Localization of complement factor H gene expression and protein distribution in the mouse outer retina

Area of Science:

• Ophthalmology
• Immunology
• Molecular Biology

Background:

• Complement factor H (Cfh) plays a crucial role in regulating the complement system.
• Understanding Cfh localization in the retina is vital for comprehending its function in ocular health and disease.

Purpose of the Study:

• To precisely determine the localization of complement factor H (Cfh) mRNA and its corresponding protein within the mouse outer retina.

Main Methods:

• Quantitative real-time PCR (qPCR) to analyze Cfh and Cfh-related (Cfhr) transcript expression in the RPE/choroid.
• In situ hybridization (ISH) using the RNAscope 2.0 FFPE assay for Cfh mRNA localization in the outer retina.
• Immunohistochemistry (IHC) and western blotting to confirm Cfh protein localization and antibody specificity.

Main Results:

• qPCR detected Cfh and Cfhr2 transcripts in the RPE/choroid; other Cfhr transcripts were not found.
• ISH revealed abundant Cfh mRNA specifically in the retinal pigment epithelium (RPE) across tested mouse strains.
• Unexpectedly, IHC localized Cfh protein to photoreceptor cells, not RPE cells, with validated antibody specificity.

Conclusions:

• Only Cfh and Cfhr2 genes are expressed in the mouse outer retina.
• Cfh mRNA is present in the RPE, but the protein is not detected there, suggesting low steady-state levels due to secretion.
• The study highlights a novel localization of Cfh protein in photoreceptors, distinct from its mRNA in RPE.