Clarin‐1 expression in adult mouse and human retina highlights a role of Müller glia in Usher syndrome

Abstract

Usher syndrome type 3 (USH3) is an autosomal recessively inherited disorder caused by mutations in the gene clarin-1 (CLRN1), leading to combined progressive hearing loss and retinal degeneration. The cellular distribution of CLRN1 in the retina remains uncertain, either because its expression levels are low or because its epitopes are masked. Indeed, in the adult mouse retina, Clrn1 mRNA is developmentally downregulated, detectable only by RT-PCR. In this study we used the highly sensitive RNAscope in situ hybridization assay and single-cell RNA-sequencing techniques to investigate the distribution of Clrn1 and CLRN1 in mouse and human retina, respectively. We found that Clrn1 transcripts in mouse tissue are localized to the inner retina during postnatal development and in adult stages. The pattern of Clrn1 mRNA cellular expression is similar in both mouse and human adult retina, with CLRN1 transcripts being localized in Müller glia, and not photoreceptors. We generated a novel knock-in mouse with a hemagglutinin (HA) epitope-tagged CLRN1 and showed that CLRN1 is expressed continuously at the protein level in the retina. Following enzymatic deglycosylation and immunoblotting analysis, we detected a single CLRN1-specific protein band in homogenates of mouse and human retina, consistent in size with the main CLRN1 isoform. Taken together, our results implicate Müller glia in USH3 pathology, placing this cell type to the center of future mechanistic and therapeutic studies to prevent vision loss in this disease.

Retinal Gene Therapy for Usher Syndrome: Current Developments, Challenges, and Perspectives

“… 99 However, several studies concluded that endogenous CLRN1 protein remains below the levels of detection of immunostaining. 47 , 49 , 97 , 100 Recently, we have generated and characterized a novel N-terminus epitope-tagged CLRN1 knock-in mouse to facilitate the detection of endogenous CLRN1 protein expression in retinal sections with a high affinity anti-HA antibody. 100 We noted a similar background labeling pattern in both HA-tagged CLRN1 knock-in and the C57BL/6J controls.…”

“… 47 , 49 , 97 , 100 Recently, we have generated and characterized a novel N-terminus epitope-tagged CLRN1 knock-in mouse to facilitate the detection of endogenous CLRN1 protein expression in retinal sections with a high affinity anti-HA antibody. 100 We noted a similar background labeling pattern in both HA-tagged CLRN1 knock-in and the C57BL/6J controls. Importantly, the HA-tagged CLRN1 in this mouse was only detectable by western blot analysis of retinal homogenates, and was expressed continuously during postnatal development and adulthood.…”

Usher syndrome: clinical features, molecular genetics and advancing therapeutics

“…Three genes underlying Usher 2 have been identi fied as USH2A (USH2A), 51 ADGRV1 (USH2C) 52 and WHRN (USH2D 65,66 Myosin VIIA is also an essen tial RPE protein, [67][68][69] and evidence suggests that clarin1 is restricted to the retinal Müller glia. 70 Various studies have indicated the involvement of Usher proteins in a range of processes, including cohesion, mechanotransduction, synaptic matu ration, and protein and organelle transport. 72,74,[88][89][90] However, missense mutations in CDH23 can also cause milder or 'atypical' Usher 1, and genotype-phe notype correlations are not always predictable.…”

“… 65 , 66 Myosin VIIA is also an essential RPE protein, 67 – 69 and evidence suggests that clarin-1 is restricted to the retinal Müller glia. 70 Various studies have indicated the involvement of Usher proteins in a range of processes, including cohesion, mechanotransduction, synaptic maturation, and protein and organelle transport.…”

Antisense Oligonucleotide- and CRISPR-Cas9-Mediated Rescue of mRNA Splicing for a Deep Intronic CLRN1 Mutation

“…In the retina, the protein is suggested to be localized in the inner segments and ribbon synapses of photoreceptors; however, recent data also suggest expression in Müller glia cells. 2 , 3 This gene encodes many different splice isoforms, whose function is also poorly understood. 4 CLRN1 mutations have an estimated prevalence of 1:100,000 individuals worldwide; however, USH3 is the major USH subtype in the Ashkenazi Jewish and the Finnish populations.…”