Towards a Mouse Model of AMD: Chimeric Complement Factor H Molecules Carrying the Y402H Variants Are Functional in vivo

Abstract

Purpose: : Complement factor H (Cfh) is a key regulator of the alternative complement pathway, and in particular, of the final steps of the complement cascade. A mutation in Cfh (Y402H) increases the risk of AMD, and seems to blunt the prophylactic benefit of AREDS supplementation and the treatment benefit of anti-VEGF agents. Still, some argue that the genetic association is the result of linkage disequilibrium with a different mutation. We are working on developing a mouse model of AMD based on this genetic risk factor.

Methods: : CFH consists of 20 short consensus repeat (SCR) subunits. Y402H localizes to SCR7. We generated transgenic lines using two transgenic constructs consisting of the human SCR6-8 sequence flanked by the mouse SCR1-5 and SCR9-20 sequences (under the ApoE promoter). The constructs code for either a tyrosine or a histidine at amino acid position 402. We bred 2^nd or 3^rd generation Cfh transgenic lines with Cfh KO mice to eliminate mouse Cfh. The resulting lines were tested for mRNA levels, protein expression and functionality (C3 ELISA). We are following these mice for the development of clinical or histological AMD-like changes.

Results: : RT PCR analysis showed that the transgenic mRNA is expressed in the liver and in the posterior segment of the eye in our mice. Western blots showed that the protein is secreted into the circulation in good levels. The chimeric protein was found to be functional as demonstrated by its ability to rescue C3, which is depleted in Cfh KO mice. Clinical and histopathology data will also be presented.

Conclusions: : We have generated transgenic mouse lines which express our chimeric Cfh molecules in the absence of mouse Cfh, and have shown that both chimeric proteins (Y and H variants) are functional. A combination of a known genetic risk factor (e.g. mutation in Cfh) with increased oxidative stress (e.g. light) may result in a model of AMD combining pathogenetic relevance and suitability for experimental work.

Keywords: age-related macular degeneration • gene/expression • oxidation/oxidative or free radical damage