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

Abstract

Mutations in CLRN1 cause 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 zebrafish clrn1. Retina of clrn1 mutant 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 in clrn1 mutants, including actin-based structures of the Muller glia and photoreceptor cells. To assess cell-specific contributions to USH3A pathology, we specifically re-expressed clrn1 in either Muller glia or photoreceptor cells. Muller glia re-expression of clrn1 prevented the elevated cell death observed in larval clrn1 mutant 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 and clrn1 mutant 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, clrn1 mutant zebrafish exhibit key pathological features of USH3A; second, Clrn1 within Muller glia plays a pivotal role in photoreceptor maintenance, with its expression requiring controlled regulation; third, the reliance of photoreceptors on Muller glia suggests a structural support mechanism, possibly through direct interactions between Muller 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 Muller glia. The role of Muller glia in maintaining photoreceptor health and contributions to USH3 pathology is understudied, in part as Clrn1 mutant 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 Muller glia. Importantly, we demonstrate that the expression of Clrn1 in Muller glia, but not rods and cones, alleviated light-induced damage in clrn1 mutant zebrafish. We also highlight that the dosage of Clrn1 in Muller glia is critical for maintaining proper photoreceptor function. These findings demonstrate the key contribution of Muller glia to USH pathology and can guide strategies for gene-replacement therapies.