Seizure susceptibility and mortality in mice that over-express amyloid precursor protein

Abstract

Alzheimer's disease and Fragile X syndrome both display synaptic phenotypes, and based on recent studies, likely share dendritic over expression of amyloid precursor protein (APP) and beta-amyloid (Abeta). In order to create a mouse model to specifically study the effects of APP and Abeta at synapses, we crossed Tg2576, which over-express human APP with the Swedish mutation (hAPPsw), with fmr-1 KO mice. The progeny, named FRAXAD, displayed increased mortality (23% by 30 days of age) compared to Tg2576 (3%) and WT and fmr-1 KO littermate controls (0%) consistent with a developmental defect. By 60 days of age, both the Tg2576 and FRAXAD mice approached a 40% mortality rate compared to 0% for WT and fmr-1 KO littermates. To understand the mechanism underlying increased mortality in APP over-expressing mice, we assessed seizure thresholds in response to pentylenetetrazol (PTZ). Both the Tg2576 and FRAXAD mice had a lower threshold to PTZ-induced seizures (average seizure score of >/=4.0) in comparison to nontransgenic littermates (average seizure score 1.9-2.9). Seizures are a major phenotype of AD, FXS, Down syndrome, autism and epilepsy, and these data suggested that developmental over-expression of dendritic APP or Abeta increased seizure susceptibility.

Keywords: FRAXAD; Fragile X mental retardation protein; amyloid; amyloid precursor protein; seizure; synapse.

Figure 1

FRAXAD mice generated more Aβ₄₀ than Tg2576. GnHCl-soluble brain lysates (A and C) and SN (B and D) from 14–16 day-old Tg2576 and FRAXAD mice were analyzed by ELISA for Aβ₄₀ (A and B) and APP/APPα (C and D). Each cohort (n=6) from backcrosses n7-n9 consisted of 3 female and 3 male mice. The difference between FRAXAD (13,571 ± 666 pg/mL/μg) and Tg2576 (11,060 ± 696 pg/mL/μg) Aβ₄₀ levels in brain homogenates was statistically significant, Student T-test: p<0.03. The difference between FRAXAD (3.63 ± 0.24 pg/mL/μg) and Tg2576 (3.40 ± 0.39 pg/mL/μg) Aβ₄₀ levels in SNs was not statistically significant, Student T-test: p=0.6. The difference between FRAXAD (25,897 ± 2047 ng/mL/μg) and Tg2576 (28,962 ± 2875 ng/mL/μg) APP/APPα levels in brain homogenate was not statistically significant, Student T-test: p=0.4. The difference between FRAXAD (80.64 ± 11.14 ng/mL/μg) and Tg2576 (97.45 ± 10.86 ng/mL/μg) APP/APPα levels in SNs was not statistically significant, Student T-test: p=0.3.

Figure 2

FRAXAD mice exhibited increased juvenile mortality compared to Tg2576. Deaths for Tg2576 and FRAXAD mice were monitored over a 378-day period (backcrosses 1–6) and the mouse longevity in days was plotted against the percent of mice surviving (60 days shown, see Supplementary Figure 1 for the 378 day period). As a few mice were removed from the colony for use in experiments, the number of mice decreased during the survival time course. At the beginning of the time course (18 days), survival was based on 105 FRAXAD mice and 91 Tg2576 mice, and by the end of the time course (60 days), survival was based on 93 FRAXAD and 80 Tg2576 mice.

Figure 3

PTZ-induced seizures in male and female WT and fmr-1 KO mice in a pure C57BL/6 background. C57BL/6 mice (8–10 weeks old) were injected intraperitoneally with 50 mg PTZ per kg body weight and seizure activity was monitored for 60 min. Average seizure scores were 2.2±0.33 (WT females, n=10), 3.13±0.19 (WT males, n=15), 2.67±0.37 (fmr-1 KO females, n=9) and 2.9±0.22 (fmr-1 KO males, n=20). The 0.93 difference in seizure grade between the WT male and female mice was statistically significant (p=0.015).

Figure 4

Male and female Tg2576 and FRAXAD mice had a lower threshold to PTZ-induced seizures. Mice from the n3-n6 backcrosses (87.5–98.4% C57BL/6) were injected with 50 mg/kg and (A) mouse strain was plotted against the average seizure score: 1.89±0.20 (WT female, n=9), 2.42±0.31 (WT male, n=12), 2.55±0.22 (frm-1 KO female, n=20), 2.85±0.27 (frm-1 KO male, n=13), 4.67±0.33 (Tg2576 female, n=6), 4.57±0.30 (Tg2576 male, n=7), 4.86±0.14 (FRAXAD female, n=7) and 4.08±0.31 (FRAXAD male, n=12). The difference between FRAXAD males and females was statistically significant, p=0.04, as was the difference between each hAPPsw transgenic mouse and its nontransgenic littermate control, p<0.007. The delta seizure scores (B) were 2.78 (WT and Tg2576 females), 2.15 (WT and Tg2576 males), 2.31 (fmr-1 KO and FRAXAD females) and 1.23 (fmr-1 KO and FRAXAD males).

Figure 5

FRAXAD female mice had a higher juvenile mortality than FRAXAD males. The survival data for the mice included in Figure 1 was plotted separating the male and female mice. Tg2576 females (n=44), Tg2576 males (n=47), FRAXAD females (n=43) and FRAXAD males (n=62).