Two-vector AAV system effectively transduces mouse brain, and GADD34 control AAV transduction does not elevate levels of BACE1 or phosphorylated eIF2α in the brain.

Abstract

<p>5XFAD or non-Tg pups were injected on postnatal day 0 into lateral ventricles with 2 µl per hemisphere containing 6.6×10<sup>10</sup> viral genomes of GADD34 CA-AAV or GADD 34 cont–AAV plus 6.9×10<sup>10</sup> viral genomes of CaMKII tTA-AAV. Mice were aged to 6 months and brains harvested for immunoblot, and immunofluorescence microscopy analysis. (A) GFP fluorescence in coronal brain sections of 6 month-old 5XFAD mice injected with GADD 34 cont–AAV (left column) or GADD34 CA-AAV (right column) shows comparable expression levels of GFP from both transduced AAV vectors. Upper row: entire hemibrain sections showing wide-spread GFP expression, especially in the hippocampus. Lower row: lower exposure of hippocampus showing cellular GFP expression. The AAV serotype 1 with the CaMKII promoter effectively drives expression in excitatory forebrain neurons, with particularly high expression in the hippocampus. Scale bar  =  1 mm (top row), 250 µm (bottom row). (B) Low exposure high magnification image of a section of the hippocampus from a mouse transduced with GADD34 CA-AAV stained with an antibody against GADD34 (red) shows high co-localization of GADD34 CA and GFP in neurons of the CA regions, indicating that GFP fluorescence is an effective proxy marker for GADD34 expression Scale bar = 250 µm. (C) 20 µg/lane of cortex or hippocampus homogenate from 6 month-old 5XFAD (+) and non-Tg (–) mice either uninjected (uninj) or GADD 34 cont–AAV injected (cont inj) were subjected to immunoblot analysis for BACE1, total eIF2α, phosphorylated (p)-eIF2α, and βIII-tubulin as a loading control. All samples were transferred onto a single piece of PVDF membrane, as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0101643#s2" target="_blank">Methods</a>, and representative blots are shown here. (D) BACE1 immunosignal intensities were normalized to those of βIII-tubulin. Phosphorylated and total eIF2α immunosignal intensities were measured and phosphorylated:total eIF2α (phospho/total eIF2α) ratio calculated. All measures are displayed as percentage of uninjected non-Tg control. Comparison of GADD34 cont–AAV injected mice with genotype-matched uninjected mice shows that there is no effect on BACE1 or p-eIF2α levels from AAV brain injection itself. n = 9–15 mice per group. Bars represent SEM. Asterisks (*) indicate significant difference from non-Tg uninj p<0.01**, p<0.001***.</p

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