Behavioural phenotype of APPC100.V717F transgenic mice over-expressing a mutant Aβ-bearing fragment...

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Behavioural Brain Research 209 (2010) 27–35

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Behavioural phenotype of APPC100.V717F transgenic mice over-expressinga mutant A�-bearing fragment is associated with reducedNMDA receptor density

Wah Chin Boona,1, Maarten van den Buuseb,∗,1, Nico Wegenerb, Sally Martinb,Hui Kheng Chuaa, Ashley I. Bushb, Colin L. Mastersb,c,Paul A. Adlardb, Qiao-Xin Lib,c,d

a Florey Neuroscience Institute, Melbourne, Australiab The Mental Health Research Institute, Parkville, Australiac Department of Pathology, The University of Melbourne, Melbourne, Australiad Centre for Neuroscience, The University of Melbourne, Melbourne, Australia

a r t i c l e i n f o

Article history:Received 18 December 2009Received in revised form 7 January 2010Accepted 9 January 2010Available online 18 January 2010

Keywords:Amyloid precursor proteinTransgenic miceLocomotor hyperactivityCognitionNMDA receptor

a b s t r a c t

The aim of this study was to characterize APPC100.V717F transgenic (TgC100.V717F) mice which over-express a mutant C100 fragment of the amyloid precursor protein. The mice were compared to TgC100wild type mice (TgC100.WT) and non-transgenic controls at 4–9 and 16–22 months of age. TgC100.V717Fmice showed behavioural hyperactivity, particularly at a younger age, as shown by increased numbers ofelevated plus maze arm entries and Y-maze arm entries, enhanced baseline locomotor activity in the openfield, and enhanced amphetamine-induced hyperlocomotion. This hyperactivity was less pronounced inTgC100.WT which only displayed significant differences to non-transgenic controls at a younger agefor the number of Y-maze arm entries and baseline locomotor activity in the open field. In addition,TgC100.V717F mice, but not TgC100.WT, demonstrated cognitive deficits, as shown by reduced sponta-neous alternation in the Y-maze and markedly reduced retention in a passive avoidance test. At an olderage, TgC100.V717F mice showed enhanced startle and increased immobility time in the forced swim test.In the TgC100.V717F mice, but not TgC100.WT, the behavioural changes were paralleled by a significantreduction in the expression of hippocampal NMDA receptor subunits types 1 and 2A. Concomitantly, wedetected axonal disruption and apoptosis in the hippocampus of TgC100.V717F mice. In conclusion, thesedata demonstrate that the mutant C100 fragment is an effector of biochemical and both cognitive andnon-cognitive behaviours. These transgenic mice may be a model for the psychotic features associatedwith early Alzheimer’s disease.

© 2010 Elsevier B.V. All rights reserved.

1. Introduction

Alzheimer’s disease (AD) is a progressive neurodegenerativedisorder that is characterized by a decline in higher order cognitivefunctions that occurs in concert with an evolving brain pathologythat most notably includes the extracellular deposition of amyloidplaques and the intracellular accumulation of neurofibrillary tan-gles. The major constituent of the amyloid plaques is �-amyloid(A�), which is produced by sequential proteolytic cleavage of theamyloid precursor protein (APP) by �-and �-secretases [1–3]. Thisbi-enzymatic cleavage process also generates the short C-terminal

∗ Corresponding author. Tel.: +61 3 93892967; fax: +61 3 93875061.E-mail address: [email protected] (M. van den Buuse).

1 They contribute equally to this work.

fragment, the amyloid intracellular domain (AICD). The �-secretasecleavage alone also generates a longer C-terminal fragment con-taining the A� peptide, variably referred to as C99, �CTF or C100,in addition to the larger soluble N-terminal fragment of APP. Manystudies support the notion that full-length A� is toxic and is respon-sible for the neurodegeneration in AD [4]. However, recent evidencealso postulates that the C100 fragment, which has been identifiedin the AD brain, is toxic to neurons [5,6].

Transgenic mice over-expressing APP or C100 (or �CTF or C99)have been created to address the role of the proteolytic productsof APP in the development of the biochemical markers and cog-nitive behaviours that occur in AD. One of the most widely usedmouse models of AD is the Tg2576 line that over-expresses humanAPPsw [7]. These animals develop plaques from ∼9 months of ageand also show concomitant deficits in a variety of cognitive andnon-cognitive behaviours, including spatial learning [7,8] and sen-

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28 W.C. Boon et al. / Behavioural Brain Research 209 (2010) 27–35

sorimotor functioning [9,10]. More recent data has demonstratedthat Tg2576 mice develop behavioural impairments well before theonset of overt extracellular plaque formation [8]. These data sug-gest that there are more subtle factors other than insoluble A�, suchas fragments or other soluble forms of A� that may be mediatingthe behavioural phenotype in the Tg2576 model.

To delineate the role of C100/A� from full-length APP, mousemodels expressing only this A�-bearing C-terminal fragment havebeen created. Some of these transgenic lines show neuronal atro-phy, plaque formation and behavioural impairment [11–16], whileothers display no obvious phenotype [17]. As all these studiesinvolve either C100 with a wild type sequence or an AD-mutationaround the �-secretase site but not with both wild type and mutantC100 in one study, it is difficult to assess whether wild type ormutated C100 is toxic. Another question is whether plaques/A�deposits or soluble A� is required for the phenotype development.

We have previously developed transgenic mice expressing wildtype C100 (TgC100.WT) and mutant C100 (TgC100.V717F) [18]. Atthe biochemical level, these mice display an intraneuronal accu-mulation of A�/C100 in the hippocampus but do not developextracellular plaques and contain only soluble oligomeric A�. TheTgC100 mice also display changes in acetylcholinesterase isoformsand glycosylation patterns similar to that detected in AD [19]. TheseTgC100 mice also display metal imbalances, mirroring the changesthat occur in the AD brain [20]. In this study we have utilized a bat-tery of behavioural paradigms to characterize these two transgeniclines. The tests utilized mirror aspects of psychosis, depression,anxiety and cognition [21,22]. Furthermore, we have undertakenpreliminary investigations into the effect of these mutations onseveral biochemical endpoints in order to elucidate potential mech-anisms for the behavioural abnormalities observed in these mice.In particular, we have examined expression levels of N-methyl-d-aspartate (NMDA) receptor subunits, the levels of which are alteredin the AD brain [23,24] and which are involved in learning andmemory processes. We also examined markers for cell death andmorphological disruption.

2. Methods

2.1. Animals and general protocol

Male TgC100.WT, TgC100.V717F and non-transgenic controls on a mixedC57Bl/6/DBA background, aged either 4–5 months (“young”) or 16–18 months(“old”), were utilized in this study. Transgenic mice had been bred to homozygos-ity and characterized as described previously [18]. The entire cohort underwent aseries of consecutive behavioural tests with 2–4 weeks interval between separatetests (see below). The sequence of behavioural testing was: elevated plus maze foranxiety-like behaviour [25], prepulse inhibition (PPI) of acoustic startle for sensori-motor gating [21,22], Y-maze spontaneous alternation for short-term memory [26],forced swim test for depression-like behaviour [27], locomotor hyperactivity forpsychosis-like behaviour [21,22], and (in only one age group) passive avoidancelearning. All procedures followed the guidelines of the Australian Code of Practicefor the Care and Use of Animals for Scientific Purposes and were approved by theanimal ethics committee of the University of Melbourne.

2.2. Elevated plus maze

The elevated plus maze is commonly used to assess the action of anxiolyticdrugs and anxiety-like behaviour [25]. We used a dimly lit grey plastic platform,elevated approximately 30 cm off the ground, with 30 cm long and 4 cm wide arms.Two opposing arms had 20 cm high walls on the side and the end. Each mouse wasplaced in the central square and the number of visits to the open and closed arms wasrecorded for 10 min by an observer [28]. The number of mice tested on the elevatedplus maze at approximately 4 and 17 months of age was: n = 13 and 10 for controls,n = 9 and 12 for TgC100.WT, and n = 11 and 11 for TgC100.V717F, respectively.

2.3. Prepulse inhibition (PPI) of acoustic startle

PPI is a measure of sensorimotor gating, which is the ability to ignore irrelevantinformation and focus attention [21,22]. Mice are tested for prepulse inhibition ofacoustic startle using a four-unit automated startle system (SR-Lab, San Diego Instru-ments, San Diego, CA, USA) as previously described [28]. The mice were placed in

perspex cylinders (5 cm diameter) on a motion-sensitive platform. Trials pseudo-randomly consisted of 20 pulse-alone trials or prepulse trials, including a 115 dBpulse-alone startle stimulus preceded 100 ms by a 20 ms prepulse of either 2, 4, 8,12, or 16 dB over the 70 dB background (i.e. 72, 74, 78, 82, 86 dB prepulses). Pre-pulse inhibition was calculated as the difference between startle responses aftera prepulse trial and those after pulse-alone trials, expressed as percentage of theresponses obtained from the pulse-alone trials. The number of mice tested in theprepulse inhibition experiment at approximately 4 and 17 months of age was: n = 13and 8 for controls, n = 8 and 11 for TgC100.WT, and n = 10 and 9 for TgC100.V717F,respectively.

2.4. Y-maze spontaneous alternation

Spontaneous alternation is a test for short-term memory which is hippocampus-dependent [26]. We used a Y-maze constructed of grey-painted timber with arms29.5 cm long × 7.5 cm wide × 15.5 cm high. There were visual cues on the wall of eacharm and elsewhere in the experimental room. Mice were placed in a randomly allo-cated start arm facing the wall. Arm entries of the mice were scored by an observerduring an 8 min trial. Alternation was defined as successive entries in different armson overlapping triplets and expressed as percentage of total number of entries. Thenumber of mice tested in the spontaneous alternation at approximately 6 and 18months of age was n = 13 and 10 for controls, n = 9 and 12 for TgC100.WT, and n = 11and 11 for TgC100.V717F, respectively.

2.5. Forced swim test

The Porsolt forced swim test for depression-like behaviour [27] involved theuse of a clear glass cylinder (height: 15 cm; diameter 11 cm) containing 10 cm ofwater with a temperature of 25 ± 1 ◦C. On two consecutive days, the animals wereindividually placed into the water for 5 min of forced swimming and immobility wasmeasured in seconds. After the forced swim period animals were allowed to dry for15 min in a warm enclosure before being transferred back to their home cages. Allmice showed the greatest amount of immobility time on the second day of testing,therefore only these data will be presented. The number of mice tested in the forcedswim test at approximately 9 and 22 months of age was: n = 13 and 8 for controls,n = 9 and 9 for TgC100.WT, and n = 11 and 10 for TgC100.V717F, respectively.

2.6. Locomotor hyperactivity

Baseline and amphetamine-induced locomotor hyperactivity are used as ananimal model of psychosis [21,22]. Mice were placed in rectangular plastic boxes(39 cm × 29 cm × 12 cm l × w × h) and were allowed 60 min of baseline exploratorylocomotor activity, after which they were injected intraperitoneally with 5 mg/kg ofamphetamine sulphate (Sigma) and behaviour was monitored for another 120 min.Behaviour was recorded on video and analysed with the Ethovision video-trackingsystem (Noldus, The Netherlands). Data for distance moved were expressed both in5 min bins and as averages of the 60 min pre-injection and 120 min post-injectiontime blocks. The number of mice tested in the locomotor hyperactivity experimentat approximately 9 and 22 months of age was: n = 9 and 7 for controls, n = 9 and 9for TgC100.WT, n = 8 and 8 for TgC100.V717F, respectively.

2.7. Passive avoidance learning

The passive avoidance task is used to assess both short- and long-term memory.Mice were tested for passive avoidance using a GEMINI Avoidance System (San DiegoInstruments, USA). The apparatus had two compartments connected to a computer,which controlled the delivery of stimuli and acquisition of latency data. A step-through ‘Trials to Criterion’ procedure was used in the training sessions on the firstday (Day 0) [29]. The animal was given a brief moderate footshock (2 mA for 2 s)whenever it entered the dark compartment. The training session finished when themouse stayed in the bright compartment for 300 s twice in a maximum of 5 trials.One and three days after the training session, mice were tested for retention up to300 s, during which no electric shock was administered. The number of mice testedin the passive avoidance experiment at approximately 18 months of age was: n = 17for controls, n = 15 for TgC100.WT, n = 18 for TgC100.V717F, respectively.

2.8. NMDA receptor subunits mRNA analysis

A separate cohort of mice (10 months, 6 mice per group) were killed by cervi-cal dislocation and the hippocampus was dissected out from the brain in RNAlater(Ambion, TX, USA). Total RNA was then extracted using the phenol–chloroformbased UltraspecTM RNA isolation system (Biotecx, TX, USA), DNase-treated(Ambion) and then quantified spectrophotometrically (NanoDrop 2000, ThermoFisher Scientific, USA). RNA quality was assessed by 1% agarose, 1XTBE gel elec-trophoresis. Reverse-transcription was performed on 500 ng of total RNA. Randomhexamers (50 �g/�l; Promega) were denatured at 72 ◦C for 5 min and annealed at4 ◦C for 5 min in a total volume of 10 �l before addition of RNAse inhibitor (40 U;Promega) and AMV-Reverse Transcriptase (20 U; Promega) 5× RT buffer and dNTPs(100 pmol/�l, Promega). cDNA was synthesized at 37 ◦C for 1 h in a 25 �l reactionvolume. Transcripts were quantified by real-time PCR (Corbett Rotorgene RG-3000;


W.C. Boon et al. / Behavioural Brain Research 209 (2010) 27–35 29

Corbett Research, Sydney, Australia) using a SYBR green PCR reaction mix andPCR primer pairs specific to NMDA receptor subunits [30]. The specificity of thePCR amplicons was confirmed by Tm (Rotorgene) and product lengths shown bygel electrophoresis. All transcript levels were normalized to cyclophilin transcriptexpression [31].

2.9. TUNEL staining

Four to six mice per group (4 sections per animal) from two ages (10 and 16–20months) were used for the TUNEL and tubulin immunohistochemistry. Apoptoticcells in 5 �m formalin-fixed coronal brain sections were labelled using the ApoptagFluorescein in situ Apoptosis Detection Kit (Chemicon) according to the manu-facturer’s instructions. Sections were deparaffinised through xylene and gradedethanol rehydration, pretreated with Proteinase K (20 �g/ml, 15 min) at room tem-perature, and washed twice with PBS. After 10 s incubation in Equilibration Buffer,TdT Enzyme was added to sections and incubated in a humidified chamber at 37 ◦Cfor 1 h. For positive control, tissue sections were treated with DNase I for 1 h at 37 ◦Cbefore addition of TdT Enzyme. For negative control, TdT enzyme was substitutedwith water. The labelling reaction was stopped by 10 min incubation in Stop/Washbuffer at room temperature followed by two PBS washes. Anti-DIG-Fluorescein con-jugate (green fluorescence) was then applied and incubated in a humidified chamberfor 30 min at room temperature. After two PBS washes, the sections were counter-stained with DAPI (1:6000) in Fluorosave mounting medium and coverslipped. Usingthis protocol, apoptotic cells are stained with a green fluorescence and nuclei withblue fluorescence.

2.10. ˇ-Tubulin immunohistochemistry

The immunoflurorescence of �-tubulin has been reported to be constant alongthe length of axons and reflects axon diameter [32]. After deparaffinizing the brainsections and rehydrating through graded ethanol solutions, sections were pre-treated with 1% Triton X-100 for 10 min at room temperature and washed twicewith PBS for 2 min. The sections were blocked with 6% normal goat serum in 0.3%Triton X-100 in PBS for 1 h at room temperature. Primary antibody mAb mouseanti-� tubulin (1:1000; Promega) was then added to the slides and incubated in ahumidified chamber overnight at room temperature. After three PBS washes, sec-ondary antibody (Goat anti-mouse, Alexa 594, 1:1000; Molecular Probes) was addedand incubated for 2 h at room temperature. Finally, the slides were washed with PBS,3× 5 min, counterstained with DAPI in Fluorosave and coverslipped.

2.11. Data analysis

All data are expressed as mean ± standard error of the mean (SEM) or as pro-portion and median score (in the case of passive avoidance behaviour). Differencesbetween the groups and between ages were compared with analysis of variance(ANOVA) using the statistical software package Systat 9.0 (SPSS Inc., USA). Passiveavoidance data were analysed by Fisher Exact test (proportion of animals whichremained passive on retention days) and Wilcoxon’s two-sample test (for retentiontime). Expression levels of NMDA receptor subunits were compared between groupsusing Student’s t-test.

3. Results

3.1. Elevated plus maze

TgC100.V717F mice were hyperactive at the younger age, butnot older age, as shown by the highest total number of arm entries(main effect of Genotype F(2,60) = 6.9, P = 0.002; main effect of AgeF(1,60) = 35.4, P < 0.001; Genotype × Age interaction F(2,60) = 10.9,P < 0.001). While these mice also tended to show the highest scoresfor the proportion of open arm visits, irrespective of age, the maineffect of Genotype (F(2,60) = 3.8, P = 0.029) for all three genotypescombined was caused by a difference between TgC100.WT andTgC100.V717F as neither of these groups differed significantly fromthe controls (Fig. 1).

3.2. Prepulse inhibition (PPI) of acoustic startle

PPI was lower in 17-month-old mice than in 4-month-old mice(main effect of Age F(1,54) = 7.6, P = 0.008). However, there were nodifferences in PPI between the genotypes at either age (Fig. 2). Incontrast, analysis of startle data revealed a significant differencebetween the genotypes (F(2,54) = 10.4, P < 0.001). Pair-wise com-parison revealed lower startle in TgC100.WT than in controls in the

Fig. 1. Total elevated plus maze number of arm entries (top panel) and %num-ber of open arm entries (bottom panel) of young (4 months) and old (17 months)TgC100.V717F (n = 11 for both age groups) compared to age-matched TgC100.WT(n = 8 and 12, respectively) and control mice (n = 13 and 10). Young TgC100.V717Fmice, but not TgC100.WT mice, showed higher total numbers of arm entries com-pared to controls (P < 0.001). This effect was not present in the older age group.

younger age-group, but not the older age group (Genotype × Ageinteraction F(1,36) = 5.5, P = 0.025). In contrast, startle amplitudewas significantly higher in TgC100.V717F than in controls (maineffect of Genotype F(1,37) = 8.7, P = 0.006).

3.3. Y-maze spontaneous alternation

Analysis of percentage spontaneous alternation (Fig. 3) of allgenotypes and both ages showed a main effect of Genotype(F(2,60) = 6.5, P = 0.003). Further analysis of data from TgC100.WTmice compared to controls showed no main effects but there was aGenotype × Age interaction (F(1,40) = 5.9, P = 0.020), reflecting thetrend for increased alternation at the younger age (P = 0.051) but notthe older age (Fig. 3). Further analysis of data from TgC100.V717Fmice vs. controls revealed a significantly reduced percentage alter-nation (F(1,41) = 8.9, P = 0.005) which was independent of age.

Analysis of the total number of entries showed signif-icant main effects of Genotype (F(2,60) = 5.1, P = 0.009) andAge (F(1,60) = 8.5, P = 0.005) and a Genotype × Age interaction(F(2,60) = 6.1, P = 0.004). Further analysis of data from TgC100.WTmice compared to controls, showed a main effect of Genotype(F(1,40) = 7.8, P = 0.008), reflecting increased locomotor activityin these mice at both ages. The results were more com-plex when TgC100.V717F were compared to controls, as therewere main effects of Genotype (F(1,41) = 8.0, P = 0.007) and ofAge (F(1,41) = 9.0, P = 0.005) and a Genotype × Age interaction(F(1,41) = 9.0, P = 0.005). The number of entries was markedly



Fig. 2. Average (Avg) prepulse inhibition of acoustic startle (PPI, top panel) andaverage startle amplitude of young (4 months) and old (17 months) TgC100.V717F(n = 11 and 9, respectively) compared to age-matched TgC100.WT (n = 8 and 11)and control mice (n = 13 and 8). Average PPI was lower in the older age groupbut not different between the three genotypes. Startle amplitudes were reducedin TgC100.WT of both ages, whereas they were significantly higher than controls inolder TgC100.V717F mice.

increased in TgC100.V717F at the younger age (P = 0.001) but notat the older age (Fig. 3).

3.4. Forced swim test

Combined analysis of immobility time of all genotypes andages revealed main effects of Genotype (F(2,54) = 15.8, P < 0.001)and Age (F(1,54) = 6.0, P = 0.017) and a Genotype × Age interaction(F(2,54) = 9.3, P < 0.001). Further analysis of data from TgC100.WTmice compared to controls, showed a significant Genotype × Ageinteraction (F(1,35) = 15.7, P < 0.001) which reflected the signifi-cantly reduced immobility scores in these mice at the younger agein contrast to the higher immobility scores in these mice at theolder age (Fig. 4). If TgC100.V717F mice were compared to controls,again there were main effects of Genotype (F(1,38) = 21.0, P < 0.001)and Age (F(1,38) = 19.2, P < 0.001) and a Genotype × Age interaction(F(1,38) = 6.9, P = 0.013). At the younger age, these mice had signif-icantly reduced immobility scores compared to controls, while atthe older age there was no significant difference (Fig. 4).

3.5. Baseline locomotor activity

Analysis of the combined data of the three genotypes at twoages (9 and 22 months, Fig. 5) revealed reduced baseline locomo-tor activity in the older group (main effect of Age F(1,43) = 46.5,P < 0.001). As expected, locomotor activity habituated over the60 min observation period (main effect of Time F(11,473) = 18.5,P < 0.001) but there were no major differences in the pattern of

Fig. 3. Spontaneous Y-maze alternation (top panel) and total entries (bottom panel)displayed by young (4 months) and old (17 months) TgC100.V717F (n = 11 for bothages) compared to age-matched TgC100.WT (n = 8 and 11) and control mice (n = 13and 10). TgC100.V717F showed a reduced percentage spontaneous alternation inboth young and old age. Bottom panel: Total number of entries were higher inTgC100.WT than control at both ages, while TgC100.V717F had higher number ofentries only at the younger age.

Fig. 4. Immobility in the forced swim test displayed by young (4 months) and old (17months) TgC100.V717F (n = 11 for both ages) compared to age-matched TgC100.WT(n = 9 at both ages) and control mice (n = 13 and 8, respectively). TgC100.WT hadreduced immobility scores at the younger age but higher scores at the older agecompared to controls. TgC100.V717F had reduced immobility scores only at youngerage.



Fig. 5. Baseline locomotor activity and amphetamine-induced locomotor hyperac-tivity analysis of young (4 months) and old (17 months) TgC100.V717F (n = 8 forboth ages) compared to age-matched TgC100.WT (n = 9 at both ages) and controlmice (n = 8 and 7, respectively). Mice were allowed 60 min of baseline activity, afterwhich they were injected with 5 mg/kg of amphetamine and monitored for another120 min. Data were expressed as 5-min scores (top panel) or total distance movedafter amphetamine treatment minus baseline distance moved before treatment(bottom panel). TgC100.V717F showed higher total distance move than TgC100.WTand control before amphetamine treatment, and also showed significantly greateramphetamine-induced locomotor hyperactivity compared to control mice.

habituation between the genotypes. There was a weak main effectof Genotype (F(2,43) = 3.5, P = 0.041) and a Genotype × Age interac-tion (F(2,43) = 4.3, P = 0.019), which were further investigated usingpair-wise ANOVAs.

TgC100.WT mice displayed slight hyperactivity during the pre-injection period compared to controls (main effect of GenotypeF(1,29) = 6.2, P = 0.019). TgC100.V717F mice also showed slighthyperactivity at 9 months of age, but not at 22 months ofage (Genotype × Age interaction F(1,27) = 11.5, P = 0.002). Totaldistance moved during the 60 min pre-injection period in con-trols, TgC100.WT and TgC100V717F was 124 ± 8, 162 ± 11 and168 ± 12 m, respectively, at 9 months of age and 97 ± 10, 110 ± 11and 83 ± 3 m, respectively, at 22 months of age.

3.6. Amphetamine-induced locomotor hyperactivity

Similar to pre-injection baseline activity, locomotor distancemoved after amphetamine injection was lower in 22-month-oldmice than in 9-month-old mice (main effect of Age F(1,43) = 12.5,P = 0.001). There was a main effect of Genotype (F(2,43) = 5.3,P = 0.009) which was further investigated using pair-wise ANOVAs.There was no significant difference between TgC100.WT and con-trols. In contrast, locomotor activity after amphetamine treatmentwas significantly higher in TgC100.V717F compared to controls atboth ages (main effect of Genotype F(1,27) = 7.0, P = 0.014, lack ofGenotype × Age interaction) (Fig. 5).

3.7. Passive avoidance learning

All mouse groups acquired passive avoidance responses inapproximately the same time: the total number of trials requiredto reach criterion was 4.1 ± 0.2 in controls, 3.8 ± 0.2 in TgC100.WTand 4.2 ± 0.2 in TgC100.V717F. Mice were then tested for retentionone and three days after the training session. The percentage num-ber of mice which remained passive during these retention trialsdeclined with time in the TgC100.V717F mice but not the controlsand TgC100.WT mice (Fig. 6). A similar result was seen when datawere expressed as the time during which the mice remained pas-sive: this score was significantly reduced three days, but not one dayafter training in the TgC100.V717F compared to the other groups(Fig. 6).

Fig. 6. Passive avoidance behaviour of 18-months old TgC100.V717F (n = 12) com-pared to age-matched TgC100.WT (n = 12) and control mice (n = 19). Mice weretraining to acquire repeated passive avoidance responses and then tested for reten-tion at Day 1 and 3 after the training sessions. Top panel depicts that the percentageof mice which remained passive decreased with time and reached significance onDay 3 only for TgC100.V717F. Bottom panel depicts that median time remaining pas-sive by these animals was significantly reduced in TgC100.V717F, suggesting loss ofmemory of the previously acquired avoidance responses.



Fig. 7. NMDA subunit transcript levels in the hippocampus of 10-month-oldTgC100.V717F compared to age-matched TgC100.WT and control mice, determinedby reverse transcription followed by real-time PCR using cyclophillin as the inter-nal control. The transcript levels of NR1 and NR2A were significantly lower inTgC100.V717F mice whereas the NR2B and NR2C expression was not differentbetween the genotypes. n = 6 for all groups.

3.8. NMDA subunit levels

The expression of hippocampal NR1 and NR2A subunits was sig-nificantly lower in TgC100.V717F, as compared to controls (Fig. 7).There was no change in these subunits in TgC100.WT animals, norwas there any difference in the expression levels of NR2B or NR2Cbetween the three groups.

3.9. ˇ-Tubulin staining

At both 10 months and 16–20 months of age, immunostainingshowed that axons in the cornu ammonis 1 (CA1) region of the hip-pocampus were disrupted in TgC100.V717F mice (Fig. 8C), whereasthose in the TgC100.WT mice (Fig. 8B) were intact and comparableto those in control animals (Fig. 8A).

3.10. TUNEL staining

In the CA1 of the hippocampus of TgC100.V717F, we detecteda small number of TUNEL-positive cells whereas no such stain-ing was detected in the hippocampus of the control animals orthe TgC100.WT mice (Fig. 8D). There appeared to be no differencebetween animals of 10 months of age or 16–20 months of age (datanot shown).

4. Discussion

The main findings of this behavioural phenotyping study werethat TgC100.V717F mice show a complex series of behaviouralchanges which can be grouped into a number of general cate-gories (Table 1). Firstly, these mice show hyperactivity, particularlyat a younger age, as shown by an increased number of ele-vated plus maze arm entries, increased number of Y-maze armentries, enhanced baseline locomotor activity in the open field,and enhanced amphetamine-induced locomotor activation. Thisbehavioural hyperactivity was less pronounced in TgC100.WT andthese animals only displayed significant differences with controlsat a younger age for the number of Y-maze arm entries and baselinelocomotor activity in the open field. It is possible that the hyper-activity in both genotypes contributed to their reduced immobilitytime in the forced swim test. Secondly, TgC100.V717F mice showeda phenotype of cognitive deficiency, as shown by reduced sponta-neous alternation in the Y-maze and markedly reduced retentionin the passive avoidance. In addition, at an older age, TgC100.V717Fmice showed enhanced startle and increased immobility time in theforced swim test. The behavioural changes were accompanied by

Table 1Overview of behavioural observations in TgC100.WT and TgC100.V717F mice com-pared to controls.

TgC100.WT TgC100.V717F

Young Old Young Old

EPM total number of arm entries – – ↑ –EPM proportion open arm visits – – – –Prepulse Inhibition – – – –Startle amplitude ↓ – – ↑Y-maze spontaneous alternation – – ↓ ↓Y-maze total number of arm entries ↑ – ↑ –Forced swim test immobility time ↓ – ↓ ↑Baseline locomotor activity ↑ ↑ ↑ –Amphetamine-induced hyperactivity – – ↑ ↑%Mice passive avoidance retention ND – ND ↓Passive avoidance retention time ND – ND ↓

↓ or ↑ indicate reduced and enhanced response, respectively, as determined bymain effect of Genotype or Genotype × Age interaction in ANOVA compared to con-trols. –, Indicates no difference with controls, EPM = elevated plus maze, ND = notdetermined.



Fig. 8. Typical �-tubulin immunostaining (panels A–C) and TUNEL-positive cell number counts in the CA1 region of the hippocampus of 10-month-old TgC100.V717Fcompared to age-matched TgC100.WT and control mice. There was axonal disruption (C) in the 10-month-old TgC100.V717F mouse hippocampus using �-tubulin as anaxonal marker (�-tubulin red; DAPI stained nuclei blue) but not Control (A) and TgC100.WT (B). Increased number of apoptotic cells were detected in TgC100.V717F transgenicmice compared to TgC100.WT (n = 5 for all 3 groups). *P < 0.05 (For interpretation of the references to colour in this figure legend, the reader is referred to the web version ofthe article.).

reduced expression of NMDA receptor subunits NR1 and NR2A, butnot NR2B or NR2C. In addition, we observed indices of disruptedaxonal morphology as revealed by �-tubulin immunoreactivity,and increased numbers of TUNEL-positive cells in the CA1 of thehippocampus in TgC100.V717F mice.

Elevated levels of baseline locomotor activity and especiallyamphetamine-induced locomotor hyperactivity, has been usedwidely as an animal model of psychotic-like behaviour [21,22].Thus, one interpretation of the increased behavioural activity ofthe TgC100.V717F mice in several of our behavioural tests couldbe, that these animals display behavioural changes with relevanceto psychosis. There is considerable literature on an association ofpsychosis with Alzheimer’s disease [33–35] although less so withother dementias, such as frontotemporal dementia [36]. Relativelylittle has been done to assess specifically psychotic features intransgenic mouse models of the disease [37] and there is consid-erable variability between studies. Tg2576 mice, which unlike theTgC100 mice develop �-amyloid neuritic plaques, were hyperactiveat baseline at an age when soluble A� levels are elevated [38,39]and while this hyperactivity diminishes across age as plaques beginto accumulate, it remains elevated compared to control animals[10]. Other AD models, such as APP23 transgenic mice with theSwedish mutation contain plaques and also displayed hyperac-tivity [40]. However, transgenic mice expressing the PS1-A246Emutation, which have increased levels of A� (42) but no plaques,were found to be normally active and showed no cognitive deficits[41]. Finally, transgenic mice expressing the C99 terminal fragmentof APP showed mild hypoactivity but no plaques [12]. Differ-ences with the C100 mice in the present study may be due to thegenetic background, differences in promoters or in brain regionswhere the transgene is expressed. In non-transgenic mice, chronicintracerebroventricular injection with �-amyloid (25–35) inducedbaseline locomotor hyperactivity and hypersensitivity to the actionof amphetamine [42]. Interestingly, similar to the TgC100.V717Fmice in the present study, �-amyloid-treated mice also showed dis-

rupted passive avoidance behaviour and spontaneous alternation[42].

In rats, amphetamine-induced locomotor hyperactivity is asso-ciated with elevated dopamine release in basal forebrain regions,such as the nucleus accumbens [43–45]. Thus, amphetamineacts on the dopamine transporter to enhance extracellular levelsof dopamine which activates post-synaptic dopamine recep-tors [46]. It would therefore be of interest to assess whetherTgC100.V717F mice display enhanced dopamine receptor levels oraltered dopamine transporter levels. However, it should be notedthat there is little evidence of changes in dopaminergic parametersin Alzheimer’s disease, unlike dementia with Lewy bodies [47].

The other main finding in TgC100.V717F was of disruptedspontaneous alternation and passive avoidance retention. Simi-lar findings have been described in a variety of transgenic modelsof Alzheimer’s disease (e.g. [10,39,42]). However, further studiesare needed in TgC100.WT and TgC100.V717F mice to assess theirperformance in other spatial memory tests, such as the Morriswatermaze. The data so far confirm that cognitive deficits can arisein the face of over-expression of APP fragments without plaqueformation. The mechanisms for these behavioural deficits may berelated to altered NMDA receptor expression and apoptotic celldeath which we observed in the TgC100.V717F mice. Hippocampallesions have previously been associated with hyperactivity [48] andmay thus contribute to the animals’ hyperactivity as well as cogni-tive deficits. Furthermore, it has been reported that A� could leadto neuronal cytoskeletal disruption in AD [49] and that it is solubleA� oligomers that catalyse such disruption [50]. Our current studiesdemonstrate that the over-expression of mutant C100 in additionto A� can also lead disruption of �-tubulin, a main constituent ofneuronal cytoskeleton [32].

Altered NMDA receptor levels have been observed in both theAD brain [51–54] and APPsw transgenic mice [55]. It has been well-described that treatment of mice with NMDA receptor antagonists,such as MK-801, causes hyperactivity [56], which is concordant



with the present results. Both TgC100 transgenic models over-express the APP C100 fragment and produce increased soluble A�,but TgC100.V717F mice also have an increased A� 42/40 ratio.Previously, it has been demonstrated that knocking down NR1expression in the hippocampus by siRNAs resulted in impairedlearning in the rat [57]. Thus, the cognitive deficiency of theTgC100.V717F mouse may be attributed to the reductions of theNR1 and NR2A subunits as well as to the axonal disruptions in itshippocampus. The over-expression of the soluble A� on its ownin TgC100.WT mice did not result in the lowering of the expres-sion of the NMDA subunits nor axonal disruption and no cognitivedeficits. Thus it can be concluded that it is the mutated C100 frag-ment that causes reduced NR1 and NR2A expression and axonaldisruptions, despite the absence of �-amyloid plaques. On the otherhand, the over-expression of soluble �-amyloid did lead to indica-tions of apoptotic cell death in the hippocampus of both transgenicmouse models regardless of the presence of mutation. In a studyusing an APP[V717I] transgenic mouse model, it has been shownthat there is decreased concentrations of NMDA-receptor subunitNR2B in post-synaptic density preparations of transgenic mice [58].In our current study, we did not detect a decrease in the expressionof the NR2B. However, the distribution of the protein may also bealtered in our TgC100.V717F further contributing to the cognitivedeficiency of the animals.

In conclusion, this study supports the notion that the C100 frag-ment bearing an Alzheimer’s disease mutation is toxic to neuronsand responsible for the cognitive and non-cognitive changes in theTgC100.V717F mice. The underlying mechanism for these effectsis as yet unclear but could be related to the reduction of NMDAreceptor subunit expression and disruption of neuronal cytoskele-ton.

Acknowledgements

This work was supported in part by NHMRC program grant#400202 and NHMRC project grant 494813 (W.C. Boon and H.K.Chua). At the time of the experiments, M. van den Buuse was sup-ported by a Griffith Senior Research Fellowship from the Universityof Melbourne, Australia.

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Behavioural phenotype of APPC100.V717F transgenic mice over-expressing a mutant Aβ-bearing fragment...

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