京都大学 / 0048 / 新制・課程博士 / 博士(農学) / 甲第22506号 / 農博第2410号 / 新制||農||1078(附属図書館) / 学位論文||R2||N5286(農学部図書室) / 京都大学大学院農学研究科応用生命科学専攻 / (主査)教授 木岡 紀幸, 教授 小川 順, 教授 矢﨑 一史 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第18145号 / 医博第3865号 / 新制||医||1002(附属図書館) / 31003 / 京都大学大学院医学研究科医学専攻 / (主査)教授 萩原 正敏, 教授 柳田 素子, 教授 稲垣 暢也 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

京都大学 / 新制・課程博士 / 博士(農学) / 甲第25331号 / 農博第2597号 / 新制||農||1105(附属図書館) / 京都大学大学院農学研究科応用生命科学専攻 / (主査)教授 木岡 紀幸, 教授 矢﨑 一史, 教授 三芳 秀人 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DGAM

Mémoire numérisé par la Division de la gestion de documents et des archives de l'Université de Montréal.

Der ABC-Transporter ABCA1 ist unmittelbar in die zelluläre Lipidhomeostasie einbezogen, in dem er die Freisetzung von Cholesterol an plasmatische Rezeptoren, wie ApoA-I, vermittelt. Trotz intensiver Untersuchungen ist dieser molekulare Mechanismus nicht verstanden. Verschiedene Studien deuten daraufhin, dass durch die Aktivität von ABCA1 bedingte Veränderungen in der Lipidphase der äußeren Hälfte der Plasmamembran (PM) wichtig für die Freisetzung des Cholesterols sind. In der vorliegenden Arbeit wird die Lipidumgebung von ABCA1 in der PM lebender Säugetierzellen unter Anwendung der Fluoreszenzlebenszeitmikroskopie von fluoreszierenden Lipidsonden untersucht. Es wurde eine breite Verteilung der Fluoreszenzlebenszeiten der Sonden gefunden, die sensitiv gegenüber Veränderungen der lateralen und transversalen Organisation der Lipide ist. Im Einklang mit Studien an riesengroßen unilamellaren Vesikeln und Plasmamembranvesikeln weisen unsere Ergebnisse die Existenz einer größeren Vielfalt submikroskopischer Lipiddomänen auf. Die FLIM-Untersuchungen an ABCA1 exprimierenden HeLa-Zellen weisen eine die Lipidphase destabilisierende Funktion des Transportes aus. Dieses wurde unterstützt durch die Lipidanalyse von Fraktionen der PM. Auf der Basis unserer Untersuchungen und früheren Daten stellen wir die Hypothese auf, dass die Exponierung von Phosphatidylserin (PS) auf der Zelloberfläche ein zentrales Ereignis der ABCA1 bedingten Veränderungen ist. Allerdings zeigen vergleichende Studien an ABCA7 exprimierenden Zellen, dass dies nicht ausreicht, um die ABCA1 verursachten Veränderungen in der Lipidpackung der PM zu erklären. Unsere Ergebnisse beweisen, dass die Fähigkeit von ABCA1, den Cholesterolefflux zu vermitteln, auf durch den Transporter bedingte Veränderungen in der LP der PM zurückzuführen sind, die unabhängig von der Bindung von ApoA-1 sind und dieser vorausgehen. Diese Veränderungen sind notwendig für die Lipidierung von ApoA-1 und der Generierung von HDL-Partikeln. / The ABCA1 transporter organizes cellular lipid homeostasis by promoting the release of cholesterol to plasmatic acceptors such as ApoA-I. Despite intensive investigation, the molecular mechanism of such a process has not yet been clarified. In the present study we report on the analysis of the ABCA1 lipid microenvironment at the plasma membrane of living cells, by a novel approach based on fluorescence lifetime imaging microscopy (FLIM). In the plasma membrane of mammalian cells, a broad fluorescence lifetime distribution sensitive to treatments interfering with the membrane lateral and transbilayer organization was found. In agreement with investigations in giant unilamellar vesicles and giant plasma membrane vesicles, our results are consistent with the existence of a large variety of submicroscopic lipid domains. Based on that, FLIM in HeLa cells expressing ABCA1 revealed the destabilizing function of the transporter on the lipid arrangement at the membrane, indicating that lipid packing was a primary target of ABCA1 activity. This was corroborated by the analysis of plasma membrane fractions isolated by density fractionation. On the basis of our analysis and previous data, we speculate that the exposure of phosphatidylserine on the cell surface is a central event for ABCA1-dependent modifications. However, a comparative study of cells expressing ABCA7, the member of the ABCA subfamily with the highest homology to ABCA1, revealed that exposure of PS alone cannot account for the detected effects. Collectively, our data suggest that the ability of ABCA1 to promote cholesterol efflux is independent and precedes its actual binding to ApoA-I. Rather, ABCA1-induced plasma membrane modifications are necessary for the lipidation of ApoA-I and the generation of high density lipoprotein particles.

Mémoire numérisé par la Division de la gestion de documents et des archives de l'Université de Montréal

Thèse numérisée par la Direction des bibliothèques de l'Université de Montréal.

L’épididyme est un organe accolé au testicule, formé d’un long tubule dont l’épithélium est constitué de 6 types cellulaires différents aux fonctions partiellement connues. Le tubule épididymaire est entouré de muscles lisses participant, par leurs contractions, à l’avancée des spermatozoïdes. Au centre du canal se retrouve le fluide épididymaire dont la composition est régulée par les absorptions et sécrétions de l’épithélium épididymaire. Les gamètes arrivant du testicule sont inaptes à assurer la fécondation. La maturation post-testiculaire des spermatozoïdes, durant leur descente dans le tubule épididymaire, va leur permettre d’acquérir la mobilité et la capacité à reconnaître et féconder un ovule (qualités regroupées sous le terme de “pouvoir fécondant”). Cette maturation se fait grâce aux interactions permanentes des spermatozoïdes avec le fluide. Les LXRs sont des régulateurs majeurs de l’homéostasie du cholestérol, un élément fondamental dans la physiologie de la reproduction puisqu’il est le précurseur des hormones stéroïdes (androgènes) et un régulateur du pouvoir fécondant des spermatozoïdes. Les souris mâles invalidées pour les 2 isoformes de ces récepteurs (lxrα;β-/-) ont une fertilité perturbée en association avec des altérations testiculaires et épididymaires. Ces mâles présentent une infertilité dès 6 mois conduisant à une stérilité complète à 9 mois. Le but de ce travail est de comprendre la relation entre la régulation de l’homéostasie du cholestérol et le phénotype épididymaire de ces souris. A l’âge de 4 mois, alors que les souris mâles lxrα;β-/- sont fertiles, le phénotype est caractérisé par une accumulation de lipides dans certains types cellulaires de l’épithélium et dans les muscles lisses entourant le tubule. Chez les mâles lxrα;β-/- âgés de 9 mois, lorsque le phénotype d’infertilité complète est installé, on observe une destructuration de la partie proximale de l’épididyme comprenant une diminution de la hauteur de l’épithélium, une perte des muscles lisses péritubulaires et la présence de spermatozoïdes non mobiles et malformés dans la lumière du tubule. Mes travaux ont permis de mettre en évidence différents phénomènes : 1/ Une nouvelle fonction d’un type cellulaire particulier, les cellules “apicales”, dans la régulation de l’homéostasie du cholestérol épididymaire. En effet, ces cellules accumulent du cholestérol en association avec la perte de “ABCA1” (ATP Binding Cassette A1), codée par un gène cible de LXR, impliquée dans l’export de l’excédent de cholestérol cellulaire. Suite à cela, ces cellules apicales entrent en apoptose et contribuent à la perte de fonction de l’épithélium. 2/ Une altération des cellules musculaires lisses péritubulaires associée au vieillissement des souris lxrα;β-/-. Ces cellules accumulent elles aussi du cholestérol et se transforment en cellules invasives, les cellules “spumeuses” migrant à travers l’épithélium. Ce phénomène est très proche des phases de développement de la pathologie artérielle liée au cholestérol et au vieillissement, l’athérosclérose. La perte des muscles lisses va limiter la progression des spermatozoïdes et accompagner la perte de fonction des cellules épithéliales qui ne seront plus aptes à effectuer des échanges avec le fluide, empêchant ainsi l’acquisition du pouvoir fécondant des spermatozoïdes. (...) / The epididymis is a long tubule which epithelium is made of 6 different cellular types with partially unknown functions. The epididymal tubule is surrounded by smooth muscles participating, by their contractions, to the sperm progression. At the center of the tubule is present the epididymal fluid which composition is regulated by the absorptions and secretions of the epithelium. The spermatozoa coming from the testis are unable to fertilize an oocyte. Post-testicular maturation of spermatozoa, during their transit through the epididymis, allows them to become motile, to recognize and to fertilize an oocyte. This maturation is due to the constant interaction of the spermatozoon with the epididymal fluid. LXR are the major sensors of cholesterol homeostasis, a fundamental element in reproductive physiology because it is the steroid hormones precursor and a regulator of the sperm fertilizing capacities. LXR deficient mice (lxrα;β-/-) have fertility disorder associated with testicular and epididymal alterations. These males present an infertility since 6 months of age leading to a complete infertility at 9 months of age. The purpose of this work is to understand the relation between cholesterol homeostasis regulation and epididymal sperm maturation. At 4 months of age, the lxrα;β-/- males are fertile but present an epididymal phenotype characterized by neutral lipid accumulations in some epithelial cell types and in the smooth muscle cells surrounding the tubule. At 9 months of age, the totally infertile lxrα;β-/- male, present a complete destructuration of the proximal epididymis associated with a complete shrinking of the epithelium height, the loss of the peritubular smooth muscle cells and the presence of immotile and abnormal spermatozoa in the cauda epididymis. My works put in light : 1/ A novel function of a particular cell type, the “apical cells”, in the regulation of the epididymal cholesterol homeostasis. These cells accumulate cholesteryl esters in association with the loss of the ATP Binding Cassette A1 “ABCA1”, a transporter implicated in the cholesterol efflux. Following this, apical cells become apoptotic participating to the epithelial function loss. 2/ An alteration of peritubular smooth muscle cells associated with the mice aging. These cells also accumulate cholesteryl esters and transdifferentiate into invasive foam cells migrating through the epithelium. This phenomenon is very similar to what is observed in the arterial pathology related to cholesterol and aging, the atherosclerosis. The loss of smooth muscle cells limit the progression of the spermatozoa in the lumen and lead to the loss of function of the epithelial cells which become unable to make any exchange with the fluid, hence disturbing spermatozoa maturation. 3/ A deleterious effect of a cholesterol enriched diet on the fertility in 3-month-old lxrα;β-/- male, which are normally fertile and without epididymal phenotype. When submitted to the diet during 4 weeks, these mice become completely infertile, showing the epididymal phenotype of the 9-month-old lxrα;β-/- male mice. The infertility of these mice is characterized by defects in spermatozoa motility, viability and functional parameters. Our results show, on the one hand, the importance of LXR in the post testicular maturation of spermatozoa and in the maintenance of the integrity and functionality of the epididymis. On the other hand, the data that we collected put in evidence the impact of the alimentation and in particular cholesterol enriched diet on the post-testicular maturation.

Formation of lipid-laden macrophage foam cells and inflammation are the central components in the initiation and progression of atherosclerosis. ABCA1 is well established as an anti-atherogenic factor that facilitates cellular cholesterol and phospholipid efflux, promotes reverse cholesterol transport, and suppresses pro-inflammatory cytokine secretion. Through these functions, ABCA1 is capable of reducing the lipid burden in atherosclerotic plaque. PKA signaling is an integral factor in promoting many anti-atherogenic functions of ABCA1; however, mechanistic aspects of PKA signaling associated with ABCA1 remain poorly defined. Thus, the first part of this study investigates the involvement of spatially regulated PKA signaling in ABCA1 activities through the use of st-Ht31, a PKA de-anchoring peptide. It appears that de-anchoring PKA robustly increases ABCA1-mediated microparticle release, one of the cholesterol efflux pathways of ABCA1, and reverses macrophage foam cell formation. These results highlight the significance of subcellular compartmentalization of PKA signaling in ABCA1 functions and present PKA de-anchoring as a potential therapeutic strategy for atherosclerotic lesion regression. The second part of this study provides evidence that ABCA1 activates PKA and promotes the secretion of anti-inflammatory IL-10, a cytokine crucial for inflammation resolution. Furthermore, we provide evidence that this elevated PKA activity is the underlying mechanism in which macrophage ABCA1 promotes M2-like inflammatory response. Our results also suggest that ABCA1 activates PKA by regulating cholesterol, which poises macrophages towards an anti-inflammatory or M2-activated phenotype. Collectively, we demonstrate that PKA signaling plays a crucial multifactorial role in anti-atherogenic functions of ABCA1.

Formation of lipid-laden macrophage foam cells and inflammation are the central components in the initiation and progression of atherosclerosis. ABCA1 is well established as an anti-atherogenic factor that facilitates cellular cholesterol and phospholipid efflux, promotes reverse cholesterol transport, and suppresses pro-inflammatory cytokine secretion. Through these functions, ABCA1 is capable of reducing the lipid burden in atherosclerotic plaque. PKA signaling is an integral factor in promoting many anti-atherogenic functions of ABCA1; however, mechanistic aspects of PKA signaling associated with ABCA1 remain poorly defined. Thus, the first part of this study investigates the involvement of spatially regulated PKA signaling in ABCA1 activities through the use of st-Ht31, a PKA de-anchoring peptide. It appears that de-anchoring PKA robustly increases ABCA1-mediated microparticle release, one of the cholesterol efflux pathways of ABCA1, and reverses macrophage foam cell formation. These results highlight the significance of subcellular compartmentalization of PKA signaling in ABCA1 functions and present PKA de-anchoring as a potential therapeutic strategy for atherosclerotic lesion regression. The second part of this study provides evidence that ABCA1 activates PKA and promotes the secretion of anti-inflammatory IL-10, a cytokine crucial for inflammation resolution. Furthermore, we provide evidence that this elevated PKA activity is the underlying mechanism in which macrophage ABCA1 promotes M2-like inflammatory response. Our results also suggest that ABCA1 activates PKA by regulating cholesterol, which poises macrophages towards an anti-inflammatory or M2-activated phenotype. Collectively, we demonstrate that PKA signaling plays a crucial multifactorial role in anti-atherogenic functions of ABCA1.