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Donepezil attenuates Aβ-associated mitochondrial dysfunction and reduces mitochondrial Aβ accumulation in vivo and in vitro
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Neuropharmacology xxx (2015) 1e8
Contents lists available at ScienceDirect
Neuropharmacology journal homepage: www.elsevier.com/locate/neuropharm
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Donepezil attenuates Ab-associated mitochondrial dysfunction and reduces mitochondrial Ab accumulation in vivo and in vitro
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Chun Yan Ye, Yun Lei, Xi Can Tang, Hai Yan Zhang*
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CAS Key Laboratory of Receptor Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, People's Republic of China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 22 October 2014 Received in revised form 19 January 2015 Accepted 12 February 2015 Available online xxx
The main purpose of the present study is to investigate the influence of donepezil, a well-known acetylcholinesterase (AChE) inhibitor, on amyloid-b (Ab)-associated mitochondrial dysfunction, in order to gain a better understanding of the neuroprotective effects of this clinically used anti-Alzheimer's disease (AD) drug. First, our study verifies the ameliorative effects of donepezil on behavioral deficits in both working memory and anxiety in APP/PS1 double transgenic mice, at a time point that AChE is not inhibited. Meanwhile, we demonstrate that donepezil enhances the resistance of brain mitochondria of APP/PS1 mice to the induction of mitochondrial permeability transition (MPT) by calcium ions. Moreover, the level of mitochondrial Ab in the brain of donepezil-treated APP/PS1 transgenic mice is significantly lower than that of vehicle-treated APP/PS1 mice. Our in vitro study using isolated mitochondria from rat brains, which is expected as an AChE-free subcellular system, further confirms the ameliorative effects of donepezil on oligomeric Ab1-42 induced mitochondrial swelling and ATP reduction. In addition, donepezil treatment also significantly blocks the Ab accumulation in the isolated mitochondria. Our study reported for the first time that the protective effects of donepezil against Ab-associated mitochondrial dysfunction are closely associated with the reduction of Ab accumulation in the mitochondria. Above observation led us to assume that, besides potent AChE inhibitory effects, other non-cholinergic mechanisms may be involved in the neuroprotective profiles of donepezil. © 2015 Elsevier Ltd. All rights reserved.
Keywords: Alzheimer's disease Donepezil Amyloid-b Oligomer Mitochondrial dysfunction
1. Introduction Alzheimer's disease (AD) is the leading cause of dementia in people over the age of 60 (Alzheimer's Association, 2014; Plassman et al., 2007), which is mainly characterized by two pathological hallmarks: extracellular deposits of amyloid b (Ab) protein and intracellular phosphorylated tau protein (Gandy and DeKosky, 2013; Risacher and Saykin, 2013; Selkoe, 1986). Although the link of Ab or tau protein to brain degeneration has remained elusive, the Ab cascade hypothesis remains as one of the dominant hypotheses for AD etiology. Nevertheless, with the fact that many therapeutic approaches towards Ab lowering/clearing fail to gain anticipated benefits in the clinical trials (Giacobini and Gold, 2013; Mangialasche et al., 2010), it is of great importance to further understand and analyze the essence of Ab cascade theory. Currently, the extracellular deposit of insoluble Ab is no longer considered as * Corresponding author. Shanghai Institute of Materia Medica, 555 Zu Chong Zhi Road, Zhangjiang Hi-Tech Park, Shanghai 201203, People's Republic of China. Tel./ fax: þ86 21 50806710. E-mail address: [email protected] (H.Y. Zhang).
the major contributor for AD pathogenesis (Goure et al., 2014; Shankar et al., 2008), whereas supports from numerous experimental paradigms have implicated the abnormal accumulation of intracellular Ab oligomers is responsible for the manifestations of AD pathology (Benilova et al., 2012; Lambert et al., 1998; Narayan et al., 2014). Growing researches have been focused on studying the association between the intracellular Ab cascade and the dysfunction of subcellular organelles especially mitochondria (Dragicevic et al., 2011; Du et al., 2008; Gao and Tang, 2006; LaFerla et al., 2007; Sarkar et al., 2014; Takuma et al., 2005). More interestingly, it is reported that mitochondrial Ab levels were positively correlated with the extent of mitochondrial dysfunction in different brain regions in APP or APP/PS1 transgenic mice, and the degree of cognitive impairment in AD transgenic mice could be linked to the extent of synaptic mitochondrial dysfunction and mitochondrial Ab levels (Dragicevic et al., 2010; Manczak et al., 2006). Hence targeting Ab-associated mitochondrial dysfunction, especially blocking mitochondrial Ab accumulation is expected as a promising approach for AD modifying. Donepezil, a classical acetylcholinesterase inhibitor (AChEI) approved by FDA, is generally known to provide only symptomatic
http://dx.doi.org/10.1016/j.neuropharm.2015.02.020 0028-3908/© 2015 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Ye, C.Y., et al., Donepezil attenuates Ab-associated mitochondrial dysfunction and reduces mitochondrial Ab accumulation in vivo and in vitro, Neuropharmacology (2015), http://dx.doi.org/10.1016/j.neuropharm.2015.02.020
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efficacy to AD patients. However, mounting evidence from preclinical and clinical studies have demonstrated that donepezil also possesses neuroprotective roles including halting the progression of brain atrophy in AD patients (Hashimoto et al., 2005). Donepezil was proved to protect against the Ab toxicity in cell and animal models (Noh et al., 2009; Romberg et al., 2011; Tang et al., 2014). Furthermore, in both Ab25-35 injected mice and Ab1-42 injected rats, donepezil could significantly ameliorate the severe oxidative stress and cognitive decline (Tang et al., 2014; Meunier et al., 2006). Since the neurotoxicity of Ab is believed to be closely linked to mitochondrial dysfunction, it is therefore of great interests to reveal whether the neuroprotection of donepezil may be attributed to the preservation of mitochondrial function as well as blocking Ab accumulation in the mitochondria. The present study was designed to 1) validate long term behavioral improvement of donepezil in transgenic mice carrying APPswe/PS1dE9 mutations (APP/PS1 mice) d a widely used AD animal model (McClean and Holscher, 2014; Cioanca et al., 2014); 2) investigate whether donepezil possesses ameliorative effects on mitochondrial dysfunction and abnormal Ab accumulation in mitochondria in APP/PS1 mice (in vivo); 3) further evaluate the molecular mechanisms of donepezil in ameliorating oligomeric Abinduced mitochondrial failure, especially focusing on its interference on Ab accumulation in isolated mitochondria from rat brains (in vitro).
randomly scattered inside beforehand) for 3 min to habituate them to the experimental environment. In stage II, individual mouse was constrained to run from the start arm to the opened goal arm containing food reward, with the other goal arm blocked by its door. Equal numbers of left and right choices were given. Both stage I and stage II lasted for 3 days. In stage III, all doors were open with both choice arms containing food. After consuming food in either of the choice arm in the first trial, the mouse was supposed to go for the other choice arm to find food in the second trial, and so forth to 8 trials (the first trial not included) in total. Mice were allowed to enjoy the food reward when choosing the correct arm. If they chose the wrong arm, they were punished by being blocked in the corner of the wrong arm for 1 min without enjoying any food. Stage III lasted for 5 days. In stage IV, the interval between two trials was enlarged to 30 s and 60 s in order to increase the memory difficulty. The correct rate of each mouse was recorded and calculated every day. Correct rate ¼ correct arm choices/total arm choices 100%. The maze was wiped with ethanol to eliminate odor between trials. Food ration was required through the whole experiment. 2.5. Elevated plus maze (EPM) behavioral test Elevated plus maze is a cross maze consists of two closed arms with walls around and two open arms without walls, which is widely adopted to assess the anxiety of animals, with anxious animals showing less entry into the open arm (Hadamitzky et al., 2014; Walf and Frye, 2007). The whole apparatus was elevated about 40 cm from the floor. Mice were individually placed at the center of the cross maze, facing the same open arm. Each mouse was given 5 min to explore the maze, with its trace recorded by a video camera. Mice that fell off the maze during the trial were not given a second chance. The maze was wiped with ethanol between trials to eliminate odor. The entry number into the open arm or the close arm of each mouse was calculated separately. 2.6. Preparation of rat brain mitochondria and drug treatment
2. Material and methods 2.1. Chemicals and preparation Donepezil hydrochloride (King-Pharm Co.Ltd., Nanjing, China) was dissolved with deionized water to 10 mM as stock solution. The drug stock solution was further diluted with deionized water (for animal study) or mitochondrial resuspension buffer (250 mM surcrose, 10 mM Tris, 2.5 mM KH2PO4, PH 7.4) (for isolated mitochondria study) to proper concentrations before usage. Ab1-42 (Millipore, USA) was prepared in 100% DMSO to 5 mM for storage at 80 C and diluted with mitochondrial resuspension buffer before usage. Unless otherwise stated, all other chemicals were purchased from Sigma (MO, USA). 2.2. Animals and drug administration APP/PS1 (APPswe/PS1dE9) double transgenic mice were purchased from Jackson's Laboratory (ME, USA) and bred in our own animal facility. The genotype was confirmed with polymerase chain reaction. Mice were maintained on a 12/12 h light-dark cycle in a temperature controlled room with free access to water and food. Five-month-old male APP/PS1 mice were randomly assigned into two groups: donepezil treated group and vehicle treated group. Age and sex matched wild type littermates were used as control. Mice were orally administrated with donepezil (1 mg/kg) or vehicle once a day for two months before subjected to behavioral tests and biomedical measurements. All behavioral and other functional tests were conducted no less than 16 h after the last drug administration. SpragueeDawley (SD) rats weighing around 250 g were purchased from Slac (Shanghai, China). All animal experiments were performed with the approval of the Animal Care and Use Committee in Shanghai Institute of Materia Medica and under the principles of Regulations of Experimental Animal Administration issued by the State Committee of Science and Technology of the People's Republic of China. 2.3. Assessment of acetylcholinesterase activity The AChE activity was assessed according to our previously described procedure (Yang et al., 2012). Briefly, 16 h after last drug administration, the cortex of APP/PS1 transgenic mice were dissected and homogenized with 75 mM phosphate buffer saline (PBS). Then 100 mL of the homogenate was added to 3.9 mL pre-warmed substrate (0.3 mM iodide-S-acetyl-thiocholine in 100 mM PBS) and incubated for 8 min at 37 C, terminated by 1 mL of 3% SDS. Finally, yellow 5-thio-2-nitrobenzoic acid was produced by the addition of 1 mL 0.2% 5, 50 -Dithiobis-(2-nitrobenzoic acid) (DTNB). The optic density value at 440 nm was measured to represent the AChE activity.
Mitochondria were isolated as previously described (Yang et al., 2012). Briefly, rats or mice were decapitated and the cortex of each mouse was rapidly removed, washed, minced, and homogenized in ice-cold isolation buffer (320 mM sucrose, 10 mM TriseHCl, 1 mM EDTAe2Kþ, 0.1% BSA, pH 7.4) with Dounce homogenizer. The homogenate was centrifuged at 1000 g for 5 min at 4 C, and then the supernatant was centrifuged at 7000 g for 10 min at 4 C. The obtained pellet was resuspended in ice-cold isolation buffer and the same centrifugation process was repeated. The crude mitochondrial pellet was re-suspended in 14% percoll solution (14% percoll, 320 mM sucrose, 0.5 mM EDTAe2Kþ, and 10 mM TriseHCl, pH 7.4) and centrifuged at 12000 g for 10 min at 4 C. The final mitochondrial pellet was resuspended in ice-cold resuspension buffer and protein concentrations were adjusted to 0.5 mg/ml. Isolated mitochondria from APP/PS1 mice were directly used for mitochondrial functional analysis, while those from rats were divided to proper aliquots for following incubation. First, 1 mM oligomeric Ab1-42 or vehicle was added into the aliquots for 10 min at 30 C, then different concentrations of donepezil or vehicle were added to the mixture to incubate for another 50 min. The mitochondrial samples were then ready for measurements of mitochondrial swelling, ATP production, as well as Ab levels and oligomeric forms. 2.7. Analysis of mitochondrial swelling Mitochondrial swelling was measured following a previously described protocol (Du et al., 2010). Briefly, after initiated by 250 mM calcium chloride and 5 mM sodium succinate, the osmotic volume changes of mitochondria were estimated by changes in light scattering at 540 nm (30 C) as monitored continuously for 40 cycles (the interval between trials was 30 s) with a microplate reader (Envision, Perkin Elmer). A decrease in absorbance indicates an increase in mitochondrial swelling. The decrease slope of the absorbance at 540 nm from cycle 1 to cycle 20 was calculated with linear regression to represent mitochondrial swelling degree. 2.8. Detection of mitochondrial ATP production The ATP production in the isolated rat cortical mitochondria was measured using a bioluminescent ATP detection kit (Promega, USA). After treatment, isolated mitochondria were immediately incubated with ATP synthesizing substrates (2.5 mM ADP, 1 mM L-()-malic acid and 1 mM pyruvate) for 5 min at 30 C before mixed with luciferin substrate and luciferase enzyme in the kit to produce bioluminesecence. The bioluminescence was assessed on a microplate reader (Envision, Perkin Elmer). The ATP level was calculated as described in the manual of the kit and normalized by control group.
2.4. T maze behavioral test
2.9. Enzyme-linked immunosorbent assay (ELISA) for Ab1-42 and Ab1-40
Food rewarded T maze is very sensitive for testing the working memory (Deacon and Rawlins, 2006; Soares et al., 2013). In the present study, food rewarded T maze was conducted according to literature (Deacon and Rawlins, 2006) with some modification. The test consisted of four stages and lasted for 13 days in total. Briefly, in stage I, mice were allowed to explore freely in the maze (sunflower seeds were
Mitochondrial pellets were lysed in 8-fold buffer A (5 M guanidine HCl, 50 mM Tris HCl, pH 8.0) for 3 h, and then centrifuged at 12,000 g for 10 min to remove insoluble matters before diluted with buffer B (Dulbecco's phosphate buffered saline with 5% BSA and 0.03% Tween-20) to appropriate concentration. The following ELISA process was under the instruction of kit manual (Invitrogen).
Please cite this article in press as: Ye, C.Y., et al., Donepezil attenuates Ab-associated mitochondrial dysfunction and reduces mitochondrial Ab accumulation in vivo and in vitro, Neuropharmacology (2015), http://dx.doi.org/10.1016/j.neuropharm.2015.02.020
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C.Y. Ye et al. / Neuropharmacology xxx (2015) 1e8 2.10. Western blot analysis The mitochondrial pellets were lysed by RIPA buffer (Beyotime, China) and mixed with equal volume of 2 Loading buffer without DTT. The protein mixture was resolved in a 4%/10%/17%/20% gradient Tricine-SDS-PAGE gel, and transferred to a nitrocellulose membrane (Whatman), followed by blocking with 5% nonfat milk for 1 h at room temperature. After overnight incubation with anti-Ab antibody (6E10, 1:1000, Covance) and the mitochondrial outer membrane marker (voltage dependent anion channel, VDAC) antibody (1:5000, Calbiochem) at 4 C, the membranes were washed 3 times in TBST (0.005% Tween-20, 50 mM Tris and 150 mM NaCl, pH 7.5) and incubated with HRP-conjugated secondary antibody for 1 h at room temperature. After TBST washing for another 3 times, the bands were detected using chemiluminescence reaction (Immobilon ECL, Millipore). Similar as the study of Walls et al. (Walls et al., 2012), the Ab level of each group is represented as the sum of all the bands detected by 6E10, with VDAC as the loading control of mitochondria. 2.11. Immunogold labeled microscopic staining Mice were anesthetized with 10% chloral hydrate (0.25 mL/30 g), before perfusion with saline and fixation buffer (4% paraformaldehyde þ 0.5% glutaraldehyde). After perfusion, the brain was further fixed for 48 h. Frontal cortex (Bregma 2.1 to 1.42) was carefully cut and trimmed into 1 mm3 pieces, postfixed in 2% osmium tetroxide for 30 min, and then dehydrated, embedded and cut into ultrathin slices. The ultrathin slices were then blocked with 1% BSA in PBS for 30 min at room temperature before incubated with anti-Ab antibody (6E10, 1:300, Covance) for 48 h at 4 C. After washing, the slices were then incubated with 15 nm gold labeled secondary antibody (1:75, British Biocell) for 3 h at room temperature. The slices were further stained with aqueous lead citrate and observed under a CM120 transmission electron microscope (Philips). Five slices from the same layers of cortical tissue (Bregma 2.1 to 1.42) of each mouse were examined and used for statistical analysis. 2.12. Statistical analysis Data were expressed as mean ± SEM. Student t test was applied in the comparisons between two groups. Multiple comparisons between model group and
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different concentrations of donepezil treated groups were analyzed by one way ANOVA, followed by Dunnett test. Differences were considered significant at p < 0.05.
3. Results 3.1. Donepezil ameliorates the Ab related behavioral deficits of APP/ PS1 mice AD-associated behavioral deficits including cognitive decline and psychiatric symptoms (Cummings et al., 1994; Mega et al., 1996; Petersen et al., 1999) are well reproduced in APP transgenic animal models (Cioanca et al., 2014; Le Cudennec et al., 2008; Yang et al., 2012). In order to evaluate the beneficial effect of donepezil on AD-related cognitive and psychiatric behavioral performances, we measured the working memory and anxiety by two widely adopted behavioral tasksdfood rewarded T maze (Colombo et al., 1996; Soares et al., 2013) and elevated plus maze (EPM) (Hadamitzky et al., 2014; Cioanca et al., 2014; Walf and Frye, 2007), respectively. To ensure the effect of donepezil in our behavioral tests was not directly associated with acute AChE inhibition, we examined the AChE activity at the same time point of our behavioral test. As shown in Fig. 1A, there are no significant differences between the AChE activity of donepezil ((62.70 ± 10.38) % of wild type) and vehicle ((79.93 ± 21.88) % of wild type) treated APP/PS1 mice (p ¼ 0.4932). As shown in Fig. 1B, vehicle treated APP/PS1 mice show decreased accuracy ((49.78 ± 6.20) %, p < 0.05 vs wild type group) in T maze as compared with wild type control
Fig. 1. Donepezil (1 mg/kg) attenuates the behavioral deficits of APP/PS1 mice. A) AChE activity of the mice 16 h after last drug administration. B) Statistical graph of T maze performance. T maze accuracy is calculated from the right arm choose percentage. EPM accuracy is represented by C) the entry number and D) time into the open arm. Values are mean ± SEM. n ¼ 8e10 per group. *p < 0.05, ***p < 0.001 vs wild type control; #p < 0.05, ##p < 0.01 vs vehicle treated APP/PS1 transgenic mice.
Please cite this article in press as: Ye, C.Y., et al., Donepezil attenuates Ab-associated mitochondrial dysfunction and reduces mitochondrial Ab accumulation in vivo and in vitro, Neuropharmacology (2015), http://dx.doi.org/10.1016/j.neuropharm.2015.02.020
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((65.55 ± 4.33) %), indicating significant working memory deficit in APP/PS1 mice, while donepezil treatment ((74.14 ± 4.20) %) significantly rescues this memory deficit (p < 0.01 vs vehicletreated APP/PS1 group). We then assessed the anxiety of APP/PS1 mice with EPM. As shown in Fig. 1C, the entry number into the open arm of vehicle treated APP/PS1 mice (0.60 ± 0.22, p < 0.001 vs wild type group) is significantly lower than wild type control (2.67 ± 0.41), while donepezil treatment markedly reverses the decrease to 2.90 ± 0.96 (p < 0.05 vs vehicle-treated APP/PS1 group). There is no obvious difference in the total entry number into open arm and close arm between the three groups (data not shown), indicating that the variations in EPM performance are not due to the individual propensity for activity of animals. As shown in Fig. 1D, the time in open arm of vehicle treated APP/PS1 mice (5.42 ± 1.89, p < 0.001 vs wild type group) is significantly lower than wild type control (26.41 ± 5.04), while donepezil treatment reverses the decrease to 21.82 ± 7.61 (p ¼ 0.0509 vs vehicle-treated APP/PS1 group). 3.2. Donepezil reduces Ca2þ-induced mitochondrial swelling To determine whether the memory improvement of 1 mg/kg donepezil consecutively administrated for 2 months on APP/PS1 transgenic mice is associated with preservation of mitochondrial function, the effect of donepezil on brain mitochondrial swelling was detected to provide an indirect measure of mitochondrial membrane permeability. In the mitochondria isolated from each mice group, the Ca2þ exposure induces a significant decrease in the optical density at 540 nm (Fig. 2A). As compared with the wild type group, the Ca2þ-induced mitochondrial swelling in the vehicle treated APP/PS1 group exhibits a much more rapid decrease during 30 min duration, while donepezil treatment shows a protective effect against the mitochondrial swelling. As shown in Fig. 2B, the mitochondria of vehicle treated APP/PS1 mice show larger swelling slope ((145.50 ± 12.09) % of wild type, p ¼ 0.08) than that of wild type control ((110.50 ± 11.04) %), reflected by the decreasing of optical density at 540 nm. Donepezil treatment significantly decreases the slope to the wild type level ((105.50 ± 12.26) %, p < 0.05). 3.3. Donepezil reduces brain mitochondrial Ab accumulation in APP/PS1 mice Mitochondria are reported as an intracellular Ab accumulating organelle and the Ab level in mitochondria is associated with the mitochondrial dysfunction (Dragicevic et al., 2010; Du et al., 2010). To investigate whether the protective effect of donepezil on mitochondrial dysfunction in vivo is attributed to mitochondrial Ab level, the amount and distribution of Ab in the brain mitochondria of APP/PS1 mice were investigated with immunogold labeled electronic microscopic technology. As shown in Fig. 3A and Fig. 3B, there are more gold particles (indicated by red arrow head, Fig. 3A) inside the mitochondria of vehicle treated APP/PS1 mice (36.45 ± 4.00 particles every 37.52 mm2, p < 0.05 vs control group) than control group (11.10 ± 5.87 particles every 37.52 mm2), while donepezil treatment significantly reduces this accumulation to 20.29 ± 2.87 particles every 37.52 mm2 (Fig. 3B, p < 0.05). We further detected the mitochondrial Ab1-40 and Ab1-42 levels in the APP/PS1 mice with ELISA. As shown in Fig. 3C and D, the accumulation of Ab1-40 (981.70 ± 165.50 pg/mg) and Ab1-42 (10721.00 ± 1869.00 pg/mg) in the mitochondria of APP/PS1 mice are both significantly higher than that in the wild type control mice (118.00 ± 15.73 pg/mg Ab1-40, 269.90 ± 59.10 pg/mg Ab1-42, p < 0.001 vs wild type mice), while donepezil treatment significantly reduces the mitochondrial Ab1-40 and Ab1-42 accumulation to
Fig. 2. Donepezil (1 mg/kg) attenuates the ex vivo mitochondrial dysfunction in APP/ PS1 transgenic mice. A) Representative swelling curve of mitochondria isolated from APP/PS1 transgenic mice and wild type mice. Swelling slope is indicated by the decrease in OD540. B) Statistical graph of mitochondrial swelling slope. Values are expressed as mean ± SEM. n ¼ 4e6 per group. #p < 0.05 vs vehicle treated APP/PS1 transgenic mice.
546.70 ± 95.60 pg/mg (p < 0.05 vs vehicle-treated APP/PS1 mice) and 4876.00 ± 729.60 pg/mg (p < 0.01 vs vehicle-treated APP/PS1 mice), respectively. 3.4. Donepezil ameliorates mitochondrial functional deficits in isolated brain mitochondria from SD rats In order to further elucidate the potential mechanisms of donepezil on Ab-associated mitochondrial dysfunction, we employed an in vitro subcellular system, isolated brain mitochondria from SD rats, to minimize the interference from AChE. To identify the integrity of isolated brain cortical mitochondria, mitochondrial ultrastructural morphology was assessed before the initiation of following experiments. Oligomeric Ab1-42 was used in this study to induce mitochondrial dysfunction, since the “toxic Ab oligomer” hypothesis has attracted considerable attention in the recent years and Ab1-42 oligomers are believed as one of the crucial drivers of mitochondrial dysfunction (Hou et al., 2014; Sun et al., 2014). First of all, we tested the beneficial effects of donepezil on Ab1-42-induced mitochondrial dysfunction. As shown in Fig. 4A and Fig. 4B and 1 mM oligomeric Ab1-42 exposure causes a dramatic drop of the optical density at 540 nm ((128.00 ± 4.40) % of control, p < 0.01), indicating severe mitochondrial swelling, while
Please cite this article in press as: Ye, C.Y., et al., Donepezil attenuates Ab-associated mitochondrial dysfunction and reduces mitochondrial Ab accumulation in vivo and in vitro, Neuropharmacology (2015), http://dx.doi.org/10.1016/j.neuropharm.2015.02.020
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Fig. 3. Donepezil (1 mg/kg) reduces the Ab accumulation in the mitochondria of APP/PS1 mice. A) Representative graph of mitochondrial Ab detected by immunogold labeled electronic microscope with 6E10 antibody. Mitochondrial Ab signals are indicated by red arrow heads. Scale bar ¼ 1 mm. B) Statistical result of immunogold staining. n ¼ 4e6 per group. C) Ab1-40 and D) Ab1-42 level in APP/PS1 transgenic mice detected by ELISA. Values are expressed as mean ± SEM. n ¼ 8e10 per group. *p < 0.05, ***p < 0.001 vs wild type control; #p < 0.05, ##p < 0.01 vs vehicle treated APP/PS1 transgenic mice. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
donepezil at 0.1 mM ((103.00 ± 6.30) % of control, p < 0.05 vs Ab group) and 1 mM ((93.70 ± 5.40) % of control, p < 0.001 vs Ab group) final concentrations could both ameliorate this mitochondrial swelling. Similarly, as shown in Fig. 4C, oligomeric Ab1-42 exposure significantly impairs the in vitro ATP producing ability of mitochondria to (90.47 ± 1.01) % of control (p < 0.01), while donepezil at 0.1 mM ((96.82 ± 2.16) % of control, p < 0.05 vs Ab group) and 1 mM ((96.28 ± 0.69) % of control, p < 0.05 vs Ab group) final concentrations could significantly ameliorate the Ab-associated mitochondrial dysfunction in the isolated mitochondria. 3.5. Donepezil reduces mitochondrial Ab overload in isolated brain mitochondria from SD rats To further investigate whether donepezil could directly inhibit the Ab accumulation in the mitochondria, we then measured the Ab1-42 level in the pellet of isolated brain mitochondria after 1 mM oligomeric Ab1-42 exposure. After being exposed to 1 mM oligomeric Ab1-42, the isolated brain mitochondria were treated with different concentrations of donepezil. As expected, Ab1-42 was prone to accumulate in the mitochondria, since we detected a high level of Abin the pellet of the mitochondria treated with oligomeric Ab1-42 (Fig. 5A and Fig. 5B, p < 0.01). Western blot result shows that the total level of Ab bands detected with 6E10 antibody in Ab1-42 incubated group was 6.39 ± 1.21 fold of control group (p < 0.01), while donepezil at 0.1 mM (3.41 ± 0.77, p < 0.01 vs Ab group) and 1 mM (4.29 ± 1.12, p < 0.05 vs Ab group) final concentrations significantly inhibit the abnormal accumulation of Ab in the mitochondria, to approximately 4 fold of control group. 4. Discussion Although the neuroprotective effects of donepezil beyond AChE inhibition, especially significant ameliorative effects on Ab-
associated neurotoxicity (Noh et al., 2009; Romberg et al., 2011; Tang et al., 2014), have attracted intense interest in recent years, the exact molecular mechanisms underlying have not yet been clarified. The major impressive characteristics of the present study include: 1) the ameliorative effect of chronic treatment with donepezil on behavioral deficits in both T-maze and elevated cross maze task in APP/PS1 mice is correlated with preserved mitochondrial function and reduced mitochondrial Ab level in the brain; 2) donepezil treatment significantly ameliorated oligomeric Ab1-42induced subcellular mitochondrial dysfunction and reduced the accumulation of Ab in the isolated brain mitochondria. As an extension of previous studies, the current study confirmed the ameliorative effects of donepezil on Ab-associated behavioral deficits and mitochondrial dysfunction by employing in vivo and in vitro models. First, in consistence with previous studies (He et al., 2009; Lok et al., 2013), our study verifies the multi-aspect behavioral deficits in APP/PS1 transgenic mice, and shows that donepezil could ameliorate the deficits of APP/PS1 mice in both food rewarded T maze and elevated plus maze. It is worth mentioning that to avoid the acute symptomatic improvement caused by acetylcholine elevation, above behaviors were assessed no less than 16 h after the last drug administration, a time point when the AChE inhibition of donepezil should have already diminished, while in previous studies functional tests were usually conducted shortly after drug administration (Noda et al., 2010; Wang et al., 2010; Winocur et al., 2011). As shown in literature (Abe et al., 2003) and our unpublished data, the maximal AChE inhibition of donepezil occurs at about 1 h after drug administration in mice and the inhibition diminishes with time passing. Above finding in the inhibitory effect of donepezil on AChE may be associated with its pharmacodynamics data, as donepezil was reported to have a half-life of about 3 h in rats (Goh et al., 2011; Nirogi et al., 2012). Therefore, the protection we observed in APP/PS1 mice may mainly indicate the neuroprotective effects of donepezil aside from its AChE inhibition.
Please cite this article in press as: Ye, C.Y., et al., Donepezil attenuates Ab-associated mitochondrial dysfunction and reduces mitochondrial Ab accumulation in vivo and in vitro, Neuropharmacology (2015), http://dx.doi.org/10.1016/j.neuropharm.2015.02.020
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Fig. 5. Donepezil (0.1 and 1 mM) reduces the Ab1-42 accumulation in isolated mitochondria from rat brains. A) Representative picture of mitochondrial Ab accumulation detected by western blot with 6E10 antibody. VDAC was used as loading control. B) Statistical graph of the total Ab signal detected in the above blot. Values are expressed as mean ± SEM. Data are from at least 4 independent experiments. Con: control; Don: donepezil. ***p < 0.001 vs control group; #p < 0.05, ##p < 0.01 vs Ab group.
Fig. 4. Donepezil (0.1 and 1 mM) ameliorates the dysfunction of isolated rat mitochondria caused by 1 mM oligomeric Ab1-42 in vitro. A) Representative swelling curve of mitochondria treated by oligomeric Ab1-42 with or without donepezil. Swelling slope is indicated by the decrease in OD540. B) Statistical graph of mitochondrial swelling slope. C) The ATP producing ability of mitochondria treated with 1 mM oligomeric Ab1-42 followed by donepezil treatment. Values are expressed as mean ± SEM. Data are from at least 4 independent experiments. Con: control; Don: donepezil. **p < 0.01 vs control group; #p < 0.05, ##p < 0.01 vs Ab group.
Ab plays a pivotal role in the mitochondrial dysfunctions, because mitochondrial deficits like oxidative stress, energy deficiency and mitochondrial depolarization were frequently seen in Ab treated cell models and Ab overexpression animal models (Dragicevic et al., 2011; Gao and Tang, 2006; Sarkar et al., 2014; Takuma et al., 2005). Therefore, reversing Ab-associated mitochondrial dysfunction may provide an opportunity to recover the cognitive and psychiatric behavioral deficits. Interestingly, the present study found that the ameliorative effect of donepezil on
behavioral deficits is positively correlated with the alleviated brain mitochondrial swelling. Moreover, mitochondrial Ab accumulation is frequently seen in AD brains (Chen and Yan, 2006; Hansson Petersen et al., 2008), and increases with age at a speed much faster than Ab accumulation in brain tissue homogenates of APP/ PS1 transgenic mice (our unpublished data). In addition, mitochondrial Ab accumulation was found to be associated with the extent of mitochondrial dysfunction in different brain regions and the degree of cognitive impairment in APP and APP/PS1 transgenic mice, while reducing mitochondrial Ab accumulation can improve mitochondrial function (Dragicevic et al., 2010; Yao et al., 2011). In the current study, we found that donepezil administration could effectively reduce the mitochondrial Ab accumulation in APP/PS1 mice, which as suggested by the above literature, may contribute to the mitochondrial functional enhancement and behavioral improvement by donepezil. This is the first report of donepezil reducing mitochondrial Ab accumulation to our knowledge. The
Please cite this article in press as: Ye, C.Y., et al., Donepezil attenuates Ab-associated mitochondrial dysfunction and reduces mitochondrial Ab accumulation in vivo and in vitro, Neuropharmacology (2015), http://dx.doi.org/10.1016/j.neuropharm.2015.02.020
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above data may further affirm the critical role of mitochondrial Ab in the disease progression. In order to minimize the influence of AChE inhibitory effect of donepezil, isolated mitochondria were then adopted in this study to further understand the molecular mechanism of donepezil. It is reported that Ab is able to accumulate in isolated mitochondria (Hansson Petersen et al., 2008) thus cause mitochondrial swelling and cytochrome c release (Kim et al., 2002; Rodrigues et al., 2000). Our result on isolated mitochondria showed that donepezil ameliorated the mitochondrial swelling and reduced ATP level on Ab1-42 injured mitochondria, as well as reducing the Ab1-42 accumulation. As an extension of previous findings, the study presented herein together with the previous reports suggest that the subcellular pharmacological benefits of donepezil on mitochondrial ATP production and mitochondrial swelling may be, at least partly, mediated through suppressing the accumulation of Ab1-42 in the mitochondria, although the precise mechanism still need to be clarified. Above results observed that donepezil had similar influences on mitochondrial dysfunction and mitochondrial Ab accumulation in both in vivo and in vitro models, which indicates that the direct effect of donepezil on isolated mitochondria may possibly contribute to the findings in the APP/PS1 transgenic mice. We also verified that donepezil could significantly ameliorate the mitochondrial swelling induced by Ab exposure in the isolated mouse mitochondria in our preliminary study. Still, further studies will be needed for making close linkage between in vivo and in vitro results, as the current in vitro studies were mainly done with isolated mitochondria from rat brains. To sum up, our research for the first time found that donepezil could reduce mitochondrial Ab accumulation in APP/PS1 mice, probably contributing to its cognitive benefits. Furthermore, we found that donepezil have direct mitochondrial protection in isolated mitochondria against Ab1-42 insult, as well as reducing mitochondrial Ab accumulation, which could be different from its formerly reported specific effect on AChE. Our data might shed more light on the clinical benefits gained by donepezil and provide useful clues for future AD drug development, however, the precise mechanisms underlying the beneficial effect of the drug remains to be explored in future study. Acknowledgments This work was supported by the National Natural Science Foundation of China (No 81173034, 81072646), the National Science & Technology Major Project “Key New Drug Creation and Manufacturing Program” of China (No. 2012ZX09301001-004), and the Ministry of Science and Technology of China (No 2011CB510004). References Abe, Y., Aoyagi, A., Hara, T., Abe, K., Yamazaki, R., Kumagae, Y., Naruto, S., Koyama, K., Marumoto, S., Tago, K., Toda, N., Takami, K., Yamada, N., Ori, M., Kogen, H., Kaneko, T., 2003. Pharmacological characterization of RS-1259, an orally active dual inhibitor of acetylcholinesterase and serotonin transporter, in rodents: possible treatment of Alzheimer's disease. J. Pharmacol. Sci. 93, 95e105. Alzheimer's Association, 2014. 2014 Alzheimer's disease facts and figures. Alzheimer's Dementia 10, 1e75. Benilova, I., Karran, E., Strooper, B., 2012. The toxic Ab oligomer and Alzheimer's disease: an emperor in need of clothes. Nat. Neurosci. 15, 349e357. Chen, X., Yan, S.D., 2006. Mitochondrial abeta: a potential cause of metabolic dysfunction in Alzheimer's disease. IUBMB Life 58, 686e694. Cioanca, O., Hritcu, L., Mihasan, M., Trifan, A., Hancianu, M., 2014. Inhalation of coriander volatile oil increased anxiolytic-antidepressant-like behaviors and decreased oxidative status in b-amyloid (1-42) rat model of Alzheimer's disease. Physiol. Behav. 131, 68e74. Colombo, G., Agabio, R., Balaklievskaia, N., Lobina, C., Reali, R., Fadda, F., Gessa, G.L., 1996. T-maze and food reinforcement: an inexpensive drug discrimination procedure. J. Neurosci. Methods 67, 83e87.
7
Cummings, J.L., Mega, M., Gray, K., Rosenberg-Thompson, S., Carusi, D.A., Gornbein, J., 1994. The neuropsychiatric inventory: comprehensive assessment of psychopathology in dementia. Neurology 44, 2308e2314. Deacon, R.M.J., Rawlins, J.N.P., 2006. T-maze alternation in the rodent. Nat. Protoc. 1, 7e12. Dragicevic, N., Mamcarz, M., Zhu, Y., Buzzeo, R., Tan, J., Arendash, G.W., Bradshaw, P.C., 2010. Mitochondrial amyloid-b levels are associated with the extent of mitochondrial dysfunction in different brain regions and the degree of cognitive impairment in Alzheimer's transgenic mice. J. Alzheimers Dis. 20 (Suppl. 2), S535eS550. Dragicevic, N., Smith, A., Lin, X., Yuan, F., Copes, N., Delic, V., Tan, J., Cao, C., Shytle, R.D., Bradshaw, P.C., 2011. Green tea epigallocatechin-3-gallate (EGCG) and other flavonoids reduce Alzheimer's amyloid-induced mitochondrial dysfunction. J. Alzheimers Dis. 26, 507e521. Du, H., Guo, L., Fang, F., Chen, D., Sosunov, A.A., McKhann, G.M., Yan, Y., Wang, C., Zhang, H., Molkentin, J.D., Gunn-Moore, F.J., Vonsattel, J.P., Arancio, O., Chen, J.X., Yan, S.D., 2008. Cyclophilin D deficiency attenuates mitochondrial and neuronal perturbation and ameliorates learning and memory in Alzheimer's disease. Nat. Med. 14, 1097e1105. Du, H., Guo, L., Yan, S., Sosunov, A.A., McKhann, G.M., Yan, S.S., 2010. Early deficits in synaptic mitochondria in an Alzheimer's disease mouse model. Proc. Natl. Acad. Sci. U. S. A. 107, 18670e18675. Gandy, S., DeKosky, S.T., 2013. Toward the treatment and prevention of Alzheimer's disease: rational strategies and recent progress. Annu Rev. Med. 64, 367e383. Gao, X., Tang, X.C., 2006. Huperzine A attenuates mitochondrial dysfunction in bamyloid-treated PC12 cells by reducing oxygen free radicals accumulation and improving mitochondrial energy metabolism. J. Neurosci. Res. 83, 1048e1057. Giacobini, E., Gold, G., 2013. Alzheimer disease therapyemoving from amyloid-b to tau. Nat. Rev. Neurol. 9, 677e686. Goh, C.W., Aw, C.C., Lee, J.H., Chen, C.P., Browne, E.R., 2011. Pharmacokinetic and pharmacodynamic properties of cholinesterase inhibitors donepezil, tacrine, and galantamine in aged and young lister hooded rats. Drug Metab. Dispos. 39, 402e411. Goure, W.F., Krafft, G.A., Jerecic, J., Hefti, F., 2014. Targeting the proper amyloid-beta neuronal toxins: a path forward for Alzheimer's disease immunotherapeutics. Alzheimers Res. Ther. 6, 42. Hadamitzky, M., Herring, A., Keyvani, K., Doenlen, R., Krugel, U., Bosche, K., Orlowski, K., Engler, H., Schedlowski, M., 2014. Acute systemic rapamycin induces neurobehavioral alterations in rats. Behav. Brain Res. 273, 16e22. Hansson Petersen, C.A., Alikhani, N., Behbahani, H., Wiehager, B., Pavlov, P.F., Alafuzoff, I., Leinonen, V., Ito, A., Winblad, B., Glaser, E., Ankarcrona, M., 2008. The amyloid b-peptide is imported into mitochondria via the TOM import machinery and localized to mitochondrial cristae. Proc. Natl. Acad. Sci. U. S. A. 105, 13145e13150. Hashimoto, M., Kazui, H., Matsumoto, K., Nakano, Y., Yasuda, M., Mori, E., 2005. Does donepezil treatment slow the progression of hippocampal atrophy in patients with Alzheimer's disease? Am. J. Psychiatry 162, 676e682. He, J., Luo, H., Yan, B., Yu, Y., Wang, H., Wei, Z., Zhang, Y., Xu, H., Tempier, A., Li, X., Li, X.M., 2009. Beneficial effects of quetiapine in a transgenic mouse model of Alzheimer's disease. Neurobiol. Aging 30, 1205e1216. Hou, Y., Ghosh, P., Wan, R., Ouyang, X., Cheng, H., Mattson, M.P., Cheng, A., 2014. Permeability transition pore-mediated mitochondrial superoxide flashes mediate an early inhibitory effect of Ab1-42 on neural progenitor cell proliferation. Neurobiol. Aging 35, 975e989. Kim, H.S., Lee, J.H., Lee, J.P., Kim, E.M., Chang, K.A., Park, C.H., Jeong, S.J., Wittendorp, M.C., Seo, J.H., Choi, S.H., Suh, Y.H., 2002. Amyloid b peptide induces cytochrome C release from isolated mitochondria. Neuroreport 13, 1989e1993. LaFerla, F.M., Green, K.N., Oddo, S., 2007. Intracellular amyloid-b in Alzheimer's disease. Nat. Rev. Neurosci. 8, 499e509. Lambert, M.P., Barlow, A.K., Chromy, B.A., Edwards, C., Freed, R., Liosatos, M., Morgans, T.E., Rozovsky, I., Trommer, B., Viola, K.L., Wals, P., Zhang, C., Finch, C.E., Krafft, G.A., Klein, W.L., 1998. Diffusible, nonfibrillar ligands derived from Ab are potent central nervous system neurotoxins. Proc. Natl. Acad. Sci. U. S. A. 95, 6448e6453. Le Cudennec, C., Faure, A., Ly, M., Delatour, B., 2008. One-year longitudinal evaluation of sensorimotor functions in APP751SL transgenic mice. Genes. Brain Behav. 7 (Suppl. 1), 83e91. Lok, K., Zhao, H., Zhang, C., He, N., Shen, H., Wang, Z., Zhao, W., Yin, M., 2013. Effects of accelerated senescence on learning and memory, locomotion and anxietylike behavior in APP/PS1 mouse model of Alzheimer's disease. J. Neurol. Sci. 335, 145e154. Manczak, M., Anekonda, T.S., Henson, E., Park, B.S., Quinn, J., Reddy, P.H., 2006. Mitochondria are a direct site of Ab accumulation in Alzheimer's disease neurons: implications for free radical generation and oxidative damage in disease progression. Hum. Mol. Genet. 15, 1437e1449. Mangialasche, F., Solomon, A., Winblad, B., Mecocci, P., Kivipelto, M., 2010. Alzheimer's disease: clinical trials and drug development. Lancet Neurol. 9, 702e716. McClean, P.L., Holscher, C., 2014. Lixisenatide, a drug developed to treat type 2 diabetes, shows neuroprotective effects in a mouse model of Alzheimer's disease. Neuropharmacology 86, 241e258. Mega, M.S., Cummings, J.L., Fiorello, T., Gornbein, J., 1996. The spectrum of behavioral changes in Alzheimer's disease. Neurology 46, 130e135.
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Meunier, J., Ieni, J., Maurice, T., 2006. The anti-amnesic and neuroprotective effects of donepezil against amyloid b25-35 peptide-induced toxicity in mice involve an interaction with the sigma1 receptor. Br. J. Pharmacol. 149, 998e1012. Narayan, P., Holmstrom, K.M., Kim, D.H., Whitcomb, D.J., Wilson, M.R., St GeorgeHyslop, P., Wood, N.W., Dobson, C.M., Cho, K., Abramov, A.Y., Klenerman, D., 2014. Rare individual amyloid-b oligomers act on astrocytes to initiate neuronal damage. Biochemistry 53, 2442e2453. Nirogi, R., Bhyrapuneni, G., Kandikere, V., Benade, V., Muddana, N., Saralaya, R., Irappanavar, S., Ponnamaneni, R., Mukkanti, K., 2012. Concurrent administration of atypical antipsychotics and donepezil: drug interaction study in rats. Eur. J. Drug Metab. Pharmacokinet. 37, 155e161. Noda, Y., Mouri, A., Ando, Y., Waki, Y., Yamada, S., Yoshimi, A., Yamada, K., Ozaki, N., Wang, D., Nabeshima, T., 2010. Galantamine ameliorates the impairment of recognition memory in mice repeatedly treated with methamphetamine: involvement of allosteric potentiation of nicotinic acetylcholine receptors and dopaminergic-ERK1/2 systems. Int. J. Neuropsychopharmacol. 13, 1343e1354. Noh, M.-Y., Koh, S.-H., Kim, Y., Kim, H.Y., Cho, G.W., Kim, S.H., 2009. Neuroprotective effects of donepezil through inhibition of GSK-3 activity in amyloid-b-induced neuronal cell death. J. Neurochem. 108, 1116e1125. Petersen, R.C., Smith, G.E., Waring, S.C., Ivnik, R.J., Tangalos, E.G., Kokmen, E., 1999. Mild cognitive impairment: clinical characterization and outcome. Arch. Neurol. 56, 303e308. Plassman, B.L., Langa, K.M., Fisher, G.G., Heeringa, S.G., Weir, D.R., Ofstedal, M.B., Burke, J.R., Hurd, M.D., Potter, G.G., Rodgers, W.L., Steffens, D.C., Willis, R.J., Wallace, R.B., 2007. Prevalence of dementia in the United States: the aging, demographics, and memory study. Neuroepidemiology 29, 125e132. Risacher, S.L., Saykin, A.J., 2013. Neuroimaging and other biomarkers for Alzheimer's disease: the changing landscape of early detection. Annu Rev. Clin. Psychol. 9, 621e648. Rodrigues, C.M., Sola, S., Silva, R., Brites, D., 2000. Bilirubin and amyloid-b peptide induce cytochrome c release through mitochondrial membrane permeabilization. Mol. Med. 6, 936e946. Romberg, C., Mattson, M.P., Mughal, M.R., Bussey, T.J., Saksida, L.M., 2011. Impaired attention in the 3xTgAD mouse model of Alzheimer's disease: rescue by donepezil (Aricept). J. Neurosci. 31, 3500e3507. Sarkar, P., Zaja, I., Bienengraeber, M., Rarick, K.R., Terashvili, M., Canfield, S., Falck, J.R., Harder, D.R., 2014. Epoxyeicosatrienoic acids pretreatment improves amyloid b-induced mitochondrial dysfunction in cultured rat hippocampal astrocytes. Am. J. Physiol. Heart Circ. Physiol. 306, H475eH484.
Selkoe, D.J., 1986. Altered structural proteins in plaques and tangles: what do they tell us about the biology of Alzheimer's disease? Neurobiol. Aging 7, 425e432. Shankar, G.M., Li, S., Mehta, T.H., Garcia-Munoz, A., Shepardson, N.E., Smith, I., Brett, F.M., Farrell, M.A., Rowan, M.J., Lemere, C.A., Regan, C.M., Walsh, D.M., Sabatini, B.L., Selkoe, D.J., 2008. Amyloid-b protein dimers isolated directly from Alzheimer's brains impair synaptic plasticity and memory. Nat. Med. 14, 837e842. Soares, J.C., Oliveira, M.G., Ferreira, T.L., 2013. Inactivation of muscarinic receptors impairs place and response learning: implications for multiple memory systems. Neuropharmacology 73, 320e326. Sun, Q., Jia, N., Wang, W., Jin, H., Xu, J., Hu, H., 2014. Protective effects of astragaloside IV against Ab1-42 neurotoxicity by inhibiting the mitochondrial permeability transition pore opening. PLoS One 9, e98866. Takuma, K., Yao, J., Huang, J., Xu, H., Chen, X., Luddy, J., Trillat, A.C., Stern, D.M., Arancio, O., Yan, S.S., 2005. ABAD enhances Abeta-induced cell stress via mitochondrial dysfunction. FASEB J. 19, 597e598. Tang, S.S., Hong, H., Chen, L., Mei, Z.L., Ji, M.J., Xiang, G.Q., Li, N., Ji, H., 2014. Involvement of cysteinyl leukotriene receptor 1 in Ab1-42-induced neurotoxicity in vitro and in vivo. Neurobiol. Aging 35, 590e599. Walf, A.A., Frye, C.A., 2007. The use of the elevated plus maze as an assay of anxietyrelated behavior in rodents. Nat. Protoc. 2, 322e328. Walls, K.C., Coskun, P., Gallegos-Perez, J.L., Zadourian, N., Freude, K., Rasool, S., Blurton-Jones, M., Green, K.N., LaFerla, F.M., 2012. Swedish Alzheimer mutation induces mitochondrial dysfunction mediated by HSP60 mislocalization of amyloid precursor protein (APP) and b-amyloid. J. Bio. Chem. 287, 30317e30327. Wang, J., Zhang, H.Y., Tang, X.C., 2010. Huperzine A improves chronic inflammation and cognitive decline in rats with cerebral hypoperfusion. J. Neurosci. Res. 88, 807e815. Winocur, G., Binns, M.A., Tannock, I., 2011. Donepezil reduces cognitive impairment associated with anti-cancer drugs in a mouse model. Neuropharmacology 61, 1222e1228. Yang, L., Ye, C.Y., Huang, X.T., Tang, X.C., Zhang, H.Y., 2012. Decreased accumulation of subcellular amyloid-beta with Improved mitochondrial function mediates the neuroprotective effect of Huperzine A. J. Alzheimers Dis. 31, 131e142. Yao, J., Du, H., Yan, S., Fang, F., Wang, C., Lue, L.F., Guo, L., Chen, D., Stern, D.M., Gunn Moore, F.J., Xi Chen, J., Arancio, O., Yan, S.S., 2011. Inhibition of amyloid-b (Ab) peptide-binding alcohol dehydrogenase-Abeta interaction reduces Ab accumulation and improves mitochondrial function in a mouse model of Alzheimer's disease. J. Neurosci. 31, 2313e2320.
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Contents lists available at ScienceDirect
Neuropharmacology journal homepage: www.elsevier.com/locate/neuropharm
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Donepezil attenuates Ab-associated mitochondrial dysfunction and reduces mitochondrial Ab accumulation in vivo and in vitro
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Chun Yan Ye, Yun Lei, Xi Can Tang, Hai Yan Zhang*
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CAS Key Laboratory of Receptor Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, People's Republic of China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 22 October 2014 Received in revised form 19 January 2015 Accepted 12 February 2015 Available online xxx
The main purpose of the present study is to investigate the influence of donepezil, a well-known acetylcholinesterase (AChE) inhibitor, on amyloid-b (Ab)-associated mitochondrial dysfunction, in order to gain a better understanding of the neuroprotective effects of this clinically used anti-Alzheimer's disease (AD) drug. First, our study verifies the ameliorative effects of donepezil on behavioral deficits in both working memory and anxiety in APP/PS1 double transgenic mice, at a time point that AChE is not inhibited. Meanwhile, we demonstrate that donepezil enhances the resistance of brain mitochondria of APP/PS1 mice to the induction of mitochondrial permeability transition (MPT) by calcium ions. Moreover, the level of mitochondrial Ab in the brain of donepezil-treated APP/PS1 transgenic mice is significantly lower than that of vehicle-treated APP/PS1 mice. Our in vitro study using isolated mitochondria from rat brains, which is expected as an AChE-free subcellular system, further confirms the ameliorative effects of donepezil on oligomeric Ab1-42 induced mitochondrial swelling and ATP reduction. In addition, donepezil treatment also significantly blocks the Ab accumulation in the isolated mitochondria. Our study reported for the first time that the protective effects of donepezil against Ab-associated mitochondrial dysfunction are closely associated with the reduction of Ab accumulation in the mitochondria. Above observation led us to assume that, besides potent AChE inhibitory effects, other non-cholinergic mechanisms may be involved in the neuroprotective profiles of donepezil. © 2015 Elsevier Ltd. All rights reserved.
Keywords: Alzheimer's disease Donepezil Amyloid-b Oligomer Mitochondrial dysfunction
1. Introduction Alzheimer's disease (AD) is the leading cause of dementia in people over the age of 60 (Alzheimer's Association, 2014; Plassman et al., 2007), which is mainly characterized by two pathological hallmarks: extracellular deposits of amyloid b (Ab) protein and intracellular phosphorylated tau protein (Gandy and DeKosky, 2013; Risacher and Saykin, 2013; Selkoe, 1986). Although the link of Ab or tau protein to brain degeneration has remained elusive, the Ab cascade hypothesis remains as one of the dominant hypotheses for AD etiology. Nevertheless, with the fact that many therapeutic approaches towards Ab lowering/clearing fail to gain anticipated benefits in the clinical trials (Giacobini and Gold, 2013; Mangialasche et al., 2010), it is of great importance to further understand and analyze the essence of Ab cascade theory. Currently, the extracellular deposit of insoluble Ab is no longer considered as * Corresponding author. Shanghai Institute of Materia Medica, 555 Zu Chong Zhi Road, Zhangjiang Hi-Tech Park, Shanghai 201203, People's Republic of China. Tel./ fax: þ86 21 50806710. E-mail address: [email protected] (H.Y. Zhang).
the major contributor for AD pathogenesis (Goure et al., 2014; Shankar et al., 2008), whereas supports from numerous experimental paradigms have implicated the abnormal accumulation of intracellular Ab oligomers is responsible for the manifestations of AD pathology (Benilova et al., 2012; Lambert et al., 1998; Narayan et al., 2014). Growing researches have been focused on studying the association between the intracellular Ab cascade and the dysfunction of subcellular organelles especially mitochondria (Dragicevic et al., 2011; Du et al., 2008; Gao and Tang, 2006; LaFerla et al., 2007; Sarkar et al., 2014; Takuma et al., 2005). More interestingly, it is reported that mitochondrial Ab levels were positively correlated with the extent of mitochondrial dysfunction in different brain regions in APP or APP/PS1 transgenic mice, and the degree of cognitive impairment in AD transgenic mice could be linked to the extent of synaptic mitochondrial dysfunction and mitochondrial Ab levels (Dragicevic et al., 2010; Manczak et al., 2006). Hence targeting Ab-associated mitochondrial dysfunction, especially blocking mitochondrial Ab accumulation is expected as a promising approach for AD modifying. Donepezil, a classical acetylcholinesterase inhibitor (AChEI) approved by FDA, is generally known to provide only symptomatic
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efficacy to AD patients. However, mounting evidence from preclinical and clinical studies have demonstrated that donepezil also possesses neuroprotective roles including halting the progression of brain atrophy in AD patients (Hashimoto et al., 2005). Donepezil was proved to protect against the Ab toxicity in cell and animal models (Noh et al., 2009; Romberg et al., 2011; Tang et al., 2014). Furthermore, in both Ab25-35 injected mice and Ab1-42 injected rats, donepezil could significantly ameliorate the severe oxidative stress and cognitive decline (Tang et al., 2014; Meunier et al., 2006). Since the neurotoxicity of Ab is believed to be closely linked to mitochondrial dysfunction, it is therefore of great interests to reveal whether the neuroprotection of donepezil may be attributed to the preservation of mitochondrial function as well as blocking Ab accumulation in the mitochondria. The present study was designed to 1) validate long term behavioral improvement of donepezil in transgenic mice carrying APPswe/PS1dE9 mutations (APP/PS1 mice) d a widely used AD animal model (McClean and Holscher, 2014; Cioanca et al., 2014); 2) investigate whether donepezil possesses ameliorative effects on mitochondrial dysfunction and abnormal Ab accumulation in mitochondria in APP/PS1 mice (in vivo); 3) further evaluate the molecular mechanisms of donepezil in ameliorating oligomeric Abinduced mitochondrial failure, especially focusing on its interference on Ab accumulation in isolated mitochondria from rat brains (in vitro).
randomly scattered inside beforehand) for 3 min to habituate them to the experimental environment. In stage II, individual mouse was constrained to run from the start arm to the opened goal arm containing food reward, with the other goal arm blocked by its door. Equal numbers of left and right choices were given. Both stage I and stage II lasted for 3 days. In stage III, all doors were open with both choice arms containing food. After consuming food in either of the choice arm in the first trial, the mouse was supposed to go for the other choice arm to find food in the second trial, and so forth to 8 trials (the first trial not included) in total. Mice were allowed to enjoy the food reward when choosing the correct arm. If they chose the wrong arm, they were punished by being blocked in the corner of the wrong arm for 1 min without enjoying any food. Stage III lasted for 5 days. In stage IV, the interval between two trials was enlarged to 30 s and 60 s in order to increase the memory difficulty. The correct rate of each mouse was recorded and calculated every day. Correct rate ¼ correct arm choices/total arm choices 100%. The maze was wiped with ethanol to eliminate odor between trials. Food ration was required through the whole experiment. 2.5. Elevated plus maze (EPM) behavioral test Elevated plus maze is a cross maze consists of two closed arms with walls around and two open arms without walls, which is widely adopted to assess the anxiety of animals, with anxious animals showing less entry into the open arm (Hadamitzky et al., 2014; Walf and Frye, 2007). The whole apparatus was elevated about 40 cm from the floor. Mice were individually placed at the center of the cross maze, facing the same open arm. Each mouse was given 5 min to explore the maze, with its trace recorded by a video camera. Mice that fell off the maze during the trial were not given a second chance. The maze was wiped with ethanol between trials to eliminate odor. The entry number into the open arm or the close arm of each mouse was calculated separately. 2.6. Preparation of rat brain mitochondria and drug treatment
2. Material and methods 2.1. Chemicals and preparation Donepezil hydrochloride (King-Pharm Co.Ltd., Nanjing, China) was dissolved with deionized water to 10 mM as stock solution. The drug stock solution was further diluted with deionized water (for animal study) or mitochondrial resuspension buffer (250 mM surcrose, 10 mM Tris, 2.5 mM KH2PO4, PH 7.4) (for isolated mitochondria study) to proper concentrations before usage. Ab1-42 (Millipore, USA) was prepared in 100% DMSO to 5 mM for storage at 80 C and diluted with mitochondrial resuspension buffer before usage. Unless otherwise stated, all other chemicals were purchased from Sigma (MO, USA). 2.2. Animals and drug administration APP/PS1 (APPswe/PS1dE9) double transgenic mice were purchased from Jackson's Laboratory (ME, USA) and bred in our own animal facility. The genotype was confirmed with polymerase chain reaction. Mice were maintained on a 12/12 h light-dark cycle in a temperature controlled room with free access to water and food. Five-month-old male APP/PS1 mice were randomly assigned into two groups: donepezil treated group and vehicle treated group. Age and sex matched wild type littermates were used as control. Mice were orally administrated with donepezil (1 mg/kg) or vehicle once a day for two months before subjected to behavioral tests and biomedical measurements. All behavioral and other functional tests were conducted no less than 16 h after the last drug administration. SpragueeDawley (SD) rats weighing around 250 g were purchased from Slac (Shanghai, China). All animal experiments were performed with the approval of the Animal Care and Use Committee in Shanghai Institute of Materia Medica and under the principles of Regulations of Experimental Animal Administration issued by the State Committee of Science and Technology of the People's Republic of China. 2.3. Assessment of acetylcholinesterase activity The AChE activity was assessed according to our previously described procedure (Yang et al., 2012). Briefly, 16 h after last drug administration, the cortex of APP/PS1 transgenic mice were dissected and homogenized with 75 mM phosphate buffer saline (PBS). Then 100 mL of the homogenate was added to 3.9 mL pre-warmed substrate (0.3 mM iodide-S-acetyl-thiocholine in 100 mM PBS) and incubated for 8 min at 37 C, terminated by 1 mL of 3% SDS. Finally, yellow 5-thio-2-nitrobenzoic acid was produced by the addition of 1 mL 0.2% 5, 50 -Dithiobis-(2-nitrobenzoic acid) (DTNB). The optic density value at 440 nm was measured to represent the AChE activity.
Mitochondria were isolated as previously described (Yang et al., 2012). Briefly, rats or mice were decapitated and the cortex of each mouse was rapidly removed, washed, minced, and homogenized in ice-cold isolation buffer (320 mM sucrose, 10 mM TriseHCl, 1 mM EDTAe2Kþ, 0.1% BSA, pH 7.4) with Dounce homogenizer. The homogenate was centrifuged at 1000 g for 5 min at 4 C, and then the supernatant was centrifuged at 7000 g for 10 min at 4 C. The obtained pellet was resuspended in ice-cold isolation buffer and the same centrifugation process was repeated. The crude mitochondrial pellet was re-suspended in 14% percoll solution (14% percoll, 320 mM sucrose, 0.5 mM EDTAe2Kþ, and 10 mM TriseHCl, pH 7.4) and centrifuged at 12000 g for 10 min at 4 C. The final mitochondrial pellet was resuspended in ice-cold resuspension buffer and protein concentrations were adjusted to 0.5 mg/ml. Isolated mitochondria from APP/PS1 mice were directly used for mitochondrial functional analysis, while those from rats were divided to proper aliquots for following incubation. First, 1 mM oligomeric Ab1-42 or vehicle was added into the aliquots for 10 min at 30 C, then different concentrations of donepezil or vehicle were added to the mixture to incubate for another 50 min. The mitochondrial samples were then ready for measurements of mitochondrial swelling, ATP production, as well as Ab levels and oligomeric forms. 2.7. Analysis of mitochondrial swelling Mitochondrial swelling was measured following a previously described protocol (Du et al., 2010). Briefly, after initiated by 250 mM calcium chloride and 5 mM sodium succinate, the osmotic volume changes of mitochondria were estimated by changes in light scattering at 540 nm (30 C) as monitored continuously for 40 cycles (the interval between trials was 30 s) with a microplate reader (Envision, Perkin Elmer). A decrease in absorbance indicates an increase in mitochondrial swelling. The decrease slope of the absorbance at 540 nm from cycle 1 to cycle 20 was calculated with linear regression to represent mitochondrial swelling degree. 2.8. Detection of mitochondrial ATP production The ATP production in the isolated rat cortical mitochondria was measured using a bioluminescent ATP detection kit (Promega, USA). After treatment, isolated mitochondria were immediately incubated with ATP synthesizing substrates (2.5 mM ADP, 1 mM L-()-malic acid and 1 mM pyruvate) for 5 min at 30 C before mixed with luciferin substrate and luciferase enzyme in the kit to produce bioluminesecence. The bioluminescence was assessed on a microplate reader (Envision, Perkin Elmer). The ATP level was calculated as described in the manual of the kit and normalized by control group.
2.4. T maze behavioral test
2.9. Enzyme-linked immunosorbent assay (ELISA) for Ab1-42 and Ab1-40
Food rewarded T maze is very sensitive for testing the working memory (Deacon and Rawlins, 2006; Soares et al., 2013). In the present study, food rewarded T maze was conducted according to literature (Deacon and Rawlins, 2006) with some modification. The test consisted of four stages and lasted for 13 days in total. Briefly, in stage I, mice were allowed to explore freely in the maze (sunflower seeds were
Mitochondrial pellets were lysed in 8-fold buffer A (5 M guanidine HCl, 50 mM Tris HCl, pH 8.0) for 3 h, and then centrifuged at 12,000 g for 10 min to remove insoluble matters before diluted with buffer B (Dulbecco's phosphate buffered saline with 5% BSA and 0.03% Tween-20) to appropriate concentration. The following ELISA process was under the instruction of kit manual (Invitrogen).
Please cite this article in press as: Ye, C.Y., et al., Donepezil attenuates Ab-associated mitochondrial dysfunction and reduces mitochondrial Ab accumulation in vivo and in vitro, Neuropharmacology (2015), http://dx.doi.org/10.1016/j.neuropharm.2015.02.020
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C.Y. Ye et al. / Neuropharmacology xxx (2015) 1e8 2.10. Western blot analysis The mitochondrial pellets were lysed by RIPA buffer (Beyotime, China) and mixed with equal volume of 2 Loading buffer without DTT. The protein mixture was resolved in a 4%/10%/17%/20% gradient Tricine-SDS-PAGE gel, and transferred to a nitrocellulose membrane (Whatman), followed by blocking with 5% nonfat milk for 1 h at room temperature. After overnight incubation with anti-Ab antibody (6E10, 1:1000, Covance) and the mitochondrial outer membrane marker (voltage dependent anion channel, VDAC) antibody (1:5000, Calbiochem) at 4 C, the membranes were washed 3 times in TBST (0.005% Tween-20, 50 mM Tris and 150 mM NaCl, pH 7.5) and incubated with HRP-conjugated secondary antibody for 1 h at room temperature. After TBST washing for another 3 times, the bands were detected using chemiluminescence reaction (Immobilon ECL, Millipore). Similar as the study of Walls et al. (Walls et al., 2012), the Ab level of each group is represented as the sum of all the bands detected by 6E10, with VDAC as the loading control of mitochondria. 2.11. Immunogold labeled microscopic staining Mice were anesthetized with 10% chloral hydrate (0.25 mL/30 g), before perfusion with saline and fixation buffer (4% paraformaldehyde þ 0.5% glutaraldehyde). After perfusion, the brain was further fixed for 48 h. Frontal cortex (Bregma 2.1 to 1.42) was carefully cut and trimmed into 1 mm3 pieces, postfixed in 2% osmium tetroxide for 30 min, and then dehydrated, embedded and cut into ultrathin slices. The ultrathin slices were then blocked with 1% BSA in PBS for 30 min at room temperature before incubated with anti-Ab antibody (6E10, 1:300, Covance) for 48 h at 4 C. After washing, the slices were then incubated with 15 nm gold labeled secondary antibody (1:75, British Biocell) for 3 h at room temperature. The slices were further stained with aqueous lead citrate and observed under a CM120 transmission electron microscope (Philips). Five slices from the same layers of cortical tissue (Bregma 2.1 to 1.42) of each mouse were examined and used for statistical analysis. 2.12. Statistical analysis Data were expressed as mean ± SEM. Student t test was applied in the comparisons between two groups. Multiple comparisons between model group and
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different concentrations of donepezil treated groups were analyzed by one way ANOVA, followed by Dunnett test. Differences were considered significant at p < 0.05.
3. Results 3.1. Donepezil ameliorates the Ab related behavioral deficits of APP/ PS1 mice AD-associated behavioral deficits including cognitive decline and psychiatric symptoms (Cummings et al., 1994; Mega et al., 1996; Petersen et al., 1999) are well reproduced in APP transgenic animal models (Cioanca et al., 2014; Le Cudennec et al., 2008; Yang et al., 2012). In order to evaluate the beneficial effect of donepezil on AD-related cognitive and psychiatric behavioral performances, we measured the working memory and anxiety by two widely adopted behavioral tasksdfood rewarded T maze (Colombo et al., 1996; Soares et al., 2013) and elevated plus maze (EPM) (Hadamitzky et al., 2014; Cioanca et al., 2014; Walf and Frye, 2007), respectively. To ensure the effect of donepezil in our behavioral tests was not directly associated with acute AChE inhibition, we examined the AChE activity at the same time point of our behavioral test. As shown in Fig. 1A, there are no significant differences between the AChE activity of donepezil ((62.70 ± 10.38) % of wild type) and vehicle ((79.93 ± 21.88) % of wild type) treated APP/PS1 mice (p ¼ 0.4932). As shown in Fig. 1B, vehicle treated APP/PS1 mice show decreased accuracy ((49.78 ± 6.20) %, p < 0.05 vs wild type group) in T maze as compared with wild type control
Fig. 1. Donepezil (1 mg/kg) attenuates the behavioral deficits of APP/PS1 mice. A) AChE activity of the mice 16 h after last drug administration. B) Statistical graph of T maze performance. T maze accuracy is calculated from the right arm choose percentage. EPM accuracy is represented by C) the entry number and D) time into the open arm. Values are mean ± SEM. n ¼ 8e10 per group. *p < 0.05, ***p < 0.001 vs wild type control; #p < 0.05, ##p < 0.01 vs vehicle treated APP/PS1 transgenic mice.
Please cite this article in press as: Ye, C.Y., et al., Donepezil attenuates Ab-associated mitochondrial dysfunction and reduces mitochondrial Ab accumulation in vivo and in vitro, Neuropharmacology (2015), http://dx.doi.org/10.1016/j.neuropharm.2015.02.020
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((65.55 ± 4.33) %), indicating significant working memory deficit in APP/PS1 mice, while donepezil treatment ((74.14 ± 4.20) %) significantly rescues this memory deficit (p < 0.01 vs vehicletreated APP/PS1 group). We then assessed the anxiety of APP/PS1 mice with EPM. As shown in Fig. 1C, the entry number into the open arm of vehicle treated APP/PS1 mice (0.60 ± 0.22, p < 0.001 vs wild type group) is significantly lower than wild type control (2.67 ± 0.41), while donepezil treatment markedly reverses the decrease to 2.90 ± 0.96 (p < 0.05 vs vehicle-treated APP/PS1 group). There is no obvious difference in the total entry number into open arm and close arm between the three groups (data not shown), indicating that the variations in EPM performance are not due to the individual propensity for activity of animals. As shown in Fig. 1D, the time in open arm of vehicle treated APP/PS1 mice (5.42 ± 1.89, p < 0.001 vs wild type group) is significantly lower than wild type control (26.41 ± 5.04), while donepezil treatment reverses the decrease to 21.82 ± 7.61 (p ¼ 0.0509 vs vehicle-treated APP/PS1 group). 3.2. Donepezil reduces Ca2þ-induced mitochondrial swelling To determine whether the memory improvement of 1 mg/kg donepezil consecutively administrated for 2 months on APP/PS1 transgenic mice is associated with preservation of mitochondrial function, the effect of donepezil on brain mitochondrial swelling was detected to provide an indirect measure of mitochondrial membrane permeability. In the mitochondria isolated from each mice group, the Ca2þ exposure induces a significant decrease in the optical density at 540 nm (Fig. 2A). As compared with the wild type group, the Ca2þ-induced mitochondrial swelling in the vehicle treated APP/PS1 group exhibits a much more rapid decrease during 30 min duration, while donepezil treatment shows a protective effect against the mitochondrial swelling. As shown in Fig. 2B, the mitochondria of vehicle treated APP/PS1 mice show larger swelling slope ((145.50 ± 12.09) % of wild type, p ¼ 0.08) than that of wild type control ((110.50 ± 11.04) %), reflected by the decreasing of optical density at 540 nm. Donepezil treatment significantly decreases the slope to the wild type level ((105.50 ± 12.26) %, p < 0.05). 3.3. Donepezil reduces brain mitochondrial Ab accumulation in APP/PS1 mice Mitochondria are reported as an intracellular Ab accumulating organelle and the Ab level in mitochondria is associated with the mitochondrial dysfunction (Dragicevic et al., 2010; Du et al., 2010). To investigate whether the protective effect of donepezil on mitochondrial dysfunction in vivo is attributed to mitochondrial Ab level, the amount and distribution of Ab in the brain mitochondria of APP/PS1 mice were investigated with immunogold labeled electronic microscopic technology. As shown in Fig. 3A and Fig. 3B, there are more gold particles (indicated by red arrow head, Fig. 3A) inside the mitochondria of vehicle treated APP/PS1 mice (36.45 ± 4.00 particles every 37.52 mm2, p < 0.05 vs control group) than control group (11.10 ± 5.87 particles every 37.52 mm2), while donepezil treatment significantly reduces this accumulation to 20.29 ± 2.87 particles every 37.52 mm2 (Fig. 3B, p < 0.05). We further detected the mitochondrial Ab1-40 and Ab1-42 levels in the APP/PS1 mice with ELISA. As shown in Fig. 3C and D, the accumulation of Ab1-40 (981.70 ± 165.50 pg/mg) and Ab1-42 (10721.00 ± 1869.00 pg/mg) in the mitochondria of APP/PS1 mice are both significantly higher than that in the wild type control mice (118.00 ± 15.73 pg/mg Ab1-40, 269.90 ± 59.10 pg/mg Ab1-42, p < 0.001 vs wild type mice), while donepezil treatment significantly reduces the mitochondrial Ab1-40 and Ab1-42 accumulation to
Fig. 2. Donepezil (1 mg/kg) attenuates the ex vivo mitochondrial dysfunction in APP/ PS1 transgenic mice. A) Representative swelling curve of mitochondria isolated from APP/PS1 transgenic mice and wild type mice. Swelling slope is indicated by the decrease in OD540. B) Statistical graph of mitochondrial swelling slope. Values are expressed as mean ± SEM. n ¼ 4e6 per group. #p < 0.05 vs vehicle treated APP/PS1 transgenic mice.
546.70 ± 95.60 pg/mg (p < 0.05 vs vehicle-treated APP/PS1 mice) and 4876.00 ± 729.60 pg/mg (p < 0.01 vs vehicle-treated APP/PS1 mice), respectively. 3.4. Donepezil ameliorates mitochondrial functional deficits in isolated brain mitochondria from SD rats In order to further elucidate the potential mechanisms of donepezil on Ab-associated mitochondrial dysfunction, we employed an in vitro subcellular system, isolated brain mitochondria from SD rats, to minimize the interference from AChE. To identify the integrity of isolated brain cortical mitochondria, mitochondrial ultrastructural morphology was assessed before the initiation of following experiments. Oligomeric Ab1-42 was used in this study to induce mitochondrial dysfunction, since the “toxic Ab oligomer” hypothesis has attracted considerable attention in the recent years and Ab1-42 oligomers are believed as one of the crucial drivers of mitochondrial dysfunction (Hou et al., 2014; Sun et al., 2014). First of all, we tested the beneficial effects of donepezil on Ab1-42-induced mitochondrial dysfunction. As shown in Fig. 4A and Fig. 4B and 1 mM oligomeric Ab1-42 exposure causes a dramatic drop of the optical density at 540 nm ((128.00 ± 4.40) % of control, p < 0.01), indicating severe mitochondrial swelling, while
Please cite this article in press as: Ye, C.Y., et al., Donepezil attenuates Ab-associated mitochondrial dysfunction and reduces mitochondrial Ab accumulation in vivo and in vitro, Neuropharmacology (2015), http://dx.doi.org/10.1016/j.neuropharm.2015.02.020
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Fig. 3. Donepezil (1 mg/kg) reduces the Ab accumulation in the mitochondria of APP/PS1 mice. A) Representative graph of mitochondrial Ab detected by immunogold labeled electronic microscope with 6E10 antibody. Mitochondrial Ab signals are indicated by red arrow heads. Scale bar ¼ 1 mm. B) Statistical result of immunogold staining. n ¼ 4e6 per group. C) Ab1-40 and D) Ab1-42 level in APP/PS1 transgenic mice detected by ELISA. Values are expressed as mean ± SEM. n ¼ 8e10 per group. *p < 0.05, ***p < 0.001 vs wild type control; #p < 0.05, ##p < 0.01 vs vehicle treated APP/PS1 transgenic mice. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
donepezil at 0.1 mM ((103.00 ± 6.30) % of control, p < 0.05 vs Ab group) and 1 mM ((93.70 ± 5.40) % of control, p < 0.001 vs Ab group) final concentrations could both ameliorate this mitochondrial swelling. Similarly, as shown in Fig. 4C, oligomeric Ab1-42 exposure significantly impairs the in vitro ATP producing ability of mitochondria to (90.47 ± 1.01) % of control (p < 0.01), while donepezil at 0.1 mM ((96.82 ± 2.16) % of control, p < 0.05 vs Ab group) and 1 mM ((96.28 ± 0.69) % of control, p < 0.05 vs Ab group) final concentrations could significantly ameliorate the Ab-associated mitochondrial dysfunction in the isolated mitochondria. 3.5. Donepezil reduces mitochondrial Ab overload in isolated brain mitochondria from SD rats To further investigate whether donepezil could directly inhibit the Ab accumulation in the mitochondria, we then measured the Ab1-42 level in the pellet of isolated brain mitochondria after 1 mM oligomeric Ab1-42 exposure. After being exposed to 1 mM oligomeric Ab1-42, the isolated brain mitochondria were treated with different concentrations of donepezil. As expected, Ab1-42 was prone to accumulate in the mitochondria, since we detected a high level of Abin the pellet of the mitochondria treated with oligomeric Ab1-42 (Fig. 5A and Fig. 5B, p < 0.01). Western blot result shows that the total level of Ab bands detected with 6E10 antibody in Ab1-42 incubated group was 6.39 ± 1.21 fold of control group (p < 0.01), while donepezil at 0.1 mM (3.41 ± 0.77, p < 0.01 vs Ab group) and 1 mM (4.29 ± 1.12, p < 0.05 vs Ab group) final concentrations significantly inhibit the abnormal accumulation of Ab in the mitochondria, to approximately 4 fold of control group. 4. Discussion Although the neuroprotective effects of donepezil beyond AChE inhibition, especially significant ameliorative effects on Ab-
associated neurotoxicity (Noh et al., 2009; Romberg et al., 2011; Tang et al., 2014), have attracted intense interest in recent years, the exact molecular mechanisms underlying have not yet been clarified. The major impressive characteristics of the present study include: 1) the ameliorative effect of chronic treatment with donepezil on behavioral deficits in both T-maze and elevated cross maze task in APP/PS1 mice is correlated with preserved mitochondrial function and reduced mitochondrial Ab level in the brain; 2) donepezil treatment significantly ameliorated oligomeric Ab1-42induced subcellular mitochondrial dysfunction and reduced the accumulation of Ab in the isolated brain mitochondria. As an extension of previous studies, the current study confirmed the ameliorative effects of donepezil on Ab-associated behavioral deficits and mitochondrial dysfunction by employing in vivo and in vitro models. First, in consistence with previous studies (He et al., 2009; Lok et al., 2013), our study verifies the multi-aspect behavioral deficits in APP/PS1 transgenic mice, and shows that donepezil could ameliorate the deficits of APP/PS1 mice in both food rewarded T maze and elevated plus maze. It is worth mentioning that to avoid the acute symptomatic improvement caused by acetylcholine elevation, above behaviors were assessed no less than 16 h after the last drug administration, a time point when the AChE inhibition of donepezil should have already diminished, while in previous studies functional tests were usually conducted shortly after drug administration (Noda et al., 2010; Wang et al., 2010; Winocur et al., 2011). As shown in literature (Abe et al., 2003) and our unpublished data, the maximal AChE inhibition of donepezil occurs at about 1 h after drug administration in mice and the inhibition diminishes with time passing. Above finding in the inhibitory effect of donepezil on AChE may be associated with its pharmacodynamics data, as donepezil was reported to have a half-life of about 3 h in rats (Goh et al., 2011; Nirogi et al., 2012). Therefore, the protection we observed in APP/PS1 mice may mainly indicate the neuroprotective effects of donepezil aside from its AChE inhibition.
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Fig. 5. Donepezil (0.1 and 1 mM) reduces the Ab1-42 accumulation in isolated mitochondria from rat brains. A) Representative picture of mitochondrial Ab accumulation detected by western blot with 6E10 antibody. VDAC was used as loading control. B) Statistical graph of the total Ab signal detected in the above blot. Values are expressed as mean ± SEM. Data are from at least 4 independent experiments. Con: control; Don: donepezil. ***p < 0.001 vs control group; #p < 0.05, ##p < 0.01 vs Ab group.
Fig. 4. Donepezil (0.1 and 1 mM) ameliorates the dysfunction of isolated rat mitochondria caused by 1 mM oligomeric Ab1-42 in vitro. A) Representative swelling curve of mitochondria treated by oligomeric Ab1-42 with or without donepezil. Swelling slope is indicated by the decrease in OD540. B) Statistical graph of mitochondrial swelling slope. C) The ATP producing ability of mitochondria treated with 1 mM oligomeric Ab1-42 followed by donepezil treatment. Values are expressed as mean ± SEM. Data are from at least 4 independent experiments. Con: control; Don: donepezil. **p < 0.01 vs control group; #p < 0.05, ##p < 0.01 vs Ab group.
Ab plays a pivotal role in the mitochondrial dysfunctions, because mitochondrial deficits like oxidative stress, energy deficiency and mitochondrial depolarization were frequently seen in Ab treated cell models and Ab overexpression animal models (Dragicevic et al., 2011; Gao and Tang, 2006; Sarkar et al., 2014; Takuma et al., 2005). Therefore, reversing Ab-associated mitochondrial dysfunction may provide an opportunity to recover the cognitive and psychiatric behavioral deficits. Interestingly, the present study found that the ameliorative effect of donepezil on
behavioral deficits is positively correlated with the alleviated brain mitochondrial swelling. Moreover, mitochondrial Ab accumulation is frequently seen in AD brains (Chen and Yan, 2006; Hansson Petersen et al., 2008), and increases with age at a speed much faster than Ab accumulation in brain tissue homogenates of APP/ PS1 transgenic mice (our unpublished data). In addition, mitochondrial Ab accumulation was found to be associated with the extent of mitochondrial dysfunction in different brain regions and the degree of cognitive impairment in APP and APP/PS1 transgenic mice, while reducing mitochondrial Ab accumulation can improve mitochondrial function (Dragicevic et al., 2010; Yao et al., 2011). In the current study, we found that donepezil administration could effectively reduce the mitochondrial Ab accumulation in APP/PS1 mice, which as suggested by the above literature, may contribute to the mitochondrial functional enhancement and behavioral improvement by donepezil. This is the first report of donepezil reducing mitochondrial Ab accumulation to our knowledge. The
Please cite this article in press as: Ye, C.Y., et al., Donepezil attenuates Ab-associated mitochondrial dysfunction and reduces mitochondrial Ab accumulation in vivo and in vitro, Neuropharmacology (2015), http://dx.doi.org/10.1016/j.neuropharm.2015.02.020
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above data may further affirm the critical role of mitochondrial Ab in the disease progression. In order to minimize the influence of AChE inhibitory effect of donepezil, isolated mitochondria were then adopted in this study to further understand the molecular mechanism of donepezil. It is reported that Ab is able to accumulate in isolated mitochondria (Hansson Petersen et al., 2008) thus cause mitochondrial swelling and cytochrome c release (Kim et al., 2002; Rodrigues et al., 2000). Our result on isolated mitochondria showed that donepezil ameliorated the mitochondrial swelling and reduced ATP level on Ab1-42 injured mitochondria, as well as reducing the Ab1-42 accumulation. As an extension of previous findings, the study presented herein together with the previous reports suggest that the subcellular pharmacological benefits of donepezil on mitochondrial ATP production and mitochondrial swelling may be, at least partly, mediated through suppressing the accumulation of Ab1-42 in the mitochondria, although the precise mechanism still need to be clarified. Above results observed that donepezil had similar influences on mitochondrial dysfunction and mitochondrial Ab accumulation in both in vivo and in vitro models, which indicates that the direct effect of donepezil on isolated mitochondria may possibly contribute to the findings in the APP/PS1 transgenic mice. We also verified that donepezil could significantly ameliorate the mitochondrial swelling induced by Ab exposure in the isolated mouse mitochondria in our preliminary study. Still, further studies will be needed for making close linkage between in vivo and in vitro results, as the current in vitro studies were mainly done with isolated mitochondria from rat brains. To sum up, our research for the first time found that donepezil could reduce mitochondrial Ab accumulation in APP/PS1 mice, probably contributing to its cognitive benefits. Furthermore, we found that donepezil have direct mitochondrial protection in isolated mitochondria against Ab1-42 insult, as well as reducing mitochondrial Ab accumulation, which could be different from its formerly reported specific effect on AChE. Our data might shed more light on the clinical benefits gained by donepezil and provide useful clues for future AD drug development, however, the precise mechanisms underlying the beneficial effect of the drug remains to be explored in future study. Acknowledgments This work was supported by the National Natural Science Foundation of China (No 81173034, 81072646), the National Science & Technology Major Project “Key New Drug Creation and Manufacturing Program” of China (No. 2012ZX09301001-004), and the Ministry of Science and Technology of China (No 2011CB510004). References Abe, Y., Aoyagi, A., Hara, T., Abe, K., Yamazaki, R., Kumagae, Y., Naruto, S., Koyama, K., Marumoto, S., Tago, K., Toda, N., Takami, K., Yamada, N., Ori, M., Kogen, H., Kaneko, T., 2003. Pharmacological characterization of RS-1259, an orally active dual inhibitor of acetylcholinesterase and serotonin transporter, in rodents: possible treatment of Alzheimer's disease. J. Pharmacol. Sci. 93, 95e105. Alzheimer's Association, 2014. 2014 Alzheimer's disease facts and figures. Alzheimer's Dementia 10, 1e75. Benilova, I., Karran, E., Strooper, B., 2012. The toxic Ab oligomer and Alzheimer's disease: an emperor in need of clothes. Nat. Neurosci. 15, 349e357. Chen, X., Yan, S.D., 2006. Mitochondrial abeta: a potential cause of metabolic dysfunction in Alzheimer's disease. IUBMB Life 58, 686e694. Cioanca, O., Hritcu, L., Mihasan, M., Trifan, A., Hancianu, M., 2014. Inhalation of coriander volatile oil increased anxiolytic-antidepressant-like behaviors and decreased oxidative status in b-amyloid (1-42) rat model of Alzheimer's disease. Physiol. Behav. 131, 68e74. Colombo, G., Agabio, R., Balaklievskaia, N., Lobina, C., Reali, R., Fadda, F., Gessa, G.L., 1996. T-maze and food reinforcement: an inexpensive drug discrimination procedure. J. Neurosci. Methods 67, 83e87.
7
Cummings, J.L., Mega, M., Gray, K., Rosenberg-Thompson, S., Carusi, D.A., Gornbein, J., 1994. The neuropsychiatric inventory: comprehensive assessment of psychopathology in dementia. Neurology 44, 2308e2314. Deacon, R.M.J., Rawlins, J.N.P., 2006. T-maze alternation in the rodent. Nat. Protoc. 1, 7e12. Dragicevic, N., Mamcarz, M., Zhu, Y., Buzzeo, R., Tan, J., Arendash, G.W., Bradshaw, P.C., 2010. Mitochondrial amyloid-b levels are associated with the extent of mitochondrial dysfunction in different brain regions and the degree of cognitive impairment in Alzheimer's transgenic mice. J. Alzheimers Dis. 20 (Suppl. 2), S535eS550. Dragicevic, N., Smith, A., Lin, X., Yuan, F., Copes, N., Delic, V., Tan, J., Cao, C., Shytle, R.D., Bradshaw, P.C., 2011. Green tea epigallocatechin-3-gallate (EGCG) and other flavonoids reduce Alzheimer's amyloid-induced mitochondrial dysfunction. J. Alzheimers Dis. 26, 507e521. Du, H., Guo, L., Fang, F., Chen, D., Sosunov, A.A., McKhann, G.M., Yan, Y., Wang, C., Zhang, H., Molkentin, J.D., Gunn-Moore, F.J., Vonsattel, J.P., Arancio, O., Chen, J.X., Yan, S.D., 2008. Cyclophilin D deficiency attenuates mitochondrial and neuronal perturbation and ameliorates learning and memory in Alzheimer's disease. Nat. Med. 14, 1097e1105. Du, H., Guo, L., Yan, S., Sosunov, A.A., McKhann, G.M., Yan, S.S., 2010. Early deficits in synaptic mitochondria in an Alzheimer's disease mouse model. Proc. Natl. Acad. Sci. U. S. A. 107, 18670e18675. Gandy, S., DeKosky, S.T., 2013. Toward the treatment and prevention of Alzheimer's disease: rational strategies and recent progress. Annu Rev. Med. 64, 367e383. Gao, X., Tang, X.C., 2006. Huperzine A attenuates mitochondrial dysfunction in bamyloid-treated PC12 cells by reducing oxygen free radicals accumulation and improving mitochondrial energy metabolism. J. Neurosci. Res. 83, 1048e1057. Giacobini, E., Gold, G., 2013. Alzheimer disease therapyemoving from amyloid-b to tau. Nat. Rev. Neurol. 9, 677e686. Goh, C.W., Aw, C.C., Lee, J.H., Chen, C.P., Browne, E.R., 2011. Pharmacokinetic and pharmacodynamic properties of cholinesterase inhibitors donepezil, tacrine, and galantamine in aged and young lister hooded rats. Drug Metab. Dispos. 39, 402e411. Goure, W.F., Krafft, G.A., Jerecic, J., Hefti, F., 2014. Targeting the proper amyloid-beta neuronal toxins: a path forward for Alzheimer's disease immunotherapeutics. Alzheimers Res. Ther. 6, 42. Hadamitzky, M., Herring, A., Keyvani, K., Doenlen, R., Krugel, U., Bosche, K., Orlowski, K., Engler, H., Schedlowski, M., 2014. Acute systemic rapamycin induces neurobehavioral alterations in rats. Behav. Brain Res. 273, 16e22. Hansson Petersen, C.A., Alikhani, N., Behbahani, H., Wiehager, B., Pavlov, P.F., Alafuzoff, I., Leinonen, V., Ito, A., Winblad, B., Glaser, E., Ankarcrona, M., 2008. The amyloid b-peptide is imported into mitochondria via the TOM import machinery and localized to mitochondrial cristae. Proc. Natl. Acad. Sci. U. S. A. 105, 13145e13150. Hashimoto, M., Kazui, H., Matsumoto, K., Nakano, Y., Yasuda, M., Mori, E., 2005. Does donepezil treatment slow the progression of hippocampal atrophy in patients with Alzheimer's disease? Am. J. Psychiatry 162, 676e682. He, J., Luo, H., Yan, B., Yu, Y., Wang, H., Wei, Z., Zhang, Y., Xu, H., Tempier, A., Li, X., Li, X.M., 2009. Beneficial effects of quetiapine in a transgenic mouse model of Alzheimer's disease. Neurobiol. Aging 30, 1205e1216. Hou, Y., Ghosh, P., Wan, R., Ouyang, X., Cheng, H., Mattson, M.P., Cheng, A., 2014. Permeability transition pore-mediated mitochondrial superoxide flashes mediate an early inhibitory effect of Ab1-42 on neural progenitor cell proliferation. Neurobiol. Aging 35, 975e989. Kim, H.S., Lee, J.H., Lee, J.P., Kim, E.M., Chang, K.A., Park, C.H., Jeong, S.J., Wittendorp, M.C., Seo, J.H., Choi, S.H., Suh, Y.H., 2002. Amyloid b peptide induces cytochrome C release from isolated mitochondria. Neuroreport 13, 1989e1993. LaFerla, F.M., Green, K.N., Oddo, S., 2007. Intracellular amyloid-b in Alzheimer's disease. Nat. Rev. Neurosci. 8, 499e509. Lambert, M.P., Barlow, A.K., Chromy, B.A., Edwards, C., Freed, R., Liosatos, M., Morgans, T.E., Rozovsky, I., Trommer, B., Viola, K.L., Wals, P., Zhang, C., Finch, C.E., Krafft, G.A., Klein, W.L., 1998. Diffusible, nonfibrillar ligands derived from Ab are potent central nervous system neurotoxins. Proc. Natl. Acad. Sci. U. S. A. 95, 6448e6453. Le Cudennec, C., Faure, A., Ly, M., Delatour, B., 2008. One-year longitudinal evaluation of sensorimotor functions in APP751SL transgenic mice. Genes. Brain Behav. 7 (Suppl. 1), 83e91. Lok, K., Zhao, H., Zhang, C., He, N., Shen, H., Wang, Z., Zhao, W., Yin, M., 2013. Effects of accelerated senescence on learning and memory, locomotion and anxietylike behavior in APP/PS1 mouse model of Alzheimer's disease. J. Neurol. Sci. 335, 145e154. Manczak, M., Anekonda, T.S., Henson, E., Park, B.S., Quinn, J., Reddy, P.H., 2006. Mitochondria are a direct site of Ab accumulation in Alzheimer's disease neurons: implications for free radical generation and oxidative damage in disease progression. Hum. Mol. Genet. 15, 1437e1449. Mangialasche, F., Solomon, A., Winblad, B., Mecocci, P., Kivipelto, M., 2010. Alzheimer's disease: clinical trials and drug development. Lancet Neurol. 9, 702e716. McClean, P.L., Holscher, C., 2014. Lixisenatide, a drug developed to treat type 2 diabetes, shows neuroprotective effects in a mouse model of Alzheimer's disease. Neuropharmacology 86, 241e258. Mega, M.S., Cummings, J.L., Fiorello, T., Gornbein, J., 1996. The spectrum of behavioral changes in Alzheimer's disease. Neurology 46, 130e135.
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Meunier, J., Ieni, J., Maurice, T., 2006. The anti-amnesic and neuroprotective effects of donepezil against amyloid b25-35 peptide-induced toxicity in mice involve an interaction with the sigma1 receptor. Br. J. Pharmacol. 149, 998e1012. Narayan, P., Holmstrom, K.M., Kim, D.H., Whitcomb, D.J., Wilson, M.R., St GeorgeHyslop, P., Wood, N.W., Dobson, C.M., Cho, K., Abramov, A.Y., Klenerman, D., 2014. Rare individual amyloid-b oligomers act on astrocytes to initiate neuronal damage. Biochemistry 53, 2442e2453. Nirogi, R., Bhyrapuneni, G., Kandikere, V., Benade, V., Muddana, N., Saralaya, R., Irappanavar, S., Ponnamaneni, R., Mukkanti, K., 2012. Concurrent administration of atypical antipsychotics and donepezil: drug interaction study in rats. Eur. J. Drug Metab. Pharmacokinet. 37, 155e161. Noda, Y., Mouri, A., Ando, Y., Waki, Y., Yamada, S., Yoshimi, A., Yamada, K., Ozaki, N., Wang, D., Nabeshima, T., 2010. Galantamine ameliorates the impairment of recognition memory in mice repeatedly treated with methamphetamine: involvement of allosteric potentiation of nicotinic acetylcholine receptors and dopaminergic-ERK1/2 systems. Int. J. Neuropsychopharmacol. 13, 1343e1354. Noh, M.-Y., Koh, S.-H., Kim, Y., Kim, H.Y., Cho, G.W., Kim, S.H., 2009. Neuroprotective effects of donepezil through inhibition of GSK-3 activity in amyloid-b-induced neuronal cell death. J. Neurochem. 108, 1116e1125. Petersen, R.C., Smith, G.E., Waring, S.C., Ivnik, R.J., Tangalos, E.G., Kokmen, E., 1999. Mild cognitive impairment: clinical characterization and outcome. Arch. Neurol. 56, 303e308. Plassman, B.L., Langa, K.M., Fisher, G.G., Heeringa, S.G., Weir, D.R., Ofstedal, M.B., Burke, J.R., Hurd, M.D., Potter, G.G., Rodgers, W.L., Steffens, D.C., Willis, R.J., Wallace, R.B., 2007. Prevalence of dementia in the United States: the aging, demographics, and memory study. Neuroepidemiology 29, 125e132. Risacher, S.L., Saykin, A.J., 2013. Neuroimaging and other biomarkers for Alzheimer's disease: the changing landscape of early detection. Annu Rev. Clin. Psychol. 9, 621e648. Rodrigues, C.M., Sola, S., Silva, R., Brites, D., 2000. Bilirubin and amyloid-b peptide induce cytochrome c release through mitochondrial membrane permeabilization. Mol. Med. 6, 936e946. Romberg, C., Mattson, M.P., Mughal, M.R., Bussey, T.J., Saksida, L.M., 2011. Impaired attention in the 3xTgAD mouse model of Alzheimer's disease: rescue by donepezil (Aricept). J. Neurosci. 31, 3500e3507. Sarkar, P., Zaja, I., Bienengraeber, M., Rarick, K.R., Terashvili, M., Canfield, S., Falck, J.R., Harder, D.R., 2014. Epoxyeicosatrienoic acids pretreatment improves amyloid b-induced mitochondrial dysfunction in cultured rat hippocampal astrocytes. Am. J. Physiol. Heart Circ. Physiol. 306, H475eH484.
Selkoe, D.J., 1986. Altered structural proteins in plaques and tangles: what do they tell us about the biology of Alzheimer's disease? Neurobiol. Aging 7, 425e432. Shankar, G.M., Li, S., Mehta, T.H., Garcia-Munoz, A., Shepardson, N.E., Smith, I., Brett, F.M., Farrell, M.A., Rowan, M.J., Lemere, C.A., Regan, C.M., Walsh, D.M., Sabatini, B.L., Selkoe, D.J., 2008. Amyloid-b protein dimers isolated directly from Alzheimer's brains impair synaptic plasticity and memory. Nat. Med. 14, 837e842. Soares, J.C., Oliveira, M.G., Ferreira, T.L., 2013. Inactivation of muscarinic receptors impairs place and response learning: implications for multiple memory systems. Neuropharmacology 73, 320e326. Sun, Q., Jia, N., Wang, W., Jin, H., Xu, J., Hu, H., 2014. Protective effects of astragaloside IV against Ab1-42 neurotoxicity by inhibiting the mitochondrial permeability transition pore opening. PLoS One 9, e98866. Takuma, K., Yao, J., Huang, J., Xu, H., Chen, X., Luddy, J., Trillat, A.C., Stern, D.M., Arancio, O., Yan, S.S., 2005. ABAD enhances Abeta-induced cell stress via mitochondrial dysfunction. FASEB J. 19, 597e598. Tang, S.S., Hong, H., Chen, L., Mei, Z.L., Ji, M.J., Xiang, G.Q., Li, N., Ji, H., 2014. Involvement of cysteinyl leukotriene receptor 1 in Ab1-42-induced neurotoxicity in vitro and in vivo. Neurobiol. Aging 35, 590e599. Walf, A.A., Frye, C.A., 2007. The use of the elevated plus maze as an assay of anxietyrelated behavior in rodents. Nat. Protoc. 2, 322e328. Walls, K.C., Coskun, P., Gallegos-Perez, J.L., Zadourian, N., Freude, K., Rasool, S., Blurton-Jones, M., Green, K.N., LaFerla, F.M., 2012. Swedish Alzheimer mutation induces mitochondrial dysfunction mediated by HSP60 mislocalization of amyloid precursor protein (APP) and b-amyloid. J. Bio. Chem. 287, 30317e30327. Wang, J., Zhang, H.Y., Tang, X.C., 2010. Huperzine A improves chronic inflammation and cognitive decline in rats with cerebral hypoperfusion. J. Neurosci. Res. 88, 807e815. Winocur, G., Binns, M.A., Tannock, I., 2011. Donepezil reduces cognitive impairment associated with anti-cancer drugs in a mouse model. Neuropharmacology 61, 1222e1228. Yang, L., Ye, C.Y., Huang, X.T., Tang, X.C., Zhang, H.Y., 2012. Decreased accumulation of subcellular amyloid-beta with Improved mitochondrial function mediates the neuroprotective effect of Huperzine A. J. Alzheimers Dis. 31, 131e142. Yao, J., Du, H., Yan, S., Fang, F., Wang, C., Lue, L.F., Guo, L., Chen, D., Stern, D.M., Gunn Moore, F.J., Xi Chen, J., Arancio, O., Yan, S.S., 2011. Inhibition of amyloid-b (Ab) peptide-binding alcohol dehydrogenase-Abeta interaction reduces Ab accumulation and improves mitochondrial function in a mouse model of Alzheimer's disease. J. Neurosci. 31, 2313e2320.
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