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Presenilin 2 mutation accelerates the onset of impairment in trace eyeblink conditioning in a mouse model of Alzheimer's disease overexpressing human mutant amyloid precursor protein
Neuroscience Letters 538 (2013) 15–19
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Presenilin 2 mutation accelerates the onset of impairment in trace eyeblink conditioning in a mouse model of Alzheimer’s disease overexpressing human mutant amyloid precursor protein Yasushi Kishimoto ∗ , Yutaka Kirino Laboratory of Neurobiophysics, Kagawa School of Pharmaceutical Sciences, Tokushima Bunri University, Sanuki, Kagawa 769-2193, Japan
h i g h l i g h t s
Tested trace eyeblink conditioning in APP/PS2 transgenic AD model (PS2Tg2576) mice. PS2Tg2576 mice aged 3 months showed normal performance in trace conditioning. Impaired trace conditioning in PS2Tg2576 mice at ages 4, 6, and 12 months. Intact trace conditioning in Tg2576 APPsw mice at age 4 months. PS2 mutation accelerates the onset of cognitive impairment in trace conditioning.
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Article history: Received 19 October 2012 Received in revised form 6 January 2013 Accepted 16 January 2013 Keywords: Aging Amyloid precursor protein Alzheimer’s disease Presenilin 2 Associative learning Trace eyeblink conditioning
a b s t r a c t Missense mutations in 2 homologous genes, presenilin 1 (PS1) and presenilin 2 (PS2), cause dementia in a subset of early-onset familial Alzheimer’s disease (FAD) pedigrees. The purpose of the present study was to investigate whether PS2 mutation accelerates the onset of trace eyeblink conditioning deficits in an Alzheimer’s disease (AD) mouse model overexpressing human amyloid precursor protein (APP) with the Swedish mutation (K670N, M671L) (Tg2576 mice). For this purpose, a double-transgenic mouse (PS2Tg2576 mice) was produced by cross-breeding transgenic mice carrying human mutant PS2 (N141I) with Tg2576 mice. Long-trace interval (trace interval = 500 ms) eyeblink conditioning was tested in the PS2Tg2576 mice at ages 3, 4, 6, and 12 months. At 3 months, PS2Tg2576 mice exhibited normal acquisition of conditioned responses (CRs) during trace eyeblink conditioning, whereas trace conditioning was significantly impaired in PS2Tg2576 mice at ages 4, 6, and 12 months. In contrast, Tg2576 mice showed intact memory performance during trace conditioning at 4 months. This cross-sectional study clearly indicates that PS2 mutation significantly accelerates the onset of cognitive impairment in associative trace eyeblink memory. © 2013 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Alzheimer’s disease (AD) is a chronic neurodegenerative disorder characterized clinically by progressive loss of cognitive abilities, especially impairment in retention of recently learned information [2,5]. The classic neuropathologic features of AD include abnormal deposition of -amyloid (A) peptides, neurofibrillary tangles, and neuronal loss in selective brain regions,
Abbreviations: A, -amyloid; AD, Alzheimer’s disease; APP, amyloid- precursor protein; CR, conditioned response; FAD, familial Alzheimer’s disease; OO, orbicularis oculi; MWM, Morris water maze; PS1, presenilin 1; PS2, presenilin 2; TI, trace interval. ∗ Corresponding author. Tel.: +81 87 894 5111; fax: +81 87 894 0181. E-mail address: [email protected] (Y. Kishimoto). 0304-3940/$ – see front matter © 2013 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.neulet.2013.01.025
including the hippocampus and cortex [10,18]. A is produced after sequential cleavage of the amyloid precursor protein (APP) by the proteases, - and ␥-secretase [8,11,29]. The highly homologous proteins presenilin 1 and 2 (PS1 and PS2) are important components of the ␥-secretase complex [4,12,14]. A mutation in 1 of these 3 genes (APP, PS1, and PS2) is responsible the approximately 5% of AD known as familial Alzheimer’s disease (FAD) [9,23]. Mouse model studies have shown that mutation of PS1 or PS2 results in acceleration of AD-like pathologies, with enhanced ␥secretase activity leading to increased A42 production [1,6,26]. Furthermore, cognitive deficits in a variety of learning tasks have been reported in PS1/APP double-transgenic mice [3,7,19,24]. In contrast, only a few studies have evaluated the cognitive effects of PS2 mutation using PS2/APP double-transgenic mice [25,28]. Indeed, no previous report has described the effects on associative hippocampal learning, including contextual fear conditioning
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or trace eyeblink conditioning, in PS2/APP double-transgenic mice. Trace eyeblink conditioning is an associative learning paradigm that requires an intact hippocampus in many species, including mice [15,21,27,34,35]. In fact, the eyeblink conditioning paradigm is advantageous over other behavioral paradigms in that it can be performed/applied in mammals, including mice and humans [33–36]. Therefore, trace eyeblink conditioning is beneficial for discussing the parallels between human diseases and rodent models of such diseases. A recent study demonstrated impairment in trace eyeblink conditioning with the 500-ms stimulus-free interval, in a mouse model of AD characterized by overexpression of human APP with the Swedish mutation (K670N, M671L) (Tg2576 mice) [16]. This study investigated the long-trace eyeblink conditioning in Tg2576 mice at ages 3, 6, and 12 months and showed that at age 3 months, the mice exhibited normal acquisition of trace eyeblink conditioning, whereas at age 6 months, they showed an impairment in trace eyeblink conditioning [16]. Hence, the purpose of the present crosssectional study was to confirm whether PS2 mutation accelerates the onset of trace eyeblink memory impairment in Tg2576 mice. 2. Methods 2.1. Animals In the present study, Tg2576 mice, APP/PS2 double-transgenic (PS2Tg2576) mice, and control mice from the same litter were used for the behavioral assays. Tg2576 mice in a C57BL6 SJL F1 background were obtained from Taconic Farms, Inc. (Hudson, NY USA) [13]. To obtain PS2Tg2576 mice, male Tg2576 mice were crossed with female PS2M1 mice, which express human presenilin-2 (PS2) proteins containing the N141I mutation in a C57BL/6JJcl background (purchased from Immuno-Biological Laboratories Co, Ltd., Fujioka, Japan) [22]. Wild-type littermates of the PS2Tg2576 mice were used as control mice. Genotype was confirmed by PCR as previously described [28]. Mice were housed in a room with a 12:12-h light–dark (LD) cycle. All mice were given ad libitum access to food and water. All animal procedures were approved by the Tokushima Bunri University animal ethics committee and were performed in accordance with the guidelines laid down by the National Institutes of Health (NIH, Bethesda, MD USA).
Fig. 1. Experimental design and schematic representation of long-trace eyeblink conditioning in AD-model mice. (A) Experimental design for the timing and period of behavioral tests. Mouse age is indicated below the schedules. Closed boxes indicate the timing and period of behavioral testing in the present study. PS2Tg2576 mice and their wild-type littermates were subjected to the trace eyeblink conditioning test at ages 3, 4, 6, and 12 months. Tg2576 mice and their wild-type littermates were also subjected to the trace eyeblink conditioning test at age 4 months. Hatched boxes indicate the timing and period of behavioral testing in a previous study, in which Tg2576 mice were tested at ages 3, 4, and 12 months [16]. Asterisks indicate the results of previous work (i.e., Tg2576 mice showed normal performance in trace eyeblink conditioning at age 3 months, whereas at ages 6 and 12 months, Tg2576 mice exhibited significant cognitive deficits in trace memory) [16]. (B) Schematic representation of stimulus timing for the long-trace eyeblink conditioning paradigm used in the present study. A stimulus-free interval (500 ms) is present between the end of the preceding CS and the US onset. Examples of three typical EMG recordings (CRs) from the orbicularis oculi (OO) muscle in the wild-type mice aged 4 months are shown under the CS–US representations.
2.3. Statistical analysis Data were statistically analyzed using a two-tailed Student’s t-test, a Tukey–Kramer test (for the number of trials required to elicit 6 CRs within 10 trials), or a repeated-measures ANOVA with post hoc Scheffé’s test (for daily CR%) using SPSS (IBM Corporation, Armonk, NY). The threshold for statistical significance was set at p < 0.05. All data are presented as mean ± SEM. 3. Results
2.2. Trace eyeblink conditioning PS2Tg2576 mice and their wild-type littermates at ages 3, 4, 6, and 12 months were used to assess long-trace eyeblink conditioning (Fig. 1). In order to compare our data with data obtained using Tg2576 mice and to enable further discussion, the present study investigated the impairment in trace eyeblink conditioning in Tg2576 mice at age 4 months (Fig. 1A) because no study has characterized the onset of cognitive decline in trace conditioning in Tg2576 mice between the ages of 3 and 6 months. Each mouse was used only once in the behavioral test. The surgery, anesthesia, and conditioning procedure were performed as previously described [15,16]. A 352-ms tone (1 kHz, 80 dB) was used as the conditioned stimulus (CS) and a 100-ms electrical shock (0.2 mA, 100 Hz, square pulses) was used as the unconditioned stimulus (US). The CS and US were each delivered separately, with a stimulus-free interval (trace interval, TI = 500 ms) (Fig. 1B). Each session consisted of 90 CS-US paired trials and 10 CS-only trials (every tenth trial). Mice underwent a total of 10 sessions (1 session per day). Electromyogram (EMG) signals were analyzed as previously described [15]. In addition to the daily conditioned response rate (CR%), the number of trials required to elicit 6 CRs within 10 trials was evaluated [16,31].
3.1. Long-trace interval eyeblink conditioning is impaired in PS2Tg2576 mice from age 4 months A long-trace eyeblink with a 500-ms TI was evaluated in PS2Tg2576 mice at 4 different ages: 3, 4, 6, and 12 months (Figs. 1 and 2). First, trace conditioning was tested in PS2Tg2576 mice at age 3 months (Fig. 2A). At age 3 months, PS2Tg2576 mice and control mice successfully acquired the CRs during the 10-day sessions and there were no differences in performance between the 2 groups (p > 0.05). In contrast, at age 4 months, PS2Tg2576 mice exhibited significant impairment in CR acquisition during trace conditioning. At age 4 months, the CR% for control mice progressively increased to approximately 60% during the 10 sessions, whereas that of PS2Tg2576 mice was 39.9% (Fig. 2B). No significant interaction effects were demonstrated between session and group [F (9, 144) = 0.93; p = 0.50], but a significant group effect was identified [F (1, 16) = 9.83; p = 0.006]. At age 4 months, the number of trials required for eliciting 6 CRs within a 10-trial block was significantly higher in PS2Tg2576 mice than in control mice (p = 0.029) (Fig. 2B). Furthermore, for trace conditioning at age 6 months (Fig. 2C), no significant interaction effects were demonstrated between session and group [F (9, 162) = 0.75; p = 0.66], but a significant group effect
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Fig. 2. Impairment of long-trace interval eyeblink conditioning in PS2Tg2576 mice from age 4 months. Long-trace interval eyeblink conditioning (TI = 500 ms) was investigated in PS2Tg2576 mice (filled circles) and their littermate control mice (empty circles) at ages 3, 4, 6, and 12 months. The daily conditioned response rate (CR%) during 10 sessions (left) and the number of trials required to exhibit 6 CRs within a 10-trial block (right) were determined. CR data from Tg2576 mice used in a previous study [16] were re-plotted for multiple comparison (CR% was indicated by empty triangles and dotted lines). (A) Trace conditioning in control (n = 9), PS2Tg2576 (n = 9), and Tg2576 (n = 9) mice at age 3 months. (B) Trace conditioning in control (n = 9) and PS2Tg2576 (n = 9) mice at age 4 months. (C) Trace conditioning in control (n = 10), PS2Tg2576 (n = 10), and Tg2576 (n = 10) mice at age 6 months. (D) Trace conditioning in control (n = 9), PS2Tg2576 (n = 10), and Tg2576 (n = 10) mice at age 12 months. * p < 0.05, ** p < 0.01 (PS2Tg2576 vs. the control group).
was again identified [F (1, 18) = 24.47; p < 0.001]. The number of trials required for eliciting 6 CRs within a 10-trial block was also significantly increased in PS2Tg2576 mice at age 6 months, compared to control mice (p = 0.007). Finally, trace conditioning was evaluated in PS2Tg2576 and control mice at age 12 months (Fig. 2D). Compared to mice aged 3–6 months, control mice at age 12 months exhibited slightly decreased improvement in CR acquisition attributable to the normal effects of aging. Therefore, at age 12 months, the difference between control and PS2Tg2576 mice was considerably reduced. However, a significant group effect was still detected [F (1, 17) = 7.01; p = 0.017], but no interaction effect was demonstrated between session and group [F (9, 153) = 0.38; p = 0.94]. The number of trials producing 6 CRs within a 10-trial block was also slightly increased in PS2Tg2576 mice at age 12 months and there was a significant difference between the 2 groups (p = 0.027). In Fig. 2A, C, and D, to directly compare trace eyeblink conditioning performance between PS2Tg2576 and Tg2576 mice, the trace eyeblink conditioning performance in Tg2576 mice aged 3, 6, and 12 months were re-plotted from our previous study [16]. The comparative analysis indicated that there was no difference in the performance between Tg2576 and PS2Tg2576 mice at any of the 3-, 6-, or 12-month ages (p > 0.05, detailed statistical analysis not shown).
development relative to the Tg2576 mice (Fig. 3, left panel). Indeed, ANOVA revealed that no significant interaction [F (18, 234) = 0.96; p = 0.503], but significant genotypic effect [F (2, 26) = 9.55; p < 0.001] in the learning curves (Fig. 3, left panel). There was no difference in the number of trials required to exhibit 6 CRs within a 10-trial block between the 3 genotypic groups (p = 0.16; Fig. 3, right panel). Thus, the present results indicated that, at age 4 months, PS2Tg2576 mice exhibited significantly lower CR% than Tg2576 mice. 4. Discussion The principal finding in the current study is that in APP/PS2 double-transgenic AD-model mice (PS2Tg2576 mice) significant cognitive dysfunction for trace eyeblink conditioning occurs as early as 4 months of age (Fig. 2). This is the first study to elucidate
3.2. At age 4 months, Tg2576 mice exhibited normal acquisition of long-trace eyeblink conditioning Next, we tested trace eyeblink conditioning in Tg2576 and control mice at age 4 months and further compared the performance of PS2Tg2576 mice at age 4 months (Fig. 3). It should be noted that the data for the PS2Tg2576 mice were the same as those shown in the previous figure (Fig. 2B). At this age, Tg2576 mice successfully acquired CRs in a similar manner to control mice, whereas PS2Tg2576 mice were significantly impaired with regard to CR
Fig. 3. Long-trace interval eyeblink conditioning is not impaired in Tg2576 mice at age 4 months. Long-trace eyeblink conditioning was tested in Tg2576 mice (filled circles; n = 10) and their littermate control mice (empty circles; n = 10) at age 4 months. The performance of PS2Tg2576 mice (empty triangles; n = 9) at the age of 4 months was re-plotted for multiple comparisons (The data are the same as those shown in Fig. 2B). The daily CR% during 10 sessions (left) and the number of trials required to exhibit 6 CRs within a 10-trial block (right) were determined. ** p < 0.01 (PS2Tg2576 mice vs. control or Tg2576 mice).
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the effect of PS2 mutation on mouse eyeblink conditioning. Considering that this study also demonstrated intact trace eyeblink conditioning in 4-month-old Tg2576 mice (Fig. 3), these results indicate that PS2 mutation accelerates the onset of cognitive impairment of associative trace eyeblink conditioning in APP transgenic AD-model mice. In the comparison of the cognitive performance between PS2Tg2576 and Tg2576 mice, significant differences were observed at 4 months of age (Fig. 3), but not at ages 3, 6, and 12 months (Fig. 2A, C, and D). At 3 months, neither Tg2576 nor PS2Tg2576 mice are impaired in trace eyeblink conditioning, but from 6 months of age, even Tg2576 mice exhibited considerable impairment. Therefore, a significant difference was no longer detectable between Tg2576 and PS2Tg2576 mice. Using both animal models and human subjects, classical eyeblink conditioning studies have contributed greatly to understanding the neurologic basis underlying learning and memory [27,34,35], particularly with respect to age-related decline in cognitive abilities under normal and disease conditions [20,36]. Indeed, eyeblink conditioning has been used to assess cognitive decline in human patients with AD [33,34,36], even though it has been reported that delay eyeblink conditioning is more susceptible to cognitive deterioration than trace eyeblink conditioning [33]. Over the last decade, several studies have investigated eyeblink conditioning in AD-model mice, including APP/PS1 doubletransgenic (PS1Tg2576) mice [3,7,30,31]. However, most attempts have failed to detect severe impairment in eyeblink conditioning using the delay or short-interval trace paradigm [3,7,31]. A recent report [16] showed that, using a 500-ms stimulus-free interval, trace eyeblink conditioning is impaired in Tg2576 mice at age 6 months (Fig. 1A). This was the first study to show significant impairment in eyeblink conditioning in AD-model mice, implying that a sufficiently long TI may be required to detect age-related cognitive deterioration in AD-model mice. The present study again confirmed that with a 500-ms TI, trace eyeblink conditioning is susceptible to age-related cognitive deterioration in AD-model mice. Of note, even control mice at age 12 months in this study exhibited considerable deterioration in trace conditioning, attributable to normal aging (Fig. 2D). This observation is consistent with previous results showing that 500-ms-TI trace eyeblink conditioning is significantly impaired as early as ∼12 months of age in wild-type mice [17]. This finding was also supported by the findings of Gruart et al. [7], who found lower CR development in 12-month-old wild-type mice than in 3-month-old wild-type mice during 500ms-TI trace eyeblink conditioning. Therefore, to detect impairment in trace eyeblink conditioning in AD-model mice, it is important to limit testing to mice that are not too old because it becomes difficult to distinguish differences in cognitive performance between older control and AD-model mice [16,17]. Toda et al., reported the early deposition of A at age 2–3 months and significant accumulation from 4 to 5 months, in brain regions, including the neocortex and the hippocampus, of PS2Tg2576 mice [28]. In these mice, this represents a substantially advanced onset of A deposits, compared to Tg2576 mice, in which A-containing neuritic plaques appear from approximately 8 months of age [13,28,32]. In addition, compared to the onset of plaques in PS1Tg2576 mice (age 6 months), the onset in PS2Tg2576 mice begins substantially earlier [6,22]. Previous studies have also shown that PS2Tg2576 mice, ages 4–5 months, exhibited cognitive impairments during the probe trial of a Morris water maze (MWM) task, indicating that PS2 mutation accelerated spatial learning impairment associated with A accumulation [28]. The current results demonstrating associative cognitive impairment at 4 months are temporally consistent with previous results showing spatial learning impairment. Moreover, using a different line of APP/PS2 double-transgenic mice (PS2APP mice)—generated by a different promoter from that
used for the PS2Tg2576 mice in this study—one study demonstrated that the onset of A plaque formation starts at age 6 months and demonstrated deterioration in cognitive abilities on the MWM or passive avoidance test from age 8 months [25]. Therefore, compared to many other mouse models of AD, PS2Tg2576 mice consistently develop the earliest onset and most rapidly progressive AD-like pathology and cognitive dysfunction. Furthermore, the present study indicates that cognitive impairment in PS2Tg2576 mice could be detectable as early as 4 months of age. The longtrace eyeblink conditioning task, using PS2Tg2576 mice, could be a suitable system for studying the molecular basis of cognitive dysfunction in AD and for screening new drug/therapy candidates for AD.
5. Conclusion These findings demonstrate age-dependent cognitive impairment in long trace eyeblink conditioning in PS2Tg2576 mice, which starts as early as 4 months of age. In contrast, at age 4 months, Tg2576 mice exhibited normal performance in trace conditioning. Taken together, these results suggest that PS2 mutation accelerates the onset of cognitive impairment in associative trace eyeblink conditioning in APP transgenic mice.
Acknowledgments This study was supported by Grants-in-Aid for Scientific Research (grant nos. 20790084 and 24590133 to Y. Kishimoto), the Takeda Science Foundation (to Y. Kishimoto), and by the Smoking Research Foundation (to Y. Kirino). We thank Ms. Junko Nakayama for technical assistance and Ms. Hisako Fukuoka for animal care.
References [1] D.R. Borchelt, T. Ratovitski, J. van Lare, M.K. Lee, V. Gonzales, N.A. Jenkins, N.G. Copeland, D.L. Price, S.S. Sisodia, Accelerated amyloid deposition in the brains of transgenic mice coexpressing mutant presenilin 1 and amyloid precursor proteins, Neuron 19 (1997) 939–945. [2] G.A. Carlesimo, M. Oscar-Berman, Memory deficits in Alzheimer’s patients: a comprehensive review, Neuropsychol. Rev. 3 (1992) 119–169. [3] M. Ewers, D.G. Morgan, M.N. Gordon, D.S. Woodruff-Pak, Associative and motor learning in 12-month-old transgenic APP + PS1 mice, Neurobiol. Aging 27 (2006) 1118–1128. [4] P. St. George-Hyslop, P.E. Fraser, Assembly of the presenilin ␥-/-secretase complex, J. Neurochem. 120 (2012) 84–88. [5] C.A. Gold, A.E. Budson, Memory loss in Alzheimer’s disease: implications for development of therapeutics, Expert Rev. Neurother. 8 (2008) 1879–1891. [6] M.N. Gordon, D.L. King, D.M. Diamond, P.T. Jantzen, K.V. Boyett, C.E. Hope, J.M. Hatcher, G. DiCarlo, W.P. Gottschall, D. Morgan, G.W. Arendash, Correlation between cognitive deficits and A deposits in transgenic APP + PS1 mice, Neurobiol. Aging 22 (2001) 377–385. ˜ J.M. Delgado-Garcia, Aged wild-type [7] A. Gruart, J.C. López-Ramos, M.D. Munoz, and APP, PS1, and APP + PS1 mice present similar deficits in associative learning and synaptic plasticity independent of amyloid load, Neurobiol. Dis. 30 (2008) 439–450. [8] C. Haass, D.J. Selkoe, Soluble protein oligomers in neurodegeneration: lessons from the Alzheimer’s amyloid -peptide, Nat. Rev. Mol. Cell Biol. 8 (2007) 101–112. [9] J. Hardy, The Alzheimer family of diseases: many etiologies, one pathogenesis? Proc. Natl. Acad. Sci. U.S.A. 94 (1997) 2095–2097. [10] J. Hardy, D. Allsop, Amyloid deposition as the central event in the aetiology of Alzheimer’s disease, Trends Pharmacol. Sci. 12 (1991) 383–388. [11] J. Hardy, D.J. Selkoe, The amyloid hypothesis of Alzheimer’s disease: progress and problems on the road to therapeutics, Science 297 (2002) 353–356. [12] A. Herreman, L. Serneels, W. Annaert, D. Collen, L. Schoonjans, B. De Strooper, Total inactivation of ␥-secretase activity in presenilin-deficient embryonic stem cells, Nat. Cell Biol. 2 (2000) 461–462. [13] K. Hsiao, P. Chapman, S. Nilsen, C. Eckman, Y. Harigaya, S. Younkin, F. Yang, G. Cole, Correlative memory deficits, A elevation, and amyloid plaques in transgenic mice, Science 274 (1996) 99–102. [14] W.T. Kimberly, W. Xia, T. Rahmati, M.S. Wolfe, D.J. Selkoe, The transmembrane aspartates in presenilin 1 and 2 are obligatory for ␥-secretase activity and amyloid -protein generation, J. Biol. Chem. 275 (2000) 3173–3178.
Y. Kishimoto, Y. Kirino / Neuroscience Letters 538 (2013) 15–19 [15] Y. Kishimoto, K. Nakazawa, S. Tonegawa, Y. Kirino, M. Kano, Hippocampal CA3 NMDA receptors are crucial for adaptive timing of trace eyeblink conditioned response, J. Neurosci. 26 (2006) 1562–1570. [16] Y. Kishimoto, I. Oku, A. Nishigawa, A. Nishimoto, Y. Kirino, Impaired long-trace eyeblink conditioning in a Tg2576 mouse model of Alzheimer’s disease, Neurosci. Lett. 506 (2012) 155–159. [17] Y. Kishimoto, M. Suzuki, S. Kawahara, Y. Kirino, Age-dependent impairment of delay and trace eyeblink conditioning in mice, NeuroReport 12 (2001) 3349–3352. [18] F.M. LaFerla, S. Oddo, Alzheimer’s disease: A, tau and synaptic dysfunction, Trends Mol. Med. 11 (2005) 170–176. [19] S. Lagadec, L. Rotureau, A. Hémar, N. Macrez, S. Delcasso, Y. Jeantet, Y.H. Cho, Early temporal short-term memory deficits in double transgenic APP/PS1 mice, Neurobiol. Aging 33 (2012), 203.e1-203.e11. ˜ J.M. [20] J.C. López-Ramos, M.T. Jurado-Parras, C. Sanfeliu, D. Acuna-Castroviejo, Delgado-García, Learning capabilities and CA1-prefrontal synaptic plasticity in a mice model of accelerated senescence, Neurobiol. Aging 33 (2012) 627.e13–627.e26. [21] J.R. Moyer Jr., R.A. Deyo, J.F. Disterhoft, Hippocampectomy disrupts trace eyeblink conditioning in rabbits, Behav. Neurosci. 104 (1990) 243–252. [22] F. Oyama, N. Sawamura, K. Kobayashi, M. Morishima-Kawashima, T. Kuramochi, M. Ito, T. Tomita, K. Maruyama, T.C. Saido, T. Iwatsubo, A. Capell, J. Walter, J. Grünberg, Y. Ueyama, C. Haass, Y. Ihara, Mutant presenilin 2 transgenic mouse: effect on an age-dependent increase of amyloid -protein 42 in the brain, J. Neurochem. 71 (1998) 313–322. [23] D.L. Price, S.S. Sisodia, Mutant genes in familial Alzheimer’s disease and transgenic models, Ann. Rev. Neurosci. 21 (1998) 479–505. [24] J. Puoliväli, J. Wang, T. Heikkinen, M. Heikkilä, T. Tapiola, T. van Groen, H. Tanila, Hippocampal A 42 levels correlate with spatial memory deficit in APP and PS1 double transgenic mice, Neurobiol. Dis. 9 (2002) 339–347. [25] J.G. Richards, G.A. Higgins, A.M. Ouagazzal, L. Ozmen, J.N. Kew, B. Bohrmann, P. Malherbe, M. Brockhaus, H. Loetscher, C. Czech, G. Huber, H. Bluethmann, H. Jacobsen, J.A. Kemp, PS2APP transgenic mice, coexpressing hPS2mut and hAPPswe, show age-related cognitive deficits associated with discrete brain amyloid deposition and inflammation, J. Neurosci. 23 (2003) 8989–9003.
19
[26] N. Sawamura, M. Morishima-Kawashima, H. Waki, K. Kobayashi, T. Kuramochi, M.P. Frosch, K. Ding, M. Ito, T.W. Kim, R.E. Tanzi, F. Oyama, T. Tabira, S. Ando, Y. Ihara, Mutant presenilin 2 transgenic mice. A large increase in the levels of A 42 is presumably associated with the low density membrane domain that contains decreased levels of glycerophospholipids and sphingomyelin, J. Biol. Chem. 275 (2000) 27901–27908. [27] R.F. Thompson, J.J. Kim, Memory systems in the brain and localization of a memory, Proc. Natl. Acad. Sci. U.S.A. 93 (1996) 13438–13444. [28] T. Toda, Y. Noda, G. Ito, M. Maeda, T. Shimizu, Presenilin-2 mutation causes early amyloid accumulation and memory impairment in a transgenic mouse model of Alzheimer’s disease, J. Biomed. Biotechnol. 2011 (2011) 617974. [29] J. Walter, C. Kaether, H. Steiner, C. Haass, The cell biology of Alzheimer’s disease: uncovering the secrets of secretases, Curr. Opin. Neurobiol. 11 (2001) 585–590. [30] J.M. Wang, C. Singh, L. Liu, R.W. Irwin, S. Chen, E.J. Chung, R.F. Thompson, R.D. Brinton, Allopregnanolone reverses neurogenic and cognitive deficits in mouse model of Alzheimer’s disease, Proc. Natl. Acad. Sci. U.S.A. 107 (2010) 6498–6503. [31] C. Weiss, P.N. Venkatasubramanian, A.S. Aguado, J.M. Power, B.C. Tom, L. Li, K.S. Chen, J.F. Disterhoft, A.M. Wyrwicz, Impaired eyeblink conditioning and decreased hippocampal volume in PDAPP V717F mice, Neurobiol. Dis. 11 (2002) 425–433. [32] M.A. Westerman, D. Cooper-Blacketer, A. Mariash, L. Kotilinek, T. Kawarabayashi, L.H. Younkin, G.A. Carlson, S.G. Younkin, K.H. Ashe, The relationship between A and memory in the Tg2576 mouse model of Alzheimer’s disease, J. Neurosci. 22 (2002) 1858–1867. [33] D.S. Woodruff-Pak, M. Papka, Alzheimer’s disease and eyeblink conditioning: 750 ms trace vs. 400 ms delay paradigm, Neurobiol. Aging 17 (1996) 397–404. [34] D.S. Woodruff-Pak, J.E. Steinmetz, Eyeblink Classical Conditioning, Volume 1: Applications in Humans, Kluwer Academic Publishers, Boston, MA, 2000. [35] D.S. Woodruff-Pak, J.E. Steinmetz, Eyeblink Classical Conditioning, Volume 2: Animal Models, Kluwer Academic Publishers, Boston, MA, 2000. [36] D.S. Woodruff-Pak, Eyeblink classical conditioning differentiates normal aging from Alzheimer’s disease, Integr. Physiol. Behav. Sci. 36 (2001) 87–108.
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Presenilin 2 mutation accelerates the onset of impairment in trace eyeblink conditioning in a mouse model of Alzheimer’s disease overexpressing human mutant amyloid precursor protein Yasushi Kishimoto ∗ , Yutaka Kirino Laboratory of Neurobiophysics, Kagawa School of Pharmaceutical Sciences, Tokushima Bunri University, Sanuki, Kagawa 769-2193, Japan
h i g h l i g h t s
Tested trace eyeblink conditioning in APP/PS2 transgenic AD model (PS2Tg2576) mice. PS2Tg2576 mice aged 3 months showed normal performance in trace conditioning. Impaired trace conditioning in PS2Tg2576 mice at ages 4, 6, and 12 months. Intact trace conditioning in Tg2576 APPsw mice at age 4 months. PS2 mutation accelerates the onset of cognitive impairment in trace conditioning.
a r t i c l e
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Article history: Received 19 October 2012 Received in revised form 6 January 2013 Accepted 16 January 2013 Keywords: Aging Amyloid precursor protein Alzheimer’s disease Presenilin 2 Associative learning Trace eyeblink conditioning
a b s t r a c t Missense mutations in 2 homologous genes, presenilin 1 (PS1) and presenilin 2 (PS2), cause dementia in a subset of early-onset familial Alzheimer’s disease (FAD) pedigrees. The purpose of the present study was to investigate whether PS2 mutation accelerates the onset of trace eyeblink conditioning deficits in an Alzheimer’s disease (AD) mouse model overexpressing human amyloid precursor protein (APP) with the Swedish mutation (K670N, M671L) (Tg2576 mice). For this purpose, a double-transgenic mouse (PS2Tg2576 mice) was produced by cross-breeding transgenic mice carrying human mutant PS2 (N141I) with Tg2576 mice. Long-trace interval (trace interval = 500 ms) eyeblink conditioning was tested in the PS2Tg2576 mice at ages 3, 4, 6, and 12 months. At 3 months, PS2Tg2576 mice exhibited normal acquisition of conditioned responses (CRs) during trace eyeblink conditioning, whereas trace conditioning was significantly impaired in PS2Tg2576 mice at ages 4, 6, and 12 months. In contrast, Tg2576 mice showed intact memory performance during trace conditioning at 4 months. This cross-sectional study clearly indicates that PS2 mutation significantly accelerates the onset of cognitive impairment in associative trace eyeblink memory. © 2013 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Alzheimer’s disease (AD) is a chronic neurodegenerative disorder characterized clinically by progressive loss of cognitive abilities, especially impairment in retention of recently learned information [2,5]. The classic neuropathologic features of AD include abnormal deposition of -amyloid (A) peptides, neurofibrillary tangles, and neuronal loss in selective brain regions,
Abbreviations: A, -amyloid; AD, Alzheimer’s disease; APP, amyloid- precursor protein; CR, conditioned response; FAD, familial Alzheimer’s disease; OO, orbicularis oculi; MWM, Morris water maze; PS1, presenilin 1; PS2, presenilin 2; TI, trace interval. ∗ Corresponding author. Tel.: +81 87 894 5111; fax: +81 87 894 0181. E-mail address: [email protected] (Y. Kishimoto). 0304-3940/$ – see front matter © 2013 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.neulet.2013.01.025
including the hippocampus and cortex [10,18]. A is produced after sequential cleavage of the amyloid precursor protein (APP) by the proteases, - and ␥-secretase [8,11,29]. The highly homologous proteins presenilin 1 and 2 (PS1 and PS2) are important components of the ␥-secretase complex [4,12,14]. A mutation in 1 of these 3 genes (APP, PS1, and PS2) is responsible the approximately 5% of AD known as familial Alzheimer’s disease (FAD) [9,23]. Mouse model studies have shown that mutation of PS1 or PS2 results in acceleration of AD-like pathologies, with enhanced ␥secretase activity leading to increased A42 production [1,6,26]. Furthermore, cognitive deficits in a variety of learning tasks have been reported in PS1/APP double-transgenic mice [3,7,19,24]. In contrast, only a few studies have evaluated the cognitive effects of PS2 mutation using PS2/APP double-transgenic mice [25,28]. Indeed, no previous report has described the effects on associative hippocampal learning, including contextual fear conditioning
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or trace eyeblink conditioning, in PS2/APP double-transgenic mice. Trace eyeblink conditioning is an associative learning paradigm that requires an intact hippocampus in many species, including mice [15,21,27,34,35]. In fact, the eyeblink conditioning paradigm is advantageous over other behavioral paradigms in that it can be performed/applied in mammals, including mice and humans [33–36]. Therefore, trace eyeblink conditioning is beneficial for discussing the parallels between human diseases and rodent models of such diseases. A recent study demonstrated impairment in trace eyeblink conditioning with the 500-ms stimulus-free interval, in a mouse model of AD characterized by overexpression of human APP with the Swedish mutation (K670N, M671L) (Tg2576 mice) [16]. This study investigated the long-trace eyeblink conditioning in Tg2576 mice at ages 3, 6, and 12 months and showed that at age 3 months, the mice exhibited normal acquisition of trace eyeblink conditioning, whereas at age 6 months, they showed an impairment in trace eyeblink conditioning [16]. Hence, the purpose of the present crosssectional study was to confirm whether PS2 mutation accelerates the onset of trace eyeblink memory impairment in Tg2576 mice. 2. Methods 2.1. Animals In the present study, Tg2576 mice, APP/PS2 double-transgenic (PS2Tg2576) mice, and control mice from the same litter were used for the behavioral assays. Tg2576 mice in a C57BL6 SJL F1 background were obtained from Taconic Farms, Inc. (Hudson, NY USA) [13]. To obtain PS2Tg2576 mice, male Tg2576 mice were crossed with female PS2M1 mice, which express human presenilin-2 (PS2) proteins containing the N141I mutation in a C57BL/6JJcl background (purchased from Immuno-Biological Laboratories Co, Ltd., Fujioka, Japan) [22]. Wild-type littermates of the PS2Tg2576 mice were used as control mice. Genotype was confirmed by PCR as previously described [28]. Mice were housed in a room with a 12:12-h light–dark (LD) cycle. All mice were given ad libitum access to food and water. All animal procedures were approved by the Tokushima Bunri University animal ethics committee and were performed in accordance with the guidelines laid down by the National Institutes of Health (NIH, Bethesda, MD USA).
Fig. 1. Experimental design and schematic representation of long-trace eyeblink conditioning in AD-model mice. (A) Experimental design for the timing and period of behavioral tests. Mouse age is indicated below the schedules. Closed boxes indicate the timing and period of behavioral testing in the present study. PS2Tg2576 mice and their wild-type littermates were subjected to the trace eyeblink conditioning test at ages 3, 4, 6, and 12 months. Tg2576 mice and their wild-type littermates were also subjected to the trace eyeblink conditioning test at age 4 months. Hatched boxes indicate the timing and period of behavioral testing in a previous study, in which Tg2576 mice were tested at ages 3, 4, and 12 months [16]. Asterisks indicate the results of previous work (i.e., Tg2576 mice showed normal performance in trace eyeblink conditioning at age 3 months, whereas at ages 6 and 12 months, Tg2576 mice exhibited significant cognitive deficits in trace memory) [16]. (B) Schematic representation of stimulus timing for the long-trace eyeblink conditioning paradigm used in the present study. A stimulus-free interval (500 ms) is present between the end of the preceding CS and the US onset. Examples of three typical EMG recordings (CRs) from the orbicularis oculi (OO) muscle in the wild-type mice aged 4 months are shown under the CS–US representations.
2.3. Statistical analysis Data were statistically analyzed using a two-tailed Student’s t-test, a Tukey–Kramer test (for the number of trials required to elicit 6 CRs within 10 trials), or a repeated-measures ANOVA with post hoc Scheffé’s test (for daily CR%) using SPSS (IBM Corporation, Armonk, NY). The threshold for statistical significance was set at p < 0.05. All data are presented as mean ± SEM. 3. Results
2.2. Trace eyeblink conditioning PS2Tg2576 mice and their wild-type littermates at ages 3, 4, 6, and 12 months were used to assess long-trace eyeblink conditioning (Fig. 1). In order to compare our data with data obtained using Tg2576 mice and to enable further discussion, the present study investigated the impairment in trace eyeblink conditioning in Tg2576 mice at age 4 months (Fig. 1A) because no study has characterized the onset of cognitive decline in trace conditioning in Tg2576 mice between the ages of 3 and 6 months. Each mouse was used only once in the behavioral test. The surgery, anesthesia, and conditioning procedure were performed as previously described [15,16]. A 352-ms tone (1 kHz, 80 dB) was used as the conditioned stimulus (CS) and a 100-ms electrical shock (0.2 mA, 100 Hz, square pulses) was used as the unconditioned stimulus (US). The CS and US were each delivered separately, with a stimulus-free interval (trace interval, TI = 500 ms) (Fig. 1B). Each session consisted of 90 CS-US paired trials and 10 CS-only trials (every tenth trial). Mice underwent a total of 10 sessions (1 session per day). Electromyogram (EMG) signals were analyzed as previously described [15]. In addition to the daily conditioned response rate (CR%), the number of trials required to elicit 6 CRs within 10 trials was evaluated [16,31].
3.1. Long-trace interval eyeblink conditioning is impaired in PS2Tg2576 mice from age 4 months A long-trace eyeblink with a 500-ms TI was evaluated in PS2Tg2576 mice at 4 different ages: 3, 4, 6, and 12 months (Figs. 1 and 2). First, trace conditioning was tested in PS2Tg2576 mice at age 3 months (Fig. 2A). At age 3 months, PS2Tg2576 mice and control mice successfully acquired the CRs during the 10-day sessions and there were no differences in performance between the 2 groups (p > 0.05). In contrast, at age 4 months, PS2Tg2576 mice exhibited significant impairment in CR acquisition during trace conditioning. At age 4 months, the CR% for control mice progressively increased to approximately 60% during the 10 sessions, whereas that of PS2Tg2576 mice was 39.9% (Fig. 2B). No significant interaction effects were demonstrated between session and group [F (9, 144) = 0.93; p = 0.50], but a significant group effect was identified [F (1, 16) = 9.83; p = 0.006]. At age 4 months, the number of trials required for eliciting 6 CRs within a 10-trial block was significantly higher in PS2Tg2576 mice than in control mice (p = 0.029) (Fig. 2B). Furthermore, for trace conditioning at age 6 months (Fig. 2C), no significant interaction effects were demonstrated between session and group [F (9, 162) = 0.75; p = 0.66], but a significant group effect
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Fig. 2. Impairment of long-trace interval eyeblink conditioning in PS2Tg2576 mice from age 4 months. Long-trace interval eyeblink conditioning (TI = 500 ms) was investigated in PS2Tg2576 mice (filled circles) and their littermate control mice (empty circles) at ages 3, 4, 6, and 12 months. The daily conditioned response rate (CR%) during 10 sessions (left) and the number of trials required to exhibit 6 CRs within a 10-trial block (right) were determined. CR data from Tg2576 mice used in a previous study [16] were re-plotted for multiple comparison (CR% was indicated by empty triangles and dotted lines). (A) Trace conditioning in control (n = 9), PS2Tg2576 (n = 9), and Tg2576 (n = 9) mice at age 3 months. (B) Trace conditioning in control (n = 9) and PS2Tg2576 (n = 9) mice at age 4 months. (C) Trace conditioning in control (n = 10), PS2Tg2576 (n = 10), and Tg2576 (n = 10) mice at age 6 months. (D) Trace conditioning in control (n = 9), PS2Tg2576 (n = 10), and Tg2576 (n = 10) mice at age 12 months. * p < 0.05, ** p < 0.01 (PS2Tg2576 vs. the control group).
was again identified [F (1, 18) = 24.47; p < 0.001]. The number of trials required for eliciting 6 CRs within a 10-trial block was also significantly increased in PS2Tg2576 mice at age 6 months, compared to control mice (p = 0.007). Finally, trace conditioning was evaluated in PS2Tg2576 and control mice at age 12 months (Fig. 2D). Compared to mice aged 3–6 months, control mice at age 12 months exhibited slightly decreased improvement in CR acquisition attributable to the normal effects of aging. Therefore, at age 12 months, the difference between control and PS2Tg2576 mice was considerably reduced. However, a significant group effect was still detected [F (1, 17) = 7.01; p = 0.017], but no interaction effect was demonstrated between session and group [F (9, 153) = 0.38; p = 0.94]. The number of trials producing 6 CRs within a 10-trial block was also slightly increased in PS2Tg2576 mice at age 12 months and there was a significant difference between the 2 groups (p = 0.027). In Fig. 2A, C, and D, to directly compare trace eyeblink conditioning performance between PS2Tg2576 and Tg2576 mice, the trace eyeblink conditioning performance in Tg2576 mice aged 3, 6, and 12 months were re-plotted from our previous study [16]. The comparative analysis indicated that there was no difference in the performance between Tg2576 and PS2Tg2576 mice at any of the 3-, 6-, or 12-month ages (p > 0.05, detailed statistical analysis not shown).
development relative to the Tg2576 mice (Fig. 3, left panel). Indeed, ANOVA revealed that no significant interaction [F (18, 234) = 0.96; p = 0.503], but significant genotypic effect [F (2, 26) = 9.55; p < 0.001] in the learning curves (Fig. 3, left panel). There was no difference in the number of trials required to exhibit 6 CRs within a 10-trial block between the 3 genotypic groups (p = 0.16; Fig. 3, right panel). Thus, the present results indicated that, at age 4 months, PS2Tg2576 mice exhibited significantly lower CR% than Tg2576 mice. 4. Discussion The principal finding in the current study is that in APP/PS2 double-transgenic AD-model mice (PS2Tg2576 mice) significant cognitive dysfunction for trace eyeblink conditioning occurs as early as 4 months of age (Fig. 2). This is the first study to elucidate
3.2. At age 4 months, Tg2576 mice exhibited normal acquisition of long-trace eyeblink conditioning Next, we tested trace eyeblink conditioning in Tg2576 and control mice at age 4 months and further compared the performance of PS2Tg2576 mice at age 4 months (Fig. 3). It should be noted that the data for the PS2Tg2576 mice were the same as those shown in the previous figure (Fig. 2B). At this age, Tg2576 mice successfully acquired CRs in a similar manner to control mice, whereas PS2Tg2576 mice were significantly impaired with regard to CR
Fig. 3. Long-trace interval eyeblink conditioning is not impaired in Tg2576 mice at age 4 months. Long-trace eyeblink conditioning was tested in Tg2576 mice (filled circles; n = 10) and their littermate control mice (empty circles; n = 10) at age 4 months. The performance of PS2Tg2576 mice (empty triangles; n = 9) at the age of 4 months was re-plotted for multiple comparisons (The data are the same as those shown in Fig. 2B). The daily CR% during 10 sessions (left) and the number of trials required to exhibit 6 CRs within a 10-trial block (right) were determined. ** p < 0.01 (PS2Tg2576 mice vs. control or Tg2576 mice).
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the effect of PS2 mutation on mouse eyeblink conditioning. Considering that this study also demonstrated intact trace eyeblink conditioning in 4-month-old Tg2576 mice (Fig. 3), these results indicate that PS2 mutation accelerates the onset of cognitive impairment of associative trace eyeblink conditioning in APP transgenic AD-model mice. In the comparison of the cognitive performance between PS2Tg2576 and Tg2576 mice, significant differences were observed at 4 months of age (Fig. 3), but not at ages 3, 6, and 12 months (Fig. 2A, C, and D). At 3 months, neither Tg2576 nor PS2Tg2576 mice are impaired in trace eyeblink conditioning, but from 6 months of age, even Tg2576 mice exhibited considerable impairment. Therefore, a significant difference was no longer detectable between Tg2576 and PS2Tg2576 mice. Using both animal models and human subjects, classical eyeblink conditioning studies have contributed greatly to understanding the neurologic basis underlying learning and memory [27,34,35], particularly with respect to age-related decline in cognitive abilities under normal and disease conditions [20,36]. Indeed, eyeblink conditioning has been used to assess cognitive decline in human patients with AD [33,34,36], even though it has been reported that delay eyeblink conditioning is more susceptible to cognitive deterioration than trace eyeblink conditioning [33]. Over the last decade, several studies have investigated eyeblink conditioning in AD-model mice, including APP/PS1 doubletransgenic (PS1Tg2576) mice [3,7,30,31]. However, most attempts have failed to detect severe impairment in eyeblink conditioning using the delay or short-interval trace paradigm [3,7,31]. A recent report [16] showed that, using a 500-ms stimulus-free interval, trace eyeblink conditioning is impaired in Tg2576 mice at age 6 months (Fig. 1A). This was the first study to show significant impairment in eyeblink conditioning in AD-model mice, implying that a sufficiently long TI may be required to detect age-related cognitive deterioration in AD-model mice. The present study again confirmed that with a 500-ms TI, trace eyeblink conditioning is susceptible to age-related cognitive deterioration in AD-model mice. Of note, even control mice at age 12 months in this study exhibited considerable deterioration in trace conditioning, attributable to normal aging (Fig. 2D). This observation is consistent with previous results showing that 500-ms-TI trace eyeblink conditioning is significantly impaired as early as ∼12 months of age in wild-type mice [17]. This finding was also supported by the findings of Gruart et al. [7], who found lower CR development in 12-month-old wild-type mice than in 3-month-old wild-type mice during 500ms-TI trace eyeblink conditioning. Therefore, to detect impairment in trace eyeblink conditioning in AD-model mice, it is important to limit testing to mice that are not too old because it becomes difficult to distinguish differences in cognitive performance between older control and AD-model mice [16,17]. Toda et al., reported the early deposition of A at age 2–3 months and significant accumulation from 4 to 5 months, in brain regions, including the neocortex and the hippocampus, of PS2Tg2576 mice [28]. In these mice, this represents a substantially advanced onset of A deposits, compared to Tg2576 mice, in which A-containing neuritic plaques appear from approximately 8 months of age [13,28,32]. In addition, compared to the onset of plaques in PS1Tg2576 mice (age 6 months), the onset in PS2Tg2576 mice begins substantially earlier [6,22]. Previous studies have also shown that PS2Tg2576 mice, ages 4–5 months, exhibited cognitive impairments during the probe trial of a Morris water maze (MWM) task, indicating that PS2 mutation accelerated spatial learning impairment associated with A accumulation [28]. The current results demonstrating associative cognitive impairment at 4 months are temporally consistent with previous results showing spatial learning impairment. Moreover, using a different line of APP/PS2 double-transgenic mice (PS2APP mice)—generated by a different promoter from that
used for the PS2Tg2576 mice in this study—one study demonstrated that the onset of A plaque formation starts at age 6 months and demonstrated deterioration in cognitive abilities on the MWM or passive avoidance test from age 8 months [25]. Therefore, compared to many other mouse models of AD, PS2Tg2576 mice consistently develop the earliest onset and most rapidly progressive AD-like pathology and cognitive dysfunction. Furthermore, the present study indicates that cognitive impairment in PS2Tg2576 mice could be detectable as early as 4 months of age. The longtrace eyeblink conditioning task, using PS2Tg2576 mice, could be a suitable system for studying the molecular basis of cognitive dysfunction in AD and for screening new drug/therapy candidates for AD.
5. Conclusion These findings demonstrate age-dependent cognitive impairment in long trace eyeblink conditioning in PS2Tg2576 mice, which starts as early as 4 months of age. In contrast, at age 4 months, Tg2576 mice exhibited normal performance in trace conditioning. Taken together, these results suggest that PS2 mutation accelerates the onset of cognitive impairment in associative trace eyeblink conditioning in APP transgenic mice.
Acknowledgments This study was supported by Grants-in-Aid for Scientific Research (grant nos. 20790084 and 24590133 to Y. Kishimoto), the Takeda Science Foundation (to Y. Kishimoto), and by the Smoking Research Foundation (to Y. Kirino). We thank Ms. Junko Nakayama for technical assistance and Ms. Hisako Fukuoka for animal care.
References [1] D.R. Borchelt, T. Ratovitski, J. van Lare, M.K. Lee, V. Gonzales, N.A. Jenkins, N.G. Copeland, D.L. Price, S.S. Sisodia, Accelerated amyloid deposition in the brains of transgenic mice coexpressing mutant presenilin 1 and amyloid precursor proteins, Neuron 19 (1997) 939–945. [2] G.A. Carlesimo, M. Oscar-Berman, Memory deficits in Alzheimer’s patients: a comprehensive review, Neuropsychol. Rev. 3 (1992) 119–169. [3] M. Ewers, D.G. Morgan, M.N. Gordon, D.S. Woodruff-Pak, Associative and motor learning in 12-month-old transgenic APP + PS1 mice, Neurobiol. Aging 27 (2006) 1118–1128. [4] P. St. George-Hyslop, P.E. Fraser, Assembly of the presenilin ␥-/-secretase complex, J. Neurochem. 120 (2012) 84–88. [5] C.A. Gold, A.E. Budson, Memory loss in Alzheimer’s disease: implications for development of therapeutics, Expert Rev. Neurother. 8 (2008) 1879–1891. [6] M.N. Gordon, D.L. King, D.M. Diamond, P.T. Jantzen, K.V. Boyett, C.E. Hope, J.M. Hatcher, G. DiCarlo, W.P. Gottschall, D. Morgan, G.W. Arendash, Correlation between cognitive deficits and A deposits in transgenic APP + PS1 mice, Neurobiol. Aging 22 (2001) 377–385. ˜ J.M. Delgado-Garcia, Aged wild-type [7] A. Gruart, J.C. López-Ramos, M.D. Munoz, and APP, PS1, and APP + PS1 mice present similar deficits in associative learning and synaptic plasticity independent of amyloid load, Neurobiol. Dis. 30 (2008) 439–450. [8] C. Haass, D.J. Selkoe, Soluble protein oligomers in neurodegeneration: lessons from the Alzheimer’s amyloid -peptide, Nat. Rev. Mol. Cell Biol. 8 (2007) 101–112. [9] J. Hardy, The Alzheimer family of diseases: many etiologies, one pathogenesis? Proc. Natl. Acad. Sci. U.S.A. 94 (1997) 2095–2097. [10] J. Hardy, D. Allsop, Amyloid deposition as the central event in the aetiology of Alzheimer’s disease, Trends Pharmacol. Sci. 12 (1991) 383–388. [11] J. Hardy, D.J. Selkoe, The amyloid hypothesis of Alzheimer’s disease: progress and problems on the road to therapeutics, Science 297 (2002) 353–356. [12] A. Herreman, L. Serneels, W. Annaert, D. Collen, L. Schoonjans, B. De Strooper, Total inactivation of ␥-secretase activity in presenilin-deficient embryonic stem cells, Nat. Cell Biol. 2 (2000) 461–462. [13] K. Hsiao, P. Chapman, S. Nilsen, C. Eckman, Y. Harigaya, S. Younkin, F. Yang, G. Cole, Correlative memory deficits, A elevation, and amyloid plaques in transgenic mice, Science 274 (1996) 99–102. [14] W.T. Kimberly, W. Xia, T. Rahmati, M.S. Wolfe, D.J. Selkoe, The transmembrane aspartates in presenilin 1 and 2 are obligatory for ␥-secretase activity and amyloid -protein generation, J. Biol. Chem. 275 (2000) 3173–3178.
Y. Kishimoto, Y. Kirino / Neuroscience Letters 538 (2013) 15–19 [15] Y. Kishimoto, K. Nakazawa, S. Tonegawa, Y. Kirino, M. Kano, Hippocampal CA3 NMDA receptors are crucial for adaptive timing of trace eyeblink conditioned response, J. Neurosci. 26 (2006) 1562–1570. [16] Y. Kishimoto, I. Oku, A. Nishigawa, A. Nishimoto, Y. Kirino, Impaired long-trace eyeblink conditioning in a Tg2576 mouse model of Alzheimer’s disease, Neurosci. Lett. 506 (2012) 155–159. [17] Y. Kishimoto, M. Suzuki, S. Kawahara, Y. Kirino, Age-dependent impairment of delay and trace eyeblink conditioning in mice, NeuroReport 12 (2001) 3349–3352. [18] F.M. LaFerla, S. Oddo, Alzheimer’s disease: A, tau and synaptic dysfunction, Trends Mol. Med. 11 (2005) 170–176. [19] S. Lagadec, L. Rotureau, A. Hémar, N. Macrez, S. Delcasso, Y. Jeantet, Y.H. Cho, Early temporal short-term memory deficits in double transgenic APP/PS1 mice, Neurobiol. Aging 33 (2012), 203.e1-203.e11. ˜ J.M. [20] J.C. López-Ramos, M.T. Jurado-Parras, C. Sanfeliu, D. Acuna-Castroviejo, Delgado-García, Learning capabilities and CA1-prefrontal synaptic plasticity in a mice model of accelerated senescence, Neurobiol. Aging 33 (2012) 627.e13–627.e26. [21] J.R. Moyer Jr., R.A. Deyo, J.F. Disterhoft, Hippocampectomy disrupts trace eyeblink conditioning in rabbits, Behav. Neurosci. 104 (1990) 243–252. [22] F. Oyama, N. Sawamura, K. Kobayashi, M. Morishima-Kawashima, T. Kuramochi, M. Ito, T. Tomita, K. Maruyama, T.C. Saido, T. Iwatsubo, A. Capell, J. Walter, J. Grünberg, Y. Ueyama, C. Haass, Y. Ihara, Mutant presenilin 2 transgenic mouse: effect on an age-dependent increase of amyloid -protein 42 in the brain, J. Neurochem. 71 (1998) 313–322. [23] D.L. Price, S.S. Sisodia, Mutant genes in familial Alzheimer’s disease and transgenic models, Ann. Rev. Neurosci. 21 (1998) 479–505. [24] J. Puoliväli, J. Wang, T. Heikkinen, M. Heikkilä, T. Tapiola, T. van Groen, H. Tanila, Hippocampal A 42 levels correlate with spatial memory deficit in APP and PS1 double transgenic mice, Neurobiol. Dis. 9 (2002) 339–347. [25] J.G. Richards, G.A. Higgins, A.M. Ouagazzal, L. Ozmen, J.N. Kew, B. Bohrmann, P. Malherbe, M. Brockhaus, H. Loetscher, C. Czech, G. Huber, H. Bluethmann, H. Jacobsen, J.A. Kemp, PS2APP transgenic mice, coexpressing hPS2mut and hAPPswe, show age-related cognitive deficits associated with discrete brain amyloid deposition and inflammation, J. Neurosci. 23 (2003) 8989–9003.
19
[26] N. Sawamura, M. Morishima-Kawashima, H. Waki, K. Kobayashi, T. Kuramochi, M.P. Frosch, K. Ding, M. Ito, T.W. Kim, R.E. Tanzi, F. Oyama, T. Tabira, S. Ando, Y. Ihara, Mutant presenilin 2 transgenic mice. A large increase in the levels of A 42 is presumably associated with the low density membrane domain that contains decreased levels of glycerophospholipids and sphingomyelin, J. Biol. Chem. 275 (2000) 27901–27908. [27] R.F. Thompson, J.J. Kim, Memory systems in the brain and localization of a memory, Proc. Natl. Acad. Sci. U.S.A. 93 (1996) 13438–13444. [28] T. Toda, Y. Noda, G. Ito, M. Maeda, T. Shimizu, Presenilin-2 mutation causes early amyloid accumulation and memory impairment in a transgenic mouse model of Alzheimer’s disease, J. Biomed. Biotechnol. 2011 (2011) 617974. [29] J. Walter, C. Kaether, H. Steiner, C. Haass, The cell biology of Alzheimer’s disease: uncovering the secrets of secretases, Curr. Opin. Neurobiol. 11 (2001) 585–590. [30] J.M. Wang, C. Singh, L. Liu, R.W. Irwin, S. Chen, E.J. Chung, R.F. Thompson, R.D. Brinton, Allopregnanolone reverses neurogenic and cognitive deficits in mouse model of Alzheimer’s disease, Proc. Natl. Acad. Sci. U.S.A. 107 (2010) 6498–6503. [31] C. Weiss, P.N. Venkatasubramanian, A.S. Aguado, J.M. Power, B.C. Tom, L. Li, K.S. Chen, J.F. Disterhoft, A.M. Wyrwicz, Impaired eyeblink conditioning and decreased hippocampal volume in PDAPP V717F mice, Neurobiol. Dis. 11 (2002) 425–433. [32] M.A. Westerman, D. Cooper-Blacketer, A. Mariash, L. Kotilinek, T. Kawarabayashi, L.H. Younkin, G.A. Carlson, S.G. Younkin, K.H. Ashe, The relationship between A and memory in the Tg2576 mouse model of Alzheimer’s disease, J. Neurosci. 22 (2002) 1858–1867. [33] D.S. Woodruff-Pak, M. Papka, Alzheimer’s disease and eyeblink conditioning: 750 ms trace vs. 400 ms delay paradigm, Neurobiol. Aging 17 (1996) 397–404. [34] D.S. Woodruff-Pak, J.E. Steinmetz, Eyeblink Classical Conditioning, Volume 1: Applications in Humans, Kluwer Academic Publishers, Boston, MA, 2000. [35] D.S. Woodruff-Pak, J.E. Steinmetz, Eyeblink Classical Conditioning, Volume 2: Animal Models, Kluwer Academic Publishers, Boston, MA, 2000. [36] D.S. Woodruff-Pak, Eyeblink classical conditioning differentiates normal aging from Alzheimer’s disease, Integr. Physiol. Behav. Sci. 36 (2001) 87–108.