient (Vim KO) 129 Svev mice by applying a drop of 0.15 N NaOH to the cornea and removing of the epithelium, according to the standard corneal alkali burn injury model. After seven days of systemic administration of WFA at a dose of 2 mg/kg/day, mice were sacrificed and eyes processed for immunohistochemical and western blot analysis. Anti-GFAP, p27Kip1, cyclin D3 and proliferating cell nuclear antigen (PCNA) antibodies were used.
Results: :
Cyclin D3 and PCNA levels were elevated in injured WT retinas and associated with extended GFAP filaments being expressed in Muller glial cells. WFA treatment potently downregulated the expression soluble vimentin and GFAP and the filamentous expression of GFAP was found to be fragmented. This phenotype was associated with downregulation of cyclin D3 and PCNA expression. In injured Vim KO mice, soluble GFAP expression was overexpressed, while GFAP filaments were severely fragmented. Injured Vim KO mice expressed increased levels of cyclin D3 and PCNA and their sustained protein levels with WFA treatment was associated with increase in the size of GFAP filaments. On the other hand, injured WT mice downregulated p27Kip1 expression and this protein's expression was reversed by WFA treatment. In Vim KO retinas, p27Kip1 expression was potently downregulated with injury and WFA treatment upregulated p27Kip1 expression.
Conclusions: :
Collectively, these findings demonstrate that WFA differentially affects the expression of critical cell cycle growth regulators in the retinas of injured WT and Vim KO mice. We associate WFA's activity on Muller cell cycle regulation to its IF-targeting activity. This is the first demonstration of a small molecule that directly targets type III IFs in vivo to control reactive gliosis in the retina.
Keywords: Muller cells • stress response • transgenics/knock-outs