京都大学 / 0048 / 新制・課程博士 / 博士(農学) / 甲第17986号 / 農博第2033号 / 新制||農||1019(附属図書館) / 学位論文||H26||N4811(農学部図書室) / 80830 / 京都大学大学院農学研究科応用生命科学専攻 / (主査)教授 植田 和光, 教授 植田 充美, 教授 三芳 秀人 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

Orientadores: Andrei Carvalho Sposito, Eliana Cotta de Faria / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-25T15:58:25Z (GMT). No. of bitstreams: 1 Virginio_VitorWilsondeMoura_M.pdf: 2355466 bytes, checksum: 77d04b1ff5936c26cd64f6d516447ff7 (MD5) Previous issue date: 2014 / Resumo: Atualmente aproximadamente 17 milhões de pessoas morrem por doenças cardiovasculares (DCV). No Brasil durante 2011 ocorreram cerca de 200 mil óbitos por DCV principalmente em decorrência da aterosclerose, classificada como uma doença imuno-inflamatória crônica que causa estreitamento luminal e gera diversas fatalidades como infarto agudo do miocárdio (IAM), acidente vascular encefálico (AVE), doença arterial periférica (DAP), entre outras. A DAP prediz outras DCV e é a principal causa de amputação devido a obstrução das artérias de membros inferiores. Análises histológicas indicam que a ruptura da placa, capa fibrosa do ateroma, erosão da placa e nódulo calcificado geram as principais fatalidades nas DCV. Até o momento não há biomarcadores plasmáticos para uso clínico afim de avaliar a estabilidade e a gravidade das placas ateroscleróticas, esse fato motiva a exploração de componentes envolvidos em doenças imuno-inflamatórias e no metabolismo lipídico tais como oxisteróis e marcadores da síntese e absorção do colesterol (MSAC). Os oxisteróis, principalmente 27-hidroxicolesterol (27-HDC) e 7-ketocolesterol estão envolvidos em mecanismos fisiopatológicos da aterosclerose e desempenham um papel na inflamação, citotoxicidade e necrose, além de funções no metabolismo lipídico. É relatado que os oxisteróis 24S- e 25-hidroxicolesterol (24S- e 25-HDC) estão envolvidos em doenças neurodegenerativas como Alzheimer. Estudos apontam uma causa em comum entre Alzheimer e aterosclerose, ambas são doenças inflamatórias crônicas, assim são necessárias mais investigações desses oxisteróis na doença aterosclerótica. A proteína de transferência de éster de colesterol (CETP) e proteína de transferência de fosfolípides (PLTP), assim como as lipoproteínas, podem mediar a transferência desses lípides. O objetivo desse trabalho foi quantificar concentrações de oxisteróis (24S-, 25-, 27-HDC e 7-ketocolesterol) e marcadores de síntese (desmosterol e latosterol) e absorção (?-sitosterol e campesterol) do colesterol em ateromas e plasma de 10 indivíduos com DAP (grupo DAP) e comparar com artérias sem ateroma de 13 voluntários (grupo CTL). Também relacionar esses esteróis com outros parâmetros como histórico clínico e proteínas de transferência de lípides. Foram realizadas análises clínicas e bioquímicas, classificações histológicas dos ateromas e quantificações de esteróis em artérias e plasma. Para interpretação dos dados obtidos foram usados testes estatísticos de comparação e correlação. Os dois grupos apresentaram uma similaridade nos dados clínicos e bioquímicos, diferenciando apenas em pressão arterial sistólica, atividade da PLTP, glicose e proteína C reativa (PCR), maiores no grupo DAP. A PCR se correlacionou positivamente com 24S-HDC, 25-HDC e 27-HDC plasmáticos. Comparação entre os grupos em relação a concentração de oxisteróis em artérias mostraram o 24S-HDC e o 27-HDC maiores no ateroma do grupo DAP, curiosamente o 25-HDC esteve maior em CTL. Quantificações de oxisteróis em plasma do grupo DAP mostrou um aumento do 25-HDC em relação ao grupo CTL. Não houve diferença significante para os MSAC. Os ateromas classificaram-se em 50% ruptura da placa, 20 % capa fibrosa do ateroma, 10% nódulo calcificado e 10% lesão fibrocálcica. Em conclusão mostramos o acumulo do 24S-HDC em placas de aterosclerose. O 27-HDC assim como outros estudos esteve em maior concentração nos ateromas indicando fortemente sua ação na patogênese dessa doença. Todos os oxisteróis sintetizados enzimaticamente dosados em plasma se correlacionaram positivamente com atividade inflamatória sistêmica, assim como 24S-HDC e 27-HDC em ateromas. Finalmente a atividade da PLTP esteve maior no grupo DAP / Abstract: Currently around 17 million people die from cardiovascular diseases (CVD). In Brazil, durant 2011 nearly 200.000 deaths occur mainly due atherosclerosis, classified as a chronic, immune-inflammatory disease that causes luminal narrowing and generates several outcomes as myocardial infarction, cerebrovascular accidents (stroke), peripheral arterial disease (PAD) and others. DAP predict other CVDs and is the leading cause of amputation due the clogged arteries in inferior members. Histological analysis indicates that plaque rupture, fibrous cap atheroma, plaque erosion and calcified nodules generate major CVD fatalities. So far, no plasma biomarkers for clinical use are available in order to evaluate the stability and extend of atherosclerotic plaques, therefore is necessary the investigation of the components involved in immune-inflammatory diseases and lipids metabolism such as oxysterols and cholesterol synthesis and absorption markers (CSAM). Oxidized sterols or oxysterols, manly 27-hidroxycholesterol and 7-ketocholesterol are involved in pathophysiological mechanisms of atherosclerosis and play a role in inflammation, apoptosis, necrosis and cytotoxicity, in addiction to lipid metabolism. Oxysterols 24S- and 25-hidroxycholesterol are involved with neurodegenerative diseases such Alzheimer¿s and resemble with atherosclerosis development since are chronical inflammatory diseases, for this reason is necessary an investigation of these oxysterols in atherosclerosis is needed. Cholesteryl ester transfer protein (CETP) and phospholipid transfer protein (PLTP) as well as lipoproteins, could mediate the transfer of the oxysterols. The aim of this work was quantified the levels of oxysterols (24S- 25-, 27- hidroxycholesterol and 7-ketocholesterol) and cholesterol synthesis (desmosterol and lathosterol) and absorption markers (?-sitosterol and campesterol) in atheroma and plaques from 10 individuals with PAD (PAD group) and compare with arteries without atheroma of 13 volunteers (CTL group). Also, relate these sterols with other parameters as clinical history, lipid transfer proteins and histological scores. Clinical and biochemical data were collected and sterols were quantified in arteries and plasma by gas chromatography coupled to a mass spectrophotometer (GCMS) and histological atheroma classification were determined. Comparison anlysis were performed using Mann-Whitney and correlations by Spearman¿s test. Both groups were not different regarding clinical and biochemical data, differencing only for systolic blood pressure, alcohol intake, PLTP activity, glucose and CRP, increased in PAD group. CRP was positively correlated with 24S-HDC, 25-HDC e 27-HDC from plasma. The comparisons of oxysterols levels in atheroma showed that 24-HDC and 27-HDC were increased in PAD group; interestingly, the 25-HDC was higher in CTL group. No significant differences were observed for CSAM between groups. The atheroma were classified as 50% plaque rupture, 20% fibrous cap, 10% calcified nodules and 10% of fibrocalcific injury. From the oxysterols from plasma of PAD group, only 25-HDC was increased compared to the CTL group. In conclusion, we demonstrated the accumulation of 24S-HDC in atherosclerotic plaques. The 27-HDC, as well as other studies, present higher levels in atheroma¿s, strongly indicating is role in the disease pathogenesis. All oxysterols measured in plasma were positively correlated with the systemic inflammatory activity. Finally, all atherosclerotic plaques were classified as lesions of advanced extension, according to the histological analysis / Mestrado / Clinica Medica / Mestre em Clinica Medica

Dysfunction of the N-methyl-D-aspartate (NMDA) receptor is thought to contribute to the cognitive deficits of many neurodegenerative diseases and psychiatric disorders. Cognitive symptoms of Alzheimer's disease can be treated with NMDA receptor antagonists or drugs targeting the cholinergic system; however, there are no effective treatments for cognitive deficits of schizophrenia or Huntington's disease. With the discovery of a potent and selective allosteric modulator of the NMDA receptor, there is the possibility of new treatments based on NMDA receptor functional-enhancement through neuroactive steroids, closely related in structure to the endogenous neurosteroid, cerebrosterol. The aim of this thesis was to examine steroidal modulation of the NMDA receptor both in vitro and in vivo. In chapter 2, NMDA receptor enhancement of both the synthetic and endogenous neuroactive steroids was assessed in neurons maintained in cell culture using calcium imaging techniques. Sulphation of the steroids greatly increased the efficacy of NMDA receptor enhancement compared to the unsulphated steroids. Chapters 3 and 4 investigate the potential for neuroactive steroids to treat cognitive impairments of Huntington's disease. Using a mouse model, tests were selected that were analogous to those in which patients are impaired; however, no impairments were found in the mouse model. Chapter 5, therefore, used a different model of cognitive impairment – namely, rats with a set-shifting impairment, as is seen in many psychiatric and neurological disorders, including Huntington's disease – to assess the effect of the synthetic steroid administration. Unfortunately, the rats did not show the expected impairment. The lack of reliable animal models compromised testing the efficacy of these promising NMDA receptor positive allosteric modulators. Nevertheless, the promising in vitro results suggest that there could still be therapeutic potential. In addition, the compound is a useful research tool for exploring NMDA receptor function in health and disease.

La famille des Astéracées est économiquement très importante, car beaucoup de ses plantessont cultivées principalement pour leurs valeurs alimentaires dont la laitue (Lactuca), lachicorée (Cichorium) et le tournesol (Heliantus annuus) cultivé pour son huile. Une despropriétés typiques de cette famille, dont fait partie le Silybum marianum, est la richesse deses huiles en divers composés naturels bénéfiques pour la santé. Actuellement et pour lesdécennies à venir, les maladies liées à l’âge constituent un problème majeur de santépublique. Prévenir leur apparition ou s'opposer à leur évolution est un objectif majeur. Dansce contexte, les activités cytoprotectrices de l'huile de graines de Silybum marianumoriginaires de différentes régions de la Tunisie ont été étudiées sur le modèled’oligodendrocytes murins 158N en utilisant le 7-cétocholestérol (7KC) comme agentcytotoxique. Le 7KC a été utilisé car il est augmenté dans le plasma, le liquide céphalorachidienet/ou certains tissus (paroi vasculaire et intestinale, rétine, cristallin, cerveau) depatients atteints de maladies liées à l’âge (maladies cardiovasculaires, maladie d'Alzheimer,dégénérescence maculaire liée à l’âge, cataracte) ou de maladies inflammatoires évolutives del’intestin. Le 24S-hydroxycholestérol (24S-OHC) a aussi été utilisé car il est augmenté dans lecerveau aux stades précoces de la maladie d’Alzheimer. Afin de déterminer la composition del'huile de Silybum marianum (acides gras, tocophérols, polyphénols, phytostérols) diversestechniques physicochimiques et de chromatographie liquide et gazeuse ont été utilisées. Lespropriétés anti-oxydantes de l'huile de Silybum marianum ont été définies par les tests KRL,FRAP et DPPH et les propriétés cytoprotectrices par des techniques complémentaires demicroscopie, de cytométrie en flux et de biochimie. Les études sur la compositionphysicochimique des huiles des graines de Silybum marianum de différentes régions de laTunisie (Bizerte, Sousse, Zaghouan) ont montré que ces dernières sont riches en Vitamine E(α-tocophérol) et sont fortement anti-oxydantes. Les acides gras majeurs sont l'acidelinoléique (C18:2) (valeurs comprises entre 57,0% et 60,3%) et l'acide oléique (C18:1) (lesvaleurs se situent entre 15,5% et 22,4%). Trois acides phénoliques ont également étéidentifiés (vanillique, p-coumarique et silybine), avec une prédominance de l'acide vanillique.Sur les cellules 158N, ces huiles atténuent la cytotoxicité du 7KC et du 24S-OHC: perted'adhérence cellulaire, altération de la membrane plasmique, dysfonctionnementmitochondrial, surproduction d’espèces réactives de l’oxygène, induction de l'apoptose(condensation et / ou fragmentation nucléaire, activation de la caspase-3 et clivage PARP) etautophagie (activation de LC3-I en LC3-II). L'atténuation des effets cytotoxiques du 7KC etdu 24S-OHC observée avec les huiles de Silybum marianum est de l'ordre de celle observéeavec l’α-tocophérol utilisé comme témoin positif. La composition chimique et les résultatsobtenus cellules 158N sont en faveur d’effets bénéfiques de l’huile de Chardon-Marie sur lasanté humaine et la prévention de certaines maladies liées à l’âge. / The family Asteraceae is economically very important because many of its plants are grownmainly for their food values including lettuce (Lactuca), chicory (Cichorium) and sunflower(Heliantus annuus) grown for its oil. One of the typical properties of this family, whichincludes Silybum marianum, is the richness of its oils in various natural compounds beneficialto health. Currently and for decades to come, age-related diseases are a major public healthproblem. Preventing their appearance or opposing their evolution is a major objective. In thiscontext, the cytoprotective activities of Silybum marianum seed oil originating from differentarea of Tunisia were studied on the 158N murine oligodendrocyte model using 7-ketocholesterol (7KC) and 24S-hydroxycholesterol (24S-OHC) as a cytotoxic agent. The 7KChas been used because it is increased in plasma, cerebrospinal fluid and / or tissues (vascularand intestinal wall, retina, lens, brain) of patients with age-related diseases (cardiovasculardiseases, Alzheimer's disease, age-related macular degeneration, cataract) or inflammatorybowel disease. The 24S-OHC was used because it is increased in the brain at the early stagesof Alzheimer’s disease. In order to determine the composition of Silybum marianum oil (fattyacids, tocopherols, polyphenols, phytosterols) various liquid and gas chromatographytechniques were used. The anti-oxidant properties of Silybum marianum oil were defined byKRL, FRAP and DPPH tests and cytoprotective properties by complementary microscopy,flow cytometry and biochemistry techniques. Studies on the physicochemical composition ofSilybum marianum seeds oils from different regions of Tunisia (Bizerte, Sousse, Zaghouan)have shown that the latter are rich in vitamin E (α-tocopherol) and are strongly antioxidant.The major fatty acids are linoleic acid (C18:2) (values between 57.0% and 60.3%) and oleicacid (C18:1) (values between 15.5% and 22.4%).. Three phenolic acids have also beenidentified (vanillic, p-coumaric and silybin), with a predominance of vanillic acid. On 158Ncells, these oils attenuate the cytotoxicity of 7KC and 24S-OHC: loss of cell adhesion,alteration of the plasma membrane, mitochondrial dysfunction, overproduction of reactiveoxygen species, induction of apoptosis (nuclear condensation and / or fragmentation). ,activation of caspase-3 and PARP cleavage) and autophagy (activation of LC3-I in LC3-II).The attenuation of cytotoxic effects of 7KC and 24S-OHC observed with Silybum marianumoils is of the order of that observed with α-tocopherol used as a positive control. The chemicalcomposition and results obtained on 158N cells are in favor of the beneficial effects of milkthistle oil on human health and on its ability to prevent some age-related diseases.

Le potassium (K+) est impliqué dans la régulation de l’excitabilité cellulaire, la régulation du cycle cellulaire, la viabilité cellulaire, la neuroprotection et le maintien des fonctions microgliales et oligodendrocytaires. Le dysfonctionnement des canaux potassiques, décrit dans plusieurs maladies neurodégénératives comme la Maladie d’Alzheimer (MA), la sclérose en plaques (SEP), la maladie de Parkinson et la maladie de Huntington, pourrait être une potentiel cible thérapeutique. Les mécanismes toxiques sous-jacents de ces pathologies neurodégénératives impliquent des oxystérols, dérivés oxydés du cholestérol, et des acides gras en relation avec le métabolisme peroxysomal. Le 7-cétocholestérol (7KC), le 24S-hydroxycholestérol (24S-OHC) et l'acide tétracosanoïque (C24: 0), souvent trouvés à des taux élevés au niveau du cerveau et dans le plasma de patients atteints de maladies neurodégénératives (MA, maladie de Nieman-Pick, SEP, maladie de Parkinson, maladie de Huntington et X-ALD conduisent une rupture de l’équilibre Redox qui aboutirait à la neurodégénérescence. Dans ce contexte, il est intéressant de déterminer l’éventuelle connexion entre environnement lipidique et homéostasie potassique. L’étude in vitro a été réalisée sur des olygodendrocytes murins 158N et les cellules microgliale BV-2. Nous avons montré que la lipotoxicité du 7KC, 24S-OHC et C24:0 implique une rétention du K+ faisant intervenir les canaux potassium voltage dépendant (Kv). Ces résultats ont montré que l'inhibition des canaux Kv conduisant à une augmentation la [K+]i contribue à la cytotoxicité du 7KC, 24S-OHC et C24:0. Nous nous sommes focalisés sur le canal Kv3.1b. La retention du K+ induite par les oxystérols (7KC et 24S-OHC) serait sous le contrôle de Kv3.1b. L’étude clinique réalisée sur du plasma de MA a révélé une corrélation négative entre le taux d’acide docosahexaénoïque (DHA) et la concentration de K+. Chez les souris transgéniques J20, modèle de la MA, l’étude de la topographie d’expression de Kv3.1b et d’Abcd3, au niveau de l’hippocampe et du cortex, a montré une baisse de l’expression de ces deux marqueurs. Dans leur ensemble, les résultats obtenus ont établi des relations entre lipotoxicité, métabolisme peroxysomal et altération de l’homéostasie potassique dans la neurodégénérescence et suggèrent une possible modulation de l’expression et de l’activité de kv3.1b dans la physiopathologie des maladies neurodégénératives. / Potassium (K+) is involved in the regulation of cellular excitability, cell cycle regulation, cell viability, neuroprotection and maintenance of microglial and oligodendrocytic functions. Potassium dysfunction, described in several neurodegenerative diseases such as Alzheimer's Disease (AD), multiple sclerosis (MS), Parkinson's disease and Huntington's disease, may be a potential therapeutic target. The underlying toxic mechanisms of these neurodegenerative pathologies involve oxysterols, which are oxidized cholesterol derivatives, and fatty acids including those associated with peroxisomal metabolism. 7-ketocholesterol (7KC), 24S-hydroxycholesterol (24S-OHC) and tetracosanoic acid (C24:0), often found at increased levels in the brain and plasma of patients with neurodegenerative diseases (Nieman-Pick disease, MS, Parkinson's disease, Huntington's disease and X-ALD) lead to a breakdown of the redox equilibrium leading to neurodegeneration. In this context, it is interesting to determine the possible connection between the lipid environment and potassium homeostasis The in vitro study was carried out on 158N murine oligodendrocytes and microglial BV-2 cells. We have shown that the lipotoxicity of 7KC, 24S-OHC and C24:0 implies retention of K+ involving the voltage dependent potassium channels (Kv). These results have shown that inhibition of Kv channels lead to an increase in [K +] i contributing to the cytotoxicity of 7KC, 24S-OHC and C24:0. The retention of K+ induced by oxysterols (7KC and 24S-OHC) would be under the control of Kv3.1b. A clinical study, on plasma of patients with Alzheimer’s disease, revealed a negative correlation between docosahexaenoic acid (DHA) and K+ concentration. In the J20 mice, a transgenic model of Alzheimer’s disease, the expression of Kv3.1b and Abcd3 was decreased in the hippocampus and cortex. Overall, the results obtained established relationships between lipotoxicity, peroxisomal metabolism and potassium homeostasis in neurodegeneration and suggest a possible modulation of the expression and activity of kv3.1b in the pathophysiology of neurodegenerative diseases. So, modulation of Kv3.1 could constitute a new therapeuthic approach against some neurodegenerative diseases.

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 glaucoma