京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第21002号 / 医博第4348号 / 新制||医||1027(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 大森 孝一, 教授 山田 亮, 教授 Shohab YOUSSEFIAN / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

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Indiana University-Purdue University at Indianapolis (IUPUI) / Human pluripotent stem cells (hPSCs) are an attractive tool for the study of different neurodegenerative diseases due to their potential to form any cell type of the body. Due to their versatility and self-renewal capacity, they have different applications such as disease modeling, high throughput drug screening and transplantation. Different animal models have helped answer broader questions related to the physiological functioning of various pathways and the phenotypic effects of a particular neurodegenerative disease. However, due to the lack of success recapitulating some targets identified from animal models into successful clinical trials, there is a need for a direct translational disease model. Since their advent, hPSCs have helped understand various disease effectors and underlying mechanisms using genetic engineering techniques, omics studies and reductionist approaches for the recognition of candidate molecules or pathways required to answer questions related to neurodevelopment, neurodegeneration and neuroregeneration. Due to the simplified approach that iPSC models can provide, some in vitro approaches are being developed using microphysiological systems (MPS) that could answer complex physiological questions. MPS encompass all the different in vitro systems that could help better mimic certain physiological systems that tend to not be mimicked by in vivo models. In this dissertation, efforts have been directed to disease model as well as to understand the intrinsic as well as extrinsic cues using two different MPS. First, we have used hPSCs with Alzheimer’s disease (AD)-related mutations to differentiate into retinal organoids and identify AD related phenotypes for future studies to identify retinal AD biomarkers. Using 5 month old retinal organoids from AD cell lines as well as controls, we could identify retinal AD phenotypes such as an increase in Aβ42:Aβ40 ratio along with increase in pTau:Tau. Nanostring analyses also helped in identification of potential target genes that are modulated in retinal AD that were related to synaptic dysfunction. Thus, using retinal organoids for the identification of retinal AD phenotypes could help delve deeper into the identification of future potential biomarkers in the retina of AD patients, with the potential to serve as a means for early identification and intervention for patients. The next MPS we used to serve to explore non-cell autonomous effects associated with glaucoma to explore the neurovascular unit. Previous studies have demonstrated the degeneration of RGCs in glaucoma due to a point mutation OPTN(E50K) that leads to the degeneration of RGCs both at morphological and functional levels. Thus, using the previous studies as a basis, we wanted to further unravel the impact of this mutation using the different cell types of the neurovascular unit such as endothelial cells, astrocytes and RGCs. Interestingly, we observed the barrier properties being impacted by the mutation present in both RGCs and astrocytes demonstrated through TEER, permeability and transcellular transport changes. We also identified a potential factor TGFβ2 that was observed to be overproduced by the OPTN E50K astrocytes to demonstrate similar effects with the exogenous addition of TGFβ2 on the barrier. Furthermore, the inhibition of TGFβ2 helped rescue some of the barrier dysfunction phenotypes. Thus, TGFβ2 inhibition can be used as a potential candidate that can be used to further study its impact in in vivo models and how that can be used in translational applications. Thus, MPS systems have a lot of applications that can help answer different physiologically relevant questions that are hard to approach using in vivo models and the further development of these systems to accentuate the aspects of neural development and how it goes awry in different neurodegenerative diseases.

Staphylococcus aureus est une bactérie responsable de nombreuses infections. Divers facteurs de virulence sont décrits comme ayant un rôle aggravant dans l’infection staphylococcique. La leucocidine de Panton-Valentine (LPV) en est un. Elle interagit par l’intermédiaire du récepteur de C5a (C5aR) avec les leucocytes et les cellules neuronales dans différents tissus, mais son action au niveau rétinien est méconnue. Nous avons recherché des cibles rétiniennes cellulaires de l’intoxination à la LPV et étudié ses conséquences cellulaires et inflammatoires précoces dans les tissus rétiniens. AINSI, deux modèles de lapins ont été créés : l'injection intravitréenne in vivo et les explants rétiniens ex vivo. Dans les deux modèles, les cellules ganglionnaires étaient les principales cibles cellulaires rétiniennes de la LPV et le seul type de neurones rétiniennes qui exprimait C5aR. Les cellules de Müller comme la microglie étaient activées. L’explant rétinien était facilement manipulé, ils peuvent servir à la recherche de la LPV sur la rétine. La LPV seule pourrait induire une inflammation rétinienne après avoir ciblé spécifiquement les cellules neuronales. / Staphylococcus aureus is responsible for many infections. It secretes various virulence factors aggravating the staphylococcal infections. Panton-Valentine leucocidin (PVL) is a virulent leukotoxin from S. aureus and presents active effects towards leukocytes and neuronal cells via the C5a receptor (C5aR). The effects of PVL on retina is little known. We explored PVL retinal cell target and early retinal inflammation and tried to find the processes of bacterial toxins aggravating bacterial endophthalmitis. We employed two different rabbit models to study the PVL effects on retina: intravitreal injection in vivo and retinal explant ex vivo. In the two models, retinal ganglion cells were the only retinal neurons which express C5aR and the major cell targets of PVL in retina. PVL induced retinal Müller and microglial cell activation. The retinal explants were easily manipulated and showed obvious cellular targets of PVL and glial cell activations, they can contribute to research the effects of PVL on retina in future. PVL alone without S. aureus could induce great retinal inflammation after targeting specifically retinal neurons.

A non-experimental quantitative, descriptive and contextual study which sought to examine the level of awareness about Diabetic Retinopathy (DR), and how aware female diabetic patients were about retinal screening as a preventative measure to eye complications and blindness was conducted. The objective of the study was to explore and describe the variables related to the awareness level of female diabetic patients about Diabetic Retinopathy and diabetic retinal screening. A convenient sample of 149 respondents was obtained. A questionnaire was used to collect data. Data was analysed using the Statistical Package for Social Sciences (SPSS), 13.0 computer software program. Results evidenced a good level of awareness about DR. Recommendations based on the findings were made for consideration in clinical practice, education and research. / Health Studies / M.P.H.

La dégénérescence maculaire liée à l’âge ou DMLA est une pathologie oculaire qui touche près d’un million de personnes en France, et représente la première cause de cécité légale dans les pays industrialisés. C’est une affection multifactorielle (environnement, génétique), dans laquelle les stress inflammatoires, métaboliques et oxydants interviennent et aboutissent à la mort des photorécepteurs. L’apparition des drusen (dépôts de matériel extracellulaire contenant de l’amyloïde-β (Aβ)), entre les cellules de l’épithélium pigmentaire de la rétine (EPR) et la membrane de Brush, représente un facteur de risque de développement de la DMLA. De plus, le 4-hydroxynonenal (4-HNE) est un marqueur de stress oxydant dans la rétine de patients de différentes pathologies dégénératives comme la DLMA. L’identification des mécanismes moléculaires et cellulaires impliqués dans les dégénérescences rétiniennes la pathogenèse de la DMLA constitue un enjeu de santé publique, puisqu’elle permettrait de développer de nouvelles stratégies thérapeutiques anti-dégénératives.Le but de mon travail de thèse a été dans un premier temps de mieux comprendre le rôle de l’Aβ dans la dégénérescence rétinienne.Nous avons montré que l’Aβ induit une activation rapide des cellules microgliales, une gliose soutenue des cellules gliales de Müller (CGM), un œdème dans la rétine interne et une apoptose des photorécepteurs. La dégénérescence des photorécepteurs est en corrélation avec une activation soutenue de PERK, impliquée dans la voie pro-apoptotique de la réponse UPR. Par ailleurs la gliose des CGM est caractérisé par une délocalisation des canaux Kir4.1, une diminution de l’expression d’AQP4 et de la glutamine synthetase (GS), et une augmentation de l’expression des canaux Kir2.1 et du transporteur GLAST1, suggérant une dérégulation de l’homéostasie rétinienne contrôlée par ces protéines. Nous avons montré que l’inhibition de la réponse inflammatoire, par l’utilisation de l’indomethacine, un inhibiteur non stéroïdien de de la cyclooxygénase (COX) 2, réverse l’effet de l’Aβ sur l’expression des canaux Kir4.1 et sur GLAST1 mais pas celle de la GS et d’AQP4, suggérant un couplage partiel entre la gliose et la réponse inflammatoire dans notre modèle d’injection sous-rétinienne d’Aβ.Dans un deuxième temps, nous nous sommes intéressés au rôle du 4-HNE dans les CGM, un produit de peroxydation lipidique, qui est produit dans la rétine sous l’effet de l’Aβ. Nous avons observé qu’un stress oxydant unique et létal induit par le 4-HNE, entraîne la mort des CGM par apoptose dépendante de l’activation des caspases. L’utilisation d’antioxydants impliqués dans la régénération du glutathion (GSH), protège contre la mort des CGM. L’analyse du transcriptome des CGM soumises au 4-HNE a permis de mettre en évidence une réponse transcriptionnelle adaptative des CGM : une activation de la défense anti-oxydante, de la réponse UPR (unfolded protein response) au stress du réticulum endoplasmique, et un phénotype anti-inflammatoire. Par ailleurs, la surexpression de l’APP (amyloid protein precursor), dont l’expression du transcrit est augmentée sous l’effet du stress oxydant dans les CGM, protège ces cellules contre la mort induite par le 4-HNE. Cette protection est associée à une augmentation des capacités anti-oxydantes et à une activation de la voie de survie de la réponse UPR. L’ensemble de nos résultats montre un rôle de l’Aβ dans la dégénérescence des photorécepteurs et indique que le métabolisme de l’APP, ainsi que les voies de survie et pro-apoptotique de la réponse UPR pourraient constituer des cibles thérapeutiques contre la dégénérescence rétinienne induite par l’Aβ ou les stress oxydants. / Age related macular degeneration (AMD) is a leading cause of blindness in western countries and affects one million people in France. Multiple risk factors (genetics, environment) are involved in the pathogenesis of AMD. In addition, the AMD pathogenesis is strongly associated with chronic oxidative stress and inflammation that ultimately lead to photoreceptor death. AMD is characterized by the formation of drusen, extracellular deposits, including amyloid-β (Aβ), between the retinal pigmented epithelium and Bruch’s membrane. Moreover, 4-hydroxynonenal (4-HNE) is an oxidative stress marker of different retinal diseases including AMD. The determination of molecular and cell mechanisms involved in retinal degeneration and the pathogenesis of AMD is required in order to develop new therapeutic anti-degenerative approaches. The aim of our study was first to investigate the role of Aβ in retinal degeneration. We demonstrated that subretinal injection of Aβ induces an early activation of microglial cells, a sustained retinal Müller glial (RMG) cells gliosis, an oedema in the internal part of retina and photoreceptors apoptosis. The photoreceptors apoptosis was correlated with a sustained activation of PERK, a kinase implicated in the pro-apoptotic pathway of UPR (unfolded protein response). In addition, RMG gliosis has been characterized by a Kir4.1 channel redistribution, a down-regulation of AQP4 and glutamine synthetase (GS) expression, and an up-regulation of Kir2.1 channel and GLAST1 transporter expression, suggesting a dysregulation of the retinal homeostasis which is controlled by these proteins. The inhibition of the inflammatory response using indomethacin, a non-steroidal and non-specific cyclooxygenase (COX) 2 inhibitor, reversed Aβ-induced Kir4.1 channel redistribution and GLAST1 up-regulation but not GS and AQP4 down-regulation, suggesting a partial coupling between gliosis and inflammatory response in retinal degeneration after subretinal injection of Aβ in mice. The second part of our study aimed to investigate the effects on RMG cells of 4-HNE, a lipid peroxidation product that is up-regulated in retina after Aβ injection. We have shown that a single lethal oxidative stress using 4-HNE induces RMG cells apoptosis associated with caspase 3 and caspase 9 activation. Pre-treatment of RMG cells with anti-oxidative molecules involved in glutathione regeneration restored cell viability. Transcriptome analysis of RMG cells treated with 4-HNE showed an adaptive transcriptional response consisting in an activation of anti-oxidative stress cell defense, activation of UPR in response to endoplasmic reticulum stress and anti-inflammatory phenotype. APP (amyloid protein precursor) overexpression, which the transcript is up-regulated in RMG cells under oxidative stress, protects from 4-HNE-induced cell death. This protection is associated with an up-regulation of anti-oxidative cell defense and an activation of the pro-survival pathway of UPR. Our study pinpoints the role of Aβ in photoreceptors degeneration and suggests that targeting APP metabolism, pro and anti-apoptotic pathways of the UPR response may hel develop selective methods against retinal degeneration implicating Aβ and oxidative stress.

Fundus kamera je široce dostupné zobrazovací zařízení, které umožňuje relativně rychlé a nenákladné vyšetření zadního segmentu oka – sítnice. Z těchto důvodů se mnoho výzkumných pracovišť zaměřuje právě na vývoj automatických metod diagnostiky nemocí sítnice s využitím fundus fotografií. Tato dizertační práce analyzuje současný stav vědeckého poznání v oblasti diagnostiky glaukomu s využitím fundus kamery a navrhuje novou metodiku hodnocení vrstvy nervových vláken (VNV) na sítnici pomocí texturní analýzy. Spolu s touto metodikou je navržena metoda segmentace cévního řečiště sítnice, jakožto další hodnotný příspěvek k současnému stavu řešené problematiky. Segmentace cévního řečiště rovněž slouží jako nezbytný krok předcházející analýzu VNV. Vedle toho práce publikuje novou volně dostupnou databázi snímků sítnice se zlatými standardy pro účely hodnocení automatických metod segmentace cévního řečiště.

Correct layering (lamination) of neurons in the central nervous system (CNS) is critical for the tissue functionality. Neuronal lamination is established during development, when the majority of neurons have to move from their birthplace to the appropriate layer, where they function. Therefore, to grasp the logic of CNS development, it is essential to understand the kinetics and modes of the variety of neuronal translocation events. Most of our knowledge about neuronal translocation has been gained using fixed tissue or ex vivo imaging, which is not ideal for such a dynamic process heavily dependent on the surrounding environment. To avoid these limitations, I combined translucent zebrafish embryos with light sheet fluorescence microscopy, which together enabled gentle in toto imaging of neuronal translocation. I studied the translocation of retinal ganglion cells (RGCs) across the developing zebrafish retina. RGCs are the first neurons that differentiate in the vertebrate retina and are born in a proliferative zone at the retinal apical side. From here, they move basally, spanning the complete apico-basal length of the tissue. They are destined to occupy the most basal layer, where their axons form the optic nerve. Although it was described that RGCs move their soma while being attached to both apical and basal sides of the retina, the kinetics and cell biological mechanisms of somal translocation remained unknown. Extracting single cell behavior of RGCs from high-resolution movies of their translocation allowed for quantitative analysis of RGC movement. I revealed that RGCs cross the retina in less than two hours in a directionally persistent manner. The movement of RGC soma is a cell autonomously generated process, which requires intact microtubules and actin-dependent basal attachment of cells for speed and efficiency. Unexpectedly, interference with somal translocation leads to a shift towards a multipolar migratory mode, previously not observed for RGCs, in which they temporarily lose both apical and basal attachment and apico-basal polarity. The multipolar mode is overall slower and less directionally persistent, but still allows RGCs to reach the basal retina. However, when RGC translocation is inhibited completely, they differentiate ectopically in the center of the retina, which in turn triggers the formation of ectopic layers of later born neurons. These results highlight the importance of establishing the basal layer of ganglion cells for ensuing retinal lamination. Overall, I generated important advances in the understanding of neuronal translocation and lamination, which might be relevant for other parts of the CNS.

Rhodopsin, der Rezeptor der visuellen Kaskade, gehört zu größten Klasse A der G-Protein-koppelnden Rezeptoren (GPCRs) und gilt als Modell-Rezeptor in der GPCR-Forschung. Über 3 % des humanen Genoms kodieren für GPCRs, doch trotz der physiologischen Bedeutung dieser Proteinfamilie sind die fundamentalen Mechanismen, mit denen diese Rezeptoren extrazelluläre Signale in das Zellinnere weiterleiten noch nicht verstanden. In der vorliegenden Dissertation werden Aspekte des Aktivierungsmechanismus von Rhodopsin sowie der Kopplung und Aktivierung des G-Proteins Transduzin untersucht. Die Arbeit ist in drei Schwerpunkte unterteilt: I. Es wurde ein in GPCR’s hochkonserviertes NPxxYx(5,6)F Motiv (Aminosäuresequenz Asn-Pro-x-x-Tyr-x(5,6)-Phe) in der siebten und achten Helix charakterisiert. In diesem konservierten Motiv sind mehrere für die Ausbildung der aktiven Rezeptorkonformation wichtige Funktionen vereint: Verknüpfung zu einem Wasserstoffbrückennetzwerk, Helixflexibilität sowie die exakte Positionierung der achten Helix. Letzteres hat nicht nur bei der Rezeptoraktivierung sondern auch bei der nachfolgenden Interaktion mit dem G-Protein eine Bedeutung. II. Anhand von chimären Rezeptoren, bei denen Teile der achten Helix durch homologe Sequenzen des beta2-adrenergen Rezeptors ausgetauscht wurden, wurde die Rolle der achten Helix bei der Rezeptor-Aktivierung und Bindung des G-Proteins untersucht. Auch bei dieser Studie wurde gezeigt, dass die exakte Positionierung der achten Helix essentiell für die Interaktion mit dem G-Protein ist. Zudem wurde ein bezüglich der G-Protein-Aktivierung funktionsfähiger chimärer Rezeptor gefunden, was auf einen übergeordneten Mechanismus bei der Aktivierung von G-Proteinen durch GPCRs hindeutet. III. Die Funktion des ß-Ionon-Rings des Retinals beim Aktivierungsmechanismus von Rhodopsin wurde an einem Retinal studiert, bei welchem Teile des Retinal-Rings fehlten (azyklisches Retinal). Auch diesem azyklischen Retinal können Eigenschaften eines partiellen Agonisten zugeschrieben werden. Beim Vergleich zu Pigmenten mit dem nativen 11-cis-Retinal wurden starke Analogien bei der initialen Energieaufnahme durch die Retinal-Isomerisierung sowie bei der Weiterleitung der Lichtenergie ins Protein gefunden. Allerdings wird die Energie schlechter auf das Protein übertragen, wodurch wesentlich weniger der aktiven G-Protein bindenden Rezeptorkonformation gebildet wird. Als wichtigste Funktion des Retinal-Rings wurde die Aufrechterhaltung der aktiven Meta-II-Konformation identifiziert. / Rhodopsin, the receptor of the visual cascade, belongs to the largest group A of G-protein coupled receptors (GPCRs) and can be seen as a model receptor in GPCR research. More than 3 % of the human genome code for GPCRs. But despite their physiological relevance, the detailed mechanism of signal transduction from extra cellular signal to different cellular pathways remains to be fully understood. Different aspects of receptor activation and the coupling and activation of the G-protein transducin are investigated in this dissertation. The thesis focuses on the following three subjects: I. A NPxxYx(5,6)F motif (amino acid sequence Asn-Pro-x-x-Tyr-x(5,6)-Phe) has been characterized for rhodopsin. It is localized in helix VII and VIII and is highly conserved throughout the GPCR family. Various roles for rhodopsin activation are combined in this motif: linkage to a hydrogen-bond network, helix flexibility and the exact positioning of helix VIII. The latter is not only relevant for the activation of the receptor but also for interaction with its G-protein. II. The role of helix VIII for receptor activation and G-protein coupling was studied on chimeric receptors, in which parts of helix VIII were exchanged against homologous sequences of the beta2 adrenergic receptor. This study confirmed the importance of helix VIII’s position for G-protein coupling. Furthermore, a chimeric receptor was found, which was fully functional concerning G-protein activation. This indicates that GPCRs might use a single, generic mechanism for G-protein activation. III. The role of the ß-ionone-ring for the activation mechanism of rhodopsin was studied by means of an acyclic retinal, which lacks four carbon atoms of the ß-ionone-ring. This modified retinal could be classified as a partial agonist for rhodopsin. Energy input by retinal isomerization and formation of the G-protein binding Meta-II conformation were found to be very similar to rhodopsin when bound to its native 11-cis-retinal. However, the lack of the ring structure resulted in a lower amount of Meta-II and a fast decay of activity. It was concluded that the main role of the ring structure is to maintain the active state of rhodopsin.