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Impairments in learning and memory accompanied by neurodegeneration in mice transgenic for the carboxyl-terminus of the amyloid precursor protein
Molecular Brain Research 66 Ž1999. 150–162
Research report
Impairments in learning and memory accompanied by neurodegeneration in mice transgenic for the carboxyl-terminus of the amyloid precursor protein Joanne Berger-Sweeney a , Donna L. McPhie b, Jill A. Arters a , Jane Greenan c , Mary Lou Oster-Granite c , Rachael L. Neve b,) a
b
Department of Biological Sciences, Wellesley College, Wellesley, MA 02181, USA Department of Genetics, HarÕard Medical School and McLean Hospital, Belmont, MA 02478, USA c DiÕision of Biomedical Sciences, UniÕersity of California, RiÕerside, CA 92521-0121, USA Accepted 29 December 1998
Abstract In Alzheimer’s disease ŽAD., a progressive decline of cognitive functions is accompanied by neuropathology that includes the degeneration of neurons and the deposition of amyloid in plaques and in the cerebrovasculature. We have proposed that a fragment of the Alzheimer amyloid precursor protein ŽAPP. comprising the carboxyl-terminal 100 amino acids of this molecule ŽAPP-C100. plays a crucial role in the neurodegeneration and subsequent cognitive decline in AD. To test this hypothesis, we performed behavioral analyses on transgenic mice expressing APP-C100 in the brain. The results revealed that homozygous APP-C100 transgenic mice were significantly impaired in cued, spatial and reversal performance of a Morris water maze task, that the degree of the impairment in the spatial learning was age-dependent, and that the homozygous mice displayed significantly more degeneration of neurons in Ammon’s horn of the hippocampal formation than did heterozygous or control mice. Among the heterozygotes, females were relatively more impaired in their spatial learning than were males. These findings show that expression of APP-C100 in the brain can cause age-dependent cognitive impairments that are accompanied by hippocampal degeneration. q 1999 Elsevier Science B.V. All rights reserved. Keywords: Neurodegeneration; Transgenic mouse; Spatial learning; Alzheimer disease; Amyloid precursor protein; Gene; Neurotoxicity; Cognition
1. Introduction All individuals with Alzheimer’s disease ŽAD. experience a progressive loss of cognitive function, resulting from a neurodegenerative process characterized classically by granulovacular degeneration, the deposition of bamyloid ŽAb . in plaques and in the cerebrovasculature, and the formation of neurofibrillary tangles in neurons. Additional pathological hallmarks of AD include degeneration of synapses w8,14,40x and decreases in cell density w39x in distinct regions of the brain. The molecular events that link these distinct pathological entities remain cryptic.
) Corresponding author. Molecular Neurogenetics Laboratory, McLean Hospital, 115 Mill Street, Belmont, MA 02478, USA. Fax: q1-617-8553793; E-mail: [email protected]
We w42x and others w11,38x have reported that the carboxyl-terminal ŽC-terminal. 100-amino acid fragment of the amyloid protein precursor ŽAPP-C100., which includes the 42-amino acid Ab peptide and 58 adjacent amino acids in the carboxyl-terminus of APP, is neurotoxic, although it is not yet clear whether intact APP-C100 or the Ab peptide that is derived from it is directly responsible for the neurotoxicity displayed by this molecule. Published in vivo models have tested the hypothesis that APP-C100 plays a causal role in AD neurodegeneration. In particular, AD-like pathology has been described in transgenic mice expressing APP-C100 w19,29,35x or APP-C104 w26x in the brain. In support of these models, such carboxyl-terminal fragments of APP have been shown to accumulate in pathological lysosomal structures in AD brain w2,25x, and in neurons expressing any of the five known familial Alzheimer’s disease ŽFAD. mutants of
0169-328Xr99r$ - see front matter q 1999 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 9 - 3 2 8 X Ž 9 9 . 0 0 0 1 4 - 5
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APP w23x. We now describe deficits in learning accompanied by profound degeneration of the hippocampal formation that occurs in the brains of transgenic mice that express APP-C100 in the brain. The appearance of some stereotypical features of AD in these transgenic mice provides support for the hypothesis that APP-C100 may be a critical contributor to the processes that lead to neurodegeneration in AD.
2. Materials and methods 2.1. Production and characterization of transgenic mice The creation of transgenic mice expressing Flag w32x epitope-tagged APP-C100 in the brain under the control of the dystrophin brain promoter has been described w29x. Reverse transcription-PCR ŽRT-PCR. was used as described w29x to assess expression of the transgene RNA. 2.2. Immunoblots and immunoprecipitations We used the following anti-peptide antibodies: 10D5 Žgift of D. Schenk., directed against amino acids 1–15 of human Ab; E1-42 w7x Žgift of B. Cummings., directed against the 42-amino acid Ab polypeptide; and C8 w37x Žgift of D. Selkoe., immunoreactive with the carboxylterminal 10 amino acids of APP. Following cervical dislocation and decapitation of the mice, brain tissue was dissected rapidly from transgenic and control mice and immediately homogenized as described w9x. Each homogenate Ž20–25 mg. was subjected to electrophoresis in 16.5% Tris-tricine SDS-polyacrylamide ŽTT-SDS-PAGE., transferred to PVDF ŽMillipore. membranes, and immunoblotted with the antibody 10D5 Ž5 mgrml. as described w27x. Immunoprecipitations on 100-ml samples of the soluble and particulate fractions of brain were carried out at 48C using standard protocols w15x. Following the final wash, the samples were subjected to 16.5% TT-SDS-PAGE, electrotransferred to PVDF membrane, and processed for immunoblot analysis with the antibody C8 Ž1:6000 dilution.. 2.3. Numbers and ages of mice analyzed behaÕiorally Ninety-four mice were included in the behavioral analysis: 31 controls, 22 heterozygotes, and 31 homozygotes. At the time of testing the heterozygous mice were between 12 and 14.5 months of age; there were 12 males and 10 females. The homozygotes were various ages ranging from 4 to 10 months: 21 four month-olds, 5 six month-olds and 5 ten month-olds. The controls consisted of 4 males and 4 females at 4–6 months of age, and 13 males and 11 females at 12–14 months of age. Mice were housed 3–4 mice per cage, separated by sex and maintained in a room
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on a 12:12 h light–dark cycle, lights on at 0600 h. Food and water were available ad libitum except during water maze testing. The mice were first habituated to the experimenter for about one week, at the end of which a neurological battery Žadapted from Paul et al. w31x. was carried out. The battery included testing of righting, grasping, and placing reflexes. The mice then were subjected to three weeks of a Morris water maze task which included cued, spatial, probe and reversal tasks. This task is used commonly to assess learning and memory in mice. Dark-cycle locomotor activity was monitored on the same days as the swim maze tasks to examine activity as a possible confound on the water maze tasks. The experimenter was blind to the transgenic status of the mice during testing. 2.4. Swim maze testing and dark cycle actiÕity measurements All of the navigation tasks were conducted in a 180 cm diameter white circular pool surrounded by distal cues w3x and filled with 21–238C water. A 103 cm diameter ring was placed in the pool, in order to adapt the size of the pool for testing mice. A 55 cm diameter ring was used for pretraining trials. The platform, a 6 = 6 cm clear acrylic square, was always placed 15 cm from the edge of the ring in one of four quadrants defined by four equally spaced, arbitrarily designated start points ŽN, S, E, and W.. The testing schedule, consisting of one day of pretraining, five days of cued trials, five days of spatial trials followed by a probe trial, and four days of reversal trials followed by a probe trial, has been described w3,4x. For the reversal task, the pool was set up and trials were run in a manner identical to that of the spatial task, except that the invisible platform was moved to a new location. The day after completion of the reversal trials, the platform was removed from the pool and again each mouse was given a probe trial for 60 s. All navigation data were tracked and analyzed using an HVS video tracking system ŽHVS Image, Hampton, England.. Additional analyses were carried out using software designed by Wolfer and Lipp w41x. The following parameters were examined in different phases of the swim maze tasks: latency to find the platform, path length, speed, floating, and percent time in former platform quadrant during the probe trials. Dark cycle locomotor activity was measured. Briefly, mice were place in a standard mouse cage surrounded by three photobeams ŽSan Diego Instruments Cage Rack Activity System. mounted approximately 2.5 cm above the floor of the cage. Horizontal activity was measured as the total number of photobeam breaks per hour over a 12 h period, starting at 1700 h. 2.5. Histological analyses We examined 45 mice, ranging in age from 4 to 14.5 months: 11 controls, 4 heterozygotes, and 30 homozygotes
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Žone histological sample was lost.. Approximately equal numbers of each sex were examined. The heterozygote mice used for histological analyses were different from those used in the behavioral studies; the homozygotes used for histological analyses were the same mice that were used in the behavioral studies. Because the preponderant expression of the dystrophin brain promoter used in the transgenic constructs is in the hippocampus w13x, we focused our histological analyses on this structure. Deeply anesthetized animals were perfused with 4% paraformaldehyde, 0.1 M phosphate buffer, pH 7.4. The brains were cut into coronal blocks, each containing the entire hippocampal field at a given level. These regions were processed through graded alcohols to Polybed 812, and 2-mm sections were cut from each block. The sections were stained with toluidine blue. Healthy cells in CA2–CA4 were counted in the toluidine blue-stained sections. For each brain, approximately fifteen 2-mm sections were cut from the hippocampus at 10-mm intervals, at the anatomical level of the hippocampus between 1.7 and 2.18 mm posterior to Bregma. Healthy cells were defined as those with round, regularly-shaped
white nuclei, that did not stain darkly with the toluidine blue. Three independent 2-mm sections, one from the beginning, one from the middle, and one from the end of the series of fifteen sections, were counted. Degeneration was also evaluated with a silver stain in selected mice; numbers of healthy cells were obtained that were comparable to those obtained with the toluidine blue stain Ždata not shown.. 2.6. Statistical analyses In one set of analyses, control Žnon-transgenic. mice were compared to heterozygote transgenic and homozygote transgenic mice. Cued, spatial and reversal navigation data were analyzed using repeated measures analysis of variance ŽANOVA., with the transgenic status Žcontrol, heterozygote and homozygote. as the main effect; the days of the trials Žsessions. were the repeated measure. Probe data and healthy cell histology counts were analyzed using factorial ANOVAs. In a second set of analyses, the control and heterozygote data were separated by sex. Two-way repeated measures ANOVAs were performed with sex and
Fig. 1. ŽA. Brain expression of the APP-Flag-C100 transgene is revealed by RT-PCR. Hybridization of a radiolabeled internal oligonucleotide to a Southern blot of the amplification products from brain RNAs of a line 18 Flag-APP-C100 transgenic mouse and a control non-transgenic sib is shown. ŽB. Immunoblots showing the presence of the 19-kDa Flag-APP-C100 fragment Žarrows. in the brains of transgenic but not of control non-transgenice mice. Lanes 1–2: 10D5 immunoblot of brain homogenates from Flag-APP-C100 line 2 mice. Lane 1 is transgene-positive, while lane 2 is transgene-negative. Lanes 3–6: C8 immunoblot of brain fractions immunoprecipitated with E1-42. 3 and 4, soluble and particulate fractions, respectively, from the brain of a line 18 non-transgenic control sib; 5 and 6, soluble and particulate fractions from the brain of a line 18 transgenic mouse. Note the presence of an immunoreactive 19-kDa band Žarrow. in the immunoprecipitated soluble fraction of the transgenic brain. Lane 7: 35 S-labeled Flag-APP-C100 made by in vitro transcriptionrtranslation in a wheat germ lysate. This sample was electrophoresed on the same gel used for the immunoblot shown in lanes 3–6, and the lane containing the sample was not moved vertically relative to the other lanes when the figure was created.
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Finally, simple regression analysis was performed to examine linear correlations between the swim maze data Žcued, spatial and reversal path lengths. and the hippocampal healthy cell counts in the homozygote mice. Specifically, the last two days of the swim tasks were combined to represent asymptotic performance on a given task and these totals were then correlated with the histological data. Asymptotic performance represents the memory component on these navigation tasks; in previous studies following cholinergic lesions, we have seen correlations between histological parameters and asymptotic performance on navigation tasks w1x. Fig. 2. Mean photobeam breaks Ž"S.E.M.. made by control, heterozygote, and homozygote mice during 12 h of locomotor activity measurements.
3. Results 3.1. Generation of flag-APP-C100 transgenic mice
transgenic status as main effects and sessions as the repeated measure. A third set of analyses were performed on the homozygote data, examining behavioral effects with age. The ANOVAs were performed comparing the four month-old homozygote group to the six-month old homozygote group and the ten month-old homozygote group. Fisher’s protected least squared differences ŽPLSDs. were used, posthoc, to reveal significant differences between groups.
We described previously w19x the generation of APPC100 transgenic mice, in which the transgene is expressed under the control of the dystrophin brain promoter. We later created transgenic mice expressing Flag-APP-C100 w29x, which is identical to APP-C100 except for the fusion of the hydrophilic Flag epitope w32x to the N-terminus of APP-C100. A 4.65 kilobase Žkb. DNA fragment containing the dystrophin brain promoter-Flag-C100 fusion gene
Fig. 3. Mean path length Ž"S.E.M.. of control, heterozygote, and homozygote mice to find the platform during five days of a cued task, five days of a spatial task, and four days of a reversal task in the Morris water maze.
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with the SV40 early region splice and polyadenylation sequences was microinjected into the male pronuclei of fertilized eggs from F2 C57BLr6J= SJLrJ hybrid mice. Eight of the offspring were positive for the transgene, and 6 of these 8 produced transgene-positive progeny.
in the particulate fraction. C-terminal fragments of APP can be detected in human brain by this immunoprecipitation strategy w9x; in contrast, similar endogenous APPC100-like fragments were not observed in the control mouse brains.
3.2. Expression of the APP-C100 transgene RNA and protein products
3.3. BehaÕioral analyses
Reverse transcription coupled with PCR ŽRT-PCR. was used to assess transgene expression in the brains of 6–7 month old Flag-APP-C100 mice; we observed the expected 650 base pair Žbp. RT-PCR fragment ŽFig. 1A. in all transgenic brain RNAs examined. The dystrophin brain promoter is a weak promoter, as indicated by the low levels of dystrophin in the brain w28x. We chose this low-level promoter for the expression of Flag-APP-C100 because of the possibility that high levels of expression of the transgene would result in lethality before embryonic development was complete. Our use of the weak dystrophin brain promoter to drive expression of the Flag-APP-C100 transgene made it difficult to detect the transgene protein product in the brains of the transgenic mice. However, we exploited the fact that the transgene was created from a human APP cDNA, and used a monoclonal antibody to human Ab Ž10D5., which does not react with mouse bAPP, to demonstrate the expression of Flag-APP-C100 protein in the line 2 mice ŽFig. 1B.. This antibody immunodetected the expected 19-kDa band in animals that were positive for the transgene Žlane 1. but not in their transgene-negative littermates Žlane 2.. APPC100 usually migrates as a 15 kDa species w21x, but addition of the Flag tag causes it to migrate with an apparent molecular weight of 19 kDa, as seen in lane 7, in which 35 S-labeled flag-APP-C100 made in vitro as described by Kozlowski et al. w21x was subjected to the same TT-SDS-PAGE conditions. Commercially available Flag antibodies work only weakly on immunoblots Žunpublished data of R.L.N.. and therefore were not suitable for the detection of the very rare transgene product. Expression of the transgene protein product in the brains of line 18 transgenic mice was demonstrated by immunoprecipitation experiments ŽFig. 1B, lanes 3–6.. The immunoaffinity purified antiserum E1-42 w7x, raised against a peptide representing the 42-amino acid human Ab fragment, was used to immunoprecipitate C-terminal derivatives of APP from soluble and insoluble fractions of transgenic brain homogenates. The precipitated proteins were subjected to TT-SDS-PAGE and probed with C8, an antibody to the C-terminal 10 amino acids of APP w37x. Again, we detected the appropriate 19-kDa protein in the soluble fraction of brains of transgenic ŽFig. 1B, lane 6. but not of control mice Žlane 4.. Since we were able to immunoprecipitate the protein from the soluble fraction only, it seems likely that soluble Flag-APP-C100 protein is more accessible to the E1-42 antibody than is the protein
3.3.1. General health and neurological battery Control and heterozygote and homozygote transgenic mice ranged in color from black to brown to agouti. No gross motor or sensory abnormalities were apparent in the mice. All of the mice exhibited normal righting, grasping and placing reflexes. None of the mice floated excessively during any of the swim trials.
Fig. 4. Mean percentage of time Ž"S.E.M.. that control, heterozygote, and homozygote mice spent in the former platform quadrant during probe trials Žplatform removed from the pool. following a. the spatial task Žprobe 1. and b. the reversal task Žprobe 2.. Ž). Denotes significantly different from control, p’s - 0.01. Žq. Denotes significantly different from control and heterozygote, p’s - 0.05.
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3.3.2. Dark-cycle locomotor actiÕity In all of the groups of mice, locomotor activity levels followed a similar habituation profile; levels were high during the first hour, decreased sharply over the next four hours and reached asymptotic levels by about five hours ŽFig. 2.. This resulted in a significant effect of hours w F Ž11,1001. s 56.5, p s 0.0001x. There were no significant differences among the groups when controls were compared to transgenic heterozygotes and transgenic homozygotes, and no significant interaction. 3.3.3. Swim maze tasks During the cued task, all mice learned the task, in that they performed better across the days of testing. Both latency w F Ž4,364. s 22.1, p s 0.0001x and path length w F Ž4,364. s 30.3, p s 0.0001x decreased significantly across days ŽFig. 3; only path length data shown graphically.. Nevertheless, significant differences were seen among the groups on the cued task for both latency w F Ž2,91. s 10.2, p s 0.001x and path length w F Ž2,91. s 9.1, p s 0.003x. Posthoc analyses revealed that the homozygotes had significantly longer latencies than controls w p s 0.0001x and heterozygotes w p s 0.004x. There was also a significant latency= transgenic status interaction w F Ž8,364. s 2.3, p s 0.02x; the learning curve for the homozygotes was steeper than that of the other two groups. The homozygotes also had longer path lengths than controls w p s 0.001x and heterozygotes w p s 0.002x. There
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were also significant differences among the groups on speed during the cued trials w F Ž2,91. s 4.2, p s 0.018x. Post hoc analyses revealed that the homozygotes swam significantly more slowly than the heterozygotes w p s 0.005x, but not the controls. There were significant differences among the groups on the spatial task for both latency and path length. All mice learned the task, in that they performed better across the days of testing. Both latency w F Ž4,364. s 38.6, p s 0.0001x and path length w F Ž4,364. s 53.3, p s 0.001x decreased significantly across the days ŽFig. 3; only path length data shown graphically.. Nevertheless, the groups differed significantly for both latency w F Ž2,91. s 4.0, p s 0.02x and path length w F Ž2,91. s 3.8, p s 0.03x. Posthoc analyses revealed that the homozygotes had significantly longer latencies than controls w p s 0.009x and heterozygotes w p s 0.03x. The homozygotes also had longer path lengths than controls w p s 0.007x, but not heterozygotes. There were no significant interactions. Swim speed during the spatial trials, in contrast to that during the cued trials, did not differ among the groups. On the reversal task, there were no significant differences among the groups on latency, path length or speed measures. All mice learned the task, in that both latency w F Ž3,273. s 44.9, p s 0.0001x and path length w F Ž3,273. s 54.5, p s 0.0001x decreased significantly across the days ŽFig. 3, only path length data shown graphically.. There was, however, a significant transgenic status = sessions
Fig. 5. Mean path length Ž"S.E.M.. of four month, six month, and ten month old homozygote mice to find the platform during five days of a cued task, five days of a spatial task, and four days of a reversal task in the Morris water maze.
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interaction for path length w F Ž6,273. s 3.6, p s 0.002x and a trend towards a transgenic status = sessions interaction for latency w F Ž6,273. s 2.0, p s 0.07x. In both cases the interaction Žor trend. was due to a flatter learning curve in the homozygote group as compared to controls and heterozygotes. As was the case for the spatial task, there were no significant differences among the groups in swim speeds. On the probe trials, there were significant differences among the groups ŽFig. 4.. On probe trial a1 Žafter the
spatial navigation trials; Fig. 4A., there were significant differences in the percent time spent in the former goal quadrant among the groups w F Ž2,92. s 8.0, p s 0.006x. Posthoc analysis revealed that control mice spent significantly more time in the former goal quadrant than either heterozygotes w p s 0.004x or homozygotes w p s 0.005x. On probe a2 Žafter the reversal navigation trials; Fig. 4B., there were also significant differences in the percent time spent in the former goal quadrant among the groups
Fig. 6. Performance measures of 12–14.5-month control and heterozygote mice of both sexes during testing in the Morris water maze. ŽA. mean path length Ž"S.E.M.. to find the platform during five days of a cued task, five days of a spatial task, and four days of a reversal task, and ŽB. mean percentage of time Ž"S.E.M.. spent in the former platform quadrant during the probe trial Žplatform removed from the pool. following the spatial task Žprobe 1.. Ž). Denotes significantly different from control, p - 0.01.
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w F Ž2,92. s 4.3, p s 0.016x. Posthoc analysis revealed that homozygotes spent significantly less time in the former goal quadrant than controls w p s 0.005x or heterozygotes w p s 0.046x. 3.3.4. Age-related behaÕioral changes within the homozygote group The majority of the significant behavioral effects were seen in the homozygotes relative to controls. Therefore, we expanded our analysis of the homozygotes by comparing the performances of different age groups Žfour-month-olds, six-month-olds and ten-month-olds. on the swim maze tasks. On the cued task, latency and path length did not differ significantly among the different age groups ŽFig. 5, only path length data shown graphically.. Cued speed, however, did differ significantly in the different age groups w F Ž2,28. s 3.6, p s 0.04x. The ten-month-old mice swam significantly slower than the four-month- and six-month-old mice. On the spatial task, latency and path length did not differ significantly among the different age groups. However, there were significant age = session interactions for latency w F Ž8,112. s 3.0, p s 0.004x and path length w F Ž8,112. s 2.4, p s 0.018x. In both cases, the ten-monthold mice were much worse at the start of the task and performance improved steadily over the five days of test-
Fig. 7. ŽA. Mean number of healthy cells Ž"S.E.M.. in hippocampal region CA3r4 of control, heterozygote, and homozygote mice. Ž). Denotes significantly different from control and heterozygote, p’s - 0.05. ŽB. Toluidine blue stain of the pyramidal cell layer of the hippocampus in 5-month-old homozygous ŽA. one-year-old heterozygous ŽB. and control ŽC. mice. The inset in panel A is a higher-power photomicrograph of the pyramidal cell layer of this mouse, at the CA2r3 border. Degenerating cells are darkly stained. The arrowhead indicates the border of CA2 at which the healthy cell counts were initiated; all healthy cells Žas defined in Section 2. between this border and the end of CA4 extending into the hilar region of the dentate gyrus were counted.
Fig. 7 Žcontinued..
ing; the four- and six-month-olds were better at the start of testing and improved only slightly during the five days of testing. There were significant differences among age groups in swim speed w F Ž2,28. s 3.4, p s 0.048x. The
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Fig. 8. Scatterplot of the number of healthy cells vs. the sum of path length measures from the last two days of Ža. the cued task, Žb. the spatial task, and Žc. the reversal task in the Morris water maze in the homozygote mice.
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ten-month-old mice swam significantly slower than fourw p s 0.02x and six-w p s 0.03x month-old mice. By the time of the reversal task, significant differences by age were no longer apparent. There were no significant differences in either latency or path length among the different age groups. There were also no significant differences in swim speed in the different age groups and no significant interactions. Additionally, there were no significant differences by age in the time spend in the former goal quadrant during either probe trial a1 or probe trial a2. 3.3.5. Sex differences in spatial performance in heterozygotes Because some investigators have suggested that gender differences exist in Alzheimer’s disease w6,10,33,34x, we compared the performance of female and male heterozygotes, relative to controls, on the swim maze tasks at one year of age. On the cued task, there were no significant differences in either latency or path length measures by transgenic status or sex ŽFig. 6a, only path length data shown graphically.. There was a significant sex difference in swim speed w F Ž1,53. s 8.0, p s 0.007x; females swam faster than males. On the spatial task, there was a trend towards a sex difference in path lengths w F Ž1,53. s 3.5, p s 0.068x. Female heterozygotes tended to have longer path lengths than the mice in other groups. In the reversal task, the sex differences in path length reached statistical significance w F Ž1,53. s 3.9, p s 0.05x. Posthoc analyses revealed that female heterozygotes had longer path lengths than female controls w p s 0.002x, whereas male heterozygotes had shorter path lengths than male controls w p s 0.02x. This led to a significant sex = transgenic status interaction w F Ž1,53. s 16.5, p s 0.0002x. On probe a1, there were significant differences by transgenic status in the amount of time spent in the former goal quadrant wFig. 6b; F Ž1,54. s 14.7, p s 0.003x. Male and female heterozygotes spent considerably less time in the quadrant that formerly contained the platform Ž36.7 " 2.1% and 38.7 " 3.7%, respectively. than did control mice, who spent about 50% of the time in the former goal quadrant. These differences had disappeared by probe a2. There were no other significant main effects of sex or significant interactions. 3.4. Neurodegeneration in the hippocampal formation of controls, heterozygotes and homozygotes Quantitative analyses of cellular degeneration in the hippocampal formation revealed significant differences in healthy cell counts in the CA2-4 layer among the controls, heterozygotes and homozygotes w F Ž2,42. s 9.6, p s 0.004x ŽFig. 7.. Posthoc analyses ŽFig. 7a. revealed that homozygotes had significantly fewer healthy hippocampal cells than did controls w p s 0.0002x or heterozygotes w p s 0.03x.
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3.5. Correlations between behaÕior and hippocampal pathology Interestingly, the behavioral and anatomical measures followed similar patterns, controls being the best Žshortest latencies and path lengths and highest numbers of healthy hippocampal cells., followed by heterozygotes and homozygotes. Correlation analyses were possible only on the homozygote animals and selected controls because this was the only group for which we had behavioral and anatomical measures from the same animals. Asymptotic performances on cued and spatial tasks did not correlate with healthy cell counts ŽFig. 8.. There was a trend w p s 0.085x, however, that long path lengths on the reversal task were associated with fewer healthy cells.
4. Discussion We describe significant deficits in cued, spatial and reversal trials in homozygous transgenic mice expressing Flag epitope-tagged APP-C100 in the brain, relative to heterozygote transgenics and controls. Analysis of the probe data suggests additionally that the homozygotes did not learn, nor did they remember, the spatial or reversal tasks as well as the heterozygote or control mice. The significant interactions between transgenic status and days of testing on the cued and reversal tasks suggest that homozygotes also are learning these tasks at different rates from controls. Although there is a cued deficit in the homozygotes, we do not believe that this deficit represents an inability of the homozygotes to see or motate. Path lengths decreased across the days of testing on all tasks, suggesting that the homozygotes can learn and hence probably can see, since there is no reason to assume that sight would improve over the days of testing. The cued deficit also does not appear to represent a general locomotor problem because Ž1. even though the homozygotes swim more slowly than other groups on the cued task, there are no swim speed deficits in the spatial or reversal task, and Ž2. there are no significant differences between homozygotes and controls in general dark cycle locomotor activity. The cued impairment could represent a motivational problem or a cued learningrmemory impairment. It is notable that other transgenic mouse models of AD w17x have cued, as well as spatial impairments on water maze tasks. The degree of impairment of spatial learning in homozygous mice appears to be age-dependent. Cued performance, however, is not impaired in an age-related fashion. Ten-month-old homozygotes learn the spatial task at a different rate from four- and six-month-olds. Interestingly, the ten-month-olds begin the spatial task at a level significantly worse than do either four- or six-month-olds. The younger mice behave as if they developed a spatial map during the cued task, which allowed them to master quickly
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the spatial task. In contrast, the older homozygous mice do not appear to have remembered what they learned in the cued task to guide performance in the spatial task. These age-related changes are no longer apparent in the reversal task. Although the heterozygous transgenic mice displayed significantly less learning and memory impairments than did the homozygotes, subtle alterations in their behavior could be seen. Significantly, the heterozygous mice evince, by the age of one year, spatial learning deficits in the absence of cued learning impairment, relative to controls. It is interesting to note that in the heterozygotes, the task that was most sensitive to their deficits was the reversal task. Additionally, the behaviorrhistology correlation that came closest to being significant was the correlation between asymptotic performance of the reversal task and healthy cell counts. These two results suggest that the reversal task, which involves unlearning one platform position and relearning a new platform position, may be particularly sensitive in detecting transgene-induced impairments in the mice. This task did not appear to be similarly sensitive to the age-related deficits in the homozygotes, perhaps because for the ten-month-old mice the spatial task resembles a ‘reversal’ task because there is little carry-over from the cued task, as noted above. There were notable sex differences in the performance of the spatial and reversal tasks by the heterozygous mice. Female heterozygotes performed more poorly than did controls of both sexes, and more poorly than male heterozygotes. In the spatial task, the heterozygous males performed well Žhad short path lengths., suggesting that they had learned the position of the hidden platform. However, when the platform was removed Žduring the probe trial a1., the performance of the heterozygous males was significantly worse than that of the other groups, in that these males spent significantly less time in the former platform quadrant than did the other groups. These males performed as though they had not learned, or they had forgotten, which quadrant the platform was in. There are two possible explanations: Ž1. The improvement of heterozygous males in performance of spatial and reversal tasks during the course of the sessions was not based on learning spatial cues; or Ž2. the heterozygous males learned the spatial position of the hidden platform, but when they did not find the platform immediately Žbecause it had been removed for the probe trial., they did not persist in searching that quadrant but instead began to look elsewhere for the platform. In this regard, it is notable that spatially directed attention is reportedly impaired in AD patients w36x. The latter explanation would also account for the ability of the heterozygous males to learn the reversal task so quickly. The data, then, suggest that the heterozygous males were not performing these spatial and reversal navigation tasks in the same way that controls were. Their performance was abnormal, but the deficit was different from that of the heterozygous females.
Neither locomotor activity nor speed nor cued navigation data suggest that the differences in path lengths seen between female and male heterozygous Flag-APP-C100 mice were due to sex differences in motor skills or motivation. We therefore conclude that the differences noted were in spatial learning abilities, and that the expression of the transgene in the brain differentially affects spatial learning and memory in the two sexes. It will be important to examine the developmental profile of the learning disabilities in the two sexes, to determine if the spatial deficits have different ages of onset, that may correlate with hormonal changes. At least some studies have shown that estrogen replacement therapy in older women may be associated with a decreased risk for AD ww16,20,30x; but see also w5xx. Our model may help us to determine the nature of the sex differences in the spatial deficits in the transgenic mice, and to determine whether these gender differences also exist in Alzheimer’s disease, as has been suggested by some investigators w6,10,33,34x. The transgenic mice demonstrating the greatest similarity in phenotype to the Flag-APP-C100 mice described here are those expressing APP-C104 w26x, which show virtually identical neuropathology to that of APP-C100 and Flag-APP-C100 transgenic mice w29x and in addition are deficient in spatial learning. The learning deficits in the homozygous Flag-APP-C100 mice also resemble those in the mice expressing the Swedish mutation of APP described by Hsiao et al. w17x. In this regard, it is notable that expression of the Swedish mutation of APP in neurons leads to significant intracellular accumulation of C100 w23x. Interestingly, mice showing the most advanced amyloid deposition w12,18x or expressing Ab 1 – 42 in the brain w22x are not reported to be impaired in learning and memory. The Flag-APP-C100 transgenic mice recreate the neurodegeneration that is one of the hallmarks of AD neuropathology. Homozygotes have the most severe pathology and most significant behavioral impairment, followed by the heterozygotes relative to controls. In addition to neurodegeneration, the Flag-APP-C100 mice display synaptic and axonal degeneration, accompanied by lysosomal abnormalities, thickened basement membranes in the cerebrovasculature, and proliferation of microglia w29x. Further studies will be necessary to determine if any of these other aspects of the transgenic pathology correlate better with behavior and shed more light on the sex differences in behavioral impairments. In any event, the demonstration in these animals of certain behavioral and anatomical sequellae similar to those seen in AD leads us to suggest that APP-C100 is a critical component of the molecular mechanism of AD neurodegeneration. Acknowledgements We thank Drs. Frederick Boyce and Ralph Nixon for helpful discussions. We also thank Dr. D.P. Wolfer for
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help in analyzing swim navigation data, Dr. U.V. Berger for help with the photomicrography, B.N. Cole for help with imaging and statistical analyses, C. Santeufemio for assistance with the histology, and B. Konieczny for photographic work. Critical reading of the manuscript by Drs. Frederick Boyce and Daniel Alkon is greatly appreciated. The work carried out at Wellesley College was supported by a Young Investigator Award to J.B.S. from the National Science Foundation ŽIBN-9458101. and the Whitehall Foundation. This research was supported by grants HD19932 ŽM.L.O.G.. and AG12954 ŽR.L.N.. from the National Institutes of Health, and by Janssen Research Foundation.
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Research report
Impairments in learning and memory accompanied by neurodegeneration in mice transgenic for the carboxyl-terminus of the amyloid precursor protein Joanne Berger-Sweeney a , Donna L. McPhie b, Jill A. Arters a , Jane Greenan c , Mary Lou Oster-Granite c , Rachael L. Neve b,) a
b
Department of Biological Sciences, Wellesley College, Wellesley, MA 02181, USA Department of Genetics, HarÕard Medical School and McLean Hospital, Belmont, MA 02478, USA c DiÕision of Biomedical Sciences, UniÕersity of California, RiÕerside, CA 92521-0121, USA Accepted 29 December 1998
Abstract In Alzheimer’s disease ŽAD., a progressive decline of cognitive functions is accompanied by neuropathology that includes the degeneration of neurons and the deposition of amyloid in plaques and in the cerebrovasculature. We have proposed that a fragment of the Alzheimer amyloid precursor protein ŽAPP. comprising the carboxyl-terminal 100 amino acids of this molecule ŽAPP-C100. plays a crucial role in the neurodegeneration and subsequent cognitive decline in AD. To test this hypothesis, we performed behavioral analyses on transgenic mice expressing APP-C100 in the brain. The results revealed that homozygous APP-C100 transgenic mice were significantly impaired in cued, spatial and reversal performance of a Morris water maze task, that the degree of the impairment in the spatial learning was age-dependent, and that the homozygous mice displayed significantly more degeneration of neurons in Ammon’s horn of the hippocampal formation than did heterozygous or control mice. Among the heterozygotes, females were relatively more impaired in their spatial learning than were males. These findings show that expression of APP-C100 in the brain can cause age-dependent cognitive impairments that are accompanied by hippocampal degeneration. q 1999 Elsevier Science B.V. All rights reserved. Keywords: Neurodegeneration; Transgenic mouse; Spatial learning; Alzheimer disease; Amyloid precursor protein; Gene; Neurotoxicity; Cognition
1. Introduction All individuals with Alzheimer’s disease ŽAD. experience a progressive loss of cognitive function, resulting from a neurodegenerative process characterized classically by granulovacular degeneration, the deposition of bamyloid ŽAb . in plaques and in the cerebrovasculature, and the formation of neurofibrillary tangles in neurons. Additional pathological hallmarks of AD include degeneration of synapses w8,14,40x and decreases in cell density w39x in distinct regions of the brain. The molecular events that link these distinct pathological entities remain cryptic.
) Corresponding author. Molecular Neurogenetics Laboratory, McLean Hospital, 115 Mill Street, Belmont, MA 02478, USA. Fax: q1-617-8553793; E-mail: [email protected]
We w42x and others w11,38x have reported that the carboxyl-terminal ŽC-terminal. 100-amino acid fragment of the amyloid protein precursor ŽAPP-C100., which includes the 42-amino acid Ab peptide and 58 adjacent amino acids in the carboxyl-terminus of APP, is neurotoxic, although it is not yet clear whether intact APP-C100 or the Ab peptide that is derived from it is directly responsible for the neurotoxicity displayed by this molecule. Published in vivo models have tested the hypothesis that APP-C100 plays a causal role in AD neurodegeneration. In particular, AD-like pathology has been described in transgenic mice expressing APP-C100 w19,29,35x or APP-C104 w26x in the brain. In support of these models, such carboxyl-terminal fragments of APP have been shown to accumulate in pathological lysosomal structures in AD brain w2,25x, and in neurons expressing any of the five known familial Alzheimer’s disease ŽFAD. mutants of
0169-328Xr99r$ - see front matter q 1999 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 9 - 3 2 8 X Ž 9 9 . 0 0 0 1 4 - 5
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APP w23x. We now describe deficits in learning accompanied by profound degeneration of the hippocampal formation that occurs in the brains of transgenic mice that express APP-C100 in the brain. The appearance of some stereotypical features of AD in these transgenic mice provides support for the hypothesis that APP-C100 may be a critical contributor to the processes that lead to neurodegeneration in AD.
2. Materials and methods 2.1. Production and characterization of transgenic mice The creation of transgenic mice expressing Flag w32x epitope-tagged APP-C100 in the brain under the control of the dystrophin brain promoter has been described w29x. Reverse transcription-PCR ŽRT-PCR. was used as described w29x to assess expression of the transgene RNA. 2.2. Immunoblots and immunoprecipitations We used the following anti-peptide antibodies: 10D5 Žgift of D. Schenk., directed against amino acids 1–15 of human Ab; E1-42 w7x Žgift of B. Cummings., directed against the 42-amino acid Ab polypeptide; and C8 w37x Žgift of D. Selkoe., immunoreactive with the carboxylterminal 10 amino acids of APP. Following cervical dislocation and decapitation of the mice, brain tissue was dissected rapidly from transgenic and control mice and immediately homogenized as described w9x. Each homogenate Ž20–25 mg. was subjected to electrophoresis in 16.5% Tris-tricine SDS-polyacrylamide ŽTT-SDS-PAGE., transferred to PVDF ŽMillipore. membranes, and immunoblotted with the antibody 10D5 Ž5 mgrml. as described w27x. Immunoprecipitations on 100-ml samples of the soluble and particulate fractions of brain were carried out at 48C using standard protocols w15x. Following the final wash, the samples were subjected to 16.5% TT-SDS-PAGE, electrotransferred to PVDF membrane, and processed for immunoblot analysis with the antibody C8 Ž1:6000 dilution.. 2.3. Numbers and ages of mice analyzed behaÕiorally Ninety-four mice were included in the behavioral analysis: 31 controls, 22 heterozygotes, and 31 homozygotes. At the time of testing the heterozygous mice were between 12 and 14.5 months of age; there were 12 males and 10 females. The homozygotes were various ages ranging from 4 to 10 months: 21 four month-olds, 5 six month-olds and 5 ten month-olds. The controls consisted of 4 males and 4 females at 4–6 months of age, and 13 males and 11 females at 12–14 months of age. Mice were housed 3–4 mice per cage, separated by sex and maintained in a room
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on a 12:12 h light–dark cycle, lights on at 0600 h. Food and water were available ad libitum except during water maze testing. The mice were first habituated to the experimenter for about one week, at the end of which a neurological battery Žadapted from Paul et al. w31x. was carried out. The battery included testing of righting, grasping, and placing reflexes. The mice then were subjected to three weeks of a Morris water maze task which included cued, spatial, probe and reversal tasks. This task is used commonly to assess learning and memory in mice. Dark-cycle locomotor activity was monitored on the same days as the swim maze tasks to examine activity as a possible confound on the water maze tasks. The experimenter was blind to the transgenic status of the mice during testing. 2.4. Swim maze testing and dark cycle actiÕity measurements All of the navigation tasks were conducted in a 180 cm diameter white circular pool surrounded by distal cues w3x and filled with 21–238C water. A 103 cm diameter ring was placed in the pool, in order to adapt the size of the pool for testing mice. A 55 cm diameter ring was used for pretraining trials. The platform, a 6 = 6 cm clear acrylic square, was always placed 15 cm from the edge of the ring in one of four quadrants defined by four equally spaced, arbitrarily designated start points ŽN, S, E, and W.. The testing schedule, consisting of one day of pretraining, five days of cued trials, five days of spatial trials followed by a probe trial, and four days of reversal trials followed by a probe trial, has been described w3,4x. For the reversal task, the pool was set up and trials were run in a manner identical to that of the spatial task, except that the invisible platform was moved to a new location. The day after completion of the reversal trials, the platform was removed from the pool and again each mouse was given a probe trial for 60 s. All navigation data were tracked and analyzed using an HVS video tracking system ŽHVS Image, Hampton, England.. Additional analyses were carried out using software designed by Wolfer and Lipp w41x. The following parameters were examined in different phases of the swim maze tasks: latency to find the platform, path length, speed, floating, and percent time in former platform quadrant during the probe trials. Dark cycle locomotor activity was measured. Briefly, mice were place in a standard mouse cage surrounded by three photobeams ŽSan Diego Instruments Cage Rack Activity System. mounted approximately 2.5 cm above the floor of the cage. Horizontal activity was measured as the total number of photobeam breaks per hour over a 12 h period, starting at 1700 h. 2.5. Histological analyses We examined 45 mice, ranging in age from 4 to 14.5 months: 11 controls, 4 heterozygotes, and 30 homozygotes
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Žone histological sample was lost.. Approximately equal numbers of each sex were examined. The heterozygote mice used for histological analyses were different from those used in the behavioral studies; the homozygotes used for histological analyses were the same mice that were used in the behavioral studies. Because the preponderant expression of the dystrophin brain promoter used in the transgenic constructs is in the hippocampus w13x, we focused our histological analyses on this structure. Deeply anesthetized animals were perfused with 4% paraformaldehyde, 0.1 M phosphate buffer, pH 7.4. The brains were cut into coronal blocks, each containing the entire hippocampal field at a given level. These regions were processed through graded alcohols to Polybed 812, and 2-mm sections were cut from each block. The sections were stained with toluidine blue. Healthy cells in CA2–CA4 were counted in the toluidine blue-stained sections. For each brain, approximately fifteen 2-mm sections were cut from the hippocampus at 10-mm intervals, at the anatomical level of the hippocampus between 1.7 and 2.18 mm posterior to Bregma. Healthy cells were defined as those with round, regularly-shaped
white nuclei, that did not stain darkly with the toluidine blue. Three independent 2-mm sections, one from the beginning, one from the middle, and one from the end of the series of fifteen sections, were counted. Degeneration was also evaluated with a silver stain in selected mice; numbers of healthy cells were obtained that were comparable to those obtained with the toluidine blue stain Ždata not shown.. 2.6. Statistical analyses In one set of analyses, control Žnon-transgenic. mice were compared to heterozygote transgenic and homozygote transgenic mice. Cued, spatial and reversal navigation data were analyzed using repeated measures analysis of variance ŽANOVA., with the transgenic status Žcontrol, heterozygote and homozygote. as the main effect; the days of the trials Žsessions. were the repeated measure. Probe data and healthy cell histology counts were analyzed using factorial ANOVAs. In a second set of analyses, the control and heterozygote data were separated by sex. Two-way repeated measures ANOVAs were performed with sex and
Fig. 1. ŽA. Brain expression of the APP-Flag-C100 transgene is revealed by RT-PCR. Hybridization of a radiolabeled internal oligonucleotide to a Southern blot of the amplification products from brain RNAs of a line 18 Flag-APP-C100 transgenic mouse and a control non-transgenic sib is shown. ŽB. Immunoblots showing the presence of the 19-kDa Flag-APP-C100 fragment Žarrows. in the brains of transgenic but not of control non-transgenice mice. Lanes 1–2: 10D5 immunoblot of brain homogenates from Flag-APP-C100 line 2 mice. Lane 1 is transgene-positive, while lane 2 is transgene-negative. Lanes 3–6: C8 immunoblot of brain fractions immunoprecipitated with E1-42. 3 and 4, soluble and particulate fractions, respectively, from the brain of a line 18 non-transgenic control sib; 5 and 6, soluble and particulate fractions from the brain of a line 18 transgenic mouse. Note the presence of an immunoreactive 19-kDa band Žarrow. in the immunoprecipitated soluble fraction of the transgenic brain. Lane 7: 35 S-labeled Flag-APP-C100 made by in vitro transcriptionrtranslation in a wheat germ lysate. This sample was electrophoresed on the same gel used for the immunoblot shown in lanes 3–6, and the lane containing the sample was not moved vertically relative to the other lanes when the figure was created.
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Finally, simple regression analysis was performed to examine linear correlations between the swim maze data Žcued, spatial and reversal path lengths. and the hippocampal healthy cell counts in the homozygote mice. Specifically, the last two days of the swim tasks were combined to represent asymptotic performance on a given task and these totals were then correlated with the histological data. Asymptotic performance represents the memory component on these navigation tasks; in previous studies following cholinergic lesions, we have seen correlations between histological parameters and asymptotic performance on navigation tasks w1x. Fig. 2. Mean photobeam breaks Ž"S.E.M.. made by control, heterozygote, and homozygote mice during 12 h of locomotor activity measurements.
3. Results 3.1. Generation of flag-APP-C100 transgenic mice
transgenic status as main effects and sessions as the repeated measure. A third set of analyses were performed on the homozygote data, examining behavioral effects with age. The ANOVAs were performed comparing the four month-old homozygote group to the six-month old homozygote group and the ten month-old homozygote group. Fisher’s protected least squared differences ŽPLSDs. were used, posthoc, to reveal significant differences between groups.
We described previously w19x the generation of APPC100 transgenic mice, in which the transgene is expressed under the control of the dystrophin brain promoter. We later created transgenic mice expressing Flag-APP-C100 w29x, which is identical to APP-C100 except for the fusion of the hydrophilic Flag epitope w32x to the N-terminus of APP-C100. A 4.65 kilobase Žkb. DNA fragment containing the dystrophin brain promoter-Flag-C100 fusion gene
Fig. 3. Mean path length Ž"S.E.M.. of control, heterozygote, and homozygote mice to find the platform during five days of a cued task, five days of a spatial task, and four days of a reversal task in the Morris water maze.
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with the SV40 early region splice and polyadenylation sequences was microinjected into the male pronuclei of fertilized eggs from F2 C57BLr6J= SJLrJ hybrid mice. Eight of the offspring were positive for the transgene, and 6 of these 8 produced transgene-positive progeny.
in the particulate fraction. C-terminal fragments of APP can be detected in human brain by this immunoprecipitation strategy w9x; in contrast, similar endogenous APPC100-like fragments were not observed in the control mouse brains.
3.2. Expression of the APP-C100 transgene RNA and protein products
3.3. BehaÕioral analyses
Reverse transcription coupled with PCR ŽRT-PCR. was used to assess transgene expression in the brains of 6–7 month old Flag-APP-C100 mice; we observed the expected 650 base pair Žbp. RT-PCR fragment ŽFig. 1A. in all transgenic brain RNAs examined. The dystrophin brain promoter is a weak promoter, as indicated by the low levels of dystrophin in the brain w28x. We chose this low-level promoter for the expression of Flag-APP-C100 because of the possibility that high levels of expression of the transgene would result in lethality before embryonic development was complete. Our use of the weak dystrophin brain promoter to drive expression of the Flag-APP-C100 transgene made it difficult to detect the transgene protein product in the brains of the transgenic mice. However, we exploited the fact that the transgene was created from a human APP cDNA, and used a monoclonal antibody to human Ab Ž10D5., which does not react with mouse bAPP, to demonstrate the expression of Flag-APP-C100 protein in the line 2 mice ŽFig. 1B.. This antibody immunodetected the expected 19-kDa band in animals that were positive for the transgene Žlane 1. but not in their transgene-negative littermates Žlane 2.. APPC100 usually migrates as a 15 kDa species w21x, but addition of the Flag tag causes it to migrate with an apparent molecular weight of 19 kDa, as seen in lane 7, in which 35 S-labeled flag-APP-C100 made in vitro as described by Kozlowski et al. w21x was subjected to the same TT-SDS-PAGE conditions. Commercially available Flag antibodies work only weakly on immunoblots Žunpublished data of R.L.N.. and therefore were not suitable for the detection of the very rare transgene product. Expression of the transgene protein product in the brains of line 18 transgenic mice was demonstrated by immunoprecipitation experiments ŽFig. 1B, lanes 3–6.. The immunoaffinity purified antiserum E1-42 w7x, raised against a peptide representing the 42-amino acid human Ab fragment, was used to immunoprecipitate C-terminal derivatives of APP from soluble and insoluble fractions of transgenic brain homogenates. The precipitated proteins were subjected to TT-SDS-PAGE and probed with C8, an antibody to the C-terminal 10 amino acids of APP w37x. Again, we detected the appropriate 19-kDa protein in the soluble fraction of brains of transgenic ŽFig. 1B, lane 6. but not of control mice Žlane 4.. Since we were able to immunoprecipitate the protein from the soluble fraction only, it seems likely that soluble Flag-APP-C100 protein is more accessible to the E1-42 antibody than is the protein
3.3.1. General health and neurological battery Control and heterozygote and homozygote transgenic mice ranged in color from black to brown to agouti. No gross motor or sensory abnormalities were apparent in the mice. All of the mice exhibited normal righting, grasping and placing reflexes. None of the mice floated excessively during any of the swim trials.
Fig. 4. Mean percentage of time Ž"S.E.M.. that control, heterozygote, and homozygote mice spent in the former platform quadrant during probe trials Žplatform removed from the pool. following a. the spatial task Žprobe 1. and b. the reversal task Žprobe 2.. Ž). Denotes significantly different from control, p’s - 0.01. Žq. Denotes significantly different from control and heterozygote, p’s - 0.05.
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3.3.2. Dark-cycle locomotor actiÕity In all of the groups of mice, locomotor activity levels followed a similar habituation profile; levels were high during the first hour, decreased sharply over the next four hours and reached asymptotic levels by about five hours ŽFig. 2.. This resulted in a significant effect of hours w F Ž11,1001. s 56.5, p s 0.0001x. There were no significant differences among the groups when controls were compared to transgenic heterozygotes and transgenic homozygotes, and no significant interaction. 3.3.3. Swim maze tasks During the cued task, all mice learned the task, in that they performed better across the days of testing. Both latency w F Ž4,364. s 22.1, p s 0.0001x and path length w F Ž4,364. s 30.3, p s 0.0001x decreased significantly across days ŽFig. 3; only path length data shown graphically.. Nevertheless, significant differences were seen among the groups on the cued task for both latency w F Ž2,91. s 10.2, p s 0.001x and path length w F Ž2,91. s 9.1, p s 0.003x. Posthoc analyses revealed that the homozygotes had significantly longer latencies than controls w p s 0.0001x and heterozygotes w p s 0.004x. There was also a significant latency= transgenic status interaction w F Ž8,364. s 2.3, p s 0.02x; the learning curve for the homozygotes was steeper than that of the other two groups. The homozygotes also had longer path lengths than controls w p s 0.001x and heterozygotes w p s 0.002x. There
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were also significant differences among the groups on speed during the cued trials w F Ž2,91. s 4.2, p s 0.018x. Post hoc analyses revealed that the homozygotes swam significantly more slowly than the heterozygotes w p s 0.005x, but not the controls. There were significant differences among the groups on the spatial task for both latency and path length. All mice learned the task, in that they performed better across the days of testing. Both latency w F Ž4,364. s 38.6, p s 0.0001x and path length w F Ž4,364. s 53.3, p s 0.001x decreased significantly across the days ŽFig. 3; only path length data shown graphically.. Nevertheless, the groups differed significantly for both latency w F Ž2,91. s 4.0, p s 0.02x and path length w F Ž2,91. s 3.8, p s 0.03x. Posthoc analyses revealed that the homozygotes had significantly longer latencies than controls w p s 0.009x and heterozygotes w p s 0.03x. The homozygotes also had longer path lengths than controls w p s 0.007x, but not heterozygotes. There were no significant interactions. Swim speed during the spatial trials, in contrast to that during the cued trials, did not differ among the groups. On the reversal task, there were no significant differences among the groups on latency, path length or speed measures. All mice learned the task, in that both latency w F Ž3,273. s 44.9, p s 0.0001x and path length w F Ž3,273. s 54.5, p s 0.0001x decreased significantly across the days ŽFig. 3, only path length data shown graphically.. There was, however, a significant transgenic status = sessions
Fig. 5. Mean path length Ž"S.E.M.. of four month, six month, and ten month old homozygote mice to find the platform during five days of a cued task, five days of a spatial task, and four days of a reversal task in the Morris water maze.
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interaction for path length w F Ž6,273. s 3.6, p s 0.002x and a trend towards a transgenic status = sessions interaction for latency w F Ž6,273. s 2.0, p s 0.07x. In both cases the interaction Žor trend. was due to a flatter learning curve in the homozygote group as compared to controls and heterozygotes. As was the case for the spatial task, there were no significant differences among the groups in swim speeds. On the probe trials, there were significant differences among the groups ŽFig. 4.. On probe trial a1 Žafter the
spatial navigation trials; Fig. 4A., there were significant differences in the percent time spent in the former goal quadrant among the groups w F Ž2,92. s 8.0, p s 0.006x. Posthoc analysis revealed that control mice spent significantly more time in the former goal quadrant than either heterozygotes w p s 0.004x or homozygotes w p s 0.005x. On probe a2 Žafter the reversal navigation trials; Fig. 4B., there were also significant differences in the percent time spent in the former goal quadrant among the groups
Fig. 6. Performance measures of 12–14.5-month control and heterozygote mice of both sexes during testing in the Morris water maze. ŽA. mean path length Ž"S.E.M.. to find the platform during five days of a cued task, five days of a spatial task, and four days of a reversal task, and ŽB. mean percentage of time Ž"S.E.M.. spent in the former platform quadrant during the probe trial Žplatform removed from the pool. following the spatial task Žprobe 1.. Ž). Denotes significantly different from control, p - 0.01.
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w F Ž2,92. s 4.3, p s 0.016x. Posthoc analysis revealed that homozygotes spent significantly less time in the former goal quadrant than controls w p s 0.005x or heterozygotes w p s 0.046x. 3.3.4. Age-related behaÕioral changes within the homozygote group The majority of the significant behavioral effects were seen in the homozygotes relative to controls. Therefore, we expanded our analysis of the homozygotes by comparing the performances of different age groups Žfour-month-olds, six-month-olds and ten-month-olds. on the swim maze tasks. On the cued task, latency and path length did not differ significantly among the different age groups ŽFig. 5, only path length data shown graphically.. Cued speed, however, did differ significantly in the different age groups w F Ž2,28. s 3.6, p s 0.04x. The ten-month-old mice swam significantly slower than the four-month- and six-month-old mice. On the spatial task, latency and path length did not differ significantly among the different age groups. However, there were significant age = session interactions for latency w F Ž8,112. s 3.0, p s 0.004x and path length w F Ž8,112. s 2.4, p s 0.018x. In both cases, the ten-monthold mice were much worse at the start of the task and performance improved steadily over the five days of test-
Fig. 7. ŽA. Mean number of healthy cells Ž"S.E.M.. in hippocampal region CA3r4 of control, heterozygote, and homozygote mice. Ž). Denotes significantly different from control and heterozygote, p’s - 0.05. ŽB. Toluidine blue stain of the pyramidal cell layer of the hippocampus in 5-month-old homozygous ŽA. one-year-old heterozygous ŽB. and control ŽC. mice. The inset in panel A is a higher-power photomicrograph of the pyramidal cell layer of this mouse, at the CA2r3 border. Degenerating cells are darkly stained. The arrowhead indicates the border of CA2 at which the healthy cell counts were initiated; all healthy cells Žas defined in Section 2. between this border and the end of CA4 extending into the hilar region of the dentate gyrus were counted.
Fig. 7 Žcontinued..
ing; the four- and six-month-olds were better at the start of testing and improved only slightly during the five days of testing. There were significant differences among age groups in swim speed w F Ž2,28. s 3.4, p s 0.048x. The
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Fig. 8. Scatterplot of the number of healthy cells vs. the sum of path length measures from the last two days of Ža. the cued task, Žb. the spatial task, and Žc. the reversal task in the Morris water maze in the homozygote mice.
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ten-month-old mice swam significantly slower than fourw p s 0.02x and six-w p s 0.03x month-old mice. By the time of the reversal task, significant differences by age were no longer apparent. There were no significant differences in either latency or path length among the different age groups. There were also no significant differences in swim speed in the different age groups and no significant interactions. Additionally, there were no significant differences by age in the time spend in the former goal quadrant during either probe trial a1 or probe trial a2. 3.3.5. Sex differences in spatial performance in heterozygotes Because some investigators have suggested that gender differences exist in Alzheimer’s disease w6,10,33,34x, we compared the performance of female and male heterozygotes, relative to controls, on the swim maze tasks at one year of age. On the cued task, there were no significant differences in either latency or path length measures by transgenic status or sex ŽFig. 6a, only path length data shown graphically.. There was a significant sex difference in swim speed w F Ž1,53. s 8.0, p s 0.007x; females swam faster than males. On the spatial task, there was a trend towards a sex difference in path lengths w F Ž1,53. s 3.5, p s 0.068x. Female heterozygotes tended to have longer path lengths than the mice in other groups. In the reversal task, the sex differences in path length reached statistical significance w F Ž1,53. s 3.9, p s 0.05x. Posthoc analyses revealed that female heterozygotes had longer path lengths than female controls w p s 0.002x, whereas male heterozygotes had shorter path lengths than male controls w p s 0.02x. This led to a significant sex = transgenic status interaction w F Ž1,53. s 16.5, p s 0.0002x. On probe a1, there were significant differences by transgenic status in the amount of time spent in the former goal quadrant wFig. 6b; F Ž1,54. s 14.7, p s 0.003x. Male and female heterozygotes spent considerably less time in the quadrant that formerly contained the platform Ž36.7 " 2.1% and 38.7 " 3.7%, respectively. than did control mice, who spent about 50% of the time in the former goal quadrant. These differences had disappeared by probe a2. There were no other significant main effects of sex or significant interactions. 3.4. Neurodegeneration in the hippocampal formation of controls, heterozygotes and homozygotes Quantitative analyses of cellular degeneration in the hippocampal formation revealed significant differences in healthy cell counts in the CA2-4 layer among the controls, heterozygotes and homozygotes w F Ž2,42. s 9.6, p s 0.004x ŽFig. 7.. Posthoc analyses ŽFig. 7a. revealed that homozygotes had significantly fewer healthy hippocampal cells than did controls w p s 0.0002x or heterozygotes w p s 0.03x.
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3.5. Correlations between behaÕior and hippocampal pathology Interestingly, the behavioral and anatomical measures followed similar patterns, controls being the best Žshortest latencies and path lengths and highest numbers of healthy hippocampal cells., followed by heterozygotes and homozygotes. Correlation analyses were possible only on the homozygote animals and selected controls because this was the only group for which we had behavioral and anatomical measures from the same animals. Asymptotic performances on cued and spatial tasks did not correlate with healthy cell counts ŽFig. 8.. There was a trend w p s 0.085x, however, that long path lengths on the reversal task were associated with fewer healthy cells.
4. Discussion We describe significant deficits in cued, spatial and reversal trials in homozygous transgenic mice expressing Flag epitope-tagged APP-C100 in the brain, relative to heterozygote transgenics and controls. Analysis of the probe data suggests additionally that the homozygotes did not learn, nor did they remember, the spatial or reversal tasks as well as the heterozygote or control mice. The significant interactions between transgenic status and days of testing on the cued and reversal tasks suggest that homozygotes also are learning these tasks at different rates from controls. Although there is a cued deficit in the homozygotes, we do not believe that this deficit represents an inability of the homozygotes to see or motate. Path lengths decreased across the days of testing on all tasks, suggesting that the homozygotes can learn and hence probably can see, since there is no reason to assume that sight would improve over the days of testing. The cued deficit also does not appear to represent a general locomotor problem because Ž1. even though the homozygotes swim more slowly than other groups on the cued task, there are no swim speed deficits in the spatial or reversal task, and Ž2. there are no significant differences between homozygotes and controls in general dark cycle locomotor activity. The cued impairment could represent a motivational problem or a cued learningrmemory impairment. It is notable that other transgenic mouse models of AD w17x have cued, as well as spatial impairments on water maze tasks. The degree of impairment of spatial learning in homozygous mice appears to be age-dependent. Cued performance, however, is not impaired in an age-related fashion. Ten-month-old homozygotes learn the spatial task at a different rate from four- and six-month-olds. Interestingly, the ten-month-olds begin the spatial task at a level significantly worse than do either four- or six-month-olds. The younger mice behave as if they developed a spatial map during the cued task, which allowed them to master quickly
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the spatial task. In contrast, the older homozygous mice do not appear to have remembered what they learned in the cued task to guide performance in the spatial task. These age-related changes are no longer apparent in the reversal task. Although the heterozygous transgenic mice displayed significantly less learning and memory impairments than did the homozygotes, subtle alterations in their behavior could be seen. Significantly, the heterozygous mice evince, by the age of one year, spatial learning deficits in the absence of cued learning impairment, relative to controls. It is interesting to note that in the heterozygotes, the task that was most sensitive to their deficits was the reversal task. Additionally, the behaviorrhistology correlation that came closest to being significant was the correlation between asymptotic performance of the reversal task and healthy cell counts. These two results suggest that the reversal task, which involves unlearning one platform position and relearning a new platform position, may be particularly sensitive in detecting transgene-induced impairments in the mice. This task did not appear to be similarly sensitive to the age-related deficits in the homozygotes, perhaps because for the ten-month-old mice the spatial task resembles a ‘reversal’ task because there is little carry-over from the cued task, as noted above. There were notable sex differences in the performance of the spatial and reversal tasks by the heterozygous mice. Female heterozygotes performed more poorly than did controls of both sexes, and more poorly than male heterozygotes. In the spatial task, the heterozygous males performed well Žhad short path lengths., suggesting that they had learned the position of the hidden platform. However, when the platform was removed Žduring the probe trial a1., the performance of the heterozygous males was significantly worse than that of the other groups, in that these males spent significantly less time in the former platform quadrant than did the other groups. These males performed as though they had not learned, or they had forgotten, which quadrant the platform was in. There are two possible explanations: Ž1. The improvement of heterozygous males in performance of spatial and reversal tasks during the course of the sessions was not based on learning spatial cues; or Ž2. the heterozygous males learned the spatial position of the hidden platform, but when they did not find the platform immediately Žbecause it had been removed for the probe trial., they did not persist in searching that quadrant but instead began to look elsewhere for the platform. In this regard, it is notable that spatially directed attention is reportedly impaired in AD patients w36x. The latter explanation would also account for the ability of the heterozygous males to learn the reversal task so quickly. The data, then, suggest that the heterozygous males were not performing these spatial and reversal navigation tasks in the same way that controls were. Their performance was abnormal, but the deficit was different from that of the heterozygous females.
Neither locomotor activity nor speed nor cued navigation data suggest that the differences in path lengths seen between female and male heterozygous Flag-APP-C100 mice were due to sex differences in motor skills or motivation. We therefore conclude that the differences noted were in spatial learning abilities, and that the expression of the transgene in the brain differentially affects spatial learning and memory in the two sexes. It will be important to examine the developmental profile of the learning disabilities in the two sexes, to determine if the spatial deficits have different ages of onset, that may correlate with hormonal changes. At least some studies have shown that estrogen replacement therapy in older women may be associated with a decreased risk for AD ww16,20,30x; but see also w5xx. Our model may help us to determine the nature of the sex differences in the spatial deficits in the transgenic mice, and to determine whether these gender differences also exist in Alzheimer’s disease, as has been suggested by some investigators w6,10,33,34x. The transgenic mice demonstrating the greatest similarity in phenotype to the Flag-APP-C100 mice described here are those expressing APP-C104 w26x, which show virtually identical neuropathology to that of APP-C100 and Flag-APP-C100 transgenic mice w29x and in addition are deficient in spatial learning. The learning deficits in the homozygous Flag-APP-C100 mice also resemble those in the mice expressing the Swedish mutation of APP described by Hsiao et al. w17x. In this regard, it is notable that expression of the Swedish mutation of APP in neurons leads to significant intracellular accumulation of C100 w23x. Interestingly, mice showing the most advanced amyloid deposition w12,18x or expressing Ab 1 – 42 in the brain w22x are not reported to be impaired in learning and memory. The Flag-APP-C100 transgenic mice recreate the neurodegeneration that is one of the hallmarks of AD neuropathology. Homozygotes have the most severe pathology and most significant behavioral impairment, followed by the heterozygotes relative to controls. In addition to neurodegeneration, the Flag-APP-C100 mice display synaptic and axonal degeneration, accompanied by lysosomal abnormalities, thickened basement membranes in the cerebrovasculature, and proliferation of microglia w29x. Further studies will be necessary to determine if any of these other aspects of the transgenic pathology correlate better with behavior and shed more light on the sex differences in behavioral impairments. In any event, the demonstration in these animals of certain behavioral and anatomical sequellae similar to those seen in AD leads us to suggest that APP-C100 is a critical component of the molecular mechanism of AD neurodegeneration. Acknowledgements We thank Drs. Frederick Boyce and Ralph Nixon for helpful discussions. We also thank Dr. D.P. Wolfer for
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help in analyzing swim navigation data, Dr. U.V. Berger for help with the photomicrography, B.N. Cole for help with imaging and statistical analyses, C. Santeufemio for assistance with the histology, and B. Konieczny for photographic work. Critical reading of the manuscript by Drs. Frederick Boyce and Daniel Alkon is greatly appreciated. The work carried out at Wellesley College was supported by a Young Investigator Award to J.B.S. from the National Science Foundation ŽIBN-9458101. and the Whitehall Foundation. This research was supported by grants HD19932 ŽM.L.O.G.. and AG12954 ŽR.L.N.. from the National Institutes of Health, and by Janssen Research Foundation.
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