Insights into Caveolin-1 Function: Quantitative Proteomic Analysis of a Novel Retina-Specific Knockout Mouse Model

Abstract

Purpose: : Caveolin-1 (Cav-1) is a lipid raft protein implicated in autoimmune uveitis, diabetic retinopathy, and glaucoma. Given its broad tissue distribution, Cav-1 functions in the retina have been difficult to elucidate. We developed a mouse model in which Cav-1 was specifically deleted in the retina (ret-CavKO) leaving other tissues Cav-1-competent. To determine retina-intrinsic Cav-1 functions, we carried out quantitative proteomic analyses of ret-CavKO.

Methods: : Ret-CavKO mice were generated by crossing Cav-1 "floxed" mice (loxP sites inserted flanking exon 2) with mice expressing Cre recombinase under control of the Chx10 promoter. Controls were floxed littermates that did not express Cre. Retinal membrane proteins were subjected to tryptic digestion and analyzed by liquid chromatography mass spectrometry (LC-MSMS, OrbiTrap). Peptides were aligned, statistically analyzed for differential abundance, and identified by database search (Ensembl, species Mus musculus). Gene Set Enrichment Analysis (KEGG pathways, gene ontologies, transcription factor binding sites) and Ingenuity Pathway Analysis identified functions and canonical pathways affected by retina-specific Cav-1 deletion. Candidate proteins were validated by Western blotting and immunohistochemistry (IHC).

Results: : Deletion efficiency assessed by Western blotting of retinal proteins indicated >70% reduction in Cav-1 in agreement with quantitative proteomics. Given that, by design, the retinal vasculature retained Cav-1 expression, we estimate the deletion efficiency to approach 90% in cells specifically targeted by Chx10-driven Cre. By IHC, Cav-1 ablation was evident in Müller glia which abundantly express Cav-1. As designed, expression was retained in retinal vasculature and RPE demonstrating specificity. Quantitative proteomics identified 1591 proteins of which 233 were differentially represented between ret-CavKO and control (p ≤ 0.05). Both KEGG and Ingenuity analyses identified transmembrane molecular transport as the major downregulated pathway in ret-CavKO. Several amino acid transporters (glutamate, glycine, glutamine) and nearly all solute carriers were downregulated. Oxidative metabolism and mitochondrial dysfunction were also significantly represented with nearly all redox carrier complexes affected.

Conclusions: : Our results suggest that Cav-1 plays a key role in Müller glial transport activities and that loss of Cav-1 in these cells leads to metabolic dysfunction in the retina.

Keywords: Muller cells • transgenics/knock-outs • proteomics