Liraglutide protects against amyloid-β protein-induced impairment of spatial learning and memory in rats

Neurobiology of Aging 34 (2013) 576 –588 www.elsevier.com/locate/neuaging

Liraglutide protects against amyloid-␤ protein-induced impairment of spatial learning and memory in rats Wei-Na Hana, Christian Hölscherb, Li Yuana, Wei Yanga, Xiao-Hui Wanga, Mei-Na Wua, Jin-Shun Qia,* a

Department of Physiology, Key Laboratory of Cellular Physiology, Ministry of Education, Shanxi Medical University, Taiyuan, PR China b School of Biomedical Sciences, University of Ulster, Coleraine, UK Received 24 October 2011; received in revised form 24 March 2012; accepted 17 April 2012

Abstract Type 2 diabetes mellitus is a risk factor of Alzheimer’s disease (AD), most likely linked to an impairment of insulin signaling in the brain. Liraglutide, a novel long-lasting glucagon-like peptide 1 (GLP-1) analog, facilitates insulin signaling and shows neuroprotective properties. In the present study, we analyzed the effects of liraglutide on the impairment of learning and memory formation induced by amyloid-␤ protein (A␤), and the probable underlying electrophysiological and molecular mechanisms. We found that (1) bilateral intrahippocampal injection of A␤25–35 resulted in a significant decline of spatial learning and memory of rats in water maze tests, together with a serious depression of in vivo hippocampal late-phase long-term potentiation (L-LTP) in CA1 region of rats; (2) pretreatment with liraglutide effectively and dose-dependently protected against the A␤25–35-induced impairment of spatial memory and deficit of L-LTP; (3) liraglutide injection also activated cAMP signal pathway in the brain, with a nearly doubled increase in the cAMP contents compared with control. These results strongly suggest that upregulation of GLP-1 signaling in the brain, such as application of liraglutide, may be a novel and promising strategy to ameliorate the learning and memory impairment seen in AD. © 2013 Elsevier Inc. All rights reserved. Keywords: Liraglutide; Amyloid-␤ protein; Spatial memory; Late-phase long-term potentiation; cAMP; GLP-l; Alzheimer disease

1. Introduction Alzheimer’s disease (AD), with about 35.6 million patients worldwide (Wimo and Prince, 2010), is a progressive and irreversible neurodegenerative disorder associated with memory loss, cognitive deterioration, and weakness of intellectual capacity (Selkoe, 2001). One critical event in the pathogenesis of AD is abundant deposits of senile plaques composed of amyloid-␤ protein (A␤). The amyloid deposits accumulate first in isocortical areas, followed by limbic and allocortical structures including entorhinal cortex and hippocampus (Arnold et al., 1991; Thal et al., 2002). The neurotoxicity of A␤ peptides has been widely reported * Corresponding author at: Department of Physiology, Key Laboratory of Cellular Physiology, Ministry of Education, Shanxi Medical University, Taiyuan, PR China. Fax: ⫹86 351 4135091. E-mail address: [email protected] (J.-S. Qi). 0197-4580/$ – see front matter © 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.neurobiolaging.2012.04.009

(Chen et al., 2000; Deshpande et al., 2006), and the A␤ hypothesis of AD has been widely accepted. It has been reported that prolonged infusion of synthetic A␤ into the brain can cause learning and memory deficits in rats (Nitta et al., 1997), including impairment of working memory and place learning in eight-arm radial maze (Stepanichev et al., 2005), Y-maze, and water maze (Maurice et al., 1996). In our previous experiments, we also found that not only the full length of A␤ molecule such as A␤1– 42 and A␤1– 40 but also the A␤ fragments including A␤25–35 and A␤31–35 could significantly impair the spatial memory (Pan et al., 2010) and hippocampal synaptic plasticity (Li et al., 2011; Wang et al., 2010) in normal rats. However, effective neuroprotective measures against A␤ neurotoxicity are still lacking. It has been reported recently that AD and type 2 diabetes mellitus (T2DM), another degenerative disease, share several common clinical and pathological characteristics. On

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the one hand, T2DM is a risk factor for developing AD in the elderly (Craft, 2007; Hölscher, 2005; Hoyer, 2004; Luchsinger et al., 2004; Perry et al., 2007), with a 1.5–2.5fold increased risk of dementia (Strachan et al., 2011). On the other hand, AD is associated with the peripheral and central insulin abnormalities, including the desensitization of insulin receptors in the brains (Craft, 2007; Hoyer, 2004; Li and Hölscher, 2007). Because the impairment of insulin system in the brain is closely related to the development of neurodegenerative disorders (Craft, 2007; Hölscher, 2005; Hoyer, 2004), it might be a well promising strategy to normalize insulin signaling in the brain for the prevention and treatment of AD. Interestingly, glucagon-like peptide 1 (GLP-1), an incretin hormone, has been reported to cross the blood– brain barrier (BBB) and facilitate insulin signaling (Gengler et al., 2012; Kastin et al., 2002); exenatide, a GLP-1 receptor agonist, could enhance neuronal progenitor proliferation in the brain of diabetic mouse (Hamilton et al., 2011) and reduce endogenous levels of A␤ in transgenic AD mice (Li et al., 2010). However, it is not completely understood what role the replacement of neurons plays in the brain, and whether these regenerative neurons are actually functionally integrated into the neuronal networks and whether the reduced A␤ level in the brain is related to the behavioral improvement in AD. Another issue is the shorter half-life of GLP-1, only several minutes in blood plasma (Deacon et al., 1995), which seriously limits its application in clinical practice. Recently, liraglutide, a novel long-lasting GLP-1 analog, has been brought in the market as therapeutics for diabetes (Holscher, 2010). First studies show that liraglutide has neuroprotective effects in an amyloid precursor protein/ presenilin 1 (APP/PS1) mouse model of AD. Plaque formation and A␤ synthesis were reduced in these mice, while memory and synaptic plasticity were preserved by the drug (McClean et al., 2011). Therefore, it is of interest to further investigate whether the A␤-induced dysfunction in central nervous system can be effectively alleviated by liraglutide. In this study, we firstly observed the effects of bilateral intrahippocampal injection of liraglutide on spatial learning and memory of rats in Morris water maze (MWM) test. Further, we investigated its possible electrophysiological mechanism by recording in vivo late-phase long-term potentiation (L-LTP) in hippocampal CA1 region of rats. The liraglutide-induced alteration of cAMP level in the brain was also examined.

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every experiment, the animals were killed with an overdose of urethane. Considering the much more solubility and the similar neurotoxicity, A␤25–35 was used in the present study. A␤25–35 was ordered from Sigma (St. Louis, MO, USA) and dissolved in saline (1 nmol/1 ␮L). Liraglutide was provided by Dr Christian Hölscher’s laboratory at Ulster University, UK, and was dissolved in saline (5 nmol/1 ␮L). All peptides were stored in dry form and dissolved before the experiments. Control rats received only saline. 2.2. Intrahippocampal injection Intrahippocampal injection was performed as previously described (Ryu and McLarnon, 2006, 2008). In brief, SD rats were anesthetized (chloral hydrate, 0.3 g/kg, i.p.) and placed in a stereotaxic apparatus (Narishige, Tokyo, Japan). Dissolved peptide (0.05–5 nmol liraglutide and/or 4 nmol A␤25–35) solution (4 ␮L) was injected into the bilateral hippocampus at the following coordinates: anteriorposterior (AP): ⫺3.0 mm, mediallateral (ML): ⫾2.2 mm, dorsoventral (DV): ⫺3.0 mm, from bregma (Paxinos and Watson, 2007), with an injection rate of 0.2 ␮L/min under the control of micropumps (KD Scientific, Inc, KDS310 Plus, USA). In coapplication group, liraglutide was injected at least 30 minutes before A␤25–35 injection. In more detail, 2 ␮L liraglutide was first injected into the hippocampus at a rate of 0.2 ␮L/min. After 5-minute retention, the syringe was removed, and 15 minutes later, A␤25–35 solution was applied at the same injection rate and the same retention time. Two weeks later, Morris water maze test and in vivo hippocampal L-LTP recording were performed. 2.3. Morris water maze task The rats were kept in a controlled room temperature (20 –24 °C) and humidity (60%– 80%) for 3 days adaptation, and then submitted to a spatial reference memory version of the water maze as described (Morris, 1984; Prediger et al., 2007; Terry, 2009). The water maze was mainly composed of a stainless steel pool and a platform in the pool for escape. The pool is 150 cm in diameter and 50-cm high, which was filled with tap water at 23 ⫾ 2 °C to avoid hypothermia. The escape platform (diameter, 14 cm; height, 29 cm) was placed at a fixed position in the center of one quadrant, 35 cm from the perimeter, and was hidden 1 cm beneath the water surface. Several landmarks were fixed to the walls of the water maze room. 2.3.1. Acquisition phase

2. Methods 2.1. Animals and drugs Adult male Sprague–Dawley (SD) rats (230 –250 g) were used in the behavioral and electrophysiological experiments. All animals were obtained from the Research Animal Center of Shanxi Medical University, with approval of the Shanxi Animal Research Ethics Committee. At the end of

The acquisition trial phase consisted of five training days (days 1–5) and four trials per day with a 20-second intertrial interval. Four points equally spaced along the circumference of the pool served as the starting position, which was randomly chosen across the four trials each day. If an animal did not reach the platform within 120 seconds, it was guided to the platform, where it had to remain for 30 seconds before being returned to its home cage. Rats were

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kept dry between trials, in a plastic holding cage filled with paper towels. The path length and escape latencies were recorded by a behavior software system (Ethovision 3.0, Noldus Information Technology, Wageningen, the Netherlands).

After the probe test, visual, motor, and motivation skills were also tested with a visible platform located opposite the original position within the pool. A cover rendered highly visible was attached to the platform to raise the surface above the water level (approximately 1.5 cm).

The fEPSPs were recorded for at least 3 hours after HFS. The averaged value of fEPSPs amplitude during 30 minutes of baseline recording was taken as 100%, and all recorded fEPSPs were normalized to this baseline value. A ⱖ 30% increase of fEPSP amplitude from the baseline was considered as a significant LTP. A biological signal processing system (Chengdu Instruments Ltd, PR China) was used to record all events. The system triggered an electronic stimulator (SEN-3301, Japan) to generate constant current pulse stimulation through a stimulus isolation unit (ss-102J, Japan). All evoked responses were stored on computer for further off-line analysis. The effects of A␤25–35 and liraglutide on the paired-pulse facilitation (PPF), a short-term enhancement of synaptic transmission in the CA1 region, were also examined. Two paired test stimuli with an interval of 50 ms were given before HFS to induce two paired fEPSPs. The change in PPF ratio, calculated by dividing the amplitude of the second fEPSP by the amplitude of the first fEPSP, is viewed to be associated with the changes in neurotransmitter release from presynaptic terminals.

2.4. In vivo hippocampal L-LTP recording

2.5. cAMP assay

Because hippocampal LTP has been linked to MWM performance (Bliss and Collingridge, 1993), an in vivo electrophysiological recording of L-LTP in hippocampal CA1 region of rats was performed after finishing the behavioral study. The rats were anesthetized with urethane (ethyl carbamate, Sigma-Aldrich, UK, 1.5 g/kg, i.p.) and placed in a stereotaxic apparatus (Narishige, Japan) for surgery and L-LTP recording. The body temperature was monitored throughout the experiment, and a heating pad was used to maintain the temperature of the animals at 37 ⫾ 0.5 °C. Small holes were drilled on the right side of the skull for inserting the reference, stimulating, and recording electrodes. A pair of parallel stimulating/recording electrodes (Sequim, WA, USA) was inserted in the hippocampus. The tip of the mono-polar recording electrode was positioned at the stratum radiatum in the CA1 region (3.4 mm posterior to bregma and 2.5 mm lateral to the midline), and the end of bipolar stimulating electrode was located at the Schaffer collateral/commissural pathway (4.2 mm posterior to bregma and 3.8 mm lateral to the midline). Field excitatory postsynaptic potentials (fEPSPs) were recorded from stratum radiatum in CA1 region of the right hippocampal hemisphere in response to stimulation of the Schaffer collateral/ commissural pathway. The electrodes were slowly lowered through the cortex and upper layers of the hippocampus into CA1 region until a negative deflecting fEPSP with a latency of approximately 10 ms was observed. Baseline fEPSPs were elicited by test stimuli at an interval of 30 seconds. L-LTP was induced by three sets of high-frequency stimulation (HFS) with 5 minutes of interval. One set of HFS contains three trains of 20 pulses with 5 ms of interstimulus interval (200 Hz), and the intertrain interval is 30 seconds.

2.5.1. Animals Each animal received saline (0.9% [w/v] NaCl) or liraglutide (25 nmol/kg bw) by i.p. injection (n ⫽ 5 per group). At 30 minutes post treatment, the mice were painlessly killed with an overdose of the barbiturate. Subsequently, animals were perfused with phosphate buffered saline (PBS) to clear the circulation of all blood. Each brain was weighed, placed in a Bijou tube, snap frozen in liquid nitrogen, and stored at ⫺80 °C until further processing and analysis.

2.3.2. Probe trial On the day after finishing the acquisition task (day 6), a probe trial was performed to assess the spatial memory (after a 24-hour delay). The platform was removed from the pool, and animals were allowed to swim freely for 120 seconds. Spatial acuity was expressed as the amount of time spent in the exact area where the escape platform was located. 2.3.3. Visible platform test

2.5.2. cAMP analysis of brain samples Each brain was treated using 0.1 M HCl for cAMP extraction. In all, 10 mL of 0.1 M HCl per g of tissue was added. Samples were sonicated and then centrifuged at 10,000 rpm for 15 minutes at 4 °C. The supernatant was poured off and used directly for measurement by enzymelinked immunosorbent assay (ELISA). Dilutions were made using the 0.1 M HCl provided in the kit. Brain cAMP levels were measured using the direct cAMP ELISA kit (Enzo Life Sciences), according to the manufacturer’s instructions. 2.6. Statistics All values were displayed as mean ⫾ standard error (SEM). The SPSS 13.0 and Sigmaplot 10.0 statistical packages were used for statistical analyses. In Morris water maze test, the latencies to reach the hidden platform and the distances swam in four trials for each rat on each day were averaged. A two-repeated measure analysis of variance (ANOVA) was used with day as the repeated measure and latency or distance swam as the dependent variables. For probe trials, data from two trials for each rat were averaged, and the

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Fig. 1. Intrahippocampal injection of A␤25–35 impaired spatial learning and memory of rats. (A and B) The average escape latencies and distances of rats in searching for the hidden platform in MWM, with a significant increase in A␤25–35 alone group at training days 2–5 compared with control group. (C and D) Histograms showing the percentage of time and distance for rats spent in the target quadrant in probe trial. Each point represents the mean ⫾ SEM. ** p ⬍ 0.01 compared with the control group. (E) Representative swimming tracks of rats searching for the underwater platform at 4th training day. (F) Typical tracks of rats in probe trails.

means between groups were compared via a one-way ANOVA. For the visible platform test, the second trial for each rat is recorded and compared between groups via one-way ANOVA. A two-repeated measures ANOVA was used for analysis of the electrophysiological experimental data. Statistical significance level was set at p ⬍ 0.05. 3. Results 3.1. Bilateral intrahippocampal injection of A␤25–35 impairs spatial learning and memory of rats As the evidence of learning and memory function of rats, the hidden platform tests and probe trials were performed to examine the ability of rats to acquire and retrieve spatial information, respectively. As expected, bilateral intrahippocampal injection of A␤25–35 (4 nmol) seriously impaired the performance of rats in hidden platform tests and probe trials. As shown in Fig. 1A, A␤25–35 did not change the escape latency in hidden platform tests on the 1st training day. However, the average escape latencies on training days

2–5 were significantly increased in A␤25–35 group (n ⫽ 16), being 47.17 ⫾ 3.99 seconds (p ⬍ 0.01), 31.10 ⫾ 3.27 seconds (p ⬍ 0.01), 18.81 ⫾ 0.89 seconds (p ⬍ 0.01), and 17.62 ⫾ 1.22 seconds (p ⬍ 0.01), respectively, larger than the values of 32.63 ⫾ 5.73 seconds, 17.87 ⫾ 1.22 seconds, 14.90 ⫾ 0.79 seconds, and 12.30 ⫾ 0.20 seconds in control group (n ⫽ 10). Similarly, as shown in Fig. 1B, the average escape distance in the hidden platform tests in A␤25–35 group increased to 726.33 ⫾ 73.22 cm (p ⬍ 0.05), 480.07 ⫾ 61.44 cm (p ⬍ 0.01), 361.24 ⫾ 48.91 cm (p ⬍ 0.01), and 262.93 ⫾ 28.85 cm (p ⬍ 0.01) on training days 2–5, respectively, significantly larger than the values of 507.41 ⫾ 72.05 cm, 223.42 ⫾ 20.67 cm, 169.00 ⫾ 9.59 cm, and 150.69 ⫾ 14.02 cm in control group. These results indicate that A␤25–35 impaired the spatial learning of rats. In probe trials, the percentages of time (Fig. 1C) and distance (Fig. 1D) elapsed or swam in target quadrant were 44.79 ⫾ 1.61% and 43.10 ⫾ 2.08%, respectively, in control group. After injection of A␤25–35, the percentages of time and distance significantly decreased compared with the control

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group (p ⬍ 0.01), with the values of 24.85 ⫾ 0.89% and 23.30 ⫾ 2.06%, respectively. The representative swimming traces of rats at the forth training day were shown in Fig. 1E, and the typical original swimming traces of rats in probe trials were shown in Fig. 1F. 3.2. Liraglutide alone did not affect normal cognitive behavior but dose-dependently prevented against A␤25–35-induced impairment of spatial learning and memory To investigate the neuroprotective function of liraglutide against A␤25–35-induced cognitive impairment, the effects of liraglutide alone on the spatial learning and memory were first observed. As shown in Fig. 2A and 2B, intrahippocampal administration of 5 nmol liraglutide (n ⫽ 15) alone did not affect the average escape latencies and distance in searching for the hidden platform, when compared with those of control group (p ⬎ 0.05). The mean escape latencies in 5 nmol liraglutide group were 87.41 ⫾ 1.52 seconds, 30.01 ⫾ 1.52 seconds, 16.98 ⫾ 0.76 seconds, 15.43 ⫾ 0.76 seconds, and 13.92 ⫾ 0.30 seconds, and the mean escape distances were 1342.35 ⫾ 159.34 cm, 419.64 ⫾ 35.44 cm, 233.42 ⫾ 18.95 cm, 198.97 ⫾ 15.67 cm, and 173.97 ⫾ 11.95 cm at 1–5 training days, respectively. Furthermore, we investigated the effects of pretreatment with different concentrations (0.05–5 nmol) of liraglutide on the A␤25–35-induced impairment of spatial learning of rats. We found that liraglutide could obviously and dose-dependently protect against the A␤25–35induced deficits in spatial cognition of rats. In hidden platform tests, both the escape latency and escape distance in coapplication of liraglutide and A␤25–35 groups were improved. As shown in Fig. 2C–F, 0.05 nmol liraglutide (n ⫽ 10) did not affect A␤25–35-induced impairment of spatial learning, with similar escape latency and similar escape distance in hidden platform test as compared with the A␤25–35 alone group (p ⬎ 0.05). However, pretreatment with 0.5 nmol or 5 nmol liraglutide significantly reversed the spatial learning impairments induced by A␤25–35 (p ⬍ 0.05) on most training days. For example, in the 0.5 nmol liraglutide plus A␤25–35 group (n ⫽ 10), the average escape latency (Fig. 2C and 2D) decreased to 40.08 ⫾ 3.37 seconds (p ⬎ 0.05), 19.31 ⫾ 1.98 seconds (p ⬍ 0.05), 15.30 ⫾ 0.83 seconds (p ⬍ 0.05), and 14.50 ⫾ 0.36 seconds (p ⬍ 0.05); the mean escape distances (Fig. 2E and 2F) decreased to 640.65 ⫾ 86.70 cm (p ⬎ 0.05), 327.40 ⫾ 27.33 cm (p ⬍ 0.05), 229.76 ⫾ 11.14 cm (p ⬍ 0.05), and 200.47 ⫾ 8.64 cm (p ⬍ 0.05) at training days 2–5, respectively. In the 5 nmol liraglutide plus A␤25–35 group (n ⫽ 10), the average escape latency and the average escape distance further decreased. As shown in Fig. 2C–F, the mean escape latencies were 35.34 ⫾ 2.51 seconds (p ⬍ 0.05), 18.03 ⫾ 1.28 seconds (p ⬍ 0.015), 15.88 ⫾ 0.79 seconds (p ⬍

0.05), and 13.32 ⫾ 0.31 seconds (p ⬍ 0.05), and the mean escape distances were 604.37 ⫾ 77.83 cm (p ⬎ 0.05), 244.97 ⫾ 23.22 cm (p ⬍ 0.05), 195.98 ⫾ 12.69 cm (p ⬍ 0.05), and 188.87 ⫾ 12.34 cm (p ⬍ 0.05) at 2–5 training days, respectively, significantly shorter than those of A␤ alone group. Compared with the group injected with the lower concentration (0.05 nmol) of liraglutide, higher concentrations (0.5 nmol and 5 nmol) of liraglutide show a faster decrease in average escape latency and average escape distance in coapplication groups. Figure 2G shows typical swimming tracks of rats pretreated with liraglutide, A␤25–35, and different concentrations of liraglutide plus A␤25–35 in searching for the hidden platform on the 4th training day. With the increase of concentration of liraglutide, the distance swam in searching for the hidden platform became shorter and shorter in the liraglutide plus A␤25–35 groups. In the probe trials (Fig. 3), the platform was removed from the pool, and rats were allowed to swim freely for 120 seconds. We found that 5 nmol liraglutide alone did not affect the memory behavior, with similar degree of swimming preference in target quadrant as control group, but 4 nmol A␤25–35 significantly decreased the percentage of time elapsed and the distance swum in the target quadrant. Interestingly, pretreatment with different concentrations of liraglutide dose-dependently prevented the A␤25–35-induced memory deficit. As shown in Fig. 3A and 3B, with the increase of liraglutide concentration in the coapplication of liraglutide and A␤25–35 groups, the time (Fig. 3A) and the distance (Fig. 3B) for rats spent in the target quadrant significantly increased. In 0.5 nmol and 5 nmol liraglutide plus A␤25–35 groups, the time percentages in the target quadrant increased to 38.01 ⫾ 1.67% and 43.22 ⫾ 0.93%, respectively, significantly larger than the value of 24.85 ⫾ 0.89% in A␤25–35 alone group (p ⬍ 0.05); the distance swum in the target quadrant increased to 37.76 ⫾ 1.56% and 40.94 ⫾ 1.61%, respectively, also significantly larger than 23.30 ⫾ 2.06% in A␤25–35 alone group (p ⬍ 0.05). Figure 3C showed typical swimming trajectory charts of rats, with greater percentage of distance in the target quadrant in higher concentrations of liraglutide plus A␤25–35 groups. The results aforementioned indicate that liraglutide could dose-dependently protect against A␤25–35-induced spatial learning and memory impairment. 3.3. Both liraglutide and A␤25–35 did not affect the vision and motor ability of rats To exclude the influence of vision and motor ability of the rats on the experimental results, we examined the escape latency of rats by performing a visible platform test after finishing the probe trials and compared the average swimming speeds in probe trials. The escape latencies were 14.04 ⫾ 0.48 seconds, 14.79 ⫾ 0.38 seconds, 14.30 ⫾ 0.43 seconds, 14.06 ⫾ 0.31 seconds, 14.89 ⫾ 0.50 seconds, and

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Fig. 2. Liraglutide alone did not affect normal spatial learning but protected against the A␤25–35-induced impairment in spatial learning in a dose-dependent manner. (A and B) Plots of latency (A) and distance (B) of rats to find the hidden platform over five consecutive training days, without significant difference between liraglutide group (n ⫽ 15) and control group (n ⫽ 10). (C–F) Plots and histograms showing the latencies (C and D) and distances (E and F) of rats to find the hidden platform over five consecutive training days in A␤25–35 alone group (n ⫽ 16) and coapplication of different dosages of liraglutide (0.05 nmol, 0.5 nmol, 5 nmol, n ⫽ 10 per group) plus A␤25–35 group. Each point represents the mean ⫾ SEM of five trials per day. The a, b, and c represent p ⬍ 0.05 compared with the A␤25–35 alone group, 0.05 nmol liraglutide plus A␤ group, and 0.5 nmol liraglutide plus A␤ group, respectively. (G) Typical swimming trajectories of rats pretreated with A␤25–35 and liraglutide separately or jointly in hidden platform tests on the 4th training day.

14.84 ⫾ 0.51 seconds (p ⬎ 0.05), and the swim speeds were 18.11 ⫾ 0.83 cm/s, 18.19 ⫾ 0.78 cm/s, 18.26 ⫾ 0.45 cm/s, 19.11 ⫾ 0.59 cm/s, 18.86 ⫾ 0.55 cm/s, and 18.56 ⫾ 0.59 cm/s (p ⬎ 0.05) in control group, A␤25–35 group, liraglutide

group, and different concentrations of liraglutide plus A␤ groups, respectively. The results indicate that both liraglutide and A␤25–35 used in the present study did not affect the vision and motor ability of rats.

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Fig. 3. Liraglutide dose-dependently prevented A␤25–35-induced deficit in spatial memory as shown in the probe trails. (A and B) Histograms showing the percentages of total time (A) and total distance (B) of rats spent in the previous target quadrant. Each column represents the mean ⫾ SEM of two trails per rat in each group. The a, b, and c represent p ⬍ 0.05 compared with the A␤25–35 alone group, 0.05 nmol liraglutide plus A␤ group, and 0.5 nmol liraglutide plus A␤ group, respectively. (C) Representative swimming tracks of rats in probe trials.

3.4. Liraglutide partly and dose-dependently prevented the A␤25–35-induced depression of hippocampal L-LTP In view of the close relationship between animal spatial memory and hippocampal synaptic plasticity, we further observed the effects of A␤25–35 and liraglutide on hippocampal L-LTP. As shown in Fig. 4A, immediately after delivering three sets of HFS, the amplitude of fEPSPs in control group (n ⫽ 6) increased abruptly to 193.88 ⫾ 7.99% from the initial control value set as 100%, remaining at 153% 3 hours after HFSs, indicating a successful induction of L-LTP in this in vivo experimental condition. Compared with control, A␤25–35 (4 nmol, n ⫽ 6) injection induced a significant suppression of L-LTP. The average standardized fEPSP amplitude in A␤25–35 group decreased to 133.75 ⫾ 6.46% (p ⬍ 0.01), 118.70 ⫾ 4.05% (p ⬍ 0.01), and 113.50 ⫾ 3.19% (p ⬍ 0.01) from 162.28 ⫾ 4.94%, 159.24 ⫾ 1.85%, and 153.41 ⫾ 2.79% in control group at 1 hour, 2 hours, and 3 hours after HFSs, respectively. Then, the effect of liraglutide alone on the L-LTP was observed. We did not find any significant change in fEPSP amplitude at 1 hour, 2 hours, and 3 hours after HFSs in liraglutide group (5 nmol, n ⫽ 6) as compared with the control group. Bar graphs in Fig. 4B show the average fEPSP amplitudes at 1 hour, 2 hours, and 3 hours post-HFSs in the three groups.

Furthermore, we investigated the effects of liraglutide on the A␤25–35-induced impairment of L-LTP by coapplication of different concentrations of liraglutide (0.05 nmol, 0.5 nmol, and 5 nmol) plus A␤25–35 (4 nmol). As shown in Fig. 5A, 0.5 nmol and 5 nmol, but not 0.05 nmol, of liraglutide significantly prevented 4 nmol A␤25–35-induced suppression of L-LTP (n ⫽ 6, per group). Bar graphs in Fig. 5B show the values of L-LTP at 1 hour, 2 hours, and 3 hours post-HFSs in A␤25–35 alone and coapplication groups. In 0.05 nmol liraglutide plus A␤25–35 group, the percentage of fEPSP amplitude was 147.64 ⫾ 6.91% (p ⬎ 0.05), 127.11 ⫾ 3.24% (p ⬎ 0.05), and 120.50 ⫾ 4.29% (p ⬎ 0.05) at 1 hour, 2 hours, and 3 hours post-HFSs, respectively, with an increase but without significant difference compared with A␤25–35 alone group. In 0.5 nmol liraglutide plus A␤25–35 group, the average fEPSP amplitude increased to 153.07 ⫾ 6.77% (p ⬍ 0.05), 145.40 ⫾ 4.08% (p ⬍ 0.05), and 140.84 ⫾ 4.81% (p ⬍ 0.05) at the same time points. In particular, in 5 nmol liraglutide plus A␤25–35 group, the average fEPSP amplitudes further increased to 161.95 ⫾ 3.63%, 154.82 ⫾ 2.91%, and 152.49 ⫾ 4.60% at 1 hour, 2 hours, and 3 hours post-HFSs, respectively, not only significantly larger than the values in A␤25–35 alone group (p ⬍ 0.05) but also indistinguishable from the values in the control group (p ⬎ 0.05) at the same time points. The results indicate that higher concentra-

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Fig. 4. The effects of A␤25–35 and liraglutide on the in vivo L-LTP in hippocampal CA1 region. (A) Scatter plots showing that liraglutide alone did not affect, but A␤25–35 significantly suppressed the HFS-induced L-LTP. Insets: typical fEPSP traces recorded 15 min before and 3 h after HFSs in three groups, calibration bars: 10 ms and 1 mV. (B) Histograms showing the average fEPSPs amplitude in three groups at different time points before and after HFSs. Each column shows the mean ⫾ SEM of fEPSPs amplitude. ** p ⬍ 0.01 compared with control group at the same time.

tions (0.5 nmol and 5 nmol) of liraglutide could effectively prevent A␤25–35-induced hippocampal L-LTP impairment. 3.5. Both A␤25–35 and liraglutide did not affect hippocampal PPF To clarify whether the presynaptic mechanism was involved in the effects of A␤ and liraglutide on synaptic

plasticity, PPF in the hippocampal CA1 region was examined in all groups immediately before HFS. After paired pulses were applied to the Schaffer collaterals, the PPF in CA1 stratum radiatum always appeared with the second fEPSP, obviously larger than the first one. The PPF ratio values were 175.02 ⫾ 4.31%, 176.83 ⫾ 4.33%, 171.27 ⫾ 3.80%, 172.24 ⫾ 3.00%, 168.19 ⫾ 3.98%, 172.62 ⫾ 3.76%

Fig. 5. Pretreatment with liraglutide dose-dependently prevented the A␤25–35-induced depression of L-LTP. (A) A time course of fEPSPs before and after HFSs in control, A␤ alone, and A␤ plus liraglutide groups. The suppressive effect of A␤25–35 on L-LTP was partly, but significantly, blocked by the pretreatment of liraglutide in a dose-dependent manner. Insets: typical fEPSP traces recorded 15 min before and 3 h after HFSs in different groups, calibration bars: 10 ms and 1 mV. (B) Histograms showing the average fEPSPs amplitude in all groups at different time points before and after HFSs. Each column shows the mean ⫾ SEM of fEPSPs amplitude. The letters a, b, and c represent p ⬍ 0.05 compared with the A␤25–35 alone group, 0.05 nmol liraglutide plus A␤ group, and 0.5 nmol liraglutide plus A␤ group at the same time, respectively.

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in control, A␤25–35, liraglutide, and different concentrations of liraglutide plus A␤ groups, respectively, indicating that the PPF in the hippocampal CA1 region was not affected by A␤ and liraglutide (p ⬎ 0.05). 3.6. Liraglutide upregulated intracellular cAMP level After injecting liraglutide (i.p., 25 nmol/kg), cAMP levels in the brain were increased. The cAMP concentration in total brain tissue showed a significant increase (p ⬍ 0.01) at 30 minutes postinjection of liraglutide, from 4.6 ⫾ 0.7 pM in control (n ⫽ 5) to 7.9 ⫾ 0.9 pM in liraglutide group (n ⫽ 5). 4. Discussion A␤ is thought to be responsible for the deficit of learning and memory in AD. The neurotoxicity of A␤, including different A␤ fragments, has been widely reported (Ferreira et al., 2007; Hölscher, 2005; Ye et al., 2004). In previous experiments, we found that A␤1– 40, A␤25–35, and even a shorter fragment A␤31–35 induced apoptosis in cultured cortical neurons (Yan et al., 1999), enhanced intracellular Ca2⫹ loading by forming new cation-selective channels (Qi and Qiao, 2001a), suppressed potassium (Ik) channels and large conductance Ca2⫹-activated potassium (BK) channels in isolated hippocampal neurons (Qi et al., 2004; Qi and Qiao, 2001b), and also impaired hippocampal early-phase LTP (E-LTP) and L-LTP in vivo (Li et al., 2011; Wang et al., 2010; Ye et al., 2004; Zhang et al., 2006, 2009). However, the effective neuroprotective treatments against A␤ neurotoxicity are not available up to now. One of the main interests in the present study was focused on the central neuroprotective effects of liraglutide, a novel long-lasting GLP-1 analog. GLP-1 is an endogenous incretin hormone (Lovshin and Drucker, 2009) released by the L cells of the distal intestinal mucosa and contains 30 amino acids with 50% sequence homology to glucagon (Impey et al., 1996). Currently, the GLP-1 analog liraglutide and the GLP-1 receptor agonist exendin-4 have been approved for treatment of T2DM (Hölscher and Li, 2010; Lovshin and Drucker, 2009). Because GLP-1 and its analogs do not affect blood glucose level in normoglycemic people, GLP-1 analogs can be given to nondiabetic patients (Gallwitz, 2006; Vella et al., 2002). Interestingly and importantly, the distribution and the effects of GLP-1 in the brain have been reported recently. For example, GLP-1 and GLP-1 receptors (GLP-1R) were expressed in the brain, including the hippocampus, a brain area involved in memory formation (During et al., 2003; Hamilton and Hölscher, 2009). GLP-1, as a post-translational product of preproglucagon (Kieffer and Habener, 1999) and a growth factor in the brain, possessed neurotrophic property and protected neurons against glutamate-induced apoptosis (Perry et al., 2002b) and oxidative injury in cultured neuronal cells (Perry et al., 2007); overexpressing GLP-1R in the hippocampus increased neurite

growth and improved learning and memory (During et al., 2003), whereas GLP-1R knockout impaired the development of hippocampal LTP and spatial learning and memory (Abbas et al., 2009). However, natural GLP-1 can be rapidly degraded by the enzyme dipeptidyl peptidase IV (DPP IV), and its half-life is only 2–3 minutes in blood plasma (Deacon et al., 1995). Liraglutide, as a GLP-1 analog, has profound resistance to DPP IV and greater biological activity than natural GLP-1 (Holscher, 2010). Therefore, liraglutide, not natural GLP-1, was used in the present study. In our recent study (Gengler et al., 2012), we investigated the postexposure effects of GLP-1 in APP/PS1 mice. In the present study, the pre-exposure effects of liraglutide were observed to examine its protective effects against A␤-induced impairments in behavior and L-LTP. This pre-exposure protective effect of liraglutide might be of great significance in the prevention of T2DM developed to AD. We first confirmed in the study that bilateral intrahippocampal injection of A␤25–35 resulted in a significant impairment of spatial learning and memory in freely moving rats. Then, the neuroprotective effects of liraglutide were investigated. We found that the long-lasting GLP-1 analog significantly protected against the A␤25–35-induced impairment of learning and memory. Intrahippocampal injection of 0.5 nmol and 5 nmol, but not 0.05 nmol, liraglutide dose-dependently prevented the A␤25–35-induced deficits in spatial cognition of rats. The escape latency and the swimming distance percentage in 5 nmol liraglutide plus A␤25–35 group were similar to the values in normal control group. The result is also supported by McClean’s study (McClean et al., 2011), in which liraglutide administration induced a reduction of A␤ plaques and an increase in neurogenesis in a mouse model of AD, APPswe/PS1deltaE9. These results suggest that liraglutide could probably play an important role in the prevention or treatment of memory loss in AD patients. Clinical trials in AD patients are required to test this hypothesis. As learning and memory are thought to be encoded by modification of synaptic strength (Bliss and Collingridge, 1993), LTP, a persistent increase in synaptic efficacy after HFS, is widely considered one of the major cellular mechanisms underling learning and memory (Bliss and Collingridge, 1993; Cooke and Bliss, 2006). LTP is classically described as two distinct temporal phases: E-LTP and LLTP. E-LTP is usually induced by a set of HFS, results from the modification of pre-existing proteins, and can last for approximately 1 hour (Barria et al., 1997); whereas L-LTP is induced by multiple HFSs, requires synthesis of new mRNAs and proteins (Impey et al., 1996), and can last for at least 3 hours (Abraham et al., 1993). In the present study, we found that liraglutide alone did not affect the basic synaptic transmission and HFS-induced L-LTP in hippocampal CA1 region, but pretreatment with liraglutide could dose-dependently and significantly prevent A␤25–35induced impairment of L-LTP. The protective effect of

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Fig. 6. Liraglutide and A␤ differentially modulate cAMP/PKA pathway. Extracellular and intracellular neurotoxic A␤ induces cytoplasmic Ca2⫹ overload by impairing nAChR, NMDAR, VDCCs, TRPC, and mitochondria and forming new cation channels. The Ca2⫹ overload strongly inhibits AC, which results in downregulation of cAMP/PKA signaling and inhibition of gene transcription initiated by phosphorylated CREB. On the contrary, liraglutide increases the intracellular cAMP/PKA level by activating GLP-1R/G protein/AC on the membrane. The upregulated cAMP/PKA signaling will promote CREB phosphorylation and gene transcription in the nucleus and enhance learning and memory behavior. PKA, protein kinase A; nAChR, nicotinic acetylcholine receptor; NMDAR, N-methyl-D-aspartic acid receptor; VDCCs, voltage-dependent calcium channels; TRPC, TRP cation channel; AC, adenylyl cyclase; CREB, cyclic AMP response element (CRE)-binding protein; ROS, reactive oxygen species.

liraglutide on L-LTP strongly supports the behavioral improvements seen in the Morris water maze test. Similar to our results, Gengler et al. (Gengler et al., 2012) reported that Val(8)GLP-1, another GLP-1 analog, also effectively prevented the impairment of E-LTP induced by A␤ and APP/ PS-1 model. These electrophysiological results of E-LTP and L-LTP studies will be helpful to explain why GLP-1 analog could prevent the behavioral impairments induced by A␤ or transgenic AD animals. One of the possible mechanisms by which liraglutide prevents against A␤-induced deficits in L-LTP and spatial cognition may be involved in the upregulation of intracellular cAMP level. It is reported that GLP-1 and GLP-1 receptor agonist could increase cAMP concentration in astrocytes (Iwai et al., 2006) and hypothalamic neurons (Dalvi et al., 2012); a maximal 1200-fold increase in cAMP levels was observed in PC12 cells within 15 minutes of stimulation with 33 g/mL GLP-1 (Perry et al., 2002b); GLP-1induced increase in cAMP levels was also found in cultured

hippocampal neurons (Perry et al., 2002a). Similarly, we also identified in the present study an upregulation of cAMP level in the whole brain tissue by liraglutide injection. These findings strongly suggest that the rapid and transient upregulation of cAMP may be an important initiating factor in the neuroprotective action of GLP-1, which probably effectively antagonizes the A␤-induced neurotoxicity in the brain. This result is consistent with our previous study of lixisenatide, a GLP-1 receptor agonist, that also crossed the BBB and enhanced cAMP levels in the brain (Hunter and Holscher, 2012). As shown in Fig. 6, extracellular and intracellular neurotoxic A␤ can induce cytoplasmic Ca2⫹ overload through many pathways such as nicotinic acetylcholine receptors (nAChRs) (Hardy and Selkoe, 2002; Mehta et al., 2012; Tran et al., 2002), N-methyl-D-aspartate (NMDA) receptors (Ferreira et al., 2012), voltage-dependent calcium channels (VDCCs) (Gibson et al., 2012), transient receptor potential channels (TRPCs) (Cacace et al., 1986), A␤-formed cation channel (Itkin et al., 2011; Qi and

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Qiao, 2001a), and mitochondria (Clementi et al., 2006; Crouch et al., 2006; Parihar and Brewer, 2010). The intracellular Ca2⫹ overload will strongly inhibit adenylyl cyclase (AC) activity (Wong et al., 1999), and thereby downregulate cAMP/protein kinase A (PKA) signaling and gene transcription initiated by phosphorylated cAMP response element-binding protein (CREB). It is reported that A␤-induced inhibition of LTP could be prevented by forskolin, an activator of AC (Wang et al., 2009), and rolipram, a phosphodiesterase inhibitor that raises cAMP (Vitolo et al., 2002), suggesting that A␤-induced LTP inhibition is involved in the impairment of the AC and cAMP/PKA signaling pathway. On the contrary, liraglutide, an effective GLP-1 analog, can upregulate intracellular cAMP/PKA signaling by binding to specific G protein-coupled receptors and activating AC (Arnold et al., 1991). The upregulated cAMP/PKA activity will promote CREB phosphorylation and gene transcription in the nucleus and finally may enhance learning and memory behavior via this route. Therefore, we propose in the study that the liraglutide-induced increase in cAMP level in the brain should be related to the neuroprotective roles of liraglutide, and the activation of cAMP/PKA signal pathway may be an important mechanism by which liraglutide prevents against the A␤-induced impairments in spatial memory and hippocampal synaptic plasticity. In addition, it is also assumed that GLP-1 receptors activate a growth factor-like signaling cascade (Holscher, 2010), increase dendritic sprouting and neuronal regeneration, and help to prevent or reduce long-term damage induced by A␤ (Perry et al., 2003; Perry and Greig, 2003, 2005). In conclusion, the present study investigated for the first time the protective effects of intracerebral administration of liraglutide against A␤25–35-induced impairments in spatial memory and hippocampal L-LTP in rats. The effective neuroprotective action of liraglutide in preventing neurotoxic A␤ strongly suggests that upregulation of GLP-1 signaling in the brain, such as application of liraglutide, may be a novel and promising strategy to ameliorate the learning and memory deficits in the neurodegenerative disease such as AD.

Disclosure statement Dr. Holscher is a named inventor on a patent application for the use of liraglutide as a treatment for neurodegenerative diseases. The remaining authors declare no actual or potential conflicts of interest. The study protocol was approved by the Shanxi Animal Research Ethics committee in Taiyuan. In addition, the ethical committee of the local institutions provided approval. The data contained in the manuscript being submitted have not been previously published, have not been submit-

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