Loss-of-Function Mutations in Rab Escort Protein 1 (REP-1) Affect Intracellular Transport in Fibroblasts and Monocytes of Choroideremia Patients

Abstract

BackgroundChoroideremia (CHM) is a progressive X-linked retinopathy caused by mutations in the CHM gene, which encodes Rab escort protein-1 (REP-1), an escort protein involved in the prenylation of Rabs. Under-prenylation of certain Rabs, as a result of loss of function mutations in REP-1, could affect vesicular trafficking, exocytosis and secretion in peripheral cells of CHM patients.Methodology/Principal FindingsTo evaluate this hypothesis, intracellular vesicle transport, lysosomal acidification and rates of proteolytic degradation were studied in monocytes (CD14+ fraction) and primary skin fibroblasts from the nine age-matched controls and thirteen CHM patients carrying 10 different loss-of-function mutations. With the use of pHrodo™ BioParticles® conjugated with E. coli, collagen I coated FluoSpheres beads and fluorescent DQ™ ovalbumin with BODYPY FL dye, we demonstrated for the first time that lysosomal pH was increased in monocytes of CHM patients and, as a consequence, the rates of proteolytic degradation were slowed. Microarray analysis of gene expression revealed that some genes involved in the immune response, small GTPase regulation, transcription, cell adhesion and the regulation of exocytosis were significantly up and down regulated in cells from CHM patients compared to controls. Finally, CHM fibroblasts secreted significantly lower levels of cytokine/growth factors such as macrophage chemoattractant protein-1 (MCP-1), pigment epithelial derived factor (PEDF), tumor necrosis factor (TNF) alpha, fibroblast growth factor (FGF) beta and interleukin (lL)-8.Conclusions/SignificanceWe demonstrated for the first time that peripheral cells of CHM patients had increased pH levels in lysosomes, reduced rates of proteolytic degradation and altered secretion of cytokines. Peripheral cells from CHM patients expose characteristics that were not previously recognized and could used as an alternative models to study the effects of different mutations in the REP-1 gene on mechanism of CHM development in human population.

Conditional Ablation of the Choroideremia Gene Causes Age-Related Changes in Mouse Retinal Pigment Epithelium

“…Consistent with multiple trafficking defects in the development of CHM, reduced lysosomal acidification and secretion of cytokines have been detected in monocytes isolated from CHM patients [8] and there is reduced melanosome movement into the apical processes of RPE cells in mouse models of CHM [9]. Furthermore cultured RPE cells acutely depleted of REP1 exhibit reduced lysosome acidification and delayed phagosome degradation [10].…”

Calcium influx blocked by SK&F 96365 modulates the LPS plus IFN-γ-induced inflammatory response in murine peritoneal macrophages

“…In the neutral pH outside and inside the cell, the light emission by pHrodo conjugated E. coli is dim and barely detectable by the fluorescent microscope or flow cytometry analysis (FACS). Once pHrodo™ dye labeled-E. coli particles are engulfed, the acidic environment within the phagolysosome causes a significant increase in fluorescent emission by the particles [19,20]. Confocal microscopy observations showed that the presence of fluorescent pHrodo inside of the phagolysosomes in the control group ( Fig.…”

Comprehensive mutation analysis (20 families) of the choroideremia gene reveals a missense variant that prevents the binding of REP1 with rab geranylgeranyl transferase

“…Since only one silent single-nucleotide polymorphism has been reported in the CHM coding region, it is conceivable that missense mutations may give rise to a phenotype distinct from CHM or they may be lethal, or they may even not be disadvantageous. To date, only two nucleotide changes that could produce missense variants have been found in CHM patients, one was actually a splicing alteration [Beaufrère et al, 1999], whereas the other one substitutes Leu 550 of REP1 with a proline Strunnikova et al, 2009]. No in vitro functional analysis of this REP1 variant was performed.…”