Deletion of classical complement component C1q exacerbates BRB dysfunction in a mouse model with impaired Norrin signaling

Abstract

Purpose : Blood-retina barrier (BRB) dysfunction can cause or drive retinal disease, for example cystoid macular edema (CME). Previously, we reported that Tspan12 endothelial-cell specific KO (ECKO) retinas display BRB defects, cystoid edema, and increased classical complement pathway components due to loss of norrin/frizzled4 signaling. Classical complement components may drive pathological changes downstream of barrier defects, or alternatively, have protective roles, for example by activating beta-catenin-dependent signaling through LRP5/6. Here, we sought to evaluate the role of the classical complement pathway in BRB dysfunction using a mouse model of pharmacologically and genetically manipulating norrin/frizzled4 signaling.

Methods : Frizzled4/LRP5 agonist (F4L5.13) was intraperitoneally injected into Tspan12 KO mice from P6 to P27 (every 3 days) to restore norrin/frizzled4 signaling and angiogenesis until P30 and subsequently observe BRB maintenance defects after discontinuation of treatment. Cohorts of WT, C1qa KO and Tspan12 KO; C1qa KO compound mutant mice (the latter treated with F4L5.13 agonist) were used for comparison. Fluorescein angiography and ERG were performed longitudinally from P30 to 12 months. Image-guided OCT was used to evaluate cystoid edema at 6 month and 9 month timepoints. Sulfo-NHS-biotin tracer was used to evaluate the retinal vascular leakage. Wholemount immunostaining was used to examine microglia number. Topflash luciferase reporter assay was used to measure canonical Wnt/beta-catenin pathway activation in cultured 293T cells.

Results : F4L5.13 administration fully restored BRB function at P30 and BRB and ERG b-wave defects gradually appeared in Tspan12 KO mice and Tspan12; C1qa compound mutant mice after the agonist administration was discontinued. Tspan12; C1qa compound deletion led to increased retinal vascular leakage of sulfo-NHS biotin, increased cystoid edema, and increase of IBA-1 positive retinal immune cells. Topflash luciferase reporter assays revealed that canonical Wnt signaling activity was reduced in 293T cells cultured with serum from C1qa KO mice vs. WT mouse serum.

Conclusions : C1qa contributes to maintaining beta-catenin-dependent signaling in retinal ECs and C1qa deletion exacerbates the pathological consequences of BRB dysfunction in Tspan12 KO mice.

This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.