Abstract for laypersons

We investigate retinal organoids regarding mechanisms and pathomechanisms of the crumbs-like 1 CRB1 protein derived from patient and control persons. Patient derived cells carry CRB1 mutations that lead amongst others, to morphological changes. These changes are patly seen in the cytoskeleton of retinal cells. We aim to elucidate these changes and finally find a possibiöity to revert these changes in the patient surrounding.

Scientific abstract

Mutations in CRB1 cause autosomal-recessive Retinitis Pigmentosa (RP), cone-rod dystrophy and Lebers Congenital Amaurosis (LCA), which are the leading cause of inherited blindness. However, a great variance in the natural history of the disease, with respect to onset, severity and progression of retinal degeneration (RD) can be observed in individual patients. Currently, no treatment is available for those patients. CRB1 is a multi-facetted protein in the human with crucial roles in structure, development and function of the retina. As member of the crumbs family, CRB1 shares a cytoplasmic domain, a transmembrane domain and a signaling peptide with the two other family members CRB2 and CRB3, but only CRB1 and 2 give rise to large extracellular domain with multiple protein domains including EGF-like, laminin A G-like domains and SH3 domains [4], indicating broad protein-protein interactions. Of note, several splice forms of CRB1 and 2 have been demonstrated, yet, no specific functions have been defined to individual variants in humans. In the human retina, CRB1 localizes to the sub-apical region of the photoreceptor (RPR) inner segment and Müller glia cells (RMG), where it is thought to be involved in the regulation of cell-cell adhesion, cell polarity, integrity of the retinal outer limiting membrane (OLM) and intracellular communication. In Drosophila, the CRB1 analogue crumbs organizes an intracellular protein scaffold that e.g. defines the assembly of a continuous zonula adherens as part of the OLM. Cellular interaction, such as e.g. the close cooperation of RMG and RPRs are a prerequisite for function and survival of the RPRs, upholding vision. In mouse retina, loss of CRB1/2 results in dysregulation of target genes for signalling pathways such as the Notch1 and YAP/Hippo signalling cascades. Furthermore, CRB1 seems to be involved in progenitor cell cycle regulation. Although it has been described that ablation of CRB1 in a mouse models leads to mild retinal disorganisation and that CRB1 is involved in the physiological integrity of RPR polarity, many aspects of CRB1 physiological function during human retinal development as well as its molecular function in human retinal cells including that in RPR remain elusive. This is partly due to the different localisation of CRB1 in human and mouse retina. While in humans CRB1 is expressed in both RPR and RMG, in mice, CRB1 is only expressed in RMG. Induced pluripotent stem cells (iPSC) provide a bona fide platform to evaluate not only human specific cell biology of CRB1, but also in context of RD. Current knowledge of CRB1 function suggests a role in organizing the development of the OLM in between RMG and RPR. Based on results of the previous funding period, we propose a role of CRB1 as an organizer of cytoskeletal structure and function in the outer retina. To study this in a human context, we will analyze established patient specific iPSC lines carrying the most common CRB1 sequence change C948Y, CRB1 knockout lines, corresponding control iPSC lines and retinal organoids (RO) derived from both. This will allow us to study CRB1 function and dysfunction in physiological and pathological organotypic surroundings