Ocular Phenotyping in the harlequin Mouse Model of Retinal Degeneration: A Framework for Therapeutic Testing

Retinal degeneration, despite devastating effects, lacks therapy. Memantine has potential for preserving vision by reducing excitotoxicity associated with reactive oxygen species (ROS). A model for memantine delivery is the oxidative stress- and retinal degeneration in harlequin (hq) mice. Wild type (WT) and hq mice received untreated or memantine-treated (30 mg/kg/ day) drinking water at 1 out to 2,4,6, 8 and 10 months (mo) of age (5 males per cohort). Retinal integrity was assessed using electroretinography and ocular coherence tomography with ROS levels and apoptosis examined postmortem. Reduced hq vision was evident at 2 mo with a slight elevation in ROS at 2 mo, central retinal photoreceptor layer thinning at 4 mo and significant apoptosis at 4 mo. Excitotoxicity was not evident. Memantine had expected effects in WT mice but did not preserve hq vision. Ocular phenotyping of hq mice revealed dry age-related macular degeneration and a valid framework for testing appropriate drugs

Understanding cone photoreceptor dystrophies : from animal models to engineered patient-derived retinal tissues

La vision est considérée comme un des sens les plus importants, prenant en charge environ 80% des perceptions que nous recevons dans notre vie quotidienne. Les photorécepteurs de type cônes sont responsables de la vision centrale de haute résolution et en couleurs, et leur dégénérescence est souvent la cause de la perte de vision dans les maladies dégénératives rétiniennes (RDs). Les RDs sont un groupe hétérogène de maladies affectant des millions de personnes dans le monde, qui pour le moment sont pour la plupart sans aucune option thérapeutique. Les modèles animaux sont extrêmement utiles pour étudier le développement ou la dégénérescence de la rétine, ainsi que pour comprendre les mécanismes moléculaires des maladies génétiques héréditaires affectant les photorécepteurs. La modélisation des maladies dégénératives et du développement peut être particulièrement difficile, spécialement dans le cas de maladies humaines rares et complexes pour lesquelles aucun modèle animal exhaustif n'est disponible. De nos jours, la génération et le maintien de modèles de maladies humaines permettant une analyse approfondie du mécanisme moléculaire représente un grand défis . La technologie des cellules souches possède un grand potentiel dans la modélisation des maladies et représente un outil puissant pour générer des modèles évolutifs, sans l’utilisation d’animaux qui peuvent illustrer plus précisément les phénotypes cliniques de maladies humaines complexes. Nous avons développé un protocole pour différencier les cellules souches pluripotentes (PSCs) en feuillets rétiniens (RSs), qui sont des tissus polarisés et multicouches contenant des photorécepteurs cône et exprimant les marqueurs spécifiques du segment externe (OS), du cilium connecteur (CC) et du noyau. En utilisant à la fois des modèles de souris et des modèles humanisés à base de cellules souches, nous avons étudié le rôle de BMI1 dans les photorécepteurs matures. La protéine du groupe Polycomb Bmi1 est connue pour ses fonctions neuroprotectrices en contrôlant la sénescence et l'apoptose, et est exprimée à la fois dans le progéniteur rétinien et les neurones, mais on en sait peu sur son rôle spécifique dans la rétine adulte. Elle a été récemment associée à des troubles neurodégénératifs d'apparition tardive, et elle pourrait avoir un rôle dans la pathologie des RDs d'apparition tardive, comme la dégénérescence maculaire liée à l'âge (DMLA). Nous avons montré que les photorécepteurs cône et les neurones bipolaires sont générés normalement mais subissent ensuite une dégénérescence rapide chez les souris Bmi1-/- par nécroptose associée à Rip3. La dégénérescence était associée à des anomalies de compactage de la chromatine, à l'activation des répétitions en tandem et au stress oxydatif. De plus, nous montrons que BMI1 est préférentiellement exprimé dans les cônes au niveau des foyers hétérochromatiques dans la rétine humaine. Son inactivation dans les cellules souches embryonnaires humaines (hESCs) a altéré la différenciation terminale du cône et a entraîné des anomalies de compactage de la chromatine, l'activation des répétitions en tandem et l'induction de P53. Ces résultats fournissent un mécanisme expliquant comment une carence en Bmi1 conduit à la dégénérescence des cônes et révèlent des fonctions biologiques conservées et des différences pour Bmi1 dans la biologie des photorécepteurs entre la souris et l'homme. En utilisant un modèle humain basé sur les cellules souches pluripotentes induites (iPSCs), nous avons ensuite étudié le processus dégénératif chez les patients atteints de ciliopathies, un groupe de maladies génétiques hétérogènes affectant les protéines impliquées dans la structure et la fonction du cil primaire, qui sont fréquemment accompagnée d'une dégénérescence rétinienne. Nous générons des feuillets rétiniens dérivés d'iPSCs à partir de patients atteints de deux ciliopathies, les syndromes de Meckel-Gruber (MKS) et de Bardet-Biedl (BBS). Les photorécepteurs ciliopathiques présentaient des altérations communes significatives dans l'expression de centaines de gènes de développement. De plus, ils ont montré plusieurs anomalies dans la formation et le maintien du cilium interne, le positionnement du centriole mère, l'activation d'une réponse au stress aux protéines mal repliées, instabilités génomiques et l'accumulation de dommages à l'ADN. Cette étude révèle comment la combinaison des technologies de reprogrammation cellulaire et d'organogenèse avec le séquençage de nouvelle génération permet d'élucider les mécanismes moléculaires et cellulaires impliqués dans les troubles dégénératifs et développementaux de la rétine humaine. La même approche, combinant la différenciation en RSs avec des techniques de séquençage du génome à large spectre, pourrait être appliquée pour modéliser de nombreuses maladies génétiques, développementales et dégénératives affectant les photorécepteurs. Il peut également aider à élucider les mécanismes moléculaires sous-jacents à ces maladies, au criblage de médicaments de composés ayant des effets thérapeutiques potentiels et à prédire les effets secondaires des médicaments.Vision is considered the most important sense, taking on about 80% of the perceptions we receive in our everyday life. Cone-photoreceptors are responsible for high-resolution central vision and color discrimination, and their degeneration is frequently the cause of vision loss in retinal degenerative diseases (RDs). RDs are a heterogeneous group of diseases affecting millions of people worldwide, which at the moment are mostly without any therapeutic option. Animal models are extremely useful in studying the retina's development or degeneration and understanding the molecular mechanisms in inherited genetic disease affecting photoreceptors. Modeling human developmental and degenerative diseases can be particularly challenging, especially in the case of rare and complex diseases where no exhaustive animal models are available. Generation of sustainable human disease models that allow in-depth analysis of the molecular mechanism is one of the big challenges nowadays. Stem cell technology holds great potential in disease modeling and represents a new powerful tool for generating scalable and animal-free models that can more accurately illustrate clinical phenotypes of complex human diseases. We developed a protocol to differentiate pluripotent stem cells (PSCs) into retinal sheets (RSs), which are polarized, multi-layered tissues containing cone photoreceptors and expressing outer segment (OS), connecting cilium (CC), and nuclear specific markers. Using both mouse and stem cells-based humanized models, we first investigate the role of BMI1 in mature photoreceptors. The Polycomb group protein Bmi1 is known for its neuroprotective functions by controlling senescence and apoptosis and is expressed in both retinal progenitor and neurons, but little is known about its specific role in the adult retina. It has been recently linked to late-onset neurodegenerative disorders, and it could have a role in the pathology of late-onset RDs, such as Age-related Macular Degeneration (AMD). We showed that cone photoreceptors and bipolar neurons are generated normally but then undergo rapid degeneration in Bmi1-/- mice through Rip3-associated necroptosis. Degeneration was associated with chromatin compaction anomalies, activation of tandem-repeats, and oxidative stress. Furthermore, we show that BMI1 is preferentially expressed in cones at heterochromatic foci in the human retina. Its inactivation in human embryonic stem cells (hESCs) impaired cone terminal differentiation and resulted in chromatin compaction anomalies, activation of tandem-repeats, and P53 induction. These findings provide a mechanism explaining how Bmi1 deficiency leads to cone degeneration and reveal conserved biological functions and differences for Bmi1 in photoreceptor biology between mouse and man. Using an induced Pluripotent Stem Cells (iPSCs) based human model, we then investigate the degenerative process in patients with ciliopathies, a group of heterogeneous genetic diseases affecting proteins involved in primary cilium structure and function frequently accompanied by retinal degeneration. We generate iPSC-derived retinal sheets from patients affected by two ciliopathies, Meckel-Gruber (MKS) and Bardet-Biedl syndromes (BBS). Ciliopathic photoreceptors displayed significant common alterations in the expression of hundreds of developmental genes. Moreover, they showed several anomalies in the formation and maintenance of cilia, the mother centriole's positioning, the activation of a stress response to misfolded proteins, genomic instabilities, and DNA damage accumulation. This study reveals how combining cell reprogramming and organogenesis technologies with next-generation sequencing enables the elucidation of molecular and cellular mechanisms involved in human retinal degenerative and developmental disorders. The same approach, combining photoreceptor sheet differentiation and wide-genome expression profile, could be applied to model many genetic, developmental, and degenerative diseases affecting photoreceptors. It may help elucidate the molecular mechanisms underlining these diseases, drug screening of compounds with potential therapeutic effects, and predict drug side effects

Leber Congenital Amaurosis and other autosomal recessive retinal dystrophies: A clinical and molecular genetic study

Leber congenital amaurosis (LCA) and the early onset retinal dystrophies (EORD) are a spectrum of autosomal recessively inherited genetic conditions affecting children who have visual impairment starting under the age of five years. There are currently 19 known genes that account for approximately two thirds of cases. Only two of these 19 genes (IMPDH 1 and CRX; not studied in this project) have been found to cause autosomal dominant LCA. This genetic heterogeneity makes the identification of these causative genes expensive and time consuming. Phenotype-genotype correlations are therefore important in directing efforts to determine the molecular cause of disease. The aims of this research project were to recruit and clinically characterise a large panel of LCA and EORD patients and to identify the underlying genetic cause of autosomal recessive disease. Patients were recruited from Moorfields Eye Hospital and Great Ormond Street Hospital. A full clinical examination was carried out. DNA samples were analysed using the Asper Ophthalmics LCA microarray chip and by direct sequencing. Large families, with several affected members, were examined using the Affymetrix gene chip arrays for regions of homozygosity and candidate gene sequencing was performed. DNA samples from 158 patients were obtained and 117 patients were examined clinically. A definitive molecular diagnosis was obtained for 26% of patients. Of the cohort of 158 patients with one or two mutated alleles identified and genotyped: RPE65 accounts for 1% of this cohort, 6% are due to mutations in CRB1, 15% are due to RDH12 mutations and 11% are due to mutations in CEP290. Two families were identified with novel CRALBP mutations. The genotype yield from the period of this research, August 2006- August 2008, is lower than that expected with newer technologies in 2014; such as next generation sequencing (NGS) or whole exome sequencing. Useful prognostic information gained will help future patients with these disorders. Patients with a molecular diagnosis may be eligible for clinical trials of gene replacement therapy

Causes of visual loss in patients with uveitis

The last major study of causes of vision loss in 600 eyes with uveitis was published over 10 years ago and there have been many advances in treatment over this time. In this thesis I undertook a study of 1594 patients (2593 eyes) with uveitis currently attending the clinic, 75% of whom were aged between 24 and 63 years. The type of uveitis, sight threatening complications that developed and treatment were followed from presentation to final follow up. At presentation, 16% of eyes had BCVA ≤ 6/18 (e.g. 6/18-6/36) and 14% of affected eyes had BCVA 6/60 or worse. At one year follow-up, we found 11% of eyes with vision loss to 6/18-6/36 and 8% of eyes with severe visual loss or blindness. In the group of eyes followed up for 10 years or more, 19% developed severe visual loss or blindness and 16% developed vision loss to 6/18-6/36. Chronic macular damage was the main cause of visual loss, accounting for both for visual impairment and for severe visual loss, accounting for 41% and 36% respectively. Cystoid macular oedema accounted for 29% in visual impairment and 19% in severe visual loss or blindness. When classified by uveitis types, CMO was the main cause of vision loss in intermediate uveitis (38%), glaucoma was the leading cause in anterior uveitis (32%), and chronic macular damage accounted for 46% in posterior/panuveitis. Additionally, I looked at the outcome and subsequent impact on vision of ocular surgery for cataract, glaucoma and vitreo-retinal procedures. Visual prognosis after cataract surgery was favourable in anterior and intermediate uveitis. Eyes which underwent glaucoma surgery had vision stabilised or slightly improved over time. The mean log MAR BCVA prior to glaucoma surgery was 0.53+/- 60, and 0.31+/- 49 at final follow-up visit. (P= 0.012). There was no statistically significant improvement in visual acuity in eyes which had undergone vitreo-retinal procedures. The mean logMAR BCVA were 1.1+/-0.82 and 0.87+/-0.80 respectively pre-operative and at last post- op visit. (P=0.28) The 3rd main results chapter looks at patients presenting with retinal vasculitis who had ischemia and the long term outcome for these eyes. Of the 106 eyes which developed ischemia, 24% had vision loss to 6/18-6/36 at presentation, 23% of these had BCVA 6/60 or worse. Chronic macular damage was the main cause of visual impairment and accounted for 36%, macular ischemia accounted for 67% of severe visual loss or blindness. I found that in most eyes with ischemia, visual loss developed early in the first 5 years and do not worsen with time