EHT treatment reduces tau phosphorylation.

<p><b>(</b>A) Representative western blots for the indicated proteins and methylated PP2A/C performed on hippocampal homogenates prepared from animals fed control diet, or diets containing 0.01 or 0.1% EHT. (B) Histogram showing average ± SEM for tubulin-normalized band intensities expressed as average percent of control band intensity from replicate western blots in A show no significant differences in expression levels for any of the indicated proteins or for methylated PP2A/C (ANOVA for: PP2A/C: F(2,22) = 0.2533, P = 0.7784; PP2A/A: F(2,22) = 0.2588, P = 0.7743; B55α: F(2,22) = 0.06221, P = 0.9399; PME-1: F(2,22) = 0.4942, P = 0.6167; LCMT-1: F(2,22) = 0.2498, P = 0.7812; methyl-PP2A/C: F(2,22) = 0.1666, P = 0.8476). (C) Representative western blots for demethylated PP2A, and total PP2A/C performed on the homogenates described in A either treated (+) or mock treated (-) with 0.5 M sodium hydroxide. (D) Histogram of average demethylated PP2A/C (± SEM) in hippocampal homogenates prepared from animals fed control, or 0.01 or 0.1% EHT containing-diets show no significant differences in demethylated PP2A/C levels (ANOVA: F(2,22) = 0.1436, P = 0.8670). Values were calculated as ratios of demethyl-PP2A/C to total PP2A/C band intensities for -NaOH treated samples from replicate western blots shown in C and expressed as percent of the average of control. (E) Representative western blots performed on hippocampal homogenates prepared from animals fed control diet, or diets containing 0.01 or 0.1% EHT for phospho-Ser396/404 (PHF1), phospho-Ser202 (CP13) together with their corresponding total tau loading controls, as well as and total tau together with its corresponding β-actin loading control. (F) Histogram showing average band intensities ± SEM for phospho-Ser396/404-tau (PHF1), phospho-Ser202-tau (CP13) normalized to corresponding total tau loading control, and total tau normalized to corresponding β-actin loading control for replicate western blots shown in H show a trend for reduced phosphorylation at these sites in EHT-treated animals (ANOVA for: PHF1: F(2,22) = 5.147, P = 0.147, Bonferroni post-hoc for PHF1 0.1% EHT vs. Control: t = 3.154, P = 0.0092; CP13: F(2,22) = 1.433, P = 0.2599; total tau: F(2,22) = 0.1268, P = 0.8815). (G and H) Representative western blots for phospho-Ser9 and total GSK3B and phospho-Ser133 and total Creb performed on hippocampal homogenates prepared from animals fed control diet, or diets containing 0.01 or 0.1% EHT. (I) Histogram showing average ± SEM of phospho-GSK3β and phospho-Creb band intensities normalized to corresponding total GSK3β and Creb respectively for replicate western blots shown in G&H show no significant effect of EHT treatment on phosphorylation at these sites (ANOVA for: P-GSK3β F(2,22) = 1.761, P = 0.1952; P-Creb: F(2,22) = 0.1776, P = 0.8385). (N = 8 control, 8 0.01% EHT and 9 0.1% EHT treated animals for each measure).</p

Chronic EHT treatment prevents Aβ-induced impairment of long-term potentiation.

<p><b>(</b>A-B) Time course of averaged Schaffer collateral fEPSP responses (± SEM) in hippocampal slices prepared from animals fed control or EHT-containing diets and treated with either vehicle or 100 nM Aβ (horizontal bar) 20 min prior to delivery of theta-burst stimulation (arrow). (A) Aβ treatment significantly reduces potentiated responses following TBS in slices prepared from animals on control diets (2-way RM-ANOVA for treatment with time and treatment as factors: F(1,29) = 8.913, P = 0.0057). (B) Mice fed diets containing 0.01% EHT are resistant to Aβ-induced LTP impairment (2-way RM-ANOVA for treatment with time and treatment as factors: F(1,26) = 0.0943, P = 0.7612). C) Input/output (2-way RM-ANOVA for treatment with stimulus and treatment as factors: F(1,57) = 0.5466, P = 0.4628) (N = 31 control, 28 0.01% EHT).</p

Chronic EHT treatment prevents Aβ-induced impairment of long-term potentiation.

<p><b>(</b>A-B) Time course of averaged Schaffer collateral fEPSP responses (± SEM) in hippocampal slices prepared from animals fed control or EHT-containing diets and treated with either vehicle or 100 nM Aβ (horizontal bar) 20 min prior to delivery of theta-burst stimulation (arrow). (A) Aβ treatment significantly reduces potentiated responses following TBS in slices prepared from animals on control diets (2-way RM-ANOVA for treatment with time and treatment as factors: F(1,29) = 8.913, P = 0.0057). (B) Mice fed diets containing 0.01% EHT are resistant to Aβ-induced LTP impairment (2-way RM-ANOVA for treatment with time and treatment as factors: F(1,26) = 0.0943, P = 0.7612). C) Input/output (2-way RM-ANOVA for treatment with stimulus and treatment as factors: F(1,57) = 0.5466, P = 0.4628) (N = 31 control, 28 0.01% EHT).</p

Acute EHT treatment prevents Aβ-induced impairment of long-term potentiation.

<p><b>(</b>A-F) Time course of averaged Schaffer collateral fEPSP responses (± SEM) in hippocampal slices prepared from slices treated with vehicle or 0, 0.0001, 0.001, 0.01, 0.1, or 1 μM EHT +/- vehicle or 100 nM Aβ (horizontal bar) 20 min prior to delivery of theta-burst stimulation (arrow). (A) Aβ treatment significantly reduces potentiated responses following TBS in slices treated with 0 μM EHT (2-way RM-ANOVA for treatment with time and treatment as factors: F(1,19) = 8.827, P = 0.0078). (B) Aβ treatment significantly reduces potentiated responses following TBS in slices treated with 0.0001 μM EHT (2-way RM-ANOVA for treatment with time and treatment as factors: F(1,11) = 6.84, P = 0.0240). (B) Aβ treatment yields a non-significant trend for reduced potentiated responses following TBS in slices treated with 0.001 μM EHT (2-way RM-ANOVA for treatment with time and treatment as factors: F(1,16) = 2.123, P = 0.1645). (D-F) Aβ treatment does not significantly reduce potentiated responses following TBS in slices treated with 0.01, 0.1, or 1 μM EHT (2-way RM-ANOVA for treatment with time and treatment as factors: For 0.01 μM EHT: F(1,18) = 0.1646, P = 0.6898; For 0.1 μM EHT: F(1.21) = 0.0034, P = 0.9543; For 1 μM EHT: F(1,15) = 0.0014, P = 0.9702). Comparison of potentiated responses in the absence of Aβ revealed no effect of EHT treatment alone on TBS-induced LTP (2-way RM-ANOVA comparisons to 0 EHT + vehicle for treatment with time and treatment as factors: For 0.0001 μM EHT: F(1,15) = 0.0132, P = 0.9099; For 0.001 μM EHT: F(1,18) = 0.0539, P = 0.8191; For 0.01 μM EHT: F(1,18) = 0.16, P = 0.6939; For 0.1 μM EHT: F(1,21) = 0.2294, P = 0.6369; For 1 μM EHT: F(1,18) = 0.7098, P = 0.4106). (G) Plot of the average potentiated responses over the last 10 min of the recordings shown in B-F for slices treated with Aβ in the presence of the indicated concentrations of EHT. The upper and lower dashed lines indicate the mean potentiated response obtained in the absence of EHT for vehicle or Aβ treated slices respectively. (N = 11 0 μM EHT + vehicle, 10 0 μM EHT + Aβ, 6 0.0001 μM EHT + vehicle, 7 0.0001 μM EHT + Aβ, 9 0.0001 μM EHT + vehicle, 9 0.0001 μM EHT + Aβ, 9 0.01 μM EHT + vehicle, 11 0.01 μM EHT + Aβ, 12 0.1 μM EHT + vehicle, 11 0.1 μM EHT + Aβ, 9 1 μM EHT + vehicle, 8 1 μM EHT + Aβ slices).</p

EHT prevents Aβ-induced impairment of spatial learning and memory in a 2-day radial arm water maze task.

<p><b>(</b>A) Average number of errors committed (± SEM) during each 3-trial training block of a 2-day radial arm water maze task for the indicated treatment groups. 2-way RM-ANOVA for day 2 (blocks 6–10) with block and group as factors: F (5,69) = 4.424, P = 0.0015 for group; F (4,276) = 25.95, P<0.0001 for block,; F (20,276) = 0.5657, P = 0.9338 for interaction. Bonferroni post-hoc comparisons of the control + vehicle group to all other treatment groups show that only the control + Aβ group is significantly different than control + vehicle group. (N = 12 control + vehicle, 13 control + Aβ, 12 0.01% EHT + vehicle, 12 0.01% + Aβ, 13 0.1% + vehicle, 13 0.1% EHT + Aβ.) (B) Plot of the average escape latency (± SEM) for the indicated treatment groups during training on a visible platform Morris water maze task reveals no significant differences between groups (2-way RM-ANOVA with trial block and treatment group as factors: F(5,69) = 0.9766, P = 0.4384 for group, F(3,207) = 72.48, P<0.0001 for block, and F(15,207) = 0.8627, P = 0.6068 for interaction). (N = 12 control + vehicle, 13 control + Aβ, 12 0.01% EHT + vehicle, 12 0.01% + Aβ, 13 0.1% + vehicle, 13 0.1% EHT + Aβ). (C) Plot of the average swim speed (± SEM) for the indicated treatment groups during training on the visible platform Morris water maze task described in B reveals no significant differences between groups (2-way RM-ANOVA with trial block and treatment group as factors: F(5,69) = 1.232, P = 0.3035 for group, F(3,207) = 28.3, P<0.0001 for block, and F(15,207) = 0.9227, P = 0.5398 for interaction).</p

List of antibodies.

<p>List of antibodies.</p