The USH3A causative gene clarin1 functions in Müller glia to maintain retinal photoreceptors

Abstract

ABSTRACTMutations inCLRN1cause Usher syndrome type IIIA (USH3A), an autosomal recessive disorder characterized by hearing and vision loss, and often accompanied by vestibular balance issues. The identity of the cell types responsible for the pathology and mechanisms leading to vision loss in USH3A remains elusive. To address this, we employed CRISPR/Cas9 technology to delete a large region in the coding and untranslated (UTR) region of zebrafishclrn1. Retina ofclrn1mutant larvae exhibited sensitivity to cell stress, along with age-dependent loss of function and degeneration in the photoreceptor layer. Investigation revealed disorganization in the outer retina inclrn1mutants, including actin-based structures of the Müller glia and photoreceptor cells. To assess cell-specific contributions to USH3A pathology, we specifically re-expressedclrn1in either Müller glia or photoreceptor cells. Müller glia re-expression ofclrn1prevented the elevated cell death observed in larvalclrn1mutant zebrafish exposed to high-intensity light. Notably, the degree of phenotypic rescue correlated with the level of Clrn1 re-expression. Surprisingly, high levels of Clrn1 expression enhanced cell death in both wild-type andclrn1mutant animals. However, rod- or cone-specific Clrn1 re-expression did not rescue the extent of cell death. Taken together, our findings underscore three crucial insights. First,clrn1mutant zebrafish exhibit key pathological features of USH3A; second, Clrn1 within Müller glia plays a pivotal role in photoreceptor maintenance, with its expression requiring controlled regulation; third, the reliance of photoreceptors on Müller glia suggests a structural support mechanism, possibly through direct interactions between Müller glia and photoreceptors mediated in part by Clrn1 protein.AUTHOR SUMMARYMutations in USH-associated genes profoundly impact patients, affecting auditory, visual, and vestibular function. While the basis of inner ear defects is reasonably well understood for USH and auditory devices can improve hearing, the mechanisms underlying photoreceptor loss are unknown, and there are no approved treatments for vision deficits. In USH3A, the affected gene,clarin1(clrn1), is predominantly expressed in Müller glia. The role of Müller glia in maintaining photoreceptor health and contributions to USH3 pathology is understudied, in part asClrn1mutant mice - the traditional experimental model used to study retinal diseases - do not phenocopy the photoreceptor loss of USH3 patients. In the present study, we developed a zebrafish model of USH3A that displays many features of the human disease. Our research shows that the loss of Clrn1 affects actin-based structures of the outer retina, including those of photoreceptor cells and Müller glia. Importantly, we demonstrate that the expression of Clrn1 in Müller glia, but not rods and cones, alleviated light-induced damage inclrn1mutant zebrafish. We also highlight that the dosage of Clrn1 in Müller glia is critical for maintaining proper photoreceptor function. These findings demonstrate the key contribution of Müller glia to USH pathology and can guide strategies for gene-replacement therapies.