Genetic reduction of eIF2α phosphorylation via eIF2α S51A knockin mutation does not block amyloid-associated BACE1 elevation in 5XFAD brain.

Abstract

<p>5XFAD mice were crossed with mice harboring the eIF2α S51A targeted replacement mutation to generate 5XFAD (+) or non-Tg (–) offspring that were either heterozygous for the eIF2α S51A knockin mutation (S/A) or wild-type (S/S). Mice were aged to 12 months, brains harvested, and homogenates prepared. 20 µg/lane of brain homogenate were subjected to immunoblot analysis for transgenic human (h) APP, BACE1, total eIF2α, and phosphorylated (p)-eIF2α. All samples were transferred onto a single piece of PVDF membrane and stained with ponceau S as a protein loading control, as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0101643#s2" target="_blank">Methods</a>. For quantification, BACE1 immunosignal intensity was normalized to ponceau S staining intensity for a given lane. Phosphorylated and total eIF2α immunosignal intensities were measured and phosphorylated:total eIF2α (phospho/total eIF2α) ratio calculated for a given lane. The means of each group were calculated and means displayed as percentage of the mean non-Tg S/S control. Both non-Tg and 5XFAD mice that were also heterozygous for the eIF2α S51A knockin mutation had a ∼40% reduction in phospho/total eIF2α ratio compared to their S/S counterparts; presumably, the phospho/total eIF2α ratios did not reach the expected 50% reduction because of a high non-specific background on the p-eIF2α immunoblot or partial compensatory increased phosphorylation of the wild type allele. Importantly, BACE1 level in 5XFAD; S/A brain showed an amyloid-associated elevation that was equivalent to that of 5XFAD; S/S brain, despite the 40% reduction in phospho/total eIF2α ratio. n = 19–30 mice per group. Bars represent SEM, asterisks (*) indicate significant changes compared to non-Tg S/S control, p<0.05*, p<0.01**, p<0.001***, NS  =  not significant, (#) indicates significant difference between 5XFAD S/S and 5XFAD S/A p<0.001 ###.</p

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