Is 24(S)-hydroxycholesterol a potent modulator of cholesterol metabolism in Müller cells? An in vitro study about neuron to glia communication in the retina

Abstract Communication between neurons and glia plays a major role in nervous tissue homeostasis. It is thought to participate in tuning cholesterol metabolism to cellular demand, which is a critical issue for neuronal health. Cholesterol is a membrane lipid crucial for nervous tissue functioning, and perturbed regulation of its metabolism has been linked to several neurodegenerative disorders. In the brain, 24(S)-hydroxycholesterol (24S-OHC) is an oxysterol synthesized by neurons to eliminate cholesterol, and 24S-OHC has been shown to regulate cholesterol metabolism in astrocytes, glial cells which provide cholesterol to neurons. In the retina, 24S-OHC is also an elimination product of cholesterol produced by neurons, especially the retinal ganglion cells. However, it is not known whether Muller cells, the major macroglial cells of the retina, play the role of cholesterol provider for retinal neurons and whether they respond to 24S-OHC signaling, similarly to brain glial cells. In the present study, primary cultures of rat Muller cells were treated with 0, 0.5 or 1.5 μM 24S-OHC for 48 hours. The levels of cholesterol, precursors and oxysterols were quantified using gas chromatography coupled to flame-ionization detection or mass spectrometry. In addition, the expression of key genes related to cholesterol metabolism was analyzed using RTq-PCR. Muller cells were shown to express many genes linked to cholesterol metabolism, including genes coding for proteins implicated in cholesterol biosynthesis (HMGCR), cholesterol uptake and export via lipoproteins (LDL-R, SR-BI, ApoE and ABACA1) and regulation of cholesterol metabolism (SREBP2 and LXRβ). Cholesterol and several of its precursors and oxidative products were present. CYP27A1, the main retinal enzyme implicated in cholesterol elimination via oxysterol production, was quantified at low transcript levels but neither of its two typical products were detected in Muller cells. Furthermore, our results demonstrate that 24S-OHC has a strong hypocholesterolemic effect in Muller cells, leading to cholesterol depletion (-37 % at 1.5 μM). This was mediated by a decrease in cholesterol synthesis, as illustrated by reduced levels of cholesterol precursors: desmosterol (-38 % at 1.5 μM) and lathosterol (-84 % at 1.5 μM), and strong downregulation of HMGCR gene expression (2.4 fold decrease at 1.5μM). In addition, LDL-R and SR-BI gene expression were reduced in response to 24S-OHC treatment (2 fold and 1.6 fold at 1.5 μM, respectively), suggesting diminished lipoprotein uptake by the cells. On the contrary, there was a dramatic overexpression of ABCA1 transporter (10 fold increase at 1.5 μM), probably mediating an increase in cholesterol efflux. Finally, 24S-OHC induced a small but significant upregulation of the CYP27A1 gene. These data indicate that Muller cells possess the necessary cholesterol metabolism machinery and that they are able to sharply adjust their cholesterol metabolism in response to 24S-OHC, a signal molecule of neuronal cholesterol status. This suggests that Muller cells could be major players of cholesterol homeostasis in the retina via neuron-glia crosstalk.