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Rajni Parthasarathy, Michael P Fautsch, David R Pepperberg; Quantification of amyloid-beta in mouse and human eye tissues. Invest. Ophthalmol. Vis. Sci. 2014;55(13):1317.
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© ARVO (1962-2015); The Authors (2016-present)
Elevated levels of amyloid-beta (Aβ) (38-43 amino acid peptide) are suspected to have toxic activity in brain degenerative diseases. Aβ’s roles in eye diseases, particularly those with degenerative characteristics such as age-related macular degeneration, glaucoma and diabetic retinopathy (1), remain unclear. As foundation for future studies addressing this topic, we determined the levels of Aβ40 and Aβ42 (two prominent Aβ forms) in posterior eye tissues of 5XFAD mice, a transgenic strain that overexpresses human Aβ42 (2). Additionally, we quantified Aβ in vitreous from human donor eyes.
Combined retina, RPE and choroid of 5XFAD mouse eyes (4-10 months of age) were supplemented with PBS and protease inhibitor cocktail 3 (Calbiochem), homogenized and centrifuged (3). Using ELISA, the recovered supernatant was analyzed for human Aβ42 (Innotest kit 80177; Innogenetics), for human/mouse Aβ40 (Covance kit 38954), and for total protein (Bradford assay). Aliquots of human vitreous were directly analyzed by ELISA and Bradford.
Aβ determinations in 10 samples of 5XFAD tissue extract yielded 38.8 ± 14.8 pg/mL Aβ42 (range: 26.4 - 59.8 pg/mL) and 139.3 ± 33.0 pg/mL Aβ40 (range:105.1 - 179.4 pg/mL). As normalized to total protein, the results were: 3.2 ± 1.2 pmol Aβ42 per g protein and 11.8 ± 2.8 pmol Aβ40 per g protein. The analyses of human vitreous yielded 53.7 ± 39.5 pg/mL Aβ42 (range:1.2 - 144.5 pg/mL; 34 eyes from 21 individuals), 537.6 ± 193.7 pg/mL Aβ40 (range: 48.2 - 918.3 pg/mL; 22 eyes from 11 individuals), and total protein-normalized concentrations of 8.7 ± 5.9 pmol Aβ42 per g protein and 83.7 ± 36.0 pmol Aβ40 per g protein.
The 5XFAD results expand knowledge of ocular Aβ levels in this transgenic strain (2) and indicate readily detectable Aβ42 and Aβ40 in the posterior eye tissues. The human vitreous data show similarity to previous determinations of Aβ42 (4) and provide the first indication of relative Aβ42 and Aβ40 concentrations. These findings will facilitate studies to examine the effects, on Aβ levels, of biomolecules with hypothesized therapeutic action in retinal degenerations. (1) Ohno-Matsui K (2011) Prog. Retin. Eye Res. 30:217-238. (2) Alexandrov PN et al. (2011) NeuroReport 22:623-627. (3) Dutescu RM et al. (2009) Graefe’s Arch. Clin. Exper. Ophthalmol. 247:1213-1221.(4) Yoneda S et al. (2005) Jpn. J. Ophthalmol. 49:106-108.
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