3xTg-AD/BDNF^+/− and 3xTg-AD/BDNF^+/+ mice have comparable levels of Aβ and tau pathology.

<p>No differences between groups were detected by ELISA in levels of Aβ_1–40 or Aβ_1–42 in soluble (<b>A</b>, <i>n = </i>4) or insoluble (<b>B</b>, <i>n = </i>4) fractions. (<b>C</b>) Representative maximum intensity projections of immunofluorescently labeled Aβ and tau in CA1 and cortex indicate a similar pattern of immunoreactivity between 3xTg-AD/BDNF^+/+ and 3xTg-AD/BDNF^+/− mice. Scale bars  = 20 μm. Western blot analyses suggest 3xTg-AD/BDNF^+/+ and 3xTg-AD/BDNF^+/− mice have similar levels of proteins related to Aβ production (<b>D–E</b>, <i>n = </i>6) and similar levels of tau and various tau phospho-epitopes (<b>F–G</b>, <i>n = </i>6). Data are presented as means ±SEM.</p

Immunohistochemical observations of Amygdalar, Cortical, and Hippocampal Neurons.

<p>Anti-HA antibody was diluted 100-fold. Immunohistochemical observations were repeated thrice and each observation gave the same result. Ten homozygous transgenic 3-month-old mice were fed B6-deficient food for 3 weeks. Transgenic experimental mice were injected with anti-HA antibody every 3 days. For details, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0008593#s2" target="_blank">Materials and Methods</a>. CTL, transgenic control mice; Ex, transgenic experimental mice.</p

3xTg-AD/BDNF^+/− mice have reduced BDNF levels but no changes in BDNF-related signaling.

<p>(<b>A–B</b>) Western blot analysis on whole brain homogenates from homozygous 3xTg-AD mice reveals no significant difference in levels of mature BDNF as compared to wildtype controls (<i>n = </i>3). Levels of proBDNF trend toward an increase in 3xTg-AD mice, although this difference is not significant (<i>p</i> = 0.16). (<b>C</b>) 3xTg-AD/BDNF^+/− mice have a 43% reduction in BDNF protein by ELISA versus 3xTg-AD/BDNF^+/+ controls (<i>n = </i>4, <i>p</i> = 0.015). (<b>D</b>) Representative western blots for levels of various BDNF-related signaling proteins in the cerebral cortex of 3xTg-AD/BDNF^+/+ and 3xTg-AD/BDNF^+/− mice (<i>n = </i>6) are shown in alternating lanes. (<b>E</b>) Quantification of bands from <i>B</i> are normalized to GAPDH levels and shown as levels relative to 3xTg-AD/BDNF^+/+ controls. Data are presented as means ±SEM.</p

Curative Effect of Anti-HA Antibody as shown by Long-Term Memory Performance.

<p>Seven-month-old male 3xTg-AD homozygous mice mice fed B6-deficient food for 3 weeks served as the control. Experimental mice were treated with anti-HA antibody every 3 days; antibody (100-fold dilution) was injected into the brain as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0008593#s2" target="_blank">Materials and Methods</a>. The figure shows average data for five male mice. To demonstrate the strong curative effect of the antibody, for comparison we show the results for 2-month-old hemizygous male mice, whose memory performance was normal. Statistically significant difference was observed after 2 trial days (<i>P</i><0.001). However after 3 trial days, a statistically significant difference was not observed (^*<i>P</i><0.001). The difference between control and experiment mice (<i>P</i><0.01). The difference between experimental mice and 7-month-old mice with normal food (n = 5).</p

The Effect on Memory Performance of HA Vaccine on Normal Feeding of 3xTg-AD Male Mice.

<p>Mice were 12 months old. Memory performance was measured via the Morris water experiment described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0008593#s2" target="_blank">Materials and Methods</a>. Statistically significant difference was observed on the third, fourth, and fifth trial days (<i>P</i><0.001). 3xTg-AD male mice were all homozygous (n = 15).</p

HA level in 3xTg-AD mice.

<p>Note: A: Vaccine treatment was started at 12 months. Three months later, urine was collected an entire day and HA level was measured. 3xTg-AD male mice were all homozygous. HA level with vaccine treatment decreased with time, indicating that vaccine efficacy became stronger with time.</p><p><b>Note: For reference, HA level in 12-month-old in 3xTg-AD male mice were observed.</b></p><p>Note: B: After Morris water maze test, these mice were sacrificed and their brains were immediately freezed in liquid nitrogen. Next, HA level was measured with the method described in Materials and Method.</p

Long-Term Memory Test in the Morris Water Maze task.

<p>Non-transgenic mice had an average score of 3 mice each day. Hemizygous transgenic control (hemizygous + B6 deficient) mice had an average score of 3 transgenic control mice each day. Transgenic experimental (hemizygous + B6 deficient + anti-HA antibody) mice had an average score of 3 transgenic experimental mice each day. 3xTg-AD mice were aged 3 months and 3 weeks. Statistically significant difference was observed after 2 trial days between hemizygous + B6 deficient and hemizygous + B6 deficient + antibody (<i>P</i><0.001) and after 3 trial days between hemizygous + B6 deficient and hemizygous + B6 deficient + antibody (<i>P</i><0.05).</p

Hematoxylin-Eosin Staining of Cortical and Hippocampal Neurons in Control and Experimental Groups.

<p>Mice were treated the same as those shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0008593#pone-0008593-g003" target="_blank">Fig. 3</a>.</p

The comparison of hippocampus major axis length between control and experiment.

<p>Control: 7 month-old hemizygous + B6 deficient.</p><p>Experimental: 7 month-old hemizygous male +B6 deficient + anti-HA antibody.</p><p>5 different sections were observed and its major axis was measured.</p><p>Average (n = 2) is shown.</p><p>Non-Tg control showed Hippocampal major axis of 10±0.5 cm.</p

Homocysteic Acid Level in 3xTg-AD mice brain at 4 months compared with Non-Tg control mice.

<p>HA level in the brain of 3xTg-AD male mice at 4 months was measured by the method described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0008593#s2" target="_blank">Materials and Methods</a>. Control was non-Tg male mice at 4 months.</p