A longitudinal multimodal in vivo molecular imaging study of the 3xTg-AD mouse model shows progressive early hippocampal and taurine loss

Abstract

The understanding of the natural history of Alzheimer's disease (AD) and temporal trajectories of in vivo molecular mechanisms requires longitudinal approaches. A behavioral and multimodal imaging study was performed at 4/8/12 and 16 months of age in a triple transgenic mouse model of AD (3xTg-AD). Behavioral assessment included the open field and novel object recognition tests. Molecular characterization evaluated hippocampal levels of amyloid β (Aβ) and hyperphosphorylated tau. Magnetic resonance imaging (MRI) included assessment of hippocampal structural integrity, blood-brain barrier (BBB) permeability and neurospectroscopy to determine levels of the endogenous neuroprotector taurine. Longitudinal brain amyloid accumulation was assessed using 11C Pittsburgh compound B positron emission tomography (PET), and neuroinflammation/microglia activation was investigated using 11C-PK1195. We found altered locomotor activity at months 4/8 and 16 months and recognition memory impairment at all time points. Substantial early reduction of hippocampal volume started at month 4 and progressed over 8/12 and 16 months. Hippocampal taurine levels were significantly decreased in the hippocampus at months 4/8 and 16. No differences were found for amyloid and neuroinflammation with PET, and BBB was disrupted only at month 16. In summary, 3xTg-AD mice showed exploratory and recognition memory impairments, early hippocampal structural loss, increased Aβ and hyperphosphorylated tau and decreased levels of taurine. In sum, the 3xTg-AD animal model mimics pathological and neurobehavioral features of AD, with early-onset recognition memory loss and MRI-documented hippocampal damage. The early-onset profile suggests temporal windows and opportunities for therapeutic intervention, targeting endogenous neuroprotectors such as taurine.

Figure 1

(A) Representative trace images of WT and 3xTg-AD mice in the open field. Locomotor activity of WT and 3xTg-AD animals was evaluated by measuring the (B) distance traveled (m) and (C speed (m/s) at different time points (4, 8, 12 and 16 months of age). Results are presented as mean ± SEM and were analyzed with the Student’s t-test; ^*P < 0.05 and ^**P < 0.01 (n _{WT at 4 months} = 21, n _{WT at 8 months} = 20, n _{WT at 12 months} = 17, n _{WT at 16 months} = 17, n _{3xTg-AD at 4 months} = 20, n _{3xTg-AD at 8 months} = 23, n _{3xTg-AD at 12 months} = 22, n _{3xTg-AD at 16 months} = 22).

Figure 2

Exploration time of WT and 3xTg-AD mice in the novel object recognition test at different time points (4, 8, 12 and 16 months of age). (A) Exploration time of both groups is compared; (B) exploration time of WT in familiar and novel object; (C) exploration time of 3xTg-AD in familiar and novel object. Results are presented as mean ± SEM and were analyzed with the Student’s t-test; ^*P < 0.05, ^**P < 0.01 and ^***P < 0.001 (n _{WT at 4 months} = 21, n _{WT at 8 months} = 20, n _{WT at 12 months} = 17, n _{WT at 16 months} = 17, n _{3xTg-AD at 4 months} = 20, n _{3xTg-AD at 8 months} = 23, n _{3xTg-AD at 12 months} = 22, n _{3xTg-AD at 16 months} = 22).

Figure 3

Number of explorations of WT and 3xTg-AD mice in the novel object recognition test at different time points (4, 8, 12 and 16 months of age). (A) Number of explorations of both groups is compared; (B) number of explorations of WT in familiar and novel object; (C) number of explorations of 3xTg-AD in familiar and novel object. Results are presented as mean ± SEM and were analyzed with the Student’s t-test; ^*P < 0.05, ^**P < 0.01 and ^***P < 0.001 (n _{WT at 4 months} = 21, n _{WT at 8 months} = 20, n _{WT at 12 months} = 17, n _{WT at 16 months} = 17, n _{3xTg-AD at 4 months} = 20, n _{3xTg-AD at 8 months} = 23, n _{3xTg-AD at 12 months} = 22, n _{3xTg-AD at 16 months} = 22).

Figure 4

Total exploration time (A) and novel object exploration time (B) of WT and 3xTg-AD mice at different time points (4, 8, 12 and 16 months of age). Results are presented as mean ± SEM and were analyzed with the Student’s t-test; ^*P < 0.05, ^**P < 0.01 and ^***P < 0.001 (n _{WT at 4 months} = 21, n _{WT at 8 months} = 20, n _{WT at 12 months} = 17, n _{WT at 16 months} = 17, n _{3xTg-AD at 4 months} = 20, n _{3xTg-AD at 8 months} = 23, n _{3xTg-AD at 12 months} = 22, n _{3xTg-AD at 16 months} = 22).

Figure 5

Increase of Aβ and p-tau protein levels in the hippocampus of 3xTg-AD mice. The protein levels of Aβ (A) and p-tau (B) in total hippocampal extracts from 3xTg-AD and age-matched WT mice were analyzed by western blotting. Representative images for Aβ, p-Tau and β-actin (loading control) for each time point (4, 8, 12 and 16 months of age) are presented. The results are expressed as percentage of age-matched WT animals and are presented as mean ± SEM of four to six animals. ^*P < 0.05 and ^**P < 0.01, different from WT, Mann–Whitney test.

Figure 6

Hippocampal volumes of WT and 3xTg-AD mice at different time points (4, 8, 12 and 16 months). Solid lines represent the regression curves showing the temporal evolution of the hippocampal volumes. Regression curves: WT,; 3xTg-AD, . V, volume; t, time. Results are presented as mean ± SEM and were analyzed with an ANOVA repeated measures (mixed-effect) followed by a Bonferroni post hoc test and a linear regression analysis (n _{WT at 4, 8, 12 and 16 months} = 4, n _{3xTg-AD at 4, 8, 12 and 16 months} = 6).

Figure 7

VBM results in a coronal view at 4 (A) and 16 months. (B) Color bar denotes the T-score magnitude, at the voxel level, with a threshold level of P < 0.05 FWE corrected (n _{WT at 4, 8, 12 and 16 months} = 6, n _{3xTg-AD at 4, 8, 12 and 16 months} = 7).

Figure 8

Perfusion peak amplitude and BBB index at 16 months of age. Right panel, BBB permeability index. Left panel, perfusion peak amplitude. Results are presented as mean ± SEM and were analyzed with an ANOVA repeated measures (mixed-effect) followed by a pairwise comparison employing a Bonferroni post hoc test; ^**P < 0.01 (n _{WT at 4, 8, 12 and 16 months} = 4, n _{3xTg-AD at 4, 8, 12 and 16 months} = 6).

Figure 9

Taurine levels of 3xTg-AD and WT mice at different time points (4, 8, 12 and 16 months of age). Results are presented as mean ± SEM and were analyzed with an ANOVA repeated measures (mixed-effect) followed by a pairwise comparison employing a Bonferroni post hoc test; ^*P < 0.05 and ^**P < 0.01(n _{WT at 4, 8, 12 and 16 months} = 4 and n _{3xTg-AD at 4, 8, 12 and 16 months} = 6).

Figure 10

Representative 1H-MRS spectra of WT (A) and 3xTg-AD (B) mice acquired at 16 months.