Macular degeneration (MD) is the loss of structure and function of the macula; the central region of the retina that provides detailed vision. Age-related macular degeneration (AMD) is the major cause of visual impairment in developed countries. However, to date, there is no effective treatment for dry AMD, the most prevalent form of the disease.
Inherited and age-related macular degenerations share clinical features. An early hallmark of these disorders is alteration of the extracellular matrix (ECM) of Bruch’s membrane, with accumulation of basal deposits and drusen between the retinal pigment epithelium (RPE) and Bruch’s membrane. 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.
Using a mouse model of the inherited EFEMP1-associated macular degeneration we demonstrated that the complement system, specifically C3, plays a key role in the formation of basal deposits (Figure 1) (1-5).
Figure1. Primary RPE cells from Efemp1R345W mice recapitulate deposit formation in vitro.
Transmission electron microscopy (TEM) (A-F) and scanning electron microscopy (SEM) (G-I) of polarized RPE monolayers grown on transwells for 2 weeks show apical microvilli, basal infoldings and tight junctions (white arrows) (A-C). B1 shows a cellular projection growing through a pore of the insert (black arrow) (notice that the pore appears wider than those shown in flat mounts due to the sectioning process). Thick deposits (white arrow) spread across the bottom side of the insert in KI cultures (E), while very thin cellular projections are barely visible in WT (D) and KIC3KO (F). SEM from bottom flatmounts shows thin cellular projections (arrows) in WT (G, J) and KIC3KO (I, L), where pores (white arrowheads) can be seen between cellular material (J, L). KI cultures present overlapping layers of deposits (H), which completely cover the pores of the insert (K). J, K, L are higher magnifications from black squares in G, H, I respectively. Scale bar A-F, 5μm, G-L, 10μm. KI = Efemp1R345W and KIC3KO = Efemp1R345W:C3-/-. Thickness of the insert = 10μm.
We are now investigating the roles of the ECM and complement system in early macular degeneration using human RPE cell culture systems. We have found that abnormal ECM, produced either by the disease causing p.R345W mutation in EFEMP1 of by aging, stimulates the activation of complement system via cleavage of complement factor 3 (C3).
Figure 2. SEM of decellularized transwells of CRISPR-engineered ARPE-19 wildtype and EFEMP1R345/R345W cultures show abnormal ECM made by mutant cells, which anchors active complement components C3b and CFH. HfRPE cells grown on the abnormal ECM make sub-RPE deposits that are visible by SEM after decellularization. The deposits are comprised of ECM proteins that are typical of basal deposits in AMD patients. Scale bars 10µm.
The data further suggested that C3 produced by RPE cells is activated likely via tick-over and deposited in excess on abnormal ECM. The fluid-phase C3-convertase stabilizes on the ECM and triggers a local chronic activation of the alternative complement pathway. Alternatively, the resulting C3a can stimulate normal RPE cells to produce basal deposits, via a process that depends on C3a binding to its cell surface receptor.
These findings demonstrate that alterations in the ECM and subsequent increase in the activation state of the complement system underlie both inherited and age-related macular degeneration. They also suggest that strategies to prevent the production or function of C3a could have potential for the treatment of early macular degeneration.
1. Fu L, Garland D, Yang Z, Shukla D, Rajendran A, Pearson E, Stone EM, Zhang K, Pierce EA. The R345W mutation in EFEMP1 is pathogenic and causes AMD-like deposits in mice. Hum Mol Genet. 2007 Oct 15;16(20):2411-22. Epub 2007 Jul 30. PMID:17666404
2. Garland DL, Fernandez-Godino R, Kaur I, Speicher KD, Harnly JM, Lambris JD, Speicher DW, Pierce EA. Mouse genetics and proteomic analyses demonstrate a critical role for complement in a model of DHRD/ML, an inherited macular degeneration. Human Molecular Genetics 2014 Jan 1;23(1):52-68. PMID:23943789
3. Fernandez-Godino R, Garland DL, Pierce EA. A Local Complement Response by RPE Causes Early-Stage Macular Degeneration. Human Molecular Genetics 2015 Oct 1;24(19):5555-69. PMID: 26199322.
5. Fernandez-Godino R, Garland DL and Pierce EA. A protocol for the isolation, culture and characterization of polarized primary RPE cells from mice. Nature protocols. 2016 Jul;11(7):1206-18. PMID: 27281648