Abstract
Purpose:
Our aim is to determine how the expression of genes by the RPE is altered by two different factors: 1. the 402H variant of complement factor H, and 2. the RD8 mutation of the Crb1 gene.
Methods:
Using the SRIRS (Simultaneous RPE cell Isolation and RNA Stabilization) technique, we isolated RPE cells from mouse eyecups of 2 year old CfhTg/mCfhKO and C57BL/6J mice, and isolated RNA from these RPE cells. We ran Illumina microarray chips on these samples, and performed a pathway analysis on the resulting genes. QPCR was used to test a subset of these genes and also an independent group of candidate genes for qPCR analysis. We also isolated RNA from the RPE (and also the retina separately) of 8m RD8+/+ vs. B6-wild type (WT) mice and performed qPCR for candidate genes.
Results:
We were able to isolate enough RNA from the RPE layer (and overlying subretinal microglia) from individual eyecups to perform Illumina and qPCR testing. QPCR confirmed Illumina findings: an increased expression of CD68 and TREM2, and a decreased expression of SNCA in CfhTg mice vs. B6 controls. Some candidate genes were also found to be increased in CfhTg/mCfhKO mice including: IP-10, NLRP3 and C4. In RD8 +/+ mice, qPCR of candidate genes revealed an increased expression of Ccl-2, CFB, C3, NF-kB and CD200R in the RPE compared to WT mice. The retina of RD8 +/+ mice showed an increased expression of oxidative stress marker HO-1, and complement components C1q and C4 compared to WT.
Conclusions:
The 402H variant of Cfh can lead to an increased expression of inflammatory markers in the RPE. The RD8 mutation, which causes a retinal degeneration, also leads to a distinct increase in inflammatory gene expression in the RPE, and an increase in oxidative stress and complement activation markers in the retina.
Keywords: 412 age-related macular degeneration •
539 genetics •
533 gene/expression