Abstract
Purpose :
Central areolar choroidal dystrophy (CACD) is an inherited retinal disease primarily affecting the macula, and is strongly linked to mutations in PRPH2. However, the molecular mechanisms underlying PRPH2-associated CACD remain largely unknown. This study aims to unravel pathological mechanisms by which a recurrent PRPH2 missense mutation (p.Arg142Trp) causes CACD by determining PRPH2 expression levels, its interactions to other PRPH2 molecules as well as to its binding partner ROM1, and its in vitro subcellular localization.
Methods :
To determine PRPH2 protein levels, HEK293T cells were transfected with PRPH2 wildtype or mutant plasmids, followed by western blot analysis. Subsequently, HEK293T cells were co-transfected with plasmids encoding wildtype or mutant PRPH2 and wildtype ROM1. A visual immunoprecipitation (VIP) assay was performed to determine PRPH2 self-, and PRPH2-ROM1 interactions. Finally, IMCD3-PRPH2 stable cell lines were generated, followed by immunocytochemistry (ICC) to determine the subcellular localization of PRPH2. The Mander’s coefficient (MC) was used to determine levels of co-localization.
Results :
Western blot analysis revealed no differences in protein expression levels between wildtype and mutant p.Arg142Trp PRPH2. The VIP-assay revealed that the mutant protein retains the ability to form homodimers with both wildtype and p.Arg142Trp proteins. Intriguingly, a reduced interaction with ROM1 was observed. Furthermore, ICC showed that wildtype PRPH2 spreads into the cytoplasm and partly localizes to the membrane (MC PRPH2-wildtype = 0.67 vs. MC PRPH2-p.Arg142Trp = 0.12), while the PRPH2-p.Arg142Trp mutant protein partly localized within the Golgi apparatus (MC PRPH2-wildtype = 0.23 vs. MC PRPH2-p.Arg142Trp = 0.60). Finally, the mutant protein was found only at the base of the cilium, while wildtype protein was observed along the entire cilium (MC PRPH2-wildtype = 0.89 vs. MC PRPH2-p.Arg142Trp = 0.15).
Conclusions :
The diminished PRPH2-ROM1 interaction, accompanied by aberrant subcellular localization of the PRPH2-p.Arg142Trp protein likely contributes to the loss of photoreceptor cells that is seen in CACD patients. Thereby, this study provides additional insights into the pathological mechanisms underlying PRPH2-associated CACD.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.