Under-prenylated rab GTPases in choroideremia RPE cells

Abstract

Purpose : Purpose: Choroideremia is an inherited retinal degeneration caused by mutations in CHM encoding rab escort protein 1 (REP-1), leading to under-prenylation of rab GTPases (rabs). The phenotype is exclusive to the retina, with evidence for primary pathology in the retinal pigment epithelial (RPE) cells, despite ubiquitous expression of CHM. However, the spectrum of under-prenylated rabs in RPE cells is unknown. We hypothesize that RPE cells contain a unique spectrum of under-prenylated rabs contributing to the retina-specific phenotype in choroideremia.

Methods : Methods: CHM^-/- induced pluripotent stem cell (iPSC)-derived RPE cells were generated by CRISPR/Cas-9 induced biallelic frameshift mutations in exon 1 leading to premature stop codons in commercially obtained iPSCs, followed by differentiation into RPE cells. Non-edited iPSC-RPE cells were used as isogenic controls. iPSC-RPE lysates were used for in vitro prenylation of rabs using exogenously added rab GGTase, REP-1, and biotinylated prenyl groups, followed by streptavidin pull down and tandem mass tag (TMT) spectrometry to determine which rabs were unprenylated in the original cell lysate. Results for selected rabs were validated by western blot.

Results : Results: Our preliminary data show 17 rabs that were under-prenylated in CHM^-/- iPSC-RPE cells. Of these, 10 show elevated expression in adult primary RPE cells compared to other tissues relevant to rab-associated disorders (brain cortex, peripheral nerve, whole blood), using data from the National Eye Institute eyeIntegration database. Rabs that show both under-prenylation in RPE cells as well as increased differential expression are involved in early endosomal traffic as well as late endosome fusion with lysosomes and lysosomal exocytosis.

Conclusions : Conclusions: These results show the spectrum of under-prenylated Rabs specific to RPE cells with REP-1 deficiency. Those with increased differential expression in RPE cells may have particular importance to RPE function and therefore to the choroideremia phenotype. This study lays the groundwork for identification of the full spectrum of under-prenylated rabs in the RPE and future investigations of specific candidate rabs that may play an important role in choroideremia pathogenesis.

This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.