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Comparative Study
. 2010 Jan 6;30(1):372-81.
doi: 10.1523/JNEUROSCI.5341-09.2010.

Many neuronal and behavioral impairments in transgenic mouse models of Alzheimer's disease are independent of caspase cleavage of the amyloid precursor protein

Julie A Harris  1 , Nino Devidze, Brian Halabisky, Iris Lo, Myo T Thwin, Gui-Qiu Yu, Dale E Bredesen, Eliezer Masliah, Lennart Mucke
Affiliations
Comparative Study

Many neuronal and behavioral impairments in transgenic mouse models of Alzheimer's disease are independent of caspase cleavage of the amyloid precursor protein

Julie A Harris et al. J Neurosci. .

Abstract

Previous studies suggested that cleavage of the amyloid precursor protein (APP) at aspartate residue 664 by caspases may play a key role in the pathogenesis of Alzheimer's disease. Mutation of this site (D664A) prevents caspase cleavage and the generation of the C-terminal APP fragments C31 and Jcasp, which have been proposed to mediate amyloid-beta (Abeta) neurotoxicity. Here we compared human APP transgenic mice with (B254) and without (J20) the D664A mutation in a battery of tests. Before Abeta deposition, hAPP-B254 and hAPP-J20 mice had comparable hippocampal levels of Abeta(1-42). At 2-3 or 5-7 months of age, hAPP-B254 and hAPP-J20 mice had similar abnormalities relative to nontransgenic mice in spatial and nonspatial learning and memory, elevated plus maze performance, electrophysiological measures of synaptic transmission and plasticity, and levels of synaptic activity-related proteins. Thus, caspase cleavage of APP at position D664 and generation of C31 do not play a critical role in the development of these abnormalities.

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Figures

Figure 1.
Figure 1.
hAPP levels are higher in hAPP–B254 mice than in hAPP–J20 mice. A, The PDGF–hAPP transgene in the hAPP–J20 line carries the Swedish and Indiana familial AD mutations (Mucke et al., 2000). An additional mutation was introduced into this transgene at the D664 caspase cleavage site to generate the hAPP–B254 line (Galvan et al., 2006). Genomic DNA was amplified and sequenced in both forward and reverse directions to verify that the D664A mutation was indeed present in the hAPP–B254 mice analyzed in this study. An A-to-C nucleotide substitution was identified, resulting in a D-to-A amino acid change at the correct position (data not shown). B, Cortical homogenates from 7- to 10-month-old transgenic mice were analyzed by Western blotting and densitometric analysis of hAPP signals. Tubulin served as a loading control. hAPP levels were ∼1.2-fold higher in hAPP–B254 than hAPP–J20 mice. n = 11 mice per genotype, *p < 0.05 versus J20, t test. Bar graph shows mean ± SEM.
Figure 2.
Figure 2.
Aβ levels in hAPP–B254 and hAPP–J20 mice. A–C, Aβ1–x and Aβ1-42 levels were measured in cortical lysates from the two transgenic lines at 2 and 3–4 months by ELISA. At 2 months, Aβ1–x (A), but not Aβ1-42 (B), was significantly higher in hAPP–B254 than hAPP–J20 mice. By 3–4 months, both Aβ1–x and Aβ1-42 levels in hAPP–B254 mice were higher than in age-matched hAPP–J20 mice and higher than in 2-month-old hAPP–B254 mice. Aβ1-42/Aβ1–x ratios (C) in hAPP–B254 and hAPP–J20 mice did not differ at 2 months but were significantly higher in hAPP–B254 mice at 3–4 months. n = 4–7 mice per genotype and age; *p < 0.05, **p < 0.005 versus J20, t test; ##p < 0.005 by ANOVA and Tukey's post hoc test. D, Immunostaining for Aβ with the 3D6 antibody at 7–10 months of age revealed a greater level of hippocampal Aβ deposition in hAPP–B254 than hAPP–J20 mice. E, The percentage area covered by 3D6-immunoreactive Aβ deposits was quantified to determine “plaque loads.” n = 11 per genotype; **p < 0.005 versus J20, t test. F, Levels of the Aβ*56 oligomer were measured in cortical homogenates from 7- to 10-month-old transgenic mice by Western blotting. G, Densitometric quantitation of Western blot signals revealed comparable levels of Aβ*56 in the two transgenic lines. H, Aβ*56/hAPP ratios were lower in hAPP–B254 than hAPP–J20 mice. n = 11 per genotype; ***p < 0.0005 versus J20, t test. All bar graphs show mean ± SEM.
Figure 3.
Figure 3.
The D664A mutation does not prevent behavioral abnormalities in the elevated plus maze or hyperactivity in an open field arena. Groups of transgenic and NTG mice from lines B254 and J20 were analyzed in the indicated behavioral paradigms at 2–3 or 5–7 months of age. A, In the elevated plus maze, hAPP–B254 mice and hAPP–J20 mice spent more time in the open arms than NTG controls. B, In the open field, hAPP–B254 mice and hAPP–J20 mice were hyperactive compared with their NTG controls. In both paradigms, hAPP–B254 trended toward impairments at 2–3 months of age, but the differences only reached significance in the 5- to 7-month-old group. n = 9 mice per group (at 2–3 months); n = 11–15 J20 mice per group and 22–23 B254 mice per group (at 5–7 months). *p < 0.05, **p < 0.005, ***p < 0.0005 versus NTG, t test. All bar graphs show mean ± SEM.
Figure 4.
Figure 4.
The D664A mutation does not prevent deficits in nonspatial recognition memory. Groups of transgenic and NTG mice from lines B254 and J20 were analyzed in the novel object recognition test at 2–3 or 5–7 months of age. hAPP–B254 mice and hAPP–J20 mice did not spend more time with a novel than with a familiar object in test sessions, in contrast to NTG controls. n = 9 mice per group (at 2–3 months); n = 11–15 J20 mice per group and 22–23 B254 mice per group (at 5–7 months). *p < 0.05, **p < 0.005 novel versus familiar object or as indicated by brackets, ANOVA with Tukey's post hoc test. All bar graphs show mean ± SEM.
Figure 5.
Figure 5.
The D664A mutation does not prevent deficits in spatial learning and memory. Two independent cohorts of 5- to 7-month-old hAPP–B254 mice, hAPP–J20 mice, and NTG controls were trained in the Morris water maze for 5 d. A, In the first cohort, hAPP–B254 and hAPP–J20 mice both had longer latencies to find a hidden platform than NTG controls (p < 0.0001, repeated measures ANOVA). Total distances of the swim paths to reach the platform were also analyzed and showed similar results as latency to platform (data not shown). B, Similar to the first cohort, a second, independent cohort of hAPP–B254 and hAPP–J20 mice also had impaired learning in the hidden platform component of the task (p < 0.0001, repeated measures ANOVA). C, In the first cohort of mice, a probe trial 24 h after the hidden task training was completed showed that all groups of mice remembered the location of the platform, spending more time in the target quadrant compared with the other quadrants of the pool. D, In the second cohort, an earlier probe trial was given 2 d after the start of hidden platform training. This probe trial revealed memory deficits in both hAPP–B254 and hAPP–J20 mice, whereas the NTG littermates from both lines already showed a significant preference for the target quadrant. n = 6–9 mice per group (cohort 1; A, C); n = 5–13 mice per group (cohort 2; B, D). *p < 0.05, **p < 0.005, ***p < 0.0005, percentage time in target quadrant versus other quadrants by t test. Graphs show mean ± SEM.
Figure 6.
Figure 6.
The D664A mutation does not prevent alterations in synaptic activity-related proteins in the dentate gyrus. A–D, Representative photomicrographs show brain sections from 7- to 10-month-old mice immunostained for calbindin (A), Fos (B), or NPY (C, D). Arrows indicate the molecular layer (C) and mossy fiber terminals in CA3 (D). E–H, Protein levels of calbindin (E) and NPY (G, H) were quantified by densitometry of immunoreactivities. Fos expression (F) was quantified by counting granule cells immunoreactive for Fos. Compared with NTG controls, both hAPP–B254 and hAPP–J20 mice had significant reductions in calbindin (E) and Fos (F). hAPP–B254 mice had significantly less calbindin expression than hAPP–J20 mice. Transgenic mice of both lines had comparable increases in NPY in the molecular layer of the dentate gyrus (G) and the mossy fiber pathway (H). n = 11 per genotype; *p < 0.05, **p < 0.005, ***p < 0.0005 versus NTG from the same line, t test; #p < 0.005 by ANOVA and Tukey's post hoc test. Graphs show mean ± SEM.
Figure 7.
Figure 7.
The D664A mutation does not prevent loss of synaptophysin and MAP-2. A–D, The percentage area of immunostained sections covered by synaptophysin (A, B) or MAP2 (C, D) immunoreactivity was quantified in the frontal cortex and outer molecular layer of the dentate gyrus. hAPP–B254 mice and hAPP–J20 mice showed comparable reductions in synaptophysin and MAP-2 in both regions. n = 9–11 mice per group. **p < 0.005, ***p < 0.0005 versus NTG from the same line, t test. Graphs show mean ± SEM.
Figure 8.
Figure 8.
The D664A mutation does not prevent deficits in baseline synaptic transmission strength in CA1. Acute hippocampal slices were obtained from 2- to 3-month-old mice and analyzed electrophysiologically. A, B, The input–output relationship along the Schaffer collateral–CA1 synapse was impaired in both hAPP–B254 mice and hAPP–J20 mice relative to NTG controls (p < 0.005, Tukey's post hoc analysis on repeated measures ANOVA; n = 6 slices from 3 mice per genotype). C, D, In contrast, synaptic strength at the medial perforant path synapses onto granule cells of the dentate gyrus was not affected in either hAPP–B254 or hAPP–J20 mice (n = 6 slices from 3 mice per genotype). Insets show example fEPSP traces for different levels of stimulation. Calibration: 10 ms, 0.5 mV.
Figure 9.
Figure 9.
The D664A mutation does not prevent deficits in synaptic plasticity in the dentate gyrus. Acute hippocampal slices were obtained from 2- to 3-month-old mice and analyzed electrophysiologically. LTP and paired-pulse ratios were analyzed at synapses in CA1 and the DG. A, B, LTP was not impaired at the Schaffer collateral–CA1 synapse in either hAPP–B254 (A) or hAPP–J20 (B) mice compared with NTG controls (n = 6–7 slices from 3 mice per genotype). C, Paired-pulse ratios at the Schaffer collateral–CA1 synapse were also unimpaired in both hAPP transgenic lines relative to NTG controls (n = 6 slices from 3 mice per genotype). D, E, LTP at the medial perforant path synapse on granule cells of the dentate gyrus was depressed in both hAPP–B254 (D) and hAPP–J20 (E) mice relative to NTG controls (p < 0.005, Tukey's post hoc analysis on repeated measures ANOVA; n = 6–7 slices from 3 mice per genotype). F, Paired-pulse ratios at the medial perforant pathway were also reduced in both hAPP–B254 and hAPP–J20 mice relative to NTG controls (***p < 0.0005 vs NTG, t test; n = 6–7 slices from 3 mice per genotype). Insets show example fEPSP traces before (black line) and after (gray line) LTP induction stimuli. Calibration: 10 ms, 0.5 mV.
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