Müller Glia–Exclusive CLRN1 Expression Drives Non–Cell-Autonomous Photoreceptor Degeneration in Usher Syndrome Type 3A

Purpose: Usher syndrome type 3A (USH3A), caused by CLRN1 mutations, leads to progressive deaf-blindness. Although CLRN1 expression has been localized to Müller glia (MG), the pathogenic mechanisms linking glial CLRN1 deficiency to photoreceptor death remain undefined. We utilized a novel large-animal model to elucidate cell-type-specific pathogenesis at single-nucleus resolution. Methods: CLRN1-/- rabbits were generated via CRISPR/Cas9. Retinal phenotyping included longitudinal optical coherence tomography (OCT), electroretinography (ERG), and histology for up to 3 years. Single-nucleus RNA sequencing (snRNA-seq) was performed at a pre-symptomatic stage (10 months, n = 3/genotype). Differentially expressed genes (DEGs) were identified using pseudobulk DESeq2 and validated via immunofluorescence. Results: CLRN1-/- rabbits exhibited progressive outer nuclear layer thinning and delayed ERG responses starting after 20 months, mimicking human clinical kinetics. The snRNA-seq revealed that CLRN1 is exclusively expressed in a discrete subset (∼24%) of MG. Mutant MG exhibited transcriptomic downregulation of the adherens junction component CTNNA2, which was validated at the protein level by immunofluorescence staining. Photoreceptors exhibit extensive non-cell-autonomous dysregulation (232 rod and 68 cone DEGs) characterized by loss of the cone synaptic organizer TENM2 and rod-specific splicing dysregulation (CWF19L2). Concurrently, inner retinal neurons mounted a robust proteostatic response via HSP90 family upregulation (HSP90AB1, HSP90AA1, HSP90B1), a defensive signature absent in vulnerable photoreceptors. Conclusions: Our findings suggest an "anchor-shield" mechanism: MG CLRN1 loss compromises the outer limiting membrane (OLM) adhesion complex ("anchor"), and photoreceptors lack the proteostatic response ("shield") seen in resilient inner neurons. Restoring CLRN1 in Müller glia or implementing photoreceptor neuroprotection represents promising therapeutic strategies for USH3A.