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Albert H. Alhatem, Nataliya Lenchik, Sarka Beranova-Giorgianni, Mikael Klingeborn, David D New, Francesco Giorgianni, Ivan Gerling, Marko Radic, Catherine Bowes Rickman, Alessandro Iannaccone; The Apolipoprotein E4 Targeted Replacement (APOE4 TR) Mouse Model of Age-Related Macular Degeneration (AMD) Exhibits Anti-Retinal Autoreactivity. Invest. Ophthalmol. Vis. Sci. 2014;55(13):1173.
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© ARVO (1962-2015); The Authors (2016-present)
To test the hypothesis that auto-antibodies (AAbs) recognizing ocular tissue antigens develop in the APOE4 TR mouse model of AMD.
APOE4 TR mice aged at least 65 weeks (~15 mo) develop an AMD-like phenotype after being fed high fat cholesterol-enriched diet (HFCD) for 8-10 wks (Malek et al. PNAS 2005; 102: 11900-05). Western blots (WBs) were performed by reacting sera collected at 24 mos from APOE4 TR-HFCD mice and control APOE4 TR mice on normal diet (ND) against retina/RPE/BM/choroid tissue lysates of adult C57BL/6 mice. Lysates (10 µg) were loaded on gels, incubated with 5µL of serum, and developed. The intensity of the bands seen on WB was quantified with the Odyssey system and compared by student’s T-test. Immunohistochemistry (IHC) was performed against anti-mouse IgG antibody by fluorescence microscopy following incubation of adult C57BL/6 mouse retina sections with mouse sera. To identify the autoantigens, APOE4 TR-HFCD sera were immunoprecipitated, followed by 2-dimension electrophoresis (2DE) and liquid chromatography-tandem mass spectrometry (LC-MS/MS), performed on spots seen on 2DEs with APOE4 TR-HFCD sera following previously reported methods (Lenchik et al. ARVO 2013, Abs. 4103).
Significantly more intensely reactive bands were observed in sera of APOE4 TR-HFCD compared to APOE4 TR-ND mice. The most robust reactivities were seen at 17kDa (p= 0.005 after 1-hr incubation, p= 0.000007 after 3 hrs) and 13kDa (p= 0.005, 1 hr; p=0.001, 3 hrs). Additional reactivities were seen after 1-hr incubation at 79kDa (p= 0.013), 47kDa (p= 0.028), and 20kDa (p=0.046). IHC studies showed moderate and diffuse staining of C57BL/6 mouse retinal sections at the outer nuclear layer level. No staining was seen with APOE4 TR-ND sera. Differentially reactive spots were also observed on 2DE between APOE4 TR-HFCD and APOE4 TR-ND sera, and IDs are being investigated by LC-MS/MS.
As we have previously shown in the serum of human AMD (Iannaccone et al. Adv. Exp. Med. Biol. 2012; 723:11-6), AAbs recognizing retinal targets develop in this animal model of AMD, suggesting a stereotyped response to AMD-like retinal degenerative events that incites a secondary autoimmune component and that has the potential to further retinal damage.
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