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Involvement of cysteinyl leukotriene receptor 1 in Aβ1–42-induced neurotoxicity in vitro and in vivo
Neurobiology of Aging 35 (2014) 590e599
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Involvement of cysteinyl leukotriene receptor 1 in Ab1e42-induced neurotoxicity in vitro and in vivo Su-Su Tang, Hao Hong*, Lan Chen, Zhen-lin Mei, Miao-jin Ji, Guo-qing Xiang, Ning Li, Hui Ji* Department of Pharmacology, China Pharmaceutical University, Nanjing, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 27 June 2013 Received in revised form 15 September 2013 Accepted 22 September 2013 Available online 23 October 2013
Accumulation of amyloid-b (Ab) is thought to be associated with the progressive neuronal death observed in Alzheimer’s disease, but the mechanisms underlying neurotoxicity triggered by Ab remain elusive. In the current study, we investigated the roles of cysteinyl leukotriene receptor 1 (CysLT1R) in Ab1e42-induced neurotoxicity in vitro or in vivo. In vitro exposure of mouse primary neurons to Ab1e42 caused a gradual increases in CysLT1R expression. In vivo bilateral intrahippocampal injection of Ab1e42 also elicited time-dependent increases of CysLT1R expression in the hippocampus and cortex of mice. The CysLT1R antagonist pranlukast not only reversed Ab1e42-induced upregulation of CysLT1R, but also suppressed Ab1e42-triggered neurotoxicity evidenced by enhanced nuclear factor-kappa B p65, activated caspase-3, decreased B-cell lymphoma-2 and cell viability and impaired memory. Furthermore, chronic treatment with pranlukast produced similar beneficial effects on memory behavior and hippocampal long-term potentiation to memantine or donepezil in intrahippocampal Ab1e42-injected mice. Our data indicate that CysLT1R is involved in Ab1e42-induced neurotoxicity, and that blockade of CysLT1R, such as application of CysLT1R antagonist, could be a novel and promising strategy for the treatment of Alzheimer’s disease. Ó 2014 Elsevier Inc. All rights reserved.
Keywords: Cysteinyl leukotriene receptor 1 Ab1e42 Neurotoxicity NF-kB Caspase-3 Bcl-2 Memory
1. Introduction Alzheimer’s disease (AD) is the most common neurodegenerative disorder that causes progressive loss of cognitive function and dementia. Amyloid-b (Ab) deposition and neurofibrillary tangle formation are the major pathogenic mechanisms that act in concert to produce memory dysfunction and decline of cognition (GrundkeIqbal et al., 1986; Selkoe, 2002). Although the precise cause of AD remains elusive, it has been suggested that neuronal loss in AD is attributed to the accumulation of toxic Ab, the constituent of extracellular plaques observed in patients with AD (Hardy and Selkoe, 2002). Ab is a cleavage product derived from amyloid precursor protein. On sequential cleavage by aspartyl proteases b-secretase and g-secretase, amyloid precursor protein generates various peptide species, including Ab1e42, a more toxic form, that is prone to oligomerization, leading to the formation of amyloid plaques (De Strooper and Annaert, 2000). The amyloid deposits
S.-S. Tang and H. Hong contributed equally to this work. * Corresponding author at: Department of Pharmacology, China Pharmaceutical University, Tong Jiaxiang, Nanjing 210009, China. Tel.: þ86 25 86185227; fax: þ86 25 83271157. E-mail addresses: [email protected] (H. Hong), [email protected] (H. Ji). 0197-4580/$ e see front matter Ó 2014 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.neurobiolaging.2013.09.036
accumulate first in isocortical areas, followed by limbic and allocortical structures including the entorhinal cortex and hippocampus (Arnold et al., 1991; Thal et al., 2002). The neurotoxicity of Ab peptides has been well documented (Deshpande et al., 2006). However, the mechanisms underlying neurotoxicity triggered by Ab are far from being elucidated. Numerous studies have demonstrated that neuroinflammatory processes contribute to the pathogenesis of AD (Daniela et al., 2010; Gorelick, 2010; Wyss-Coray, 2006; Zotova et al., 2010). It has been shown that inflammatory cytokines, chemokines, proteases, and reactive oxygen species can augment Ab formation (Daniela et al., 2010; Giunta et al., 2008; Guo et al., 2002; Jimenez et al., 2008). Treatment that blocks the inflammatory responses to Ab reverses Abinduced memory deficits (O’Hare et al., 2011; Russo et al., 2012; Wang et al., 2012). Cysteinyl leukotrienes (CysLTs), including LTC4, LTD4, and LTE4, are inflammatory lipid mediators derived from the 5lipoxygenase pathway of the arachidonic acid metabolism (Singh et al., 2010). The role of 5-lipoxygenase in the pathogenesis of AD has been demonstrated (Chu and Praticò, 2011a, 2011b, 2012; Chu et al., 2012; Manev et al., 2011). Pharmacologic characterizations have suggested the existence of at least 2 types of CysLT receptors, designated as cysteinyl leukotriene receptor 1 (CysLT1R) and cysteinyl leukotriene receptor 2 (CysLT2R), based on the potency of agonists and antagonists (Singh et al., 2010). In more recent years, the focus on
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CysLT1R has intensified, because its novel pathophysiological role has emerged in brain damage, such as cerebral ischemia (Zhang et al., 2006), traumatic brain injury (Dinq et al., 2007; Zhang et al., 2004), experimental autoimmune encephalomyelitis (Wang et al., 2011), and more. We recently reported that intracerebral infusions of LTD4 impaired memory, with obvious expression of CysLT1R in the hippocampus and cortex of mice, and LTD4 also induced Ab generation by a CysLT1R-mediated b-secretase pathway in vivo (Tang et al., 2013) and in vitro (Wang et al., 2013). Next, we speculated that Ab-induced neurotoxicity is most likely to be involved in CysLT1R. The aim of the current study was, therefore, to investigate the roles of CysLT1R in Ab1e42-induced neurotoxicity in primary neurons or in mice. 2. Methods 2.1. Animals and drugs Male Institute of Cancer Research (ICR) mice (approximately 3 months old, weighing 25e30 g; Yangzhou University Medical Center Yangzhou, China), and double transgenic (APPswe, PSEN1dE9) mice and age- and strain-matched wild-type control mice (Institute of Laboratory of Animal Science of Chinese Academy of Medical Sciences & Peking Union Medical College) were used for the experiments. All experiments were carried out according to the National Institutes of Health Guide for the Care and Use of Laboratory Animals (Laevis et al., 1996). The procedures were approved by the Animal Care and Use Committee of China Pharmaceutical University. All animals were maintained on a 12-hour light/12-hour dark cycle with free access to water and standard laboratory chow. Ab1e42 and pranlukast were purchased from Sigma Aldrich (St. Louis, MO). Pranlukast for oral administration was purchased from Jiangsu Hengrui Medicine Co, Ltd (Lianyungang, Jiangsu, China). Memantine was purchased from H. Lundbeck A/S (Copenhagen, Denmark). Donepezil (Aricept) was purchased from Eisai China, Inc (Suzhou, China). Antibodies were purchased from different companies: anti-CysLT1R from Santa Cruz Biotechnology, Inc (Heidelberg, Germany); anti-nuclear factor-kappa B p65 (NF-kB p65), antiprocaspase-3 or cleaved caspase-3, and anti-B-cell lymphoma2 (Bcl-2) from Cell Signaling Technology, Inc (Boston, MA); and antib-actin and secondary antibodies from Bioworld Technology Co, Ltd (Minneapolis, MN). All other chemicals were of analytical grade and were commercially available. Ab1e42 was reconstituted in phosphate-buffered saline (pH 7.4) at a concentration of 410 pmol/5 mL, and was aggregated by incubation at 37 C for 7 days before administration (Russo et al., 2012). 2.2. Primary cortical neuron culture Primary cortical neurons were prepared from embryonic brains (E16) of ICR mice as described previously (Cissé et al., 2011). Briefly, the cortices were digested with papain. Cells were plated in poly-L-lysine-coated 24- or 6-well plates at densities of approximately 2 104 cells per well and 1 105 cells per well, respectively, and maintained in serum-free Neurobasal Medium supplemented with B27 and antibiotics. Half the medium was changed after 5 days in culture. Cells were used after 5e11 days in culture. More than 90% of the cells were neurons, as determined by staining with an antibody against the neuron-specific marker Microtubule-associated protein-2 (MAP2) (data not shown). The cultured primary neurons were treated with Ab1e42 for 48 hours, followed by pranlukast treatment for 24 hours, and the treated neurons were collected for assays of cell viability, CysLT1R, NF-kB p65, caspase-3, and Bcl-2.
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2.3. Stereotaxic intrahippocampal Ab infusion ICR mice were anesthetized with an intraperitoneal injection of 350 mg/kg chloral hydrate and then immobilized on a stereotactic frame (SR-5; Narishige, Tokyo, Japan). The dura was exposed, and a glass micropipette connected to a microinjection pump (Dakumar Machinery, Sweden) was inserted into the left and right parietal cortices at a site 2.0 mm caudal to the bregma, 1.5 mm from the midline, and 2.0 mm below the dural surface (Paxinos and Franklin, 2003). Phosphate-buffered saline (5 mL) with or without Ab1e42 (410 pM/mouse) was injected bilaterally into the hippocampus (1 mL/min for all infusions). The micropipettes were left in place for 5 minutes to minimize backflux of liquid. After 3 days, the selective CysLT1R antagonist pranlukast was injected intrahippocampally (1.5 ng/mouse). Another 3 days later, one part of the mice was submitted to the Morris water maze (MWM) task for 6 days, and other part of the mice was sacrificed by cervical dislocation, and their brain tissues were taken for assay of CysLT1R, NF-kB p65, caspase-3, and Bcl-2. 2.4. Western blot analysis The neurons were washed with phosphate-buffered saline and homogenized in lysis buffer (10 mM Tris-HCl [pH 7.4], 150 mM NaCl, 1 mM ethylenediamine tetraacetic acid, 1% Triton X-100) containing phenylmethanesulfonyl fluoride. The homogenates were rocked at 4 C for 30 minutes and then centrifuged at 13,000g at 4 C for 30 minutes to remove cell debris and collect the resulting supernatant. Mouse hippocampus and cortex were chopped into small pieces, homogenized in 0.5 mL radio immunoprecipitation assay buffer. The dissolved proteins were collected from the supernatant after centrifugation at 12,000g for 15 minutes. Protein concentrations were determined using Coomassie blue-based assay reagent, and then the expression of CysLT1R, NFkB p65, caspase-3, or Bcl-2 was assessed. Protein extracts were separated by sodium dodecylsulfate-polyacrylamide gel electrophoresis, and were then transferred onto a polyvinylidene Fluoride membrane. The membrane was blocked with 5% skim milk in Tris buffer saline and then incubated at 4 C overnight with respective primary antibodies for anti-CysLT1R antibody (1:1000), anti-NF-kB p65 antibody (1:1000), anti-procaspase-3 or cleaved caspase-3 antibody (1:1000), anti-Bcl-2 anti-body (1:1000), or b-actin (inner control, 1:2000). After washing with Tris buffered saline with Tween, the membranes were incubated with a horseradish peroxidase-conjugated secondary antibody (1:5000) for 2 hours at room temperature. The antibody-reactive bands were visualized using enhanced chemiluminescence detection reagents and a gel imaging system (Tanon Science & Technology Co, Ltd, Shanghai, China). 2.5. Reverse transcription-polymerase chain reaction assay Total RNA was extracted from the mouse hippocampal and cortical tissues using Trizol reagents following the procedures described in the manufacturer’s instructions. For complementary DNA synthesis, aliquots of total RNA (2 mg) were mixed with 0.2 mg random hexamer primer, 20 U RNasin, 1 mM/L deoxy-ribonucleoside triphosphate (dNTP), and 200 U Moloney-murine leukemia virus reverse transcriptase in 20 mL of the reverse reaction buffer. The mixture was incubated at 25 C for 10 minutes, 42 C for 60 minutes, and then at 72 C for 10 minutes to inactivate the reverse transcriptase. Polymerase chain reaction (PCR) was performed on an Eppendorf Master Cycler (Eppendorf, Hamburg, Germany). The mixture contained: 1 mL reverse transcription-complementary DNA template dissolved in 20 mL reaction mixture containing 1 PCR
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buffer, 200 mM/L dNTP, 1.5 mM/L MgCl2, 20 pM of each primer, and 0.5 U Taq DNA polymerase. Cycling parameters were as follows: 94 C for 1 minute, followed by 33 cycles at 94 C for 30 seconds, at 63 C for 30 seconds, and at 72 C for 1 minute, with a final extension step of 72 C for 7 minutes. The abundance of transcripts in complementary DNA samples was measured by reverse transcription-PCR with the following primers: mouse CysLT1R forward 50 -ATTCCTGGAGAACATGAATGG-30 and reverse 50 -CATTGTTCTGCACTGTAGATG AG-30 (1062 bp, nucleotides 419e1480 in NM_021476.4; GenBank), and b-actin forward 50 -TCTTGGGTATGGAATCCTGTG-30 and reverse 50 -ATCTCCTTCTGCATCCTGTCA-30 (154 bp, nucleotides 876e1029 in NM_007393.3; GenBank). The amplification products were separated by electrophoresis on 2% agarose gel containing ethidium bromide and were then photographed. The optical density of the bands was determined by an image analysis system (Tanon Science & Technology Co, Ltd). 2.6. Behavioral assessment of cognitive function by the Morris water maze (MWM) task
Bilaney) were implanted at coordinates 2 mm caudal to the bregma, 1.4 mm from the midline, and 1.5 mm below the dural surface for the recording electrode; for the stimulating electrode, 3.8 mm caudal to the bregma, 3.0 mm from the midline, and 1.5 mm below the dural surface. Population spike (PS) were evoked by a squarewave constant current pulse stimulation of 0.1 m second duration at a frequency of 1/60 Hz. Before each experiment, input/output curves were generated to determine the maximal amplitude of the PS, and the intensity of stimulus was set at a level that evoked a PS at 55%e65% of the maximum amplitude. The amplitude of the PS was measured every 5 minutes. LTP was induced by applying highfrequency stimulation (HFS) of 8 trains of 400-msecond stimuli at 400 Hz repeated 3 times at 10-second intervals. All recordings and stimulations were performed using an online, computerized oscilloscope stimulator and data analysis interface system. The percentage of the ratio of absolute PS amplitude to the basal value was used to represent the level of PS amplitude. LTP was measured as normalized PS amplitude (as a percentage). 2.8. Statistical analysis
Spatial memory was assessed by the MWM, which consisted of 5 days of training (visible and invisible platform training sessions) and a probe trial on day 6. This was carried out as described previously (Tang et al., 2013). Mice were trained individually in a circular pool (diameter, 120 cm; height, 50 cm) filled to a depth of 30 cm with water maintained at 25 C. The maze was located in a lit room with visual cues. An escape platform (diameter, 9 cm) was placed in the center of 1 quadrant of the pool. The platform’s position was fixed throughout the training sessions; the starting points were pseudorandomized for each trial, with the animals facing toward the wall. Each mouse was trained individually in both visible-platform (days 1e2) and hidden-platform (days 3e5) versions. Visible-platform training was performed for baseline differences in vision and motivation; the escape platform was placed 1 cm below the surface of the water and was indicated by a small flag (height, 5 cm). The hiddenplatform version evaluates spatial learning and was used to determine the retention of memory to find the escape platform. During the training, the escape platform was placed 1 cm below the surface of the water and the flag was removed. The escape platform was always in the same place. On each day, the animal was subjected to 4 trials with a 1-hour interval between trials. Each trial lasted for 90 seconds unless the animal reached the escape platform. The time (escape latency) that elapsed until the mouse reached the escape platform was noted. If an animal failed to find the platform within 90 seconds, the test was ended and the animal was navigated gently to the platform by hand for 30 seconds. On day 6, the escape platform was removed and the probe trial started, during which animals had 90 seconds to search for the escape platform. The time spent in the target quadrant (i.e., the quadrant where the escape platform was located previously) and the number of platform location crossings were recorded. Data for escape latency, time spent in the target quadrant, number of platform location crossings, and swim speed were collected by the video tracking equipment and processed by a computer equipped with an analysis management system (Viewer 2 Tracking Software; Ji Liang Instruments, China). 2.7. In vivo hippocampal long-term potentiation (LTP) recording Mice were anesthetized with an intraperitoneal injection of 1.5 g/kg urethane and then immobilized on a stereotactic frame (SR-5; Narishige). The electrophysiological recording procedure was performed as described in a previous study (Yang et al., 2011). A stimulating electrode was placed in the perforant path, and a recording glass pipette was inserted into the dentate gyrus of the dorsal hippocampus. Electrodes (tungsten with Teflon coating;
All data were distributed normally and are presented as means standard error of the mean. In the case of single-mean comparison, data were analyzed by Student’s t-test. In case of multiple mean comparisons, the data were analyzed by analysis of variance and Newman-Keuls posttest, or 2-way repeated-measures analysis of variance followed by Bonferroni multiple comparison tests. A p value less than 0.05 was regarded to reflect a significant difference. 3. Results 3.1. Ab exposure upregulates CysLT1R expression in vitro or in vivo Initially, we tentatively investigated that effect of graded concentrations of Ab1e42, a more neurotoxic Ab species, on the expression of CysLT1R in primary cultured neurons using Western blot assay. Surprisingly, neurons treated with Ab1e42 (1e20 mM) for 48 hours showed obvious increases in plasma membrane CysLT1R protein, with a maximum effect at 10 mM or 20 mM of Ab1e42 (Fig. 1A and B). A time-dependent study showed that Ab1e42 at 10 mM increased CysLT1R expression graduallydan effect that was statistically significant at 48 hours and 72 hours compared with untreated neurons (Fig. 1C and D). We also performed bilateral stereotaxic intrahippocampal infusions of Ab1e42 in mice, and then detected CysLT1R expression of the hippocampus and cortex. Similarly, Ab1e42 caused significant increases in CysLT1R expression in both the hippocampus and cortex of mice on days 3, 5, and 7 instead of 1 or 2 days after infusion (p < 0.01; Fig. 1EeH). These data indicate an interesting phenomenon that Ab may upregulate CysLT1R expression in vitro and in vivo. To identify the CysLT1R potentially involved in AD pathophysiology, we examined brain CysLT1R protein in a transgenic (APPswe, PSEN1dE9) mouse model of Ab accumulation, which displays distinct pathologic characteristics. Lysates of hippocampus or cortex from these mice at the age of 1, 4, and 7 months were subjected to Western blot analysis. Intriguingly, CysLT1R protein displayed progressive increases in the cortex and hippocampus with age (p < 0.05 or p < 0.01), but no obvious changes were observed in those of age- and strain-matched wild-type control mice (p > 0.05; Fig. E1). 3.2. Blockade of CysLT1R reverses Ab1e42-induced CysLT1R expression upregulation Next, we wondered whether blockade of CysLT1R influences Ab1e42-triggered CysLT1R expression. As shown in Fig. 2, blockade
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Fig. 1. Amyloid-b (Ab)1e42 exposure induces cysteinyl leukotriene receptor 1 (CysLT1R) expression in vitro and in vivo. (AeD) CysLT1R levels are shown after cultured primary neurons were treated with Ab1e42 at 1, 10, and 20 mM for 48 hours (A, B) or with Ab1e42 at 10 mM for 12, 24, 48, and 72 hours (C, D). (EeH) CysLT1R levels were displayed in the hippocampus (E, F) and cortex (G, H) on days 1e7 after Ab1e42 (410 pmol/mouse) was injected bilaterally into the hippocampus of mice. b-actin was used as a loading control. Results are expressed as mean standard error of the mean (n ¼ 3e5). * p < 0.01 versus vehicle group. Abbreviations: d, day; h, hour.
of CysLT1R by applying the selective CysLT1R antagonist pranlukast after Ab1e42 exposure reversed CysLT1R protein expression and messenger RNA expression in vivo (Western blot (WB): F(2,8) ¼ 23.806, p ¼ 0.001 for the hippocampus and F(2,8) ¼ 18.208, p ¼ 0.003 for the cortex [Fig. 2A and B]; reverse transcription-PCR: F(2,8) ¼ 18.403, p ¼ 0.003 for the hippocampus and F(2,8) ¼ 13.059, p ¼ 0.007 the for cortex [Fig. 2C and D]) and in vitro (WB: F(5,17) ¼ 6.035, p ¼ 0.005; Fig. E2), although pranlukast alone did not affect CysLT1R expression.
subunits, and promoted an apoptotic profile of gene expression (Valerio et al., 2006). We were curious whether Ab1e42-activated NF-kB is involved in CysLT1R. Consistent with previous reports, our data showed that Ab1e42 activated NF-kB pathway characterized by p65 subunit increment. Interestingly, treatment of the selective CysLT1R antagonist pranlukast was able to block Ab1e42-activated NF-kB signaling in vivo (F(2,8) ¼ 26.034, p ¼ 0.001 for the hippocampus and F(2,8) ¼ 14.616, p ¼ 0.005 for the cortex [Fig. 3A and B]) and in vitro (F(5,17) ¼ 6.035, p ¼ 0.005; Fig. E3).
3.3. Blockade of CysLT1R suppresses Ab1e42-activated NF-kB signaling
3.4. Blockade of CysLT1R inhibits Ab1e42-triggered caspase-3 activation and Bcl-2 downregulation
It was reported that Ab1e42 activated the NF-kB pathway by selectively inducing the nuclear translocation of p65 and p50
Mounting evidence suggests that the activation of apoptotic pathways is a crucial event in Ab-induced neurotoxicity (Awasthi
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Fig. 2. Blockade of cysteinyl leukotriene receptor 1 (CysLT1R) reverses amyloid-b (Ab)1e42-induced CysLT1R upregulation in the hippocampus and cortex of mice. The CysLT1R antagonist pranlukast (Pran; 0.75 ng/side) was injected bilaterally into the hippocampus 3 days after intrahippocampal microinfusion of Ab1e42 (205 pM/side). (A, C) Three days after that, CysLT1R protein (A) or messenger RNA (C) in the hippocampus and cortex was detected by Western blot or reverse transcription-polymerase chain reaction, respectively. (B, D) Quantification of CysLT1R (B, D) is expressed as the ratio (as a percentage) of VehþVeh. b-actin was used as a loading control. Results are expressed as means standard error of the mean (n ¼ 3e5). * p < 0.05. y p < 0.01 versus Ab1e42 þ Veh for hippocampus. z p < 0.05. x p < 0.01 versus Ab1e42 þ Veh for cortex. Abbreviation: Veh, vehicle.
et al., 2005; Rohn et al., 2008; Stadelmann et al., 1999). To evaluate whether the blockade of CysLT1R plays an antiapoptotic role in Ab1e42-induced neurotoxicity, we examined caspase-3, which is known to be a crucial mediator of apoptosis through its protease activity, and Bcl-2, an antiapoptotic protein, by Western blot analysis. Quantification of Western blot revealed that Ab1e42 led to caspase-3 activation, as indicated by an increase in the ratio of caspase-3 fragment to procaspase-3, and led to a significant reduction in antiapoptotic protein Bcl-2 in the hippocampus and cortex of mice compared with the vehicle. In addition, treatment with Ab1e42 plus pranlukast was able to attenuate caspase-3 activation (F(2,8) ¼ 8.144, p ¼ 0.020 for the hippocampus and
F(2,8) ¼ 15.121, p ¼ 0.005 for the cortexe [Fig. 4A and B]) and elevate Bcl-2 levels (F(2,8) ¼ 12.801, p ¼ 0.007 for the hippocampus and F(2,8) ¼ 18.399, p ¼ 0.003 for the cortex [Fig. 4C and D]) compared with Ab1-42 plus vehicle. Consistent with an in vivo study, an in vitro study also showed that treatment with pranlukast at 0.1 mM or 10 mM reversed Ab1e42-triggered caspase-3 activation and Bcl-2 downregulation (F(5,17) ¼ 26.720, p < 0.001 for caspase-3 [Fig. E4A and B]; F(5,17) ¼ 7.896, p ¼ 0.002 for Bcl-2 [Fig. E4C and D]). To determine further the role of CysLT1R in Ab1e42-induced cytotoxicity, we used an 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide reduction assay and a lactate dehydrogenase
Fig. 3. Blockade of cysteinyl leukotriene receptor 1 (CysLT1R) suppresses amyloid-b (Ab)1e42-activated nuclear factor-kappa B (NF-kB) signaling in the hippocampus and cortex of mice. (A) Immunoblot of NF-kB p65 detected by Western blot. (B) Quantification of NF-kB p65 expressed as the ratio (as a percentage) of VehþVeh. Administration of Ab1e42 and pranlukast (Pran) was the same as that noted in the caption of Fig. 2. Values shown are means standard error of the mean (n ¼ 4). *p < 0.01 versus Ab1e42 þ Veh for hippocampus. y p < 0.05. z p < 0.01 versus Ab1e42 þ Veh for cortex. Abbreviation: Veh, vehicle.
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Fig. 4. Blockade of cysteinyl leukotriene receptor 1 (CysLT1R) attenuates amyloid-b (Ab)1e42-triggered caspase-3 activation and B-cell lymphoma-2 (Bcl-2) downregulation in the hippocampus and cortex of mice. The treatment procedure is as described in the caption for Fig. 2. (A, C) Precaspase-3 and cleaved caspase-3 (A) or Bcl-2 (C) were determined by Western blot using respective antibodies. (B) Caspase-3 activation was expressed as the ratio of caspase-3 fragment to procaspase-3. (D) Quantification of Bcl-2 was expressed as the ratio (as a percentage) of VehþVeh. Values shown are means standard error of the mean (n ¼ 4). * p < 0.05. y p < 0.01 versus Ab1e42 þ Veh for hippocampus. z p < 0.05. x p < 0.01 versus Ab1e42 þ Veh for cortex. Abbreviation: Pran, pranlukast; Veh, vehicle.
release assay to observe mitochondrial activity and the number of dead or plasma membrane-damaged cells. The results showed that pranlukast (0.1 mM or 10 mM) rescued neurons from Ab1e42-induced neurotoxicity (Fig. E5).
3.5. Blockade of CysLT1R reverses Ab1e42-induced cognitive deficit in mice To determine the role of CysLT1R in an Ab1e42-induced cognitive deficit, MWM tests were performed to evaluate the effect of pranlukast on intrahippocampal Ab1e42-injected mice. We first assessed the performance of mice in a nonspatial visible-platform variant of the MWM to test for baseline differences in vision and motivation among groups. Mice in each group exhibited similar escape latency in the visible-platform test, suggesting no influence of Ab1e42 or the CysLT1R antagonist pranlukast on vision or basal motivation of mice (F(2,199) ¼ 2.560, p ¼ 0.080; Fig. 5A). We then tested the mice in the spatial hidden-platform variant, and data showed that mice treated with Ab1e42 plus vehicle displayed increased escape latencies compared with those treated with vehicle plus vehicle (p < 0.001; Fig. 5B), which were reversed by pranlukast (p < 0.001; Fig. 5B; 4 trials per day for 3 days; F(2,299) ¼ 30.832, p < 0.001). In the probe trial, a putative measure of spatial learning and memory retention, all mice showed preference for the target quadrant, with the exception of mice in Ab1e42 plus vehicle group, which displayed a significant decrease in the time spent in the target quadrant (p < 0.05; Fig. 5C) and the number of platform location crossings (p < 0.05; Fig. 5D) compared with those of the vehicle plus vehicle group. In contrast, mice in the Ab1e42 plus pranlukast group showed significant increases in both indices compared with those in the Ab1e42 plus vehicle group (p < 0.05 for the time, p < 0.05 for the number; Fig. 5C and D). In addition, all mice showed similar swim speeds during the tests (Fig. 5F). These data suggest that blockade of CysLT1R reverses an Ab1e42-induced cognitive deficit in mice.
3.6. Chronic treatment of CysLT1R antagonist, N-methyl-Daspartate receptor antagonist and acetylcholinesterase inhibitor produce similar effects on the behavior and hippocampal LTP in an experimental model of AD To reveal the potential value of CysLT1R for treatment of AD, we performed a comparison study of the CysLT1R antagonist pranlukast with the N-methyl-D-aspartate (NMDA) receptor antagonist memantine and acetylcholinesterase (AChE) inhibitor donepezil that were approved by the Food and Drug Administration for treatment of AD. Intrahippocampal Ab1e42-injected mice were administered pranlukast (0.6 mg/kg), memantine (20 mg/kg), and donepezil (2 mg/kg) intragastrically for 14 consecutive days, and then subjected to the MWM test. Results showed that mice in each group exhibited similar escape latencies in the visible-platform test (F(4,389) ¼ 0.772, p ¼ 0.544; Fig. 6A), suggesting no difference in vision or basal motivation among the 5 groups. In the spatial hidden-platform variant, Ab1e42-injected mice treated with pranlukast, memantine, and donepezil exhibit progressively decreased escape latencies during training (4 times a day) compared with the Ab1e42-injected mice treated with vehicle (F(4,599) ¼ 6.647, p < 0.001, Fig. 6B). During the probe trial, a putative measure of spatial learning and memory retention, only the Ab1e42-injected mice treated with vehicle failed to favor the target platform location (p < 0.05 or p < 0.01). The Ab1e42-injected mice treated with pranlukast, memantine, and donepezil showed significant increases in both the time spent in the target quadrant and in the number of platform location crossings during the final probe trial (F(4,39) ¼ 6.745, p < 0.001 for the time; F(4,39) ¼ 4.488, p ¼ 0.005 for the number; Fig. 6CeE). No differences were observed in the performance of mice treated with 3 drugs in place navigation (F(2,359) ¼ 1.350, p ¼ 0.261) or in the probe trial (F(2,23) ¼ 0.035, p ¼ 0.965 and F(2,23) ¼ 0.201, p ¼ 0.820). There were no differences in swimming speed among groups during the MWM test (Fig. 6F), and these drugs had no effects on learning and memory in unimpaired mice (data not
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Fig. 5. Blockade of cysteinyl leukotriene receptor 1 (CysLT1R) rescues amyloid-b (Ab)1e42-induced spatial learning and memory deficits in mice. Mice received bilateral intrahippocampal injection of Ab1e42 (205 pM/side) 3 days later, followed by a single bilateral intrahippocampal injection of the CysLT1R antagonist pranlukast (Pran; 0.75 ng/side). Mice were tested in the Morris water maze task on 3 day after Pran injection. Day 0 indicates performance on the first trail; subsequent points represent an average of all daily trails. (A) No differences were found in the escape latency among all groups during the visible platform test (days 1-2). (B) Changes in escape latency to reach the hidden platform during acquisition trials (days 3-5). (CeE) The time spent in the target quadrant (C), the number of platform location crossings (D), and the representative swim paths of individual mice in each group during the probe trial test on day 6 (E) are presented. (F) Average swimming speed on each day of testing is also shown. Values shown are means standard error of the mean (n ¼ 8e10). * p < 0.05 versus Ab1e42 þ Veh group. Abbreviation: Veh, vehicle.
shown). In addition, LTP recordings showed that hippocampal LTP was significantly inhibited in Ab1e42-injected mice (142.12 1.65%) after the tetanic stimulation persisting for at least 60 minutes compared with controls (173.55 3.70%, p < 0.001), and was significantly increased in Ab1e42-injected mice with chronic treatment of pranlukast, memantine, or donepezil (171.73 3.59%, 177.19 2.56%, and 173.91 2.12%, p < 0.001; Fig. 6G and H). No differences were observed in the ameliorative effects of 3 drugs on hippocampal LTP in Ab1e42-injected mice (F(2,35) ¼ 1.414, p ¼ 0.257). We also used liquid chromatography-mass spectrometry/ mass spectrometry analysis to confirm its presence in the brain after oral administration of pranlukast (data not shown). These data suggest that the CysLT1R antagonist produced similar
improvements as the NMDA receptor antagonist or AChE inhibitor in Ab1e42-induced memory impairment. 4. Discussion Our knowledge of the mechanisms underlying the action of Ab remains limited despite the abundance of evidence pointing to an essential role of Ab in AD pathophysiology. Explicating the molecular mechanisms that underlie neuronal apoptosis during AD is therefore critical for unraveling the pathophysiology of the disease. Our study clearly shows that Ab1e42, the most toxic and proaggregate form of Ab, induced increases in CysLT1R expression in a dose- or timedependent manner in primary neurons, and bilateral hippocampal
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Fig. 6. Chronic treatment with the cysteinyl leukotriene receptor 1 (CysLT1R) antagonist pranlukast (Pran) improves amyloid-b (Ab)1e42-induced memory impairment and hippocampal LTP depression such as the N-methyl-D-aspartate (NMDA) receptor antagonist memantine (Mem) or acetylcholinesterase (AChE) inhibitor donepezil (Don). Animals received bilateral intrahippocampal injections of Ab1e42 (205 pM/side), and they were treated daily with intragastric administration of Pran (0.6 mg/kg), Mem (20 mg/kg), and Don (2 mg/kg). After 14 days, mice were tested in the Morris water maze task or hippocampal long-term potentiation (LTP). (A) No differences were found in the escape latency among all groups during visible platform test (days 1-2). (B) Changes in escape latency to reach the hidden platform during acquisition trials (days 3-5). (CeE) The time spent in the target quadrant (C), the number of platform location crossings (D), and the representative swim paths of individual mice in each group during the probe trial test on day 6 (E) are presented. (F) Average swimming speed on each day of testing is also shown. High-frequency stimulation (HFS) was given at the end of a 30-minute baseline recording of population spike (PS). (G, H) The time course of PS after HFS is plotted (G), and the average PS at 60 minutes post-HFS is summarized as columns (H). Values shown are expressed as means standard error of the mean (n ¼ 8e10). * p < 0.05. y p < 0.01 versus Ab1e42 þ Veh. Abbreviation: Veh, vehicle.
injection of Ab1e42 in ICR mice also caused increases in CysLT1R expression in the hippocampus and cortex. This upregulation was coupled with neurotoxicity, as evidenced by increased NF-kB p65, activated caspase-3, decreased Bcl-2, and lowered cellular viability as well as impaired memory. More important, blockade of CysLT1R reversed Ab1e42-induced CysLT1R upregulation and inhibited Ab1e42-induced neurotoxicity in vitro and in vivo. Furthermore,
chronic treatment with the CysLT1R antagonist pranlukast (0.6 mg/ kg), NMDA receptor antagonist memantine (20 mg/kg) and AChE inhibitor donepezil (2 mg/kg) produced similar beneficial effects on memory behavior and hippocampal LTP in intrahippocampal Ab1e42-injected mice. In addition, age-dependent increases of CysLT1R were observed in both the hippocampus and the cortex of the transgenic (APPswe, PSEN1dE9) AD mouse model. Collectively,
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these findings strongly suggest that CysLT1R is involved in Ab1e42induced neurotoxicity, and raise the possibility that CysLT1R is an important player in the progression of AD. CysLT1R is a high-affinity G protein-coupled receptor for the pro-inflammatory mediator LTD4, which is implicated in many inflammatory conditions (Funk, 2001; Serhan et al., 1996). The pathophysiological role of CysLT1R in several inflammatory conditions is well documented, with a general emphasis on asthma, and during the past 25 years a considerable effort has been made to identify and develop receptor antagonists to improve asthma management, limit its morbidity, and reduce the side effects of current medications (Singh et al., 2010). It has been reported that CysLT1R mediates astrocyte proliferation (Ciccarelli et al., 2004; Huang et al., 2008), traumatic brain injury, brain cryoinjury and brain tumors (Dinq et al., 2007; Zhang et al., 2004), and acute neuronal injury after focal cerebral ischemia in mice (Zhang et al., 2006). Neurons are believed to be the major source of Ab that has a causal role in AD (Palop and Mucke, 2010). We used exogenous Ab1e42 to mimic Ab release of neurons and found a novel pathophysiological link between Ab and CysLT1R. Interestingly, basal expression of CysLT1R is pretty weak in neurons, and neurons treated with Ab1e42 displayed a significant increase in CysLT1R expression. This effect was abolished completely by the selective CysLT1R antagonist pranlukast. These results imply that CysLT1R might be an inducible receptor with more pathophysiological significance in the brain. How Ab1e42 triggers CysLT1R upregulation remains to be elucidated. It is possible that Ab1e42 first induces the upregulation of 5-lipoxygenase (Firuzi et al., 2008), then the resulting CysLTs (such as LTD4) induce CysLT1R expression, which may be responsible for the observed memory deficit (Tang et al., 2013). It has been reported that CysLT1Rmediated signaling refers to the NF-kB pathway in some peripheral cells (Kawano et al., 2003; Suzuki et al., 2008; Thompson et al., 2006). Recently, CysLT1R-mediated NF-kB signaling was also observed in neurons (Wang et al., 2013). Ab has been shown to activate NF-kB (Kaltschmidt et al., 1997). Activated NF-kB was detected in AD brains (Boissier et al., 1997; Terai et al., 1996), and in neuronal and glial nuclei proximal to early-stage plaques (Ferrer et al., 1998; Kaltschmidt et al., 1997). Ab1e42 activated the NF-kB pathway by selectively inducing the nuclear translocation of p65 and p50 subunits, and promoted an apoptotic profile of gene expression (Valerio et al., 2006). Numerous studies have implicated apoptosis as a major pathway of cell death in AD (Culmsee and Landshamer, 2006), and inhibiting proapoptotic protein caspase-3 or activating antiapoptotic protein Bcl-2 in neurons of patients with AD could be an effective means of treating this disease (Awasthi et al., 2005; Rohn et al., 2008). Targeting the NF-kB pathway might be useful to counteract the establishment or progression of AD through prevention of neuronal cell death. The current study shows that blockade of CysLT1R suppressed Ab1-42triggered NF-kB signaling as well as neuronal apoptosis marked by activated caspase-3 and decreased Bcl-2 in vivo or in vitro. These findings suggest that CysLT1R is involved in Ab1e42-activated the NFkB pathway and subsequent cellular apoptosis. Further study showed that Ab1e42-injected mice treated either acutely or chronically with the CysLT1R antagonist pranlukast displayed significant improvement in cognitive deficit as evaluated by the MWM test. Even more exciting, CysLT1R antagonist pranlukast produced significant improvement in memory impairment and hippocampal LTP inhibition in intrahippocampal Ab1e42-injected mice, and the effects were similar to those of NMDA receptor antagonist memantine or AChE inhibitor donepezil that is used clinically as a treatment of AD. These results strongly support that CysLT1R plays a critical role in Ab1e42-induced neurotoxicity. Overall, our study demonstrates for the first time the contributions of CysLT1R to Ab1e42-induced neurotoxicity. These appear to
be that Ab1e42 induces CysLT1R activation, leading to NF-kB signaling enhancement, which triggers caspase-3 activation and Bcl-2 inactivation, producing apoptosis and cognitive impairment. Thus, CysLT1R could be a novel target for developing new drugs in the treatment of AD. In addition, CysLT1R antagonists such as pranlukast, zafirlukast, and montelukast used clinically for the treatment of asthma might be beneficial for AD. Disclosure statement None of authors have financial interests to disclose. Acknowledgements This work was supported, in part, by grants from the National Natural Science Foundation of China (81273497). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.neurobiolaging. 2013.09.036. References Arnold, S.E., Hyman, B.T., Flory, J., Damasio, A.R., Van Hoesen, G.W., 1991. The topographical and neuroanatomical distribution of neurofibrillary tangles and neuritic plaques in the cerebral cortex of patients with Alzheimer’s disease. Cereb. Cortex 1, 103e116. Awasthi, A., Matsunaga, Y., Yamada, T., 2005. Amyloid-beta causes apoptosis of neuronal cells via caspase cascade, which can be prevented by amyloid betaderived short peptides. Exp. Neurol. 196, 282e289. Boissier, E.F., Hunot, S., Faucheux, B., Duyckaerts, C., Hauw, J.J., Agid, Y., Hirsch, E.C., 1997. Nuclear translocation of NF-kappaB in cholinergic neurons of patients with Alzheimer’s disease. Neuroreport 8, 2849e2852. Chu, J., Praticò, D., 2011a. 5-Lipoxygenase as an endogenous modulator of amyloid b formation in vivo. Ann. Neurol. 69, 34e46. Chu, J., Praticò, D., 2011b. Pharmacologic blockade of 5-lipoxygenase improves the amyloidotic phenotype of an Alzheimer’s disease transgenic mouse model involvement of g-secretase. Am. J. Pathol. 178, 1762e1769. Chu, J., Praticò, D., 2012. Involvement of 5-lipoxygenase activating protein in the amyloidotic phenotype of an Alzheimer’s disease mouse model. J. Neuroinflamm. 14, 127e136. Ciccarelli, R., D’Alimonte, I., Santavenere, C., D’Auro, M., Ballerini, P., Nargi, E., Buccella, S., Nicosia, S., Folco, G., Caciagli, F., Di Iorio, P., 2004. Cysteinyl-leukotrienes are released from astrocytes and increase astrocyte proliferation and glial fibrillary acidic protein via Cys-LT1 receptors and mitogen-activated protein kinase pathway. Eur. J. Neurosci. 20, 1514e1524. Cissé, M., Halabisky, B., Harris, J., Devidze, N., Dubal, D.B., Sun, B., Orr, A., Lotz, G., Kim, D.H., Hamto, P., Ho, K., Yu, G.Q., Mucke, L., 2011. Reversing EphB2 depletion rescues cognitive functions in Alzheimer model. Nature 469, 47e52. Culmsee, C., Landshamer, S., 2006. Molecular insights into mechanisms of the cell death program: role in the progression of neurodegenerative disorders. Curr. Alzheimer Res. 3, 269e283. Daniela, L.K., Norbert, M., 2010. Neuroinflammation, microglia and implications for anti-inflammatory treatment in Alzheimer’s disease. Int. J. Alzheimers Dis. 10, 4061e4069. De Strooper, B., Annaert, W., 2000. Proteolytic processing and cell biological functions of the amyloid precursor protein. J. Cell Sci. 113, 1857e1870. Deshpande, A., Mina, E., Glabe, C., Busciglio, J., 2006. Different conformations of amyloid beta induce neurotoxicity by distinct mechanisms in human cortical neurons. J. Neurosci. 26, 6011e6018. Dinq, Q., Fang, S.H., Zhou, Y., Zhang, L.H., Zhang, W.P., Chen, Z., Wei, E.Q., 2007. Cysteinyl leukotriene receptor 1 partially mediates brain cryoinjury in mice. Acta Pharmacol. Sin. 28, 945e952. Ferrer, I., Marti, E., Lopez, E., Tortosa, A., 1998. NF-kB immunoreactivity is observed in association with beta A4 diffuse plaques in patients with Alzheimer’s disease. Neuropathol. Appl. Neurobiol. 24, 271e277. Firuzi, O., Zhuo, J., Chinnici, C.M., Wisniewski, T., Praticò, D., 2008. 5-Lipoxygenase gene disruption reduces amyloid-beta pathology in a mouse model of Alzheimer’s disease. FASEB J. 22, 1169e1178. Funk, C.D., 2001. Prostaglandins and leukotrienes: advances in eicosanoid biology. Science 294, 1871e1875. Giunta, B., Fernandez, F., Nikolic, W.V., Obregon, D., Rrapo, E., Town, T., Tan, J., 2008. Inflammaging as a prodrome to Alzheimer’s disease. J. Neuroinflamm. 5, 1e15. Gorelick, P.B., 2010. Role of inflammation in cognitive impairment: results of observational epidemiological studies and clinical trials. Ann. N Y Acad. Sci. 1207, 155e162.
S.-S. Tang et al. / Neurobiology of Aging 35 (2014) 590e599 Grundke-Iqbal, I., Iqbal, K., Tung, Y.C., Quinlan, M., Wisniewski, H.M., Binder, L.I., 1986. Abnormal phosphorylation of the microtubule-associated protein tau (tau) in Alzheimer cytoskeletal pathology. Proc. Natl. Acad. Sci. U.S.A 83, 4913e4917. Guo, J.T., Yu, J., Grass, D., de Beer, F.C., Kindy, M.S., 2002. Inflammation-dependent cerebral deposition of serum amyloid a protein in a mouse model of amyloidosis. J. Neurosci. 22, 5900e5909. Hardy, J., Selkoe, D.J., 2002. The amyloid hypothesis of Alzheimer’s disease: progress and problems on the road to therapeutics. Science 297, 353e356. Huang, X.J., Zhang, W.P., Li, C.T., Shi, W.Z., Fang, S.H., Lu, Y.B., Chen, Z., Wei, E.Q., 2008. Activation of CysLT receptors induces astrocyte proliferation and death after oxygen-glucose deprivation. Glia 56, 27e37. Jimenez, S., Baqlietto-Varqas, D., Caballero, C., Moreno-Gonzalez, I., Torres, M., Sanchez-Varo, R., Ruano, D., Vizuete, M., Gutierrez, A., Vitorica, J., 2008. Inflammatory response in the hippocampus of PS1M146L/APP751SL mouse model of Alzheimer’s disease: age-dependent switch in the microglial phenotype from alternative to classic. J. Neurosci. 28, 11650e11661. Kaltschmidt, B., Uherek, M., Volk, B., Baeuerle, P.A., Kaltschmidt, C., 1997. Transcription factor NF-kappaB is activated in primary neurons by amyloid beta peptides and in neurons surrounding early plaques from patients with Alzheimer’s disease. Proc. Natl. Acad. Sci. U.S.A 94, 2642e2647. Kawano, T., Matsuse, H., Kondo, Y., Machida, I., Saeki, S., Tomari, S., Mitsuta, K., Obase, Y., Fukushima, C., Shimoda, T., Kohno, S., 2003. Cysteinyl leukotrienes induce nuclear factor-kB activation and RANTES production in a murine model of asthma. J. Allergy Clin. Immunol. 112, 369e374. Laevis, Tropicials, X., 1996. Environment, Housing, and Management, in: National Research Council, eighth ed. The National Academic Press, Washington, D. C., pp. 80e103. Manev, H., Chen, H., Dzitoyeva, S., Manev, R., 2011. Cyclooxygenases and 5-lipoxygenase in Alzheimer’s disease. Prog. Neuropsychopharmacol. Biol. Psychiatry 35, 315e319. O’Hare, E., Scopes, D.I., Treherne, J.M., Monaghan, J., Palmer, P.M., Amijee, H., Kim, E.M., 2011. Novel anti-inflammatory compound SEN1176 alleviates behavioral deficits induced following bilateral intrahippocampal injection of aggregated amyloid-b1-42. J. Alzheimers Dis. 25, 219e229. Palop, J.J., Mucke, L., 2010. Amyloid-b induced neuronal dysfunction in Alzheimer’s disease: from synapses toward neural networks. Nat. Neurosci. 13, 812e818. Paxinos, G., Franklin, K.B.J., 2003. The Mouse Brain in Stereotaxic Coordinates, second ed. Academic Press, San Diego, CA. Rohn, T.T., Vyas, V., Hernandez-Estrada, T., Nichol, K.E., Christie, L.A., Head, E., 2008. Lack of pathology in a triple transgenic mouse model of Alzheimer’s disease after overexpression of the anti-apoptotic protein Bcl-2. J. Neurosci. 28, 3051e3059. Russo, I., Caracciolo, L., Tweedie, D., Choi, S.H., Greig, N.H., Barlati, S., Bosetti, F., 2012. 3,60 -Dithiothalidomide, a new TNF-a synthesis inhibitor, attenuates the effect of Ab1-42 intracerebroventricular injection on hippocampal neurogenesis and memory deficit. J. Neurochem. 122, 1181e1192. Selkoe, D.J., 2002. Deciphering the genesis and fate of amyloid beta-protein yields novel therapies for Alzheimer disease. J. Clin. Invest. 11, 1375e1381. Serhan, C.N., Haeggström, J.Z., Leslie, C.C., 1996. Lipid mediator networks in cell signaling: update and impact of cytokines. FASEB J. 10, 1147e1158. Singh, R.K., Gupta, S., Dastidar, S., Ray, A., 2010. Cysteinyl leukotrienes and their receptors: molecular and functional characteristics. Pharmacology 85, 336e349.
599
Stadelmann, C., Deckwerth, T.L., Srunivasan, A., Bancher, C., Bruck, W., Jellinger, K., Lassmann, H., 1999. Activation of caspase-3 in single neurons and autophagic granules of granulovacuolar degeneration in Alzheimer’s disease: evidence for apoptotic cell death. Am. J. Pathol. 155, 1459e1466. Suzuki, S., Takeuchi, K., Ishinaga, H., Basbaum, C., Majima, Y., 2008. Leukotriene D4 upregulates MUC2 gene transcription in human epithelial cells. Pharmacology 81, 221e228. Tang, S.S., Wang, X.Y., Hong, H., Long, Y., Li, Y.Q., Xiang, G.Q., Jiang, L.Y., Zhang, H.T., Liu, L.P., Miao, M.X., Hu, M., Zhang, T.T., Hu, W., Ji, H., Ye, F.Y., 2013. Leukotriene D4 induces cognitive impairment through enhancement of CysLT1R-mediated amyloid-b generation in mice. Neuropharmacology 65, 182e192. Terai, K., Matsuo, A., McGeer, P.L., 1996. Enhancement of immunoreactivity for NFkappa B in the hippocampal formation and cerebral cortex of Alzheimer’s disease. Brain Res. 735, 159e168. Thal, D.R., Rüb, U., Orantes, M., Braak, H., 2002. Phases of Ab deposition in the human brain and its relevance for the development of AD. Neurology 58, 1791e1800. Thompson, C., Cloutier, A., Boss, Y., Thivierge, M., Gouill, C.L., Larivée, P., McDonald, P.P., Stankova, J., Rola-Pleszczynski, M., 2006. CysLT1 receptor engagement induces activator protein-1- and NF-kappaB-dependent IL-8 expression. Am. J. Respir. Cell Mol. Biol. 35, 697e704. Valerio, A., Boroni, F., Benarese, M., Sarnico, I., Ghisi, V., Bresciani, L.G., Ferrario, M., Borsani, G., Spano, P., Pizzi, M., 2006. NF-kappaB pathway: a target for preventing beta-amyloid (Abeta)-induced neuronal damage and Abeta42 production. Eur. J. Neurosci. 23, 1711e1720. Wang, L., Du, C., Lv, J., Wei, W., Cui, Y., Xie, X., 2011. Antiasthmatic drugs targeting the cysteinyl leukotriene receptor 1 alleviate central nervous system inflammatory cell infiltration and pathogenesis of experimental autoimmune encephalomyelitis. J. Immunol. 187, 2336e2345. Wang, X.Y., Tang, S.S., Hu, M., Long, Y., Li, Y.Q., Liao, M.X., Ji, H., Hong, H., 2013. Leukotriene D4 induces amyloid-b generation via CysLT1R-mediated NF-kB pathways in primary neurons. Neurochem. Int. 62, 340e347. Wang, C., Yang, X.M., Zhuo, Y.Y., Zhou, H., Lin, H.B., Cheng, Y.F., Xu, J.P., Zhang, H.T., 2012. The phosphodiesterase-4 inhibitor rolipram reverses Ab-induced cognitive impairment and neuroinflammatory and apoptotic responses in rats. Int. J. Neuropsychopharmacol. 9, 1e18. Wyss-Coray, T., 2006. Inflammation in Alzheimer disease: driving force, bystander or beneficial response? Nat. Med. 12, 1005e1015. Yang, J., Hu, Z.L., Jiang, B., Ni, L., Jin, Y., Chen, J.G., Wang, F., 2011. Effect of chloramineT on long-term potentiation at synapses between perforant path and dentate gyrus in hippocampus of rats in vivo. Neurotoxicology 32, 199e205. Zhang, W.P., Hu, H., Zhang, L., Dinq, W., Yao, H.T., Chen, K.D., Sheng, W.W., Chen, Z., Wei, E.Q., 2004. Expression of cysteinyl leukotriene receptor 1 in human traumatic brain injury and brain tumors. Neurosci. Lett. 363, 247e251. Zhang, Y.J., Zhang, L., Ye, Y.L., Fang, S.H., Zhou, Y., Zhang, W.P., Lu, Y.B., Wei, E.Q., 2006. Cysteinyl leukotriene receptors CysLT1 and CysLT2 are upregulated in acute neuronal injury after focal cerebral ischemia in mice. Acta Pharmacol. Sin. 27, 1553e1560. Zotova, E., Nicoll, J.A., Kalaria, R., Holmes, C., Boche, D., 2010. Inflammation in Alzheimer’s disease: relevance to pathogenesis and therapy. Alzheimers Res. Ther. 2, 1e9.
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Neurobiology of Aging journal homepage: www.elsevier.com/locate/neuaging
Involvement of cysteinyl leukotriene receptor 1 in Ab1e42-induced neurotoxicity in vitro and in vivo Su-Su Tang, Hao Hong*, Lan Chen, Zhen-lin Mei, Miao-jin Ji, Guo-qing Xiang, Ning Li, Hui Ji* Department of Pharmacology, China Pharmaceutical University, Nanjing, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 27 June 2013 Received in revised form 15 September 2013 Accepted 22 September 2013 Available online 23 October 2013
Accumulation of amyloid-b (Ab) is thought to be associated with the progressive neuronal death observed in Alzheimer’s disease, but the mechanisms underlying neurotoxicity triggered by Ab remain elusive. In the current study, we investigated the roles of cysteinyl leukotriene receptor 1 (CysLT1R) in Ab1e42-induced neurotoxicity in vitro or in vivo. In vitro exposure of mouse primary neurons to Ab1e42 caused a gradual increases in CysLT1R expression. In vivo bilateral intrahippocampal injection of Ab1e42 also elicited time-dependent increases of CysLT1R expression in the hippocampus and cortex of mice. The CysLT1R antagonist pranlukast not only reversed Ab1e42-induced upregulation of CysLT1R, but also suppressed Ab1e42-triggered neurotoxicity evidenced by enhanced nuclear factor-kappa B p65, activated caspase-3, decreased B-cell lymphoma-2 and cell viability and impaired memory. Furthermore, chronic treatment with pranlukast produced similar beneficial effects on memory behavior and hippocampal long-term potentiation to memantine or donepezil in intrahippocampal Ab1e42-injected mice. Our data indicate that CysLT1R is involved in Ab1e42-induced neurotoxicity, and that blockade of CysLT1R, such as application of CysLT1R antagonist, could be a novel and promising strategy for the treatment of Alzheimer’s disease. Ó 2014 Elsevier Inc. All rights reserved.
Keywords: Cysteinyl leukotriene receptor 1 Ab1e42 Neurotoxicity NF-kB Caspase-3 Bcl-2 Memory
1. Introduction Alzheimer’s disease (AD) is the most common neurodegenerative disorder that causes progressive loss of cognitive function and dementia. Amyloid-b (Ab) deposition and neurofibrillary tangle formation are the major pathogenic mechanisms that act in concert to produce memory dysfunction and decline of cognition (GrundkeIqbal et al., 1986; Selkoe, 2002). Although the precise cause of AD remains elusive, it has been suggested that neuronal loss in AD is attributed to the accumulation of toxic Ab, the constituent of extracellular plaques observed in patients with AD (Hardy and Selkoe, 2002). Ab is a cleavage product derived from amyloid precursor protein. On sequential cleavage by aspartyl proteases b-secretase and g-secretase, amyloid precursor protein generates various peptide species, including Ab1e42, a more toxic form, that is prone to oligomerization, leading to the formation of amyloid plaques (De Strooper and Annaert, 2000). The amyloid deposits
S.-S. Tang and H. Hong contributed equally to this work. * Corresponding author at: Department of Pharmacology, China Pharmaceutical University, Tong Jiaxiang, Nanjing 210009, China. Tel.: þ86 25 86185227; fax: þ86 25 83271157. E-mail addresses: [email protected] (H. Hong), [email protected] (H. Ji). 0197-4580/$ e see front matter Ó 2014 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.neurobiolaging.2013.09.036
accumulate first in isocortical areas, followed by limbic and allocortical structures including the entorhinal cortex and hippocampus (Arnold et al., 1991; Thal et al., 2002). The neurotoxicity of Ab peptides has been well documented (Deshpande et al., 2006). However, the mechanisms underlying neurotoxicity triggered by Ab are far from being elucidated. Numerous studies have demonstrated that neuroinflammatory processes contribute to the pathogenesis of AD (Daniela et al., 2010; Gorelick, 2010; Wyss-Coray, 2006; Zotova et al., 2010). It has been shown that inflammatory cytokines, chemokines, proteases, and reactive oxygen species can augment Ab formation (Daniela et al., 2010; Giunta et al., 2008; Guo et al., 2002; Jimenez et al., 2008). Treatment that blocks the inflammatory responses to Ab reverses Abinduced memory deficits (O’Hare et al., 2011; Russo et al., 2012; Wang et al., 2012). Cysteinyl leukotrienes (CysLTs), including LTC4, LTD4, and LTE4, are inflammatory lipid mediators derived from the 5lipoxygenase pathway of the arachidonic acid metabolism (Singh et al., 2010). The role of 5-lipoxygenase in the pathogenesis of AD has been demonstrated (Chu and Praticò, 2011a, 2011b, 2012; Chu et al., 2012; Manev et al., 2011). Pharmacologic characterizations have suggested the existence of at least 2 types of CysLT receptors, designated as cysteinyl leukotriene receptor 1 (CysLT1R) and cysteinyl leukotriene receptor 2 (CysLT2R), based on the potency of agonists and antagonists (Singh et al., 2010). In more recent years, the focus on
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CysLT1R has intensified, because its novel pathophysiological role has emerged in brain damage, such as cerebral ischemia (Zhang et al., 2006), traumatic brain injury (Dinq et al., 2007; Zhang et al., 2004), experimental autoimmune encephalomyelitis (Wang et al., 2011), and more. We recently reported that intracerebral infusions of LTD4 impaired memory, with obvious expression of CysLT1R in the hippocampus and cortex of mice, and LTD4 also induced Ab generation by a CysLT1R-mediated b-secretase pathway in vivo (Tang et al., 2013) and in vitro (Wang et al., 2013). Next, we speculated that Ab-induced neurotoxicity is most likely to be involved in CysLT1R. The aim of the current study was, therefore, to investigate the roles of CysLT1R in Ab1e42-induced neurotoxicity in primary neurons or in mice. 2. Methods 2.1. Animals and drugs Male Institute of Cancer Research (ICR) mice (approximately 3 months old, weighing 25e30 g; Yangzhou University Medical Center Yangzhou, China), and double transgenic (APPswe, PSEN1dE9) mice and age- and strain-matched wild-type control mice (Institute of Laboratory of Animal Science of Chinese Academy of Medical Sciences & Peking Union Medical College) were used for the experiments. All experiments were carried out according to the National Institutes of Health Guide for the Care and Use of Laboratory Animals (Laevis et al., 1996). The procedures were approved by the Animal Care and Use Committee of China Pharmaceutical University. All animals were maintained on a 12-hour light/12-hour dark cycle with free access to water and standard laboratory chow. Ab1e42 and pranlukast were purchased from Sigma Aldrich (St. Louis, MO). Pranlukast for oral administration was purchased from Jiangsu Hengrui Medicine Co, Ltd (Lianyungang, Jiangsu, China). Memantine was purchased from H. Lundbeck A/S (Copenhagen, Denmark). Donepezil (Aricept) was purchased from Eisai China, Inc (Suzhou, China). Antibodies were purchased from different companies: anti-CysLT1R from Santa Cruz Biotechnology, Inc (Heidelberg, Germany); anti-nuclear factor-kappa B p65 (NF-kB p65), antiprocaspase-3 or cleaved caspase-3, and anti-B-cell lymphoma2 (Bcl-2) from Cell Signaling Technology, Inc (Boston, MA); and antib-actin and secondary antibodies from Bioworld Technology Co, Ltd (Minneapolis, MN). All other chemicals were of analytical grade and were commercially available. Ab1e42 was reconstituted in phosphate-buffered saline (pH 7.4) at a concentration of 410 pmol/5 mL, and was aggregated by incubation at 37 C for 7 days before administration (Russo et al., 2012). 2.2. Primary cortical neuron culture Primary cortical neurons were prepared from embryonic brains (E16) of ICR mice as described previously (Cissé et al., 2011). Briefly, the cortices were digested with papain. Cells were plated in poly-L-lysine-coated 24- or 6-well plates at densities of approximately 2 104 cells per well and 1 105 cells per well, respectively, and maintained in serum-free Neurobasal Medium supplemented with B27 and antibiotics. Half the medium was changed after 5 days in culture. Cells were used after 5e11 days in culture. More than 90% of the cells were neurons, as determined by staining with an antibody against the neuron-specific marker Microtubule-associated protein-2 (MAP2) (data not shown). The cultured primary neurons were treated with Ab1e42 for 48 hours, followed by pranlukast treatment for 24 hours, and the treated neurons were collected for assays of cell viability, CysLT1R, NF-kB p65, caspase-3, and Bcl-2.
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2.3. Stereotaxic intrahippocampal Ab infusion ICR mice were anesthetized with an intraperitoneal injection of 350 mg/kg chloral hydrate and then immobilized on a stereotactic frame (SR-5; Narishige, Tokyo, Japan). The dura was exposed, and a glass micropipette connected to a microinjection pump (Dakumar Machinery, Sweden) was inserted into the left and right parietal cortices at a site 2.0 mm caudal to the bregma, 1.5 mm from the midline, and 2.0 mm below the dural surface (Paxinos and Franklin, 2003). Phosphate-buffered saline (5 mL) with or without Ab1e42 (410 pM/mouse) was injected bilaterally into the hippocampus (1 mL/min for all infusions). The micropipettes were left in place for 5 minutes to minimize backflux of liquid. After 3 days, the selective CysLT1R antagonist pranlukast was injected intrahippocampally (1.5 ng/mouse). Another 3 days later, one part of the mice was submitted to the Morris water maze (MWM) task for 6 days, and other part of the mice was sacrificed by cervical dislocation, and their brain tissues were taken for assay of CysLT1R, NF-kB p65, caspase-3, and Bcl-2. 2.4. Western blot analysis The neurons were washed with phosphate-buffered saline and homogenized in lysis buffer (10 mM Tris-HCl [pH 7.4], 150 mM NaCl, 1 mM ethylenediamine tetraacetic acid, 1% Triton X-100) containing phenylmethanesulfonyl fluoride. The homogenates were rocked at 4 C for 30 minutes and then centrifuged at 13,000g at 4 C for 30 minutes to remove cell debris and collect the resulting supernatant. Mouse hippocampus and cortex were chopped into small pieces, homogenized in 0.5 mL radio immunoprecipitation assay buffer. The dissolved proteins were collected from the supernatant after centrifugation at 12,000g for 15 minutes. Protein concentrations were determined using Coomassie blue-based assay reagent, and then the expression of CysLT1R, NFkB p65, caspase-3, or Bcl-2 was assessed. Protein extracts were separated by sodium dodecylsulfate-polyacrylamide gel electrophoresis, and were then transferred onto a polyvinylidene Fluoride membrane. The membrane was blocked with 5% skim milk in Tris buffer saline and then incubated at 4 C overnight with respective primary antibodies for anti-CysLT1R antibody (1:1000), anti-NF-kB p65 antibody (1:1000), anti-procaspase-3 or cleaved caspase-3 antibody (1:1000), anti-Bcl-2 anti-body (1:1000), or b-actin (inner control, 1:2000). After washing with Tris buffered saline with Tween, the membranes were incubated with a horseradish peroxidase-conjugated secondary antibody (1:5000) for 2 hours at room temperature. The antibody-reactive bands were visualized using enhanced chemiluminescence detection reagents and a gel imaging system (Tanon Science & Technology Co, Ltd, Shanghai, China). 2.5. Reverse transcription-polymerase chain reaction assay Total RNA was extracted from the mouse hippocampal and cortical tissues using Trizol reagents following the procedures described in the manufacturer’s instructions. For complementary DNA synthesis, aliquots of total RNA (2 mg) were mixed with 0.2 mg random hexamer primer, 20 U RNasin, 1 mM/L deoxy-ribonucleoside triphosphate (dNTP), and 200 U Moloney-murine leukemia virus reverse transcriptase in 20 mL of the reverse reaction buffer. The mixture was incubated at 25 C for 10 minutes, 42 C for 60 minutes, and then at 72 C for 10 minutes to inactivate the reverse transcriptase. Polymerase chain reaction (PCR) was performed on an Eppendorf Master Cycler (Eppendorf, Hamburg, Germany). The mixture contained: 1 mL reverse transcription-complementary DNA template dissolved in 20 mL reaction mixture containing 1 PCR
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buffer, 200 mM/L dNTP, 1.5 mM/L MgCl2, 20 pM of each primer, and 0.5 U Taq DNA polymerase. Cycling parameters were as follows: 94 C for 1 minute, followed by 33 cycles at 94 C for 30 seconds, at 63 C for 30 seconds, and at 72 C for 1 minute, with a final extension step of 72 C for 7 minutes. The abundance of transcripts in complementary DNA samples was measured by reverse transcription-PCR with the following primers: mouse CysLT1R forward 50 -ATTCCTGGAGAACATGAATGG-30 and reverse 50 -CATTGTTCTGCACTGTAGATG AG-30 (1062 bp, nucleotides 419e1480 in NM_021476.4; GenBank), and b-actin forward 50 -TCTTGGGTATGGAATCCTGTG-30 and reverse 50 -ATCTCCTTCTGCATCCTGTCA-30 (154 bp, nucleotides 876e1029 in NM_007393.3; GenBank). The amplification products were separated by electrophoresis on 2% agarose gel containing ethidium bromide and were then photographed. The optical density of the bands was determined by an image analysis system (Tanon Science & Technology Co, Ltd). 2.6. Behavioral assessment of cognitive function by the Morris water maze (MWM) task
Bilaney) were implanted at coordinates 2 mm caudal to the bregma, 1.4 mm from the midline, and 1.5 mm below the dural surface for the recording electrode; for the stimulating electrode, 3.8 mm caudal to the bregma, 3.0 mm from the midline, and 1.5 mm below the dural surface. Population spike (PS) were evoked by a squarewave constant current pulse stimulation of 0.1 m second duration at a frequency of 1/60 Hz. Before each experiment, input/output curves were generated to determine the maximal amplitude of the PS, and the intensity of stimulus was set at a level that evoked a PS at 55%e65% of the maximum amplitude. The amplitude of the PS was measured every 5 minutes. LTP was induced by applying highfrequency stimulation (HFS) of 8 trains of 400-msecond stimuli at 400 Hz repeated 3 times at 10-second intervals. All recordings and stimulations were performed using an online, computerized oscilloscope stimulator and data analysis interface system. The percentage of the ratio of absolute PS amplitude to the basal value was used to represent the level of PS amplitude. LTP was measured as normalized PS amplitude (as a percentage). 2.8. Statistical analysis
Spatial memory was assessed by the MWM, which consisted of 5 days of training (visible and invisible platform training sessions) and a probe trial on day 6. This was carried out as described previously (Tang et al., 2013). Mice were trained individually in a circular pool (diameter, 120 cm; height, 50 cm) filled to a depth of 30 cm with water maintained at 25 C. The maze was located in a lit room with visual cues. An escape platform (diameter, 9 cm) was placed in the center of 1 quadrant of the pool. The platform’s position was fixed throughout the training sessions; the starting points were pseudorandomized for each trial, with the animals facing toward the wall. Each mouse was trained individually in both visible-platform (days 1e2) and hidden-platform (days 3e5) versions. Visible-platform training was performed for baseline differences in vision and motivation; the escape platform was placed 1 cm below the surface of the water and was indicated by a small flag (height, 5 cm). The hiddenplatform version evaluates spatial learning and was used to determine the retention of memory to find the escape platform. During the training, the escape platform was placed 1 cm below the surface of the water and the flag was removed. The escape platform was always in the same place. On each day, the animal was subjected to 4 trials with a 1-hour interval between trials. Each trial lasted for 90 seconds unless the animal reached the escape platform. The time (escape latency) that elapsed until the mouse reached the escape platform was noted. If an animal failed to find the platform within 90 seconds, the test was ended and the animal was navigated gently to the platform by hand for 30 seconds. On day 6, the escape platform was removed and the probe trial started, during which animals had 90 seconds to search for the escape platform. The time spent in the target quadrant (i.e., the quadrant where the escape platform was located previously) and the number of platform location crossings were recorded. Data for escape latency, time spent in the target quadrant, number of platform location crossings, and swim speed were collected by the video tracking equipment and processed by a computer equipped with an analysis management system (Viewer 2 Tracking Software; Ji Liang Instruments, China). 2.7. In vivo hippocampal long-term potentiation (LTP) recording Mice were anesthetized with an intraperitoneal injection of 1.5 g/kg urethane and then immobilized on a stereotactic frame (SR-5; Narishige). The electrophysiological recording procedure was performed as described in a previous study (Yang et al., 2011). A stimulating electrode was placed in the perforant path, and a recording glass pipette was inserted into the dentate gyrus of the dorsal hippocampus. Electrodes (tungsten with Teflon coating;
All data were distributed normally and are presented as means standard error of the mean. In the case of single-mean comparison, data were analyzed by Student’s t-test. In case of multiple mean comparisons, the data were analyzed by analysis of variance and Newman-Keuls posttest, or 2-way repeated-measures analysis of variance followed by Bonferroni multiple comparison tests. A p value less than 0.05 was regarded to reflect a significant difference. 3. Results 3.1. Ab exposure upregulates CysLT1R expression in vitro or in vivo Initially, we tentatively investigated that effect of graded concentrations of Ab1e42, a more neurotoxic Ab species, on the expression of CysLT1R in primary cultured neurons using Western blot assay. Surprisingly, neurons treated with Ab1e42 (1e20 mM) for 48 hours showed obvious increases in plasma membrane CysLT1R protein, with a maximum effect at 10 mM or 20 mM of Ab1e42 (Fig. 1A and B). A time-dependent study showed that Ab1e42 at 10 mM increased CysLT1R expression graduallydan effect that was statistically significant at 48 hours and 72 hours compared with untreated neurons (Fig. 1C and D). We also performed bilateral stereotaxic intrahippocampal infusions of Ab1e42 in mice, and then detected CysLT1R expression of the hippocampus and cortex. Similarly, Ab1e42 caused significant increases in CysLT1R expression in both the hippocampus and cortex of mice on days 3, 5, and 7 instead of 1 or 2 days after infusion (p < 0.01; Fig. 1EeH). These data indicate an interesting phenomenon that Ab may upregulate CysLT1R expression in vitro and in vivo. To identify the CysLT1R potentially involved in AD pathophysiology, we examined brain CysLT1R protein in a transgenic (APPswe, PSEN1dE9) mouse model of Ab accumulation, which displays distinct pathologic characteristics. Lysates of hippocampus or cortex from these mice at the age of 1, 4, and 7 months were subjected to Western blot analysis. Intriguingly, CysLT1R protein displayed progressive increases in the cortex and hippocampus with age (p < 0.05 or p < 0.01), but no obvious changes were observed in those of age- and strain-matched wild-type control mice (p > 0.05; Fig. E1). 3.2. Blockade of CysLT1R reverses Ab1e42-induced CysLT1R expression upregulation Next, we wondered whether blockade of CysLT1R influences Ab1e42-triggered CysLT1R expression. As shown in Fig. 2, blockade
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Fig. 1. Amyloid-b (Ab)1e42 exposure induces cysteinyl leukotriene receptor 1 (CysLT1R) expression in vitro and in vivo. (AeD) CysLT1R levels are shown after cultured primary neurons were treated with Ab1e42 at 1, 10, and 20 mM for 48 hours (A, B) or with Ab1e42 at 10 mM for 12, 24, 48, and 72 hours (C, D). (EeH) CysLT1R levels were displayed in the hippocampus (E, F) and cortex (G, H) on days 1e7 after Ab1e42 (410 pmol/mouse) was injected bilaterally into the hippocampus of mice. b-actin was used as a loading control. Results are expressed as mean standard error of the mean (n ¼ 3e5). * p < 0.01 versus vehicle group. Abbreviations: d, day; h, hour.
of CysLT1R by applying the selective CysLT1R antagonist pranlukast after Ab1e42 exposure reversed CysLT1R protein expression and messenger RNA expression in vivo (Western blot (WB): F(2,8) ¼ 23.806, p ¼ 0.001 for the hippocampus and F(2,8) ¼ 18.208, p ¼ 0.003 for the cortex [Fig. 2A and B]; reverse transcription-PCR: F(2,8) ¼ 18.403, p ¼ 0.003 for the hippocampus and F(2,8) ¼ 13.059, p ¼ 0.007 the for cortex [Fig. 2C and D]) and in vitro (WB: F(5,17) ¼ 6.035, p ¼ 0.005; Fig. E2), although pranlukast alone did not affect CysLT1R expression.
subunits, and promoted an apoptotic profile of gene expression (Valerio et al., 2006). We were curious whether Ab1e42-activated NF-kB is involved in CysLT1R. Consistent with previous reports, our data showed that Ab1e42 activated NF-kB pathway characterized by p65 subunit increment. Interestingly, treatment of the selective CysLT1R antagonist pranlukast was able to block Ab1e42-activated NF-kB signaling in vivo (F(2,8) ¼ 26.034, p ¼ 0.001 for the hippocampus and F(2,8) ¼ 14.616, p ¼ 0.005 for the cortex [Fig. 3A and B]) and in vitro (F(5,17) ¼ 6.035, p ¼ 0.005; Fig. E3).
3.3. Blockade of CysLT1R suppresses Ab1e42-activated NF-kB signaling
3.4. Blockade of CysLT1R inhibits Ab1e42-triggered caspase-3 activation and Bcl-2 downregulation
It was reported that Ab1e42 activated the NF-kB pathway by selectively inducing the nuclear translocation of p65 and p50
Mounting evidence suggests that the activation of apoptotic pathways is a crucial event in Ab-induced neurotoxicity (Awasthi
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Fig. 2. Blockade of cysteinyl leukotriene receptor 1 (CysLT1R) reverses amyloid-b (Ab)1e42-induced CysLT1R upregulation in the hippocampus and cortex of mice. The CysLT1R antagonist pranlukast (Pran; 0.75 ng/side) was injected bilaterally into the hippocampus 3 days after intrahippocampal microinfusion of Ab1e42 (205 pM/side). (A, C) Three days after that, CysLT1R protein (A) or messenger RNA (C) in the hippocampus and cortex was detected by Western blot or reverse transcription-polymerase chain reaction, respectively. (B, D) Quantification of CysLT1R (B, D) is expressed as the ratio (as a percentage) of VehþVeh. b-actin was used as a loading control. Results are expressed as means standard error of the mean (n ¼ 3e5). * p < 0.05. y p < 0.01 versus Ab1e42 þ Veh for hippocampus. z p < 0.05. x p < 0.01 versus Ab1e42 þ Veh for cortex. Abbreviation: Veh, vehicle.
et al., 2005; Rohn et al., 2008; Stadelmann et al., 1999). To evaluate whether the blockade of CysLT1R plays an antiapoptotic role in Ab1e42-induced neurotoxicity, we examined caspase-3, which is known to be a crucial mediator of apoptosis through its protease activity, and Bcl-2, an antiapoptotic protein, by Western blot analysis. Quantification of Western blot revealed that Ab1e42 led to caspase-3 activation, as indicated by an increase in the ratio of caspase-3 fragment to procaspase-3, and led to a significant reduction in antiapoptotic protein Bcl-2 in the hippocampus and cortex of mice compared with the vehicle. In addition, treatment with Ab1e42 plus pranlukast was able to attenuate caspase-3 activation (F(2,8) ¼ 8.144, p ¼ 0.020 for the hippocampus and
F(2,8) ¼ 15.121, p ¼ 0.005 for the cortexe [Fig. 4A and B]) and elevate Bcl-2 levels (F(2,8) ¼ 12.801, p ¼ 0.007 for the hippocampus and F(2,8) ¼ 18.399, p ¼ 0.003 for the cortex [Fig. 4C and D]) compared with Ab1-42 plus vehicle. Consistent with an in vivo study, an in vitro study also showed that treatment with pranlukast at 0.1 mM or 10 mM reversed Ab1e42-triggered caspase-3 activation and Bcl-2 downregulation (F(5,17) ¼ 26.720, p < 0.001 for caspase-3 [Fig. E4A and B]; F(5,17) ¼ 7.896, p ¼ 0.002 for Bcl-2 [Fig. E4C and D]). To determine further the role of CysLT1R in Ab1e42-induced cytotoxicity, we used an 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide reduction assay and a lactate dehydrogenase
Fig. 3. Blockade of cysteinyl leukotriene receptor 1 (CysLT1R) suppresses amyloid-b (Ab)1e42-activated nuclear factor-kappa B (NF-kB) signaling in the hippocampus and cortex of mice. (A) Immunoblot of NF-kB p65 detected by Western blot. (B) Quantification of NF-kB p65 expressed as the ratio (as a percentage) of VehþVeh. Administration of Ab1e42 and pranlukast (Pran) was the same as that noted in the caption of Fig. 2. Values shown are means standard error of the mean (n ¼ 4). *p < 0.01 versus Ab1e42 þ Veh for hippocampus. y p < 0.05. z p < 0.01 versus Ab1e42 þ Veh for cortex. Abbreviation: Veh, vehicle.
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Fig. 4. Blockade of cysteinyl leukotriene receptor 1 (CysLT1R) attenuates amyloid-b (Ab)1e42-triggered caspase-3 activation and B-cell lymphoma-2 (Bcl-2) downregulation in the hippocampus and cortex of mice. The treatment procedure is as described in the caption for Fig. 2. (A, C) Precaspase-3 and cleaved caspase-3 (A) or Bcl-2 (C) were determined by Western blot using respective antibodies. (B) Caspase-3 activation was expressed as the ratio of caspase-3 fragment to procaspase-3. (D) Quantification of Bcl-2 was expressed as the ratio (as a percentage) of VehþVeh. Values shown are means standard error of the mean (n ¼ 4). * p < 0.05. y p < 0.01 versus Ab1e42 þ Veh for hippocampus. z p < 0.05. x p < 0.01 versus Ab1e42 þ Veh for cortex. Abbreviation: Pran, pranlukast; Veh, vehicle.
release assay to observe mitochondrial activity and the number of dead or plasma membrane-damaged cells. The results showed that pranlukast (0.1 mM or 10 mM) rescued neurons from Ab1e42-induced neurotoxicity (Fig. E5).
3.5. Blockade of CysLT1R reverses Ab1e42-induced cognitive deficit in mice To determine the role of CysLT1R in an Ab1e42-induced cognitive deficit, MWM tests were performed to evaluate the effect of pranlukast on intrahippocampal Ab1e42-injected mice. We first assessed the performance of mice in a nonspatial visible-platform variant of the MWM to test for baseline differences in vision and motivation among groups. Mice in each group exhibited similar escape latency in the visible-platform test, suggesting no influence of Ab1e42 or the CysLT1R antagonist pranlukast on vision or basal motivation of mice (F(2,199) ¼ 2.560, p ¼ 0.080; Fig. 5A). We then tested the mice in the spatial hidden-platform variant, and data showed that mice treated with Ab1e42 plus vehicle displayed increased escape latencies compared with those treated with vehicle plus vehicle (p < 0.001; Fig. 5B), which were reversed by pranlukast (p < 0.001; Fig. 5B; 4 trials per day for 3 days; F(2,299) ¼ 30.832, p < 0.001). In the probe trial, a putative measure of spatial learning and memory retention, all mice showed preference for the target quadrant, with the exception of mice in Ab1e42 plus vehicle group, which displayed a significant decrease in the time spent in the target quadrant (p < 0.05; Fig. 5C) and the number of platform location crossings (p < 0.05; Fig. 5D) compared with those of the vehicle plus vehicle group. In contrast, mice in the Ab1e42 plus pranlukast group showed significant increases in both indices compared with those in the Ab1e42 plus vehicle group (p < 0.05 for the time, p < 0.05 for the number; Fig. 5C and D). In addition, all mice showed similar swim speeds during the tests (Fig. 5F). These data suggest that blockade of CysLT1R reverses an Ab1e42-induced cognitive deficit in mice.
3.6. Chronic treatment of CysLT1R antagonist, N-methyl-Daspartate receptor antagonist and acetylcholinesterase inhibitor produce similar effects on the behavior and hippocampal LTP in an experimental model of AD To reveal the potential value of CysLT1R for treatment of AD, we performed a comparison study of the CysLT1R antagonist pranlukast with the N-methyl-D-aspartate (NMDA) receptor antagonist memantine and acetylcholinesterase (AChE) inhibitor donepezil that were approved by the Food and Drug Administration for treatment of AD. Intrahippocampal Ab1e42-injected mice were administered pranlukast (0.6 mg/kg), memantine (20 mg/kg), and donepezil (2 mg/kg) intragastrically for 14 consecutive days, and then subjected to the MWM test. Results showed that mice in each group exhibited similar escape latencies in the visible-platform test (F(4,389) ¼ 0.772, p ¼ 0.544; Fig. 6A), suggesting no difference in vision or basal motivation among the 5 groups. In the spatial hidden-platform variant, Ab1e42-injected mice treated with pranlukast, memantine, and donepezil exhibit progressively decreased escape latencies during training (4 times a day) compared with the Ab1e42-injected mice treated with vehicle (F(4,599) ¼ 6.647, p < 0.001, Fig. 6B). During the probe trial, a putative measure of spatial learning and memory retention, only the Ab1e42-injected mice treated with vehicle failed to favor the target platform location (p < 0.05 or p < 0.01). The Ab1e42-injected mice treated with pranlukast, memantine, and donepezil showed significant increases in both the time spent in the target quadrant and in the number of platform location crossings during the final probe trial (F(4,39) ¼ 6.745, p < 0.001 for the time; F(4,39) ¼ 4.488, p ¼ 0.005 for the number; Fig. 6CeE). No differences were observed in the performance of mice treated with 3 drugs in place navigation (F(2,359) ¼ 1.350, p ¼ 0.261) or in the probe trial (F(2,23) ¼ 0.035, p ¼ 0.965 and F(2,23) ¼ 0.201, p ¼ 0.820). There were no differences in swimming speed among groups during the MWM test (Fig. 6F), and these drugs had no effects on learning and memory in unimpaired mice (data not
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Fig. 5. Blockade of cysteinyl leukotriene receptor 1 (CysLT1R) rescues amyloid-b (Ab)1e42-induced spatial learning and memory deficits in mice. Mice received bilateral intrahippocampal injection of Ab1e42 (205 pM/side) 3 days later, followed by a single bilateral intrahippocampal injection of the CysLT1R antagonist pranlukast (Pran; 0.75 ng/side). Mice were tested in the Morris water maze task on 3 day after Pran injection. Day 0 indicates performance on the first trail; subsequent points represent an average of all daily trails. (A) No differences were found in the escape latency among all groups during the visible platform test (days 1-2). (B) Changes in escape latency to reach the hidden platform during acquisition trials (days 3-5). (CeE) The time spent in the target quadrant (C), the number of platform location crossings (D), and the representative swim paths of individual mice in each group during the probe trial test on day 6 (E) are presented. (F) Average swimming speed on each day of testing is also shown. Values shown are means standard error of the mean (n ¼ 8e10). * p < 0.05 versus Ab1e42 þ Veh group. Abbreviation: Veh, vehicle.
shown). In addition, LTP recordings showed that hippocampal LTP was significantly inhibited in Ab1e42-injected mice (142.12 1.65%) after the tetanic stimulation persisting for at least 60 minutes compared with controls (173.55 3.70%, p < 0.001), and was significantly increased in Ab1e42-injected mice with chronic treatment of pranlukast, memantine, or donepezil (171.73 3.59%, 177.19 2.56%, and 173.91 2.12%, p < 0.001; Fig. 6G and H). No differences were observed in the ameliorative effects of 3 drugs on hippocampal LTP in Ab1e42-injected mice (F(2,35) ¼ 1.414, p ¼ 0.257). We also used liquid chromatography-mass spectrometry/ mass spectrometry analysis to confirm its presence in the brain after oral administration of pranlukast (data not shown). These data suggest that the CysLT1R antagonist produced similar
improvements as the NMDA receptor antagonist or AChE inhibitor in Ab1e42-induced memory impairment. 4. Discussion Our knowledge of the mechanisms underlying the action of Ab remains limited despite the abundance of evidence pointing to an essential role of Ab in AD pathophysiology. Explicating the molecular mechanisms that underlie neuronal apoptosis during AD is therefore critical for unraveling the pathophysiology of the disease. Our study clearly shows that Ab1e42, the most toxic and proaggregate form of Ab, induced increases in CysLT1R expression in a dose- or timedependent manner in primary neurons, and bilateral hippocampal
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Fig. 6. Chronic treatment with the cysteinyl leukotriene receptor 1 (CysLT1R) antagonist pranlukast (Pran) improves amyloid-b (Ab)1e42-induced memory impairment and hippocampal LTP depression such as the N-methyl-D-aspartate (NMDA) receptor antagonist memantine (Mem) or acetylcholinesterase (AChE) inhibitor donepezil (Don). Animals received bilateral intrahippocampal injections of Ab1e42 (205 pM/side), and they were treated daily with intragastric administration of Pran (0.6 mg/kg), Mem (20 mg/kg), and Don (2 mg/kg). After 14 days, mice were tested in the Morris water maze task or hippocampal long-term potentiation (LTP). (A) No differences were found in the escape latency among all groups during visible platform test (days 1-2). (B) Changes in escape latency to reach the hidden platform during acquisition trials (days 3-5). (CeE) The time spent in the target quadrant (C), the number of platform location crossings (D), and the representative swim paths of individual mice in each group during the probe trial test on day 6 (E) are presented. (F) Average swimming speed on each day of testing is also shown. High-frequency stimulation (HFS) was given at the end of a 30-minute baseline recording of population spike (PS). (G, H) The time course of PS after HFS is plotted (G), and the average PS at 60 minutes post-HFS is summarized as columns (H). Values shown are expressed as means standard error of the mean (n ¼ 8e10). * p < 0.05. y p < 0.01 versus Ab1e42 þ Veh. Abbreviation: Veh, vehicle.
injection of Ab1e42 in ICR mice also caused increases in CysLT1R expression in the hippocampus and cortex. This upregulation was coupled with neurotoxicity, as evidenced by increased NF-kB p65, activated caspase-3, decreased Bcl-2, and lowered cellular viability as well as impaired memory. More important, blockade of CysLT1R reversed Ab1e42-induced CysLT1R upregulation and inhibited Ab1e42-induced neurotoxicity in vitro and in vivo. Furthermore,
chronic treatment with the CysLT1R antagonist pranlukast (0.6 mg/ kg), NMDA receptor antagonist memantine (20 mg/kg) and AChE inhibitor donepezil (2 mg/kg) produced similar beneficial effects on memory behavior and hippocampal LTP in intrahippocampal Ab1e42-injected mice. In addition, age-dependent increases of CysLT1R were observed in both the hippocampus and the cortex of the transgenic (APPswe, PSEN1dE9) AD mouse model. Collectively,
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these findings strongly suggest that CysLT1R is involved in Ab1e42induced neurotoxicity, and raise the possibility that CysLT1R is an important player in the progression of AD. CysLT1R is a high-affinity G protein-coupled receptor for the pro-inflammatory mediator LTD4, which is implicated in many inflammatory conditions (Funk, 2001; Serhan et al., 1996). The pathophysiological role of CysLT1R in several inflammatory conditions is well documented, with a general emphasis on asthma, and during the past 25 years a considerable effort has been made to identify and develop receptor antagonists to improve asthma management, limit its morbidity, and reduce the side effects of current medications (Singh et al., 2010). It has been reported that CysLT1R mediates astrocyte proliferation (Ciccarelli et al., 2004; Huang et al., 2008), traumatic brain injury, brain cryoinjury and brain tumors (Dinq et al., 2007; Zhang et al., 2004), and acute neuronal injury after focal cerebral ischemia in mice (Zhang et al., 2006). Neurons are believed to be the major source of Ab that has a causal role in AD (Palop and Mucke, 2010). We used exogenous Ab1e42 to mimic Ab release of neurons and found a novel pathophysiological link between Ab and CysLT1R. Interestingly, basal expression of CysLT1R is pretty weak in neurons, and neurons treated with Ab1e42 displayed a significant increase in CysLT1R expression. This effect was abolished completely by the selective CysLT1R antagonist pranlukast. These results imply that CysLT1R might be an inducible receptor with more pathophysiological significance in the brain. How Ab1e42 triggers CysLT1R upregulation remains to be elucidated. It is possible that Ab1e42 first induces the upregulation of 5-lipoxygenase (Firuzi et al., 2008), then the resulting CysLTs (such as LTD4) induce CysLT1R expression, which may be responsible for the observed memory deficit (Tang et al., 2013). It has been reported that CysLT1Rmediated signaling refers to the NF-kB pathway in some peripheral cells (Kawano et al., 2003; Suzuki et al., 2008; Thompson et al., 2006). Recently, CysLT1R-mediated NF-kB signaling was also observed in neurons (Wang et al., 2013). Ab has been shown to activate NF-kB (Kaltschmidt et al., 1997). Activated NF-kB was detected in AD brains (Boissier et al., 1997; Terai et al., 1996), and in neuronal and glial nuclei proximal to early-stage plaques (Ferrer et al., 1998; Kaltschmidt et al., 1997). Ab1e42 activated the NF-kB pathway by selectively inducing the nuclear translocation of p65 and p50 subunits, and promoted an apoptotic profile of gene expression (Valerio et al., 2006). Numerous studies have implicated apoptosis as a major pathway of cell death in AD (Culmsee and Landshamer, 2006), and inhibiting proapoptotic protein caspase-3 or activating antiapoptotic protein Bcl-2 in neurons of patients with AD could be an effective means of treating this disease (Awasthi et al., 2005; Rohn et al., 2008). Targeting the NF-kB pathway might be useful to counteract the establishment or progression of AD through prevention of neuronal cell death. The current study shows that blockade of CysLT1R suppressed Ab1-42triggered NF-kB signaling as well as neuronal apoptosis marked by activated caspase-3 and decreased Bcl-2 in vivo or in vitro. These findings suggest that CysLT1R is involved in Ab1e42-activated the NFkB pathway and subsequent cellular apoptosis. Further study showed that Ab1e42-injected mice treated either acutely or chronically with the CysLT1R antagonist pranlukast displayed significant improvement in cognitive deficit as evaluated by the MWM test. Even more exciting, CysLT1R antagonist pranlukast produced significant improvement in memory impairment and hippocampal LTP inhibition in intrahippocampal Ab1e42-injected mice, and the effects were similar to those of NMDA receptor antagonist memantine or AChE inhibitor donepezil that is used clinically as a treatment of AD. These results strongly support that CysLT1R plays a critical role in Ab1e42-induced neurotoxicity. Overall, our study demonstrates for the first time the contributions of CysLT1R to Ab1e42-induced neurotoxicity. These appear to
be that Ab1e42 induces CysLT1R activation, leading to NF-kB signaling enhancement, which triggers caspase-3 activation and Bcl-2 inactivation, producing apoptosis and cognitive impairment. Thus, CysLT1R could be a novel target for developing new drugs in the treatment of AD. In addition, CysLT1R antagonists such as pranlukast, zafirlukast, and montelukast used clinically for the treatment of asthma might be beneficial for AD. Disclosure statement None of authors have financial interests to disclose. Acknowledgements This work was supported, in part, by grants from the National Natural Science Foundation of China (81273497). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.neurobiolaging. 2013.09.036. References Arnold, S.E., Hyman, B.T., Flory, J., Damasio, A.R., Van Hoesen, G.W., 1991. The topographical and neuroanatomical distribution of neurofibrillary tangles and neuritic plaques in the cerebral cortex of patients with Alzheimer’s disease. Cereb. Cortex 1, 103e116. Awasthi, A., Matsunaga, Y., Yamada, T., 2005. Amyloid-beta causes apoptosis of neuronal cells via caspase cascade, which can be prevented by amyloid betaderived short peptides. Exp. Neurol. 196, 282e289. Boissier, E.F., Hunot, S., Faucheux, B., Duyckaerts, C., Hauw, J.J., Agid, Y., Hirsch, E.C., 1997. Nuclear translocation of NF-kappaB in cholinergic neurons of patients with Alzheimer’s disease. Neuroreport 8, 2849e2852. Chu, J., Praticò, D., 2011a. 5-Lipoxygenase as an endogenous modulator of amyloid b formation in vivo. Ann. Neurol. 69, 34e46. Chu, J., Praticò, D., 2011b. Pharmacologic blockade of 5-lipoxygenase improves the amyloidotic phenotype of an Alzheimer’s disease transgenic mouse model involvement of g-secretase. Am. J. Pathol. 178, 1762e1769. Chu, J., Praticò, D., 2012. Involvement of 5-lipoxygenase activating protein in the amyloidotic phenotype of an Alzheimer’s disease mouse model. J. Neuroinflamm. 14, 127e136. Ciccarelli, R., D’Alimonte, I., Santavenere, C., D’Auro, M., Ballerini, P., Nargi, E., Buccella, S., Nicosia, S., Folco, G., Caciagli, F., Di Iorio, P., 2004. Cysteinyl-leukotrienes are released from astrocytes and increase astrocyte proliferation and glial fibrillary acidic protein via Cys-LT1 receptors and mitogen-activated protein kinase pathway. Eur. J. Neurosci. 20, 1514e1524. Cissé, M., Halabisky, B., Harris, J., Devidze, N., Dubal, D.B., Sun, B., Orr, A., Lotz, G., Kim, D.H., Hamto, P., Ho, K., Yu, G.Q., Mucke, L., 2011. Reversing EphB2 depletion rescues cognitive functions in Alzheimer model. Nature 469, 47e52. Culmsee, C., Landshamer, S., 2006. Molecular insights into mechanisms of the cell death program: role in the progression of neurodegenerative disorders. Curr. Alzheimer Res. 3, 269e283. Daniela, L.K., Norbert, M., 2010. Neuroinflammation, microglia and implications for anti-inflammatory treatment in Alzheimer’s disease. Int. J. Alzheimers Dis. 10, 4061e4069. De Strooper, B., Annaert, W., 2000. Proteolytic processing and cell biological functions of the amyloid precursor protein. J. Cell Sci. 113, 1857e1870. Deshpande, A., Mina, E., Glabe, C., Busciglio, J., 2006. Different conformations of amyloid beta induce neurotoxicity by distinct mechanisms in human cortical neurons. J. Neurosci. 26, 6011e6018. Dinq, Q., Fang, S.H., Zhou, Y., Zhang, L.H., Zhang, W.P., Chen, Z., Wei, E.Q., 2007. Cysteinyl leukotriene receptor 1 partially mediates brain cryoinjury in mice. Acta Pharmacol. Sin. 28, 945e952. Ferrer, I., Marti, E., Lopez, E., Tortosa, A., 1998. NF-kB immunoreactivity is observed in association with beta A4 diffuse plaques in patients with Alzheimer’s disease. Neuropathol. Appl. Neurobiol. 24, 271e277. Firuzi, O., Zhuo, J., Chinnici, C.M., Wisniewski, T., Praticò, D., 2008. 5-Lipoxygenase gene disruption reduces amyloid-beta pathology in a mouse model of Alzheimer’s disease. FASEB J. 22, 1169e1178. Funk, C.D., 2001. Prostaglandins and leukotrienes: advances in eicosanoid biology. Science 294, 1871e1875. Giunta, B., Fernandez, F., Nikolic, W.V., Obregon, D., Rrapo, E., Town, T., Tan, J., 2008. Inflammaging as a prodrome to Alzheimer’s disease. J. Neuroinflamm. 5, 1e15. Gorelick, P.B., 2010. Role of inflammation in cognitive impairment: results of observational epidemiological studies and clinical trials. Ann. N Y Acad. Sci. 1207, 155e162.
S.-S. Tang et al. / Neurobiology of Aging 35 (2014) 590e599 Grundke-Iqbal, I., Iqbal, K., Tung, Y.C., Quinlan, M., Wisniewski, H.M., Binder, L.I., 1986. Abnormal phosphorylation of the microtubule-associated protein tau (tau) in Alzheimer cytoskeletal pathology. Proc. Natl. Acad. Sci. U.S.A 83, 4913e4917. Guo, J.T., Yu, J., Grass, D., de Beer, F.C., Kindy, M.S., 2002. Inflammation-dependent cerebral deposition of serum amyloid a protein in a mouse model of amyloidosis. J. Neurosci. 22, 5900e5909. Hardy, J., Selkoe, D.J., 2002. The amyloid hypothesis of Alzheimer’s disease: progress and problems on the road to therapeutics. Science 297, 353e356. Huang, X.J., Zhang, W.P., Li, C.T., Shi, W.Z., Fang, S.H., Lu, Y.B., Chen, Z., Wei, E.Q., 2008. Activation of CysLT receptors induces astrocyte proliferation and death after oxygen-glucose deprivation. Glia 56, 27e37. Jimenez, S., Baqlietto-Varqas, D., Caballero, C., Moreno-Gonzalez, I., Torres, M., Sanchez-Varo, R., Ruano, D., Vizuete, M., Gutierrez, A., Vitorica, J., 2008. Inflammatory response in the hippocampus of PS1M146L/APP751SL mouse model of Alzheimer’s disease: age-dependent switch in the microglial phenotype from alternative to classic. J. Neurosci. 28, 11650e11661. Kaltschmidt, B., Uherek, M., Volk, B., Baeuerle, P.A., Kaltschmidt, C., 1997. Transcription factor NF-kappaB is activated in primary neurons by amyloid beta peptides and in neurons surrounding early plaques from patients with Alzheimer’s disease. Proc. Natl. Acad. Sci. U.S.A 94, 2642e2647. Kawano, T., Matsuse, H., Kondo, Y., Machida, I., Saeki, S., Tomari, S., Mitsuta, K., Obase, Y., Fukushima, C., Shimoda, T., Kohno, S., 2003. Cysteinyl leukotrienes induce nuclear factor-kB activation and RANTES production in a murine model of asthma. J. Allergy Clin. Immunol. 112, 369e374. Laevis, Tropicials, X., 1996. Environment, Housing, and Management, in: National Research Council, eighth ed. The National Academic Press, Washington, D. C., pp. 80e103. Manev, H., Chen, H., Dzitoyeva, S., Manev, R., 2011. Cyclooxygenases and 5-lipoxygenase in Alzheimer’s disease. Prog. Neuropsychopharmacol. Biol. Psychiatry 35, 315e319. O’Hare, E., Scopes, D.I., Treherne, J.M., Monaghan, J., Palmer, P.M., Amijee, H., Kim, E.M., 2011. Novel anti-inflammatory compound SEN1176 alleviates behavioral deficits induced following bilateral intrahippocampal injection of aggregated amyloid-b1-42. J. Alzheimers Dis. 25, 219e229. Palop, J.J., Mucke, L., 2010. Amyloid-b induced neuronal dysfunction in Alzheimer’s disease: from synapses toward neural networks. Nat. Neurosci. 13, 812e818. Paxinos, G., Franklin, K.B.J., 2003. The Mouse Brain in Stereotaxic Coordinates, second ed. Academic Press, San Diego, CA. Rohn, T.T., Vyas, V., Hernandez-Estrada, T., Nichol, K.E., Christie, L.A., Head, E., 2008. Lack of pathology in a triple transgenic mouse model of Alzheimer’s disease after overexpression of the anti-apoptotic protein Bcl-2. J. Neurosci. 28, 3051e3059. Russo, I., Caracciolo, L., Tweedie, D., Choi, S.H., Greig, N.H., Barlati, S., Bosetti, F., 2012. 3,60 -Dithiothalidomide, a new TNF-a synthesis inhibitor, attenuates the effect of Ab1-42 intracerebroventricular injection on hippocampal neurogenesis and memory deficit. J. Neurochem. 122, 1181e1192. Selkoe, D.J., 2002. Deciphering the genesis and fate of amyloid beta-protein yields novel therapies for Alzheimer disease. J. Clin. Invest. 11, 1375e1381. Serhan, C.N., Haeggström, J.Z., Leslie, C.C., 1996. Lipid mediator networks in cell signaling: update and impact of cytokines. FASEB J. 10, 1147e1158. Singh, R.K., Gupta, S., Dastidar, S., Ray, A., 2010. Cysteinyl leukotrienes and their receptors: molecular and functional characteristics. Pharmacology 85, 336e349.
599
Stadelmann, C., Deckwerth, T.L., Srunivasan, A., Bancher, C., Bruck, W., Jellinger, K., Lassmann, H., 1999. Activation of caspase-3 in single neurons and autophagic granules of granulovacuolar degeneration in Alzheimer’s disease: evidence for apoptotic cell death. Am. J. Pathol. 155, 1459e1466. Suzuki, S., Takeuchi, K., Ishinaga, H., Basbaum, C., Majima, Y., 2008. Leukotriene D4 upregulates MUC2 gene transcription in human epithelial cells. Pharmacology 81, 221e228. Tang, S.S., Wang, X.Y., Hong, H., Long, Y., Li, Y.Q., Xiang, G.Q., Jiang, L.Y., Zhang, H.T., Liu, L.P., Miao, M.X., Hu, M., Zhang, T.T., Hu, W., Ji, H., Ye, F.Y., 2013. Leukotriene D4 induces cognitive impairment through enhancement of CysLT1R-mediated amyloid-b generation in mice. Neuropharmacology 65, 182e192. Terai, K., Matsuo, A., McGeer, P.L., 1996. Enhancement of immunoreactivity for NFkappa B in the hippocampal formation and cerebral cortex of Alzheimer’s disease. Brain Res. 735, 159e168. Thal, D.R., Rüb, U., Orantes, M., Braak, H., 2002. Phases of Ab deposition in the human brain and its relevance for the development of AD. Neurology 58, 1791e1800. Thompson, C., Cloutier, A., Boss, Y., Thivierge, M., Gouill, C.L., Larivée, P., McDonald, P.P., Stankova, J., Rola-Pleszczynski, M., 2006. CysLT1 receptor engagement induces activator protein-1- and NF-kappaB-dependent IL-8 expression. Am. J. Respir. Cell Mol. Biol. 35, 697e704. Valerio, A., Boroni, F., Benarese, M., Sarnico, I., Ghisi, V., Bresciani, L.G., Ferrario, M., Borsani, G., Spano, P., Pizzi, M., 2006. NF-kappaB pathway: a target for preventing beta-amyloid (Abeta)-induced neuronal damage and Abeta42 production. Eur. J. Neurosci. 23, 1711e1720. Wang, L., Du, C., Lv, J., Wei, W., Cui, Y., Xie, X., 2011. Antiasthmatic drugs targeting the cysteinyl leukotriene receptor 1 alleviate central nervous system inflammatory cell infiltration and pathogenesis of experimental autoimmune encephalomyelitis. J. Immunol. 187, 2336e2345. Wang, X.Y., Tang, S.S., Hu, M., Long, Y., Li, Y.Q., Liao, M.X., Ji, H., Hong, H., 2013. Leukotriene D4 induces amyloid-b generation via CysLT1R-mediated NF-kB pathways in primary neurons. Neurochem. Int. 62, 340e347. Wang, C., Yang, X.M., Zhuo, Y.Y., Zhou, H., Lin, H.B., Cheng, Y.F., Xu, J.P., Zhang, H.T., 2012. The phosphodiesterase-4 inhibitor rolipram reverses Ab-induced cognitive impairment and neuroinflammatory and apoptotic responses in rats. Int. J. Neuropsychopharmacol. 9, 1e18. Wyss-Coray, T., 2006. Inflammation in Alzheimer disease: driving force, bystander or beneficial response? Nat. Med. 12, 1005e1015. Yang, J., Hu, Z.L., Jiang, B., Ni, L., Jin, Y., Chen, J.G., Wang, F., 2011. Effect of chloramineT on long-term potentiation at synapses between perforant path and dentate gyrus in hippocampus of rats in vivo. Neurotoxicology 32, 199e205. Zhang, W.P., Hu, H., Zhang, L., Dinq, W., Yao, H.T., Chen, K.D., Sheng, W.W., Chen, Z., Wei, E.Q., 2004. Expression of cysteinyl leukotriene receptor 1 in human traumatic brain injury and brain tumors. Neurosci. Lett. 363, 247e251. Zhang, Y.J., Zhang, L., Ye, Y.L., Fang, S.H., Zhou, Y., Zhang, W.P., Lu, Y.B., Wei, E.Q., 2006. Cysteinyl leukotriene receptors CysLT1 and CysLT2 are upregulated in acute neuronal injury after focal cerebral ischemia in mice. Acta Pharmacol. Sin. 27, 1553e1560. Zotova, E., Nicoll, J.A., Kalaria, R., Holmes, C., Boche, D., 2010. Inflammation in Alzheimer’s disease: relevance to pathogenesis and therapy. Alzheimers Res. Ther. 2, 1e9.