Expression and subcellular localization of USH1C/harmonin in the human retina

Abstract

Purpose : The human Usher syndrome (USH) is the most frequent cause of inherited combined deaf-blindness. USH1C encodes for the scaffold protein harmonin organizing the USH protein interactome. We aimed to decipher the expression profiles of harmonin isoforms and analyzed harmonin’s subcellular localization in the cells of the human retina.

Methods : We analyzed and quantified the expression of harmonin isoforms in human retina by RT-PCR, RNAseq and Western blotting. Harmonin localization was studied in human and non-human primate retinas by immunofluorescence (LM) and immunoelectron (EM) microscopy. Protein-protein interactions were analyzed by co-immunoprecipitations and in situ proximity ligation assays.

Results : Although at least 20 harmonin isoforms were identified in the human retina, harmonin-a1 is the most frequently expressed variant. Bulk RNAseq of retinal cell populations revealed not only abundant expression in Müller glia cells, but also in retinal neurons including photoreceptor cells. LM and EM revealed harmonin localization in endfeet and apical microvilli of Müller glia cells. In photoreceptor cells, we found harmonin at adhesion complexes in the outer limiting membrane and calyceal processes of the inner segment. In addition, harmonin was detected in the synaptic pedicles of cones. In contrast, in rods, it was most abundant in the outer segments (ROS).

Conclusions : Our data are valuable for both gene therapy development and retinal cell biology. We identified harmonin-a1 as the most abundant harmonin isoform in human Müller glial and photoreceptor cells. Therefore, for gene therapy, we propose retinal delivery of harmonin-a1 using a vector that transduces both cell types. We hypothesize that harmonin organizes protein networks associated with the actin cytoskeleton in those cells. Of note, harmonin in ROS may participate in disk staking by interacting with actin and rhodopsin. Defects in harmonin might cause disruption of these protein networks and thus of retinal cell maintenance, leading to the retinal pathophysiology phenotype of USH1C.

This is a 2021 ARVO Annual Meeting abstract.