Elucidating the Impact of C3b Degradation Products on Microglial Activation and Neurodegeneration in the Pathogenesis of Age-Related Macular Degeneration (AMD)

• Applicant: Professor Dr. Simon J. Clark
• Subject Area: Ophthalmology ; Biochemistry ; Immunology
• Term: since 2024
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 538134976

Age-related macular degeneration (AMD) is a degenerative eye disease causing blindness in the elderly population. The role of complement over-activation in AMD has given rise to many complement targeting therapeutics being developed. However, existing therapeutics have limited efficacy, probably because they have been solely focused on switching off complement turnover rather than also addressing the downstream opsonization effect of C3b breakdown products on retinal tissue remodelling through microglia/RPE mediated phagocytosis. Remarkably, we still do not fully understand the effect of each opsonin on microglia or RPE cell molecular pathways, and therefore how best to ‘switch-off’ the subsequent excessive tissue remodelling observed in AMD. Prof. Simon Clark aims to investigate the intricate molecular consequences on microglia/RPE after engagement with each C3b breakdown opsonin (i.e. C3b>iC3b>C3dg>C3d each showing decreasing opsonisation potency). To achieve this, phagocytic assays comprising microglia cultures with and without complement receptor 3 (CR3: the main receptor for opsonin) will be established and used to examine changes in microglial phagocytic activity by feeding C3b, iC3b, C3dg, and C3d opsonized microspheres and analyzing by live cell imaging and flow cytometry. This will determine the most potent opsonin for microglial mediated phagocytosis and explore whether inhibiting CR3 can reduce microglial activation. RNAseq and multiplex ELISAs will provide a comprehensive evaluation on how these opsonin influence significant changes in gene/protein and signalling pathways that will enhance our understanding of the microglial-mediated molecular mechanisms underlying AMD progression. The inclusion of RPE cells in a co-culture system will show us how microglia, stimulated by each opsonin, have consequential effects on RPE cells both in terms of morphology and functionality, including photoreceptor outer segment (POS) phagocytosis. Finally, to validate the findings, immunohistochemistry experiments will be performed using an array of bespoke opsonin-specific antibodies in early to late AMD human donor eye tissue samples. These results will elucidate how best to address complement-mediated opsonization and subsequent excessive tissue remodelling and turnover that could contribute to a greater slowing of the retinal degeneration observed with current therapeutic strategies.

• DFG Programme: Research Grants