Altered Erp29 and Htra1 Expression in Cultured Retinal Pigment Epithelial (RPE) Cells of Ccl2/Cx3cr1 Deficient Mice - A Model of Age-Related Macular Degeneration

Abstract

Purpose:: Age-related macular degeneration (AMD) is the leading cause of vision loss in the developed world. We developed a Ccl2/Cx3cr1 deficientmouse model with high penetrance and early onset AMD pathology including: drusen formation, RPE hypopigmentation, lipofuscin accumulation, photoreceptor degeneration, and choroidal neovascularization. Given the critical role of the RPE in AMD, we aimed to compare expression of proteins in cultured RPE that may mediate AMD pathogenesis. Erp29 is an endoplasmic reticulum protein that functions as an escort chaperone and in protein folding. Proteomics data in our lab indicated that Erp29 precursor was differentially expressed in Ccl2/Cx3cr1 deficient retinas, and RT-PCR confirmed lower expression of Erp29 in the retina of Ccl2/Cx3cr1 deficient mice compared to controls. The Htra1 gene encodes a secreted serine protease. Recent human molecular epidemiological studies reveal that a risk allele in Htra1 is associated with AMD, particularly the wet form.

Methods:: RPE cells were isolated from Ccl2 deficient, Cx3cr1 deficient, Ccl2/Cx3cr1 deficient, and wild-type mice under dissecting microscope. The cells were cultured in 10% serum containing DMEM at 37^oC - 5% CO₂ for three days. At this time, mRNA isolation from the cultured RPE cells was performed with TRIzol reagent and real-time RT PCR was performed with TaqMan gene expression assay using an Applied Biosystems real time PCR system.

Results:: The cultured RPE cells of Ccl2/Cx3cr1 deficientmice expressed 0.74-fold Erp29 that of wild-type, while no difference was found between Cx3cr1 deficient mice compared to wild-type, whereas Ccl2 deficient mice expressed 3.99-fold Erp29 that of wild type. The Htra1 level in cultured RPE of Ccl2/Cx3cr1 deficient mice was 2.10-fold increased as compared to wild-type. Cultured RPE of the two single knockout mice also showed higher expression of Htra1 mRNA relative to wild-type.

Conclusions:: Low Erp29 levels in Ccl2/Cx3cr1 deficientmice may contribute to the AMD pathology by resulting in accumulation of unfolded protein in the affected RPE. The normal Erp29 levels in Cx3cr1 deficient mice and mildly increased levels in Ccl2 deficient mice may result in their milder AMD phenotype than Ccl2/Cx3cr1 deficientmice. Increased RPE levels of Htra1 transcripts suggest that it may also play a role in pathogenesis of the AMD-phenotype of all three strains.

Keywords: retinal degenerations: cell biology • retinal pigment epithelium • age-related macular degeneration