Дисертації з теми "CYP4A11"

Afin de s'adapter à son environnement chimique, l'organisme a développé au cours de l'évolution des systèmes enzymatiques capables de transformer de nombreuses molécules étrangères ou xénobiotiques (médicaments, composés toxiques, carcinogènes...), le plus souvent de nature hydrophobe, en métabolites suffisamment hydrophiles pour être plus facilement excrétés par voie urinaire et/ou biliaire. Certaines de ces enzymes sont également impliquées dans des processus cataboliques ou de biosynthèses de composés endogènes (acides gras, rétinoïdes, stéroïdes, prostaglandines...). Ces enzymes jouent ainsi un rôle fondamental à la fois dans la défense de l'organisme face à son environnement chimique et dans des processus physiologiques essentiels. On comprend dès lors que s'il existe, chez certains individus, des anomalies de séquence ou de structure des gènes codant pour ces enzymes, une partie de la population présentera une susceptibilité particulière à certaines molécules de l'environnement, voire des dysfonctionnements de certaines réactions biologiques indispensables. Les travaux de cette thèse s'inscrivent dans cette démarche. Dans un premier temps, ils ont consisté à évaluer la nature et l'étendue de la variabilité de la séquence nucléotidique de trois gènes codants pour les enzymes CYP4A11, CYP4A22 et la Glycine N-acyltransférase (GLYAT). Dans un deuxième temps, les analyses fonctionnelles des variations de séquence identifiées ont été abordées par des approches in silico et in vitro. Les cytochromes P450 CYP4A11 et CYP4A22, participent à la biotransformation de composés endogènes et sont impliqués plus particulièrement dans la voie d'activation de l'acide arachidonique. Des travaux récents suggèrent que des anomalies génétiques de ces enzymes constituent des facteurs de susceptibilité à l'hypertension artérielle chez l'homme. Nous avons ainsi analysé les variations de séquence du gène CYP4A11 et CYP4A22 dans des échantillons d'ADN provenant de volontaires sains. Au total, 26 polymorphismes ont été identifiés et 5 nouveaux CYP4A* allèles ont été caractérisés pour chaque isoforme CYP4A. Les structures 3D des protéines CYP4A ont été construites et validées pour l'analyse de l'impact des mutations identifiées. Bien que des travaux supplémentaires soient nécessaires pour confirmer le lien entre le polymorphisme génétique du CYP4A11 et du CYP4A22 et l'hypertension artérielle, ce travail représente la première description et caractérisation du polymorphisme génétique des isoformes CYP4A dans une population Française. De plus, nous avons mise en évidence une variabilité interethnique de ce polymorphisme génétique dans différentes populations testées. La glycine N-acyltransférase ou GLYAT est une enzyme impliquée dans la détoxication de xénobiotiques contenant un groupement carboxylique par conjugaison d'un résidu de glycine. Sept variations de séquence de la GLYAT ont été identifiées et quatre nouveaux GLYAT* allèles ont été caractérisés. La localisation des certaines mutations dans des structures secondaires très conservées de la protéine suggère un impact sur l'activité catalytique de cette enzyme. Bien que les conséquences cliniques potentielles de ces variations restent encore à étudier, ces résultats seront utiles pour de futures études d'association de ce polymorphisme génétique de la GLYAT avec les altérations de détoxications de xénobiotiques contenant un groupement carboxylique comme l'aspirine, certains pesticides ou le toluène.

Thesis (Ph. D.)--University of North Carolina at Chapel Hill, 2006.
Title from electronic title page (viewed Oct. 10, 2007). "... in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the School of Pharmacy." Discipline: Pharmacy; Department/School: Pharmacy.

La maladie de Huntington (MH) est une maladie génétique autosomique dominante, causée par une augmentation du nombre de CAG sur le gène de la huntingtin. L’homéostasie du cholestérol cérébral est altérée dans la MH. L’expression de CYP46A1, enzyme neuronale catabolisant le cholestérol dans le cerveau, est diminuée dans le putamen des patients et dans le striatum de souris modèles de la MH. Après restauration de l’expression de CYP46A1 dans le striatum des souris Knock-In zQ175, les capacités locomotrices sont améliorées, l’agrégation de la huntingtine mutée est diminuée, l’atrophie neuronale est limitée et le métabolisme du cholestérol est stimulé. Une nouvelle signature transcriptionnelle est induite par CYP46A1, avec une restauration des voies impliquées dans l'autophagie, le protéasome, la communication synaptique et le transport axonal, connues pour leur dysfonctionnement dans la MH. CYP46A1 améliore la transmission synaptique et augmente la densité en épines synaptiques. L'élimination des agrégats par autophagie et par le protéasome est augmentée avec CYP46A1. Enfin, le transport de BDNF et de TrkB est amélioré par CYP46A1 dans un modèle in vitro de la MH. Ces résultats révèlent l'effet pléiotrope et bénéfique de la régulation du métabolisme du cholestérol dans la MH. Pour approfondir cette étude, une technique de tri cellulaire a été mise au point, afin de séparer les neurones et les astrocytes à partir de striatum de souris, pour étudier les régulations transcriptomique et lipidomique de ces deux populations. Cette étude permettra d’identifier de nouvelles cibles impliquées dans la neuroprotection par CYP46A1 et présentant un intérêt thérapeutique dans la MH
Huntington’s disease (HD) is an autosomal dominant neurodegenerative disease caused by abnormal CAG expansion on huntingtin’s gene. Recently, altered brain cholesterol homeostasis has been implicated in HD. Particularly, the expression of CYP46A1, the rate-limiting enzyme for cholesterol degradation in the brain, is decreased in patients’ putamen and in the striatum of HD mouse models. We restored CYP46A1 expression into the striatum of the zQ175 mice. Behavioral, neuropathological and molecular tests were performed and showed an improvement of locomotor activity and histological landmarks. Cholesterol homeostasis was restored with an increase of cholesterol degradation and synthesis. CYP46A1 induced a new transcriptional signature, with restoration of pathways involved in autophagy, proteasome, synaptic communication and axonal transport, which are known to be dysfunctional in HD. CYP46A1 improved synaptic transmission and spine density in the striatum of the zQ175 mice. Aggregate clearance mediated by autophagy and proteasome was increased after CYP46A1 expression. Finally, BDNF and TrkB transport were enhanced by CY46A1 in HD in vitro models. Overall, CYP46A1 restoration alleviates the zQ175 pathological phenotype through a global compensation. To gain further insights into CYP46A1 neuroprotection, a cell sorting strategy was set up to study the transcriptomic and lipidomic signature in purified neurons and astrocytes. This method will lead to a greater understanding of cell-type-specific regulations and cell communication. Altogether, this project gave new insights into the potential application of CYP46A1 restoration as a therapeutic strategy in HD

The mouse cytochrome P450 4a family of genes is poorly characterised. This thesis describes a detailed study of these genes. Using primers derived from exon 1 of the mouse cyp3a10 cDNA sequence, a 1.4kb genomic fragment was cloned which contained the promoter region of the gene. Additionally, a lambda genomic library was screened with probes derived from the partial cDNA clones of the mouse cyp4a10 and cyp4a12. Originally, two classes of phage lambda clones were isolated. One clone, lambda 16, was partially sequenced and was found to contain the 3' end of the cyp4a12 gene. The second clone, lambda 4, was subcloned and found to be distinct from previously known murine cyp4a genes. This novel gene has been designated cyp4a14. To obtain the full cyp4a14 gene, the genomic library was rescreened. Lambda clone 12 was subsequently isolated from the library, which was found to contain exons 1 to 5 of Cyp4a14. In order to study the promoter region of cyp4a14, a pcr-based approach was undertaken to clone 4.4 kb upstream of exon 1 of the gene and a 1.2kb fragment has been sequenced. Cyp4a14 RNA was also found to be highly induced by a peroxisome proliferator, methylclofenapate. Primer extension analyses were performed to determine the start of transcription of Cyp4a14, which was mapped to a single T nucleotide 26bp upstream of the putative start site of protein translation. The promoter region of Cyp4a14 is highly similar to the corresponding regions of the rat CYP4A2 genes. however, similarity is dramatically reduced about 350bp upstream of the start of transcription, which coincides with the presence of two 358bp repeats in teh CYP4A2 gene. The promoter also contains a highly conserved 19bp element which is also found in the promoter regions of the CYP4A1 and CYP4A2 genes. Cyp4a14 is a novel member of the murine Cyp4a subfamily. It contains 12 exons and is highly similar to the rat CYP4A2/3 genes. However, there is high conservation in exon 3 between Cyp4a14 and cyp4a3, which makes it marginally more related to this gene. In addition, cyp4a14 shows very high similarity in exons 4,8, 11 and 12 with other known CYP4A genes. Correspondingly, the peptide sequences encoded by these exons are highly conserved. Exon 8 encodes a 16 amino acid motif, LRAEVDTFMGEGHDTT, which is highly conserved in the CYP4 family. Exons 22 and 12 encode the well known RNCIG motif, containing the haem-binding domain, which is the proposed signature for a P450 protein. Exon 4 in the CYP4A genes encodes for a highly conserved, but previously unreported motif, HRRMLLTPGFHYDIL. Primary sequence alignments indicate that these motifs map very close to or within predicted substrate recognition sites and thus could have an important role in the enzyme activities of the CYP4A enzyme. The identification of Cyp4a14 also enables the analysis of the evolution of the murine Cyp4a subfamily. The mouse has 4 genes in the Cyp4a subfamily; however, the closely related rat has four CYP4A members. As the rat CYP4A2, which is very similar to CYP4AA3, was not to have a homologue in the mouse, it must have arisen after the mouse lineage had diverged from the rat in evolutionary history. As all three mouse Cyp4a genes are significantly more similar to their equivalents in the rat than to other murine Cyp4a genes, the gene duplication events giving rise to these three genes must have occurred before the mouse had diverged from the rat. Phylogenetic analyses of the CYP4A, CYP4B and CYp4F subfamilies demonstrate that CYP4A genes are more similar to the CYP4B family than to the CYP4F subfamily. This suggests that the gene duplication event giving rise to the CYP4A and CYP4B genes must be a more recent event than that giving rise to the CYP4F subfamily. In addition, the CYP4A subfamily of genes is more divergent than the CYP4B subfamily across species.

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 [...]

Cytochrome P450s (P450s) play a major role in the metabolism of endogenous and exogenous chemicals. P450-metabolism generally converts compounds into more hydrophilic derivatives, which can then be excreted. In contrast, it can lead to the formation of reactive intermediates, which can attack cellular macromolecules leading to mutagenesis. The cytochrome P450 4A (CYP4A) subfamily encodes proteins involved in lipid metabolism. The CYP4A1, CYP4A2 and CYP4A3 genes are expressed constitutively in the adult rat, elevated expression being seen after administration of a hypolipidemic drug, clofibrate. The interest in the CYP4A subfamily is partly related to the apparent close association between CYP4A1 induction and the phenomenon of sustained peroxisome proliferation. It is also believed that the CYP4A proteins may be involved in the production of physiologically important renal metabolites. The aim of this thesis was a systematic study of the ontogeny and inducibility of CYP4A1 mRNA and protein levels in embryonic, fetal and post-natal rats. Constitutive and induced hepatic expression of the CYP4A1, CYP4A2 and CYP4A3 mRNAs was demonstrated in the 24 day, 10.5 day and day 1 neonates, in addition to the 18.5 day fetal expression. Renal expression was not detected prenatally, and was only apparent in the day 1 neonates following induction. The mRNAs were also present in induced adult brown fat; induced and control adult and 24 day neonatal small intestine; induced 18.5 day fetal placenta and, induced and control 11.5 day embryonic decidua. The demonstration of inducible CYP4A1 gene expression is potentially important, as in conjunction with peroxisome proliferation, it may result in an increased susceptibility of the tissue to carcinogenesis. The P450 inductive response may also play an important role in elevating the rate of metabolism of foreign compounds to detoxified forms, or in some cases to harmful reactive intermediates. If this were to occur during pregnancy it could potentially have important consequences for the developing embryo/fetus.

Introduction Cytochromes P450 (CYPs) are the major family of enzymes responsible for detoxification and metabolism of a wide range of both endogenous and xenobiotics chemicals in living organisms. The use of CYPs to activate prodrugs to cytotoxins selectively in tumours has been explored including AQ4N, Phortress and Aminoflavone. CYP1A1, CYP1B1, CYP2W1, and CYP4F11 have been identified as expressed in tumour tissue and surrounding stroma at high frequency compared to most normal tissues. Aim is to investigate the differential metabolism of novel chloromethylindoline by high frequency expressed CYPs in tumours. This differential may be exploited to elicit a selective chemotherapeutic effect by metabolising inert small molecules to potent cytotoxins within the tumour environment. Materials and Methods Sensitive and specific LC/MS/MS techniques have been developed to investigate the metabolism of chloromethylindolines. Recombinant enzymes and transfected cell lines were used to investigate the metabolic profiles with a focus on production of the cytotoxic derivatives of chloromethylindolines. Results Detailed metabolic studies show that (1-(Chloromethyl)-1,2-dihydropyrrolo [3,2-e]indol-3(6H)-yl)(5-methoxy-1H-indol-2-yl) methanone (ICT2700) and other chloromethylindolines are converted by CYP1A1 mediated hydroxylation at the C-5 position leading to highly potent metabolites. In vitro cytotoxicity studies showed differentials of up to 1000-fold was achieved between CYP1A1 activated compared to the non-metabolised parent molecules. The reactivity of metabolites of ICT2700 was also explored using glutathione as a nucleophile. The metabolites were identified by a combination of LC/MS and LC MS/MS techniques. Investigations using mouse and human liver microsomes show that a large number of metabolites are created though none were shown to be associated with a potential anticancer effect. Studies focused on CYP2W1 show that this isoform metabolised ICT2706 to a cytotoxic species and a pharmacokinetic study showed a good distribution of ICT2706 into mouse tissues including tumour. However metabolism of ICT2726 by CYP2W1 resulted only in a non-toxic metabolite profile and may have potential as a biomarker for functional CYP2W1 in tissues. Preliminary studies show that palmitic acid hydroxylation is a useful marker of functional CYP4F11. Summary and conclusion The in vitro results show that the chloromethylindolines are a novel class of agent with potential as prodrugs that following specific hydroxylation by CYP1A1 and CYP2W1 are converted to ultra-potent cytotoxins. Other metabolites are also evident which are not cytotoxic. Studies in vivo show that selected chloromethylindolines possess a good pharmacokinetic profile and show potential as prodrug anticancer agents that require activation by CYP1A1 or CYP2W1. The methods, results, progress and suggestions for future work are presented in this thesis.

Cytochrome P450 (P450 or CYP) is an enzyme system involved in the oxygenation of a wide range of endogenous compounds as well as foreign chemicals and drugs. This thesis describes investigations of P450-catalyzed oxygenation of prostaglandins, linoleic and arachidoniacids.

The formation of bisallylic hydroxy metabolites of linoleic and arachidonic acids was studied with human recombinant P450s and with human liver microsomes. Several P450 enzymes catalyzed the formation of bisallylic hydroxy metabolites. Inhibition studies and stereochemic analysis of metabolites suggest that the enzyme CYP1A2 may contribute to the biosynthesis of bisallylic hydroxy fatty acid metabolites in adult human liver microsomes.

19R-Hydroxy-PGE and 20-hydroxy-PGE are major components of human and ovine semen, respectively. They are formed in the seminal vesicles, but the mechanism of their biosynthesis is unknown. Reverse transcription-polymerase chain reaction using degenerate primers for mammalian CYP4 family genes, revealed expression of two nov P450 genes in human and ovine seminal vesicles. The full coding regions of the genes were cloned and the enzymes were expressed in a yeast system. The human enzyme was designated CYP4F8 and the ovine enzyme was designated CYP4F21. Comparison of their deduced protein sequenceshowed that they had 74 % amino acid identity. Recombinant CYP4F8 oxygenated two prostaglandin endoperoxides (PGH₁ and PGH₂) and three stable PGH₂ analogues int19-hydroxy metabolites. Oxygenation of these substrates was mirrored when incubated with microsomes isolated from human seminal vesicles. These results suggest that CYP4F8 is present in human seminal vesicles and that 19R-hydroxy-PGE is formed by CYP4F8-catalyze 19R-hydroxylation of PGH₁ and PGH₂, followed by PGE synthase-catalyzed isomerization. Studies of catalytic properties of recombinant CYP4F21 suggest that 20-hydroxy-PGE may be formed by similar mechanisms in ovine seminal vesicles. CYP4F8 is the first enzyme shown to hydroxylate prostaglandin endoperoxides.

La maladie d’Alzheimer (MA) se caractérise par une perte mnésique progressive et au plan neuropathologique par le dépôt extracellulaire de plaques amyloïdes, résultant de l’agrégation de peptides amyloïdes (Aβ), et par l’apparition d’une dégénérescence neurofibrillaire (DNF) constituée d’agrégats intraneuronaux de protéines Tau hyper et anormalement phosphorylées. L’évolution des déficits cognitifs des patients est particulièrement corrélée à la progression spatio-temporelle de la DNF. A l’heure actuelle, il n’existe aucun traitement curatif de la maladie. Le cholestérol joue un rôle central dans la physiopathologie de la MA. En particulier, l’allèle ε4 du gène de l’apolipoprotéine E, transporteur cérébral essentiel du cholestérol, est le principal facteur de risque génétique des formes sporadiques de la MA. De nombreuses études in vitro montrent qu’une surcharge en cholestérol induit la production d’Aβ pathogènes et qu’inversement, une déplétion en cholestérol entraîne une diminution de la voie amyloïde. Le cholestérol ne peut pas passer librement la barrière hémato-encéphalique (BHE). Le cholestérol cérébral est exclusivement synthétisé in situ. Le cholestérol cérébral en excès doit être exporté dans la circulation sanguine pour être métabolisé. Pour franchir la BHE, sa conversion en 24(S)-hydroxycholestérol est nécessaire, étape contrôlée par la cholestérol 24-hydroxylase (CYP46A1). Deux précédents travaux de thèse effectués dans le laboratoire ont permis de mettre en évidence des connexions étroites entre le métabolisme du cholestérol et la MA in vivo. La surexpression intracérébrale de CYP46A1 dans un modèle murin amyloïde à l’aide d’un vecteur viral adéno-associé (AAV) a conduit à la diminution de la production d’Aβ, des plaques amyloïdes et à l’amélioration des performances mnésiques des animaux. A l’inverse, l’inhibition de l’expression de CYP46A1 dans l’hippocampe de souris sauvages induit la production d’Aβ, la phosphorylation de Tau et des défauts mnésiques chez la souris. L’objectif de mon travail de doctorat a été de déterminer s’il existait un lien direct entre CYP46A1 et la pathologie Tau et si la modulation du métabolisme du cholestérol pourrait avoir un effet bénéfique sur la pathologie Tau associée à la MA. Pour répondre à ces questions, le modèle murin THY-Tau22, qui développe une pathologie Tau de type Alzheimer, a été utilisé. Cette pathologie, essentiellement hippocampique, est évolutive et associée à des déficits mnésiques. Dans l’hippocampe des souris THY-Tau22, le cholestérol libre total n’est pas modifié, alors que l’expression protéique de CYP46A1 est diminuée, et en conséquence le contenu en 24(S)-hydroxycholestérol. L’expression protéique de CYP46A1 dans l’hippocampe est également réduite dans un autre modèle murin de pathologie Tau, le modèle THY-Tau30. Ainsi, la pathologie Tau semble être à l’origine de la diminution de l’expression protéique de CYP46A1. Afin de déterminer si la surexpression de CYP46A1 chez la souris THY-Tau22 pouvait améliorer son phénotype biochimique, neuropathologique et clinique, un vecteur AAV codant pour CYP46A1 a été injecté dans l’hippocampe de souris THY-Tau22 âgées de trois mois et demi. Deux mois et demi après injection, la surexpression de CYP46A1 chez les souris THY-Tau22 induit une restauration de la concentration hippocampique en 24(S)-hydroxycholestérol et une augmentation de l’expression des gènes impliqués dans la synthèse du cholestérol, et plus particulièrement dans la voie du mévalonate. Deux mois et demi et cinq mois et demi post-injection, la surexpression de CYP46A1 entraîne une restauration complète des performances mnésiques des animaux qui s’accompagne d’un rétablissement de la dépression à long terme, de la longueur des dendrites secondaires, de la densité synaptique et de l’expression des gènes d’activité précoce dans l’hippocampe. (...)
Alzheimer’s disease (AD) is characterized by a progressive memory loss and neuropathologically by senile plaques and neurofibrillary tangles (NFTs). Senile plaques are constituted of extracellular amyloid peptide (Aβ) deposits while NFTs result from the accumulation and the aggregation of intracellular hyperphosphorylated Tau proteins. Spatiotemporal progression of NFTs particularly correlates with cognitive impairments. To date, there is no curative treatment for this disease. Cholesterol plays a central role in AD physiopathology. Indeed, the ε4 allele of the apolipoprotein E, the brain’s principal cholesterol-carrier protein, is the main genetic risk factor for sporadic forms of AD. Numerous in vitro studies have shown that cholesterol overload induces production of pathogenic Aβ and conversely, cholesterol depletion causes a reduction of the amyloidogenic pathway. In adult, brain cholesterol is exclusively synthesized in situ. Brain cholesterol is not able to freely cross the blood brain barrier and its major exportable form is 24(S)-hydroxycholesterol generated by the cholesterol 24-hydroxylase (CYP46A1). Two previous thesis works in this laboratory highlighted narrow connections between cholesterol metabolism and AD in vivo. The intracerebral overexpression of CYP46A1 mediated by an adeno-associated viral (AAV) vector, in a murine amyloid model, led to the decrease of Aβ production, senile plaques and improvement of memory abilities. At the opposite, hippocampal CYP46A1 inhibition in wild-type (WT) mice induced Aβ production, Tau phosphorylation and memory impairments. The aim of this thesis work was to determine whether there was a direct link between CYP46A1 and Tau pathology and whether cholesterol metabolism modulation could have a beneficial effect on AD-like Tau pathology. In order to answer these questions, the THY-Tau22 mouse model, that develops AD-like Tau pathology, was used. In this model, the pathology mainly occurs in the hippocampus, it is progressive, and associated with memory deficits. In THY-Tau22 mice, total free cholesterol in the hippocampus was unchanged, whereas both CYP46A1 protein expression and 24(S)-hydroxycholesterol content were decreased. Furthermore, we also demonstrated that CYP46A1 protein expression was reduced in another murine model of Tau pathology, the THY-Tau30 model. Therefore, it may suggest that Tau pathology can be responsible for CYP46A1 decrease. We next determined whether CYP46A1 overexpression in the THY-Tau22 mouse could improve its biochemical, clinical and neuropathologic phenotype. For this purpose, an AAV vector encoding CYP46A1 was injected in the hippocampus of 3.5-month-old WT and THY-Tau22 mice. Two and a half months after injection, hippocampal CYP46A1 overexpression in THY-Tau22 mice induced restoration of hippocampal 24(S)-hydroxycholesterol content and increased expression of genes involved in cholesterol synthesis, more particularly in the mevalonate pathway. Two and a half and five and a half months post-injection, CYP46A1 overexpression resulted in a complete restoration of memory abilities and was accompanied by restoration of long-term depression, length of secondary dendrites, synaptic density and expression of immediate-early genes in hippocampus. Despite this, abnormal Tau hyperphosphorylation and gliosis, that characterizes this model, remained unchanged after CYP46A1 overexpression. Altogether, these results suggest a direct connection between Tau pathology and CYP46A1 in vivo. In other words, Tau pathology could lead to memory deficits via CYP46A1 decrease. These data, together with the fact that CYP46A1 overexpression can modulate the amyloid pathology in mice, suggest that CYP46A1 may be a relevant therapeutic target for AD

Bei der adjuvanten Immuntherapie soll das Immunsystem von Tumorpatienten gezielt gegen Mikrometastasen aktiviert werden, die nach der operativen Entfernung des Primärtumors im Körper verbleiben. Tumor-assoziierte Antigene (TAA) spielen dabei die zentrale Rolle. Um der Heterogenität eines Tumors in der Expression einzelner TAAs Rechnung zu tragen, werden in modernen Vakzinierungsstrategien Pools von verschiedenen TAAs eingesetzt. Für das Mammakarzinom sind aber bislang nur wenige TAAs bekannt. Auf der Suche nach unbekannten Genen mit Mamma- bzw. Mammakarzinom-restringierter Expression in der Transkriptomdatenbank GeneExpress® wurde ein EST gefunden, das nur in 2 % der getesteten weibl. Normalgewebe ohne Mamma mit einer marginalen mittleren Expression von 16 FE (Fluoreszenzeinheit) detektiert wurde, aber in 63 % der Mammanormalgewebe (159 FE) und in 61 % der Mammakarzinome (339 FE) exprimiert wurde. Das korrespondierende UniGene-Cluster gab erste Hinweise auf die Zugehörigkeit dieses neuen Gens zu der Cytochrom P450 (CYP) Familie. Durch computergestützte Homologievergleiche mit verwandten Mitgliedern dieser Familie in Kombination mit RT-PCR konnte die cDNA-Sequenz dieses neuen CYP ermittelt werden: Durch Amplifikation der Gesamtlängen-cDNA wurden drei Transkripte in der Mammakarzinomzelllinie SK-BR-3 gefunden, die von zwei neuen CYP Genen stammen, CYP4Z1 und dem Pseudogen CYP4Z2P. Die cDNA von CYP4Z1 kodiert für ein 505 As großes Protein, das aufgrund von Homologien einer neuen Subfamilie innerhalb der CYP4 Familie zugeordnet werden kann. Sowohl die Sequenz als auch die vorhergesagte Sekundärstruktur zeigen alle charakteristischen Merkmale eines funktionellen Mitglieds dieser Familie. Aufgrund einer Nonsensemutation in Exon 8 kodiert die cDNA von CYP4Z2P (1436 bp) für ein verkürztes, nichtfunktionelles P450 von 340 As, das zu 96% identisch mit P450 4Z1 ist. Außerdem wurde in SK-BR-3 eine Spleißvariante von CYP4Z1 identifiziert. CYP4Z1 (50,8 kb) und CYP4Z2P (57,3 kb) liegen auf Chromosom 1p33-p34.1 und bestehen aus 12 Exons mit konservierten Exon-Intron-Grenzen. CYP4Z2P ist aus einer inversen Duplikation von CYP4Z1 hervorgegangen. Mittels Realtime RT-PCR mit cDNA von 17 Normalgeweben von gepoolten Spendern und von Mammakarzinomen konnte die auf Brustdrüsengewebe restringierte Expression beider Gene demonstriert werden: Die Expression von CYP4Z1 war in Mammakarzinomgewebe 3,6-mal höher als in Mammanormalgewebe, 60-mal höher als in Lunge und 84-mal höher als in Leber. Alle anderen getesteten weibl. Normalgewebe zeigten eine noch geringere Expression. Ein ähnlich stringentes Expressionsprofil ergab die Analyse von CYP4Z2P, allerdings mit einer deutlich niedrigeren Expressionsstärke. Das Mamma-restringierte Expressionsverhalten von CYP4Z1 wurde durch einen ?Cancer-Profiling-Array? (241 Tumor-/Normalgewebepaare von 13 Gewebetypen) bestätigt. Damit konnte gezeigt werden, dass CYP4Z1 bei 52 % der getesteten 50 Brustkrebspatientinnen im Tumor versus peritumoralem Normalgewebe überexprimiert war. Mit einem spezifischen Kaninchen-Antiserum konnte die Expression von P450 4Z1 Protein sowohl in CYP4Z1-transduzierten Zelllinien als auch in Mammagewebeproben mittels Western-Blot nachgewiesen werden. Konfokale Laser-Scanning Mikroskopie von MCF-7 Zellen, die das Fusionsprotein CYP4Z1-EGFP exprimierten, und die subzelluläre Fraktionierung der CYP4Z1-Transduktanten zeigten P450 4Z1 als integrales Membranprotein des endoplasmatischen Retikulums (ER). Für die Lokalisation und die Zurückhaltung im ER ohne Recycling aus dem Prä-Golgiapparat sind die ersten 32 As erforderlich, was Studien mit unterschiedlichen Deletionsmutanten aus der N-terminalen Sequenz von 4Z1 zeigten. Die Entdeckung eines neuen Mamma-spezifisch exprimierten P450 Enzyms eröffnet neue Möglichkeiten sowohl in Hinsicht auf eine Immuntherapie von Brustkrebs als auch für die Entwicklung neuer Chemotherapeutika, die spezifisch durch P450 4Z1 am Tumorort umgesetzt werden können.

Introduction: Cytochromes P450 (CYPs) are the major family of enzymes responsible for detoxification and metabolism of a wide range of both endogenous and xenobiotics chemicals in living organisms. The use of CYPs to activate prodrugs to cytotoxins selectively in tumours has been explored including AQ4N, Phortress and Aminoflavone. CYP1A1, CYP1B1, CYP2W1, and CYP4F11 have been identified as expressed in tumour tissue and surrounding stroma at high frequency compared to most normal tissues. Aim is to investigate the differential metabolism of novel chloromethylindoline by high frequency expressed CYPs in tumours. This differential may be exploited to elicit a selective chemotherapeutic effect by metabolising inert small molecules to potent cytotoxins within the tumour environment. Materials and Methods: Sensitive and specific LC/MS/MS techniques have been developed to investigate the metabolism of chloromethylindolines. Recombinant enzymes and transfected cell lines were used to investigate the metabolic profiles with a focus on production of the cytotoxic derivatives of chloromethylindolines. Results: Detailed metabolic studies show that (1-(Chloromethyl)-1,2-dihydropyrrolo [3,2-e]indol-3(6H)-yl)(5-methoxy-1H-indol-2-yl) methanone (ICT2700) and other chloromethylindolines are converted by CYP1A1 mediated hydroxylation at the C-5 position leading to highly potent metabolites. In vitro cytotoxicity studies showed differentials of up to 1000-fold was achieved between CYP1A1 activated compared to the non-metabolised parent molecules. The reactivity of metabolites of ICT2700 was also explored using glutathione as a nucleophile. The metabolites were identified by a combination of LC/MS and LC MS/MS techniques. Investigations using mouse and human liver microsomes show that a large number of metabolites are created though none were shown to be associated with a potential anticancer effect. Studies focused on CYP2W1 show that this isoform metabolised ICT2706 to a cytotoxic species and a pharmacokinetic study showed a good distribution of ICT2706 into mouse tissues including tumour. However metabolism of ICT2726 by CYP2W1 resulted only in a non-toxic metabolite profile and may have potential as a biomarker for functional CYP2W1 in tissues. Preliminary studies show that palmitic acid hydroxylation is a useful marker of functional CYP4F11. Summary and conclusion: The in vitro results show that the chloromethylindolines are a novel class of agent with potential as prodrugs that following specific hydroxylation by CYP1A1 and CYP2W1 are converted to ultra-potent cytotoxins. Other metabolites are also evident which are not cytotoxic. Studies in vivo show that selected chloromethylindolines possess a good pharmacokinetic profile and show potential as prodrug anticancer agents that require activation by CYP1A1 or CYP2W1. The methods, results, progress and suggestions for future work are presented in this thesis.

Aquesta tesi es centra bàsicament en l'estudi i caracterització de tres gens de ratolí, que comparteixen la característica d'expressar-se a ronyó i d'estar regulats pels andrògens, els quals estimulen la seva expressió renal a nivell de mRNA.
El primer d'ells correspon al gen Cyp4b1 -representant dels citocroms P450, enzims implicats en el metabolisme oxidatiu tan de substàncies endògenes com en la detoxificació de fàrmacs i xenobiòtics. S'ha estudiat la seva expressió en diversos teixits murins i, mitjançant hibridació in situ, s'ha localitzat la distribució del seus transcrits en el ronyó. En aquest darrer teixit, s'ha investigat l'efecte de les hormones andrògenes en la seva expressió, utilitzant ratolins castrats i control de vàries soques. A nivell genòmic, s'ha clonat el seu gen i les dues primeres kb de promotor del Cyp4b1, determinant la seva seqüència, la seva organització genòmica i els límits exó/intró corresponents. Amb el fragment de promotor aïllat s'han preparat construccions reporteres, amb les quals s'han realitzat assaigs de transfecció transitòria per tal d'analitzar i avaluar els possibles elements reguladors presents, tan a nivell d'expressió basal en línies renals, com a nivell d'inducció i capacitat de resposta androgènica.
Els dos gens restants descrits -Oatp1 i Oatp-d/MJAM- eren prèviament desconeguts, pel que s'ha hagut de clonar i seqüenciar el seu cDNA sencer en aquest treball. Un cop identificats s'ha vist que ambdós corresponien a membres de la família murina dels transportadors d'anions orgànics, OATPs, proteïnes poliespecífiques de transport amb una ampli ventall de substrats que pot incloure des de sals biliars, esteroides conjugats i eicosanoides, fins a drogues i xenobiòtics orgànics. Al igual que pel Cyp4b1, s'ha estudiat la seva distribució tissular a nivell de missatger, i també la dependència androgènica de la seva expressió renal en diferents soques murines.
Per Oatp1 en particular, s'ha realitzat una caracterització preliminar a nivell funcional determinant la seva afinitat per alguns substrats, amb assaigs de transport en oòcits de Xenopus laevis. A nivell genòmic, s'ha identificat temptativament la seqüència de la possible regió 5' adjacent del seu gen.
Finalment, per Oatp-d (prèviament anomenat MJAM), s'han realitzat diversos estudis comparatius entre rosegadors i humans, que inclouen el mapatge cromosòmic del gen en ratolins i del seu corresponent ortòleg en rates. El posicionament dels gens Oatp-d de rata i OATP-D -l'ortòleg humà- en sengles regions genòmiques on han estat situats possibles loci genètics d'influència en hipertensió, ha fet que exploréssim a nivell preliminar una possible implicació d'aquest gen en els processos esmentats. S'ha abordat a través de comparar la seqüències dels cDNA corresponents i l'expressió renal de Oatp-d en rates de les soques WKY(normotenses) i SHR (hipertenses genètiques).
El present treball hauria d'englobar-se dins el marc d'una aproximació als mecanismes moleculars específics reguladors de l'expressió gènica, per part dels andrògens.
This thesis is centered on the study and characterization of three murine genes with kidney androgen-regulated expression. The androgenic hormones stimulate their renal expression at the level of mRNA.
The first gene corresponds to Cyp4b1, a member of cytochrome P450 family (enzymes implied in the oxidative metabolism of endogenous substances and also in detoxification of diverse drugs and xenobiotics. The expression-pattern of Cyp4b1 was analyzed in several mouse tissues and, by means of in situ hybridization, the distribution of its mRNA was located into the kidney. The effect of androgenic hormones on its renal expression was evaluated, using castrated and intact control mice of different strains. At genomic level, the organization of the Cyp4b1 structural gene was determined as well as its promoter region (about 2-kb of its proximal promoter being cloned and sequenced). With the promoter fragment isolated, it has been prepared several reporter constructs, to test the presence of putative regulatory elements in the promoter, in transient transfection experiments. The level of basal expression of those constructs in renal cell lines and the responsiveness to androgen was also evaluated.
For the remaining two genes described (Oatp1 and Oatp-d/MJAM) that were previously unknown, it has been cloned and sequenced his corresponding full-length cDNAs. The both genes identified belongs to the murine organic anion transporting-polypeptide family (Oatp's), polyspecific organic anion transport proteins with broad range of substrates, which can include biliar salts, conjugated steroids and eicosanoids, organic drugs and xenobiotics. Like for the Cyp4b1, its tissue distribution at messenger level has studied, and also the androgenic dependency of its renal expression in several mouse strains.
For mouse Oatp1, it has been characterized at functional level, determining his affinity for some anionic substrates in transport studies using Xenopus oocytes. At genomic level, it has been identified tentatively the 5'-flanking region of their gene.
Finally, for Oatp-d (previously known as MJAM), several comparative studies between rodents and humans have been made, including the chromosomal mapping of the gene in mice and its corresponding orthologous in rats. The localization of the rat and human OATP-D genes in genomic regions where they have been described genetic loci with putative influence on hypertension, makes that we considered the analysis of the Oatp-d implication, at preliminary level, in the mentioned processes. These studies consisted in the sequencing and comparation of the corresponding cDNA full coding-regions (and also its renal expression), between Oatp-d in WKY(normotensive) and SHR (spontaneous hypertensive) rats.
The present work would have to be included within the general frame of the study of molecular mechanisms of kidney androgen gene-regulation mechanisms.
Esta tesis se centra básicamente en el estudio y caracterización de tres genes murinos que comparten la característica de expresarse y estar regulados por los andrógenos en el riñón, los cuales estimulan su expresión renal a nivel de mRNA. El primero de ellos corresponde al gen Cyp4b1 -representante de los citocromos P450, enzimas implicadas en el metabolismo oxidativo tanto de sustancias endògenas como en la detoxificación de fármacos y xenobióticos. Se ha estudiado su expresión en varios tejidos murinos y, mediante hibridación in situ, localizado la distribución de sus transcritos en el riñón. En este último tejido, también se ha investigado el efecto sobre su expresión de las hormonas andrógenas, utilizando ratones castrados y control de diferentes cepas. A nivel genómico, ha sido clonado el gen del Cyp4b1 así como las dos primeras kb de promotor, determinándose su secuencia, su organización genómica y las intersecciones exón/intrón correspondientes. Con el fragmento de promotor aislado han sido preparadas diversas construcciones reporteras, con las cuales se han realizado ensayos de transfección transitoria para evaluar los posibles elementos reguladores presentes en el promotor, tanto a nivel de expresión basal en líneas renales, como a nivel de inducción y capacidad de respuesta androénica.
Los dos genes restantes descritos -Oatp1 y Oatp-d/MJAM- eran previamente desconocidos, por lo cual se ha clonado y secuenciado su cDNA entero a partir de riñón. Uno vez identificados los correspondientes cDNAs, se ha visto que ambos correspondían a miembros de la familia murina de los transportadores de aniones orgánicos, OATPs, proteínas poliespecíficas de transporte con una amplio abanico de sustratos que puede incluir desde sales biliares, esteroides conjugados y eicosanoides, hasta drogas y xenobióticos orgánicos. Al igual que para el Cyp4b1, se ha estudiado su distribución tisular de ambos genes a nivel de mensajero, y también la dependencia androgénica de su expresión renal en diferentes cepas murinas. Para Oatp1 en particular, se ha caracterizado a nivel funcional de manera preliminar, determinando su afinidad por algunos sustratos mediante ensayos de transporte en oocitos de Xenopus laevis. A nivel genómico, ha sido identificada tentativamente la secuencia de la región 5' adyacente de su gen.
Finalmente, por Oatp-d (previamente denominado MJAM), se han realizado varios estudios computacionales comparativos entre roedores y humanos, que incluyen el mapage cromosómico del gen en ratones y de su correspondiente ortólogo en ratas. El posicionamiento de los genes Oatp-d de rata y OATP-D -su ortólogo humano- en sendas regiones genómicas dónde se han descrito posibles loci genéticos con posible influencia sobre la hipertensión, ha hecho que exploráramos a nivel preliminar una posible implicación de este gen en los procesos mencionados. Dichos estudios se han abordado a través de la comparación de la secuencias de los cDNA correspondientes (y también su expresión renal) entre Oatp-d en ratas de las cepas WKY(normotensas) y SHR (hipertensas genéticas).
El presente trabajo debería englobarse dentro del marco general de una aproximación a los mecanismos moleculares específicos reguladores de la expresión génica renal, por parte de los andrógenos.

Le vieillissement normal s’accompagne d’une diminution du contenu du cholestérol cérébral. Au contraire, une accumulation de cholestérol est associée aux processus toxiques dans plusieurs pathologies dégénératives (maladie d’Huntington, maladie de Parkinson, épilepsie, maladie de Niemann Pick de type C, maladie d’Alzheimer). De plus, les parallèles étroits existent entre la physiopathologie moléculaire de la maladie d’Alzheimer et celle de la maladie de Niemann Pick de type C, maladie de l’homéostasie du cholestérol. Ainsi on retrouve dans ces deux pathologies une hyperphosphorylation de la protéine Tau, associée à une augmentation des endosomes élargis et à la production de peptides A. L’ensemble de ces éléments évoque le rôle potentiel de la surcharge en cholestérol cérébral comme facteur favorisant le développement de la maladie d’Alzheimer. L’objectif de mon travail de doctorat a été de déterminer si une surcharge en cholestérol in vivo dans les neurones de l’hippocampe, région précocement touchée par la maladie d’Alzheimer, pouvait être à l’origine de processus neurotoxiques et de modifications biochimiques et neuropathologiques proches de ceux qui sont observés dans cette pathologie. La quasi-totalité du cholestérol cérébral est synthétisée in situ, la barrière hémato-encéphalique ne permettant qu’un apport minime du cholestérol périphérique. L’excès de cholestérol est exporté de la circulation sanguine sous la forme du 24-hydroxycholestérol, un métabolite produit exclusivement dans les neurones par la cholestérol-24-hydroxylase codée par le gène Cyp46a1.La surcharge en cholestérol a été induite in vivo par inhibition de la cholestérol-24-hydroxylase, dans l’hippocampe par une stratégie d’ARN interférence délivré par une injection stéréotaxique d’un vecteur AAV5. Nous avons étudié, d’une part la capacité d’une accumulation de cholestérol à induire chez la souris normale, un phénotype clinique et neuropathologique proche de la maladie d’Alzheimer et d’autre part si cette même accumulation de cholestérol neuronal pouvait aggraver ou compléter le phénotype Alzheimer d’un modèle murin de la maladie, la souris APP23. L’injection du vecteur AAV5-shCYP46A1 dans la stratum lacunosum moleculare de l’hippocampe conduit à une inhibition significative de l'expression du gène Cyp46a1, associée à une diminution de la concentration du 24-hydroxycholestérol et une augmentation du contenu en cholestérol dans les neurones de l’hippocampe, 3 semaines après l’injection. En réponse à cet excès de cholestérol, des mécanismes régulateurs permettent de diminuer, d’une part l’import et d’augmenter l’export du cholestérol de la cellule et d’autre part d’augmenter le contenu en phosphatidylcholine afin de rétablir un ratio phospholipide/cholestérol physiologique. Cependant, l'accumulation majeure de cholestérol intracellulaire conduit, 3 semaines après l’injection, à une activation de la réponse UPR (Unfolded Protein Response ou stress du réticulum endoplasmique) caractérisée par l'expression des gènes codant les facteurs XBP1s, ATF6, GRP78 associée à celles des protéines PERK phosphorylée, CHOP et caspase 12, entraînant l'activation des caspases 9 et 3. Elle est associée à la phosphorylation des protéines GSK3 (Tyr216) et Tau (Thr231). En parallèle, l’augmentation du cholestérol induit, 3 semaines après l’injection, une augmentation de l’expression de la protéine Rab5 (marqueur des endosomes précoces) et une relocalisation de la protéine APP dans les fractions de radeaux lipidiques associées à l'activation de la voie amyloïdogénique (production des fragments-CTF et des peptides A42). L’étude lipidomique met en évidence, 4 semaines après l’injection, une augmentation du contenu en céramide à longues chaînes et à une augmentation des gangliosides. Tous ces éléments aboutissent à un processus de perte neuronale associée à un recrutement des astrocytes dès la quatrième semaine après l’injection
An increasing number of arguments suggest a close and complex link between cholesterol metabolism and neurodegenerative diseases, particularly with Alzheimer’s disease. Normal ageing is associated with a decrease of brain cholesterol content. Conversly, accumulation of brain cholesterol is associated with several neurodegenerative diseases (Huntington disease, Parkinson disease, epilepsy, Niemann Pick C disease, Alzheimer’s disease). Moreover, close connections exist between molecular physiopathology of AD and that of Niemann Pick, a disease of cholesterol homeostasis. Altogether, these results suggest that cholesterol overload might play a role, as an initiating factor for the development of AD.In the brain, cholesterol metabolism is tightly controlled. In adults, cholesterol is mainly synthetized by astrocytes, then shuttles to neurons where it is used. All cholesterol excess must be eliminated. Cholesterol cannot cross freely the blood-brain-barrier. To be metabolized, brain cholesterol must be converted in 24-hydroxy-cholesterol by the cholesterol-24 hydroxylase enzyme, coded by CYP46A1 gene. The objective of my PhD project was to determine if cholesterol accumulation in vivo in hippocampal neurons, a region early involved in AD pathology, could trigger neurotoxic processes with biochemical and neuropathological modifications close to what is observed in AD. Cholesterol overload in vivo was induced by inhibiting cholesterol 24-hydroxylase enzyme activity, using an RNA interference strategy. Stereotactic injection of an AAV5- shCYP46A1 vector in the stratum lacunosum moleculare of the hippocampus led to significant and rapid (as soon as 3 weeks after injection) inhibition of the Cyp46a1 gene in the hippocampus with an absence of RNA interference off-target effect. This inhibition was associated with a decrease of 24-hydroxycholesterol content and an increase of the cholesterol content. In response to this cholesterol excess, cell control mechanisms were initiated leading to decrease import and increase export of cholesterol, accompanied with an increase of phosphatidylcholine content to restore a physiological ratio of phospholipide/cholesterol. However, major accumulation of cholesterol led to neuronal death with activation of caspases 9 et 3, suggesting an apoptotic process. The cholesterol overload drives to an endoplasmic reticulum stress, with activation of the unfolded protein response (UPR) and expression of spliced XBP1, ATF6, GRP78, phosphorylated PERK, CHOP and caspase 12. These modifications were associated with phosphorylation of GSK3 (Tyr 216) and tau (Thr 231) proteins. In parallel, cholesterol accumulation led to increased expression of Rab5 (early endosome marker) and relocalization of APP in rafts domains associated to activation of amyloid pathway (production of -CTF fragments and A42 peptides). Lipidomic analysis showed an increase of ceramides and gangliosides content. All these modifications were associated with neuronal death 4 weeks after injection and astrocytosis, leading to an EEG theta rhythm accelerated to beta frequencies, memory deficits and hippocampal atrophy. In a mouse model of Alzheimer disease, the APP23 mouse, cholesterol accumulation led to major aggravation of the phenotype, with increased production of A peptides, occurring of tau phosphorylation and UPR response, leading to accelerated neuronal death. Altogether, these results suggest a direct link between cholesterol accumulation in the brain and Alzheimer’s disease. Brain cholesterol accumulation could seed the sows to the development of Alzheimer’s pathology. Reducing cerebral cholesterol could thus be a relevant therapeutic strategy to prevent the development, or at least slow down the evolution of the pathology in Alzheimer’s disease

Cytochrome P450 enzymes (P450s) are involved in cancer development and treatment due to their roles in the oxidative metabolism of various endogenous (e.g. oestrogen) and exogenous (e.g. tamoxifen) compounds. It is well-known that intermediate P450 metabolites derived from oestrogen metabolism are associated with breast carcinogenesis. The main aim of this project was to profile the cytochrome P450 and P450-regulatory nuclear receptor mRNAs in a series of breast cancer cell lines (BCCs) and compare this profile with normal breast cells. This study used the qualitative reverse transcriptasepolymerase chain reaction (RT-PCR) to detect mRNA expression of target genes. Results showed CYP1B1, CYP2D6, CYP2J2, CYP2R1, CYP2U1 and CYP4X1 mRNA to be present in all cell lines. CYP2A6, CYP2C8, CYP2C18, CYP2F1 and CYP4Z1 mRNA were expressed in oestrogen receptor (ER)-positiveCaucasian and ER-negative Afro- Caribbean BCCs. Although no differences in P450 mRNA were observed between the different ethnic groups, these preliminary findings suggest potential similarities in the ERpositive Caucasian and ER-negative Afro-Caribbean BCCs which warrant further investigation The CYP4Z1 PCR product was identified as two distinct bands. Specific primer sets were used to demonstrate potential intron retention in CYP4Z1. Using established in vitro models for the study of regulatory mechanisms of CYP4Z1, T47D and ZR-75-1 breast cancer cell lines were used to determine the appropriate nuclear receptors (i.e. progesterone receptor, glucocorticoid receptor or peroxisome proliferator-activated receptor alpha ). These findings suggest that there may be an alternative receptor mechanism involved in CYP4Z1 mRNA induction in these cells. In conjunction, pre-treatment of these two cell lines with the RNA synthesis inhibitor actinomycin D followed by the agonists showed a significant reduction (p < 0.05) of CYP4Z1 mRNA levels and inhibited CYP4Z1 induction by either progesterone, dexamethasone or pirinixic acid, indicating that these agonists have effects on CYP4Z1 mRNA transcription or stability. In contrast, cycloheximide differentially affected the level of CYP4Z1 mRNA induction by these agonists. Taken together, these results suggest that CYP4Z1 mRNA induction in T47D and ZR-75-1 is mediated through differential cell type specific regulatory mechanisms and there is evidence for differential regulation of the splice variants.

The present investigation is devoted to studies on porcine members of the cytochrome P450 4A (CYP4A) and CYP8B1 subfamilies, which are involved in bile acid biosynthesis and fatty acid metabolism.

Hyocholic acid is considered to fulfil the requirements for trihydroxy bile acids in the domestic pig (Sus scrofa) in the absence of cholic acid. Hyocholic acid is a 6α-hydroxylated product of chenodeoxycholic acid and the enzyme catalyzing the 6α-hydroxylation was cloned and found to be an atypical member of the CYP4A subfamily. The primary structure of this porcine enzyme, designated CYP4A21, shows about 75% overall sequence identity to members of the CYP4A subfamily expressed in rabbit and man. Divergent amino acids in a “signature sequence” in the active site of all hitherto known CYP4A fatty acid hydroxylases, were found to be important determinants for the 6α-hydroxylase activity of CYP4A21.

Two homologous CYP4A fatty acid hydroxylases, designated CYP4A24 and CYP4A25, expressed in pig liver and kidney were cloned. These two cDNAs encode proteins of 504 amino acids similar to CYP4A21. The overall identity between CYP4A24 and CYP4A25 is 97% compared to 94% identity to CYP4A21. Whereas CYP4A21 clearly deviates regarding structural features and catalytic activity it is more difficult to establish whether CYP4A24 and CYP4A25 are distinct enzymes or allelic variants of a single enzyme.

Cloning of the CYP4A21 gene showed a conserved organization compared to CYP4A genes in other species. A segment of the CYP4A24 gene was also cloned and comparison with the CYP4A21 gene revealed an extensive sequence identity also within introns as well as within the proximal promoter regions. This indicates that CYP4A21 and CYP4A fatty acid hydroxylases have a common origin and evolved by gene duplication. The CYP4A21 and CYP4A fatty acid hydroxylases, however, show distinct patterns of expression.

The key enzyme in cholic acid biosynthesis, CYP8B1, was markedly expressed in fetal pig liver compared to livers from young pigs. The opposite was shown for the expression of CYP4A21. An apparently conserved pig CYP8B1 gene was cloned and was intronless, similar to CYP8B1 genes from other species. The pig gene encoded a protein of 501 amino acids with 81% identity to CYP8B1 expressed in rabbit and man. Unlike other CYP8B1 genes, the pig promoter lacked a TATA-box. This might offer one explanation for the unusual expression pattern, which appears to be restricted to pig fetal life.

Cytochrom P450 (CYP)-Enzyme hydroxylieren und epoxydieren Arachidonsäure (AA) zu bioaktiven Metaboliten wie 20-Hydroxyeicosatetraensäure (20-HETE) und Epoxyeicosatriensäuren (EETs). Diese CYP-abhängigen Eicosanoide fungieren als Mediatoren bei der Regulation der Gefäß-, Nieren- und Herzfunktion. Hauptziel der vorliegenden Arbeit war es, die Identität der 20-HETE bildenden CYP-Isoformen in der Mausniere aufzuklären. Ein weiterer Schwerpunkt war die Bestimmung von Veränderungen im Metabolismus CYP-abhängiger Eicosanoide in Tiermodellen des akuten Nieren- und Herzversagens. Zur Identifizierung der 20-HETE bildenden CYP-Isoform wurde die Substrat- und Wirkungsspezifität von Cyp4a10, Cyp4a12a, Cyp4a12b und Cyp4a14, sowie ihre geschlechts- und stammspezifische Expression charakterisiert. Die Ergebnisse dieser Arbeit zeigen, dass Cyp4a12a die 20-HETE Synthase der Mausniere ist. Cyp4a12a wird durch Androgene induziert und seine Expressionshöhe ist für geschlechts- und stammspezifische Unterschiede in der 20-HETE Bildung verantwortlich. Im Rattenmodell des Ischämie/Reperfusions (I/R)-induzierten Nierenschadens wird eine 20-HETE Freisetzung durch I/R induziert. Wir konnten zeigen, dass der I/R-Schaden durch Hemmung der 20-HETE Bildung signifikant reduziert wird. Im Rattenmodell der Herzinsuffizienz (SHHF) ist das Herzversagen mit einer Variante des EPHX2 Gens assoziiert. EPHX2 kodiert für die lösliche Epoxidhydrolase (sEH), die den Abbau von EETs katalysiert. Wir konnten zeigen, dass die Genvariation zu signifikant höheren sEH-Aktivitäten im Herzen (3-fachen) und in der Niere (30-fachen) führt, im Vergleich zu Rattenstämmen, die keine Herzinsuffizienz entwickeln. Die vorliegende Arbeit unterstreicht die pathophysiologische Bedeutung von Veränderungen im Metabolismus von 20-HETE und EETs. Daher erscheint es vielversprechend, den CYP-Eicosanoid Stoffwechsel als neuen Angriffspunkt für die pharmakologische Behandlung kardiovaskulärer Erkrankungen zu erschließen.
Cytochrome P450 (CYP) enzymes hydroxylate and epoxidize arachidonic acid (AA) to bioactive metabolites such as 20-hydroxyeicosatetraenoic acid (20-HETE) and epoxyeicosatrienoic acids (EETs). These CYP-dependent eicosanoids serve as mediators in the regulation of vascular, renal and cardiac function. The main objective of the present study was to identify the 20-HETE producing CYP isoforms in the mouse kidney. Another focus was to determine changes in the metabolism of CYP-dependent eicosanoids in animal models of acute renal and heart failure. To identify the 20-HETE producing CYP-isoform the substrate and reaction specificity of Cyp4a10, Cyp4a12a, Cyp4a12b and Cyp4a14, as well as their sex- and strain-specific expression were characterized. The present study shows that Cyp4a12a is the predominant AA hydroxylase in the mouse kidney. Cyp4a12a is induced by androgens and its expression determines the sex and strain-specific differences in 20-HETE generation. In a rat model of renal ischemia/reperfusion (I/R) injury, I/R triggered the release of 20-HETE and we were able to ameliorate renal injury by pharmacological inhibition of 20-HETE production. In a rat model of heart failure (spontaneously hypertensive heart failure rats, SHHF) the heart failure phenotype is associated with a variant of the EPHX2 gene. EPHX2 is coding for the soluble epoxide hydrolase (sEH) which catalyze the degradation of EETs. We found that the gene variation leads to significantly higher sEH activities in the heart (3-fold) and in the kidney (30-fold) compared to rat strains not prone to the development of heart failure. The present study emphasizes the pathophysiological relevance of changes in the biosynthesis and degradation of 20-HETE and EETs. Therefore, it appears promising to develop the CYP-eicosanoid pathway as a novel clinical target for the treatment of cardiovascular diseases.

Niemann-Pick type C (NPC) disease is a rare neurogenerative disorder characterized by accumulation of cholesterol and other lipids in late endosomes/lysosomes. There are still limited therapeutic options available for NPC, but as it causes motor and intellectual impairment and premature death there is a high interest in the development of therapeutic strategies for this disease. CYP46A1 is a key enzyme involved in cholesterol homeostasis, which converts cholesterol to 24 (S)-hydroxycholesterol, being responsible for half of cholesterol turnover and the major brain cholesterol elimination pathway. Interestingly, increasing the expression or activity of the CYP46A1 resulted in beneficial effects in several animal models for neurodegenerative diseases. In this work, we aimed to study whether increased CYP46A1 expression would improve disease-associated biochemical alterations and disease phenotype in Npc1tm(I1061T) mice, that have a knock-in of the NPC1I1061T mutation, the most common mutation in NPC patients. For that, we used adeno-associated virus gene mediated therapy with a vector encoding CYP46A1, resulting in CYP46A1 ectopic expression. Increased expression of CYP46A1 led to a positive increment in weight gain in female mice in later stages of the disease, nevertheless the same was not observed in males, probably because of the phenotypic heterogeneity observed between Npc1tm(I1061T) animals. CYP46A1 expression could not significantly prevent extensive Purkinje cell loss in the cerebellum, nor it ameliorated motor function, as assessed by the rotarod test and gait analysis. Nevertheless, molecular and biochemical analysis suggest that CYP46A1 ectopic expression is able to ameliorate important features of NPC disease, such as cholesterol homeostasis, lysosomal function and neuroinflammation. Indeed, CYP46A1 restores 3-hydroxy-3-methylglutaryl-coenzyme A reductase and cathepsin D mRNA levels, together with reduction in the accumulation of lipidated microtubule-associated protein 1A/1B light chain 3B (LC3-II) indicating restoration of cholesterol homeostasis and improvement of the cellular intracellular traffic. Moreover, CYP46A1 expression could decrease astrogliosis and microgliosis in NPC male mice cerebellum. Overall, our promising results suggest that an earlier intervention, before the onset of the disease, can eventually open a new therapeutic avenue for this disease.

Liver X receptor (LXR), farnesoid X receptor (FXR) and peroxisome proliferator-activated receptor (PPAR) are adopted orphan nuclear receptors that function as lipid sensors. These receptors respond to cellular lipid levels and regulate the expression of target gene. Previously, it was demonstrated that low doses of chlordecone (CD) pretreatment disturbed exogenous cholesterol distribution and cellular lipid transport, storage and metabolism pathway. The aim of this study was to determine whether low doses of CD affect nuclear receptor (LXR��/FXR or PPAR��)-mediated lipid homeostasis. Thus, hepatic microsomal protein contents of cytochrome P450 7a (cyp7a, regulated by LXR��/FXR), and P450 4a1 (cyp4a1, regulated by PPAR��) were determined in male C57BL/6N mice, fed AIN76 or AIN93M diet, received CD (2.5, 5.0 or 15 mg CD/kg body weight). Western blot analysis was used for protein measurements using appropriate antibodies. Cyp7a and cyp4a1 protein levels were confirmed by enzyme activities, cholesterol 7��-hydroxylase and lauric acid hydroxylase activities, respectively. Plasma total cholesterol and triglycerides, body and liver weights were also measured in these dose-response experiments. Plasma total cholesterol and triglycerides levels from animals fed AIN 93M diet were significantly lower than those from animals fed AN 76 diet. However, neither total plasma cholesterol nor triglycerides levels were changed in CD-treated mice fed AN 76 or AN 93M diet. Cyp7a protein level or its enzyme activity was not altered by CD treatment. Likewise, cyp4a1 protein level or its activity was not affected by CD treatment. In summary, the results of the present study do not support the hypothesis that CD treatment alters nuclear receptor (LXR��/FXR or PPAR��)-mediated lipid homeostasis.
Graduation date: 2002

Tese de doutoramento, Farmácia (Biologia Celular e Molecular), Universidade de Lisboa, Faculdade de Farmácia, 2016
Cholesterol is an essential molecule in central nervous system physiology and cell signalling, with a wide range of roles, such as being an essential component of cell membranes, lipid rafts and myelin sheets, or serving as a precursor for neurosteroids. The brain relies mainly on de novo cholesterol synthesis and eliminates cholesterol through its conversion to 24(S)-hydroxycholesterol (24OHC), which readily crosses the blood brain barrier into circulation. The enzyme that catalyzes this reaction is the neuronal-specific cholesterol 24-hydroxylase (CYP46A1). Knockout-mice for Cyp46a1 exhibit inactivation of brain cholesterol synthesis, which is accompanied by deficiencies in learning and memory. It has been suggested that the cognitive deficits might be due to a reduction in the levels of intermediaries of the mevalonate pathway, namely isoprenoids. However, it has never been shown if modulation of CYP46A1 expression could effectively affect prenylation, a post-translation modification critical for membrane association of signalling proteins with fundamental roles in cell biology, such as small guanosine triphosphate-binding proteins (sGTPases). Hence, we started by assessing the effect of CYP46A1 expression on the activation of sGTPses in neuronal cells. We observed that increased expression of CYP46A1 enhanced prenylation and activation of sGTPases of the Rho and Rab family, and that this effect was dependent on the activation of the mevalonate pathway. Since sGTPases control a wide variety of functions in the cell, a great number of cellular pathways might be modulated by CYP46A1. Indeed, we have shown that CYP46A1 overexpression leads to a decrease in Liver X Receptor (LXR) transcriptional activity and in mRNA levels of LXR-target genes involved in cholesterol efflux, in a prenylation-dependent manner. These results highlight a novel regulatory axis in neurons; under conditions of membrane cholesterol reduction by increased CYP46A1 expression, neurons enhance isoprenoid synthesis and sGTPase prenylation. Moreover our results also showed that CYP46A1 triggers an increase in neuronal dendritic outgrowth and dendritic protrusion density, and elicits in vitro and in vivo increase of synaptic proteins in crude synaptosomal fractions. Strikingly, in neurons, these effects were abolished by pharmacological inhibition of geranylgeranyl transferase I (GGTase-I) activity. Furthermore, CYP46A1 expression increased tropomyosin-related kinase (Trk) receptors phosphorylation, its interaction with GGTase-I, and the activity of GGTase-I. This interaction Trk-GGTase-I was shown to be crucial for the enhanced dendritic outgrowth mediated by CYP46A1. Cholesterol supplementation studies indicated that cholesterol reduction by CYP46A1 is the necessary trigger for these effects. Taking into account the role of CYP46A1 in cholesterol elimination and neuronal function, we hypothesized that it could be a potential therapeutic target in Niemann-Pick type C disease (NPC), a lysosomal storage disorder, characterized by cholesterol accumulation in the late endosomes/lysosomes (LE/L) compartment, leading to progressive neurodegeneration. Upon ectopic expression of CYP46A1 in human NPC fibroblasts, we observed a reduction in cholesterol accumulation in the LE/L compartment, which was accompanied by partial normalization of the expression levels of several genes involved in cholesterol homeostasis. We used the chemical compound U18666A to mimic the NPC phenotype in neuronal cells, and observed that CYP46A1 overexpression protects and reverts cholesterol accumulation induced by U18666A, but also inhibits the increase in reactive oxygen species and lipid peroxidation. Further experiments led us to conclude that CYP46A1 induces the expression of the antioxidant enzyme heme-oxygenase-1 (HO-1), and that the activity of this enzyme contributes to CYP46A1-mediated protection against oxidative stress. This work contributes to a further understanding of how cholesterol influences the brain, namely how the cholesterol-metabolizing enzyme, cholesterol 24S-hydroxylase affects neuronal development and function, and highlights the role of this enzyme as a therapeutic target in NPC, a disorder that has very limited therapeutic options.

Tese de doutoramento, Farmácia (Biologia Celular e Molecular), Universidade de Lisboa, Faculdade de Farmácia, 2011
Cholesterol has a crucial role in central nervous system physiology and cell signaling, and many studies correlate alterations in brain cholesterol homeostasis with neurodegenerative diseases. The CYP46A1 gene codes for the cholesterol 24-hydroxylase (CYP46A1), a cytochrome P450 specifically expressed in neurons, that is responsible for the majority of brain cholesterol turnover. However, despite its physiological importance, the molecular mechanisms underlying the human CYP46A1 expression had not been characterized. Therefore, our work aimed to identify regulatory elements and factors involved the CYP46A1 brain-specific expression, and further assess if CYP46A1 transcription is regulated by epigenetic modifications, such as DNA methylation. Moreover, we aimed to identify a human cell model that could be a valuable tool for the study of cholesterol homeostasis in human neurons. In our initial studies, we cloned and characterized the human CYP46A1 promoter. Functional deletion analysis, over-expression studies, site-directed mutagenesis and gel-shift assays identified that not only Sp transcription factors control CYP46A1 transcription, but are most probably responsible for cell type specificity. To test our hypothesis that an increase in the (Sp3+Sp4)/ Sp1 ratio would result in CYP46A1 transcriptional activation, we differentiated Ntera2/clone D1 (NT2) human teratocarcinoma cells into post-mitotic neurons (NT2N). We demonstrated for the first time a significant increase in CYP46A1 mRNA and protein levels in a human cell culture, and identified a concomitant decrease in the levels of Sp1 associated with the proximal promoter of this gene. Nevertheless, we did not observe any conserved pattern in Sp protein binding to other Sp-regulated gene promoters, suggesting that Sp-DNA binding and transcriptional activity is highly dependent on the neuronal chromatin context. Moreover, we showed that throughout NT2 differentiation, HMG-CoA Abstract xxiv synthase, HMG-CoA reductase, SREBP2 and LDLr, key players in brain cholesterol homeostasis, present expression profiles that mimic what is thought to occur in vivo. Our results also suggest that progenitor cells eliminate cholesterol in the form of 27-hydroxycholesterol while neurogenesis induces a shift to the 24-hydroxylase-dependent elimination pathway. Finally, we showed that the demethylating agent 5’-Aza-2’-deoxycytidine (DAC) is a CYP46A1 inducer. Surprisingly, bisulfite sequencing analysis revealed that the CYP46A1 core promoter is completely unmethylated in both human brain and non-neuronal human tissues where CYP46A1 is not expressed. We demonstrated that DAC induces CYP46A1 expression, in a DNA methylation- independent mechanism, by decreasing Sp3/HDAC binding to the proximal promoter. Collectively, our results provide new insights on the regulatory circuits that control CYP46A1 transcription, and contribute to the identification of potential therapeutic approaches that can modulate CYP46A1 expression.
Fundação para a Ciência e a Tecnologia:SFRH/BD/27660/2006; PPCDT/SAU-MMO/55919/2004 and PTDC/SAU-GMG/64176/2006 (to Professor Elsa Margarida Teixeira Rodrigues) from FCT and FEDER.
Fundação para a Ciência e Tecnologia (FCT),(SFRH/BD/27660/2006), Lisbon, Portugal; PPCDT/SAU-MMO/55919/2004; PTDC/SAU-GMG/64176/2006. FCT; FEDER.

Tesina (Grado en Ciencias Biológicas)--Universidad Nacional de Córdoba. Facultad de Ciencias Exactas, Físicas y Naturales. Lugar de Trabajo: Instituto de Investigación Médica Mercedes y Martín Ferreyra. INIMEC-CONICET-U.N.C. 2017. 56 h.; ils.; grafs.; tabls. Contiene Referencias Bibliográficas.
Durante el envejecimiento se producen cambios en la composición lipídica de membranas neuronales. En la mayoría de los casos, estas alteraciones lipídicas han sido relacionadas a la aparición de problemas cognitivos, tanto en envejecimiento regular como patológico. Específicamente, en el laboratorio hemos observado que el envejecimiento de neuronas hipocampales está asociado a un incremento en los niveles de la enzima colesterol-24-hidroxilasa o CYP46, lo cual produce una disminución del colesterol de membrana, y resulta en una reducida función sináptica y pérdida de memoria. Sin embargo, el aumento de los niveles de esta enzima con el envejecimiento ha sido analizado en muestras de hipocampo total, sin realizar distinción entre las distintas subregiones hipocampales. Tampoco se habían analizado hasta el momento cambios de los niveles de CYP46 con el envejecimiento en otras regiones del cerebro. Por lo tanto, en el presente trabajo se analizaron los niveles de CYP46 mediante inmunohistoquímica, comparando secciones sagitales de cerebros pertenecientes a ratones jóvenes y viejos. En animales viejos, se observó aumento en los niveles de la enzima en hipocampo, cerebelo y corteza, áreas estrechamente relacionadas a aprendizaje y memoria. Además, mediante inmunofluorescencia se determinó, en cultivos primarios de neuronas hipocampales, la distribución subcelular de CYP64 cuando esta se encuentra en niveles normales y cuando se encuentra sobreexpresada. Por último, se compararon dos herramientas destinadas a la detección y estudio de la distribución de colesterol en neuronas hipocampales.

Tese de doutoramento, Farmácia (Biologia Celular e Molecular), Universidade de Lisboa, Faculdade de Farmácia, 2012
Apart from being an essential component of cellular membranes and having a role as a signaling molecule, cholesterol is also the precursor of several bioactive molecules that include bile acids, steroid hormones, oxysterols and vitamin D. In the brain constant levels of this sterol are required for normal functioning and the homeostasis is maintained, in part, by an efficient blood-brain barrier that prevents exchanges with lipoprotein cholesterol from circulation. For that reason, de novo and in situ synthesis occur to meet cholesterol needs in the central nervous system (CNS), which despite the low synthesis rate must be excreted at some degree in order to keep its steady state. The conversion of cholesterol into 24(S)-hydroxycholesterol, by the neuronalspecific cytochrome P450 cholesterol 24-hydroxylase (CYP46A1) has been described as the major elimination mechanism. The main goal of this work was to characterize the effect of histone deacetylase (HDAC) inhibition in the transcriptional regulation of CYP46A1 gene and the molecular mechanism underlying such effect. We started by demonstrating that the inhibition of HDAC activity by trichostatin A (TSA), valproic acid and sodium butyrate cause a potent induction of both CYP46A1 promoter activity and endogenous expression. Indeed, we have shown for the first time that TSA induces an overall increase in histone acetylation levels at CYP46A1 proximal promoter, as a result of the detachment of HDACs and recruitment of histone acetyltransferases (HAT), in a process dependent on Sp3 transcription factor decreased binding to particular cis-elements. This change in chromatin structure culminates in the recruitment of RNA polymerase II and CYP46A1 gene activation. Nevertheless, the fact that histone deacetylation was evident at a time point when the HDAC/HAT ratio should still favor acetylation, led us to investigate if mechanisms besides histone hyperacetylation could participate in the TSA mediated derepression of CYP46A1 gene. Interestingly, we identified the participation of the mitogen-activated kinase kinase (MEK)-extracellular signal-regulated kinase (ERK) signaling pathway in the CYP46A1 response to TSA. A decrease in ERK1/2 phosphorylation levels was observed after TSA treatment concomitantly with a decrease in Sp3 binding activity. Inhibition of protein phosphatase activity by pre-treatment with okadaic acid (OA) completely reversed these changes, and impaired the TSA-mediated CYP46A1 activation without affecting promoter histone hyperacetylation. Our results also show that TSA treatment induces the dissociation of phosphorylated ERK1/2 from the CYP46A1 promoter and specifically from the Sp3-containing DNA fragments. This suggests that in the context of the CYP46A1 promoter phosphorylated Sp3 acts as a transcriptional repressor being responsible for the recruitment of co-repressor complexes, with and without HDAC activity. Moreover, our work highlights the importance of the MEK-ERK signaling pathway in the control of brain cholesterol elimination. The importance of CYP46A1 in cholesterol homeostasis and the drastic effect of HDAC inhibitors (HDACi) in its expression, lead us to evaluate if these compounds can affect the expression of other key players in neuronal cholesterol metabolism. In the last part of our work we have identified TSA as a cholesterol-lowering molecule, by modulating the transcription of other genes involved in cholesterol metabolism in human neuroblastoma cells, namely by up-regulating genes that control cholesterol efflux and dow-regulating genes involved in cholesterol synthesis and uptake, thus leading to an overall decrease in total cholesterol content. Moreover, we have shown that TSA is also able to partially reverse the increased cholesterol content and the transcriptional changes, induced by pathological lysosomal accumulation of intracellular cholesterol. Overall, these results clarify the role of HDACi in the modulation of CYP46A1 gene transcription as well as other key genes in cholesterol metabolism, comprising a significant contribution in the elucidation of the molecular mechanism involved in the transcriptional regulation of CYP46A1 gene and emphasizing the idea of HDAC inhibition as a promising therapeutic tool in neurodegenerative disorders with impaired cholesterol metabolismo.
O colesterol é uma molécula essencial à vida. Para além de desempenhar um papel crucial na estrutura das membranas celulares, através da regulação da permeabilidade e fluidez das mesmas, o colesterol é também percursor de inúmeras moléculas de extrema relevância biológica tais como os ácidos biliares, os oxisteróis, as hormonas esteroides e a vitamina D, podendo também atuar como uma molécula sinalizadora. No encéfalo, o colesterol está maioritariamente na forma não esterificada e encontra-se associado às bainhas de mielina e às membranas plasmáticas dos neurónios e células da glia. O colesterol é também essencial à formação e propagação de sinapses, ao crescimento dendrítico e à estabilidade dos microtúbulos. Além disso, o sistema nervoso central (SNC) necessita de níveis constantes de colesterol para um correto funcionamento, sendo que a homeostasia é assegurada, em parte, pela barreira hematoencefálica que impede trocas com o colesterol em circulação. Por esta razão, todo o colesterol presente no encéfalo deriva de uma síntese de novo e in situ. Apesar de no SNC do adulto a síntese de colesterol ser reduzida, uma fração necessita de ser excretada, de forma a manter os níveis de colesterol constantes. A conversão do colesterol em 24(S)- hidroxicolesterol, pelo enzima 24(S)-hidroxilase (CYP46A1), foi descrita como o principal mecanismo de eliminação. O produto enzimático (oxisterol), contrariamente ao colesterol, atravessa com facilidade a barreira hemato-encefálica, entra em circulação e é posteriormente eliminado no fígado, completando desta forma o processo de transporte reverso do colesterol. Nos últimos anos, um grande número de estudos aponta para uma relação entre alterações no metabolismo do colesterol e o desenvolvimento de doenças neurodegenerativas, como a doença de Alzheimer, a doença de Huntington ou a doença de Niemann-Pick tipo C. Para além disso, inúmeras evidências sugerem que a indução do CYP46A1 poderá ter consequências benéficas, nomeadamente na doença de Alzheimer. De facto a sobre-expressão do CYP46A1 poderá ter um efeito inibitório direto sobre a produção do péptido β-amilóide, um efeito indireto relacionado com a diminuição dos níveis de colesterol das membranas neuronais, ou por aumentar a ativação dos genes alvo do receptor nuclear liver X receptor. O principal objectivo deste trabalho foi a caracterização do efeito da inibição das desacetilases de histonas (HDAC) na regulação da transcrição do gene CYP46A1, bem como dos mecanismos moleculares subjacentes a esse mesmo efeito. Na primeira parte deste trabalho, começámos por demonstrar que a inibição da atividade das HDACs através do tratamento com os inibidores tricostatina A (TSA), ácido valpróico e butirato de sódio, causam uma drástica indução da atividade do promotor e da expressão endógena do gene CYP46A1. Observámos também, pela primeira vez, que o tratamento com TSA induz um aumento significativo dos níveis de acetilação das histonas do promotor próximo do gene CYP46A1, sendo que este efeito resulta da diminuição da presença de HDACs e do recrutamento de acetiltransferases de histonas (HAT). Estudos anteriores do nosso grupo identificaram como essenciais na expressão basal do gene CYP46A1 factores de transcrição da família Sp e, neste estudo, demonstrámos que as proteínas Sp1 e Sp4 são também cruciais para a ativação do CYP46A1 pelo TSA. No entanto o efeito do TSA parece depender da diminuição da ligação do factor de transcrição Sp3 a elementos de resposta específicos do promotor, uma vez que uma diminuição de ligação desta proteína está relacionada com a diminuição de HDACs presentes no promotor. De uma forma geral, as modificações na estrutura da cromatina e no complexo proteico na região do promotor próximo induzidas pelo TSA culminam no recrutamento da RNA polimerase II e na consequente ativação do gene CYP46A1. A evidência de que o nível de acetilação das histonas a determinado momento não reflete a razão entre os níveis dos enzimas HDAC/HAT presentes no promotor, conduziu-nos à investigação de mecanismos independentes da hiperacetilação de histonas que pudessem contribuir para a ativação do gene CYP46A1 pelo TSA. De facto, na segunda parte do nosso trabalho identificámos a participação da via de sinalização da cinase de proteínas ativada por mitogénios (MAPK) na resposta do CYP46A1 ao TSA. Através do pré-tratamento com inibidores químicos específicos bem como a utilização de dominantes negativos da cinase terminal desta via, a cinase de proteínas regulada por sinais extracelulares (ERK), concluímos que a inibição da atividade deste enzima potencia o efeito do TSA, contrariamente ao efeito da inibição das fosfatases de proteína, através do pré-tratamento com ácido ocadáico, que bloqueia quase na totalidade a ativação do gene CYP46A1 pelo TSA. A análise dos níveis de fosforilação da ERK1/2 demonstrou que o TSA induz uma diminuição do nível de ativação destas cinases, bem como da atividade de ligação da proteína Sp3 ao DNA, efeitos esses que são revertidos pelo pré-tratamento com ácido ocadáico, sem que o nível de hiperacetilação das histonas do promotor seja afetado. Os nossos resultados demonstram ainda que o tratamento com TSA induz a dissociação da forma fosforilada da ERK1/2 do promotor do CYP46A1, especificamente, dos fragmentos de DNA que contêm o factor de transcrição Sp3, sugerindo que no contexto do promotor do CYP46A1 a proteína Sp3 atua como um repressor da transcrição e é responsável pelo recrutamento de complexos co-repressores, com e/ou sem atividade HDAC. O TSA ao modular a ativação da ERK1/2 regula indiretamente a atividade deste factor de transcrição e consequentemente a expressão do CYP46A1. De facto, a replicação do efeito do ácido ocadáico e da inibição da atividade da ERK1/2 na atividade do promotor e expressão basal do gene CYP46A1 evidencia também a importância desta via de sinalização intracelular no controlo da eliminação de colesterol do encéfalo. A importância do CYP46A1 na homeostasia do colesterol bem como o efeito drástico dos inibidores das HDACs na sua expressão, conduziu à avaliação do efeito destes compostos na expressão de genes chave no metabolismo do colesterol em células neuronais. Como tal, na última parte deste trabalho demonstrámos a capacidade do TSA de diminuir os níveis de colesterol em células de neuroblastoma humano, através da regulação da expressão de genes envolvidos no metabolismo do colesterol, nomeadamente, através da indução de genes envolvidos no controlo do efluxo (transportadores ATP-binding cassette) e catabolismo (CYP46A1), e repressão de genes essenciais para a síntese (3-metilglutaril Coenzima A redutase) e captação de colesterol (receptor de lipoproteínas de baixa densidade), culminado desta forma na diminuição do conteúdo total de colesterol. O tratamento com o composto U18666A, que mimetiza o fenótipo da doença de Niemann-Pick tipo C, caracterizado pela acumulação patológica de colesterol nos lisossomas, resultou no aumento dos níveis de colesterol em células de neuroblastoma humano, bem com no aumento da expressão de genes envolvidos na síntese e captação de colesterol e diminuição dos genes responsáveis pelo efluxo. No entanto, o tratamento com TSA reverteu todos esses efeitos, contribuindo para a correção das perturbações no metabolismo do colesterol. Apesar de já ter sido demonstrado o efeito benéfico da inibição das HDACs em fibroblastos de pacientes com a doença de Niemann-Pick tipo C, este trabalho demonstra pela primeira vez a capacidade destes compostos de reverter, ao nível da transcrição, os efeitos da acumulação de colesterol nos lisossomas. Em conclusão, os resultados apresentados nesta tese identificam os inibidores das HDACs como agentes farmacológicos que poderão eventualmente vir a ser usados na indução da transcrição do gene CYP46A1, bem como na modelação da transcrição de genes que participam de forma relevante no metabolismo do colesterol no encéfalo. Para além disso constitui uma contribuição significativa para a compreensão dos mecanismos moleculares responsáveis pela regulação da transcrição do gene CYP46A1, identificando a via das MAPKs como umas das vias de sinalização responsáveis pelo controlo do catabolismo do colesterol no encéfalo. A compreensão dos mecanismos moleculares controlados pelos inibidores das HDACs, e envolvidos na homeostasia do colesterol no encéfalo, poderá de facto constituir uma plataforma para o desenvolvimento de possíveis intervenções farmacológicas que vão da neurodegenerescência ao cancro.
Fundação para a Ciência e a Tecnologia ( FCT, SFRH/BD/41848/2007, projetos PPCDT/SAU-MMO/55919/2004, PTDC/SAU-GMG/64176/2006) e FEDER (Fundo Europeu de Desenvolvimento Regional)

The brain possesses 20% of whole-body cholesterol, becoming the richest cholesterol organ in our body. Due to the Blood Brain Barrier, the cholesterol has its own way to synthesize and degrade cholesterol, in order to maintain its homeostasis. Cholesterol is synthesized through de novo synthesis in the brain and transported out of the brain in the form of oxysterols, more specifically 24-hydroxycholesterol (24-OHC). This process is acomplished through CYP46A1, a protein of the CYP450 family. Alterations in cholesterol homeostasis can lead to metabolic abnormalities, such as obesity, inflammation and insulin resistance. Previous studies in our laboratory showed that alterations in the expression of Cyp46A1 gene (more specifically silencing) with different diets, rich and poor in fat, had impacts in whole-body homeostasis. Taking these results into account, the main objective of this study was to evaluate the impact of modulation of Cyp46A1 gene in the main metabolic organs (liver, pancreas, and adipose tissue), through silencing and overexpressing CYP46A1 in the arcuate nucleus of the hypothalamus. This modulation was made in C57BL/6J mice fed with two distinct diets, one rich in fat (HF diet) and one poor in fat (CHOW diet). The data obtained from microscopy and tissue quantification made it possible to see the physiological changes resultant from the modulation of CYP46A1. It was possible the phenomenon BAT “whithening”, an increase in the size of Langerhans islets and lipidic hypertrophy. RT-qPCR was also realized to evaluate the mRNA levels of the different targets in the hypothalamus. The results obtained from RT-qPCR, showed that modulation of Cyp46A1 affected the mRNA levels of the different targets tested (POMC, NPY and TNF-alpha). All this data confirmed our hypothesis, that the modulation of CYP46A1 do alter the whole-body metabolism. Further studies must be conducted to continue this project, in order to investigate if this gene could be a target for genetic therapies.
Um dos grandes problemas de saúde atuais da nossa sociedade é a obesidade. Esta condição é um dos principais sintomas da síndrome metabólica sendo consequência, para além da ausência de exercício físico, de uma dieta desequilibrada e rica em gorduras. A obesidade gera várias consequências no corpo, sendo uma delas a inflamação, podendo esta ocorrer no tecido adiposo, fígado e até no cérebro. O cérebro é o órgão do corpo humano que possui maior teor de colesterol, cerca de 20% do colesterol total corporal. Este é necessário para a formação das bainhas de mielina e membranas celulares. O cérebro possui uma barreira protetora, altamente seletiva que protege o órgão de substâncias tóxicas que possam ser encontradas no sangue, a barreira hematoencefálica. Esta barreira, impede também o colesterol de entrar ou sair do cérebro, pelo que o cérebro possui um metabolismo do colesterol altamente controlado. Este equilíbrio é mantido através da síntese de novo, conversão e efluxo. Para que o colesterol possa ser transportado para fora do cérebro, é necessário que seja previamente convertido em oxisterol, um derivado hidrolisado do colesterol, que por sua vez é capaz de atravessar a barreira hematoencefálica. Esta conversão é possível através da enzima Cyp46a1, que converte o colesterol em colesterol-24-hidroxilase, para que possa ser eliminado no fígado. Diversos estudos mostraram que alterações na homeostasia do metabolismo do colesterol poderiam levar à obesidade e problemas relacionados (resistência à insulina e diabetes tipo 2). Tendo isto em conta, o principal objetivo deste estudo foi investigar os efeitos da modulação do Cyp46A1 no cérebro no metabolismo corporal. Para tal, utilizouse uma amostragem de 69 ratinhos C57BL/6J, onde 45 ratinhos foram expostos a um silenciamento da expressão do gene Cyp46A1 e 24 ratinhos a uma sobre expressão deste gene. Para tal, foi realizada uma injeção estereotáxica bilateral no núcleo arqueado do hipotálamo, com os respetivos grupos virais: a sobre expressão, AAV-Cyp46A1 e o silenciamento, AAV-shCyp46A1. Para verificar também a influência da dieta, os ratinhos foram divididos em seis grupos contendo duas dietas diferentes: AAVCyp46A1, AAVshCyp46A1 e grupo controlo (não injetados) com uma dieta rica contendo 60% de gordura (denominada High Fat Diet, HF) e AAVCyp46A1, AAV-shCyp46A1 e grupo controlo com uma dieta que continha 10% de gordura (denominada Low Fat control diet, CHOW). Os resultados obtidos neste estudo, revelaram que a modulação do gene Cyp46a1 no núcleo arqueado do hipotálamo afetou a morfologia de vários órgãos metabólicos, levando a um aumento no tamanho das ilhotas de Langerhans no pâncreas, a uma acumulação de gotículas lipídicas no fígado e também a alterações na estrutura do tecido adiposo, nomeadamente hipertrofia. Estes resultados foram também comprovados através da quantificação dos respetivos tecidos. Foram também realizados vários RT-qPCR, de forma a verificar se a modulação do gene Cyp46a1 alterou os níveis de mRNA do próprio Cyp46A1 e de outros importantes mediadores metabólicos do núcleo arqueado do hipotálamo, entre eles o POMC e o NPY. Foi possível verificar uma diminuição dos níveis de mRNA do Cyp46A1 no grupo silenciado quando comparado com o grupo controlo, embora esta não seja significativa. Por outro lado, verificou-se que os níveis de NPY, tanto o grupo AAV-shCyp46A1 como o AAV-Cyp46A1 com a dieta HF possuem aumentos significativos nos níveis de mRNA, quando comparados com o grupo de animais não injetados. Foram estudados também os níveis de mediadores de inflamação, nomeadamente do TNF-alfa. Neste alvo, observou-se claramente um aumento nos níveis de mRNA nos grupos AAV-Cyp46A1 e AAV-shCyp46A1 que possuíam a dieta HF, em relação aos grupos controlo. Os resultados obtidos sugerem então um papel fundamental do gene Cyp46a1 no metabolismo corporal, para além do papel desempenhado a nível cerebral. No entanto, será necessário realizar mais estudos de forma a tentar compreender de forma mais completa o papel desta proteína no metabolismo corporal.

Cholesterol metabolism is tightly controlled in the brain, through an equilibrium of cholesterol de novo synthesis and cholesterol efflux. Most of the cholesterol in the brain is in myelin sheaths to insulate axons and to maintain their morphology and synaptic transmission. The importance of cholesterol in the brain is highlighted by the fact that dysfunctions in its metabolism homeostasis are correlated with different neurodegenerative disorders. The hypothalamus, specially the ARC, is the principal brain region in the control of whole-body energy homeostasis, having a crucial role in metabolic organ regulation. This project goal was to silence the expression of the Cyp46a1 mouse gene in the hypothalamus of C57BL/6J wild-type mice fed with Chow and HFD, and to investigate its impact in the whole-body metabolism homeostasis. As the ARC is implicated in the control of whole-body energy metabolism, and oxysterols levels are altered in obesity, we hypothesize that the silencing of Cyp46a1 gene could lead to an obesity and TIIDM phenotypes. In this study, C57BL/6J wild-type mice (n=45) were divided into two groups corresponding to two different diets. One group (n=24) had access to a Chow containing 10% of fat and the other group (n=21) had access to an HFD containing 60% of fat. This study was conducted during 12 weeks. In the 4th week, the two groups of mice (Chow and HFD) were divided into four subgroups, from which two were submitted to the stereotaxic injection delivering the AAV of the serotype 5, with the shRNA targeting the mouse Cyp46a1 gene in each side of the ARC and two remained non-injected, as control. The silencing of Cyp46a1 mouse gene in the ARC of mice fed with Chow and HFD leads to an increase of BW, changes in food and water intake, to a reduction of glucose tolerance, of insulin sensitivity and leads also to modifications in several metabolic organs, comparatively to the non-injected animals. In fact, in the Chow AAV5-shCyp46a1 animals, the impact of the silencing Cyp46a1 gene appears to mimic an HFD effect, whereas in HFD AAV5-shCyp46a1 mice this silencing exacerbates the phenotype of obesity. These results could suggest an important role of the cholesterol metabolism in the brain, specially of the Cyp46a1 enzyme in the control of whole-body homeostasis. Finally, additional studies are needed to continue this project and it would be interesting to perform the overexpression of Cyp46a1 in the ARC to investigate if this gene could be a potential target to further genetic therapies.
No cérebro, o metabolismo do colesterol é extremamente controlado através do equilíbrio entre a síntese de novo, conversão e efluxo do colesterol. Quando a síntese do colesterol excede a sua necessidade no cérebro, ocorre o processo hidroxilação do colesterol em 24-hidroxicolesterol (24-OHC) através da ação da enzima CYP46A1, uma enzima codificada pelo gene CYP46A1. O oxisterol 24-OHC, ao contrário do colesterol, possui a capacidade de atravessar a barreira hematoencefálica contribuindo desta forma para a homeostasia do colesterol no cérebro. Este está maioritariamente localizado nas bainhas de mielina, de modo a isolar os axónios e manter a sua morfologia e transmissão sináptica. Diversas doenças neurodegenerativas, como a doença de Alzheimer e a doença de Huntington, foram correlacionados com disfunções na homeostasia do colesterol no cérebro, destacando desta forma a sua importância no organismo. O hipotálamo, especialmente o núcleo arqueado, é a região do cérebro responsável pela regulação da homeostasia energética corporal, apresentando um papel crucial no equilíbrio entre o consumo e o gasto energético. O objetivo deste projeto foi silenciar a expressão do gene Cyp46a1 no hipotálamo de ratinhos C57BL/6J wild-type alimentados com uma dieta de controlo com baixo teor em gordura (Chow – low fat control diet) e com uma dieta com alto teor em gordura (HFD - high fat diet) e investigar o seu impacto na homeostasia energética corporal. Uma vez que o núcleo arqueado está implicado regulação da homeostasia energética corporal e que os níveis de oxisteróis encontram-se alterados na obesidade, criou-se a hipótese de que o silenciamento do gene Cyp46a1 poderia resultar em um fenótipo de obesidade e de diabetes mellitus do tipo 2. Desta forma, os ratinhos C57BL/6J (n=45) foram divididos em dois grupos correspondentes a dietas distintas. Um dos grupos (n=24) teve acesso a uma dieta Chow, contendo 10% de gordura, e o outro grupo (n=21) teve acesso a uma dieta HFD, contendo 60% de gordura. Este estudo foi conduzido durante o período de 12 semanas, no qual, na quarta semana os dois grupos (Chow e HFD) foram divididos em quatro subgrupos. Dois grupos foram submetidos à injeção estereotáxica bilateral no ARC, constituindo os grupos tratados, (Chow AAV5-shCyp46a1 e HFD AAV5-shCyp46a1) e os restantes grupos não foram submetidos à injeção estereotáxica, constituindo os grupos de controlo. O silenciamento do gene Cyp46a1 no núcleo arqueado dos ratinhos C57BL/6J alimentados com uma dieta Chow resultou em um aumento no peso corporal, na ingestão de alimentos e de água, na redução da tolerância à glucose, na sensibilidade à insulina e ainda, em modificações em vários órgãos metabólicos. Da mesma forma, o silenciamento do gene Cyp46a1 dos ratinhos C57BL/6J alimentados com uma dieta HFD resultou em um aumento do peso corporal, em uma diminuição da ingestão de alimentos, da sensibilidade à insulina e em modificações nos órgãos metabólicos. O silenciamento do gene Cyp46a1 no núcleo arqueado dos ratinhos C57BL/6J parece modificar a morfologia do tecido adiposo branco, do tecido adiposo castanho e do fígado, nos animais submetidos à injeção estereotáxica. Os grupos tratados apresentaram importantes modificações na acumulação lipídica nestes órgãos, bem como modificações nos níveis de proteína. Além disso, o silenciamento do gene Cyp46a1 resulta em modificações no comportamento nos ratinhos C57BL/6J alimentados com dietas Chow e uma HFD. Nos animais Chow AAV5-shCyp46a1, o impacto do silenciamento parece mimetizar o efeito de uma HFD, enquanto nos ratinhos HFD AAV5-shCyp46a1 esse silenciamento parece exacerbar o fenótipo da obesidade, uma vez que estes animais já se encontravam metabolicamente desregulados. Os resultados podem desta forma sugerir um papel importante do metabolismo do colesterol no cérebro, especialmente da enzima CYP46A1 no controlo da homeostasia energética corporal. Finalmente, estudos adicionais ainda são necessários e seria interessante realizar a sobre expressão do gene Cyp46a1, no núcleo arqueado, para investigar se este gene poderá ser um potencial alvo terapêutico.

Diet-induced obesity causes a central inflammatory process in the brain, more specifically in the arcuate nucleus of the hypothalamus; in addition to substantially altering cholesterol homeostasis in the brain. The brain is one of the richest organs in cholesterol and cholesterol homeostasis has proved to be very important not only to maintain healthy brain physiology, but also directly influencing the whole-body homeostasis. In addition, several metabolic disorders are correlated with different neurodegenerative diseases. Cholesterol in the brain is converted primarily to 24-hydroxycholesterol (24-OHC) by CYP46A1. Changes in oxysterol metabolism have been correlated with obesity. Previous studies from our laboratory have identified CYP46A1 as a relevant therapeutic target, not only for Machado de Joseph disease, but also for other neurodegenerative diseases. One of these studies revealed that the silencing of the expression of the Cyp46a1 gene (using AAV5-shCyp46A1), in the hypothalamus of C57BL/6J mice fed a low fat diet (Chow) (control diet) and a high fat diet (HFD), had a profound impact on the dysregulation of the entire physiological process of body homeostasis. In this sense, additional studies became necessary to demonstrate that the effect of silencing the Cyp46a1 gene in the arcuate nucleus was specific, discarding the effect of the surgical procedure or viral vectors administration. Thus, in this study, our main objective was to control the effect of Cyp46a1 silencing on the hypothalamus (arcuate nucleus). For this, through stereotaxic surgery, a control gene (GFP) was delivered by AAV vectors in the arcuate nucleus; both in animals fed with Chow ration and those fed with HFD ration. The project's main hypothesis is that the GFP protein does not interfere in the physiology of the hypothalamus and consequently in the metabolism of the whole-body, regardless of diet. Our data confirm that stereotaxic surgery and GFP expression did not alter the homeostasis of the hypothalamus and, consequently, there was no change in the whole-body metabolism of the mice.
A epidemia do excesso de peso e obesidade representa um dos mais importantes problemas de saúde pública do século XXI. A obesidade induzida por dieta causa um processo inflamatório central no cérebro, mais especificamente no núcleo arqueado do hipotálamo. O núcleo arqueado é uma região especial do cérebro responsável pela regulação da homeostasia energética corporal, possuindo uma importância crucial na manutenção do equilíbrio entre consumo e gasto energético. Na obesidade há disfunção hipotalâmica e esta disfunção pode alterar substancialmente a homeostasia do colesterol no cérebro. O cérebro é um dos órgãos mais ricos em colesterol e a sua homeostasia do colesterol é muito importante para manter a fisiologia cerebral saudável e também influencia diretamente a homeostasia do resto do organismo. Além disso, o facto de os distúrbios metabólicos estarem correlacionados com diferentes doenças neurodegenerativas, como: doença de alzheimer, doença de Parkinson e doença de Huntington. O colesterol no cérebro é convertido principalmente em 24S-hidroxicolesterol pela enzima Cyp46a1. Alterações no metabolismo do oxisterol foram correlacionadas com a obesidade. Estudos do nosso laboratório identificaram o CYP46A1 como um alvo terapêutico relevante, não apenas para a doença Machado de Joseph, mas também para outras doenças neurodegenerativas. Um desses estudos, revelou que o silenciamento da expressão do gene Cyp46a1 (utilizando-se AAV5-shCyp46A1), no hipotálamo (núcleo arqueado), de morganhos C57BL/6J alimentados com dieta com baixo teor de gordura (Chow – low fat control diet) (dieta controlo) e com uma dieta com alto teor de gordura (HFD – high fat diet), tem impacto profundo na desregulação de todo o processo fisiológico da homeostasia corporal destes ratinhos. Tem ainda grande impacto no perfil fisiológico dos órgãos metabólicos e alterações na morfologia estrutural de cada um deles, como: WAT, BAT, fígado, pâncreas, entre outros. Desta forma, os resultados deste estudo sugerem que o silenciamento do Cyp46a1 nos animais Chow AAV5-shCyp46a1 e HFD AAV-shCyp46a1 resulta em um fenótipo de obesidade, alterações nos níveis de oxisteróis cerebrais e diabetes mellitus tipo 2; além de modificações significativas no comportamento. Os resultados sugerem o papel importante do gene Cyp46a1 na manutenção do metabolismo do colesterol no cérebro e no controlo da homeostasia energética corporal. Diante destas observações, estudos adicionais tornaram-se necessários para comprovar o efeito do silenciamento do gene Cyp46a1 no núcleo arqueado, descartando o efeito do procedimento cirúrgico. Desta forma, neste estudo, o nosso principal objetivo foi controlar o efeito do silenciamento de Cyp46a1 no hipotálamo (núcleo arqueado). Para tal, por meio de cirurgia estereotáxica foi realizada a injeção de um gene controlo (GFP) mediado por vetores AAV no núcleo arqueado; tanto em morganhos alimentados com dieta Chow, como em morganhos alimentados com dieta HFD. A hipótese do projeto é que a proteína GFP não interfere na fisiologia do hipotálamo e consequentemente no metabolismo de todo o corpo, independentemente da dieta. Este estudo foi conduzido durante um período de 12 semanas; no início do estudo os morganhos (C57BL/6J) foram divididos aleatoriamente em dois grupos: Chow (controlo) e HFD. A partir deste momento, cada grupo foi alimentado com a dieta específica por 12 semanas. Na quarta semana do estudo, os dois grupos foram submetidos a injeção estereotáxica bilateral no núcleo arqueado. Os resultados demonstraram que a proteína GFP não interferiu na fisiologia do hipotálamo (núcleo arqueado) e que as injeções estereotáxicas (AAVGFP) não causaram inflamação comprometedora que tenha alterado a fisiologia e o fenótipo alcançado através exclusivamente da dieta administrada (Chow e HFD) aos grupos dos morganhos utilizados. O fenótipo obeso foi previsivelmente alcançado pela indução de dieta gordurosa apenas nos morganhos HFD, assim como, a alteração da morfologia de vários tecidos metabólicos (WAT, BAT, pâncreas e fígado) e houve uma grande acumulação de lípidos nos órgãos. Os morganhos controlo (dieta Chow) não desenvolveram obesidade, não apresentaram alterações na morfologia dos tecidos metabólicos (WAT, BAT, pâncreas e fígado) e também não apresentaram modificações no acúmulo lipídico dos órgãos. Como este estudo é um controlo importante para o projeto de silenciamento do gene Cyp46a1 realizado anteriormente, os resultados aqui apresentados confirmam que a cirurgia estereotáxica e a expressão de GFP não alteram a homeostasia do hipotálamo e consequentemente não há alteração no metabolismo corporal dos morganhos.