Thèse Etude de la Structure - Fonction des Opsines dans la Rétinopathie Associée au Syndrome d'Alstrom H/F - Doctorat.Gouv.Fr

Établissement : Université Paris-Saclay GS Santé et médicaments École doctorale : Innovation thérapeutique : du fondamental à l'appliqué Laboratoire de recherche : Institut des Cellules souches pour le traitement et l'étude des maladies monogéniques Direction de la thèse : Christelle MONVILLE Début de la thèse : 2026-10-01 Date limite de candidature : 2026-05-29T23:59:59 Le syndrome d'Alström (SA) est une maladie multisystémique monogénique récessive caractérisée par une perte auditive et visuelle, une obésité, un diabète de type 2, une cardiomyopathie et une insuffisance hépatique et rénale progressive. Les symptômes affectant la vision apparaissent dans les premières semaines après la naissance et conduisent progressivement à une perte totale de la vue. À l'heure actuelle, il n'existe aucun remède contre cette maladie, et seules des solutions permettant d'atténuer les effets des symptômes peuvent être proposées.
En collaboration avec le Dr Liu de l'université HUST de Wuhan, Chine, notre projet scientifique a pour objectif d'étudier les structures et les fonctions des récepteurs opsines associés aux anomalies rétiniennes dans le SA. Les récepteurs opsines appartiennent à la famille des récepteurs couplés aux protéines G et sont utilisés par les photorécepteurs pour convertir la lumière en signaux électriques.
Au sein d'I-Stem, nous avons obtenu différents clones présentant des mutations pathologiques ou de novo à l'aide de systèmes d'édition génomique associés à CRISPR/Cas9. Nous avons caractérisé ces clones modèles en cherchant à identifier des marqueurs phénotypiques spécifiques au sein des hiPSCs. Nous avons différencié nos lignées cellulaires en organoïdes neurorétiniens afin d'étudier le développement des cellules rétiniennes au sein de ces structures, en nous concentrant particulièrement sur les photorécepteurs. Nous avons pu observer l'absence ou la réduction de l'expression des opsines caractéristiques des cônes et des bâtonnets dans les organoïdes dérivés de hiPSC mutantes ALMS1. De plus, ces organoïdes ont présenté une mort cellulaire accrue par rapport aux organoïdes dérivés de lignées hiPSC saines. Cela suggère que les photorécepteurs dégénèrent au cours de la différenciation au sein des organoïdes. Les mécanismes par lesquels les mutations du gène ALMS1 conduisent à cette dégénérescence restent flous, mais un mauvais repliement ou un dysfonctionnement de l'opsine pourraient être en cause.
Le laboratoire du Dr Liu se concentre principalement sur la structure et la fonction des récepteurs membranaires, notamment les récepteurs couplés aux protéines G (GPCR) et les canaux ioniques, ainsi que sur les résultats physiologiques de ces récepteurs et leur pharmacologie moléculaire. En outre, son laboratoire étudie également le mécanisme de régulation de ces récepteurs membranaires sur le vieillissement et les maladies liées au vieillissement, en utilisant C. elegans comme modèle. Grâce à cette collaboration, nous souhaitons créer un modèle C. elegans de la maladie d'Alström et étudier la structure/fonction des opsines à l'aide de techniques biophysiques.

The retina is a light sensitive tissue located on the inner surface of the posterior portion of the eye. Light is captured by the human retina, which converts it into electrical signals that are interpreted into vision. The retina is made up of several well-organized layers. The deepest layer contains the cones and rods that make up the photoreceptors. Rods are responsible for seeing in areas with low lighting, whereas cones are responsible for color vision and function in bright light. Anatomically, photoreceptors are separated into outer and inner segments. Organelles are found in the inner segment, whereas photopigments in the outer segment allow individual photons to be captured. Light is converted by photoreceptors into electrical signals that are transmitted to other inner layer components. A monolayer of epithelial cells is formed by the retinal pigmented epithelium (RPE) cells (Yang, Zhou, and Li 2021). They are the outermost layer of the retina, with their basolateral membrane facing the choroid and their apical surface facing the outer segments of the photoreceptors. In addition to being vital for maintaining vision, photoreceptor survival, light absorption, the exchange of biological material between the choroid and photoreceptors, and cellular paracrine communication, these cells are also necessary for maintaining photoreceptor homeostasis (Dalvi, Galloway, and Singh 2019a).
Alström Syndrome (ALMS) is an autosomal recessive and monogenic disease, with a prevalence of 1-0/1,000,000 in Europe and North America. Given that ALMS is a monogenic illness, its development is caused by a single gene. The Alström Syndrome 1 gene (ALMS1) has 268 variations that have been found in ALMS patients. These variants might be heterozygous or homozygous. Nonsense or frameshift mutations account for more than 95% of the mutations. The majority of ALMS's symptoms, which are multisystemic in nature, appear during birth or in early infancy. Cone-rod dystrophy, ***** resistance, obesity, type 2 diabetes mellitus, liver failure, renal dysfunction, and hearing loss are some of these symptoms. The ALMS is considered as Retinal Dystrophy (RDD) because it leads to specific type of RDD called cones-rods dystrophy and this project focuses specifically on the visual symptoms and cones-rods retinal dystrophy which means that loss of cone cells started first followed by the loss of rod cells (Choudhury et al. 2021a; Dassie et al. 2021). The ALMS1 protein plays roles in endosomal trafficking, cell migration, intraciliary transport, and cell cycle regulation. Because of the autosomal recessive nature of ALMS, both copies of the ALMS1 gene must be mutated for the condition to occur. Mutation of the ALMS1 gene results in the production of a truncated protein that has no function or reduced function compared to the original gene. ALMS is classified as a ciliopathy disorder. Ciliopathies are a family of genetic disorders that affect the primary cilia and their functions. Primary cilia are present in all eukaryotic cells, particularly in epithelial cells. The retina compartment in the eye includes many cells such as Retina Pigment Epithelium (RPE) cells, photoreceptors, horizontal cells, and bipolar cells. Primary cilia are present on the surface of RPE cells and modified primary cilia (specialized cilia) are present in photoreceptors. The cilia have roles mostly similar to the ALMS1 protein, in cellular signal transduction, cell cycle regulation, and protein trafficking, because the ALMS1 protein is normally present in ciliated cells, particularly in centrosomes and basal bodies (Choudhury et al. 2021b; Marshall et al. 2011). Previous studies on cell line and mouse models revealed that the ALMS1 mutation in ALMS is associated with a spectrum of cilia abnormalities that affect both primary cilia and specialized cilia in RPE cells and photoreceptors, respectively (Álvarez-Satta et al. 2021; Heydet et al. 2013). There is no specific treatment for ALMS; the available therapy management aims to treat the symptoms and complications (Beqiri-Jashari et al. 2022; Choudhury et al. 2021b; Marshall et al. 2011).
There are few studies addressing the impact of ALMS1 mutation on retinal dystrophy in cell line or mouse models. We lack a human cell model for ALMS to understand the pathological mechanisms of visual symptoms in ALMS. The emergence of Human Pluripotent Stem Cells (hiPSCs) technology provides the possibility to access biological samples for the retina and choroid by using Yamanaka factors, which are reprogramming factors able to generate hiPSCs from different sources such as keratinocytes, fibroblasts, lymphocytes, adipocytes, cord blood cells, and T cells.
The mechanisms by which mutations in ALMS1 lead to this degeneration remain unclear but Opsine misfolding or un functioning could be involved. The main objectives of this thesis will be to study ospine structure, localization and function in cellular and animal models (C.elegans) of Alstrom syndrome.
Study opsin receptors structures and functions associated with retinal defects in the Alstrom Syndrom using human pluripotent stem cells and C elegans models. Our research utilizes five different human induced pluripotent stem cell (hiPSC) models, each carrying unique mutations in the ALMS1 gene. Three of these cell lines were generated from an isogenic wild-type background, with targeted mutations introduced at exons 3, 5, and 16 using either CRISPR/Cas9 or base editing techniques. In addition, we developed a hiPSC line from patient-derived fibroblasts that carry a mutation in exon 8. This work was carried out by our retinopathies research group in collaboration with the GIGA Institute. To create a genetically matched control, we corrected the exon 8 mutation in the patient-derived hiPSCs using CRISPR/Cas9, resulting in a fifth cell line, the corrected version of the patient ALMS1 line. Altogether, these models allow us to systematically investigate the effects of different ALMS1 mutations on retinal cell function, providing a comprehensive platform to study disease mechanisms and potential therapeutic strategies. In collaboration with Dr Liu's laboratory we want to set up a C.elegans Alstrom model in order to validate some mechanisms in a more physiological model.

Etudiant motivée ayant une connaissance de culture cellulaire, biologie cellulaire et moléculaire.