Cholestérol-24S-hydroxylase (CYP46A1) et homéostasie du cholestérol dans la rétine en conditions physiologiques et pathologiques

Le cholestérol est le principal stérol présent dans la rétine. Dans sa forme libre, le cholestérol est distribué dans toutes les couches cellulaires de la rétine, alors que le cholestérol estérifié s accumule essentiellement à la base de l épithélium pigmentaire rétinien. La capacité intrinsèque de la rétine à synthétiser le cholestérol paraît limitée, ce qui implique nécessairement que des voies extra-rétiniennes participent activement à suppléer la rétine en cholestérol. Les cellules gliales de Müller contribueraient à l apport de cholestérol aux neurones de la rétine, en particulier pour la formation des synapses. Les conséquences délétères d une accumulation ou à l inverse d un déficit en cholestérol dans les neurones sur leur survie souligne l importance de maintenir l équilibre entre l apport et la néosynthèse du cholestérol d une part et son élimination d autre part. Pour cela, la rétine neurale a en particulier la capacité de convertir, pour l éliminer, le cholestérol en 24S-hydroxycholestérol. En effet, le transport du 24S-hydroxycholestérol au travers des membranes est facilité par la présence d un groupe hydroxyle supplémentaire, lui conférant une polarité plus importante par rapport au cholestérol. L enzyme qui catalyse cette réaction est la cholestérol-24S-hydroxylase (CYP46A1). Des liens ont été établis entre CYP46A1, 24S-hydroxycholestérol et processus neurodégénératifs dans le cerveau, suggérant un rôle potentiel dans certaines pathologies comme la maladie d Alzheimer. CYP46A1 est exprimée dans la rétine neurale, et plus particulièrement dans les cellules ganglionnaires de la rétine. Le rôle de CYP46A1 dans la rétine reste pour l instant inconnu. Cependant, par analogie avec le cerveau, nous pouvons supposer une fonction dans le contrôle de l homéostasie du cholestérol dans les neurones et envisager une association avec des pathologies dégénératives de la rétine comme la Dégénérescence Maculaire Liée à l Âge (DMLA) ou le glaucome. Dans ce contexte, l objectif de nos travaux a consisté à évaluer le rôle de la cholestérol-24S-hydroxylase dans la rétine en conditions physiologiques et pathologiques. Par une approche clinique, nous avons trouvé qu un polymorphisme génétique dans CYP46A1 était un facteur de risque de glaucome (Risque relatif=1,26, intervalle de confiance à 95%=1,006-1,574, p<0,05) (Fourgeux et al. 2009, Invest Ophthalmol Vis Sci 50:5712-7). Par contre, ce polymorphisme génétique n a pas été retrouvé, en tant que tel, comme facteur de risque chez des patients DMLA, mais pourrait l être chez les patients non porteurs d allèles à risque dans les gènes CFH et LOC388715 (Fourgeux et al. 2012, Invest Ophthalmol Vis Sci 53:7026-33). Deux approches expérimentales nous ont permis de suggérer qu il existe un lien entre le stress des cellules de la rétine et le 24S-hydroxycholestérol. En effet, dans une étude in vivo faite chez le rat, après avoir reproduit une caractéristique principale du glaucome par l augmentation de la pression intraoculaire, nous avons suggéré le rôle crucial de la glie dans le maintien de l expression de CYP46A1 au cours de la neurodégénérescence de la rétine (Fourgeux et al. 2012, Acta Ophthalmol, Sep 23 ; doi: 10.1111/j.1755-3768.2012.02490.x.). Enfin, l inhibition pharmacologique de l activité CYP46A1 dans la rétine par le voriconazole injecté in vivo chez le rat nous a permis de mettre en évidence que la diminution du contenu en 24S-hydroxycholestérol de la rétine était associée à une dysfonction des cellules ganglionnaires, évaluée par électrorétinographie. En parallèle, nous avons observé une activation gliale, dont l amplitude était amplifiée par l inhibition de l activation microgliale induite par la minocycline [...]Cholesterol is the major sterol found in the retina. In its free form, cholesterol is present in all cell layers of the retina, whereas cholesteryl esters mainly accumulate at the basement of the retinal pigment epithelium. The intrinsic capacity of the retina to synthetize cholesterol appears limited. Some extra-retinal pathways actively participate to cholesterol uptake to the retina. Müller glial cells may contribute to cholesterol supply to retinal neurons, especially for synaptic formation. Cholesterol accumulation or conversely deficiency have deleterious consequences on neuron survival. Maintaining the equilibrium between cholesterol supply and neosynthesis in the one hand and cholesterol elimination in the other hand is crucial. For that purpose, the inner retina converts cholesterol into 24S-hydroxycholesterol. The transport of 24S-hydroxycholesterol across membranes is facilitated by the addition of the hydroxyle group to cholesterol at position 24 of carbon chain since it renders cholesterol more hydrophilic. CYP46A1 (cholesterol 24S-hydroxylase) is the enzyme which catalyzes this reaction. Some links between CYP46A1, 24S-hydroxycholesterol and neurodegenerative processes have been reported in the brain, suggesting a potential role in several pathologies such as Alzheimer s disease. CYP46A1 is expressed in the neural retina and specifically in retinal ganglion cells. The contribution of CYP46A1 in the retina remains unknown. Moreover by analogy with the brain, we can suggest a function for CYP46A1 in the regulation of cholesterol homeostasis in retinal neurons. Possible associations between CYP46A1 and Age-related Macular Degeneration (AMD) and glaucoma were suspected. In this context, we aimed to evaluate the role of CYP46A1 in the retina in physiological and pathological conditions. Through a clinical approach, we found that a genetic polymorphism in CYP46A1 was a risk factor for glaucoma (Odd Ratio = 1.26 ; 95% CI=1.006-1.574, p<0.05) (Fourgeux et al. 2009, Invest Ophthalmol Vis Sci 50:5712-7). By contrast, this genetic polymorphism was not found as a risk factor in AMD patients, but may become an additional risk factor in patients who do not carry risk allele in CFH and LOC387715 genes (Fourgeux et al. 2012, Invest Ophthalmol Vis Sci 53:7026-33). Two experimental approaches suggested that a link between retinal stress and 24S-hydroxycholesterol does exist. Indeed, in a rat model of glaucoma of elevated intraocular pressure, we suggested the crucial role of CYP46A1 in maintaining CYP46A1 expression in the course of retinal neurodegeneration (Fourgeux et al. 2012, Acta Ophthalmol, Sep 23; doi: 10.1111/j.1755-3768.2012.02490.x.). Pharmacological inhibition of CYP46A1 activity in the retina by voriconazole administered in vivo in the rat highlighted that the decrease in retinal 24S-hydroxycholesterol levels was associated with RGC dysfunction evaluated by electroretinography. In parallel, we observed glial activation in which magnitude was exacerbated when microglia activation was inhibited by minocycline at the same time.In conclusion, by a dual clinical and experimental approach, our works suggest a crucial role for CYP46A1 in maintaining cholesterol homeostasis in the retina in physiological and pathological conditions. Müller glial cell intervention in this process may be suspected especially in pathological conditions of glaucomaDIJON-BU Doc.électronique (212319901) / SudocSudocFranceF

Participation des facteurs nutritionnels et environnementaux au vieillissement de la rétine et aux rétinopathies liées à l'âge

Chez l Homme, le vieillissement de la rétine peut aboutir à des pathologies telles que la dégénérescence maculaire liée à l âge (DMLA) ou la rétinopathie diabétique (RD). Il semble qu une alimentation riche en acides gras polyinsaturés à longue chaîne (AGPI-LC), notamment en oméga-3 comme l EPA et le DHA, soit potentiellement protecteur vis-à-vis du développement de la DMLA et de l insulinorésistance (IR), principal facteur de risque de la RD. Dans ce contexte, nous avons tenter d évalué 1- l impact de facteurs endogènes et environnementaux générateurs de stress oxydatif, de produits terminaux de glycation (PTG) ou d insulinorésistance sur la fonction et le vieillissement de la rétine, et 2- l adaptation de la rétine à un régime riche en AGPI-LC oméga-3 dans modèle murin de vieillissement de la rétine humaine, la souris ApoB100,LDLR-/-.Les animaux soumis à un stress oxydatif et à des PTG présentent une altération de la fonction rétinienne associée à une accumulation de cellules microgliales et/ou macrophages dans la rétine externe. L IR induit une modulation de gènes impliqués dans le métabolisme des lipides, l inflammation et dans la synthèse de facteurs nucléaires. Une alimentation riche en AGPI-LC oméga-3 induit une amélioration de l incorporation d acides gras oméga-3 dans la rétine et la modulation du gène codant le récepteur aux LDL dans la rétine neurosensorielle.En conclusion, nos travaux montrent une adaptation de la rétine d une part à des conditions propices au vieillissement de la rétine et l insulinorésistance, et d autre part à un régime alimentaire riche en acides gras oméga-3 et pauvre en oméga-6, reconnu comme protecteur du vieillissement de la rétineWith advanced age, aging of the human retina can evolve into pathological forms, age-related macular degeneration (AMD) or diabetic retinopathy (DR). Meanwhile, epidemiological studies suggest that a diet rich in long-chain omega-3 polyunsaturated fatty acids (LC-PUFA) such as EPA and DHA, potentially protects against the development of AMD and insulin resistance, the main risk factor for DR. Within this context, our research objectives were to assess: 1 - the impact of endogen and environmental factors, known to trigger oxidative stress, advanced glycation end-products (AGEs) or insulin resistance, on the function and aging of the retina, and 2 - the response of the retina to a omega-3 LC-PUFA-enriched diet in a murine model of aging of the human retina, the ApoB100,LDLR-/- mouse.The animals exposed to oxidative stress and AGEs showed an alteration of the retinal function associated with an accumulation of microglial cells and/or macrophages in the outer retina. The insulin resistance induced a modulation of the expression of genes coding proteins involved in lipid metabolism, inflammation and nuclear factors. An omega-3 LC-PUFA-enriched diet improved the incorporation of omega-3 LC-PUFA in the retina and modulated the expression of the LDL-receptor gene in the neurosensory retina.In conclusion, our works reported the adaptive response of the retina to environmental and endogenous factors known to promote aging of the retina. It included the impairment of the retinal function, and the modulation of gene expression. Our data also gave a better understanding of the effects of omega-3 LC-PUFA against aging of the retinaDIJON-BU Doc.électronique (212319901) / SudocSudocFranceF

Short-Term Long Chain Omega3 Diet Protects from Neuroinflammatory Processes and Memory Impairment in Aged Mice

Regular consumption of food enriched in omega3 polyunsaturated fatty acids (ω3 PUFAs) has been shown to reduce risk of cognitive decline in elderly, and possibly development of Alzheimer's disease. Docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) are the most likely active components of ω3-rich PUFAs diets in the brain. We therefore hypothesized that exposing mice to a DHA and EPA enriched diet may reduce neuroinflammation and protect against memory impairment in aged mice. For this purpose, mice were exposed to a control diet throughout life and were further submitted to a diet enriched in EPA and DHA during 2 additional months. Cytokine expression together with a thorough analysis of astrocytes morphology assessed by a 3D reconstruction was measured in the hippocampus of young (3-month-old) and aged (22-month-old) mice. In addition, the effects of EPA and DHA on spatial memory and associated Fos activation in the hippocampus were assessed. We showed that a 2-month EPA/DHA treatment increased these long-chain ω3 PUFAs in the brain, prevented cytokines expression and astrocytes morphology changes in the hippocampus and restored spatial memory deficits and Fos-associated activation in the hippocampus of aged mice. Collectively, these data indicated that diet-induced accumulation of EPA and DHA in the brain protects against neuroinflammation and cognitive impairment linked to aging, further reinforcing the idea that increased EPA and DHA intake may provide protection to the brain of aged subjects

Differential effect of maternal diet supplementation with α-Linolenic adcid or n-3 long-chain polyunsaturated fatty acids on glial cell phosphatidylethanolamine and phosphatidylserine fatty acid profile in neonate rat brains

<p>Abstract</p> <p>Background</p> <p>Dietary long-chain polyunsaturated fatty acids (LC-PUFA) are of crucial importance for the development of neural tissues. The aim of this study was to evaluate the impact of a dietary supplementation in n-3 fatty acids in female rats during gestation and lactation on fatty acid pattern in brain glial cells phosphatidylethanolamine (PE) and phosphatidylserine (PS) in the neonates.</p> <p>Methods</p> <p>Sprague-Dawley rats were fed during the whole gestation and lactation period with a diet containing either docosahexaenoic acid (DHA, 0.55%) and eicosapentaenoic acid (EPA, 0.75% of total fatty acids) or α-linolenic acid (ALA, 2.90%). At two weeks of age, gastric content and brain glial cell PE and PS of rat neonates were analyzed for their fatty acid and dimethylacetal (DMA) profile. Data were analyzed by bivariate and multivariate statistics.</p> <p>Results</p> <p>In the neonates from the group fed with n-3 LC-PUFA, the DHA level in gastric content (+65%, P < 0.0001) and brain glial cell PE (+18%, P = 0.0001) and PS (+15%, P = 0.0009) were significantly increased compared to the ALA group. The filtered correlation analysis (P < 0.05) underlined that levels of dihomo-γ-linolenic acid (DGLA), DHA and n-3 docosapentaenoic acid (DPA) were negatively correlated with arachidonic acid (ARA) and n-6 DPA in PE of brain glial cells. No significant correlation between n-3 and n-6 LC-PUFA were found in the PS dataset. DMA level in PE was negatively correlated with n-6 DPA. DMA were found to occur in brain glial cell PS fraction; in this class DMA level was correlated negatively with DHA and positively with ARA.</p> <p>Conclusion</p> <p>The present study confirms that early supplementation of maternal diet with n-3 fatty acids supplied as LC-PUFA is more efficient in increasing n-3 in brain glial cell PE and PS in the neonate than ALA. Negative correlation between n-6 DPA, a conventional marker of DHA deficiency, and DMA in PE suggests n-6 DPA that potentially be considered as a marker of tissue ethanolamine plasmalogen status. The combination of multivariate and bivariate statistics allowed to underline that the accretion pattern of n-3 LC-PUFA in PE and PS differ.</p

24S‐hydroxycholesterol: cellular effects and variations in brain diseases

The adult brain exhibit a characteristic cholesterol homeostasis, with low synthesis rate and active catabolism. Brain cholesterol turnover is possible thanks to the action of the enzyme Cytochrome P450 46A1 (CYP46A1) or 24-cholesterol hydroxylase, that transforms cholesterol into 24S-hydroxycholesterol (24S-HC). But before crossing the blood-brain barrier (BBB), this oxysterol that is the most abundant in the brain can act locally, affecting the functioning of neurons, astrocytes, oligodendrocytes, and vascular cells. The first part of this review addresses different aspects of 24S-HC production and elimination from the brain. The second part concentrates in the effects of 24S-HC at the cellular level, describing how this oxysterol affects cell viability, amyloid beta production, neurotransmission, and transcriptional activity. Finally, the role of 24S-HC in Alzheimer, Huntington and Parkinson diseases, multiple sclerosis and amyotrophic lateral sclerosis, as well as the possibility of using this oxysterol as predictive and/or evolution biomarker in different brain disorders is discussed.Fil: Sodero, Alejandro Omar. Pontificia Universidad Católica Argentina "Santa María de los Buenos Aires". Instituto de Investigaciones Biomédicas. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Biomédicas; Argentin

A study on the impact of AL-FEC techniques on TV over IP Quality of Experience

In this contribution, an evaluation of the effectiveness of Application Layer-Forward Error Correction (AL-FEC) scheme in video communications over unreliable channels is presented. In literature, several AL-FEC techniques for reducing the effect of noisy transmission on multimedia communication have been adopted. Recently, their use has been proposed for inclusion in TV over IP broadcasting international standards. The objective of the analysis performed in this paper is to verify the effectiveness of AL-FEC techniques in terms of perceived Quality of Service (QoS) and more in general of Quality of Experience (QoE), and to evaluate the trade-off between AL-FEC redundancy and video quality degradation for a given packet loss ratio. To this goal, several channel error models are investigated (random i.i.d. losses, burst losses, and network congestions) on test sequences encoded at 2 and 4 Mbps. The perceived quality is evaluated by means of three quality metrics: the full-reference objective quality metric NTIA-VQM combined with the ITU-T Rec. G.1070, the full-reference DMOS-KPN metric, and the pixel-wise error comparison performed by using the PSNR distortion measure. A post-processing synchronization between the original and the reconstructed stream has also been designed for improving the fidelity of the performed quality measures. The experimental results show the effectiveness and the limits of the Application Layer protection schemes. © 2011 Battisti et al; licensee Springer

Spatial Distribution of the Pathways of Cholesterol Homeostasis in Human Retina

The retina is a light-sensitive tissue lining the inner surface of the eye and one of the few human organs whose cholesterol maintenance is still poorly understood. Challenges in studies of the retina include its complex multicellular and multilayered structure; unique cell types and functions; and specific physico-chemical environment.We isolated specimens of the neural retina (NR) and underlying retinal pigment epithelium (RPE)/choroid from six deceased human donors and evaluated them for expression of genes and proteins representing the major pathways of cholesterol input, output and regulation. Eighty-four genes were studied by PCR array, 16 genes were assessed by quantitative real time PCR, and 13 proteins were characterized by immunohistochemistry. Cholesterol distribution among different retinal layers was analyzed as well by histochemical staining with filipin. Our major findings pertain to two adjacent retinal layers: the photoreceptor outer segments of NR and the RPE. We demonstrate that in the photoreceptor outer segments, cholesterol biosynthesis, catabolism and regulation via LXR and SREBP are weak or absent and cholesterol content is the lowest of all retinal layers. Cholesterol maintenance in the RPE is different, yet the gene expression also does not appear to be regulated by the SREBPs and varies significantly among different individuals.This comprehensive investigation provides important insights into the relationship and spatial distribution of different pathways of cholesterol input, output and regulation in the NR-RPE region. The data obtained are important for deciphering the putative link between cholesterol and age-related macular degeneration, a major cause of irreversible vision loss in the elderly

Plasma 24S-hydroxycholesterol levels in elderly subjects with late onset Alzheimer's disease or vascular dementia: a case-control study

<p>Abstract</p> <p>Background</p> <p>In central nervous system cholesterol cannot be degraded but is secreted into circulation predominantly in the form of its polar metabolite 24(<it>S</it>)-hydroxycholesterol (24S-OH-Chol). Some studies suggested an association between 24S-OH-Chol metabolism and different neurological diseases including dementia. A possible decrease in 24S-OH-Chol plasma levels has been reported late onset Alzheimer's disease (LOAD) and vascular dementia (VD), but results of previous studies are partially contradictory.</p> <p>Methods</p> <p>By high-speed liquid chromatography/tandem mass spectrometry we evaluated the plasma levels of 24S-OH-Chol in a sample of 160 older individuals: 60 patients with LOAD, 35 patients with VD, 25 subjects affected by cognitive impairment no-dementia (CIND), and 40 (144 for genetics study) cognitively normal Controls. We also investigated the possible association between PPARgamma Pro12Ala polymorphism and dementia or 24S-OH-Chol levels.</p> <p>Results</p> <p>Compared with Controls, plasma 24S-OH-Chol levels were higher in LOAD and lower in VD; a slight not-significant increase in CIND was observed (ANOVA p: 0.001). A positive correlation between 24S-OH-Chol/TC ratio and plasma C reactive protein (CRP) levels was found in the whole sample, independent of possible confounders (multiple regression p: 0.04; r²: 0.10). This correlation was strong in LOAD (r: 0.39), still present in CIND (r: 0.20), but was absent in VD patients (r: 0.08). The PPARgamma Pro12Ala polymorphism was not associated with the diagnosis of LOAD, VD, or CIND; no correlation emerged between the Ala allele and 24S-OH-Chol plasma levels.</p> <p>Conclusions</p> <p>Our results suggest that plasma 24S-OH-Chol levels might be increased in the first stages of LOAD, and this phenomenon might be related with systemic inflammation. The finding of lower 24S-OH-Chol concentrations in VD might be related with a more advanced stage of VD compared with LOAD in our sample, and/or to different pathogenetic mechanisms and evolution of these two forms of dementia.</p

Lipid Composition of the Human Eye: Are Red Blood Cells a Good Mirror of Retinal and Optic Nerve Fatty Acids?

International audienceBACKGROUND: 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