Impaired cellular calcium homeostasis in CD mice.

<p>(a) The mean frequency of calcium oscillations in CD hippocampal neurons was significantly lower (<i>t</i>₂₀₀ = 5.526, <i>p</i><0.0001) when compared to the mean frequency of WT neurons n = 103–130 cells per genotype. (b) Amplitude (<i>p</i> = 0.0028) and (c) duration (<i>p</i> = 0.0063) of calcium transients were significantly higher in CD neurons when compared to WT neurons. (d) Differences in hippocampal mRNA expression genes related to Ca²⁺ signaling levels of <i>Trpc3</i> were slightly lower in CD mice (<i>p</i> = 0.045), while <i>Ryr2</i> and <i>Ryr3</i> levels were significantly higher in CD mice when compared to WT mice (<i>p</i> = 0.0017 and <i>p</i> = 0.0071, respectively). No significant differences in mRNA expression of other genes related to Ca²⁺ signaling (<i>Cacna1c</i>, <i>Atp2a2</i> and <i>Trpc6</i>) were found. (e) TRPC3 subcellular localization in CD mice primary cultures of hippocampal neurons is not significantly altered compared with WT cells (F_1,77 = 0.3922, <i>p</i> = 0.5331). n = 16–23 cells per genotype. <i>p</i> values are shown with asterisks (genotype) indicating values that are significantly different (two-way ANOVA with Bonferroni <i>post hoc</i> test or unpaired <i>t</i>-test with Welch’s correction). *<i>p</i><0.05; **<i>p</i><0.01; ***<i>p</i><0.001. White, WT; Black, CD. Plain, water. Data are presented as the mean ± SEM.</p

NADPH-oxidase and NO pathways were not affected by EGCG.

<p>(a) DHE staining in cardiac tissue showed a ≈20% increase in the general oxidative stress levels in CD mice (<i>p</i> = 0.04). (b) CD animals showed reduced expression levels of <i>Ncf1</i> (<i>p</i> = 0.0008) as previously described, but expression of other oxidative stress-related genes of the NADPH-oxidase and NO pathways was not affected. n = 3–4 mice per group. <i>p</i> values are shown with asterisks (genotype) indicating values that are significantly different (Unpaired <i>t</i>-test with Welch’s correction). *<i>p</i><0.05; ***<i>p</i><0.001. White, WT; Black, CD. Plain, water. Data are presented as the mean ± SEM.</p

EGCG treatment prevents the cardiac hypertrophy observed in CD mice.

<p>(a) Comparison of the heart/body weight ratios obtained from each genotype showed that cardiac hypertrophy present in CD mice is not observed after EGCG treatment. A two-way ANOVA indicated a significant interaction between genotypes and treatment (F_1,46 = 7.237, <i>p</i> = 0.0099), with a significant effect of genotype (F_1,46 = 6.751, <i>p =</i> 0.0125) but with treatment effect only in CD mice (<i>p</i><0.01, Bonferroni <i>post hoc</i> test). n = 10–15 mice per group. (b) Representative images of hematoxylin-eosin stained transverse sections of heart samples. We can appreciate the thickening of the left ventricular wall of the control CD animals. (c) CD mice cardiomyocytes are significantly larger than in WT animals, while CD animals treated with EGCG show normal cell size. A two-way ANOVA indicated a significant interaction between genotypes and treatment (F_1,12 = 24.05, <i>p</i> = 0.0004), with a significant effect of genotype (F_1,12 = 14.15, <i>p</i> = 0.0027) and treatment effect in CDs (<i>p</i><0.01, Bonferroni <i>post hoc</i> test) but also in WTs (<i>p</i><0.05, Bonferroni <i>post hoc</i> test). n = 4 mice/group. <i>p</i> values are shown with asterisks (genotype) or hashes (treatment) indicating values that are significantly different (Two-way ANOVA with Bonferroni <i>post hoc</i> test). *,^#<i>p</i><0.05; **,^##<i>p</i><0.01; ***,^###<i>p</i><0.001. White, WT; Black, CD. Plain, water; Striped, EGCG. Data are presented as the mean ± SEM.</p

EGCG does not influence brain abnormalities present in CD mice.

<p>(a) Brain weight of water or EGCG fed CD mice was significantly reduced when compared to WT mice. A two-way ANOVA indicated a significant effect of genotype (F_1,37 = 40.72, <i>p</i><0.0001) but no effect of treatment (F_1,37 = 0.3766, <i>p</i> = 0.5432). (b) Dendrite length of CA1 neurons was measured in stratum radiatum (SR). A two-way ANOVA indicated a significant effect of genotype (F_1,37 = 15.98, <i>p</i> = 0.0003) but not effect of treatment (F_1,37 = 1.728, <i>p</i> = 0.1968) (c) Dendrite length of CA1 neurons was measured in stratum oriens (SO). A two-way ANOVA indicated a deleterious effect of treatment in the case of WT mice (F_1,37 = 22.92, <i>p</i><0.0001 with <i>p</i><0.001 Bonferroni <i>post hoc</i> test). (d) EGCG treatment did not increase the number of spines present in apical dendrites of CD neurons. A two-way ANOVA indicated a significant effect of genotype (F_1,37 = 13.13, <i>p</i> = 0.0009) without treatment effect (F_1,37 = 1.722, <i>p</i> = 0.1976). n = 8–13 mice per group. <i>p</i> values are shown with asterisks (genotype) or hashes (treatment) indicating values that are significantly different (two-way ANOVA with Bonferroni <i>post hoc</i> test). *,^#<i>p</i><0.05; **,^##<i>p</i><0.01; ***,^###<i>p</i><0.001. White, WT; Black, CD. Plain, water; Striped, EGCG fed mice. Data are presented as the mean ± SEM.</p

EGCG restores oxidative stress status via NRF2 pathway.

<p>(a) Representative images of cardiomyocytes cultures showed that NRF2 nuclear levels were decreased in CD cardiomyocytes in comparison with WT cells. After EGCG-treatment both genotypes increased the nuclear proportion of NRF2. Blue, DAPI; red, NRF2. (b) Histograms show the quantification of figures showed in (a). A clear interaction effect between genotype and treatment could be observed (F_1,756 = 34,51, <i>p</i><0.0001). n = 145–235 cardiomyocytes per group. (c) <i>Nqo1</i> expression levels were downregulated in CD cardiac tissue (<i>p</i><0.01, Bonferroni post hoc test) and significantly higher after EGCG treatment (<i>p</i><0.05, Bonferroni <i>post hoc</i> test). n = 4 mice/group. <i>p</i> values are shown with asterisks (genotype) or hashes (treatment) indicating values that are significantly different (two-way ANOVA or one-way ANOVA with Bonferroni <i>post hoc</i> test). *^,#<i>p</i><0.05, **^,##<i>p</i><0.01; ***^,###<i>p</i><0.001. White, WT; Black, CD. Plain, water; Striped, EGCG. Data are presented as the mean ± SEM.</p

Effect of EGCG on hippocampal synaptic plasticity markers.

<p>(a) EGCG treatment normalized <i>Bdnf</i> mRNA levels in CD animals. A two-way ANOVA indicated a significant interaction between genotypes and treatment (F_1,38 = 5.512, <i>p</i> = 0.0242), with a significant effect of genotype (F_1,38 = 6.599, <i>p</i> = 0.0143) and treatment (F_1,38 = 30.47, <i>p</i><0.0001). (b) EGCG treatment did not normalize the high <i>Pik3r1</i> mRNA levels observed in the hippocampus of CD mice. A two-way ANOVA indicated a significant effect of treatment (F_1,24 = 7.406, <i>p</i> = 0.0119), since EGCG treatment upregulated <i>Pik3r1</i> mRNA levels in WT animals. n = 6–13 per group. <i>p</i> values are shown with asterisks (genotype) or hashes (treatment) indicating values that are significantly different (two-way ANOVA with Bonferroni <i>post hoc</i> test). *,^#<i>p</i><0.05; **,^##<i>p</i><0.01; ***,^###<i>p</i><0.001. White, WT; Black, CD. Plain, water; Striped, EGCG. Data are presented as the mean ± SEM.</p

EGCG does not influence sociability or anxiety-related behavior in CD mice.

<p>(a) In a direct social test, CD mice fed with green tea behaved in the same way that water fed CD mice towards an intruder mice. A two-way ANOVA indicated a significant main effect of genotype (F_1,37 = 19.86, <i>p</i><0.0001), without effect of treatment (F_1,37 = 0.00044, <i>p</i> = 0.9834). n = 7–15 per group. White, WT; Black, CD. Plain, water; Striped, EGCG. Data are presented as the mean ± SEM. (b) Anxiety-like behavior was evaluated in the marble-burying test. A three-way ANOVA revealed no interaction between genotype, treatment and time (F_3,128 = 1.380, <i>p</i> = 0.252), with a significant main effect of genotype (F_1,128 = 306.684, <i>p</i><0.0001) and time (F_3,128 = 17.170, <i>p</i><0.0001) but no effect of treatment (F_1,128 = 0.182, <i>p</i> = 0.670). n = 8–10 per group. Squares, WT; Circles, CD. White and black, water; Green, EGCG fed mice. <i>p</i> values are shown with asterisks (genotype effect) indicating values that are significantly different in a two-way ANOVA with Bonferroni <i>post hoc</i> test (***<i>p</i><0.001).</p

Presence of activated PKR in human brain tissue.

<p>The presence of activated PKR -autophosphorylated at Thr446- was studied in human brain sections (temporal lobe) from one non-demented control and one AD patient by immunohistochemistry (A). Colocalization of BACE1 and p-PKR(T446) in neurons from an AD patient. All brain sections analysed were positive for HSV1 infection as previously reported <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0021456#pone.0021456-Wozniak3" target="_blank">[39]</a> (B).</p

Biochemical pathway linking HSV1 infection and AD.

<p>Despite the viral ability to circumvent host defensive mechanisms, including PKR activation, HSV1 activates PKR <i>in vitro</i> and <i>in vivo</i>. Activated PKR increases eIF2-alpha phosphorylation levels, leading to BACE1 translation de-repression, BACE1 protein up-regulation and Aß production (left track; continuous line). In the right track (dotted line) we present the pharmacological and biological tools that we used to study this pathway: poly (I∶C), to mimic the effect of viral dsRNA; a specific imidazolo-oxindole compound that acts as a potent PKR inhibitor; and a 5′UTR-luc reporter construct used for the evaluation of the translational effect exerted by the PKR-eIF2-alpha pathway over BACE1 5′UTR. Also, we have utilized the PP1c inhibitor salubrinal, which prevents the dephosphorylation of eIF2-alpha.</p

HSV1 infection activates PKR.

<p>SH-SY5Y cells were infected with HSV1 (1 pfu/cell, 24 h) or were not infected (controls). Immunocytochemistry analysis was carried with the following Abs: anti-p-PKR, anti-p-eIF2-alpha and anti-BACE1 (A). Protein extracts of SH-SY5Y cells infected with HSV1 (3 pfu/cell, 24 h) and uninfected cells (controls) were analysed by Western blotting using the following Abs: anti-BACE1, anti-p-PKR, anti-PKR, anti-p-eIF2-alpha, anti- eIF2-alpha and anti-tubulin. Bands were quantified by densitometric analysis. Results are expressed as the mean ± SEM of 3–4 independent experiments. * p<0.05, ** p<0.01, *** p<0.0005 by Student's <i>t</i> test (B). Sections from mouse dorsal ganglion root (DRG) were obtained from HSV1-infected mice. Contralateral uninfected ganglia were used as controls. Immunohistochemistry analysis was carried out to detect p-PKR (C).</p