Modulation of Muller cell membrane organization by 24S-hydroxycholesterol

Purpose Muller glial cells (MGC) are involved in the retinal homeostasis and they are sustaining cholesterol homeostasis. A lack or an excess of cholesterol in neurons can cause neuro-degeneration. Cholesterol is eliminated from the retina mainly as 24S-hydroxycholesterol (24S-OHC). 24S-OHC is produced by 24S-hydroxylase (CYP46A1) in retinal ganglion cells (RGC) and is overexpressed during glaucoma. the objective of this study was to determine whether 24S-OHC triggers MGC membrane dynamics involving lipid rafts and contribute to gliosis at early and late time points. Methods MGCs were grown in vitro from retinas of young rats. They were treated with 24S-OHC (10 μM) for 2 min or 6h. Lipid rafts of MGC membranes were obtained after 1% lubrol lysis and 20h-ultracentrifugation at 180,000 g in a sucrose gradient. The expression of caveolin-1, Flotillin-1, GFAP, Vimentin, Connexin-30, Connexin-43, phosphorylated and non phosphorylated p38 and p42-44 MAPK was analyzed by Western-blotting. High-performance liquid chromatography coupled with mass spectrometry (LC-MS) was used to estimate the levels of ganglioside GM3 (monosialodihexosylganglioside), and protein pathways were analyzed by nanoLC-MS/MS in raft and non raft fractions from MGCs after treatment with 24S-HOC. Results Cholesterol, sphingomyelin and saturated fatty acids C15:0, C16:0, C17:0 and C18:0 were enriched in the rafts fractions in MGCs. Structural proteins, Caveolin-1 and flotillin-1, and functional proteins, Connexin-30 and -43, were localized in the MGCs rafts. Ganglioside GM3 showed a characteristic enrichment in the raft fractions. Protein implicated in adhesion or oxidative stress pathways in the raft and non-raft fractions were up and down-regulated by treatment with 24S-OHC. Conclusions Our data showed that 24S-OHC induced early changes in protein distribution in raft microdomains.