Effects of 24s-hydroxycholesterol on primary glial Müller cells. New insights on Müller cells function and cholesterol homeostasis in the retina

Abstract

Purpose: Müller cells are macroglial cells in the retina. These cells express various proteins known to undergo glutamate clearance, which dysfunction may be involved in the loss of retinal ganglion cells (RGC) in the course of glaucoma. In neurons and especially in RGC, 24S-hydroxycholesterol (24SOH) is a metabolite produced by conversion of cholesterol by cholesterol 24S-hydroxylase (CYP46A1). This process participates to cholesterol homeostasis by facilitating the removal of cholesterol from neurons. 24SOH might be a signal molecule ensuring the cross-talk between neurons and glia, and putatively between RGC and glia. The aim of our study was to emphasize the response of primary Müller cells to 24SOH.Methods: Retinas from 10-12 day post-natal Long Evans rats were dissected after enzymatic digestion and seeded into culture dishes. Cells were grown in DMEM (5mM glucose and 2mM glutamine) + 10% FCS at 37°C. At passage 4, Müller cells were incubated with 10, 50 and 100µM 24SOH for 24h. By means of comparison, Müller cells were triggered with 2, 10, 20 ng/µL IL1β and 25mM glucose. Cellular viability was assessed by MTT test. Cholesterol and 24SOH were measured in cell pellets and culture supernatants by isotope dilution gas chromatography-mass spectrometry using internal deuterated standard. Cytokines and chemokines were measured by Luminex® in cell pellets and culture supernatants. The expression of genes involved in lipid metabolism and glial activation was analyzed by RT-qPCR.Results: Cholesterol synthesis was reduced by 39% (10µM 24SOH) and 65% (50 and 100µM 24SOH) compared to controls and did not vary in cells incubated with IL1β and high glucose. The analysis of 24SOH in cells and supernatants revealed the capacity of Müller cells to metabolize 24SOH. MCP-1 and VEGF were increased in cells incubated with 24SOH only (from x1.75 at 10µM compared to controls to x4 at 50 and 100µM) but not in culture supernatants. 24SOH up-regulated the expression of CYP46A1, Ccl-2 (MCP-1), Slc1a2 (EAAT2), GFAP and genes involved in cholesterol transport.Conclusions: 24SOH, used in our in vitro study to mimic the stress of RGC, was found to be actively metabolized by Müller cells and to reduce cholesterol biosynthesis therein. This process may contribute to cholesterol homeostasis in the retina and to Müller cells reactivity in the course of RGC loss and glaucoma