Expression and subcellular localization of USH1C/harmonin in human retina provides insights into pathomechanisms and therapy.

Usher syndrome (USH) is the most common form of hereditary deaf-blindness in humans. USH is a complex genetic disorder, assigned to three clinical subtypes differing in onset, course and severity, with USH1 being the most severe. Rodent USH1 models do not reflect the ocular phenotype observed in human patients to date; hence, little is known about the pathophysiology of USH1 in the human eye. One of the USH1 genes, USH1C, exhibits extensive alternative splicing and encodes numerous harmonin protein isoforms that function as scaffolds for organizing the USH interactome. RNA-seq analysis of human retinae uncovered harmonin_a1 as the most abundant transcript of USH1C. Bulk RNA-seq analysis and immunoblotting showed abundant expression of harmonin in Müller glia cells (MGCs) and retinal neurons. Furthermore, harmonin was localized in the terminal endfeet and apical microvilli of MGCs, presynaptic region (pedicle) of cones and outer segments (OS) of rods as well as at adhesive junctions between MGCs and photoreceptor cells (PRCs) in the outer limiting membrane (OLM). Our data provide evidence for the interaction of harmonin with OLM molecules in PRCs and MGCs and rhodopsin in PRCs. Subcellular expression and colocalization of harmonin correlate with the clinical phenotype observed in USH1C patients. We also demonstrate that primary cilia defects in USH1C patient-derived fibroblasts could be reverted by the delivery of harmonin_a1 transcript isoform. Our studies thus provide novel insights into PRC cell biology, USH1C pathophysiology and development of gene therapy treatment(s).

SANS (USH1G) regulates pre-mRNA splicing by mediating the intra-nuclear transfer of tri-snRNP complexes.

Splicing is catalyzed by the spliceosome, a compositionally dynamic complex assembled stepwise on pre-mRNA. We reveal links between splicing machinery components and the intrinsically disordered ciliopathy protein SANS. Pathogenic mutations in SANS/USH1G lead to Usher syndrome-the most common cause of deaf-blindness. Previously, SANS was shown to function only in the cytosol and primary cilia. Here, we have uncovered molecular links between SANS and pre-mRNA splicing catalyzed by the spliceosome in the nucleus. We show that SANS is found in Cajal bodies and nuclear speckles, where it interacts with components of spliceosomal sub-complexes such as SF3B1 and the large splicing cofactor SON but also with PRPFs and snRNAs related to the tri-snRNP complex. SANS is required for the transfer of tri-snRNPs between Cajal bodies and nuclear speckles for spliceosome assembly and may also participate in snRNP recycling back to Cajal bodies. SANS depletion alters the kinetics of spliceosome assembly, leading to accumulation of complex A. SANS deficiency and USH1G pathogenic mutations affects splicing of genes related to cell proliferation and human Usher syndrome. Thus, we provide the first evidence that splicing dysregulation may participate in the pathophysiology of Usher syndrome.

Mucolipidosis type IV protein TRPML1-dependent lysosome formation.

Lysosomes are dynamic organelles that undergo cycles of fusion and fission with themselves and with other organelles. Following fusion with late endosomes to form hybrid organelles, lysosomes are reformed as discrete organelles. This lysosome reformation or formation is a poorly understood process that has not been systematically analyzed and that lacks known regulators. In this study, we quantitatively define the multiple steps of lysosome formation and identify the first regulator of this process.

The Usher syndrome 1C protein harmonin regulates canonical Wnt signaling.

Human Usher syndrome (USH) is the most common form of hereditary combined deaf-blindness. USH is a complex genetic disorder, and the pathomechanisms underlying the disease are far from being understood, especially in the eye and retina. The USH1C gene encodes the scaffold protein harmonin which organizes protein networks due to binary interactions with other proteins, such as all USH proteins. Interestingly, only the retina and inner ear show a disease-related phenotype, although USH1C/harmonin is almost ubiquitously expressed in the human body and upregulated in colorectal cancer. We show that harmonin binds to ß-catenin, the key effector of the canonical Wnt (cWnt) signaling pathway. We also demonstrate the interaction of the scaffold protein USH1C/harmonin with the stabilized acetylated ß-catenin, especially in nuclei. In HEK293T cells, overexpression of USH1C/harmonin significantly reduced cWnt signaling, but a USH1C-R31* mutated form did not. Concordantly, we observed an increase in cWnt signaling in dermal fibroblasts derived from an USH1C R31*/R80Pfs*69 patient compared with healthy donor cells. RNAseq analysis reveals that both the expression of genes related to the cWnt signaling pathway and cWnt target genes were significantly altered in USH1C patient-derived fibroblasts compared to healthy donor cells. Finally, we show that the altered cWnt signaling was reverted in USH1C patient fibroblast cells by the application of Ataluren, a small molecule suitable to induce translational read-through of nonsense mutations, hereby restoring some USH1C expression. Our results demonstrate a cWnt signaling phenotype in USH establishing USH1C/harmonin as a suppressor of the cWnt/ß-catenin pathway.

Early disruption of photoreceptor cell architecture and loss of vision in a humanized pig model of usher syndromes.

Usher syndrome (USH) is the most common form of monogenic deaf-blindness. Loss of vision is untreatable and there are no suitable animal models for testing therapeutic strategies of the ocular constituent of USH, so far. By introducing a human mutation into the harmonin-encoding USH1C gene in pigs, we generated the first translational animal model for USH type 1 with characteristic hearing defect, vestibular dysfunction, and visual impairment. Changes in photoreceptor architecture, quantitative motion analysis, and electroretinography were characteristics of the reduced retinal virtue in USH1C pigs. Fibroblasts from USH1C pigs or USH1C patients showed significantly elongated primary cilia, confirming USH as a true and general ciliopathy. Primary cells also proved their capacity for assessing the therapeutic potential of CRISPR/Cas-mediated gene repair or gene therapy in vitro. AAV-based delivery of harmonin into the eye of USH1C pigs indicated therapeutic efficacy in vivo.