A mouse model of amyloid beta oligomers: their contribution to synaptic alteration, abnormal tau phosphorylation, glial activation, and neuronal loss in vivo

Abstract

Although amyloid beta (Abeta) oligomers are presumed to cause synaptic and cognitive dysfunction in Alzheimer's disease (AD), their contribution to other pathological features of AD remains unclear. To address the latter, we generated APP transgenic mice expressing the E693Delta mutation, which causes AD by enhanced Abeta oligomerization without fibrillization. The mice displayed age-dependent accumulation of intraneuronal Abeta oligomers from 8 months but no extracellular amyloid deposits even at 24 months. Hippocampal synaptic plasticity and memory were impaired at 8 months, at which time the presynaptic marker synaptophysin began to decrease. Furthermore, we detected abnormal tau phosphorylation from 8 months, microglial activation from 12 months, astrocyte activation from 18 months, and neuronal loss at 24 months. These findings suggest that Abeta oligomers cause not only synaptic alteration but also other features of AD pathology and that these mice are a useful model of Abeta oligomer-induced pathology in the absence of amyloid plaques.

Figure 1.

APP_E693Δ-Tg mice do not exhibit extracellular amyloid deposits but do display intraneuronal Aβ accumulation. A, B, Levels of expression of human APP in APP_E693Δ-Tg mice. Brain homogenates of Tg mice were subjected to Western blotting to examine levels of APP expression. A, Human APP was probed with 6E10 antibody specific to human APP/Aβ. Comparison among the Tg2576 mice, APP_E693Δ-Tg mice, APP_WT-Tg line 1, and non-Tg littermates was performed at 12 months, while that between the APP_E693Δ-Tg mice and APP_WT-Tg line 3 was performed at 8 months. B, Human and mouse APP in APP_E693Δ-Tg mice and non-Tg littermates were stained at 12 months with C40 antibody recognizing the C-terminal region of human and mouse APP. C–R, Brain Aβ burden in APP_E693Δ-Tg mice. Brain sections of 24-month-old Tg mice were stained with β001 antibody to the N-terminal region of Aβ. Tg2576 mice (C–F) displayed abundant amyloid plaques in cerebral cortex (D) and hippocampus (E, CA3 region) but not in cerebellum (F), whereas APP_E693Δ-Tg mice (G–J), APP_WT-Tg mice (K–N), and non-Tg littermates (O–R) exhibited no extracellular amyloid deposits in any regions examined; cerebral cortex (H, L, P), hippocampal CA3 region (I, M, Q), and cerebellum (J, N, R). The APP_E693Δ-Tg mice did, however, exhibit intraneuronal staining of Aβ in these regions. CTX, Cerebral cortex; HC, hippocampus; CBL, cerebellum. Scale bar, 30 μm.

Figure 2.

Age-dependent accumulation of intraneuronal Aβ oligomers in APP_E693Δ-Tg mice. A–L, Brain sections of 4 (A, E)-, 8 (B, F)-, 12 (C, G)-, and 24 (D, H)-month-old APP_E693Δ-Tg mice were stained with Aβ oligomer-selective antibody NU-1. Intraneuronal Aβ oligomers first appeared at 8 months in the cerebral cortex (A–D) and hippocampus (E–H, the CA3 region) and accumulated in age-dependent fashion. No NU-1 staining was observed in APP_WT-Tg mice (I, J) or non-Tg littermates (K, L) even at 24 months; cerebral cortex (I, K) and hippocampal CA3 region (J, L). Scale bars, 30 μm. M, Oligomer formation of Aβ in APP_E693Δ-Tg mice was confirmed by immunoprecipitation/Western blotting analysis. Brain homogenates of 24-month-old Tg mice were fractionated by 4-step ultracentrifugation into TBS-, Triton X-100-, SDS-, and FA-soluble fractions. Aβ in each fraction was immunoprecipitated with 6E10 and stained with β001. Aβ dimers, and possibly trimers, were detected in the FA-extracted fraction from APP_E693Δ-Tg mice but only slightly in APP_WT-Tg mice. Std, Standard; TS, TBS. N–Q, Brain sections of 24-month-old Tg mice were stained with the amyloid-binding dye thioflavin S. Tg2576 mice exhibited abundant extracellular staining due to parenchymal and vascular amyloid deposits in cerebral cortex (N) and hippocampus (O), whereas APP_E693Δ-Tg mice exhibited no thioflavin S staining in these regions; cerebral cortex (P) and hippocampal CA3 region (Q). However, APP_E693Δ-Tg mice exhibited very weak and somewhat diffuse staining within neurons (Q, arrowhead). CTX, Cerebral cortex; HC, hippocampus. Scale bars: N, P, 100 μm; O, Q, 20 μm.

Figure 3.

Age-dependent decrease in synaptophysin in APP_E693Δ-Tg mice. A–L, Brain sections of 4 (A, E, I)-, 8 (B, F, J)-, 12 (C, G, K)-, and 24 (D, H, L)-month-old Tg mice were stained with antibody to the presynaptic marker synaptophysin. All images were taken from the hippocampal CA3 region. Unlike non-Tg littermates (A–D) and APP_WT-Tg mice (E–H), the APP_E693Δ-Tg mice (I–L) exhibited age-dependent decrease in synaptophysin in the hippocampus from 8 months. Scale bars, 30 μm. M, Synaptophysin fluorescence intensity in 30 μm × 60 μm area of the hippocampal CA3 region was quantified using the NIH ImageJ software and shown in arbitrary units (AU). Each bar represents the mean ± SEM (n = 3). *p = 0.0258 versus NonTg; p = 0.0244 versus APP_WT-Tg, **p = 0.0052 versus NonTg; p = 0.0092 versus APP_WT-Tg, ***p = 0.0140 versus NonTg; p = 0.0387 versus APP_WT-Tg.

Figure 4.

Impairment of synaptic plasticity in APP_E693Δ-Tg mice. Synaptic functions of Tg mice were examined by in vivo electrophysiology at 8 months. Population spikes were recorded in the granular cell body layer of the dentate gyrus in response to stimulation of the perforant path. A, Basal synaptic transmission was examined by preparing I/O curves with increasing stimulus intensities. No significant difference was observed among the non-Tg littermates, APP_WT-Tg, and APP_E693Δ-Tg mice (n = 4 for each group). B, Short-term synaptic plasticity was studied by testing PPF. Compared with non-Tg littermates, both APP_E693Δ-Tg and APP_WT-Tg mice exhibited significantly reduced PPF; the reduction was larger in APP_E693Δ-Tg mice than in APP_WT-Tg mice. *p = 0.0275 versus NonTg, **p = 0.0002 versus NonTg; but not significant versus APP_WT-Tg (n = 7 for APP_E693Δ-Tg and NonTg; n = 5 for APP_WT-Tg). C, Long-term synaptic plasticity was investigated by measuring LTP, which was elicited by delivering HFS to the perforant path. Typical population spikes at 0 and 120 min after HFS are shown. Compared with non-Tg littermates, both APP_E693Δ-Tg and APP_WT-Tg mice exhibited significant impairment of LTP, which was more severe in the APP_E693Δ-Tg mice than the APP_WT-Tg mice. *p = 0.0093 versus NonTg, **p = 0.0003 versus NonTg; but not significant versus APP_WT-Tg, when compared 5–120 min after HFS (n = 5 for APP_E693Δ-Tg and NonTg; n = 4 for APP_WT-Tg). All values are the mean ± SEM.

Figure 5.

Impairment of memory in APP_E693Δ-Tg mice. Spatial reference memory of Tg mice was assessed by the Morris water maze at 8 months. A, Mice were trained to swim to the hidden platform for 6 consecutive days. Each point represents the mean latency of five trials per day ± SEM (n = 9 for APP_E693Δ-Tg; n = 8 for NonTg and APP_WT-Tg). The APP_E693Δ-Tg mice exhibited significantly longer escape latencies than the APP_WT-Tg mice and non-Tg littermates. The APP_WT-Tg mice exhibited slightly, but not significantly, longer escape latencies than the non-Tg littermates. *p < 0.0001 versus NonTg; p = 0.0010 versus APP_WT-Tg. B, At day 7, retention of memory was assessed by a probe trial for 30 s with the platform removed. Each bar represents the mean time occupancy ± SEM in the target quadrant (TQ), adjacent quadrants (AQ1, AQ2), or opposite quadrant (OQ). The APP_E693Δ-Tg mice spent significantly shorter time in the target quadrant than the APP_WT-Tg mice and non-Tg littermates did. *p = 0.0265 versus NonTg; p = 0.0149 versus APP_WT-Tg.

Figure 6.

Abnormal tau phosphorylation in APP_E693Δ-Tg mice. A–P, Brain sections of 8 (E, M)-, 12 (F–H, N–P)-, and 24 (A–D, I–L)-month-old Tg mice were stained with antibodies reactive to pathological tau, PHF-1 (A–H), and MC1 (I–P). These antibodies stained dystrophic neurites around amyloid plaques in the Tg2576 mice at 24 months (A, I). The APP_WT-Tg mice (B–D, J–L; C, K, hippocampal CA3 region; D, L, cerebral cortex) exhibited no staining with these antibodies even at 24 months. In contrast, APP_E693Δ-Tg mice (E–H, M–P) began to display PHF-1-positive and MC1-positive hippocampal mossy fibers (E, M, CA3 region) from 8 months. At 12 months, immunoreactivity was more evident (G, O, the CA3 region), and the cingulum was also stained with PHF-1 but not MC1 (H, P, the cerebral cortex). CTX, Cerebral cortex; HC, hippocampus; mf, mossy fibers; cg, cingulum. Scale bars, 30 μm.

Figure 7.

Glial activation in APP_E693Δ-Tg mice. A–T, Brain sections of 8 (E, I, M, Q)-, 12 (F, J, N, R)-, 18 (G, K, O, S)-, and 24 (A–D, H, L, P, T)-month-old Tg mice were stained with antibodies to Iba-1 (A, B, E–L) and GFAP (C, D, M–T), which are markers of microglia and astrocytes, respectively. All images were taken from the hippocampal CA3 region, except those of the Tg2576 mice, which were obtained from cerebral cortex. The Tg2576 mice (A, C) at 24 months exhibited massive staining with these antibodies around amyloid plaques, while the non-Tg littermates (B, D) exhibited no staining at 24 months. The APP_WT-Tg mice (E–H, M–P) possessed no Iba-1-positive cells and only a few GFAP-positive cells at 24 months. In contrast, the APP_E693Δ-Tg mice (I–L, Q–T) displayed Iba-1-positive cells from 12 months and GFAP-positive cells from 18 months in both the hippocampus and cerebral cortex. Scale bar, 30 μm.

Figure 8.

Neuronal loss in APP_E693Δ-Tg mice. A–C, E–G, Brain sections of Tg mice were stained with an antibody to NeuN, a marker of mature neurons. Compared with non-Tg littermates (A, E) and APP_WT-Tg mice (B, F), APP_E693Δ-Tg mice (C, G) exhibited significant decrease in NeuN-positive cells in the hippocampal CA3 region, but no decrease in cerebral cortex at 24 months; hippocampal CA3 region (A–C) and cerebral cortex (E–G). No significant difference was observed between non-Tg littermates and APP_WT-Tg mice at 24 months. D, NeuN-positive cells in the pyramidal cell layer of the hippocampal CA3 region were counted within 900 μm from its end toward the dentate gyrus in the photographs. *p = 0.0044 versus NonTg; p = 0.0121 versus APP_WT-Tg (n = 4). CTX, Cerebral cortex; HC, hippocampus. Scale bars, 100 μm.