We crossed Tg2576 mice, which over-express hAPPsw, with fmr-1 KO mice, which lack FMRP. Our goal was to generate a novel mouse model for AD research that specifically over-expressed hAPPsw and Aβ at synapses. The progeny, named FRAXAD, generated 23% more Aβ40 compared to Tg2576 in GnHCl-solublized, whole brain lysates prepared from 14–16 day-old mice (; p<0.03) and equivalent Aβ40 in SN samples (). APP/APPα levels appeared slightly higher in Tg2576 mice in both whole homogenates and SNs, but were not statistically different from the FRAXAD (). Aβ42 levels were below/at the detection limits of the ELISA assay (30 pg/mL). Thus, this data demonstrates that FRAXAD mice, which over-express hAPPsw in an fmr-1 KO background, produce significantly more Aβ40 by 2 weeks of age than Tg2576.
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 (more ...)
FRAXAD mice were viable and fertile although we did observe increased juvenile mortality. Longevity studies revealed a 23% death rate in the FRAXAD mice by 30 days of age compared to 3% for the Tg2576 (). By 60 days of age, both strains approached a 40% death rate. The WT and fmr-1
KO littermates had a 100% survival during this timeframe. The survival of the Tg2576 and FRAXAD was similar from 54 days out to one year (Supplementary Figure 1
), and overt signs of deteriorated health did not precede death. Necropsy analysis of FRAXAD mice (1–3 months old) indicated no gross morphological changes in the organs compared with WT and KO controls.
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 (more ...)
We observed spontaneous, repetitive behaviors in approximately 3% of the FRAXAD and Tg2576 mice. These behaviors started at about 6 months of age and were manifested as wild running or myoclonic jumping. We also observed home-cage seizure activity in a small percentage of Tg2576 and FRAXAD mice. Several transgenic mouse lines, which over-express familial mutation of APP, display tonic-clonic seizures in their home-cages, which have not been previously reported for Tg2576 mice. Thus, we surmised that a lower threshold to seizure induction could account for the increased mortality in the Tg2576 and FRAXAD mice compared to non-transgenic littermate controls. Therefore, we assessed seizure threshold after pentylenetetrazole (PTZ) injection in 8–10 week old mice. Seizures were graded on a five-point scale (Supplementary Table 1
and Supplementary Videos 1
To establish a baseline for seizure scores, we assessed seizures in WT and fmr-1 KO mice in a pure C57BL/6 background ( and and ). Mice were injected with 50 mg PTZ per kg body weight intraperitoneally, an established dose that generates grade 3 seizures in WT male mice, and seizure activity was monitored for 60 min. We observed an average seizure grade of 2.2 in WT female, 3.13 in WT male, 2.67 in fmr-1 KO female and 2.9 in fmr-1 KO male mice in a pure C57BL/6 background (, ). The 0.93 increase in seizure grade between the WT male and female mice was statistically significant (p=0.015). The male and female fmr-1 KO mice had average seizure scores intermediate between the male and female WT mice and were not statistically different. In general, the female mice exhibited lower grade seizures as well as longer latency times to reach grades 1–3, albeit the latency times were not statistically different from the males (). There was a higher tendency in the female mice to “walk off” the seizures resulting in stronger, lower stage seizures, while the males tended to “freeze-up” and enter higher stage seizures. While the female mice had less severe seizures, they had longer recovery times than the males (). Overall, PTZ (50 mg/kg) induced convulsive grade 3 seizures in 80% of WT male mice and 40–50% of WT female and fmr-1 KO male and female mice without causing any deaths ().
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 (more ...)
Seizure scores in WT and fmr-1 KO mice in a pure C57BL/6 background
Latency time to seizure in WT and fmr-1 KO in a pure C57BL/6 background
With our seizure baseline established, we assessed PTZ-induced seizures in Tg2576, FRAXAD and littermate controls. These mice were from n3-n6 backcrosses (87.5–98.4% C57BL/6). In the non-transgenic littermate controls, the average seizure scores ranged from 1.89–2.85 with trends for slightly reduced seizure thresholds in males compared to females and in fmr-1 KO compared to WT mice, albeit not statistically significant differences (WT female 1.89, WT male 2.42, KO female 2.55 and KO male 2.85) (, ). Thus, in a mixed background, average seizure scores were slightly lower than in the pure C57BL/6 background but maintained a trend whereby male mice were somewhat more sensitive to seizure induction than females.
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 (more ...)
In the Tg2576 and FRAXAD mice, PTZ induced an average seizure score greater than or equal to 4 for males and females of both strains (Tg2576 female 4.67, Tg2576 male 4.57, FRAXAD female 4.86 and FRAXAD male 4.08) (, ). Comparison of the hAPPsw-over-expressing mice (average seizure scores 4.08–4.86) to nontransgenic littermates (1.89–2.85) was statistically significant in all cases. Therefore, mice over-expressing hAPPsw, and likely proteolytic products of hAPPsw, show elevated rates of spontaneous and PTZ-induced seizures.
Seizure scores in Tg2576, FRAXAD and littermate controls
KO male mice had the highest average seizure score (2.85) of the nontransgenic controls and the FRAXAD males had the lowest average seizure score (4.08) of the hAPPsw transgenic strains. Thus, while the other hAPPsw over-expressing strains were 2+ seizure grades higher than their nontransgenic counterparts (WT and Tg2576 females Δ seizure score = 2.78, males = 2.15, fmr-1
KO and FRAXAD females = 2.31), the delta seizure score for the FRAXAD and fmr-1
KO male mice was 1.23 (). The percentage of mice exhibiting grades 3, 4 and 5 seizures versus strain and gender is given in Supplementary Figures 2A, 2B and 2C
, respectively. The lower average seizure score for the FRAXAD males can be attributed to a substantial decrease in the number of mice exhibiting grade 5 seizures (42%) compared to the female and male Tg2576 and female FRAXAD, 83, 71 and 86%, respectively, as well as to a small decrease in the number of grade 3 seizures. However, while the FRAXAD males exhibited the lowest percentage of grade 5 seizures of the hAPPsw over-expressing mice, the mice in this group that did reach grade 5 displayed the shortest latency time to death (335 sec ± 82) ().
Latency time to seizure in Tg2576, FRAXAD and littermate controls
Of note, seizure threshold was assessed at 8–10 weeks of age when the mice are sexually mature and survival of both strains is equivalent, 68–70% survival rate (8 weeks old) and 47–53% survival rate (10 weeks old). It would be difficult to assign seizure grades in mice at 29 days of age, which coincides with the maximum difference in mortality rate (19.9%) between the strains, due to the smaller body size and increased activity of juvenile mice. Hence, the mice injected with PTZ for these seizure studies were the healthiest ones in that they survived to age 8–10 weeks of age. As there was a large gender-based difference in average seizure scores between the male and female FRAXAD mice (females: 4.86 versus males: 4.08, p=0.04), we graphed the longevity data from based on gender (). At 29 days of age, the death rates are: 18% (FRAXAD males), 28% (FRAXAD females) and 2% Tg2576 males and females. The FRAXAD males, which exhibit a higher tolerance to PTZ-induced seizures, also have an increased survival rate from weaning to postnatal week 10 compared with FRAXAD females.
Figure 5 FRAXAD female mice had a higher juvenile mortality than FRAXAD males. The survival data for the mice included in was plotted separating the male and female mice. Tg2576 females (n=44), Tg2576 males (n=47), FRAXAD females (n=43) and FRAXAD males (more ...)
These mice were weaned at postnatal day 18. To test if the increased juvenile mortality in FRAXAD mice was due to a developmental delay that could be circumvented by extra time on the mother's milk, we also weaned mice at 21 days and assessed survival for the following 2 weeks (). Between 21 and 34 days of age, there was 0%, 14.3%, 4.5% and 9.1% mortality in FRAXAD males, FRAXAD females, Tg2576 males and Tg2576 females weaned at 21 days, respectively, compared to 18%, 27.9%, 4.3% and 6.8% for mice weaned at 18 days. Thus, three extra days on the mother's milk substantially reduced juvenile mortality in both male and female FRAXAD mice while not decreasing mortality in the Tg2576. These data suggest that (1) there is a developmental defect or delay in the FRAXAD, which is more pronounced in the female mice, and (2) the survival rate of juvenile mice weaned at 21 days is FRAXAD male > Tg2576 male > Tg2576 female > FRAXAD female, which inversely correlates with PTZ-induced seizure sensitivity in these strains ( and ).
Tg2576 & FRAXAD mortality between days 21–34 after weaning