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. Author manuscript; available in PMC: 2013 Dec 24.
Published in final edited form as: Arch Ital Biol. 2012 Mar;150(1):5–14.

Chronic ramelteon treatment in a mouse model of Alzheimer’s disease

James T McKenna 1,*,1, Michael A Christie 1,*,1, Brianne A Jeffrey 1,1,2, John G McCoy 1,1,3, Eunho Lee 1,1,4, Nina P Connolly 1,1,5, Chris P Ward 1,1,6, Robert E Strecker 1,1
PMCID: PMC3872073  NIHMSID: NIHMS533919  PMID: 22786833

Abstract

Prior research has reported beneficial effects of melatonin in rodent models of Alzheimer’s disease (AD). This study evaluated the effect of ramelteon (Rozerem®, a melatonin receptor agonist) on spatial learning & memory and neuropathological markers in a transgenic murine model of AD (the B6C3-Tg(APPswe,PSEN1dE9)85Dbo/J transgenic mouse strain; hereafter ‘AD mice’). Three months of daily ramelteon treatment (~3mg/kg/day), starting at 3 months of age, did not produce an improvement in the cognitive performance of AD mice (water maze). In contrast to wild-type control mice, AD mice did not show any evidence of having learned the location of the escape platform. The cortex and hippocampus of AD mice contained significant quantities of beta-amyloid plaques and PARP-positive (poly ADP ribose polymerase) cells, indicating apoptosis. Six months of ramelteon treatment, starting at 3 months of age, did not produce any change in these neuropathological markers. The ability of long term melatonin treatment to improve cognition and attenuate neuropathology in AD mice did not generalize to this dosage of ramelteon.

Keywords: melatonin, Alzheimer’s disease, amyloid plaques, apoptosis, maze learning, ramelteon

INTRODUCTION

Alzheimer’s disease (AD) is the most common cause of dementia in the elderly, and a widely prescribed form of treatment for AD is cholinesterase inhibitors (Birks, 2006). However, these drugs treat only the cognitive symptoms of AD, and many patients experience only modest benefits when taking them. Therefore, better treatments are needed to stave off the cognitive decline in AD patients.

Mouse models of AD are now widely available (Bornemann and Staufenbiel, 2000; Radde et al., 2008; http://www.alzforum.org/res/com/tra/default.asp). Transgenic mice (AD mice) expressing the human amyloid precursor protein gene show neurobiological damage and cognitive deficits in learning and memory tasks at about 6 months of age (Bornemann and Staufenbiel, 2000; Morgan, 2003; German and Eisch, 2004; Radde et al., 2008). Treatment with the hormone melatonin reduced the deficits in learning and memory of AD mice, as well as decreased neuropathological markers such as the beta amyloid protein (Matsubara et al., 2003; Feng et al., 2004). Pharmacological effects of melatonin can be produced either via activation of melatonin receptors, or via melatonin’s potent antioxidant properties which can protect nuclear and mitochondrial DNA (Pierrefiche and Laborit, 1995; Reiter et al., 2000; Feng et al., 2004). The structure of ramelteon, a melatonin M1/M2 receptor agonist, resembles melatonin; hence, ramelteon may also slow the development of AD. Herein we used chronic treatment with a low dose of ramelteon (3 mg/kg/day; Rozerem®, a selective M1 and M2 agonist; Takeda Pharmaceuticals, Deerfield, Illinois, USA), comparable to that investigated in clinical studies for treatment of human sleep disorders (i.e., Richardson et al., 2008), in order to determine if activation of the melatonin receptor by ramelteon is sufficient to produce beneficial effects, similar to those reported for melatonin in AD mice.

A previous investigation reported that long term melatonin treatment attenuated deficits in learning and memory, as well as decreased neuropathological markers in the B6C3-Tg(APPswe,PSEN1dE9)85Dbo/J AD mice strain (Feng et al., 2004). Hence, this AD mouse strain was used here. The cognitive performance of these AD mice treated with ramelteon was assessed with a water maze spatial reference memory task, which has been previously shown to be disrupted in AD mice (Hirai et al., 2005). One hallmark feature of Alzheimer’s disease is the development of beta-amyloid plaques and subsequent apoptosis in the brain (Su et al., 1994; Calhoun et al., 1998; Selkoe, 2001; Duyckaerts et al., 2009). Therefore, we also analyzed these neuropathological markers in the prefrontal cortex (PFC), and dorsal hippocampus (HIPP), two brain regions important for memory and cognition. We tested animals in the water maze at the 6 month age, i.e., the 3 month treatment time point. Following behavioral studies, neuropathological markers were then assessed at the 9 month age, 6 month treatment time point.

METHODS

The B6C3-Tg(APPswe,PSEN1dE9)85Dbo/J strain of AD mice and their wild-type litter mates were used (Jackson Laboratories, Bar Harbor, Maine, USA). Mice were housed under constant temperature and 12:12 light:dark cycle (7AM:7PM), with food and water available ad libitum. All experiments conformed to U.S. Veterans Administration, Harvard University, and U.S. National Institutes of Health guidelines on the ethical use of animals. All measures were taken to minimize the number of animals used and their suffering, and were carried out in accordance with the National Institute of Health Guide for the Care and Use of Laboratory Animals (NIH Publications No. 80-23), revised 1996.

Mice consumed ramelteon in their drinking water, calculated to achieve an average dose of ~3mg/kg/day, based on the average daily water consumption of these mice. This dose of ramelteon was selected because it was comparable to the dose of ramelteon previously used in clinical studies. New drug solution and water bottles were made up each week. Although desirable, a dose response using different doses of ramelteon was not possible due to limited availability of the compound. Control animals received an equal volume of distilled water throughout the experiment.

Behavioral analysis was performed in AD mice following 3 months of ramelteon treatment (i.e., at 6 months of age). Spatial reference memory was assessed in the Morris Water Maze. The pool was 48″ in diameter and filled with water that was rendered opaque with non-toxic white water-based poster paint, in order to prevent the mice from visually locating the submerged target platform. The pool was divided into four equal quadrants and the escape platform was located in the center of a quadrant, and at least 5″ from the pool wall. The room in which the pool sat had numerous spatial extra-maze cues. The 4.5″ diameter collapsible platform was lowered to the floor of the pool or raised to enable the mouse to escape the water. In the raised position the platform was just submerged beneath the water of the pool (by 1/8th inch) so that it could not be seen, but the mouse could climb on it to escape from the water. When the platform was retracted it sat 9″ beneath the surface of the water, making it impossible for the mice to use it to escape. The mice were tracked in the water maze by video tacking software system (‘EzVideo Multitrack System’; Accuscan Instruments Inc., Columbus, Ohio, USA) that computed all behavioral measures at the end of each swim/trial. The four treatment groups for behavioral studies were: 1) wild-type mice, treated with vehicle (distilled water; n=18); 2) wild-type mice, treated with ramelteon (3 mg/kg/day for three months; n=12); 3) transgenic AD mice, treated with vehicle (distilled water; n=14); and 4) transgenic AD mice, treated with ramelteon (3 mg/kg/day for three months; n=19).

The platform location remained constant throughout testing (four trials a day), for five days (20 trials total) of testing. During the first trial of each day (11 AM) the platform location was marked by a flag to facilitate learning, but the flag was absent in the subsequent (3) trials each day. The start location for each trial was randomly varied (with the exception that it couldn’t be in the same quadrant as the hidden platform) to ensure mice wouldn’t adopt egocentric swimming strategies (e.g., always swim in the same direction relative to its starting position). Mice were allowed 60 s to find the hidden platform before being guided to the platform by the experimenter. The mouse remained on the platform for approximately 15 s, and then was returned to a holding cage for 10 min until the next trial. One hour after the last training trial each day, the platform was lowered to the bottom of the pool and mice were given a probe trial with a 30 s free swim. To prevent extinction (i.e., the mouse learning that the platform is not present, and thus the mouse stops searching for the platform), the platform was raised after 30 s of the probe trial, and the mouse was given an additional 30 s to find the platform.

Histological analysis was performed in AD mice following 6 months of ramelteon treatment (i.e., at 9 months of age), following behavioral evaluation. The two treatment groups for neuropathological assessment were: 1) transgenic AD mice, treated with vehicle (distilled water; n=5); and 2) transgenic AD mice, treated with ramelteon (3 mg/kg/day for six months; n=6). Mice were anesthetized, perfused transcardially with saline, followed with a 10% formalin solution. Brains were removed, and postfixed in formalin overnight. The following day, brains were transferred to a sucrose solution for up to 24 hrs. Brains were cut on a cryostat, in 30 μm slices, into one of four wells. One set of sections through the frontal cortex and rostral forebrain (including the dorsal hippocampus) was then processed for beta-amyloid plaque labeling, while another set (well of tissue) was reacted for PARP (poly ADP ribose polymerase), indicating apoptosis.

For beta-amyloid plaque labeling, sections were treated with hot citrate buffer for 30 minutes (85°C). Tissue was then transferred to the primary antibody (rabbit polyclonal to beta amyloid; 1:500; Abcam, Cambridge, MA, USA), diluted in Tris-buffered saline with 0.5% Triton-X 100 and incubated overnight at room temperature. The following day, tissue was rinsed 2X in Tris-buffered saline with 0.5% Triton X-100, and transferred to the secondary antibody solution (donkey anti-rabbit IgG; 1:400; Jackson Immunoresearch, West Grove, Pennsylvania, USA) for two hours at room temperature. Tissue was rinsed 3X, and incubated for 30 minutes at room temperature in Cy3 fluorophore (red; 1:3000; Jackson Immunoresearch, West Grove, Pennsylvania, USA). Sections were rinsed, mounted onto slides, and coverslipped using Vectashield (Vector Laboratories, Burlingame, California, USA).

For assessment of apoptosis with PARP labeling, a PARP antibody particular to the 85 kDa fragment of cleaved PARP was used for light microscopy immunohistochemistry. Sections were washed 2X in phosphate-buffered saline (PBS), and blocked in a solution of 3% normal donkey serum (NDS), 0.5% triton X-100 in PBS, at room temperature for one hour. Tissue was incubated overnight (4°C) in a solution of primary antibody for PARP diluted in a solution of 1% NDS, 0.3% Triton X-100 in PBS. More specifically, the primary antibody is described as rabbit (polyclonal) anti-PARP (214/215) cleavage site specific antibody (1:500; Invitrogen, Carlsbad, California, USA). The next day, tissue was washed 2X in PBS, and incubated in secondary antibody (donkey anti-rabbit IgG; 1:400; Jackson Immunoresearch, West Grove, Pennsylvania, USA) for 1 hr at room temperature, diluted in a mixture of 1% NDS, 0.3% Triton X-100 in PBS. Tissue was washed 2X in PBS, and incubated for one hour at room temperature in Avidin-Biotin Complex prepared in PBS (ABC Standard Kit; Vector Laboratories, Burlingame, California, USA). Tissue was washed 2X in PBS, and stained in a chromogen diaminobenzidine/nickel solution (Vector DAB Peroxidase Substrate Kit; Vector Laboratories, Burlingame, California, USA) for 2 minutes. Tissue was rinsed, mounted onto slides, and coverslipped with Cytoseal-60 (Thermo Scientific, Hudson, New Hampshire, USA).

Microscopic analysis with Neurolucida (Version 8; Microbrightfield, Williston, Vermont, USA) was performed on one selected coronal section of the prefrontal cortex (~ +2.0mm anterior to bregma) and one of the dorsal hippocampus (~ −2.1mm posterior to bregma) from each brain, allowing comparisons between treatment groups. Cellular mapping was superimposed onto coronal schematic sections adapted from a mouse brain atlas (Franklin and Paxinos, 2008). Microphotographs were taken for illustrative purposes.

RESULTS

Behavior

In the spatial reference memory dependent task, wild-type mice that received the vehicle-control learned the task, as depicted in Figure 1. This learning is indicated by their spending longer in the target quadrant during the probe trial following the fifth (final) day (37.8% ± 6.2%, mean ± SEM), compared to either the AD mice groups, and, importantly, longer than the 25% ‘random swim’ cutoff (t-test; p<0.01). Analysis of probe trial performance at the end of each of the first four days revealed no difference between groups. Both vehicle-control and ramelteon-treated AD mice spent less than 25% of their time in the target quadrant on the final day of testing (vehicle-control AD mice: 19.2%, ±8.0%, ramelteon-treated AD mice: 16.3%, ±4.3%). Although the reduced performance of the ramelteon-treated AD mice was not quite significant compared to the 25% chance level of performance (p=0.076), their poor performance on day 5 of training clearly indicated a lack of spatial reference learning and memory. Of note, AD mice spent more time in the starting location quadrant, which likely contributed to the reduced average time the AD mice spent in the target quadrant to below 25%. While the wild-type mice that had also been treated with ramelteon for three months spent 30.0% (±7.3%) of their time in the target quadrant, this was not significantly different from the 25% random cutoff mark (p=0.27).

Figure 1.

Figure 1

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Spatial reference memory was evaluated with the Morris Water Maze, comparing both wild-type and AD mice, treated with either vehicle (distilled water for 3 months) or drug (ramelteon, 3 mg/kg/day for 3 months). Mice in the transgenic groups (both vehicle and drug-treated) and the wild-type drug-treated group spent approximately 25% (or less) of their time in the target quadrant, and were thus assumed to be swimming randomly, suggesting they had not learned the platform location. In contrast, mice in the wild-type vehicle-treated group spent more time in the target quadrant, which is interpreted in water-maze studies as evidence that the mice had learned the position of the platform. Asterisk indicates a significant difference comparing wild-type vehicle-treated to the 25% chance level of performance. Data presented as mean±SEM.

Histology

Subregions of the prefrontal cortex (PFC) were examined, specifically: 1) the medial prefrontal cortex (mPFC), included the secondary motor, cingulate (area 1), prelimbic, infralimbic, and dorsal peduncular cortices; and 2) the dorsolateral prefrontal cortex (dlPFC), included the primary motor, agranular insular (dorsal), agranular insular (ventral), lateral orbital, and ventral orbital cortices, as well as the claustrum. The dorsal hippocampus region was also analyzed (HIPP). No beta amyloid plaques were detected in wild-type animals. However, in AD mice beta amyloid plaques were quite numerous in both PFC and HIPP following either ramelteon or vehicle treatment. As depicted in Figure 2, there was not an obvious visual difference between the amount of beta-amyloid plaques in PFC of vehicle (A) vs. ramelteon-treated AD mice (B), as well as of the HIPP in the vehicle (C) vs. ramelteon-treated AD mice (D). In Figure 3, photographic examples of PARP-positive PFC and HIPP labeling in two representative cases is also provided: vehicle (PFC, A and HIPP, C) vs. ramelteon-treated AD mice (PFC, B and HIPP, D). The PARP antibody stain labeled both beta-amyloid plaques and individual apoptotic cells. We were unable to detect any significant difference of amount of PARP-positive cells between vehicle vs. ramelteon-treated AD mice. PARP-positive cells were absent in wild-type vehicle-treated sections.

Figure 2.

Figure 2

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Photomicrographs and schematic depiction of beta-amyloid plaques in the secondary motor and cingulate cortices of the prefrontal cortex (A, B) and hippocampus (C,D) of vehicle (distilled water; A, C) and ramelteon-treated (B, D) AD mice (at 9 months of age, after 6 months of treatment). Schematic templates adapted from Franklin and Paxinos (2008). Findings indicate substantial plaques following either treatment. Scale bar = 100 μm.

Figure 3.

Figure 3

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Chronic treatment with ramelteon did not alter apoptosis in AD mice. Photomicrographs are of PARP staining, indicating apoptosis, in the PFC (A-B) and hippocampus (C-D) of vehicle (distilled water; A, C) and ramelteon-treated (B, D) AD mice. Findings indicate substantial PARP labeling, indicating apoptosis, following either vehicle or ramelteon treatment. Scale bar = 100 μm.

The histograms in Figure 4 summarize the number of beta-amyloid plaques and PARP-positive cells documented in AD mice for the three areas analyzed (mPFC, dlPFC, and HIPP). A comparison of total counts in the PFC was also reported as total prefrontal cortex (tPFC), determined as the sum of mPFC and dlPFC cell counts. There was no significant difference in the number of beta-amyloid plaques when comparing transgenic-vehicle and transgenic-ramelteon treated counts in the mPFC (t-test; vehicle n=5 vs. ramelteon n=6), dlPFC (vehicle n=5 vs. ramelteon n=6), tPFC (vehicle n=5 vs. ramelteon n=6), or HIPP (vehicle n=4 vs. Ramelteon n=5). Furthermore, there was no significant difference in the amount of PARP-positive cells of AD mice, when comparing sections of the mPFC (vehicle n=4 vs. ramelteon n=5), dlPFC (vehicle n=4 vs. ramelteon n=5), tPFC (vehicle n=4 vs. ramelteon n=5), or HIPP (vehicle n=3 vs. ramelteon n=3).

Figure 4.

Figure 4

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Summary graphs of the number of beta-amyloid plaques (A) and PARP-positive cells (B) documented for the three areas analyzed (mPFC, dlPFC, and HIPP) in AD mice. Also, a comparison of total counts in the PFC was reported as total prefrontal cortex (tPFC), determined as the sum of mPFC and dlPFC cell counts. There were no significant differences in the number of beta-amyloid plaques or PARP-positive cells when comparing vehicle (distilled water) vs. ramelteon-treated AD mice counts. Data presented as mean ± SEM.

DISCUSSION

Chronic ramelteon treatment did not improve cognitive performance of AD mice in a water maze spatial reference learning and memory task. Ramelteon also had no effect on behavioral measures in wild-type control mice. The latter finding is consistent with published work reporting that neither melatonin nor ramelteon improved learning and memory in normal rodents (Hirai et al., 2005). Beta-amyloid plaques and PARP-positive (presumed apoptotic) cells were abundant in the cortex and hippocampus of AD mice, but were not seen in the brains of wild-type mice. Chronic ramelteon treatment had no significant effect on the number of beta-amyloid plaques and PARP-positive cells when comparing transgenic-vehicle treated vs. transgenic-ramelteon treated tissue. Thus the dosage of ramelteon used in this study, being consistent with the dose-range prescribed to treat sleep disorders in humans, did not improve or ameliorate cognitive deficits and deleterious neuropathological changes in this AD mouse model.

Melatonin treatment has been shown to improve learning and memory and attenuate neuropathological markers in AD mouse models (Su et al., 1994; Matsubara et al., 2003; Feng et al., 2004). Reduced nocturnal melatonin secretion has been reported in human AD (Pandi-Perumal et al., 2005), and an increase in M1 and a decrease in M2 receptors in the hippocampus has been reported in post mortem analysis of human AD (Savaskan et al., 2002, 2005). Although the hippocampus normally has many M2 receptors, transgenic mice, with M2 receptor knock out, had impaired memory, as well as reduced hippocampal long-term potentiation, a brain mechanism that is known to be important for memory consolidation (Larson et al., 2006).

The mechanism by which melatonin may reduce the symptoms in AD mice is unknown. One possibility is that melatonin produces beneficial effects in AD mice via brain melatonin receptors. A second possibility is that melatonin’s neuroprotective effects are due to its antioxidant properties (Pierrefiche and Laborit, 1995; Reiter et al., 2000; Feng et al., 2004). Melatonin and other antioxidants have beneficial effects in transgenic rodent models of AD (Matsubara et al., 2003; Feng et al., 2004; reviewed in Pratico, 2008). However, the translation of antioxidant therapy to human AD clinical trials has not been particularly successful (Sano et al., 1997; Petersen et al., 2005; reviewed in Pratico, 2008), likely due to the large inter-individual variation in human AD which makes it very difficult to detect relatively small beneficial effects of antioxidant therapy in the face of a devastating neurodegenerative disorder.

The very poor cognitive performance of the AD mice was not fully anticipated. The onset of symptoms in these AD mice usually occurs at 5 to 7 months of age (Bornemann and Staufenbiel, 2000; Morgan, 2003; Radde et al., 2008). Subtle behavioral and neuropathological effects may occur at even younger ages, and starting drug intervention at an earlier developmental time point in the course of the neurodegenerative disorder could produce a better outcome (e.g., cognitive testing at 3 months of age instead of the 6 months used herein). This prediction leads to the suggestion that ramelteon treatment may prevent the onset of AD symptoms, as opposed to reverse the symptoms/signs of AD after onset, which may be addressed in future studies.

The low dose of ramelteon used herein (~3mg/kg/day in the drinking water) is thought to act predominantly as a MT1 and MT2 receptor agonist, and this dose is comparable to that previously investigated in clinical studies (i.e., Richardson et al., 2008). At this dose, ramelteon is an effective melatonin receptor agonist. Unfortunately, this dosage did not improve cognition or neuropathological markers in this AD mouse model. This study does not rule out the possibility that higher doses of ramelteon may produce an improvement in cognition and neuropathological markers in AD mouse models, and future investigations would be advised to use a dose response curve that includes different doses of ramelteon (e.g., 30 mg/kg/day) than the dose used herein (3mg/kg/day).

In conclusion, the ability of long term melatonin treatment to reduce neurological markers and reduce deficits in learning and memory in AD mice does not generalize to this dosage of the pharmacological melatonin receptor agonist ramelteon.

Acknowledgments

We thank Jeremy Beech, Yunren Bolortuya, Gina Ciovacco, and Jessin Varghese for technical assistance and John Franco for care of the animals. This study was supported by a grant from Takeda Pharmaceuticals North America, Inc. EL was supported by a fellowship from SK Holdings. The authors have indicated no financial conflicts of interest. Salary support for some of the investigators was from the U.S. Dept. of Veterans Affairs Medical Research and NIH.

LIST OF ABBREVIATIONS

AD

Alzheimer’s Disease

AD mice

B6C3-Tg(APPswe,PSEN1dE9)85Dbo/J transgenic mouse strain

dlPFC

Dorsolateral prefrontal cortex

HIPP

Hippocampus

kg

Kilogram

M1

Melatonin-1 receptor

M2

Melatonin-2 receptor

mg

Milligram

mPFC

Medial prefrontal cortex

PARP

Poly ADP ribose polymerase

PFC

Prefrontal cortex

SEM

Standard error of the mean

tPFC

Total prefrontal cortex

Footnotes

Authors’ contributions: *JTM and *MAC contributed equally to this work. RES, CPW and JTM designed the study and wrote the research proposal; JTM supervised and analyzed the histological work and wrote the paper; MAC supervised and analyzed the water maze work and wrote the paper; NPC, JGM and EL conducted the water maze experiments; BAJ performed histological and microscopic work; RES designed research, analyzed data, and wrote the paper.

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