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Manisha Dagar, Anil Kumar Chekuri, Pooja Biswas, Shikha Pachauri, Donita Garland, Radha Ayyagari; The role of the Complement pathway in L-ORD pathology caused by mutations in CTRP5.. Invest. Ophthalmol. Vis. Sci. 2022;63(7):1949 – F0367.
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Late-onset retinal degeneration (L-ORD) is an autosomal dominant disorder caused by mutations in the C1q-tumor necrosis factor-5 (CTRP5/C1QTNF5) gene. The purpose of this study was to understand the pathology of L-ORD by analysis of the proteome of the Retinal Pigment Epithelium-Choroid (RPE-Ch) tissue in mouse models carrying the S163R Ctrp5 mutation. The current study mainly focuses on components of the complement pathway as a function of age.
The proteomes of the RPE-Ch, which include Bruch’s membrane (BrM), were analyzed in wild-type (WT), heterozygous S163R Ctrp5 mutation knock-in (Ki/WT), and homozygous knock-in (Ki/Ki) mice of approximately 5 and 18 months (m) of age. The mass spectral data were analyzed using MaxQuant software for the identification and quantification of the levels of peptides in the RPE-Ch. The proteins identified in the analysis were further experimentally validated in the RPE-Ch tissue of 5 and 18 m old mutant and WT mice by immunohistochemistry (IHC) and western blotting.
Components of the three complement pathways were present in the WT and mutant mouse proteome of RPE-Ch. Five components of the classical pathway were observed to increase over 2 fold (p<.05) in WT and mutant mice with age. C4b was increased 2.7 and 3.9 fold with age in WT and Ki/WT, respectively. Both C1qb and C1qc were increased 2 and 3 fold with age in WT and Ki/WT, respectively. CFH, which is involved in the alternative pathway increased 1.4 fold (p<.05) with age in the WT mice and was higher in the Ki/WT but it did not reach significance. IHC was used to assess the levels of the components of the alternative complement pathway CFB, CFH, and C3, specifically in BrM. At 18 m of age, the levels of each protein were higher in mutant compared to WT mice. These observations were further validated using western blot analysis. CFB showed an increase by 2.1 fold in Ki/WT and 2.9 fold in Ki/Ki compared to WT. CFH increased by 1.35 fold in Ki/WT and 1.5 fold in Ki/Ki.
Proteome analysis of RPE-Ch revealed an increase in members of the classical complement pathway with age in the WT and L-ORD mouse models. The levels of components of the alternative pathway (CFB, CFH, and C3) were increased in BrM-Ch of mutant mice at 18 m. This study provides insight and support for the potential role of the complement system in L-ORD pathology.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.
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