Inherited and age-related macular degenerations (AMDs) are important causes of vision loss. An early hallmark of these disorders is the formation of sub-retinal pigment epithelium (RPE) basal deposits. A role for the complement system in MDs was suggested by genetic association studies, but direct functional connections between alterations in the complement system and the pathogenesis of MD remain to be defined. We used primary RPE cells from a mouse model of inherited MD due to a p.R345W mutation in EGF-containing fibulin-like extracellular matrix protein 1 (EFEMP1) to investigate the role of the RPE in early MD pathogenesis. Efemp1(R345W) RPE cells recapitulate the basal deposit formation observed in vivo by producing sub-RPE deposits in vitro. The deposits share features with basal deposits, and their formation was mediated by EFEMP1(R345W) or complement component 3a (C3a), but not by complement component 5a (C5a). Increased activation of complement appears to occur in response to an abnormal extracellular matrix (ECM), generated by the mutant EFEMP1(R345W) protein and reduced ECM turnover due to inhibition of matrix metalloproteinase 2 by EFEMP1(R345W) and C3a. Increased production of C3a also stimulated the release of cytokines such as interleukin (IL)-6 and IL-1B, which appear to have a role in deposit formation, albeit downstream of C3a. These studies provide the first direct indication that complement components produced locally by the RPE are involved in the formation of basal deposits. Furthermore, these results suggest that C3a generated by RPE is a potential therapeutic target for the treatment of EFEMP1-associated MD as well as AMD.

Macular degenerations, inherited and age related, are important causes of vision loss. Human genetic studies have suggested perturbation of the complement system is important in the pathogenesis of age-related macular degeneration. The mechanisms underlying the involvement of the complement system are not understood, although complement and inflammation have been implicated in drusen formation. Drusen are an early clinical hallmark of inherited and age-related forms of macular degeneration. We studied one of the earliest stages of macular degeneration which precedes and leads to the formation of drusen, i.e. the formation of basal deposits. The studies were done using a mouse model of the inherited macular dystrophy Doyne Honeycomb Retinal Dystrophy/Malattia Leventinese (DHRD/ML) which is caused by a p.Arg345Trp mutation in EFEMP1. The hallmark of DHRD/ML is the formation of drusen at an early age, and gene targeted Efemp1(R345W/R345W) mice develop extensive basal deposits. Proteomic analyses of Bruch's membrane/choroid and Bruch's membrane in the Efemp1(R345W/R345W) mice indicate that the basal deposits comprise normal extracellular matrix (ECM) components present in abnormal amounts. The proteomic analyses also identified significant changes in proteins with immune-related function, including complement components, in the diseased tissue samples. Genetic ablation of the complement response via generation of Efemp1(R345W/R345W):C3(-/-) double-mutant mice inhibited the formation of basal deposits. The results demonstrate a critical role for the complement system in basal deposit formation, and suggest that complement-mediated recognition of abnormal ECM may participate in basal deposit formation in DHRD/ML and perhaps other macular degenerations.

Mouse models are powerful tools for the study of ocular diseases. alterations in the morphology and function of the retinal pigment epithelium (RPE) are common features shared by many ocular disorders. We report a detailed protocol to collect, seed, culture and characterize RPE cells from mice. We describe a reproducible method that we previously developed to collect and culture murine RPE cells on transwells as functional polarized monolayers. the collection of RPE cells takes ~3 h, and the cultures mimic in vivo RPE cell features within 1 week. this protocol also describes methods to characterize the cells on transwells within 1–2 weeks by transmission and scanning electron microscopy (teM and seM, respectively), immunostaining of vibratome sections and flat mounts, and measurement of transepithelial electrical resistance. the RPE cell cultures are suitable to study the biology of the RPE from wild-type and genetically modified strains of mice between the ages of 10 d and 12 months. the RPE cells can also be manipulated to investigate molecular mechanisms underlying the RPE pathology in the numerous mouse models of ocular disorders. Furthermore, modeling the RPE pathology in vitro represents a new approach to testing drugs that will help accelerate the development of therapies for vision-threatening disorders such as macular degeneration (MD).

The design of efficient therapies for age-related macular degeneration (AMD) is limited by our understanding of the pathogenesis of basal deposits, which form between retinal pigment epithelium (RPE) and Bruch's membrane (BrM) early in disease, and involve activation of the complement system. To investigate the roles of BrM, RPE and complement in an AMD, we generated abnormal extracellular matrix (ECM) using CRISPR-edited ARPE-19 cells. We introduced to these cells the p.R345W mutation in EFEMP1, which causes early-onset macular degeneration. The abnormal ECM binds active complement C3 and causes the formation of basal deposits by normal human fetal (hf)RPE cells. Human fetal RPE (hfRPE) cells grown on abnormal ECM or BrM explants from AMD donors show chronic activation of the alternative complement pathway by excessive deposition of C3b. This process is exacerbated by impaired ECM turnover via increased matrix metalloproteinase-2 activity. The local cleavage of C3 via convertase-independent mechanisms can be a new therapeutic target for early AMD.

CNV mapping substantially increased the genetic diagnostic rate of IRDs, detecting genetic causality in 18% of previously unsolved cases. Extending the search to other structural variations will probably demonstrate an even higher contribution to genetic causality of IRDs.Genet Med advance online publication 13 October 2016.