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Resume La retine est le tissu neurosensoriel de l’œil qui assure la transduction visuelle, c’est-a-dire le codage de l’information lumineuse en influx nerveux. Le terme « retine » regroupe l’association de la retine neurale et de l’epithelium pigmentaire retinien qui repose sur la membrane de Bruch. L’association physique de ces deux partenaires et leurs interactions metaboliques sont des elements indispensables au bon fonctionnement de la retine, a la fois en termes d’apport en nutriments et de maintien de l’homeostasie. Des dereglements de ces equilibres sont associes au vieillissement et a des pathologies comme la degenerescence maculaire liee a l’âge, premiere cause de malvoyance apres 50 ans dans les populations occidentales. La retine neurale est composee de plusieurs types cellulaires, incluant la glie dont le role est crucial pour la biosynthese du cholesterol dans la retine. Le cholesterol est le principal sterol de la retine. Il y est present au niveau cellulaire essentiellement en tant que cholesterol libre, et d’esters de cholesterol au niveau extracellulaire dans la membrane de Bruch. L’accumulation d’esters de cholesterol a ce niveau est un marqueur du vieillissement de la retine et de la diminution de sa fonction. Soumis a l’influence alimentaire et synthetise localement, le cholesterol est egalement regule via ses interactions avec les differentes populations cellulaires de la retine, et en particulier celles qui font intervenir les communications neurones/glie via la cholesterol 24S-hydroxylase (CYP46A1). Des liens physiopathologiques entre le metabolisme du cholesterol et les glaucomes ou la degenerescence maculaire liee a l’âge (DMLA) sont avances.

International audience; Reward-processing impairment is a common symptomatic dimension of several psychiatric disorders. However, whether the underlying pathological mechanisms are common is unknown. Herein, we asked if the decrease in the n-3 polyunsaturated fatty acid (PUFA) lipid species, consistently described in these pathologies, could underlie reward-processing deficits. We show that reduced n-3 PUFA biostatus in mice leads to selective motivational impairments. Electrophysiological recordings revealed increased collateral inhibition of dopamine D2 receptor-expressing medium spiny neurons (D2-MSNs) onto dopamine D1 receptor-expressing MSNs in the nucleus accumbens, a main brain region for the modulation of motivation. Strikingly, transgenically preventing n-3 PUFA deficiency selectively in D2-expressing neurons normalizes MSN collateral inhibition and enhances motivation. These results constitute the first demonstration of a causal link between a behavioral deficit and n-3 PUFA decrease in a discrete neuronal population and suggest that lower n-3 PUFA biostatus in psychopathologies could participate in the etiology of reward-related symptoms.

International audience

International audience; In this investigation, we aim to study, for the first time, the effect of the growing area on tocopherols, carotenoids and fatty acid content of Pistacia lentiscus fixed oil. Fruits were harvested from eight different sites located in the north and the centre of Tunisia. Tocopherols, carotenoids and fatty acid content of the fixed oils were determined. The highest carotenoid content was exhibited by Feija oil (10.57 mg/kg of oil). Oueslatia and Tabarka oils displayed the highest alpha-tocopherol content (96.79 and 92.79 mg/kg of oil, respectively). Three major fatty acids were determined: oleic, palmitic and linoleic acids. Oleic acid was the main fatty acid presenting more than 50% of the total fatty acid content. Kebouche oil presented the highest oleic acid content (55.66%). All these results highlight the richness of carotenoids, tocopherols and unsaturated fatty acids in P. lentiscus seed oil and underscore the nutritional value of this natural product.

The oxidised derivatives of phytosterols (oxyphytosterols) were identified in plasma samples from thirteen healthy human volunteers, using MS. All the samples contained noticeable quantities of (24R)-5b,6b-epoxy-24-ethylcholestan-3b-ol (b-epoxysitostanol) and (24R)-ethylcholestan-3b,5a,6b-triol (sitostanetriol) and also trace levels of (24R)-5a,6a-epoxy-24-ethylcholestan-3b-ol (a-epoxysitostanol), (24R)-methylcholestan-3b,5a,6b-triol (campestanetriol) and (24R)-ethylch olest-5-en-3b-ol-7-one(7-ketositosterol). The amounts of these oxyphytosterols in plasma varied from 4·8 to 57·2 ng/ml. There are two possibilities concerning the origin of these compounds. First, they could come from the small amounts of oxyphytosterols in food. Second, they could originate from the in vivo oxidation of phytosterols in plasma. Very few data actually exist concerning these compounds. Their identification in human samples suggests that further research is necessary in this field. Oxyphytosterols: Phytosterols: Human plasma: Mass spectrometry

WOS:000320433100013 ; http://link.springer.com/journal/11746; International audience; Seeds from 12 Acacia cyanophylla ecotypes, harvested in Tunisia, were examined for their seed oil contents of carotenoids, tocopherols and phytosterols. The average carotenoid content (lutein and zeaxanthin) was ca. 102 mg kg(-1) of total extracted lipids. Lutein (ca. 97 mg kg(-1) of total extracted lipids) was usually more abundant than zeaxanthin (ca. 5 mg kg(-1) of total extracted lipids). The mean total tocopherol content was ca. 704 mg kg(-1) of total extracted lipids. The main isomer was alpha-tocopherol, with more than 75 % of total tocopherols (ca. 528 mg kg(-1) of total extracted lipids), followed by gamma-tocopherol (ca. 168 mg kg(-1) of total extracted lipids) and delta-tocopherol (ca. 86 mg kg(-1) of total lipids). High levels of phytosterols (ca. 7.8 g kg(-1) of total extracted lipids) were detected, among which beta-sitosterol was the most abundant (47 %). All these results highlight the richness of carotenoids, tocopherols and sterols in A. cyanophylla seed oil, and imply that this species might constitute a potential resource for the development of functional foods.

Purpose: Our previous studies suggested that CYP46A1 and 24S-hydroxycholesterol. (24SOH) may be associated with glaucoma. Loss of CYP46A1-expressing retinal. ganglion cells is involved in the activation of glia, and therefore possibly in the. disbalance of cholesterol. In this context, the purpose of our present work was to. emphasize the glial and longitudinal CYP46A1 expression after an interventional. glaucoma-related stress triggered by elevated intraocular pressure (IOP). Methods: Sprague–Dawley rats were submitted to laser photocoagulation of the. trabecular meshwork, limbus and episcleral veins in one eye to induce elevated IOP. Rats were euthanized at days 3, 14, 30 and 60 (n = 10 per time-point), and 24SOH. was measured in plasma and retina by gas chromatography–mass spectrometry. CYP46A1 was quantified by Western blotting. Glial activation was assessed by glial. fibrillary acidic protein immunoreactivity in Western blots and retinal cryosections. Results: Sustained high IOP was observed in experimental eyes from day 1 to day. 21. Plasma MCP-1 and ICAM-1, quantified using multiplex assay kits, were. increased at day 3. Glial activation was observed bilaterally at all time-points, at. lower levels in contralateral eyes than in experimental eyes. In experimental retinas,. CYP46A1 expression showed a transient increase at day 3 and then returned to. baseline. Plasma and retinal 24SOH peaked at day 14 and 30, respectively. Conclusions: These data show that CYP46A1 expression was induced early in. response to retinal stress but remained constant at late time-points, reinforcing the. constitutive role of CYP46A1 in maintaining cholesterol balance in neuronal tissues.

International audience; BACKGROUND: The assessment of blood lipids is very frequent in clinical research as it is assumed to reflect the lipid composition of peripheral tissues. Even well accepted such relationships have never been clearly established. This is particularly true in ophthalmology where the use of blood lipids has become very common following recent data linking lipid intake to ocular health and disease. In the present study, we wanted to determine in humans whether a lipidomic approach based on red blood cells could reveal associations between circulating and tissue lipid profiles. To check if the analytical sensitivity may be of importance in such analyses, we have used a double approach for lipidomics. METHODOLOGY AND PRINCIPAL FINDINGS: Red blood cells, retinas and optic nerves were collected from 9 human donors. The lipidomic analyses on tissues consisted in gas chromatography and liquid chromatography coupled to an electrospray ionization source-mass spectrometer (LC-ESI-MS). Gas chromatography did not reveal any relevant association between circulating and ocular fatty acids except for arachidonic acid whose circulating amounts were positively associated with its levels in the retina and in the optic nerve. In contrast, several significant associations emerged from LC-ESI-MS analyses. Particularly, lipid entities in red blood cells were positively or negatively associated with representative pools of retinal docosahexaenoic acid (DHA), retinal very-long chain polyunsaturated fatty acids (VLC-PUFA) or optic nerve plasmalogens. CONCLUSIONS AND SIGNIFICANCE: LC-ESI-MS is more appropriate than gas chromatography for lipidomics on red blood cells, and further extrapolation to ocular lipids. The several individual lipid species we have identified are good candidates to represent circulating biomarkers of ocular lipids. However, further investigation is needed before considering them as indexes of disease risk and before using them in clinical studies on optic nerve neuropathies or retinal diseases displaying photoreceptors degeneration.

Purpose Oxidative Stress (OS) plays a critical role in the pathogenesis of age-related macular degeneration (AMD), especially by targeting the retinal pigment epithelium (RPE). Dietary habits with high consumption of docosahexaenoic acid (DHA) have been shown to prevent the development and evolution of AMD. Nevertheless, it is still unclear how DHA affects AMD. Our study aimed to investigate the involvement of the PI3K/Akt and m-TOR/p70-p85S6K pathways in human RPE cells after induction of OS, and then to assess the effect of DHA in the signaling pathways and in the protection against RPE cell death. Methods For this purpose, we used ARPE-19 cells exposed to the prooxidant agent, tert-butyl hydroperoxide (t-BHP). Results We found that exposing cells to t-BHP (400µM) showed complete inhibition of Akt and p70/p85S6K active forms. However in cells enriched with DHA (20µM) and then exposed to t-BHP (400µM), we demonstrated that Akt and p85S6K, but not p70S6K, remained phosphorylated for a longer time after stress. In addition there was a 2.6-fold decrease in the number of necrotic cells after 48hours of t-BHP treatment, as assessed by flow cytometry. Conclusion Our study suggests that 1/ PI3K/Akt and m-TOR/P70-p85S6K pathways play an important role in OS, 2/DHA protects RPE cells from apoptosis and necrosis triggered by OS by enhancing the phosphorylation of Akt and p85S6K.

Oxysterols, found in some commonly consumed foods, can induce a wide range of cytotoxic effects, which have been extensively studied. On the other hand, the side effects of phytosterols and oxyphytosterols are less well-known. Over the past few years, different types of foods have been enriched with phytosterols on the basis of the properties of these compounds that reduce circulating cholesterol levels in certain experimental conditions. It is therefore important to gain better knowledge of the risks and benefits of this type of diet. In this study, conducted in human monocytic U937 cells, the ability of phytosterols (sitosterol, campesterol) and oxyphytosterols (7 beta-hydroxysitosterol, 7-ketositosterol) to induce cell death, polar lipid accumulation, and pro-inflammatory cytokine (MCP-1; IL-8) secretion was determined and compared to that of oxysterols (7-ketocholesterol, 7 beta-hydroxycholesterol). Phytosterols and oxyphytosterols had no significant effects on the parameters studied; only 7 beta-hydroxysitosterol slightly increased cell death, whereas at the concentration used (20 mu g/mL), strong cytotoxic effects were observed with the oxysterols. With sitosterol, campesterol, and 7-ketositosterol, IL-8 secretion was decreased, and with campesterol the intracellular polar lipid level was reduced. The data show that phytosterols and oxyphytosterols have no oxysterol-like side effects, and they rather argue in favor of phytosterols' beneficial effects.

The retina is one of the vertebrate tissues with the highest content in polyunsaturated fatty acids (PUFA). A large proportion of retinal phospholipids, especially those found in photoreceptor membranes, are dipolyunsaturated molecular species. Among them, dipolyunsaturated phosphatidylcholine (PC) molecular species are known to contain very-long-chain polyunsaturated fatty acids (VLC-PUFA) from the n-3 and n-6 series having 24-36 carbon atoms (C24-C36) and four to six double bonds. Recent interest in the role played by VLC-PUFA arose from the findings that a protein called elongation of very-long-chain fatty acids 4 (ELOVL4) is involved in their biosynthesis and that mutations in the ELOVL4 gene are associated with Stargardt-like macular dystrophy (STD3), a dominantly inherited juvenile macular degeneration leading to vision loss. The aim of the present study was to develop an HPLC-ESI-MS/MS method for the structural characterisation and the quantification of dipolyunsaturated PC molecular species containing VLC-PUFA and validate this methodology on retinas from bovines and human donors. Successful separation of phosphatidylethanolamine (PE), phosphatidylinositol (PI), phosphatidylserine (PS), PC, lyso-phosphatidylcholine (LPC) and sphingomyelin (SM) was achieved using a silica gel column and a gradient of hexane/isopropanol/water containing ammonium formate as a mobile phase. A complete structural characterisation of intact phosphatidylcholine species was obtained by collision-induced dissociation (CID) in the negative mode. Fatty acid composition and distribution can be clearly assigned based on the intensity of sn-2/sn-1 fragment ions. The PC species were characterised on bovine retina, 28 of which were dipolyunsaturated PC species containing one VLC-PUFA (C24-C36) with three to six double bonds. VLC-PUFA was always in the sn-1 position while PUFA at the sn-2 position was exclusively docosahexaenoic acid (DHA, C22:6n-3). Most of these VLC-PUFA-containing dipolyunsaturated PCs were detected and quantified in human retinas. The quantitative analysis of the different PC molecular species was performed in the positive mode using precursor ion scanning of m/z 184 and 14:0/14:0-PC and 24:0/24:0-PC as internal standards. The relationship between the MS peak intensities of different PC species and their carbon chain length was included for calibration. The main compounds represented were those having VLC-PUFA with 32 carbon atoms (C32:3, C32:4, C32:5 and C32:6) and 34 carbon atoms (C34:3, C34:4, C34:5 and C34:6). Dipolyunsaturated PCs with 36:5 and 36:6 were detected but in smaller quantities. In conclusion, this new HPLC-ESI-MS/MS method is sensitive and specific enough to structurally characterise and quantify all molecular PC species, including those esterified with VLC-PUFA. This technique is valuable for a precise characterisation of PC molecular species containing VLC-PUFA in retina and may be useful for a better understanding of the pathogenesis of STD3.

International audience; Purpose: Aging is associated with an accumulation of cholesterol esters in the Bruch membrane. Cholesterol esters are prone to undergo oxidation and generate oxysterols that have cytotoxic and proinflammatory properties. We investigated the effects of three oxysterols on mitochondrial dysfunctions, inflammation, and oxidative stress in primary cultures of porcine retinal pigment epithelial (RPE) cells. Methods: RPE cells were incubated with oxysterols (50 M of 24-hydroxycholesterol, 25-hydroxycholesterol, or 7-ketocholesterol) for 24 hr and 48 hr. Oxysterol content was determined in cells and in corresponding media by gas chromatography. Mitochondrial activity was measured by mitochondrial dehydrogenase activity. The intracellular formation of reactive oxygen species in RPE cells was detected by using the fluorescent probe DCFH-DA. IL-8 was assayed in the supernatants by ELISA, and the corresponding cellular transcripts were semiquantified by RT-PCR. Results: Analyses of the oxysterols content in the RPE cells and corresponding media suggested a high rate of cellular uptake, although some differences were observed between 7-ketocholesterol on the one hand and 24-hydroxycholesterol and 25-hydroxycholesterol on the other hand. All oxysterols induced slight mitochondrial dysfunctions but a significant 2- to 4-fold increase in reactive oxygen species (ROS) production compared with the control. They also enhanced IL-8 gene expression and IL-8 protein secretion in the following decreasing order: 25-hydroxycholesterol > 24-hydroxycholesterol > 7-ketocholesterol. Conclusions: We conclude that in confluent primary porcine RPE cells, 24-hydroxycholesterol, 25-hydroxycholesterol, and 7-ketocholesterol are potent inducers of oxidation and inflammation

The purpose of this study was to evaluate the oxidative capacities and the rate of energy synthesis in isolated mitochondria extracted from normal and post-ischemic myocardium. Isolated rat hearts were perfused according to the working mode with a Krebs Heinseleit buffer containing glucose (11 mM), insulin (10 IU/l) and caprylic acid (25 microM). After a 15 min perfusion in normoxic conditions, the hearts were subjected to a 20 min local zero-flow ischemia followed by a 20 min reperfusion. During the perfusion, the aortic and coronary flows, the aortic pressure and the electrocardiogram were monitored. At the end of the reperfusion period, the non-ischemic and ischemic zones (NIZ and IZ, respectively) were separated and the mitochondria were harvested from each zone. The oxygen uptake and the rate of energy production of the NIZ and IZ mitochondria were then assessed with palmitoylcarnitine as substrate in 2 buffers differing in their free calcium concentration (0.041 and 0.150 microM). Ischemia provoked a 50% reduction of coronary and aortic flows. The reperfusion of the IZ allowed the partial recovery of coronary flow, but the aortic flow decreased beneath its ischemic value because of the occurrence of severe arrhythmias, stunning and probably hibernation. The IZ mitochondria displayed a lower rate of oxygen consumption, whatever the buffer free calcium concentration. Conversely, their rate of energy production was increased, indicating that their metabolic efficiency was improved as compared to NIZ mitochondria. This might be due to the mitochondrial calcium overload persisting during reperfusion, to the activation of the inner membrane Na+/Ca2+ exchange and to a significant mitochondrial swelling. On the other hand, the presence of an elevated free calcium concentration in the respiration buffer provoked some energy wasting characterized by a constant AMP production. This was attributed to some accumulation of acetate and the activation of the energy-consuming acetylCoA synthetase. In conclusion, ischemia and reperfusion did not alter the membrane integrity of the mitochondria but improved their metabolic efficiency. Nevertheless, these in vitro results can not reflect the mitochondrial function in the reperfused myocardium. The mitochondrial calcium overload reported to last during reperfusion in the cardiomyocytes might mimic the free calcium-induced reduction of metabolic efficiency observed in vitro in the present study. The resulting energy wasting might be responsible for the contractile abnormalities noticed in the reperfused myocardium.