Cholesterol metabolism and glaucoma: Modulation of Muller cell membrane organization by 24S-hydroxycholesterol

G Model CPL 4557 No. of Pages 13

Chemistry and Physics of Lipids xxx (2017) xxx–xxx

Contents lists available at ScienceDirect

Chemistry and Physics of Lipids journal homepage: www.elsevier.com/locate/chemphyslip

Cholesterol metabolism and glaucoma: Modulation of Muller cell membrane organization by 24S-hydroxycholesterol Ségolène Gamberta,* , Pierre-Henry Gabrielleb , Elodie Massona , Elise Leger-Charnaya , Arthur Ferrerrob , Arthur Vanniera , Clément Gendraulta , Méline Lachota , Catherine Creuzot-Garchera,b , Alain Brona,b , Stéphane Gregoirea , Laurent Leclerea , Lucy Martinea , Géraldine Lucchic , Caroline Truntzerc , Delphine Pecqueurc , Lionel Bretillona a

Centre des Sciences du GoÛt et de l’Alimentation, AgroSup Dijon, CNRS, INRA, Université Bourgogne Franche-Comté, Dijon, France Department of Ophthalmology, University Hospital, Dijon, France c Clinical Innovation Proteomic Platform, 15 Boulevard Maréchal de Lattre de Tassigny, BP37013, F-21070 Dijon Cedex, France b

A R T I C L E I N F O

Article history: Received 27 April 2017 Received in revised form 19 May 2017 Accepted 23 May 2017 Available online xxx Keywords: 24S-hydroxycholesterol Muller cells Raft microdomain

A B S T R A C T

Glaucoma is a progressive and irreversible blinding neuropathy that is characterized by the loss of retinal ganglion cells (RGCs). Muller Glial Cell (MGC) activation is induced in retinal gliosis. MGCs are the most numerous glial cells in the retina and one of their roles is to sustain cholesterol homeostasis. 24Shydroxycholesterol (24S-OHC) is one of the form of cholesterol elimination from the retina and is overexpressed during glaucoma. The objective of this study was to determine whether 24S-OHC triggers MGC membrane dynamics involving lipid rafts and contributes to gliosis at early and late time points. A proteomic analysis was carried out by nanoLC–MS/MS in raft and non-raft fractions from MGCs after treatment with 24S-OHC (10 mM). The expression of structural and functional proteins was further analyzed by Western-blotting, as well as the levels of GM3 ganglioside by LC–MS. Cholesterol, sphingomyelin, saturated fatty acids and ganglioside GM3 are enriched in the rafts fractions in MGCs. Caveolin-1, flotillin-1, connexin-30 and -43 are localized in the MGCs rafts. Proteins implicated in adhesion or oxidative stress pathways in raft fractions were up and down-regulated by the treatment. Our data showed that 24S-OHC induced early changes in protein distribution in raft microdomains; however, further studies are needed to better characterize the surrounded mechanisms. © 2017 Elsevier B.V. All rights reserved.

1. Introduction Glaucoma is a complex genetic and multifactorial disease leading to blindness. Glaucoma is defined as a chronic optical neuropathy characterized by retinal neuro-degeneration with the

Abbreviations: CYP46A1, 24S-hydroxylase; DMEM, Dulbecco’s modified eagle medium; ERKs 1/2, regulating kinases of extra-cellular signal; FBS, fetal bovine serum; GC/MS, gas chromatography/mass spectrometry; GFAP, glial acidic fibrillary protein; 24S-OHC, 24S-hydroxycholesterol; IOP, intraocular pressure; LC–MS, liquid chromatography coupled with mass spectrometry; MAPK, mitogen activated protein kinase; MGC, Muller glial cell; RGCs, retinal ganglion cells. * Corresponding author. Permanent address: CSGA – site INRA, 17 rue Sully, BP 86510, 21065 Dijon Cedex, France. E-mail address: [email protected] (S. Gambert).

progressive death of retinal ganglion cells (RGCs) by apoptosis. The rise in intraocular pressure (IOP), the first known but not essential risk factor, is still the only known pathogenic event and is treated by drugs or surgery. Muller glial cells (MGCs) are highly specialized glial cells that interact intimately with retinal neurons during embryogenesis and adulthood. In vascularized retinae, MGCs may be involved in the control of angiogenesis, and the regulation of retinal blood flow (Bringmann et al., 2006). MGC bodies are located in the inner nuclear layer of the neurosensory retina and project irregularly thick and thin processes all around every type of cell of the neuroretina. MGC functions include the development and maintenance of water and ion homeostasis, neurotransmitter recycling and neurotrophic factor support (Reichenbach and Bringmann, 2013). These functions essential for neuronal health involve a

http://dx.doi.org/10.1016/j.chemphyslip.2017.05.007 0009-3084/© 2017 Elsevier B.V. All rights reserved.

Please cite this article in press as: S. Gambert, et al., Cholesterol metabolism and glaucoma: Modulation of Muller cell membrane organization by 24S-hydroxycholesterol, Chem. Phys. Lipids (2017), http://dx.doi.org/10.1016/j.chemphyslip.2017.05.007

G Model CPL 4557 No. of Pages 13

2

S. Gambert et al. / Chemistry and Physics of Lipids xxx (2017) xxx–xxx

symbiotic relationship in which MGCs supply energy and neurotransmitters and destroy neural waste products (Bringmann et al., 2006). Another well-known characteristic of MGCs is their ability to become activated in response to a retinal insult (Bignami and Dahl, 1979; Kim et al., 1998; Chen and Weber, 2002; Luna et al., 2010; Xue et al., 2006a). Upregulation of the MGCs intermediate filament protein, glial acidic fibrillary protein (GFAP), has been considered the most sensitive early indicator of glial activation following retinal injury (Bringmann et al., 2006). Acute retinal injury, induced for example by ischemia, laser treatment ou photic injury, leads to GFAP upregulation within the first 24 h. This is characterized by a transient increase in transcription, after which the GFAP protein can be detected for several weeks (Kim et al., 1998; Chen and Weber, 2002; Luna et al., 2010; Xue et al., 2006a; Humphrey et al., 1997). Sustained injury and progressive disease, such as untreated retinal detachment, hereditary degeneration and glaucoma, lead to the propagation of this activation into retinal gliosis, a condition in which MGCs continuously overexpress GFAP, hypertrophy, and in some conditions proliferate and migrate (Lewis et al., 1989; Ekstrom et al., 1988; Tezel et al., 2003; Ghosh and Johansson, 2012). Retinal gliosis is a common feature of glutamate excito-toxicity; ischemia-induced oxidative stress; and lack of neurotrophic factors (Bringmann et al., 2006). Retinal gliosis is characterized by diverse specific and non-specific cellular responses to a pathological mechanism such as hypertrophy and proliferation of MGCs, overexpression of intermediate filament proteins: nestin, vimentin and GFAP (Bringmann and Reichenbach, 2001; Lewis and Fisher, 2003), activation of the regulating kinases of extra-cellular signals (ERKs 1/2) (Akiyama et al., 2002), changes in the expression of glutamine synthetase (Iandiev et al., 2006) or dedifferentiation of MGCs. At the beginning, MGCs activation is protective and helps RGCs to survive by amplifying the metabolic support capabilities of MGCs: MGCs protect neurons via a release of neurotrophic factors, the uptake and degradation of the excitotoxin, glutamate, and the secretion of antioxidant glutathione (Bringmann et al., 2006, 2009; Oku et al., 2002). Nevertheless, if the retinal gliosis persists, MGC activation becomes harmful because of the loss of homeostatic functions and uncoupling from retinal neurone functioning due to MGC dedifferentiation. It is thus crucial to decipher the cellular mechanisms leading to retinal gliosis in glaucoma in order to better understand its pathogenesis. Free cholesterol is the second most abundant type of lipid in the neuroretina, after phospholipids (Bretillon et al., 2008). Neurons can synthesize cholesterol only in a limited extent and under the control of glial cells (Pfrieger and Ungerer, 2011). A significant part of cholesterol in the neuroretina originates from blood lipoproteins. Cholesterol elimination from the neuroretina is supported by 3 CYP450 enzymes: CYP11A1, CYP27A1 and CYP46A1 (Wang et al., 2004). The most important pathway of its elimination in RGCs is via the formation of 24S-OHC (or Cerebrosterol), which is catalyzed by CYP46A1 (cholesterol-24S-hydroxylase). Overexpression of CYP46A1 has been associated with glaucoma. Indeed, the experimental increase in IOP in rats leads to overexpression of CYP46A1 and overproduction of 24S-OHC, which is associated with GFAP activation, a marker of retinal gliosis in glaucoma (Fourgeux et al., 2009). In the plasma membrane, dynamic microdomain structures, called lipid rafts (Singer and Nicolson, 1972), comprise planar lipid rafts (which are rich in flotillin and GM3 ganglioside) and caveolae lipid rafts (which are rich in caveolin and GM1 ganglioside). Lipid rafts float in the plasma membrane and constitute platforms for multiple cell signaling events (Bevers et al., 1999) involved in cell adhesion, cell migration, inflammation, immunity reactions or the initiation of atherosclerotic plaques. The regulation of these cell

signaling pathways is dependent on raft composition, which can be modified by the connection of ligands, hormones or cytokines to their receptors and to the recruitment of proteins in rafts (Callera et al., 2011; Noghero et al., 2012). It is reported that small modifications of cholesterol structure have moderately deleterious effects on the ability to support raft formation, like oxidation of cholesterol to generate oxysterols with different physiochemical properties and may be considered as potent modulators of lipid raft formation. Oxysterols, like 7b-hydroxycholesterol or 7 ketocholesterol, interact with lipid rafts (Wang et al., 2004). The aim of our study was to determine whether 24S-OHC alters MGCs membrane dynamics involving lipid rafts and thereby induces their activation. 2. Materials and methods All animals used in this experiment were handled according to the regulations of the Institutional Animal Care Committee (University of Burgundy, Dijon, France). 2.1. Culture and treatment of primary Muller cells Eyeballs from young (postnatal day 8–12) Long Evans rats were enucleated and the retinas were dissected free after soaking the globes overnight in 5% CO2 and 95% air in growth medium (Dulbecco’s Modified Eagle Medium) (DMEM) (glucose 1 g/L, with L-glutamine, red phenol and 10% fetal bovine serum (FBS)) with 10 mg/mL of Penicillin-streptomycin. The retinas were digested with collagenase at 70 U/mL and trypsin at 0.1% (m/v), dissociated and maintained in a stationary culture in 10% serum supplemented growth medium. After initial primary outgrowth, the culture medium was replaced every 72 h. A purified flat Müller cell population was obtained by shaking to rinse off other unneeded types of cells. Only confluent cultures that had been passaged twice were used for each independent experiment. Muller cells were treated with 24S-hydroxycholesterol (24S-OHC) 10 mM (diluted in ethanol 96%) for two minutes or six hours. 2.2. Extraction of MGCs lipid rafts Lipid rafts are membrane microdomains that are resistant to dissolution in detergent. Briefly, 20
106 cells were washed with PBS, then lysed on ice for 30 min in 1 mL MBS buffer (Sigma Aldrich, MO) containing 1% (w/v) Lubrol WX (Serva, Paris, France). Lysates were homogenized using a Dounce homogenizer and mixed with 2 mL of 90% sucrose solution to obtain a final concentration of 45% sucrose. The sucrose density gradient of 4 mL of sucrose at 45%, 5 mL of sucrose at 35% and 3 mL of sucrose at 5% (5–35-45% of sucrose) was prepared in MBS buffer. The gradient was centrifuged as reported previously (Brown and Rose, 1992). Twelve fractions of 1 mL were collected from the top to the bottom, vortexed and stored at 80  C before lipid and protein analysis. 2.3. Determination of protein composition MGCs lipid rafts by Western blot First, 12 mL of each fraction were mixed with a 4X Laemmli sample buffer, boiled for 5 min, separated on a 12% polyacrylamide SDS-containing gel and transferred onto a nitrocellulose membrane (Whatman, NJ). After blocking non-specific binding sites for 1 h with 5% non-fat milk in PBS-Tween (PBS with 0.1% Tween 20), the membrane was incubated overnight at 4  C with anti-flotillin-1 (Santa Cruz, CA), anti-caveolin-1 (Transduction Laboratories, CA), anti-GFAP (Thermo Scientific), anti-vimentin (Santa Cruz BT), anticonnexin-30 (BD transduction labs), anti-connexin-43 (BD transduction labs), anti-MAPK p42-44 or anti-p38 (CS technology)

Please cite this article in press as: S. Gambert, et al., Cholesterol metabolism and glaucoma: Modulation of Muller cell membrane organization by 24S-hydroxycholesterol, Chem. Phys. Lipids (2017), http://dx.doi.org/10.1016/j.chemphyslip.2017.05.007

G Model CPL 4557 No. of Pages 13

S. Gambert et al. / Chemistry and Physics of Lipids xxx (2017) xxx–xxx

primary antibody (1:1000), washed twice with PBS-Tween and incubated for 1 h at 4  C with horseradish peroxidase-conjugated goat anti-mouse or anti-rabbit antibodies (1:1000) (Santa Cruz, CA). The membrane was washed twice with PBS-Tween and revealed using an ECL detection kit (Amersham, NJ) and autoradiography. Densitometry was performed for each fraction of each protein of interest and normalized by total proteins using stainfree gel technology. 2.4. Lipid analysis Lipids were extracted from the sucrose fractions using the Folch method (Folch et al., 1957). 2.4.1. Cholesterol, sphingomyelin and phosphatidylcholine analysis Cholesterol and sphingomyelin from total lipid extracts were analyzed by gas chromatography/mass spectrometry (GC/MS) method (Edwards et al., 2011) and phosphatidylcholine from total lipid extracts was analyzed by liquid chromatography/mass spectrometry (LC/MS) method (Brugger et al., 1997). 2.4.2. Fatty acids analysis For fatty acid analysis, total lipids were subjected to transmethylation of the fatty acids using boron trifluoride in methanol according to Morrison and Smith (Morrison and Smith, 1964). Fatty acid methyl esters were subsequently extracted with hexane and analyzed using gas chromatography on a Hewlett Packard Model 5890 gas chromatograph (Palo Alto, CA, USA) using a CPSIL-88 column (100 m, 0.25 mm i.d., 0.20-mm film thickness, Varian, Les Ulis, France) equipped with a flame ionization detector. Hydrogen was used as the carrier gas (inlet pressure, 210 kPa). The oven temperature was held at 60  C for 5 min, increased to 165  C at15  C/min and held for 1 min and then to 225  C at 2  C/min and finally held at 225  C for 17 min. The injector and the detector were maintained at 250  C. Fatty acid methyl esters were identified by comparison with commercial and synthetic standards (Sigma Aldrich, L’Isle d’Abeau, France). The data were processed using the EZChrom Elite software (Agilent Technologies, Massy, France) and reported as a percentage of total fatty acids. The distribution of lipids (phospholipids, triacylglycerols, free fatty acids, free cholesterol, and cholesteryl esters) in Muller cells (n = 3) was determined using a combination of thin-layer chromatography on silica gel-coated quartz rods and flame ionization detection (Iatroscan system, Iatron, Tokyo, Japan), according to Ackman’s technique (Ackman, 1981) and as published by our group (Bretillon et al., 2008). The values obtained for each compound were corrected according to their response factor using specific calibration curves, as previously published (Sebedio et al., 1987). Data are reported as a percentage of total lipids in each sample. 2.4.3. Extraction, separation and purification of gangliosides and analysis by liquid chromatography coupled to mass spectrometry Alternatively, total lipids were extracted from 0.8 mL of samples (fractions 2, 3, 4, 5, 9, 10, 11, 12), overnight at 4  C, with 10 volumes (8 mL) of CHCl3/CH3OH (1:1, v/v). 0.25 mg internal deuterated GM3 standard [2H3-GM3] (N-omega-CD3-Octadecanoyl monosialoganglioside GM3) (Matreya LLC, USA) was added to fractions 2 and 9. The residual pellets obtained after centrifugation were then reextracted three times with solvent mixtures as previously described (Sibille et al., 2016). The total lipid extracts of fractions 2 to 5 and fractions 9 to 12 were pooled to make lipid extracts from rafts and non-rafts, respectively. Gangliosides (GGs) were separated from other lipids by phase partition and purified on a C18 silica gel column. GG extracts were finally re-dissolved in 1 mL CHCl3/ CH3OH (2:1, v/v) and stored, under nitrogen, at 20  C until further analyses.

3

GG separation was achieved under Hydrophilic Interaction liquid Chromatography (HILIC) before analysis by a Triple Quadrupole mass spectrometer (TSQ Quantum UltraTM, Thermo Scientific, USA) operated in the negative ion mode. The GG profile of Muller cells was almost exclusively composed of GM3. For quantification, data were acquired in Selected Reaction Monitoring (SRM) of [M-H] ! 290, a characteristic N-Acetylneuraminic acid (sialic acid), with precursor and product ion pairs selected based on previously published data (Sibille et al., 2016). The total intensity of GM3 was calculated as the sum of all detected peak areas that corresponded to the different molecular species of GM3. The intensity was then normalized with the intensity of the deuterated GM3 internal standard in the sample.

2.5. Proteomics 2.5.1. Proteomics analysis For each condition, the four top sucrose fractions were pooled as were the eight remaining fractions in two different samples (respectively raft and non-raft fractions) for proteomics analysis. To eliminate sucrose and other components that could interfere with the proteomics analysis, sucrose fractions were diluted at least 8-fold with buffer containing 1 mL SDS 1%. Diluted samples were ultracentrifuged at 39,000 rpm for 1 h, at 4  C. Pellets were precipitated with cold acetone. Samples were assayed for their protein concentrations using the BCA Protein Assay Kit (Pierce, Rockforf, IL, USA). Briefly, pellets were resuspended in PBS (50 mM Na2HPO4, 50 mM NaH2PO4, 140 mM NaCl2, pH 7.4) containing 0.25% Triton X100 (Fluka, Sigma-Aldrich). Samples and standards were mixed with working reagent and incubated for 45 min at 37  C. Absorbances were measured at 562 nm. Proteins were resolubilized with extraction MES buffered saline with 1% (p/v) Lubrol WX. 10 mg of protein extract were separated using 1D polyacrylamide gel electrophoresis to purify the protein samples before the proteomic analysis steps. The 1 cm length was cut into spots. Reduction/alkylation was achieved by incubating the excised spots successively in 10 mM tris(2carboxyethyl)phosphine/0.1 M NH4HCO3 for 30 min at 37  C, and in 55 mM iodoacetamide/0.1 M NH4HCO3 for 20 min. Peptide fragments were obtained after digestion with a solution of 40 mM NH4HCO3/10% ACN containing 5 ng of trypsin (Promega) for 3 h at 37  C. The resulting peptides were acidified with 0.1% formic acid. Peptides were extracted from the polyacrylamide gel spots by two successive incubations for a few minutes in ACN with sonication. Digests were concentrated by evaporation. 1.8 mg of peptides were separated with the nano RSLC system (ThermoScientific) fitted with a C18 analytical column. Samples were injected in triplicates. A 120-min gradient was used and peptides were analyzed by nano LC–MS/MS using an LTQ-Orbitrap Elite mass spectrometer equipped with the Advion TriVersa NanoMate nanospray source. Full-scan spectra from a mass/charge ratio of 400 to one of 1.700 at a mass resolution of 120.000 with a full width at half maximum were acquired in the Orbitrap mass spectrometer. From each full-scan spectrum, the 20 ions with the highest intensity were selected for fragmentation in the ion trap. The acquired data were searched against UniProt using Mascot, Comet, SpectraST and X!Tandem. Peptide and protein identification was validated using Peptide and ProteinProphet software (Nesvizhskii et al., 2003). Peptides that were not identified in at least two of the three technical replicates injected per sample were excluded. Retention times were then aligned and peptide intensity extracted using MASIC software (Monroe et al., 2008). A linear mixed model was then used to select differential proteins (Clough et al., 2012). Proteins identified with a fold change higher than 2 and lower than 0.5, with an FDR at 5%, were kept.

Please cite this article in press as: S. Gambert, et al., Cholesterol metabolism and glaucoma: Modulation of Muller cell membrane organization by 24S-hydroxycholesterol, Chem. Phys. Lipids (2017), http://dx.doi.org/10.1016/j.chemphyslip.2017.05.007

G Model CPL 4557 No. of Pages 13

4

S. Gambert et al. / Chemistry and Physics of Lipids xxx (2017) xxx–xxx

overall (the prominent GG in Muller cells). However, we observed a significant enrichment in GM3 in raft fractions compared to nonraft fractions (p < 0.05) (Fig. 2). Western Blots were realized on each of the fractions of rafts obtained after the various treatments (2 min or 6 h with the 24SOHC or with the ethanol 0.5%). Studied proteins were caveolin-1, flotillin-1, GFAP, vimentin, connexins-30 and 43, MAPK p38 and p42-44 and phosphorylated MAPK p38 and p42-44.

2.5.2. Post analysis of proteomics results Proteins were classified according to the Gene Ontology (GO) hierarchy using the Universal Protein Resource (UniProt) retrieval system (http://www.uniprot.org/). The “ID mapping” module for the UniProt was used to transform the GO number into a UniProt code and to associate them with corresponding gene names, GO categories and IDs, molecular functions, subcellular location and tissue specificity. “Cellular Component” enrichment was analyzed using the String database and Slim and a highest confidence score of 0.9. To investigate the distribution of proteins between the raft and the non-raft fractions in each experimental group, we did a comparative analysis of the proteins identified in raft and non-raft fractions using a Venn diagram analysis using Venny sotware (http://bioinfogp.csic.es/tools/venny/).

3.2.2. Caveolin-1 and flotillin-1, markers of lipid rafts Caveolin-1, a structural protein of rafts, is found within rafts, as confirmed in our study (fractions 3–5, Fig. 3). Whatever the treatment time (2 min or 6 h), no significant difference between the control and the treated groups was observed. The distribution between raft and non-raft fractions remained unchanged whatever the treatment duration. The treatment did not modify the localization of caveolin-1. Flotillin-1 is a structural protein of raft microdomains and is mainly expressed in lipid raft fractions 3, 4, 5, just like caveolin-1 and is almost absent from other fractions (Fig. 4). Whatever the treatment time, 2 min or 6 h, there was no effect of the treatment with 24S-OHC on flotinllin-1 localization.

2.6. Statistical analysis Continuous variables were tested for normal distribution with the use of the Kolmogorov-Smirnov test. Data are expressed as mean values  standard error of the mean (SEM) or as median with interquartile range [IQR], as appropriate. Between-group comparisons were performed using the unpaired t–test or the MannWhitney U test and Kruskal-Wallis test for continuous data, as appropriate. A p value of <0.05 was considered statistically significant. All analyses were performed using NCCS statistical software.

Raft Non Raft

GM3 (ratio/control)

2

3. Results 3.1. Cholesterol, sphingomyelin and phosphatidylcholine distribution in MGCs The lipid profile resulting from the use of Lubrol shows a characteristic enrichment in cholesterol and sphingomyelin in the raft fractions and of phosphatidylcholine in the non-raft fractions (Fig. 1).

3.2.1. GM3 quantification We have explored the GG composition of Muller cells after treatment with 24S-OHC and we found no increase in the GM3

*

*

*

1

0

3.2. Effect of 24S-OHC on lipidic and proteic markers of rafts

*

Control Treated 2 min

Control

Treated 6h

Fig. 2. GM3 (ratio/control) in Muller cells (n = 6) in control and treated groups (2 min and 6 h). The total intensity of GM3 was normalized by the signal of the internal stantdard. * = p < 0.05.

Relative distribution (%)

50

40 Cholesterol Sphingomyelin Phosphatidylcholine

30

20

10

0 1

2

3

4

Raft

5

6

7

8

9

10

11

12

Fractions

Non Raft

Fig. 1. Relative distribution of cholesterol, sphingomyelin and phosphatidylcholine in the 12 membrane fractions (fractions 2 to 5: rafts, fractions 6 to 12: non-rafts) in Muller cells (n = 3).

Please cite this article in press as: S. Gambert, et al., Cholesterol metabolism and glaucoma: Modulation of Muller cell membrane organization by 24S-hydroxycholesterol, Chem. Phys. Lipids (2017), http://dx.doi.org/10.1016/j.chemphyslip.2017.05.007

G Model CPL 4557 No. of Pages 13

S. Gambert et al. / Chemistry and Physics of Lipids xxx (2017) xxx–xxx

5

Fig. 3. Caveolin-1 localization in 12 fractions of Muller cells, in controls at 2 min (A) and 6 h (B) and in 24S-OHC-treated cells at 2 min (C) and 6 h (D) (n = 3).

Fig. 4. Flotillin-1 localization in 12 fractions of Muller cells, in controls at 2 min (A) and 6 h (B) and in 24S-OHC-treated cells at 2 min (C) and 6 h (D)(n = 3).

3.3. Effect of 24S-OHC on proteic markers of MGC activation 3.3.1. GFAP and vimentin, intermediate filament proteins These proteins contribute to the maintenance of cell shape and to the anchoring of cellular organelles. Treatment with 24S-OHC slightly promoted the localization of GFAP in raft domains after 2 min and 6 h (Fig. 5) but it is not statistically significant. Vimentin is an intermediate filament protein of the cytoskeleton of Muller cells. It is a marker of glial activation and is coexpressed with GFAP. It is present mainly in fractions 4–5 (raft) and 8–12 (non-raft) (Fig. 6). We found no difference in vimentin distribution following treatment with 24S-OHC.

3.3.2. Connexins-30 and 43, markers of interaction between astrocytes and Muller cells Connexins-30 and 43 are members of the connexin family (Soroceanu et al., 2001), which are essential to form gaps between astrocytes and Muller cells. They are 30- and 43 kDa membrane proteins, respectively. Interestingly, connexins-30 and 43 were clearly found mainly within rafts. This specific localization was not affected by 24S-OHC treatment (Figs. 7 and 8).

3.3.3. Mitogen activated protein kinase p38 and p42-44, markers of cell migration and proliferation Mitogen activated protein kinase p38 is implicated in cell migration. We observed no incresase in the phosphorylated form of p38 in raft fractions in response to 24S-OHC (0.8 and 0.4 in fraction 4 (raft) at 2 min and 6 h in controls respectively vs 0.9 and 0.6 in fraction 4 at 2 min and 6 h of treatment respectively, Fig. 9). p42-44 is implicated in cell proliferation. There was no difference between the treated group and the control group with respect to the proportion of active form of the protein p42/44 in all of the fractions. As for p38, it appeared to be preferentially located within the non-raft fractions of MGCs. 24S-OHC did not stimulate the p42-44 MAPK pathway. We observed a tendency towards an increase in activated protein (P-p42-44/p42-44) in raft fractions (1.7 and 3.4 in fraction 4 at 2 min and 6 h respectively vs 2.5 and 3.2 in fraction 4 at 2 min and 6 h respectively compared to 0.7 and 0.7 in fraction 10 at 2 min and 6 h respectively vs 0.8 and 2.1 in fraction 10 at 2 min and 6 h respectively, Fig. 10). 3.4. Fatty acid composition of Muller cell membranes To obtain a macroscopic view of the changes in lipid composition in response to treatment, we also performed total

Fig. 5. GFAP localization in 12 fractions of Muller cells, in controls at 2 min (A) and 6 h (B) and in 24S-OHC-treated cells at 2 min (C) and 6 h (D)(n = 3).

Please cite this article in press as: S. Gambert, et al., Cholesterol metabolism and glaucoma: Modulation of Muller cell membrane organization by 24S-hydroxycholesterol, Chem. Phys. Lipids (2017), http://dx.doi.org/10.1016/j.chemphyslip.2017.05.007

G Model CPL 4557 No. of Pages 13

6

S. Gambert et al. / Chemistry and Physics of Lipids xxx (2017) xxx–xxx

Fig. 6. Vimentin localization in 12 fractions of Muller cells, in controls at 2 min (A) and 6 h (B) and in 24S-OHC-treated cells at 2 min (C) and 6 h (D) (n = 3).

Fig. 7. Connexin-43 localization in 12 fractions of Muller cells, in controls at 2 min (A) and 6 h (B) and in 24S-OHC-treated cells at 2 min (C) and 6 h (D) (n = 3).

Fig. 8. Connexin-30 localization in 12 fractions of Muller cells, in controls at 2 min (A) and 6 h (B) and in 24S-OHC-treated cells at 2 min (C) and 6 h (D) (n = 3).

lipids analysis of Muller cells in the same samples from control and treated cells where lipid rafts were obtained. The GC-FID analysis provided a qualitative profile of fatty acids in different membrane fractions extracted from Muller cells in vivo and treated or not with 24S-OHC. Three experiments on raft and non-raft domains were obtained from three different cultures treated or not for 2 min or 6 h with 24S-OHC. Fractions 2 to 5 containing raft microdomains and the fractions 9 to 12 non-raft domains were each pooled before lipid extraction. Whatever the group, the main fatty acids were 16:0, 18:0, 18:1n9, 18:1n-7, 18:2n-6, 20:4n-6, 22:5n-3 and 22:6n-3. The distribution of these fatty acids 16:0, 18:0, 18:2n-6, 22:5n-3 and 22:6n-3 within rafts did not vary according to the treatment or its duration. The results were similar in all three experiments. Proportions of dma16:0 were significantly greater in the nonraft fractions than in the raft fractions in control (2 min) and treated (6 h) cells and in the non-raft fractions in the 6 h-treated cells compared to the non-raft fractions in 2 min-treated cells (p = 0.037). The distribution of C18:1n-9 was different between

non-raft fractions in the 6 h-control cells compared to non-raft fractions in the 6 h-treated cells (p = 0.05). Proportions of C20:0 were significantly greater in the raft fractions in the 2 min-treated cells than in 2 min-control cells (p = 0.05). Proportions of C20:2n-6 and C20:4n-6 were significantly greater in the non-raft fractions in the 2 min-treated cells than in the 2 min-control cells (p = 0.05), this difference was not found in the 6 h-treated cells. The distribution of C22:1n-9 was different between non-raft fractions in the 2 min-treated cells compared to non-raft fractions in the 6 htreated cells (p = 0.05). The distribution of C22:5 n-3 and C22:6n-3 was not different between raft and non-raft fractions whatever the treatment (Table 1). 3.5. Proteomics analysis of Muller cells Proteins with significant changes were identified using fold change (<0.5 and >2-fold) criteria (Table 2). Highly coordinated changes were evident in this analysis; the expression levels of multiple proteins within a pathway were increased or decreased. Examples of regulated pathways include up-regulation of Guanine

Please cite this article in press as: S. Gambert, et al., Cholesterol metabolism and glaucoma: Modulation of Muller cell membrane organization by 24S-hydroxycholesterol, Chem. Phys. Lipids (2017), http://dx.doi.org/10.1016/j.chemphyslip.2017.05.007

G Model CPL 4557 No. of Pages 13

S. Gambert et al. / Chemistry and Physics of Lipids xxx (2017) xxx–xxx

7

Fig. 9. p38 and phospho-p38 localization in 12 fractions of Muller cells, in controls at 2 min (A) and 6 h (B) and in 24S-OHC-treated cells at 2 min (C) and 6 h (D) (n = 3).

Fig. 10. p42-44 and phospho-p42-44 localization in 12 fractions of Muller cells, in controls at 2 min (A) and 6 h (B) and in 24S-OHC-treated cells at 2 min (C) and 6 h (D) (n = 3).

nucleotide-binding protein (G protein) beta polypeptide 4 (FC = 150.2 in raft vs 38.8 in non-raft fractions), NADH dehydrogenase (Ubiquinone) 1 beta subcomplex (7 and 4) (FC = 31.2 in raft vs 8.9 in non-raft fractions and FC = 12.5 in raft), Inositol 1,4,5-trisphosphate receptor type 1 (FC = 24.3 in raft and 3.3 in non-raft fractions),

Mitogen-activated protein kinase 1 (FC = 2.1 in non-raft fractions) and down-regulation of NADH dehydrogenase (ubiquinone) Fe-S protein 1 (FC = 0.47 in raft and 0.44 in non-raft fractions), p21 protein (Cdc42/Rac)-activated kinase 2 (FC = 0.11 in raft and 0.22 in non-raft fractions), NADH dehydrogenase (Ubiquinone) 1 beta

Please cite this article in press as: S. Gambert, et al., Cholesterol metabolism and glaucoma: Modulation of Muller cell membrane organization by 24S-hydroxycholesterol, Chem. Phys. Lipids (2017), http://dx.doi.org/10.1016/j.chemphyslip.2017.05.007

G Model CPL 4557 No. of Pages 13

8

S. Gambert et al. / Chemistry and Physics of Lipids xxx (2017) xxx–xxx

Table 1 Fatty acid composition of Muller cells in controls and treated cells at 2 min and 6 h in raft and non-raft fractions (% of total lipids/pooled fractions) (n = 3). Controls 2 min Raft

Controls 2 min Non-Raft

Treated 2 min Raft

Treated 2 min Non-Raft

Fatty Acid

Mean

[IQR]

Mean

[IQR]

Mean

[IQR]

Mean

[IQR]

14:0 15:0 dma16:0 16:0 16:1n–9 16:1n–7 17:0 dma18:0 dma18:1n–9 dma18:1n–7 18:0 18:1t 18:1n–9 18:1n–7 18:2n–6 20:0 20:1n–9 18:3n–3 20:2n–6 20:3n–9 22:0 20:3n–6 22:1n–9 20:4n–6 24:0 20:5n–3 24:1n–9 22:4n–6 22:5n–3 22:6n–3

1.22 0.82 $ 3.02 $ 21.39 1.19 0.98 1.01 2.07 $ 1.81 0.91 25.95 1.73 12.86 3.92 3.08 0.72 @ 0.42 0.32 0.30 $ 1.02 0.41 $ 1.68 0.22 5.29 0.74 0.56 0.61 $ 0.82 2.71 2.23

[0.75–1.48] [0.73–0.88] [2.82–3.18] [19.13–24.41] [0.88–1.61] [0.44–1.57] [0.9–1.22] [1.69–2.33] [1.42–2.15] [0.71–1.05] [19.75–31.59] [1.18–2.01] [10.61–14.9] [3.05–4.8] [2.55–3.63] [0.3–1.13] [0.31–0.57] [0.27–0.42] [0.11–0.51] [0.65–1.44] [0.23–0.6] [1.3–1.91] [0.17–0.24] [4.29–7.23] [0.47–1.02] [0.5–0.67] [0.31–0.87] [0.53–1.19] [2.19–3.33] [1.72–2.67]

0.68 0.44 4.15 19.97 0.69 0.81 0.64 3.79 2.02 1.07 28.91 1.86 13.33 2.93 2.44 0.49 0.35 0.27 0.84 ! 0.56 0.21 1.12 0.42 5.17 0.58 0.7 0.22 0.84 2.43 2.07

[0.4–0.88] [0.27–0.53] [3.73–4.46] [15.24–24.36] [0.59–0.75] [0.56–0.97] [0.56–0.68] [2.99–4.83] [1.86–2.26] [0.86–1.21] [28.44–29.2] [1.23–2.21] [11.96–15.69] [2.62–3.47] [1.53–3.09] [0.44–0.55] [0.25–0.41] [0.21–0.35] [0.25–1.87] [0.53–0.6] [0.16–0.28] [0.78–1.63] [0.27–0.63] [4.1–6.85] [0.43–0.66] [0.42–1.03] [0.15–0.35] [0.71–1.1] [2.05–3.1] [1.59–2.8]

1.07 0.71 3.34 # 19.6 1.17 1.04 0.9 2.32 2.17 # 1.1 # 23.42 1.7 14.87 4.48 3.19 0.29 # 0.47 0.3 0.24 0.82 0.24 1.84 0.24 # 6.14 # 0.77 0.55 0.4 1.11 3.02 2.48

[0.77–1.55] [0.56–0.9] [2.98–3.95] [16.49–22.65] [0.89–1.46] [0.82–1.46] [0.84–0.99] [1.75–3.25] [1.99–2.31] [0.9–1.39] [21.97–25.15] [1.16–2.06] [14.52–15.08] [4.3–4.69] [2.75–3.44] [0.25–0.33] [0.36–0.66] [0.26–0.39] [0.17–0.35] [0.69–0.94] [0.18–0.3] [1.71–1.95] [0.16–0.31] [5.2–6.96] [0.54–1.04] [0.32–0.77] [0.33–0.52] [1.05–1.19] [2.93–3.17] [2.34–2.56]

1.17 0.65 3.19 £ 21.63 1.32 1.34 1.14 2.85 1.61 0.94 25.35 1.82 14.99 2.73 3.73 0.71 0.4 0.35 0.24 0.63 0.24 1.21 0.48 £ 4.31 0.59 0.62 0.49 1.04 2.3 1.95

[0.42–2.53] [0.34–1.19] [2.24–3.86] [12.01–32.15] [0.47–2.75] [0.5–3.01] [0.61–2.15] [1.5–3.77] [0.86–2.22] [0.4–1.55] [19.98–29.59] [1.39–2.14] [14.34–16] [2.09–3.05] [1.49–6.52] [0.5–1.1] [0.28–0.63] [0.28–0.41] [0.19–0.31] [0.31–0.98] [0.12–0.32] [0.52–1.98] [0.2–0.81] [2.86–5.15] [0.15–0.94] [0.45–0.87] [0.11–0.74] [0.46–1.89] [1.37–3.18] [1.28–2.41]

total AGS total AGMI total AGPI total DMA

52.26 21.92 18.0 7.80 $

[45.17–60.55] [17.76–25.33] [14.38–21.64] [7.28–8.27]

51.92 20.62 16.44 11.03

[47.11–56.24] [18.31–23.63] [14.11–20.45] [9.86–12.49]

47.01 24.36 19.68 8.93

[44.98–49.57] [24.17–24.48] [18.62–20.8] [7.62–10.9]

51.47 23.56 16.37 8.59

[44.66–58.04] [22.71–23.99] [14.24–20.34] [5–11]

total n–6 total n–3 n–6/n–3

11.17 5.82 1.91

[9.05–13.89] [4.68–6.79] [1.76–2.05]

10.41 5.47 1.93

[8.83–12.68] [4.60–7.17] [1.77–2.09]

12.52 6.35 1.98

[11.93–13.59] [5.87–6.65] [1.81–2.08]

10.52 5.22 2.17

[8.53–12.49] [3.38–6.87] [1.58–3.12]

Controls 6 h Raft

Controls 6 h Non–Raft

Treated 6 h Raft

Treated 6 h Non–Raft

Fatty Acid

Mean

[IQR]

Mean

[IQR]

Mean

[IQR]

Mean

[IQR]

14:0 15:0 dma16:0 16:0 16:1n–9 16:1n–7 17:0 dma18:0 dma18:1n–9 dma18:1n–7 18:0 18:1t 18:1n–9 18:1n–7 18:2n–6 20:0 20:1n–9 18:3n–3 20:2n–6 20:3n–9 22:0 20:3n–6 22:1n–9 20:4n–6 24:0 20:5n–3 24:1n–9 22:4n–6 22:5n–3 22:6n–3

1.26 0.82 3.14 21.77 1.33 1.24 1.12 1.92 x 1.98 0.89 x 23.6 1.32 15.05 4.27 x 3.,07 0.46 0.45 0.27 0.14 0.76 0.2 1.72 x 0.2 5.53 0.76 0.5 0.45 0.83 2.66 2.29

[0.7–1.77] [0.75–0.94] [2.9–3.26] [20.46–24.37] [1.24–1.44] [0.95–1.43] [0.85–1.48] [1.83–1.98] [1.86–2.12] [0.87–0.92] [22.19–25.32] [1.14–1.59] [13.92–15.82] [3.91–4.56] [2.43–3.68] [0.22–0.82] [0.32–0.57] [0.24–0.31] [0.1–0.19] [0.68–0.8] [0.16–0.26] [1.5–1.93] [0.13–0.31] [4.33–6.78] [0.6–1.05] [0.18–0.71] [0.28–0.58] [0.55–1.05] [2.37–3] [2.11–2.53]

0.8 0.57 4.02 19.95 1.28 0.79 1.02 3.83 1.77 0.81 29.87 1.5 12.6 m 3.03 2.63 0.55 0.37 0.34 0.29 1.37 0.26 1.02 0.34 4.53 0.79 0.56 0.31 0.63 2.2 1.96

[0.33–1.11] [0.28–1] [3.27–5.13] [15.71–27.18] [0.7–2.37] [0.63–0.89] [0.69–1.29] [3.1–4.27] [1.51–2.23] [0.7–0.91] [27.84–32.31] [1.14–1.82] [10.25–15.14] [1.67–4.26] [1.11–3.68] [0.38–0.72] [0.21–0.48] [0.26–0.49] [0.21–0.34] [0.54–2.92] [0.15–0.35] [0.27–1.66] [0.22–0.43] [3.47–6.15] [0.44–0.97] [0.18–0.75] [0.16–0.46] [0.4–0.89] [1.83–2.64] [1.47–2.21]

1.25 0.78 2.93* 20.63 1.32 1.29 1.29 1.85 * 1.92 0.93 23.98 1.14 15.08 4.31 3.63 0.34 0.44 0.33 0.14 1.86 * 0.2 1.67 0.2 5.32 0.76 0.46 0.43 0.85 2.51 2.16

[0.89–1.72] [0.63–0.73] [2.52–3.28] [18.83–23.29] [1.23–1.46] [1.04–1.49] [1.07–1.43] [1.51–2.29] [1.71–2.02] [0.8–1.06] [22.73–25.08] [1.111.17] [13.98–17.22] [3.97–4.87] [2.82–4.64] [0.28–0.38] [0.33–0.61] [0.26–0.42] [0.11–0.15] [0.76–3.95] [0.16–0.24] [1.56–1.88] [0.14–0.3] [4.64–6.01] [0.58–1.05] [0.25–0.66] [0.28–0.52] [0.73–0.99] [2.36–2.78] [2.04–2.24]

0.74 0.45 4.33 20.76 0.77 1.02 0.79 3.74 1.72 0.78 28.46 1.31 15.31 3.48 2.3 0.52 0.41 0.33 0.21 0.55 0.13 1.2 0.25 4.4 0.52 0.54 0.25 0.67 2.15 1.94

[0.68–0.82] [0.38–0.52] [3.87–4.93] [16.88–25.24] [0.64–0.97] [0.82–1.2] [0.57–1.08] [3.05–4.21] [1.65–1.85] [0.67–0.86] [25.93–29.82] [1.14–1.58] [13.84–16.35] [2.38–4.33] [1.3–3.11] [0.3–0.7] [0.26–0.53] [0.3–0.36] [0.14–0.28] [0.43–0.61] [0.1–0.16] [0.71–1.47] [0.18–0.36] [3.64–5.41] [0.33–0.71] [0.2–0.8] [0.12–0.45] [0.54–0.74] [1.82–2.33] [1.53–2.34]

Please cite this article in press as: S. Gambert, et al., Cholesterol metabolism and glaucoma: Modulation of Muller cell membrane organization by 24S-hydroxycholesterol, Chem. Phys. Lipids (2017), http://dx.doi.org/10.1016/j.chemphyslip.2017.05.007

G Model CPL 4557 No. of Pages 13

S. Gambert et al. / Chemistry and Physics of Lipids xxx (2017) xxx–xxx

9

Table 1 (Continued) Controls 6 h Raft

Controls 6 h Non–Raft

Treated 6 h Raft

Treated 6 h Non–Raft

Fatty Acid

Mean

[IQR]

Mean

[IQR]

Mean

[IQR]

Mean

[IQR]

total AGS total AGMI total AGPI total DMA

49.99 24.32 x 17.77 7.92

[45.95–52.97] [22.72–25.27] [16.11–20.94] [7.58–8.13]

53.82 20.22 15.54 10.43

[46.54–61.7] [16.86–23.73] [9.93–18.65] [8.69–11.51]

49.23 24.27 18.93 7.62 *

[47.39–51.28] [22.65–26.78] [17.19–21.78] [6.54–8.65]

52.36 22.79 14.28 10.58

[48.87–58.03] [19.89–24.77] [10.64–16.76] [9.58–11.46]

total n–6 total n–3 n–6/n–3

11.29 5.72 1.97

[9.8–13.63] [5.13–6.51] [1.78–2.09]

9.1 5.07 1.76

[5.65–12.11] [3.74–5.88] [1.51–2.06]

11.61 5.47 2.12

[10.78–12.5] [5.33–5.55] [1.94–2.35]

8.77 4.96 1.77

[6.33–11] [3.88–5.83] [1.54–2.13]

$

significantly different for control 2 min non-raft (p < 0.05). significantly different for treated 2 min non-raft (p < 0.05). x significantly different for control 6 h non-raft (p < 0.05). * significantly different for treated 6 h non-raft (p < 0.05). £ significantly different for treated 6h non-raft (p < 0.05). m significantly different for treated 6h non-raft (p < 0.05). @ significantly different for treated 2 min raft (p < 0.05). ! significantly different for control 6 h non-raft (p < 0.05). #

subcomplex (4) (FC = 0.10 in non-raft fractions, respectively) (Table 2). In total, there were 184 proteins detected in both analyses (pooled raft and non-raft fractions). Functional and localization annotations were assigned for 184 proteins that were identified in both analyses using the information in the Uniprot database (http://www.uniprot.org, in the public domain). A search for membrane raft proteins in the Uniprot database yielded 127 unique proteins, of which 17 proteins were listed as adhesion proteins and 8 as oxidative stress proteins, for example. A search for non-raft proteins in the Uniprot database yielded 150 unique proteins, of which 18 proteins are listed as adhesion proteins and 9 as oxidative stress proteins, for example (Table 3). A great majority of adhesion proteins identified were downregulated: Collagen, type I, alpha 2 (FC = 0.45 in raft and 0.18 in non-raft fractions), Thrombospondin 1 (FC = 0.39 in raft and 0.35 in non-raft fractions), Talin 1 (FC = 0.39 in raft and 0.03 in non-raft fractions) or Vinculin (FC = 0.21 in raft and 0.06 in non-raft fractions), for example. Two adhesion proteins identified were upregulated in raft but downregulated in non-raft fractions, Catenin delta 1 (FC = 3.4 in raft and 0.08 in non-raft fractions) and Golgi glycoprotein 1 (FC = 3.3 in raft and 0.14 in non-raft fractions). Inversely, one adhesion protein identified, Mitogen-activated protein kinase 1, was downregulated in raft but upregulated in non-raft fractions (FC = 0.1 in raft and 2.1 in non-raft fractions) (Table 2). 4. Discussion The present work is a descriptive study in which we have examined early reactions in Muller cells after treatment with 24SOHC by mapping the lipid distribution and the expression patterns of structural proteins (caveolin-1, flotillin-1), of intermediate filament proteins and migration and proliferation pathway proteins. Our study aimed at understanding the implication of the 24S-OHC in the genesis of the retinal gliosis in particular in glaucoma neuropathy. The existence of lipid alterations in glaucoma has been poorly studied. In the present work, we isolated and characterized highly purified lipid rafts from Muller cells. At physiological temperatures, biological membranes are typically in a liquid-disordered state, but certain interactions between specific lipids lead to the formation of microdomains, which are organized in liquid-ordered domains called lipid rafts (Pike, 2004; Pathak and London, 2015). Studies have shown that, under specific conditions, small rafts can stabilize and grow into larger platforms. The growth of these platforms is likely to be due to specific protein-protein and

protein-lipid interactions, leading to the lateral segregation of molecular components in the cellular membrane (Pike, 2006). The presence of specific proteins, such as flotillins or caveolins, are thought to promote the stability of microdomains (Hanzal-Bayer and Hancock, 2007) and these characteristics fit perfectly well with the widely assumed composition of lipid rafts in the membranes of cells, including nerve cells (Brown and London, 2000; Pike, 2003, 2009). Our data confirmed these observations. We showed that protein markers flotillin-1 and caveolin-1 were detected in fractions 3–4, which correspond to the lipid correspond to the lipid raft fractions. Our data showed that lipid rafts of MGCs were markedly enriched in sphingomyelin, cholesterol and saturated fatty acids compared with non-raft fractions. Some oxysterols generated by the oxidation of the cholesterol have physico-chemical properties allowing them to modulate the formation of the lipid rafts (Wang et al., 2004). Cholesterol 24Shydroxylase (CYP46A1) converts the cholesterol into a more hydrophilic compound, the 24S-OHC. Our HPLC–MS analysis of the MGCs GGs allowed us to highlight higher levels of GM3 ganglioside in the raft fractions compared to non-raft fractions. This is consistent with literature data, since GGs are believed to participate in the maintenance of raft organization through their specific physical properties and their mainly saturated FA composition. There was no effect of the treatment with 24SOHC on GM3 membrane distribution. The glycosphingolipid GM3 is a characteristic component of planar raft microdomains and any modification in its composition within rafts would suggest a possible modification of rafts, thereby inducing the dysfunction of certain membrane proteins or the stimulation of cellular signaling pathways. Plasma membranes are cell structures where permanently and temporarily associated proteins exert their functions, which at the same time are affected by the surrounding lipid environment (Goni, 2002). It is known that the major fatty acids in different cell types are saturated (C16:0 and C18:0) and unsaturated (C18:1 n-9) fatty acids (>60% total fatty acids in the human brain) (Fabelo et al., 2011) and we confirmed that in Muller cells the major saturated fatty acids are C16:0 and C18:0. The length and degree of unsaturation of natural fatty acids have been associated with their impact on membrane lipid structures (Epand et al., 1991). Changes in the physicochemical properties of membranes, such as lateral pressure (Lopez et al., 2012), membrane fluidity (Lenaz, 1987) or phase behavior (Escriba et al., 1995), regulate the localization and activity of relevant signaling proteins, and result in the regulation of gene expression and the reversion of the pathological state within the cell (Escriba, 2006). Also, lipid alterations may affect the structure and functions of lipid rafts,

Please cite this article in press as: S. Gambert, et al., Cholesterol metabolism and glaucoma: Modulation of Muller cell membrane organization by 24S-hydroxycholesterol, Chem. Phys. Lipids (2017), http://dx.doi.org/10.1016/j.chemphyslip.2017.05.007

G Model CPL 4557 No. of Pages 13

10

S. Gambert et al. / Chemistry and Physics of Lipids xxx (2017) xxx–xxx

Table 2 Proteins with FC>2 and <0.5, their pathways (ORA innateDB, in Gene Ontology, KEGG and Wikipathway)), in raft and non-raft treated/control Muller cells. Raft Treated/ Control Fold Change

Non-Raft Treated/ Control Fold Change

Protein name

Pathways

Gene Symbol

Guanine nucleotide-binding protein (G protein) beta polypeptide 4

glucose-pyruvate/glutamate-glutamine/ aquaporin/ MAPK/purine oxidative stress

Gnb4

150.2

38.8

Ndufb7

31.2

8.9

glucose-pyruvate/calcium/ MAPK/VEGF oxidative stress

Itpr1

24.3

3.3

Ndufa4

12.5

0.10

Sdha Gnb1

9.4 7.1

0.02 0.07

Uqcrc1 Itgb3 Ctnnd1 Glg1 Psmd13 Cspg4 Atp2a2

5.2 4.8 3.4 3.3 2.5 2.5 2.4

0.03 3.7 0.08 0.14 0.01 0.06 0.16

Dld Ndufs1

2.1 0.47

0.06 0.44

Col1a2 Col5a2 Thbs1 Tln1 Prkcsh Hsp90aa1 Anxa1 Flna Vcl Psmd11 Ppp1ca Itga1 Col6a1 Flnb Suclg2 Atp5b

0.45 0.40 0.39 0.39 0.37 0.36 0.32 0.29 0.21 0.16 0.16 0.14 0.14 0.13 0.08 0.07

0.18 0.23 0.35 0.03 0.08 0.01 0.03 0.02 0.06 0.08 0.02 0.09 0.02 0.08 0.02 0.01

Psmc3 Actn1

0.06 0.06

0.02 0.02

oxidative stress

Ndufa4l2

2.9

7.8

VEGF/adhesion/MAPK adhesion glucose-pyruvate

Mapk1 Col1a1 Ddost

0.01 0.27 0.06

2.1 0.38 0.37

hsp-proteasome/NF-kB glucose-pyruvate/adhesion/VEGF adhesion oxidative stress

Psmd1 Iqgap1 Col5a1 Eif2s1

0.02 0.23 0.04 0.03

0.35 0.33 0.30 0.30

purine/MAPK/calcium glucose-pyruvate VEGF/adhesion/hsp-proteasome calcium adhesion oxidative stress

Gnaq Pgm1 Pak2 Plcd1 Capn2 Ppa1

0.25 0.11 0.11 0.05 0.13 0.01

0.29 0.25 0.22 0.21 0.15 0.07

NADH dehydrogenase (Ubiquinone) 1 beta subcomplex, 7, 18 kDa Inositol 1,4,5-trisphosphate receptor type 1 (=IP-3-R)

NADH dehydrogenase (Ubiquinone) 1 beta subcomplex, 4, 9 kDa Succinate dehydrogenase complex, subunit A, flavoprotein (Fp) glucose-pyruvate/oxidative stress Guanine nucleotide binding protein (G protein), beta glucose-pyruvate/glutamate-glutamine/ polypeptide 1 aquaporin/ MAPK/purine Ubiquinol-cytochrome c reductase core protein I oxidative stress Integrin, beta 3 (platelet glycoprotein IIIa, antigen CD61) VEGF/adhesion/MAPK Catenin (Cadherin associated protein), delta 1 adhesion/VEGF Golgi glycoprotein 1 adhesion/TNF Proteasome (prosome, macropain) 26S subunit, non-ATPase 13 hsp-proteasome/TNF/NF-KB Chondroitin sulfate proteoglycan 4 glucose-pyruvate calcium ATPase, Ca + + transporting, cardiac muscle, slow twitch 2 (Sarcoplasmic/endoplasmic reticulum calcium ATPase 2 (=SERCA2)) glucose-pyruvate Dihydrolipoamide dehydrogenase NADH dehydrogenase (ubiquinone) Fe-S protein 1, 75 kDa oxidative stress (NADH-coenzyme Q reductase) adhesion Collagen, type I, alpha 2 Collagen, type I, alpha 2 adhesion Thrombospondin 1 adhesion Talin 1 adhesion/MAPK Protein kinase C substrate 80K-H glucose-pyruvate Heat shock protein 90 kDa alpha (cytosolic), class A member 1 VEGF Annexin A1 MAPK Filamin A alpha adhesion Vinculin adhesion/VEGF Proteasome (prosome, macropain) 26S subunit, non ATPase, 11 NF-kB/hsp-proteasome Protein phosphatase 1, catalytic subunit, alpha isozyme adhesion Integrin, alpha-1 adhesion Collagen, type VI, alpha 1 adhesion Filamin B, beta adhesion Succinate-CoA ligase, GDP-forming, beta subunit glucose-pyruvate ATP synthase, H+ transporting, mitochondrial F1 complex, oxidative stress beta polypeptide NF-kB/hsp-proteasome Proteasome (prosome, macropain) 26S subunit, ATPase Actinin, alpha 1 adhesion NADH dehydrogenase (ubiquinone) 1 alpha subcomplex, 4-like 2 Mitogen-activated protein kinase 1 Collagen, Type I, alpha 1 Dolichyl-diphosphooligosaccharide-protein glycosyltransferase Proteasome (prosome, macropain) 26S subunit, non-ATPase, 1 IQ motif containing GTPase activating protein 1 Collagen, type V, alpha 1 Eukaryotic translation initiation factor 2, subunit 1 alpha, 35 kDa Guanine nucleotide binding protein (G protein), q polypeptide Phosphoglucomutase 1 p21 protein (Cdc42/Rac)-activated kinase 2 Phospholipase C, delta 1 Calpain 2 (m/II) large subunit Pyrophosphatase (inorganic) 1

which, in glaucoma, may enable Muller cells to affect signaling mechanisms such as those involved in cell growth and apoptosis. Through the lateral segregation of membrane-associated proteins, rafts also play a role in the regulation of protein-protein interactions. On the other hand, the clustering of proteins within rafts statistically favors their interaction. The inclusion of a particular protein within rafts prevents its interaction with

proteins located outside rafts or in distinct sub-populations of rafts. This situation results in the inhibition of signaling complex assembly and thus the subsequent activation of cascade events (Sonnino et al., 2014; Villar et al., 2016). The main proteins involved in the mechanisms of retinal gliosis of MGCs are intermediate filament proteins: GFAP, vimentin, nestin. These essential fibrillary proteins of the cytoskeleton provide the link between actin

Please cite this article in press as: S. Gambert, et al., Cholesterol metabolism and glaucoma: Modulation of Muller cell membrane organization by 24S-hydroxycholesterol, Chem. Phys. Lipids (2017), http://dx.doi.org/10.1016/j.chemphyslip.2017.05.007

G Model CPL 4557 No. of Pages 13

S. Gambert et al. / Chemistry and Physics of Lipids xxx (2017) xxx–xxx

11

Table 3 Up and down-regulation of proteins in different pathways in raft and non-raft fractions in Muller cells. Decreased protein expression

Increased protein expression

Pathways

Raft Treated/Control

Non-Raft Treated/Control

Raft Treated/Control

Non-Raft Treated/Control

Adhesion Glucose-pyruvate Oxidative stress VEGF MAPK hsp-Proteasome/NF-kB Calcium Purine TNF Aquaporin Glutamate-glutamine

15 5 4 5 3 3 2 1 0 0 0

16 8 7 5 4 4 3 2 2 1 0

2 6 4 3 4 1 2 1 2 2 2

2 2 2 3 3 0 1 1 0 1 1

filaments and microtubules. Their function is to maintain cell shape, anchor cellular organelles and ensure cell integrity. MGCs express, in particular these three types of intermediate filament proteins. Glial fibrillar acid protein or GFAP is a marker of glial cells and of glial activation. It is a 51 kDa intermediate filament protein and presents three transmembrane domains. This protein has been widely identified as a marker of glial cells (Muller cells and astrocytes) and of the glial activation (Reeves et al., 1989; Okada et al., 1990). During retinal gliosis, vimentin and GFAP are quickly upregulated, and are thus sensitive and early indicators of this process in vivo. If the retinal gliosis persists the process of dedifferentiation begins, nestin is then produced by the cell and appears as indicator of this late stage in the process of gliosis (Bringmann et al., 2009). Indeed, a previous study of our team showed that the peaks of GFAP expression in an experimental rat model of glaucoma occurred several days after hypertonia (Fourgeux et al., 2012). During the present experiment, we were not able to highlight any overexpression of GFAP in the treated group compared with the control group. We were probably at a too early stage to detect any difference in GFAP expression between the two groups. At this low dose of 24S-OHC, there was no massive induction of gliosis. Nestin is only expressed in a process of massive or persistent gliosis lasting several days with the dedifferentiation of the MGCs, which re-expresses nestin, which is normally expressed by neuroprogenitor cells (Fischer and Omar, 2005; Xue et al., 2006b). That’s in line with earlier reports by our group Fourgeux et al., (2009), Filomenko et al., (2015) suggest that the plasma levels of 24S-OHC do not differ in control and glaucoma patients, and that circulating 24S-OHC levels had no correlation with the retinopathy. Gap junctions are involved in intercellular mechanisms that allow the transfer of intercellular molecules without extracellular passage. These junctions allow, for example, MGCs to modulate the neuronal activity by releasing ATP (adenosine tri-phosphate) via connexin channels (Newman, 2015). We observed that connexins30 and 43 were preferentially expressed in raft fractions in MGCs, showing a likely interaction of this protein with raft microdomains, as reported in the literature (Schubert et al., 2002; Strale et al., 2012). The mitogen activated protein kinase (MAPK) intracellular signaling pathway is divided into three groups: ERK 1/2 (extracellular signal-regulated kinase) P42-44, P38 and JNK/SAPK (Stress activated protein kinase) and is a very important transfer pathway. The pathway is activated by phosphorylation (Robbs et al., 2013). The ERK pathway induces the expression of caveolin-1, thereby allowing the cell cycle to achieve G1/S phase transition, and is also closely related to cell proliferation and growth inhibition (Zhu et al., 2011; So and Croft, 2012). Studies have shown that MAPK plays an important role in maintaining ERK1/2 activation and cell cycle regulation. The ERK pathway is mainly

involved in mechanisms that stimulate proliferation and growth, while the P38 pathway is activated by extracellular signals related to cellular stress, thereby allowing cellular migration (Bringmann et al., 2009; Cargnello and Roux, 2011). Therefore, we analyzed MAPK and p-MAPK levels to reflect protein redistribution in the signaling pathway following exposure to 24S-OHC. We found that MAPK protein appeared in non-raft fractions whatever the time or treatment. p-MAPK appeared in both non-raft and raft fractions, whatever the time or treatment. We observed a slight relocalization of p42-44 and p38 to rafts when these proteins were activated. Activation of the P42-44 pathway was also observed. This pathway has been clearly identified in numerous retinal diseases and was highlighted in particular in retinal gliosis during glaucoma (Geller et al., 2001; Tezel et al., 2003; Akiyama et al., 2002). However, the p38 and p42-44 MAPK pathways did not seem to be significantly activated by 24S-OHC at this stage, probably because retinal gliosis had not yet started. The proteomic analysis confirmed that MAPK was up-regulated in the non-raft fractions. The various signaling proteins known to be associated with raft domains include G-proteins (Li et al., 1995) or Src family tyrosine kinases (Shenoy-Scaria et al., 1994). There are several examples of signaling proteins that appear to localize in membrane raft regions under specific conditions. For example, Ras is known to associate with rafts only in its inactive GDP-bound state (Li et al., 1995). In contrast, the muscarinic receptor is reported to move into these domains upon binding to an agonist but not an antagonist (Feron et al., 1997). It has been suggested that caveolae can act as scaffolds for pre-assembled signaling complexes which can be readily activated following cell stimulation. The available data demonstrate that the function of Ca2+ signaling proteins depends on the integrity of cholesterol-rich rafts (Okamoto et al., 1998). Furthermore, as discussed above, several components involved in Ca2+ signaling have been localized to rafts and our proteomics analysis results confirmed these studies. Several examples of regulated MAPK, adhesion or oxidative stress pathways include the upregulation in rafts of Guanine nucleotide-binding protein (G protein) beta polypeptide 4 (FC = 150), NADH dehydrogenase (Ubiquinone) 1 beta subcomplex (7 and 4) (FC = 31.2 and 12.5 respectively), Inositol 1,4,5-trisphosphate receptor type 1 (FC = 24.3) and the down-regulation of NADH dehydrogenase (ubiquinone) Fe-S protein 1 (FC = 0.47), p21 protein (Cdc42/Rac)-activated kinase 2 (FC = 0.11). With no significant changes observed in most of the proteins studied in this work, in treated and control cells, further studies are needed to better characterize the surrounded mechanisms. Conclusion-perspectives Based on the studies reviewed above, lipid composition appears to be a determinant factor of membrane architecture, especially

Please cite this article in press as: S. Gambert, et al., Cholesterol metabolism and glaucoma: Modulation of Muller cell membrane organization by 24S-hydroxycholesterol, Chem. Phys. Lipids (2017), http://dx.doi.org/10.1016/j.chemphyslip.2017.05.007

G Model CPL 4557 No. of Pages 13

12

S. Gambert et al. / Chemistry and Physics of Lipids xxx (2017) xxx–xxx

protein localization and microdomain formation but 24S-OHC does not activate MGC by a raft dynamic. It would be interesting to study a later stage of retinal gliosis to understand the mechanisms involved. The proteomics analysis allowed us to target certain proteins, others are yet to explore. Corroboration of these results will help confirm that impaired cholesterol homeostasis resulting from RGCs damage in glaucoma could be implicated in the activation of retinal gliosis, which could eventually lead to blindness, and help improve our understanding of the complex mechanisms of this optical neuropathy. Conflicts of interests The authors declare that there are no conflicts of interest. References Ackman, R.G., 1981. Flame ionization detection applied to thin-layer chromatography on coated quartz rods. Methods Enzymol. 72, 205–252. Akiyama, H., Nakazawa, T., Shimura, M., Tomita, H., Tamai, M., 2002. Presence of mitogen-activated protein kinase in retinal Muller cells and its neuroprotective effect ischemia-reperfusion injury. Neuroreport 13, 2103–2107. Bevers, E.M., Comfurius, P., Dekkers, D.W., Zwaal, R.F., 1999. Lipid translocation across the plasma membrane of mammalian cells. Biochim. Biophys. Acta 1439, 317–330. Bignami, A., Dahl, D., 1979. The radial glia of Muller in the rat retina and their response to injury:An immunofluorescence study with antibodies to the glial fibrillary acidic (GFA) protein. Exp. Eye Res. 28, 63–69. Bretillon, L., Thuret, G., Gregoire, S., Acar, N., Joffre, C., Bron, A.M., Gain, P., CreuzotGarcher, C.P., 2008. Lipid and fatty acid profile of the retina, retinal pigment epithelium/choroid, and the lacrimal gland, and associations with adipose tissue fatty acids in human subjects. Exp. Eye Res. 87, 521–528. Bringmann, A., Reichenbach, A., 2001. Role of Muller cells in retinal degenerations. Front. Biosci. 6, E72–92. Bringmann, A., Pannicke, T., Grosche, J., Francke, M., Wiedemann, P., Skatchkov, S.N., Osborne, N.N., Reichenbach, A., 2006. Muller cells in the healthy and diseased retina. Prog. Retin. Eye Res. 25, 397–424. Bringmann, A., Iandiev, I., Pannicke, T., Wurm, A., Hollborn, M., Wiedemann, P., Osborne, N.N., Reichenbach, A., 2009. Cellular signaling and factors involved in Muller cell gliosis: neuroprotective and detrimental effects. Prog. Retin. Eye Res. 28, 423–451. Brown, D.A., London, E., 2000. Structure and function of sphingolipid- and cholesterol-rich membrane rafts. J. Biol. Chem. 275, 17221–17224. Brown, D.A., Rose, J.K., 1992. Sorting of GPI-anchored proteins to glycolipid-enriched membrane subdomains during transport to the apical cell surface. Cell 68, 533– 544. Brugger, B., Erben, G., Sandhoff, R., Wieland, F.T., Lehmann, W.D., 1997. Quantitative analysis of biological membrane lipids at the low picomole level by nanoelectrospray ionization tandem mass spectrometry. Proc. Natl. Acad. Sci. U. S. A. 94, 2339–2344. Callera, G.E., Yogi, A., Briones, A.M., Montezano, A.C., He, Y., Tostes, R.C., Schiffrin, E. L., Touyz, R.M., 2011. Vascular proinflammatory responses by aldosterone are mediated via c-Src trafficking to cholesterol-rich microdomains: role of PDGFR. Cardiovasc. Res. 91, 720–731. Cargnello, M., Roux, P.P., 2011. Activation and function of the MAPKs and their substrates, the MAPK-activated protein kinases. Microbiol. Mol. Biol. Rev. 75, 50–83. Chen, H., Weber, A.J., 2002. Expression of glial fibrillary acidic protein and glutamine synthetase by Muller cells after optic nerve damage and intravitreal application of brain-derived neurotrophic factor. Glia 38, 115–125. Clough, T., Thaminy, S., Ragg, S., Aebersold, R., Vitek, O., 2012. Statistical protein quantification and significance analysis in label-free LC–MS experiments with complex designs. BMC Bioinf. 13 (Suppl. 16), S6. Edwards, S.H., Kimberly, M.M., Pyatt, S.D., Stribling, S.L., Dobbin, K.D., Myers, G.L., 2011. Proposed serum cholesterol reference measurement procedure by gas chromatography-isotope dilution mass spectrometry. Clin. Chem. 57, 614–622. Ekstrom, P., Sanyal, S., Narfstrom, K., Chader, G.J., van Veen, T., 1988. Accumulation of glial fibrillary acidic protein in Muller radial glia during retinal degeneration. Invest. Ophthalmol. Vis. Sci. 29, 1363–1371. Epand, R.M., Epand, R.F., Ahmed, N., Chen, R., 1991. Promotion of hexagonal phase formation and lipid mixing by fatty acids with varying degrees of unsaturation. Chem. Phys. Lipids 57, 75–80. Escriba, P.V., Sastre, M., Garcia-Sevilla, J.A., 1995. Disruption of cellular signaling pathways by daunomycin through destabilization of nonlamellar membrane structures. Proc. Natl. Acad. Sci. U. S. A. 92, 7595–7599. Escriba, P.V., 2006. Membrane-lipid therapy: a new approach in molecular medicine. Trends Mol. Med. 12, 34–43. Fabelo, N., Martin, V., Santpere, G., Marin, R., Torrent, L., Ferrer, I., Diaz, M., 2011. Severe alterations in lipid composition of frontal cortex lipid rafts from Parkinson's disease and incidental Parkinson's disease. Mol. Med. 17, 1107–1118.

Feron, O., Smith, T.W., Michel, T., Kelly, R.A., 1997. Dynamic targeting of the agoniststimulated m2 muscarinic acetylcholine receptor to caveolae in cardiac myocytes. J. Biol. Chem. 272, 17744–17748. Filomenko, R., Fourgeux, C., Bretillon, L., Gambert-Nicot, S., 2015. Oxysterols: influence on plasma membrane rafts microdomains and development of ocular diseases. Steroids 99, 259–265. Fischer, A.J., Omar, G., 2005. Transitin, a nestin-related intermediate filament, is expressed by neural progenitors and can be induced in Muller glia in the chicken retina. J. Comp. Neurol. 484, 1–14. Folch, J., Lees, M., Sloane Stanley, G.H., 1957. A simple method for the isolation and purification of total lipides from animal tissues. J. Biol. Chem. 226, 497–509. Fourgeux, C., Martine, L., Bjorkhem, I., Diczfalusy, U., Joffre, C., Acar, N., CreuzotGarcher, C., Bron, A., Bretillon, L., 2009. Primary open-angle glaucoma: association with cholesterol 24S-hydroxylase (CYP46A1) gene polymorphism and plasma 24-hydroxycholesterol levels. Invest. Ophthalmol. Vis. Sci. 50, 5712– 5717. Fourgeux, C., Martine, L., Pasquis, B., Maire, M.A., Acar, N., Creuzot-Garcher, C., Bron, A., Bretillon, L., 2012. Steady-state levels of retinal 24S-hydroxycholesterol are maintained by glial cells intervention after elevation of intraocular pressure in the rat. Acta Ophthalmol. 90, e560–567. Geller, S.F., Lewis, G.P., Fisher, S.K., 2001. FGFR1, signaling, and AP-1 expression after retinal detachment: reactive Muller and RPE cells. Invest. Ophthalmol. Vis. Sci. 42, 1363–1369. Ghosh, F., Johansson, K., 2012. Neuronal and glial alterations in complex long-term rhegmatogenous retinal detachment. Curr. Eye Res. 37, 704–711. Goni, F.M., 2002. Non-permanent proteins in membranes: when proteins come as visitors (Review). Mol. Membr. Biol. 19, 237–245. Hanzal-Bayer, M.F., Hancock, J.F., 2007. Lipid rafts and membrane traffic. FEBS Lett. 581, 2098–2104. Humphrey, M.F., Chu, Y., Mann, K., Rakoczy, P., 1997. Retinal GFAP and bFGF expression after multiple argon laser photocoagulation injuries assessed by both immunoreactivity and mRNA levels. Exp. Eye Res. 64, 361–369. Iandiev, I., Tenckhoff, S., Pannicke, T., Biedermann, B., Hollborn, M., Wiedemann, P., Reichenbach, A., Bringmann, A., 2006. Differential regulation of Kir4.1 and Kir2.1 expression in the ischemic rat retina. Neurosci. Lett. 396, 97–101. Kim, I.B., Kim, K.Y., Joo, C.K., Lee, M.Y., Oh, S.J., Chung, J.W., Chun, M.H., 1998. Reaction of Muller cells after increased intraocular pressure in the rat retina. Exp. Brain Res. 121, 419–424. Lenaz, G., 1987. Lipid fluidity and membrane protein dynamics. Biosci. Rep. 7, 823– 837. Lewis, G.P., Fisher, S.K., 2003. Up-regulation of glial fibrillary acidic protein in response to retinal injury: its potential role in glial remodeling and a comparison to vimentin expression. Int. Rev. Cytol. 230, 263–290. Lewis, G.P., Erickson, P.A., Guerin, C.J., Anderson, D.H., Fisher, S.K., 1989. Changes in the expression of specific Muller cell proteins during long-term retinal detachment. Exp. Eye Res. 49, 93–111. Li, S., Okamoto, T., Chun, M., Sargiacomo, M., Casanova, J.E., Hansen, S.H., Nishimoto, I., Lisanti, M.P., 1995. Evidence for a regulated interaction between heterotrimeric G proteins and caveolin. J. Biol. Chem. 270, 15693–15701. Lopez, D.J., Egido-Gabas, M., Lopez-Montero, I., Busto, J.V., Casas, J., Garnier, M., Monroy, F., Larijani, B., Goni, F.M., Alonso, A., 2012. Accumulated bending energy elicits neutral sphingomyelinase activity in human red blood cells. Biophys. J. 102, 2077–2085. Luna, G., Lewis, G.P., Banna, C.D., Skalli, O., Fisher, S.K., 2010. Expression profiles of nestin and synemin in reactive astrocytes and Muller cells following retinal injury: a comparison with glial fibrillar acidic protein and vimentin. Mol. Vis. 16, 2511–2523. Monroe, M.E., Shaw, J.L., Daly, D.S., Adkins, J.N., Smith, R.D., 2008. MASIC: a software program for fast quantitation and flexible visualization of chromatographic profiles from detected LC-MS(/MS) features. Comput. Biol. Chem. 32, 215–217. Morrison, W.R., Smith, L.M., 1964. Preparation of fatty acid methyl esters and dimethylacetals from lipids with boron fluoride–methanol. J. Lipid Res. 5, 600– 608. Nesvizhskii, A.I., Keller, A., Kolker, E., Aebersold, R., 2003. A statistical model for identifying proteins by tandem mass spectrometry. Anal. Chem. 75, 4646–4658. Newman, E.A., 2015. Glial cell regulation of neuronal activity and blood flow in the retina by release of gliotransmitters. Philos. Trans. R Soc. Lond. B Biol. Sci. 370. Noghero, A., Perino, A., Seano, G., Saglio, E., Lo Sasso, G., Veglio, F., Primo, L., Hirsch, E., Bussolino, F., Morello, F., 2012. Liver X receptor activation reduces angiogenesis by impairing lipid raft localization and signaling of vascular endothelial growth factor receptor-2. Arterioscler. Thromb. Vasc. Biol. 32, 2280– 2288. Okada, M., Matsumura, M., Ogino, N., Honda, Y., 1990. Muller cells in detached human retina express glial fibrillary acidic protein and vimentin. Graefes Arch. Clin. Exp. Ophthalmol. 228, 467–474. Okamoto, T., Schlegel, A., Scherer, P.E., Lisanti, M.P., 1998. Caveolins, a family of scaffolding proteins for organizing preassembled signaling complexes at the plasma membrane. J. Biol. Chem. 273, 5419–5422. Oku, H., Ikeda, T., Honma, Y., Sotozono, C., Nishida, K., Nakamura, Y., Kida, T., Kinoshita, S., 2002. Gene expression of neurotrophins and their high-affinity Trk receptors in cultured human Muller cells. Ophthalmic Res. 34, 38–42. Pathak, P., London, E., 2015. The effect of membrane lipid composition on the formation of lipid ultrananodomains. Biophys. J . 109, 1630–1638. Pfrieger, F.W., Ungerer, N., 2011. Cholesterol metabolism in neurons and astrocytes. Prog. Lipid Res. 50, 357–371. Pike, L.J., 2003. Lipid rafts: bringing order to chaos. J. Lipid Res. 44, 655–667.

Please cite this article in press as: S. Gambert, et al., Cholesterol metabolism and glaucoma: Modulation of Muller cell membrane organization by 24S-hydroxycholesterol, Chem. Phys. Lipids (2017), http://dx.doi.org/10.1016/j.chemphyslip.2017.05.007

G Model CPL 4557 No. of Pages 13

S. Gambert et al. / Chemistry and Physics of Lipids xxx (2017) xxx–xxx Pike, L.J., 2004. Lipid rafts: heterogeneity on the high seas. Biochem. J 378, 281–292. Pike, L.J., 2006. Rafts defined: a report on the keystone symposium on lipid rafts and cell function. J. Lipid Res. 47, 1597–1598. Pike, L.J., 2009. The challenge of lipid rafts. J. Lipid Res. 50 (Suppl), S323–S328. Reeves, S.A., Helman, L.J., Allison, A., Israel, M.A., 1989. Molecular cloning and primary structure of human glial fibrillary acidic protein. Proc. Natl. Acad. Sci. U. S. A. 86, 5178–5182. Reichenbach, A., Bringmann, A., 2013. New functions of Muller cells. Glia 61, 651– 678. Robbs, B.K., Lucena, P.I., Viola, J.P., 2013. The transcription factor NFAT1 induces apoptosis through cooperation with Ras/Raf/MEK/ERK pathway and upregulation of TNF-alpha expression. Biochim. Biophys. Acta 1833, 2016–2028. Schubert, A.L., Schubert, W., Spray, D.C., Lisanti, M.P., 2002. Connexin family members target to lipid raft domains and interact with caveolin-1. Biochemistry 41, 5754–5764. Sebedio, J.L., Astorg, P.O., Septier, C., Grandgirard, A., 1987. Quantitative analyses of polar components in frying oils by the iatroscan thin-layer chromatographyflame ionization detection technique. J. Chromatogr. 405, 371–378. Shenoy-Scaria, A.M., Dietzen, D.J., Kwong, J., Link, D.C., Lublin, D.M., 1994. Cysteine3 of Src family protein tyrosine kinase determines palmitoylation and localization in caveolae. J. Cell Biol. 126, 353–363. Sibille, E., Berdeaux, O., Martine, L., Bron, A.M., Creuzot-Garcher, C.P., He, Z., Thuret, G., Bretillon, L., Masson, E.A., 2016. Ganglioside profiling of the human retina: comparison with other ocular structures, brain and plasma reveals tissue specificities. PLoS One 11, e0168794. Singer, S.J., Nicolson, G.L., 1972. The fluid mosaic model of the structure of cell membranes. Science 175, 720–731.

13

So, T., Croft, M., 2012. Regulation of the PKCtheta-NF-kappaB axis in t lymphocytes by the tumor necrosis factor receptor family member OX40. Front. Immunol. 3, 133. Sonnino, S., Aureli, M., Grassi, S., Mauri, L., Prioni, S., Prinetti, A., 2014. Lipid rafts in neurodegeneration and neuroprotection. Mol. Neurobiol. 50, 130–148. Strale, P.O., Clarhaut, J., Lamiche, C., Cronier, L., Mesnil, M., Defamie, N., 2012. Downregulation of Connexin43 expression reveals the involvement of caveolin-1 containing lipid rafts in human U251 glioblastoma cell invasion. Mol. Carcinog. 51, 845–860. Tezel, G., Chauhan, B.C., LeBlanc, R.P., Wax, M.B., 2003. Immunohistochemical assessment of the glial mitogen-activated protein kinase activation in glaucoma. Invest. Ophthalmol. Vis. Sci. 44, 3025–3033. Villar, V.A., Cuevas, S., Zheng, X., Jose, P.A., 2016. Localization and signaling of GPCRs in lipid rafts. Methods Cell Biol. 132, 3–23. Wang, J., Megha, London, E., 2004. Relationship between sterol/steroid structure and participation in ordered lipid domains (lipid rafts): implications for lipid raft structure and function. Biochemistry 43, 1010–1018. Xue, L.P., Lu, J., Cao, Q., Hu, S., Ding, P., Ling, E.A., 2006a. Muller glial cells express nestin coupled with glial fibrillary acidic protein in experimentally induced glaucoma in the rat retina. Neuroscience 139, 723–732. Xue, L.P., Lu, J., Cao, Q., Kaur, C., Ling, E.A., 2006b. Nestin expression in Muller glial cells in postnatal rat retina and its upregulation following optic nerve transection. Neuroscience 143, 117–127. Zhu, H., Li, H., Han, Z., Shao, Y., Wang, Y., Kong, X., 2011. Identification of a spliced gene from duck enteritis virus encoding a protein homologous to UL15 of herpes simplex virus 1. Virol. J. 8, 156.

Please cite this article in press as: S. Gambert, et al., Cholesterol metabolism and glaucoma: Modulation of Muller cell membrane organization by 24S-hydroxycholesterol, Chem. Phys. Lipids (2017), http://dx.doi.org/10.1016/j.chemphyslip.2017.05.007