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Differential and Altered Spatial Distribution of Complement Expression in Age-Related Macular Degeneration
John T Demirs1, Junzheng Yang1, Maura A Crowley1
1Department of Ophthalmology, Novartis Institutes for Biomedical Research, Cambridge, Massachusetts, United States.
Insights
Complement dysregulation drives age-related macular degeneration (AMD). Complement mRNA and protein are highest in the RPE/choroid, suggesting this as a key target for geographic atrophy (GA) treatments.
Area of Science:
- Ophthalmology
- Immunology
- Genetics
Background:
- Age-related macular degeneration (AMD) pathogenesis involves complement pathway dysregulation.
- The role of locally synthesized complement components in AMD requires further investigation.
Purpose of the Study:
- To profile complement expression in postmortem eyes with and without AMD.
- To determine if locally synthesized complement components contribute to AMD.
Main Methods:
- Analysis of postmortem fundus images and histological sections to grade AMD severity.
- TaqMan and RNAscope/in situ hybridization to detect complement mRNA.
- Meso scale discovery assay and Western blot to measure complement protein levels.
Main Results:
- Complement mRNA and protein levels were significantly higher (15- to 100-fold) in macular retinal pigment epithelium (RPE)/choroid compared to neural retina.
- Modest elevations in complement mRNA and protein were observed in vitreous and neural retina of eyes with geographic atrophy (GA), but not early/intermediate AMD.
- Complement mRNAs (C3, CFB, CFH, CFI, C1QA) were prominent in GA areas, with C3 mRNA found in microglia/macrophages.
Conclusions:
- The RPE/choroid is the primary ocular site for complement, making it a key target for GA and earlier AMD treatment strategies.
- Local production of complement mRNA in the outer retina and increased complement activation are characteristic of GA.
Purpose:
Dysregulation of the alternative complement pathway is a major pathogenic mechanism in age-related macular degeneration. We investigated whether locally synthesized complement components contribute to AMD by profiling complement expression in postmortem eyes with and without AMD.
Methods:
AMD severity grade 1 to 4 was determined by analysis of postmortem acquired fundus images and hematoxylin and eosin stained histological sections. TaqMan (donor eyes n = 39) and RNAscope/in situ hybridization (n = 10) were performed to detect complement mRNA. Meso scale discovery assay and Western blot (n = 31) were used to measure complement protein levels.
Results:
The levels of complement mRNA and protein expression were approximately 15- to 100-fold (P < 0.0001-0.001) higher in macular retinal pigment epithelium (RPE)/choroid tissue than in neural retina, regardless of AMD grade status. Complement mRNA and protein levels were modestly elevated in vitreous and the macular neural retina in eyes with geographic atrophy (GA), but not in eyes with early or intermediate AMD, compared to normal eyes. Alternative and classical pathway complement mRNAs (C3, CFB, CFH, CFI, C1QA) identified by RNAscope were conspicuous in areas of atrophy; in those areas C3 mRNA was observed in a subset of IBA1+ microglia or macrophages.
Conclusions:
We verified that RPE/choroid contains most ocular complement; thus RPE/choroid rather than the neural retina or vitreous is likely to be the key site for complement inhibition to treat GA or earlier stage of the disease. Outer retinal local production of complement mRNAs along with evidence of increased complement activation is a feature of GA.
