Peripherin-2 and Rom-1 Have Opposing Effects on Rod Outer Segment Targeting of Two Misfolded Peripherin-2 Mutants in Rods

Abstract

Purpose : The tetraspanins peripherin-2 and Rom-1 can form homo- and heteromeric complexes in photoreceptor outer segments (OS). Mutations in peripherin-2, but not in Rom-1, lead to autosomal dominant retinitis pigmentosa (adRP) and the vast majority of these mutations is located in the D2 loop domain. How different complexes assembled by various combinations of wild type (per^WT), mutant peripherin-2 (per^MT) or Rom-1, which can be formed in heterozygous patients, contribute to the pathophysiology of adRP remained elusive so far. Here, focusing on the two adRP-linked peripherin-2 D2 loop mutants, per^P210L and per^C214S, we analyzed the binding characteristics, subunit assembly, subcellular localization, and rod OS targeting of per^WT-per^MT, and Rom-1-per^MT complexes in HEK293 cells and in murine rod photoreceptors.

Methods : Binding characteristics were analyzed by co-immunoprecipitation, protease accessibility assay, peptide competition assay, and quantitative FRET measurements. Subunit assembly of the single complexes was addressed by sucrose density gradient centrifugation. Rod OS targeting and subcellular localization of the single complexes was examined on retinal slices from wild type mice injected with AAV-vectors expressing different combinations of per^WT, per^MT, and Rom-1.

Results : per^P210L and per^C214S are misfolded and lead to decreased binding to per^WT and Rom-1 due to impaired binding kinetics. The per^WT-per^MT binding was stronger affected when compared to the Rom-1-per^MT combination. In contrast to per^P210L, per^C214S was mislocalized throughout the rod OS in presence of transgenic per^WT. Both mutants preferentially formed non-covalent dimers with per^WT and Rom-1. However, only per^WT-per^MT complexes could be targeted to rod OS.

Conclusions : We show that per^WT-per^MT dimers can be targeted to rod OS. Moreover, we unravel unexpected opposing roles of per^WT and Rom-1 in the pathophysiology of peripherin-2 mutants. We postulate a novel function of Rom-1 in rods with high relevance for the design of future gene-based treatments of peripherin-2-linked adRP.

This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.

 

Rod OS targeting of per^MT dimeric complexes. The rod photoreceptor localization of different per^MT-containing dimers (per^WT-per^MT, Rom-1-per^MT and per^MT only) is symbolized by a dashed rectangle. The position of the D2 loop is highlighted in red and the D2 loop misfolding caused by per^MT is symbolized by an incision.