Quantification of amyloid-beta in mouse and human eye tissues

Abstract

Purpose: 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.

Methods: 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.

Results: 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.

Conclusions: 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.

Keywords: 412 age-related macular degeneration • 494 degenerations/dystrophies • 763 vitreous