Liver X receptor agonist treatment significantly affects phenotype and transcriptome of APOE3 and APOE4 <i>Abca1</i> haplo-deficient mice

<div><p>ATP-binding cassette transporter A1 (ABCA1) controls cholesterol and phospholipid efflux to lipid-poor apolipoprotein E (APOE) and is transcriptionally controlled by Liver X receptors (LXRs) and Retinoic X Receptors (RXRs). In APP transgenic mice, lack of <i>Abca1</i> increased Aβ deposition and cognitive deficits. <i>Abca1</i> haplo-deficiency in mice expressing human APOE isoforms, increased level of Aβ oligomers and worsened memory deficits, preferentially in APOE4 mice. In contrast upregulation of <i>Abca1</i> by LXR/RXR agonists significantly ameliorated pathological phenotype of those mice. The goal of this study was to examine the effect of LXR agonist T0901317 (T0) on the phenotype and brain transcriptome of APP/E3 and APP/E4 <i>Abca1</i> haplo-deficient (APP/E3/Abca1^+/- and APP/E4/Abca1^+/-) mice. Our data demonstrate that activated LXRs/RXR ameliorated APOE4-driven pathological phenotype and significantly affected brain transcriptome. We show that in mice expressing either APOE isoform, T0 treatment increased mRNA level of genes known to affect brain APOE lipidation such as <i>Abca1</i> and <i>Abcg1</i>. In both APP/E3/Abca1^+/- and APP/E4/Abca1^+/- mice, the application of LXR agonist significantly increased ABCA1 protein level accompanied by an increased APOE lipidation, and was associated with restoration of APOE4 cognitive deficits, reduced levels of Aβ oligomers, but unchanged amyloid load. Finally, using Gene set enrichment analysis we show a significant APOE isoform specific response to LXR agonist treatment: Gene Ontology categories “Microtubule Based Process” and “Synapse Organization” were differentially affected in T0-treated APP/E4/Abca1^+/- mice. Altogether, the results are suggesting that treatment of APP/E4/Abca1^+/- mice with LXR agonist T0 ameliorates APOE4-induced AD-like pathology and therefore targeting the LXR-ABCA1-APOE regulatory axis could be effective as a potential therapeutic approach in AD patients, carriers of <i>APOEε4</i>.</p></div

APOE isoform-specific effect on gene expression.

<p><b>A,</b> Comparison between APP/E4/Abca1^+/- (vehicle plus T0) and APP/E3/Abca1^+/- mice (vehicle plus T0). The volcano plot shows differential gene expression between APP/E4/Abca1^+/- and APP/E3/Abca1^+/- mice. Data were analyzed using EdgeR and the volcano plots are built using p < 0.05 cut-off. Up- and Down-regulated genes are represented in red and blue respectively. On <b>B</b> and C are shown genes that are up- or down-regulated in APP/E4/Abca1^+/- mice B, shown is RNA-seq result for genes that are significantly upregulated in APP/E4/Abca1^+/- vs APP/E3/Abca1^+/- mice. <b>C,</b> shown is RNA-seq result for genes that are significantly down-regulated in APP/E4/Abca1^+/- vs APP/E3/Abca1^+/- mice. *, p < 0.05, **, p < 0.01, ***, p < 0.001.</p

T0 treatment significantly decreased soluble Aβ oligomers, but not amyloid plaque pathology in APP/E4/Abca1^+/- mice.

<p>Amyloid plaque pathology of mice shown on <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0172161#pone.0172161.g001" target="_blank">Fig 1</a> was assessed by immunohistochemistry and ELISA. <b>A,</b> Brain sections were stained with X-34 to visualize compact fibrillary amyloid plaques in vehicle and T0 treated APP/E3/Abca1^+/- and APP/E4/Abca1^+/- mice. Representative images of X-34 staining were captured at 10× magnification. <b>B,</b> X-34 positive amyloid plaques were analyzed by two-way ANOVA. There is no interaction between <i>APOE</i> genotype and T0 treatment and a significant main effect of <i>APOE</i> genotype (F (1, 55) = 34.7, p < 0.0001), but not of T0 treatment. N = 14–16 mice per group. N.S., not significant. <b>C,</b> Brain sections were stained with anti-Aβ antibody, 6E10, to visualize diffuse and compact (total) amyloid plaques in vehicle and T0 treated APP/E3/Abca1^+/- and APP/E4/Abca1^+/- mice. Representative images of 6E10 staining are shown (10× magnification). <b>D,</b> 6E10 positive amyloid plaque load analyzed by two-way ANOVA. There is no interaction between <i>APOE</i> genotype and T0 treatment and a significant main effect of <i>APOE</i> genotype (F(1, 19) = 4.41, p = 0.049), but not of T0 treatment. N = 5–6 mice per group. N.S., not significant. <b>E,</b> T0 treatment significantly decreases Aβ oligomers in APP/E4/Abca1^+/- mice. RIPA fraction was evaluated for soluble Aβ by Aβ oligomer ELISA. Analysis by two-way ANOVA revealed an interaction between <i>APOE</i> genotype and T0 treatment (F(1, 32) = 4.82, p = 0.036). Sidak’s post-test demonstrated a significant difference between vehicle treated APP/E3/Abca1^+/- and APP/E4/Abca1^+/- mice (***, p<0.001) and T0 and vehicle treated APP/E4/Abca1^+/- mice (*, p<0.05). N = 6–10 mice per group. <b>F,</b> T0 has no effect on full-length APP. For all panels the data are means ±SEM.</p

T0 treatment increases ABCA1 protein level and APOE lipidation.

<p>ABCA1, APOE and APOJ protein levels were determined by SDS-PAGE and APOE lipidation by Native PAGE. <b>A,</b> Representative image of ABCA1 protein level is shown above the graph. T0 significantly affected ABCA1 protein level. Analysis by two-way ANOVA shows no interaction between <i>APOE</i> genotype and T0 treatment. There is a significant main effect of T0 treatment (F(1, 34) = 26.12, p < 0.0001), but not of <i>APOE</i> genotype. Sidak’s post-test shows a significant difference between T0 and vehicle treated APP/E3/Abca1^+/- and APP/E4/Abca1^+/- mice. N = 8–10 mice per group. <b>B,</b> T0 treatment did not affect APOE or APOJ protein levels. N = 9–10 mice per group. <b>C,</b> APOE lipidation state in APP/E3/Abca1^+/- and APP/E4/Abca1^+/- mice. Representative images of APOE lipidation are shown: upper panel—male mice; lower panel—female mice (Fem). Arrows are indicative of lipidated APOE migrating at 12 nm. D, Quantification of native gel. Sidak’s post-test shows a significant difference between T0 and vehicle treated APP/E3/Abca1^+/- and APP/E4/Abca1^+/- mice. N = 4 mice per group. *, p < 0.05, ***, p<0.001.</p

Transcriptional analysis of T0 treated six month old APP/E3/Abca1^+/- and APP/E4/Abca1^+/- mice.

<p>We used total RNA extracted from cortices of APP/E3/Abca1^+/- APP/E4/Abca1^+/- male mice treated with T0 or vehicle and shown on Fug.1 and 2. <b>A,</b> Principle component analysis (PCA) plot shows two dimensional comparison (PC1 vs PC2) of <i>APOE</i> genotype and T0 treatment in APP/E3/Abca1^+/- (4 mice for vehicle and 5 for T0 treatment) and APP/E4/Abca1^+/- mice (5 mice per group). <b>B</b> and <b>D,</b> The volcano plots show differential gene expression between T0 treated APP/E3/Abca1^+/- (<b>B</b>) and APP/E4/Abca1^+/- (<b>D</b>) mice when compared to their vehicle treated counterparts using EdgeR RNA-sequencing results analysis. Significant up-regulated genes are represented in red, significantly down-regulated genes are represented in blue; the cut off is at p<0.05. Up-regulated genes represent target genes of T0 treatment. <b>C</b> and <b>E,</b> qPCR validation of upregulated genes in T0 treated APP/E3/Abca1^+/- and APP/E4/Abca1^+/- mice from the volcano plot analysis. For C and E, N = 12 mice per group. qPCR values are mean ± SEM. Analysis were performed by student <i>t</i>-test. *, p<0.05, **, p<0.01, ****, p<0.0001.</p

APOE isoform-specific effect on gene expression in APP/E3/Abca1^+/- and APP/E4/Abca1^+/- mice.

<p>Comparison between T0 treated APP/E4/Abca1^+/- and APP/E3/Abca1^+/- mice. Heat-maps provided by GSEA analysis were used to identify and rank the top 50 up-regulated genes (<b>A</b>) and top 50 down regulated genes (<b>B</b>) in APP/E4/Abca1^+/- mice. <b>C,</b> Bubble plot shows top ranked “biological process” (BP) differentially affected by T0 treatment in APP/E4/Abca1^+/- vs APP/E3/Abca1^+/- mice. The gene lists were derived from edgeR output tables and included expression data for all transcripts. Color indicates nominalized p-value. Significant BP are represented in red to purple shades (p<0.05 and FDR≤0.25). Size of bubble indicates the number of significant genes in each represented BP. GSEA enrichment score curves and corresponding heat-maps show BP significantly enriched in T0 treated APP/E4/Abca1^+/- mice, <b>D</b> and <b>E,</b> “Microtubule Based Process”. <b>D,</b> GSEA analysis provided a heat-map (right) and enrichment score (left) for this category. <b>E,</b> RNA-seq results of significantly changed mRNA expression levels of representative genes from category “Microtubule Based Process”. <b>F-G,</b> “Synapse Organization and Biosynthesis”. <b>F,</b> GSEA analysis provided a heat-map (right) and enrichment score (left). <b>G,</b> RNA-seq results of significantly changed mRNA expression levels of representative genes from category “Synapse Organization and Biosynthesis”. *, p < 0.05, **, p < 0.01, ***, p < 0.001.</p

T0 treatment restores cognition in APP/E4/Abca1^+/- mice.

<p>5-month-old APP/E3/Abca1^+/-, APP/E4/Abca1^+/- and non-transgenic mice were treated with T0 and vehicle (Veh) for one month and assessed at 6 months of age. Cognitive function was evaluated with novel object recognition (A and B) and contextual fear conditioning behavioral paradigms (C and D). <b>A,</b> T0 affected the performance of APP transgenic mice in the novel object recognition test. Analysis by two-way ANOVA shows no interaction between <i>APOE</i> genotype and T0 treatment with a significant main effects of <i>APOE</i> genotype (F(1, 51) = 7.44, p < 0.01) and T0 treatment (F(1, 51) = 4.45, p< 0.05). <b>B,</b> T0 treatment did not affect the performance of non-APP littermates. Effects of <i>APOE</i> genotype (F(1, 45) = 1.9) and T0 treatment (F(1, 45) = 0.002). <b>C,</b> LXR agonist significantly improved the performance of APP/E4/Abca1^+/- mice in contextual fear conditioning paradigm. Analysis by two-way ANOVA shows no interaction between <i>APOE</i> genotype and T0 treatment and significant main effects of T0 treatment (F(1, 51) = 5.94, p = 0.018) and <i>APOE</i> genotype (F(1, 51) = 10.6, p = 0.002). Sidak’s multiple comparison test shows a significant difference between T0 and vehicle treated APP/E4/Abca1^+/- mice (*, p < 0.05). <b>D,</b> T0 also affected the behavior of non-APP controls in the contextual fear conditioning behavior paradigm. Analysis by two-way ANOVA shows no interaction and significant main effects of T0 treatment (F(1, 45) = 4.47, p = 0.03) and <i>APOE</i> genotype (F(1, 45) = 4.49, p = 0.04). T0 had no effect on APP (E) and non-APP mice (F) during the cued phase of fear conditioning. For all panels, N = 11–15 male and females mice per group. Data represented as means ±SEM.</p