Expression and subcellular localization ofUSH1C/harmonin in human retina provides insights into pathomechanisms and therapy

Abstract

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 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 interaction of harmonin with OLM molecules in PRCs and MGCs and with 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).

The Usher syndrome 1C protein harmonin regulates canonical Wnt signaling

“…We show this with the present interaction assays in mammalian cells and in situ by PLAs, confirming previous in vitro data (Johnston et al, 2004;Nagel-Wolfrum et al, 2022). The binary interaction of harmonin and β-catenin is mediated by binding of the C-terminal tail of β-catenin with its type I PBM (DTDL) (DuChez et al, 2019) to PDZ domains 1 and 3 on harmonin, with a slight preference for PDZ3 (Nagel-Wolfrum et al, 2022). Binding of the C-terminal PBM of βcatenin to PDZ domains has been previously described for interactions with other PDZ domain-containing proteins such as MAGI-1 and LIN7 (Dobrosotskaya and James, 2000;Perego et al, 2000).…”

“…The scaffold protein harmonin is encoded by the USH1C gene (ENSG00000006611; OMIM 276904) (Bitner-Glindzicz et al, 2000;Verpy et al, 2000) which consists of 28 exons, and alternative splicing of USH1C results in numerous splice variants, which are grouped based on their domain composition into three major splice groups a, b, and c (Nagel- Wolfrum et al, 2022). Although USH1C/harmonin is almost ubiquitously expressed in humans, it functions as a key organizer of USH protein networks predominantly in the mechanosensitive hair cells of the inner ear and in retinal cells, namely photoreceptor cells and Müller glia cells, but also in brush border microvilli of intestinal epithelia (Wolfrum, 2011;Crawley et al, 2016;Li et al, 2016;Nagel-Wolfrum et al, 2022). Harmonin molecules can harbor up to three PDZ (named after PSD-95, DLG, and ZO-1) domains (Figure 1A) that are capable of binding of all other known USH proteins, but also other proteins which are often associated with membrane proteins and cytoskeletal proteins, such as JAM-B, rhodopsin or filamin-A (Reiners et al, 2006;Wolfrum, 2011;Nagel-Wolfrum et al, 2022).…”

“…The scaffold protein harmonin is encoded by the USH1C gene (ENSG00000006611; OMIM 276904 ) ( Bitner-Glindzicz et al, 2000 ; Verpy et al, 2000 ) which consists of 28 exons, and alternative splicing of USH1C results in numerous splice variants, which are grouped based on their domain composition into three major splice groups a, b, and c ( Nagel-Wolfrum et al, 2022 ). Although USH1C /harmonin is almost ubiquitously expressed in humans, it functions as a key organizer of USH protein networks predominantly in the mechanosensitive hair cells of the inner ear and in retinal cells, namely photoreceptor cells and Müller glia cells, but also in brush border microvilli of intestinal epithelia ( Wolfrum, 2011 ; Crawley et al, 2016 ; Li et al, 2016 ; Nagel-Wolfrum et al, 2022 ). Harmonin molecules can harbor up to three PDZ (named after PSD-95, DLG, and ZO-1) domains ( Figure 1A ) that are capable of binding of all other known USH proteins, but also other proteins which are often associated with membrane proteins and cytoskeletal proteins, such as JAM-B, rhodopsin or filamin-A ( Reiners et al, 2006 ; Wolfrum, 2011 ; Nagel-Wolfrum et al, 2022 ).…”

“…Although USH1C /harmonin is almost ubiquitously expressed in humans, it functions as a key organizer of USH protein networks predominantly in the mechanosensitive hair cells of the inner ear and in retinal cells, namely photoreceptor cells and Müller glia cells, but also in brush border microvilli of intestinal epithelia ( Wolfrum, 2011 ; Crawley et al, 2016 ; Li et al, 2016 ; Nagel-Wolfrum et al, 2022 ). Harmonin molecules can harbor up to three PDZ (named after PSD-95, DLG, and ZO-1) domains ( Figure 1A ) that are capable of binding of all other known USH proteins, but also other proteins which are often associated with membrane proteins and cytoskeletal proteins, such as JAM-B, rhodopsin or filamin-A ( Reiners et al, 2006 ; Wolfrum, 2011 ; Nagel-Wolfrum et al, 2022 ). Recently, we also confirmed the interaction of harmonin with β -catenin via binding of β -catenin´s type-I PDZ binding motif (DTDL = PBM) at the end of the C-terminal domain to the PDZ1 and PDZ3 domains of harmonin ( Figure 1A ; Nagel-Wolfrum et al, 2022 ) as previously suggested ( Johnston et al, 2004 ).…”

“…Harmonin molecules can harbor up to three PDZ (named after PSD-95, DLG, and ZO-1) domains ( Figure 1A ) that are capable of binding of all other known USH proteins, but also other proteins which are often associated with membrane proteins and cytoskeletal proteins, such as JAM-B, rhodopsin or filamin-A ( Reiners et al, 2006 ; Wolfrum, 2011 ; Nagel-Wolfrum et al, 2022 ). Recently, we also confirmed the interaction of harmonin with β -catenin via binding of β -catenin´s type-I PDZ binding motif (DTDL = PBM) at the end of the C-terminal domain to the PDZ1 and PDZ3 domains of harmonin ( Figure 1A ; Nagel-Wolfrum et al, 2022 ) as previously suggested ( Johnston et al, 2004 ). When USH1C is truncated due to the R31* or the R80Pfs*69 ( Figure 1A ) this particular interaction should be lost.…”