Neurotoxicity Research

, Volume 17, Issue 4, pp 421–431

A New Tacrine–Melatonin Hybrid Reduces Amyloid Burden and Behavioral Deficits in a Mouse Model of Alzheimer’s Disease

Authors

  • Carlos Spuch
    • Neuroscience LaboratoryUniversity Hospital
    • Neurodegenerative Diseases Biomedical Research Center (CIBERNED)
  • Desiree Antequera
    • Neuroscience LaboratoryUniversity Hospital
    • Neurodegenerative Diseases Biomedical Research Center (CIBERNED)
  • M. Isabel Fernandez-Bachiller
    • Instituto de Química Médica (CSIC)
  • M. Isabel Rodríguez-Franco
    • Instituto de Química Médica (CSIC)
    • Neuroscience LaboratoryUniversity Hospital
    • Neurodegenerative Diseases Biomedical Research Center (CIBERNED)
Article

DOI: 10.1007/s12640-009-9121-2

Cite this article as:
Spuch, C., Antequera, D., Isabel Fernandez-Bachiller, M. et al. Neurotox Res (2010) 17: 421. doi:10.1007/s12640-009-9121-2

Abstract

Alzheimer’s disease (AD) is a progressive degenerative disorder characterized by the presence of amyloid deposits, neurofibrillary tangles and neuron loss. Emerging evidence indicates that antioxidants could be useful either for the prevention or treatment of AD. Tacrine and melatonin are well-known drugs which act as an acetylcholinesterase inhibitor and a free radical scavenger, respectively. In this study, we evaluated the effects of a new tacrine–melatonin hybrid on behavior and the biochemical and neuropathologic changes observed in amyloid precursor protein/presenilin 1 (APP/Ps1) transgenic mice. Our findings showed that direct intracerebral administration of this hybrid decreased amyloid β peptide (Aβ)-induced cell death and amyloid burden in the brain parenchyma of APP/Ps1 mice. This reduction in Aβ pathology was accompanied by a recovery in cognitive function. Since this tacrine–melatonin hybrid apparently reduces brain Aβ and behavioral deficits, we believe this drug has remarkable and significant neuroprotective effects and might be considered a potential therapeutic strategy in AD.

Keywords

Transgenic miceCytotoxicityAlzheimer’s diseaseAmyloidHybrid

Introduction

Alzheimer’s disease (AD) is a complex neurodegenerative disorder of the central nervous system characterized by three hallmark pathological lesions: amyloid plaques, neurofibrillary tangles and neuronal loss. AD patients develop a progressive loss of cholinergic synapses in brain regions associated with higher mental functions, mainly located in the hippocampus and neocortex. Cholinergic deficits seem to occur in parallel with structural losses, including cortical synaptic loss (Scheff et al. 1990; Samuel et al. 1994; Tiraboschi et al. 2000) death and/or atrophy of basal forebrain cholinergic neurons (Wu et al. 2005; Geula et al. 2008).

Recently, several pharmacological strategies have emerged, including cholinergic and non-cholinergic interventions (Holzgrabe et al. 2007). Some clinical studies describe that patients treated with cholinesterase inhibitors do not show the widespread cortical atrophic changes associated with AD, providing evidence of neuroprotection by cholinesterase inhibitors (Krishnan et al. 2003; Modrego 2006). As a result, the effectiveness of cholinesterase inhibitors in neuroprotection has been discussed in several reports (Raschetti et al. 2007; Raina et al. 2008).

Oxidative stress occurs early in the progression of AD, significantly before the development of neurofibrillary tangles and senile plaques (Zhu et al. 2007; Moreira et al. 2008). Thus, drugs that specifically scavenge oxygen radicals could be useful for the prevention as well as the treatment of AD (Huang et al. 2004; Zhang et al. 2006), and several antioxidants have been tested in several trials (Quinn et al. 2003; Hager et al. 2007).

Tacrine is a potent non-selective inhibitor of both acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE). Nowadays, a tacrine-based beta amyloid modulator called NP-61 is in clinical trials phase I for AD (García-Palomero et al. 2008). Melatonin is a pineal neurohormone whose levels decrease with aging, and notably in AD patients (Liu et al. 1999). It has been demonstrated that melatonin is a potent antioxidant and free radical scavenger that also stimulates several endogenous antioxidative enzymes, improves mitochondrial energy metabolism, reduces neurofilament hyperphosphorylation, plays a neuroprotective role against amyloid β (Aβ) (Acuña-Castroviejo et al. 2007; Masilamoni et al. 2008), and different neurodegenerative diseases (Srinivasan et al. 2005). Moreover, clinical trials have shown that melatonin promotes early changes in the hippocampus related to memory retrieval, suggesting that this endogenous molecule plays an important role in verbal memory consolidation (Gorfine et al. 2007).

Here, we investigated whether the tacrine-melatonin hybrid N-(2-(1H-indol-3-yl) ethyl)-7-(1,2,3,4-tetrahydroacridin-9-ylamino) heptanamide can directly alter the deposition of Aβ and its neurotoxic effects in APP/Ps1 transgenic mice. Our study is based on the work of Rodriguez-Franco’s group in the field of AD (Martinez et al. 2000; Rodríguez-Franco et al. 2001, 2005; Rodríguez-Franco and Fernández-Bachiller 2002) focuses on dual acting drugs, capable of combining AChE inhibition and antioxidant properties in a single small molecule. They have developed this new tacrine–melatonin hybrid, which is a potent inhibitor of human AChE and shows a high oxygen radical absorbance capacity (Rodríguez-Franco et al. 2006; Fernández-Bachiller et al. 2009). We demonstrate that the intracerebroventricular (ICV) delivery of this hybrid results in a significant reduction in Aβ burden and alleviates behavioral impairment in APP/Ps1 mice.

Material and Methods

Cell Cultures

Primary hippocampal and cortical neuronal cell cultures from Wistar rat embryos were obtained as described (Gonzalez de la Vega et al. 2001) except that E18 rat embryos were used. Neuronal cells were suspended in Neurobasal™ medium (Invitrogen/GIBCO™) supplemented with B27 (Invitrogen/GIBCO™), 2 mM glutamine, 100 U/ml penicillin, 100 μg/ml streptomycin and 0.25 mg/ml amphotericin B, and plated at a density of 2.5 × 105 cells per square centimeter onto poly-l-lysine (1 mg/ml; Sigma-Aldrich Co.) coated multi-well plates. Cultures were maintained at 37°C in a humidified atmosphere containing 5% CO2, and cultivated for 7 days prior to experimentation.

Cortical primary astrocyte cultures were prepared from P3-5 Wistar rats as described (Pons and Torres-Aleman 2000). The astroglial cells were grown in Dulbecco’s modified Eagle’s medium (Sigma-Aldrich Co.), supplemented with 10% fetal calf serum, 2 mM glutamine, 100 U/ml penicillin, 100 μg/ml streptomycin and 0.25 mg/ml amphotericin B, in a humidified atmosphere of 95% air and 5% CO2 at 37°C. After 15 days, glial cells were harvested in a 0.25% trypsin, 1 mM EDTA solution. Cells from the second and third passage were used for the experiments.

Both cell cultures were treated during 24 and 48 h with 50 μg/ml tacrine–melatonin hybrid previously dissolved in dimethyl sulfoxide, and then diluted in a sterile culture medium, and 5 μg/ml Aβ42 (Anaspec, Inc.) previously dissolved in 0.1 M acetic acid, and afterwards in sterile distilled water, as described (Carro et al. 2002; Dietrich et al. 2008).

Organotypic Slice Cultures

Sagittal brain slices were cultured as previously described (Nuñez and Buño 1999). Briefly, APP/Ps1 were deeply anesthetized with an intraperitoneal injection of sodium pentobarbital (35 mg/kg) and decapitated immediately after disappearance of the pinch reflex. The brain was rapidly removed and submerged in a vial containing cold (4°C) artificial cerebrospinal fluid (ACSF). The composition of the ACSF was as follows (in mM): 124 NaCl, 2.69 KCl, 1.25 KH2PO4, 2 MgSO4, 26 NaHCO3, 2 CaCl2, and 10 D-glucose. The ACSF was maintained at pH 7.4 by bubbling with carbogen (95% O2, 5% CO2). Sagittal slices (350 μm) were cut with a tissue chopper and placed into six-well plates (Nunc) with 1 ml of Neurobasal™ medium. Cultures were kept for 21 days at 37°C in a 5% CO2 atmosphere.

After the treatments, slice explants were fixed with 4% paraformaldehyde in 0.1 M phosphate buffer pH 7.4, and Congo Red and thioflavin-S (both from Sigma-Aldrich Co.) stains were used for verification and quantification of Aβ deposits, according to established procedures.

Animals

Nine-month male double-transgenic amyloid precursor (APP/Ps1) mice, a cross between the Tg2576 (overexpressing human APP695) and mutant Ps1 (M146L) mice, were used. Non-mutant littermates were used as the control transgenic group. All animals were handled and cared for in accordance with the European Community Council Directive (86/609/EEC). The tacrine–melatonin hybrid (2 μl per mouse, 50 μg/ml) was stereotaxically injected in each lateral ventricle (brain coordinates [mm from bregma]: 0.6 posterior, 1.1 lateral, 2 ventral) with a 10 μl syringe at 1 μl/min. Six weeks after injections, animals were fixed by transcardial perfusion, either with saline buffer, or 4% paraformaldehyde in 0.1 M phosphate buffer pH 7.4, for biochemical and immunohistochemical analysis, respectively.

Cell Death Quantification

After incubation for 24 h with Aβ42 (5 μg/μl), 7–8 days in vitro neurons were harvested with 0.25% trypsin, fixed in 70% methanol, washed several times, incubated for 1 h with 10 mg/ml ribonuclease (Sigma-Aldrich Co.), and stained with 1 mg/ml propidium iodide (Sigma-Aldrich Co.). Finally, samples were analyzed by flow cytometry (FACScan) using CellQuest software (Becton Dickinson). Cells with less Propidium Iodide staining (hence a lower DNA content than during G1 phase) were defined as apoptotic cells (sub-G1 peak).

DNA fragmentation from in vivo (APP/Ps1 mice) and in vitro (neuronal cell cultures) samples undergoing apoptosis was also detected using a Cell Death Detection ELISAPLUS kit (Roche Diagnostics), as described previously (Vargas et al. 2008).

Cell Viability Quantification

Neurons (2.5 × 105 cells/cm2) were treated with Aβ42 (5 μg/μl), in the presence or absence of the tacrine–melatonin hybrid, at 37°C for 24 h. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide dye (Roche Diagnostics) reduction assay, reflecting mitochondrial succinate dehydrogenase activity, was employed.

Immunoassays

Western blot analysis and immunoprecipitation were performed as described previously (Carro et al. 2002). Western blot membranes were re-blotted with unrelated proteins (βIII-tubulin) as an internal standard and to normalize for protein load. Densitometric analysis was performed using ImageJ software (NIH Image). For detection of Aβ deposits, brain sections from APP/Ps1 mice were pre-incubated in 88% formic acid and immunostained, as reported previously (Carro et al. 2002). To determine Aβ burden in APP/Ps1 mice, we used an antibody that recognizes both endogenous (murine) and transgenic (human) Aβ peptides (MBL International). Stereological analysis was done, and results were expressed as the percentage of brain area covered with amyloid. Amyloid deposits in brain parenchyma were also evaluated using thioflavin-S and Congo Red staining.

The following primary antibodies were used: rabbit polyclonal anti-caspase-3 and anti-caspase-9 (Cell Signaling Technology, Inc.), and mouse monoclonal βIII-tubulin (Promega). The secondary antibody used was HRP-conjugated (BioRad Laboratories). Levels of human endogenous Aβ40 and Aβ42 in cortical and hippocampal fractions from APP/Ps1 mice were determined using human specific ELISA kits (Biosource International, Invitrogen), according to the manufacturer’s instructions.

Behavioral Testing

After adaptation to handling, behavioral tests were conduced over an 11-day period. On each day, spontaneous alternation was evaluated first, followed by the elevated plus maze (days 4 and 5), and the object recognition test (days 10–11), as previously described (Lalonde et al. 2004). Spontaneous alternation was tested using the T-maze. The number of alternations and errors (entries to previously visited arms), and the time to complete each session were recorded. In the elevated plus-maze test, the time spent in the different compartments of the maze (open and closed arms) and the number of entries into the arms were measured. The open/total arm entries and duration ratios were then calculated. These two parameters were taken as measures of an anxiety-related behavior.

Briefly, mice were habituated to an open-field box with black vertical walls and a white floor. The next day, the animals were placed in the same box and submitted to a 10 min acquisition trial. During this trial, mice were placed individually in the open field in the presence of object A (marble or dice), and the time spent exploring object A (when the animal’s snout was directed toward the object at a distance <1 cm) was measured. During a 10-min retention trial (second trial), which was performed 3 h later, a novel object (object B: marble or dice) was placed together with the familiar object (object A) in the open field. The time (tA and tB) the animal spent exploring the two objects was recorded. The recognition index, defined as the ratio of the time spent exploring the novel object, over the time spent exploring both objects, [(tB/(tA + tB)) × 100] was used to measure non-spatial memory.

Results

The Tacrine–Melatonin Hybrid Reduces Aβ Deposits in Organotypic Slice Cultures of APP/Ps1 Mice

In order to determine whether the tacrine–melatonin hybrid was able to modulate brain Aβ accumulation in APP/Ps1 mice, we treated organotypic brain slices from these transgenic with 50 μg/ml of this compound. Congo Red was used to detect cortical amyloid deposits. We observed that the addition of the tacrine–melatonin hybrid reduced Aβ deposits 9 and 21 days after the administration (Fig. 1a).
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Fig. 1

The tacrine–melatonin hybrid reduces Aβ deposits in organotypic slices from month-old APP/Ps1 mice. a Amyloid deposits (black asterisks) in organotypic slice cultures, detected using light microscopy, were significantly reduced (upper panels) 9 and (bottom panels) 21 days after hybrid addition to the cultures (scale bar = 50 μm). b After 21 days of tacrine–melatonin hybrid addition to the organotypic slices, fewer and smaller amyloid deposits stained with thioflavin-S (white arrows) were observed in the cerebral cortex (Cx) in comparison with the organotypic slices from untreated APP/Ps1 mice (upper panels scale bar = 50 μm; bottom panels scale bar = 25 μm). c In vitro tacrine–melatonin hybrid treatment also reduced amyloid deposits stained with thioflavin-S (white arrows) in the hippocampus (Hip) of these organotypic slices (upper panels scale bar = 50 μm; bottom panels scale bar = 25 μm). LV lateral ventricle

Aβ accumulation in brain parenchyma was also evaluated using thioflavin-S. With this technique, fewer and smaller deposits were identified in the cerebral cortex (Fig. 1b) and hippocampus (Fig. 1c) using organotypic brain slices of APP/Ps1 mice after in vitro treatment for 21 days with the tacrine–melatonin hybrid.

The tacrine–melatonin hybrid modulates brain Aβ clearance in APP/Ps1 mice. Stereological analysis of brain amyloid deposits stained with an antibody that recognizes the endogenous (murine) as well as the transgenic (human) Aβ peptides (MBL International) (Fig. 2a, top panel) indicated that ICV treatment for 6 weeks with the tacrine–melatonin hybrid reduced the percentage of brain parenchyma stained with the anti-amyloid antibody. Aβ staining in APP/Ps1 mice treated with the tacrine–melatonin hybrid showed a 54% decrease in the cerebral cortex (P < 0.05, Fig. 2b) and a 50% decrease in the hippocampus (P < 0.01, Fig. 2b). Congo Red-stained brain tissue also revealed a decrease in the number and size of Aβ deposits in the cerebral cortex and hippocampus of APP/Ps1 (Fig. 2a, bottom panel). Further analysis with thioflavin-S (Fig. 2c) yielded similar results in both brain areas.
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Fig. 2

The tacrine–melatonin hybrid decreases brain Aβ burden in APP/Ps1 mice. a Amyloid deposits in the cerebral cortex (Cx) of APP/Ps1 mice are reduced by hybrid treatment. Murine and human Aβ were detected with an antibody that recognizes both Aβ forms (top panels). Amyloid deposits (black asterisks) were also detected with Congo Red staining (bottom panels) (scale bar = 50 mm). b Aβ burden was decreased in the cerebral cortex (Cx) and hippocampus (Hip) of tacrine–melatonin hybrid-treated APP/Ps1 mice. Brain Aβ burden represents the percentage of area covered by Aβ immunoreactivity (data are expressed as mean ± SEM, * P < 0.05, ** P < 0.01 versus untreated APP/Ps1 mice, n = 5–7 per group). c Microphotographs illustrate the presence of thyoflavin-S-stained amyloid deposits (white arrows) in the brain of APP/Ps1 mice, which seem to be reduced after hybrid treatment (scale bar = 1 mm)

To better quantify the changes in Aβ40 and Aβ42 levels after administration of the tacrine–melatonin hybrid, we measured both Aβ forms using an ELISA assay, in the same groups of animals. Confirming the results obtained with immunostaining assays, APP/Ps1 mice treated with the tacrine–melatonin hybrid showed a reduction of 43 and 42% in Aβ40 levels in the cerebral cortex and hippocampus, respectively (Table 1). In addition, Aβ42 levels also decreased in both brain areas by 48% after hybrid administration (Table 1).
Table 1

Amyloid levels in APP/Ps1 mice treated with the tacrine–melatonin hybrid compound

 

40 (% control)

42 (% control)

Cerebral cortex

46.63 ± 29*

51.78 ± 13.5*

Hippocampus

48.62 ± 25*

51.94 ± 14.75*

Data are expressed as mean ± SEM (* P < 0.05)

The Tacrine–Melatonin Hybrid Modulates Aβ-Induced Cell Death

Using a Cell Death Detection ELISAPLUS kit, we found a significant reduction in cell death in the cerebral cortex (P < 0.05, Fig. 3a) and hippocampus (P < 0.05, Fig. 3b) of hybrid-treated APP/Ps1 mice versus untreated transgenic mice. Accordingly, Western blot analysis revealed a significant decrease in caspase-3 expression in cerebral cortex samples from APP/Ps1 mice, whereas caspase-9 levels were only slightly reduced (Fig. 3c).
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Fig. 3

a The tacrine–melatonin hybrid modulates cell death in APP/Ps1 mice. Cell death was reduced in a the cerebral cortex and b hippocampus of hybrid-treated APP/Ps1 mice (data are expressed as mean ± SEM, * P < 0.05 versus untreated APP/Ps1 mice). c Representative Western blots show that caspase-3 and -9 levels were modified in the cerebral cortex of hybrid-treated APP/Ps1 mice, respectively (data are expressed as mean ± SEM, * P < 0.05 versus untreated APP/Ps1 mice)

In addition, when the tacrine–melatonin hybrid was added to primary neuronal cell cultures, it significantly reversed Aβ42-induced apoptotic cell death, as measured by flow cytometry (P < 0.05, Fig. 4a), and by Cell Death Detection ELISAPLUS kit (P < 0.05, Fig. 4b). Addition of the tacrine–melatonin hybrid to these neuronal cell cultures resulted in a reversion of the Aβ42-induced increase in caspase-3 expression (P < 0.01, Fig. 4c). The effects on caspase-9 levels, although less intense, were still substantial, as they were significantly reduced 48 h after treatment administration (P < 0.05, Fig. 4c).
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Fig. 4

The tacrine–melatonin hybrid modulates cell death in neuronal cell cultures. a Hybrid treatment protects against Aβ42-induced cell death in neuronal cell cultures as measured by flow cytometry and b by Cell Death Detection ELISAPLUS kit (data are expressed mean ± SEM, * P < 0.05 versus untreated cell cultures). c Representative Western blots show a hybrid-induced decrease in the expression of caspase-3 and -9 in neuronal cell cultures, 24 and 48 h after treatments (data are expressed mean ± SEM, * P < 0.05, ** P < 0.01 versus untreated cell cultures). d42 reduced survival of cultured neuronal cells, and the tacrine–melatonin hybrid addition restores this effect (data are expressed mean ± SEM, * P < 0.05 versus untreated cell cultures). e Cell survival of astrocyte and neuronal cell cultures was unaffected after hybrid treatment (data are expressed mean ± SEM)

We also examined the effect of Aβ42 on cell survival in primary neuronal cell cultures (Fig. 4d). Twenty-four hours after administration of the tacrine–melatonin hybrid, the negative effects on Aβ42-induced cell survival were reversed (Fig. 4d). In order to evaluate possible secondary effects of this drug on cell survival, we tested the hybrid on primary astrocyte and neuronal cell cultures. We did not find any toxic response in both primary cell cultures after tacrine–melatonin hybrid treatment (Fig. 4e).

The Tacrine–Melatonin Hybrid Alleviates Behavioral Deficits in APP/Ps1 Mice

Next, we determined whether hybrid-treated APP/Ps1 mice showed memory recovery. In the T-maze, the mean ± SEM spontaneous alternation rate of non-transgenic control mice was significantly higher than that of the APP/Ps1 group (60% ± 4.4 versus 45% ± 4.2, P < 0.05), whereas hybrid-treated APP/Ps1 mice showed approximately the same rate than the untreated transgenic group (47.5% ± 3.1%). Choice trial latencies of APP/Ps1 mice were significantly increased compared with non-transgenic control mice and were reduced after tacrine–melatonin hybrid administration (P < 0.05, Fig. 5a).
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Fig. 5

The tacrine–melatonin hybrid modulates cognition in APP/Ps1 mice. a Increased latencies in the spontaneous alternation of APP/Ps1 mice in the T-maze were partially normalized after hybrid treatment. b In the novel-object recognition task, recognition memory is expressed as exploratory preference in the retention test. The recognition index, representing exploratory preference, was reduced in APP/Ps1 mice compared with control mice, and recovered after tacrine–melatonin hybrid treatment (data are expressed as mean ± SEM, * P < 0.05, n = 5–7 per group)

Cognitive capacity was assessed using a paradigm of non-spatial visual recognition memory by subjecting the animals to a “novel object” recognition task, which is known to depend on hippocampal activity. Briefly, after the mice became familiarized with a given object, they were tested for retention by confronting them with a novel object, next to and in addition to the familiar one. After 3 h of testing, we observed that retention in APP/Ps1 mice was significantly impaired relative to the control non-transgenic group, while in the hybrid-treated APP/Ps1 mice, impaired cognition was alleviated (P < 0.05, Fig. 5b).

On day 1 of the elevated plus-maze test (Table 2), APP/Ps1 mice spent more time in the open arms than non-transgenic controls, as previously described (Lalonde et al. 2004). This effect, shown as open/total entries ratio, was elevated in the APP/Ps1 group and partially reduced after tacrine–melatonin hybrid treatment. On day 2 the behavioral tendency was similar, and the drug was only able to decrease the open/total duration ratio in APP/Ps1 mice (Table 2).
Table 2

Data are expressed as mean ± SEM open and enclosed arms visits and duration in the elevated plus-maze by non-transgenic controls, APP/Ps1 mice, and tacrine–melatonin hybrid-treated APP/Ps1 mice for 2 days of testing

Measures

Controls

APP/Ps1

APP/Ps1 + hybrid

Day 1

 Open/total arms (%)

  Entries

35.5 ± 2.2

63.33 ± 2.6a

42.9 ± 1b

  Duration

27.9 ± 4.5

18.69 ± 2.2

13.45 ± 1.6

Day 2

 Open/total arms (%)

  Entries

12.28 ± 1.4

50 ± 7a

53.33 ± 2.3a

  Duration

1.63 ± 0.71

23.56 ± 1.2a

12.07 ± 1.3b

aP < 0.05 versus non-transgenic control mice

bP < 0.05 versus APP/Ps1 mice

Discussion

The development of drugs for the treatment of AD should focus on the pathological events associated with neurodegeneration, such as oxidative stress, inflammation or disturbances in growth factor signaling. Melatonin has been found to have an essentially neuroprotective effect against Aβ-induced neurotoxicity. Tacrine, an AChE inhibitor, has demonstrated a modest improvement in the cognitive function of AD patients. It is worth considering the use of melatonin and tacrine in clinical trials as an adjunct to standard therapy in the treatment of this disease. Using combinations of antioxidants (melatonin), and AChE or BuChE inhibitors (tacrine), both of them with anti-apoptotic properties, will probably become a requirement in the strategies for protection against (Engelhart et al. 2002; Luchsinger et al. 2003; Polidori 2004), and thus melatonin, tacrine, or its derived analogs could be explored as a therapeutic approaches in AD.

In the present study, we investigated whether a dual-acting drug, capable of combining AChE inhibition and antioxidant properties in a single small molecule, could alter Aβ and amyloid burden in APP/Ps1 mice, a model of AD amyloidosis. We observed that therapy with a tacrine–melatonin hybrid for 6 weeks alleviated the behavioral impairment in APP/Ps1 mice and reduced total brain Aβ load. Treatment with this hybrid also ameliorated other AD-related disturbances, such as apoptotic cell death.

We first confirmed that addition of the tacrine–melatonin hybrid to organotypic brain slices of APP/Ps1 mice lead to a robust reduction in Aβ deposits. In this regard, it is important to indicate that in APP/Ps1 mice, dense Aβ deposits were already present at 9 months of age. Secondly, by direct ICV injection of this hybrid in APP/Ps1 mice, we observed a significant decrease in brain Aβ levels, in accordance with our in vitro results. Several studies have showed that administration of melatonin to APP695 transgenic mice is associated with a reduction in brain Aβ levels and deposition (Matsubara et al. 2003; Feng et al. 2004). The underlying mechanism of this brain amyloid reduction is still not clear, but according to our results, it might involve a direct action of the tacrine–melatonin hybrid on the metabolism of β-amyloid precursor protein (Song and Lahiri 1997; Lahiri 1999). This hypothesis is supported by the action of melatonin on the regulation of α-secretase expression (Gutierrez-Cuesta et al. 2008), and the inhibition of β-secretase (BACE-1) activity by tacrine (Fu et al. 2008). Moreover, this reduction in Aβ burden, measured by both immunohistochemistry and ELISA assays, was accompanied by a significant reduction in apoptotic cell death in the hybrid-treated APP/Ps1 mice. This finding is not surprising in view of the ability of Aβ40 and Aβ42 to induce cell death (Yao et al. 2005; Vargas et al. 2008).

Significant neuronal loss, a common pathological feature of AD patients, was found in the hippocampus of these animals, agreeing with previous studies (Christensen et al. 2008). In our study, we found that tacrine–melatonin hybrid significantly reversed Aβ-induced cytotoxicity, both in APP/Ps1 mice and primary neuronal cultures, as measured by DNA fragmentation and flow cytometry. This neuroprotective effect could be mediated, at least in part, by melatonin, which is capable of reducing the apoptotic-related factor caspase-3 in APP transgenic mice (Feng et al. 2006). On the other hand, the other component of the hybrid, tacrine, attenuates Aβ-induced neuronal apoptosis by regulating L-type voltage-dependent Ca2+ channels in primary neuronal cultures (Fu et al. 2006). Based on these facts, our results strongly suggest that the tacrine–melatonin hybrid protects neurons from Aβ-induced apoptosis by a mechanism which may involve modulation of caspase-3 and -9 expression.

While amyloid plaques and neurofibrillary tangles are hallmark anatomical features of AD, their contribution to cognitive loss, the clinically relevant aspect of AD, and the reason they are usually first diagnosed is far from clear. The most important therapeutic goal in AD is to ameliorate cognitive disturbances. In addition to amnesia, patients with AD exhibit neuropsychiatric symptoms such as apathy, sometimes accompanied by depression, dysphoria, and social withdrawal (Chung and Cummings 2000; Frisoni et al. 1999). However, the opposite pattern consisting of agitation, restlessness, disinhibition, and euphoria is frequently reported as well (Chung and Cummings 2000). The impairment in spontaneous alternation seen in APP/Ps1 mice may be interpreted in several ways, including loss of motivation to explore, inhibitory control or spatial orientation, symptoms similar to those described in Alzheimer’s dementia (Chung and Cummings 2000; Frisoni et al. 1999). On the other hand, the results obtained in the elevated plus-maze are consistent with the hypothesis that APP/Ps1 mice exhibit disinhibitory tendencies. All these behaviors may mimic those seen in some patients with AD.

Using the object recognition test, we investigated visual short-term memory, which is severely affected in AD. Indeed, AD patients typically present short-term memory deficits before long-term memory capacity declines as the disease progresses (Germano and Kinsella 2005). We found that in APP/Ps1 mice, in which cognition is already compromised, ICV injection of the tacrine–melatonin hybrid alleviated these behavioral deficits. In a previous study, it was demonstrated that long-term application of melatonin alleviates memory impairments in APP transgenic mice (Feng et al. 2004). It is important to emphasize that our results were observed only 6 weeks after hybrid treatment, suggesting a rapid and effective action.

Acknowledgments

There is no conflict of interest with other people or organizations. This work was supported by grants from Fondo de Investigación Sanitaria (FIS) (CP04/00179, PI060155), Fundación Investigación Médica Mutua Madrileña (2006.125), CIBERNED, and Ministerio de Educación y Ciencia (BFU2006-07430/BFI and SAF2006-01249). We thank Dr. Washington Buño and Dr. David Fernandez de Sevilla for expert help in brain organotypic culture. We thank Dr. Ximena Alvira-Botero for editorial assistance.

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© Springer Science+Business Media, LLC 2009