Op-power in diabetic retinopathy

The present study is mainly concerned with the role of the OPs in diagnosing early diabetic retinopathy. In 1966 Simonsen had already found a clear relation between background diabetic retinopathy and the disappearance of OPs at the ascending limb of the b-wave of the ERG. Many investigators have studied this relationship ever since, but because of a deficiency in a quantification measure of the OPs, the clinical application has been unsatisfactory. Recent developments involving digitalising of the ERG signal and the accompanying related increase in signal processing techniques, nowadays offer us the opportunity of developing a reliable system of OP quantification. Diabetic retinopathy is characterized by multiple vascular lesions of the eye fundus. The clinical course is quite variable, but one or another feature may predominate the fundus picture at a given time. The knowledge of the pathogenesis in diabetic retinopathy remains incomplete as yet; as does the precise cell localisation of OPs and their relation to diabetic retinopathy. As long as this knowledge is incomplete a classification and measurement system of the retinal function in this disease will be defective

Pigmentation and regeneration of the zebrafish retinal pigment epithelium

The Retinal Pigment Epithelium (RPE) is a specialized monolayer of pigmented cells in the back of the eye that forms a functional unit with photoreceptor cells in the retina and is critical for photoreceptor function and eye development. As a flat sheet of progenitor cells develops, a series of complex morphogenetic changes occur which require coordinated changes in cellular morphology, cell cycle and motility. To aid the study of these processes, I have generated ten novel GAL4-inducible zebrafish transgenics that enable the tissue-specific modulation of Rho GTPase activity and demonstrated the utility of these lines in studying eye development. Diseases disrupting the normal pigmentation of the RPE, known as albinism, cause defects in retinal development and vision. Although many different alleles of albinism have been identified, it is likely that undiscovered loci responsible for causing albinism exist. To learn more about the genetic underpinnings of albinism, I have characterized two novel zebrafish albino mutants and found they result from mutations in the gene encoding N ethyl maleimide-sensitive factor B (nsfb) and have established a role for nsfb in the maturation of pigment in zebrafish RPE. Finally, diseases affecting the adult RPE have dire consequences for vision. Geographic Age-related Macular Degeneration (AMD) is the third leading cause of blindness worldwide, and occurs when atrophy of the RPE causes irreversible death of underlying photoreceptors. Despite advances in stem-cell based RPE replacement therapies, very little is known about the process by which RPE cells can successfully regenerate and integrate into damaged retinal tissue. To study this process, I established a novel zebrafish model of AMD whereby specific ablation of the RPE leads to rapid degeneration of underlying photoreceptors. Using this model, I demonstrated for the first time that the zebrafish RPE is capable of regenerating after widespread damage and provide evidence that RPE ablation provokes a robust proliferative response during which cells from the periphery move into the injury site and contribute to regeneration, and that these cells likely derive from unablated RPE. This model provides a platform for supporting the development of AMD therapies.Cellular and Molecular Biolog

Human embryonic stem cells for retinal repair : preclinical in vitro and in vivo studies for the treatment of age-related macular degeneration with human embryonic stem cell-derived retinal pigment epithelial cells

Age-related macular degeneration (AMD) is the major cause of vision loss in the industrialized countries in people above sixty years of age. The dry advanced form of the disease, also termed as geographic atrophy (GA), is characterized by the progressive death of retinal pigment epithelial cells (RPE) and consequent loss of the adjacent photoreceptor (PR) layer, leading to an impaired visual function. Since AMD has a multifactorial cause, including both genetic and epigenetic factors, a potential treatment for retinal regeneration relies on the generation of either autologous or allogeneic RPE and PR cells from human pluripotent stem cells (hPSC) in vitro. The overall aim of this thesis was to develop both in vitro and in vivo methods and models to move forward a stem-cell based replacement therapy for patients suffering from dry advanced forms of AMD. Specifically, we first developed a spontaneous, xeno-free and defined protocol to derive RPE from human embryonic stem cells (hESC-RPE) that acquired specific morphological and functional characteristics of native RPE. Additionally, we developed a large-eyed model (rabbit eye) with relevant pre-clinical imaging and surgical advantages when compared to other more commonly used rodent models. In fact, both the subretinal injections of PBS or the chemical NaIO3 created a retinal degeneration phenotype very similar to the lesion present in GA patients with RPE damage and PR loss. A next logical step was to evaluate the behavior of the hESC-RPE in such models of degeneration. From these studies, we first showed that hESC-RPE can rescue the neuroretina from further damage induced at the moment of subretinal injection, and second, that hESC-RPE are not able to integrate in areas of profound retinal degeneration caused by a 7-day pre-injection of either PBS or NaIO3, therefore supporting the idea of an early treatment. The use of allogeneic hESC as a transplantable source comes together with the forthcoming rejection of the donor cells. We then sought to create universal cells that lack HLA-I (hESC-RPEB2M-/- using CRISPR-Cas9 technology) able to evade the host adaptive immune system. Upon co-culture with T-cells under stimulatory conditions, the engineered hESC-RPEB2M-/- dampened CD8+ T-cell proliferation and when mixed with natural killer (NK) cells, a cytotoxic response was triggered. Furthermore, after transplantation of the hESC-RPEB2M-/- in the rabbit xenogeneic model, early stage rejection was reduced and the appearance of anti-human antibodies rejection associated with late rejection was delayed. Altogether, the studies described in this thesis show evidence that allogeneic replacement therapy using subretinal injection of hESC-RPE in suspension can be a successful treatment if (i) the derived cells retain native RPE cell properties; (ii) the cells are transplanted early enough so the subretinal milieu supports their integration; and (iii) the cells can be engineered so that they can evade the host immune system and consequent graft rejection

Caractérisation structurale et de liaison membranaire de rétinol déshydrogénases

Les rétinol déshydrogénases ou RDHs sont des oxydoréductases inhérentes à l’accomplissement de la fonction visuelle de la rétine. Elles sont en effet impliquées dans le cycle visuel rétinien. Suite à l’absorption de la lumière par le pigment visuel des photorécepteurs, la rhodopsine, la RDH8 est la première enzyme qui va intervenir dans le cycle visuel après la libération du chromophore de la rhodopsine, le tout-trans rétinal. Ainsi, la RDH8 détoxifie les photorécepteurs car le tout-trans rétinal est une espèce très réactive qui peut induire des dommages à la rétine. La RDH11, quant à elle, agit de concert avec la RDH5 au niveau de la dernière étape du cycle visuel dans l’épithélium pigmentaire rétinien en transformant le 11-cis rétinol en 11-cis rétinal, qui sera réacheminé vers les photorécepteurs pour régénérer le pigment visuel. Toutefois, la structure tertiaire des RDHs n’a encore jamais été résolue. Ces enzymes sont néanmoins reconnues pour être associées aux membranes cellulaires par leur segment N- et/ou C-terminal. Nous avons alors entrepris ce travail afin de caractériser la structure de ces enzymes et mieux comprendre leur interaction avec les membranes. Nous avons étudié dans un premier temps différentes portions du segment N- et C-terminal de la RDH11 et la RDH8 respectivement, par différentes méthodes spectroscopiques. Nous avons alors observé que les segments de ces deux enzymes agissent par deux modes d’action totalement différents. La RDH11 ferait appel à un segment N-terminal transmembranaire qui adopte une conformation hélicale peu importe sa longueur, alors que la RDH8 utiliserait un segment C-terminal qui adopte une structure secondaire variable selon la longueur et dont la liaison est périphérique à la membrane. En plus, la liaison de la RDH8 par son segment C-terminal serait potentiellement facilitée par une ou plusieurs acylations situées au niveau de certaines cystéines. Les mesures de pression d’insertion maximale ont permis de comparer les interactions entre des segments de longueur variable en N-terminal de la RDH11 et en C-terminal de la RDH8 avec des monocouches de différents phospholipides. Ainsi, nous avons déterminé les interactions les plus favorables pour chacun de ces segments. Nous nous sommes focalisés par la suite sur l’étude de l’enzyme RDH8 et la comparaison de ses propriétés structurales, de stabilité et de liaison membranaire avec celles de sa forme tronquée RDH8t, dépourvue de son segment en C-terminal. Notons que nous avons mis au point un protocole adapté pour surexprimer et purifier la RDH8 et sa forme tronquée. À notre connaissance, il s’agit ici des premiers travaux de recherche rapportant la surexpression et la purification d’une RDH8 (bovine) complète dans un système procaryote (E. coli). Nous avons alors constaté que les deux formes de la RDH8, complète et tronquée, comprenaient majoritairement des hélices α en plus de la présence de feuillets β, en accord avec le motif de Rossmann fold suggéré dans la littérature pour cette famille d’enzymes. Il s’est avéré également que le segment C-terminal a un impact sur la stabilité de la RDH8 comme démontré par les mesures du contenu en structure secondaire de ces protéines en fonction des conditions de stockage et dans les expérimentations de dénaturation thermique. Enfin, les mesures de pression d’insertion maximale (PIM) et de synergie ont démontré que le segment C-terminal facilitait la liaison membranaire de la forme complète par rapport à la forme tronquée, notamment dans le contexte de phospholipides portant une tête polaire chargée négativement. L’interaction membranaire de la RDH8 pourrait donc impliquer des interactions électrostatiques. Des expériences de spectroscopie de fluorescence ont permis de confirmer l’implication du segment C-terminal dans la liaison de la RDH8 avec des bicouches lipidiques grâce à la présence de deux résidus tryptophanes uniquement dans son segment C-terminal.In the retina, retinol dehydrogenases (RDHs) play a crucial role in the visual cycle allowing a good vision. The first step of the visual cycle is taking place in photoreceptors where RDH8 is located and then in the retinal pigmented epithelium (RPE) where RDH11 can be found. RDH11 is likely anchored to membranes by means of its N-terminal segment whereas RDH8 has been postulated to be membrane bound via its C-terminal segment. So, to better evaluate the role of the N-terminal segment of RDH11 and the C-terminal segment of RDH8 in the membrane binding of these proteins, different variants (Long and Short) of the aforementioned segments have been studied. In addition, mutations of the C-terminal segment of RDH8 have been introduced to monitor their interaction with lipid monolayers or bilayers. We have thus analyzed the secondary structure content of these segments by conventional spectroscopic methods such as circular dichroism (CD) and attenuated total reflectance (ATR) infrared spectroscopy whereas their interaction with phospholipids have been mainly monitored by surface pressure measurements when using monolayers and fluorescence spectroscopy for bilayers. Overall, we found that the N-terminal segment of RDH11 adopts an α-helix conformation acting as a transmembrane domain. Values of maximum insertion pressure (MIP) and synergy suggested a preferential binding of the RDH11 Long-peptide to phosphoethanolamine, which are abundant in the RPE. In the case of RDH8, our findings suggest an important role of the long C-terminal segment in membrane binding, which is supported by its helical content and the larger values of MIP and synergy. We also compared the behavior of RDH8 and its truncated form (RDH8t, without its C-terminal segment) to better understand the involvement of this segment in membrane binding. Thus, both enzymes have been expressed in E. coli, purified by affinity chromatography and studied by the spectroscopic methods mentioned above and by using MIP and synergy measurements. RDH8 and RDH8t display a secondary structure content in agreement with their predicted Rossmann fold. Interestingly, the removal of the C-terminal segment decreased the temporal and thermal stability of these enzymes. In addition, this segment contributes to protein-lipid interaction especially in presence of negatively charged phospholipids likely through electrostatic interactions. The involvement of the C-terminal segment of the RDH8 in its membrane anchoring has been further confirmed by fluorescence measurements of its two Trp residues located in this segment. The present characterization of RDH8 is a first step paving the way for the elucidation of its structural and functional features to gain a better understanding of its role within the visual cycle and investigating mechanisms of retinal pathogenesis

Towards experimental therapies for retinal degenerative diseases

This thesis describes part of the preclinical road that is essential in developing experimental therapies for retinal degenerative diseases such as age-related macular degeneration (AMD) and a specific type of retinitis pigmentosa (RPE-RP). The retinal pigment epithelium (RPE) plays a significant role in the pathology of both diseases. Indeed, patients of all ages can be affected by conditions involving (primarily) the RPE. This thesis is focused on RPE disease pathology, illustrated by the complex retinal disease AMD and a specific genetic form of the monogenic disorder RP. Many experimental therapeutic strategies are being developed to treat AMD and RPE-RP; however, gene therapy and cell-replacement therapy can be considered important strategies for these diseases, especially because of the curative nature of these two treatment modalities. In this thesis, we first used a systematic approach to identify and analyze all preclinical studies that have been published regarding RPE cell-replacement strategy to treat retinal degenerative diseases (Chapter 2). We next used a genome-editing technique to create a new animal model for an RPE-RP subtype and characterized the model in-depth (Chapter 3). Additionally, we describe an induced preclinical model for AMD and its in-depth characterization (Chapter 4). As a final step, we describe the generation of a 3D-bio-printed tissue recapitulating the RPE and underlying tissues and its transplantation and integration into rat eyes (Chapter 5)

Electrophysiological investigation of age-related macular degeneration

Age-related macular degeneration (AMD) affects 12.7 million people in Europe and North America (Klein et al., 1995 Klein et al. 1999). As a combination of decreasing birth rate and increasing longevity alter the demographic of the population, the impact of this disease can only increase. This places an immense burden, not only on the individuals afflicted by the condition, but on the financial resources of society as a whole. Unfortunately, treatment for AMD is still very restricted, and even our understanding of the pathogenesis of the disease is far from complete One concern in tackling the growing problem of AMD is that methods used in the assessment of the condition are limited, usually based on fundus appearance and visual acuity. The aim of this study was to develop a battery of electrophysiological tests which would be sensitive to the most subtle changes in retinal function in AMD. Such tests may aid diagnosis, provide a more sensitive measure of disease progression, and allow an early identification of phenotypic subtypes. Protocols were included for the recording of the focal rod ERG, the focal cone ERG, the S-cone ERG and the dynamic focal cone ERG, along with psychophysical tests of colour vision and dark adaptation. These tests were then applied to 31 subjects with ARM (12 with bilateral ARM, 11 with unilateral wet AMD and 8 with unilateral dry AMD), and 28 controls. In the analysis of ERG amplitudes a ratio of focal to full-field amplitude was introduced as a novel means of reducing intersubject variability in response. This was found to increase the accuracy of all tests in distinguishing between subject groups. The greatest separation between ARM and control groups was provided by the dynamic tests of visual function i.e. rod-cone break time of the dark adaptation function, and time constant of recovery of the dynamic focal cone ERG. The time to rod-cone break also showed potential in identifying subjects at increased risk of exudative retinal changes. Subjects were assigned to groups in this study on the basis of fundus appearance. However, individuals within each subject group showed a range of retinal function which belied the homogeneity of retinal signs. This raises the question of whether 'form' or 'function' should form the basis of classification and assessment of individuals with ARM and AMD.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Electrophysiological investigation of age-related macular degeneration

Age-related macular degeneration (AMD) affects 12.7 million people in Europe and North America (Klein et al., 1995 Klein et al. 1999). As a combination of decreasing birth rate and increasing longevity alter the demographic of the population, the impact of this disease can only increase. This places an immense burden, not only on the individuals afflicted by the condition, but on the financial resources of society as a whole. Unfortunately, treatment for AMD is still very restricted, and even our understanding of the pathogenesis of the disease is far from complete One concern in tackling the growing problem of AMD is that methods used in the assessment of the condition are limited, usually based on fundus appearance and visual acuity. The aim of this study was to develop a battery of electrophysiological tests which would be sensitive to the most subtle changes in retinal function in AMD. Such tests may aid diagnosis, provide a more sensitive measure of disease progression, and allow an early identification of phenotypic subtypes. Protocols were included for the recording of the focal rod ERG, the focal cone ERG, the S-cone ERG and the dynamic focal cone ERG, along with psychophysical tests of colour vision and dark adaptation. These tests were then applied to 31 subjects with ARM (12 with bilateral ARM, 11 with unilateral wet AMD and 8 with unilateral dry AMD), and 28 controls. In the analysis of ERG amplitudes a ratio of focal to full-field amplitude was introduced as a novel means of reducing intersubject variability in response. This was found to increase the accuracy of all tests in distinguishing between subject groups. The greatest separation between ARM and control groups was provided by the dynamic tests of visual function i.e. rod-cone break time of the dark adaptation function, and time constant of recovery of the dynamic focal cone ERG. The time to rod-cone break also showed potential in identifying subjects at increased risk of exudative retinal changes. Subjects were assigned to groups in this study on the basis of fundus appearance. However, individuals within each subject group showed a range of retinal function which belied the homogeneity of retinal signs. This raises the question of whether 'form' or 'function' should form the basis of classification and assessment of individuals with ARM and AMD