DBA/2J Genetic Background Exacerbates Spontaneous Lethal Seizures but Lessens Amyloid Deposition in a Mouse Model of Alzheimer’s Disease

<div><p>Alzheimer’s disease (AD) is a leading cause of dementia in the elderly and is characterized by amyloid plaques, neurofibrillary tangles (NFTs) and neuronal dysfunction. Early onset AD (EOAD) is commonly caused by mutations in amyloid precursor protein (APP) or genes involved in the processing of APP including the presenilins (e.g. PSEN1 or PSEN2). In general, mouse models relevant to EOAD recapitulate amyloidosis, show only limited amounts of NFTs and neuronal cell dysfunction and low but significant levels of seizure susceptibility. To investigate the effect of genetic background on these phenotypes, we generated <i>APP^swe</i> and <i>PSEN1^de9</i> transgenic mice on the seizure prone inbred strain background, DBA/2J. Previous studies show that the DBA/2J genetic background modifies plaque deposition in the presence of mutant APP but the impact of <i>PSEN1^de9</i> has not been tested. Our study shows that DBA/2J.<i>APP^swePSEN1^de9</i> mice are significantly more prone to premature lethality, likely to due to lethal seizures, compared to B6.<i>APP^swePSEN1^de9</i> mice—70% of DBA/2J.<i>APP^swePSEN1^de9</i> mice die between 2-3 months of age. Of the DBA/2J.<i>APP^swePSEN1^de9</i> mice that survived to 6 months of age, plaque deposition was greatly reduced compared to age-matched B6.<i>APP^swePSEN1^de9</i> mice. The reduction in plaque deposition appears to be independent of microglia numbers, reactive astrocytosis and complement C5 activity.</p></div

<i>Apoe</i> expression is reduced in the parietal cortex and CA1 of aged mice.

<p>(A) Representative sections of the FPC from young and aged brains hybridized with an <i>Apoe</i> riboprobe. (B) Quantification of <i>Apoe</i> hybridized area in the cortex shows a significant decrease of <i>Apoe</i> expression in the aged mice. (C) Representative sections of the hippocampal CA1 region from young and aged brains hybridized with an <i>Apoe</i> riboprobe. (D) <i>Apoe</i> hybridized area in the CA1 shows a significant decrease of <i>Apoe</i> expression in the aged mice. (E) Representative sections of the CC from young and aged brains hybridized with <i>Apoe</i> riboprobe. (F) A significant increase of <i>Apoe</i> hybridized area is measured on the aged CC. (G) Representative images of <i>Apoe</i> in situ hybridization in the cortex and CC showing decreased expression of <i>Apoe</i> in the cortex and increased expression in the CC in aged mice compared with young mice. (H) No differences in APOE protein levels were found by western blotting of whole brains from young and aged mice. Values in (B, D, and F) are relative mean <u>+</u> SEM to the young values, <i>n</i> = 6 per group. In (D) **<i>p</i> < 0.001 and in (F) *<i>p</i> = 0.0364 by unpaired <i>t</i> test. Scale Bars: 600 μm (A), 50 μm (C), 100 μm (E and G). The data used to make this figure can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002279#pbio.1002279.s001" target="_blank">S1 Dataset</a>.</p

Schematic illustration of age-related changes in the neurovascular unit that are prevented by exercise.

<p>In the aged cortex of sedentary mice, neurovascular dysfunction is evident by decreased numbers of pericytes, decline in BM coverage, and increased transcytosis on endothelial cells. Expression of AQP4 in astrocyte endfeet and down-regulation of <i>Apoe</i> (purple) are also found as part of the age-related dysfunction of the neurovascular unit. In addition, decrease in synaptic proteins such as synaptophysin (SYN) is found in aged neurons. The number of proinflammatory IBA1⁺ microglia/monocytes expressing high levels of <i>C1qa</i> RNA is also increased in the aged cortex and HP indicating age-related neuroinflammation in aged mice. These age-related changes were successfully prevented by 6 months of voluntary running during aging, indicating the important contribution of physical activity on preservation of cerebrovascular function during aging.</p

Exercise reduces <i>C1qa</i>⁺ microglia/monocytes in the aged cortex.

<p>(A) Representative sections from an aged sedentary and aged runner mouse immunostained with PDGFRβ (magenta) for pericytes and IBA1 (green) for microglia/monocyte cells. (B) Quantification of IBA1⁺ cells in aging, aged sedentary (Sd), and aged runner (Rn) mice shows a decrease in microglia/monocyte density in the aged runners versus the aged sedentary mice. (C) Correlation between PDGFRβ⁺ cells and IBA1⁺ cells (R² = 0.49, <i>p</i> = 0.01). Decreased number of pericytes correlates with increasing number of microglia/monocyte cells. (D) Representative sections of the cortex hybridized with <i>C1qa</i> riboprobe and coimmunostained with IBA1 (green) in young, aged Sd and aged Rn mice. (E) Quantification of <i>C1qa</i>/IBA1⁺ (magenta bars) and IBA1⁺ (green bars) cells in the cortex of young, aged Sd, and aged Rn mice showing a significant increase in <i>C1qa</i>⁺/IBA1 positive cells in aged Sd mice and a significant decline of these cells in the aged runner mice. A new quantification of IBA1⁺ cells (different from B) was performed for the analysis of <i>C1qa</i>⁺/IBA1 cells. (F) Representative sections of the hippocampal CA1 hybridized with <i>C1qa</i> riboprobe and coimmunostained with IBA1 (green) in young, aged Sd, and aged Rn mice. (G) <i>C1q</i>/IBA1⁺ (magenta bars) and IBA1⁺ (green bars) cells in the hippocampal CA1 are significantly increased in aged Sd mice and significantly reduced in aged Rn mice. In panels (B, E, and G) values are mean <u>+</u> SEM, <i>n =</i> 4 per group. In (B) **<i>p</i> = 0.0032 and *<i>p</i> = 0.0148, in (E) **<i>p =</i> 0.0065 and 0.0044, ***<i>p =</i> 0.0006 and *<i>p</i> = 0.0194 and in (G) ***<i>p <</i> 0.0006,***<i>p</i> = 0.0003, **<i>p =</i> 0.0025 and *<i>p</i> = 0.0302 by ANOVA followed by Tukey’s posthoc tests. Scale Bars: 50 μm. The data used to make this figure can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002279#pbio.1002279.s001" target="_blank">S1 Dataset</a>.</p

Transcriptional profiling predicts age-related neurovascular dysfunction.

<p>(A) The number of DE genes in three brain regions (FPC/CC; HP; RB) comparing 21-month-old mice to 4-month-old control mice. (B) Pathways overrepresented in the DE genes from the FPC/CC region. For further details and the overrepresented pathways from other regions, see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002279#pbio.1002279.s016" target="_blank">S4 Table</a>. (C) Genes relevant to the neurovascular unit were generally down-regulated. (D) DE genes from the FPC/CC region in the ECM-receptor interaction pathway were down-regulated (green). (E–F) Fibrin intra- and extravascular deposits (red) were significantly increased in aged cortex compared to young cortex. In (<b>F</b>) values are relative mean <u>+</u> Standard Error of the Mean (SEM) to the young values, <i>n</i> = 6 mice per group, **<i>p</i> = 0.0073 by unpaired <i>t</i> test. Scale Bars: 50 μm. The data used to make this figure can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002279#pbio.1002279.s001" target="_blank">S1 Dataset</a>.</p

Astrocytic AQP4 is decreased in aged cortical astrocytes.

<p>(A) AQP4 immunoreactivity (blue) and protein levels (B) are significantly decreased in the aged cortex. (C) Astrocyte reactivity is increased in the cortex of aged mice determined by increased immunoreactivity of astrocytic GFAP (green) when compared with young mice. (D) Electron micrographs showing examples of astrocyte endfeet (As, white region surrounding the vessels) abnormalities such as swelling and big vacuoles (*) in aged mice. Astrocyte endfeet abnormalities were not observed in all cases in aged mice and never seen in young mice. Values in (B) are relative mean <u>+</u> SEM to the young values, <i>n</i> = 4 per group. **<i>p</i> < 0.005 (B) by unpaired <i>t</i> test. Scale Bars: 50 μm (A and C) and 2 μm (D). DP = degenerated pericyte, EC = endothelial cell and BV = blood vessel. The data used to make this figure can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002279#pbio.1002279.s001" target="_blank">S1 Dataset</a>.</p

No overt difference in glial responses in plaque regions in D2.<i>APB</i>^<i>Tg</i> compared to B6.<i>APB</i>^<i>Tg</i> mice.

<p>(<b>A-D</b>) Representative images of IBA1⁺ cells localized to Thio S-labeled plaques in the superior cortex from B6.<i>APB</i>^<i>Tg</i> (A, B) and D2.<i>APB</i>^<i>Tg</i> (C, D) mice. (<b>E</b>) No significant differences were observed in the number of IBA1⁺ cells surrounding plaques in D2.<i>APB</i>^<i>Tg</i> compared to B6.<i>APB</i>^<i>Tg</i> mice (p = 0.65). (<b>F-G</b>) There was also no obvious difference in the level of astrocyte reactivity (judged by levels of GFAP staining) in regions of plaques in D2.<i>APB</i>^<i>Tg</i> compared to B6.<i>APB</i>^<i>Tg</i> mice. NS = Not significant. Scale bars: A, C = 100μm; B, D, F, G = 20μm.</p

C5 sufficiency does not affect disease state in D2.<i>APB</i>^<i>Tg</i> mice.

<p>(<b>A-C</b>) Plaque deposition is unchanged in D2.<i>APB</i>^<i>Tg</i>.<i>C5</i>^<i>B6</i> (<i>C5</i>^<i>B6</i>) mice compared to D2.<i>APB</i>^<i>Tg</i> (C5^D2) mice (p = 0.251). Similarly to D2.<i>APB</i>^<i>Tg</i> mice, D2.<i>APB</i>^<i>Tg</i>.<i>C5</i>^<i>B6</i> show significantly less plaque deposition compared to B6.<i>APB</i>^<i>Tg</i> (B6) mice (p = 0.001). (<b>D-F</b>) The number of IBA1⁺ cells surrounding plaques in not different in D2.<i>APB</i>^<i>Tg</i>.<i>C5</i>^<i>B6</i> (<i>C5</i>^<i>B6</i>) mice compared to either D2.<i>APB</i>^<i>Tg</i> (C5^D2) or B6.<i>APB</i>^<i>Tg</i> (B6) mice (p = 0.354 and p = 0.087 respectively). (<b>G-I</b>) No significant difference was observed in NeuN⁺ cells in the cortex in D2.<i>APB</i>^<i>Tg</i>.<i>C5</i>^<i>B6</i> (<i>C5</i>^<i>B6</i>) mice compared to D2.<i>APB</i>^<i>Tg</i> (C5^D2, p = 0.44). Scale bars: A, B = 100μm; D, E = 20μm; G, H = 50μm.</p

No overt changes in cortical neurons in D2.<i>APB</i>^<i>Tg</i> mice.

<p>(<b>A-D</b>) NeuN+ cells were counted in B6 (A), B6.<i>APB</i>^<i>Tg</i> (B), D2 (C) and D2.<i>APB</i>^<i>Tg</i> (D) mice. Representative images are shown and all mice showed normal neuronal morphology in three different regions of the cortex. (E) No significant difference was observed in pTau aggregates (AT8 antibody, red) in D2.<i>APB</i>^<i>Tg</i> mice (E) compared to B6.<i>APB</i>^<i>Tg</i> mice. (F) NeuN+ cells were counted in each of the 4 cohorts, in 3 discrete cortical areas (4 mice per cohort). No statistically significant difference is seen between the different cohorts (p = 0.053). Scale bars: A-D = 50μm, E = 10μm.</p

Exercise improves behavior and neuroplasticity in aged mice.

<p>(A) Experimental strategy for voluntary running experiments (Sd = sedentary, Rn = Runner, Sac = sacrifice). (B) Representative image of a mouse running in an electronic wheel and quantification of running distance/night for young and aged mice after 6 months showing no differences between groups. (C) Significant deficits in grip strength found in aged sedentary mice were prevented by voluntary running. (D) Representative images of nests scored 3 and 5 are shown. Nest construction behavior was preserved in aged running mice but not in aged sedentary mice. (E) No statistically significant changes were found in burrowing behavior, however mice in the aging group (3/7) and in the aged sedentary group (2/6) did not engage in this activity, while all young and runner aged mice were able to perform it. (F) <i>Arc</i> (purple) in situ hybridization in aged sedentary and aged runner mice after 2 h of burrowing test. Low levels of <i>Arc</i> expression are found in the cortex of a mouse that did not do the burrowing test. (G) The fraction of NeuN⁺<i>Arc</i>^<i>+</i> neurons is significantly higher in the aged runners when compared with aged sedentary mice. No changes in the number of cortical NeuN⁺ neurons between aged sedentary and running mice. In panels B–E and G, values are mean <u>+</u> SEM, <i>n</i> = 7 per group in B–E, and <i>n</i> = 5 per group in G. In (C) **<i>p</i> = 0.0012 ***<i>p</i> = 0.0006, in (D) <i>*p</i> = 0.0165 and 0.0217 respectively, and in (G) <i>*p</i> = 0.0282 by ANOVA followed by Tukey’s posthoc tests. Scale Bar: 50 μm. The data used to make this figure can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002279#pbio.1002279.s001" target="_blank">S1 Dataset</a>.</p