Neurochemistry International 62 (2013) 340–347

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Neurochemistry International

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Leukotriene D4 induces amyloid-b generation via CysLT1R-mediated NF-jBpathways in primary neurons

Xiao Yun Wang 1, Su Su Tang 1, Mei Hu, Yan Long, Yong Qi Li, Ming Xing Liao, Hui Ji, Hao Hong ⇑Department of Pharmacology, Laboratory for Diabetes & Brain Diseases, China Pharmaceutical University, Nanjing 210009, China

a r t i c l e i n f o

Article history:Received 4 September 2012Received in revised form 29 December 2012Accepted 4 January 2013Available online 11 January 2013

Keywords:LTD4CysLT1RNF-jBBACE1Ab

0197-0186/$ - see front matter � 2013 Elsevier Ltd. Ahttp://dx.doi.org/10.1016/j.neuint.2013.01.002

⇑ Corresponding author. Address: Department of Pceutical University, Tong Jiaxiang, Nanjing 2100083207491.

E-mail address: haohongchina@hotmail.com (H. H1 Both the authors are co-first authors.

a b s t r a c t

Although the pathogenesis of sporadic Alzheimer’s disease (AD) is not clearly understood, neuroinflam-mation has been known to play a role in the pathogenesis of AD. To investigate a functional link betweenthe neuroinflammation and AD, the effect of leukotriene D4 (LTD4), an inflammatory lipid mediator, wasstudied on amyloid-b generation in vitro. Application of LTD4 to cell monolayers at concentrations up to40 nM LTD4 caused increases in the Ab releases. Concentrations P40 nM LTD4 decreased neuronalviability. Application of 20 nM LTD4 caused a significant increase in Ab generation, as assessed by ELISAor Western blotting, without significant cytotoxicity. At this concentration, exposure of neurons to LTD4for 24 h produced maximal effect in the Ab generation, and significant increases in the expressions ofcysteinyl leukotriene 1 receptor (CysLT1R) and activity of b- or c-secretase with complete abrogationby the selective CysLT1R antagonist pranlukast. Exposure of neurons to LTD4 for 1 h showed activationof NF-jB pathway, by assessing the levels of p65 or phospho-p65 in the nucleus, and either CysLT1Rantagonist pranlukast or NF-jB inhibitor PDTC prevented the nuclear translocation of p65 and theconsequent phosphorylation. PDTC also inhibited LTD4-induced elevations of b- or c-secretase activityand Ab generation in vitro. Overall, our data show for the first time that LTD4 causes Ab production byenhancement of b- or c-secretase resulting from activation of CysLT1R-mediated NF-jB signalingpathway. These findings provide a novel pathologic link between neuroinflammation and AD.

� 2013 Elsevier Ltd. All rights reserved.

1. Introduction proteases, and reactive oxygen species, has been observed in the

The key pathological event in Alzheimer’s disease (AD) is theprogressive accumulation of amyloid-b (Ab) in the brain. Ab de-rives from sequential cleavage of the b-amyloid precursor protein(APP) by the b- and c-secretase. The b-secretase (b-site amyloidprecursor protein cleaving enzyme, BACE1) cleaves the ectodomainof APP, producing an APP C-terminal fragment, which is furthercleaved to Ab1–40 and Ab1–42 (Selkoe, 2001). Accumulating evi-dences strongly suggested that b- or c-secretase-dependent pro-duction of Ab is the key factor in the pathogenesis of AD(Fukumoto et al., 2002; Fukumoto et al.,2004; Haass, 2004). There-fore, cellular factors that affect the secretase-dependent produc-tion of Ab may be good targets for development of drugs toprevent and treat AD.

It has been repeatedly reported that inflammation is importantin the pathogenesis of AD. Chronic inflammation, which isindicated by accumulation of microglia around senile plaquesand elevated levels of inflammatory cytokines, chemokines,

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harmacology, China Pharma-9, China. Tel./fax: +86 25

ong).

brains of AD patients (Townsend and Praticò, 2005). Furthermore,trauma to the brain and ischemia, both of which can activateinflammation, are major factors for AD (Ikonomovic et al., 2004).

Cysteinyl leukotrienes (CysLTs) including LTC4, LTD4, and LTE4are inflammatory lipid mediators derived from the 5-Lipoxygenase(5-LO) pathway of the arachidonic acid metabolism. 5-LO catalyzesthe conversion of arachidonic acid to the unstable intermediate,leukotriene A4 (LTA4), which is then conjugated to reduced gluta-thione by leukotriene C4 synthase, forming LTC4. After transporta-tion to the extracellular space by an ATP-dependent transporter(multidrug resistance-associated protein) that recognizes its gluta-thione moiety, LTC4 is converted to leukotriene D4 (LTD4) andthen to the terminal product leukotriene E4 (LTE4) by a dipepti-dase (Orning and Hammarström, 1980). LTE4 is metabolized bythe formation of N-acetyl derivatives to lose biological activity,and then excreted by kidneys. The actions of CysLTs are mediatedby activating the corresponding receptors. The LTD4 is the highestintrinsic activity among CysLTs (Singh et al., 2010). Pharmacologi-cal characterizations have suggested the existence of at least twotypes of cysteinyl leukotriene receptors, designated as cysteinylleukotriene receptor 1 (CysLT1R) and cysteinyl leukotriene recep-tor 2 (CysLT2R), based on potency of agonists and antagonists(Bäck et al., 2011). CysLT1R has been extremely successful target

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for asthma, and its antagonist such as pranlukast, montelukast, andetc. is currently used as a treatment for asthma.

It has been shown that 5-LO and leukotrienes in the central ner-vous system (CNS) have neuromodulatory and neuroendocrinefunctions (Lammers et al., 1996; Morris and Rodger, 1998; Piomelli,1994) and play an important role in aging-associated neurodegener-ative diseases (Chinnici et al., 2006; Uz et al.,1998). Levels of 5-LOgene transcription are significantly increased in the hippocampus,cortex, and brain stem of Tg2576 mice, an animal model of AD(Firuzi et al., 2008). Consistent with these, 5-LO immunoreactivityis increased in the hippocampus of patients with AD (Ikonomovicet al., 2008). Moreover, Ab deposition is reduced in the brainof Tg2576� 5-LO�/� mice. Similar results were also observedin vitro. Based on the studies described above, it is reasonable tohypothesize that CysLTs, the derivates of 5-LO, contribute to Abgeneration and pathogenesis of AD. Thus, in the present studieswe investigated whether LTD4 induces Ab production via CysLT1

R-mediated NF-jB pathway activation in primary cultured neurons.

2. Materials and methods

2.1. Materials

LTD4, pranlukast and 3-(4,5-dimethythiazole-2-yl)-2,5-diphe-nyl-tetrazolium bromide (MTT) were purchased from SigmaAldrich (St-Louis, Mississippi, USA). Eagle’s MEM and fetal calfserum (FCS) were from Thermo Electron Corporation (Waltham,MA) and ELISA assay kit for Ab was from Immuno-BiologicalLaboratories Co. Ltd (Gunma, Japan). Antibodies were purchasedfrom different companies: anti-Ab1–42 from Abcam Ltd. (Hongkong,China), anti-CysLT1R from Santa Cruz Biotechnology, Inc.(Heidelberg, Germany), anti-BACE1 and anti-NF-jB p65 or NF-jBphosphor-p65 from Cell Signaling Technology, Inc. (Boston,Massachusetts, USA), anti- b-actin was from Boster BiotechnologyCo., Ltd (Wuhan, China), secondary antibodies from Bioworld Tech-nology Co., Ltd (Minneapolis, Minnesota, USA). b- and c-secretasekits were obtained from R&D systems (Wiesbaden, Germany).Lactate dehydrogenase (LDH) assay kit was provided by JianchenBiotech (Nanjing, China). All other chemicals were of analyticalgrade and commercially available.

2.2. Mouse primary cortical neuron cultures

The cerebral cortex was aseptically excised from fetal ICR mice(14–15 days of gestation) (Yangzhou University Medical Center,Yangzhou, China) and put in Eagle’s MEM solution containing10% heat-inactivated FCS and 2 mM L-glutamine and 24 mMNaHCO3. The neuronal cells were dispersed by pipetting, passedthrough a meshed filter (pore size: 180 lm), and placed on plasticcoverslips, or 6- or 24-well plates coated with poly-L-lysine. Thecells were cultured in the same medium at 37 �C in a humidified5% CO2 atmosphere for early 8 days, and thereafter in the mediumcontaining 10% heat-inactivated horse serum (HS) instead of FCSfor 6 days. The culture medium was changed every other day. Onthe 6th day after plating, the cells were treated with cytosine b-D-arabinofuranoside hydrochloride (Sigma–Aldrich) at 10 lM toremove non-neuronal cells. The cells were used for experimentsafter the culture for 9–14 days. One hour after the culture mediumcontaining 10% HS was renewed, the cells were stimulated withLTD4 for the indicated times and concentrations. Inhibitors wereadded to the former 10% HS-containing medium 1 h before stimu-lation and also applied to the latter 1% HS-containing medium afterstimulation.

2.3. Cytotoxicity assay

Mitochondrial activity was measured with the MTT assay. Neu-ronal cells (1 � 105 cells/well) grown on 24-well culture plateswith 500 ll of defined medium were treated with 5–40 nM LTD4for 24 or 48 h. MTT assay measured the ability of cells to metabo-lize MTT. At the end of the treatment period, 300 ll of culture med-ia were removed from each well and 20 lL of MTT solution (5 mg/ml) was added. After a 4 h incubation, 200 ll of solubilization solu-tion (10% sodium dodecyl sulfate (SDS) in HCl 0.01 M) was addedto the wells. After a further 24 h incubation at 37 �C, 100 ll of solu-tion were transferred to 96-well microtiter plates, and the absorp-tion value at 540 nm was measured in an automatic microtiterreader (Victor 1420, Perkin Elmer Life Science, Boston, MA, USA).

To supplement the MTT assay, we used a commercially-avail-able detection kit to measure lactate dehydrogenase (LDH) activityreleased from mouse primary cortical neurons. After cells had beenexposed to different concentrations of LTD4 for 24 or 48 h, totalculture medium was collected and the amount of LDH releasedby the cells was determined according to the manufacturer’s pro-tocol. The absorbance of samples was read at 440 nm. LDH leakagewas expressed as the percentage of control.

2.4. ELISA assay

Media from the mouse primary cortical neurons culture werecollected, and then briefly spun to remove cell debris. The amountof total Ab in conditioned medium was determined as described inthe manufacturer’s instructions of Ab assay kit. In short, 50 ll ofsample was added into the precoated plate and was incubatedfor 30 min at 37 �C in the dark. After washing each well of the pre-coated plate with washing buffer, 50 ll of labeled antibody solu-tion was added and the mixture was incubated for 30 min at37 �C in the dark. After washing, chromogen was added and themixture was incubated for 15 min at 37 �C in the dark. After theaddition of stop solution, the resulting color was recorded at450 nm using a microplate absorbance reader.

2.5. Western blotting analysis

Neurons were washed with PBS and homogenized in lysis buffer(10 mM Tris–HCl (pH 7.4), 150 mM NaCl, 1 mM EDTA, 1% Triton X-100) containing PMSF. The homogenates were rocked at 4 �C for30 min, centrifuged at 13,000g at 4 �C for 30 min to remove celldebris and collect the resulting supernatant. Equal amounts of pro-tein (30 lg) were subjected to 9% SDS polyacrylamide gel electro-phoresis, and separated products were transferred to PVDFmembranes. These membranes were then blocked with 5% skimmilk in 10 mM Tris–HCl (pH 7.5), 150 mM NaCl, and 0.1% Tween20 for 1 h at room temperature. These membranes were incubatedwith primary antibodies, namely, the anti-CysLT1 antibody (1:500),anti-Ab1–42 antibody (1:500), anti-BACE1 antibody (1:1000), oranti-actin antibody (1:500). The membranes were washed, andthen incubated with the appropriate secondary antibody conju-gated to horseradish peroxidase. Immunoreaction signals werevisualized with ECLTM or ECL plus TM Western blotting detectionreagent and exposed to the LAS-3000 Mini Bio-imaging AnalyzerSystem (Clubio, Gel Catcher2850). Signal intensity was determinedusing MultiGauge software (FUJIFILM).

For detection of nuclear NF-jB p65 and phospho- NF-jB p65,we also prepared cellular nuclear extracts using the nuclear extractkit (Byotime, Jiangsu, China). Briefly, the cells were washed withice cold PBS and incubated with reagent A for 15 min on ice. After10 ll reagent B was added, cell suspensions were vortexed for 5 s,kept on ice for 1 min, and vortexed for 5 s again. All subsequentcentrifugation were carried out at 4 �C. Following centrifugation

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at 15,000g for 5 min, the supernatant contained mostly cytoplas-mic constituents. The pellet was resuspended with nuclear proteinextract reagent and kept on ice for 30 min. Supernatants werecollected by centrifugation at 15,000g for 10 min, which is nuclearextracts. The protein concentration of the samples was determinedby the Bradford method, aliquots were added to SDS loading bufferand stored at �80 �C until used in 12% SDS–PAGE gel electrophore-sis. Anti-NF-jB p65 antibody (1:1000) or anti-NF-jB phosho-p65antibody (1:1000) was employed for assay.

2.6. RT-PCR

Total RNA was extracted from primary neuron. Trizol reagentsfollowing the procedures described in manufacturer’s instructions.For the cDNA synthesis, aliquots of total RNA (2 lg) were mixedwith 0.2 lg random hexamer primer, 20 U RNasin, 1 mM dNTP,and 200 UM-MuLV reverse transcriptase in 20 ll of the reversereaction buffer. The mixture was incubated at 25 �C for 10 min,42 �C for 60 min, and then at 72 �C for 10 min to inactivate the re-verse transcriptase. PCR was performed on an Eppendorf MasterCycler (Eppendorf, Hamburg, Germany). The mixture contained:1 ll RT-cDNA template dissolved in 20 ll reaction mixture con-taining 1� PCR buffer, 200 lmol/l dNTP, 1.5 mM MgCl2, 20 pM ofeach primer, and 0.5 U Taq DNA polymerase. Cycling parameterswere as below: 94 �C for 1 min, followed by 33 cycles of 94 �C for

A

B

Fig. 1. Dose–effect relationship of LTD4-stimulated Ab releases in mouse primarycultured cortical neurons (A). Neurons were treated with different concentrations ofLTD4 at 37 �C for 24 h and then ELISA assay was used to examine total Ab levels inthe culture medium. Time-effect relationship of LTD4-stimulated Ab releases inneurons (B). Neurons were treated with 20 nM LTD4 at 37 �C for different time andthen ELISA assay was used to examine total Ab levels in the culture medium. Dataare given as mean ± SEM (n = 4). ⁄P < 0.05, ⁄⁄P < 0.01 vs control.

30 s, 63 �C for 30 s, and 72 �C for 1 min, with a final extension stepof 72 �C for 7 min. The abundance of transcripts in cDNA sampleswas measured by RT-PCR with the following primers: mouseCysLT1R forward 50-ATTCCTGGAGAACATGAATGG-30 and reverse50-CATTGTTCTGCACTGTAGATGAG-30 (1062 bp, nucleotides 419–1480 in NM_021476.4, GeneBank), b-actin forward 50-TCTTGG-GTATGGAATCCTGTG-30 and reverse 50-ATCTCCTTCTGCATCCTGT-CA-30 (154 bp, nucleotides 876–1029 in NM_007393.3, GeneBank).The amplification products were separated by electrophoresis on a2% agarose gel containing ethidium bromide and photographed.The optical density of the bands was determined by an imageanalysis system (Bio-Rad, Richmond, CA, USA).

2.7. b- or c secretase activity assays

We performed assay of b- or c-secretasa activity using a com-mercially available kits, as reported previously (Lee et al., 2008;Wang et al., 2007). Briefly, after centrifugation of cell lysate at13,000g for 15 min, 50 ll of cell lysate was mixed with 50 ll ofreaction buffer. The mixture was incubated for 1 h in the dark at37 �C after 5 ll of substrate was added. Substrate conjugated tothe reporter molecules EDANS and DABCYL was cleaved by secre-tase and released a fluorescent signal. This fluorescence was mea-sured using a Fluostar galaxy fluorometer (excitation at 355 nmand emission at 510 nm) equipped with Felix software (BMG Lab-technologies, Offenburg, Germany). Results were expressed as ratio(in percentage) of control.

2.8. Statistical analysis

All values were displayed as means ± standard error (SEM). Forstatistical analysis, SPSS 14.0 (IBM) was used. Statistical signifi-cances of difference among groups were determined by one-wayanalysis of variance (ANOVA) and Fisher’s post hoc test for multiplecomparisons. P value < 0.05 was considered as statisticallysignificant.

3. Results

3.1. Effects of LTD4 on Ab release

Initially, we observed that effect of graded concentrations ofLTD4 on total Ab levels in culture medium in cultured neurons,using ELISA assay. The data showed that treatment of neurons withLTD4 (5.0–40.0 nM) for 24 h increased total Ab levels in culturemedium, with a maximum effects at 20 nM of LTD4 (Fig. 1A).Time-dependent study showed that LTD4 at 20 nM gradually in-creased release of Ab, an effect being statistically significant at24 h and 48 h, compared with PBS-treated neurons (as a vehiclecontrol) (Fig. 1B). Although some non-specific Ab release wasdetectable at 12–48 h, specific release of Ab caused by LTD4, asevaluated by subtracting the amount of Ab released in the vehi-cle-treated neurons from that in the LTD4-treated neurons,reached a plateau at 24 h and did not further increase. The signif-icant effect of 20 nM LTD4 was obtained at 24 h.

3.2. Viability of mouse primary neuron exposed to LTD4

Mitochondrial activity determined by the MTT assay was usedin this study as a measurement of cell viability. As shown inFig. 2A, there was a significant decrease in cell viability in the mon-olayers following treatment with 40 nM LTD4 for 24 h (19.2%reduction). More decreases were observed following 40 nM LTD4treatment for 48 h. To investigate LTD4 cytotoxicity further, theLDH assay, another indicator of cell toxicity, was performed. LDH

Fig. 2. Viability of mouse primary neuron exposed to LTD4. Cells grown toconfluency were maintained in the medium containing 1% HS and then treated with0, 5, 10, 20 or 40 nM LTD4 for 24 or 48 h. Cytotoxicity was evaluated by the MTT andLDH assays (A and B). Each experiment was repeated four times. Data aremeans ± SEM. ⁄P < 0.05 vs. control.

A

B

C

Fig. 3. Pranlukast blocked LTD4-induced Ab generation in neurons. Effects of LTD4(20 nM) alone or in combination with pranlukast (Pran, 0.6 lM) on the secreted Abwere measured by ELISA assays (A). The levels of Ab1–42 were detected by Westernblotting, and b-actin was used as a loading control (B). Quantification of Ab1–42

expressed as the ratio (in percentage) of Ab1–42/actin (C). Values shown areexpressed as means ± SEM; n = 3. ⁄P < 0.05, ⁄⁄P < 0.01 vs. PBS plus LTD4 group.

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is a stable cytoplasmic enzyme present in all cells, and it is rapidlyreleased into the cell culture supernatant fluid upon damage of theplasma membrane. An increase in the number of dead or plasmamembrane-damaged cells results in an increase in LDH activityin the culture supernatant fluid. As shown in Fig. 2B, LTD4-inducedcytotoxicity determined by the LDH assay was similar to thatdetermined by the MTT assay. All further investigations regardingthe effects of LTD4 on neurons were thus restricted to the use of20 nM LTD4, a concentration deemed to be non-toxic as there wereno signs of cell damage or loss of viability, and maximal effectswere apparent for 24 h.

3.3. CysLT1R antagonist inhibited LTD4-induced Ab production inprimary neurons

To determine whether CysLT1R mediates LTD4-induced Ab pro-duction, effects of pranlukast, a selective CysLT1R antagonist, onLTD4-induced-Ab production was investigated. We found that pra-nlukast significantly blocked LTD4-induced Ab releases in the cul-ture medium whereas pranlukast alone didn’t have effects(Fig. 3A). Western blotting showed that Ab1–42, a more neurotoxicAb species, markedly increased in the neuronal cells exposed toLTD4 for 24 h, and this effect was reversed by pranlukast (Fig. 3Band C). These results suggest that LTD4-induced Ab production isinvolved in cysLT1R in neuronal cells.

3.4. NF-jB inhibition blocked LTD4-induced Ab production in primaryneurons

Previous studies showed that CysLT1R-ligand interactions trig-gered NF-jB activation in human epithelial cells (Suzuki et al.,2008), and NF-jB activation was involved in Ab production (Ait-Ghezala et al., 2007). Thus, we observed effect of NF-jB signalingon LTD4-induced Ab production in primary neurons. The ELISA as-says showed that PDTC, a NF-jB inhibitor, significantly blockedLTD4-induced Ab releases in the culture medium whereas PDTCalone didn’t have significant effects (Fig. 4A). This effect is consis-tent with the Ab1–42 detected by Western blotting (Fig. 4B and C),suggesting that LTD4-induced Ab production is involved in NF-jBactivation in neurons.

3.5. CysLT1R antagonist blocked LTD4-induced CysLT1R expression inprimary neurons

To further determine role of CysLT1R in LTD4-induced Ab for-mation, we used Western blotting and RT-PCR to detect expressionof CysLT1R in the primary neurons. It was shown in Fig. 5 that thelevels of CysLT1R protein and mRNA were significantly increased at24 h after exposure to 20 nM LTD4. The effect was prevented byadding pranlucast to the medium. The basal level of CysLT1R pro-tein or mRNA was not altered significantly by pranlucast(Fig. 5A–C). In addition, we also detected CysLT2R expression using

B

A

C

Fig. 4. PDTC prevented LTD4-induced Ab generation in neurons. Effects of LTD4(20 nM) alone or in combination with PDTC (50 lM) on the secreted Ab weremeasured by ELISA assays (A). The levels of Ab1–42 were detected by Westernblotting. b-actin was used as a loading control (B). Quantification of Ab1–42

expressed as the ratio (in percentage) of Ab1–42/actin (C). Values shown areexpressed as means ± SEM; n = 3. ⁄P < 0.05, ⁄⁄P < 0.01 vs. PBS plus LTD4 group.

A

B

C

Fig. 5. Pranlukast blocked LTD4-induced increases in CysLT1R protein and mRNAlevels in primary neurons. (A) The protein levels of CysLT1R were detected by theWestern blotting. b-actin was used as a loading control. (B) Quantification ofCysLT1R expressed as the ratio (in percentage) of CysLT1R/actin. (C) The mRNAlevels of CysLT1R were measured by RT-PCR. Values shown are expressed asmeans ± SEM; n = 3. ⁄⁄P < 0.01 vs. PBS plus LTD4 group.

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Western blotting. No significant changes were observed in neuronstreated with LTD4 or pranlucast (data not shown).

3.6. CysLT1R antagonist inhibited LTD4-stimulated NF-jB activation inprimary neurons

To confirm that LTD4-induced CysLT1R activation results inNF-jB activation in primary neurons, effect of pranlukast onLTD4-induced NF-jB activation was investigated. NF-jB was acti-vated in our studies by assessing the movement of p65 from thecytosol to the nucleus. Expression of NF-jB p65 protein was clearlyincreased in nuclear extracts of the primary neurons at 1 h follow-ing LTD4 stimulation, and this effect was clearly suppressed bypranlukast (Fig. 6A and B). Also, obvious phosphorylation ofNF-jB p65 was observed since this is required for the transcrip-tional activation of NF-jB (Fig. 6A and C). In addition, PDTC, aNF-jB inhibitor, significantly inhibited NF-jB activation inducedby LTD4 (Fig. 6D–F). These data suggest that LTD4 triggers NF-jBactivation via CysLT1R in primary neurons.

3.7. LTD4 enhanced b- and c-secretase activity via CysLT1R-mediatedNF-jB pathway in primary neurons

It has been shown in recent study that NF-jB is a transcriptionfactor of regulating the expression of BACE1 that is a key rate-limiting enzyme that initiates Ab formation (Buggia-Prevot et al.,

2008). To clarify whether CysLT1R-mediated NF-jB activationupregulates the expression of BACE1, we examined effects of theselective CysLT1R antagonist or the NF-jB inhibitor on BACE1expression. As shown in Fig. 7, the expression of BACE1 weresignificantly increased after LTD4 stimulation for 24 h, eitherpranlukast or PDTC blocked LTD4-induced BACE1 expression(Fig. 7A–D). The data from b- secretase activity showed similarresults (Fig. 7E and F). In addition, we analyzed c-secretase activ-ity. The activity of c-secretase was also significantly increased inthe neurons treated with LTD4 alone and pranlukast or PDTC pro-hibited the increase of c-secretase activity induced by LTD4(Fig. 7E and F). These data indicated that LTD4 enhances eitherb- secretase or c-secretase via CysLT1R-mediated NF-jB pathwayin primary neurons.

4. Discussion

Epidemiological and genetic evidences have shown that aninflammatory process may contribute to AD pathology. However,the exact relationship and mechanisms are not clear. Our datademonstrated that LTD4, an inflammatory lipid mediator, inducedgenerations of Ab in primary cultured neurons, concomitant withCysLT1R upregulation, NF-jB activation, and b- or c-secretaseenhancement, and these effects were completely blocked byCysLT1R antagonist pranlukast. Moreover, a NF-jB signaling inhib-itor, PDTC, also inhibited LTD4-induced increases of Ab generationand b- or c-secretase. These results suggest that LTD4 causes Abproduction by enhancement of b- or c-secretase resulting fromactivation of CysLT1R-mediated NF-jB signaling pathway.

LTD4 belongs to the bioactive lipid group known as cysLTs andthe functional role of cysLTs in peripheral cells has already beencharacterized. Several studies demonstrated that cysLTs are signif-icant regulators of leukocyte trafficking, vascular permeability,smooth muscle tone and cell growth (Walch et al., 2000; Bisgaard,2001; Sala and Folco, 2001). In the CNS, it is known that thesesubstances are released as a consequence of brain damage. LTD4exerts its effects mainly through two different G protein-coupled

D

E

F

Fig. 6. (continued)

A

B

C

Fig. 6. Involvement of CysLT1R in activation of NF-jB pathway induced by LTD4.Neurons were stimulated with 20 nM of LTD4 for 1 h in the absence or presence ofthe CysLT1R antagonist pranlukast (0.6 lM) (A–C). Neurons were stimulated with20 nM of LTD4 for 1 h in the absence or presence of the NF-jB inhibitor PDTC(50 lM) (D–F). The protein levels of NF-jB p65 protein or phospho-p65 protein ofthe nuclear fraction were detected by Western blotting. Values shown areexpressed as means ± SEM; n = 3. ⁄P < 0.05, ⁄⁄P < 0.01 vs. PBS plus LTD4 group.

A

B

C

Fig. 7. LTD4 enhanced b- or c-secretase via CysLT1R-mediated NF-jB pathway.Neurons were pretreated with 0.6 lM of pranlukast or PDTC for 1 h, thenstimulated with 20 nM of LTD4 for 24 h, and the protein levels of BACE1 expressionwas detected by Western blotting (A–D). b- or c-secretase activity was assessed byusing commercially available assay kits (E and F). Values shown are expressed asmeans ± SEM, n = 3–5. ⁄P < 0.05, ⁄⁄P < 0.01 vs. PBS plus LTD4 group.

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receptors, CysLT1R and CysLT2R (Lynch et al., 1999; Heise et al.,2000). The human CysLT1R has been shown to be most highly ex-pressed in spleen, peripheral blood leukocytes and little expressionin brain (Bäck et al., 2011; Capra, 2004). But in the CNS, CysLT2RmRNA is highly expressed in several regions of the brain, such asthe hypothalamus, thalamus, putamen, pituitary and medulla(Hu et al., 2005). The difference of their expression in brain mightimply that CysLT1R has more significance of pathology than CysLT2R.It has been reported that CysLT1R mediates astrocyte proliferation(Ciccarelli et al., 2004; Huang et al., 2008), brain cryoinjury, braintumors and acute neuronal injury after focal cerebral ischemia inmice (Ding et al., 2007; Zhang et al., 2004; Zhang et al., 2006).

Neurons are believed to be the major source of Ab that has cau-sal role in AD (Palop and Mucke, 2010). We used exogenous LTD4to mimic LTD4 release of neurons and found a novel pathophysio-logical role of LTD4 and CysLT1R in vitro. The investigations in thisstudy were undertaken using a concentration of LTD4 that did notaffect cell viability. The cultured mouse neurons appeared to beable to withstand a concentration of LTD4 as high as 20 nM for24 h without any sign of damage, and a concentration of 20 nMLTD4 produced the maximal effects for total Ab release in culturemedium. Lowered viability was, however, seen at and above40 nM for 24 h or 48 h, and at 20 nM for 48 h. The levels of Ab1–

42, a more neurotoxic Ab species, also were increased after thechallenge of primary neurons with LTD4 at 20 nM for 24 h. Wetentatively observed effects of selective CysLT1R antagonist or

D

E

Fβ γ

β γ

Fig. 7. (continued)

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NF-jB signaling inhibitor on LTD4-induced Ab generation. The datashowed that both selective CysLT1R antagonist pranlukast andNF-jB signaling inhibitor PDTC significantly prevented Ab increaseinduced by LTD4 in neuronal cells. To further confirm role ofCysLT1R in LTD4-induced Ab generation, we detected CysLT1Rexpression by Western blot and RT-PCR. Interestingly, basalexpression of CysLT1R is pretty weak in neurons, and neurons trea-ted with LTD4 displayed significant increase in the protein ormRNA of CysLT1R. This effect was completely by pranlukast. Theremight be a feedback mechanism, in which CysLT1R activation withLTD4 can promote expression of CysLT1R gene like the receptor foradvanced glycation end products (RAGE) (Cho et al., 2009). It wasreported that CysLT1R-mediated signaling refers to NF-jB pathwayin some peripheral cells (Kawano et al., 2003; Thompson et al.,2006; Suzuki et al., 2008), and consequence of LTD4 stimulationwas the phosphorylation of IjBa followed by the proteolysis ofIjBa protein and simultaneous activation of the p65 NF-jB, asdemonstrated by phosphorylation on serine 536 (Thompsonet al., 2006; Kawano et al., 2003). As we expected, LTD4 treatmenttriggered NF-jB signaling characterized by increases in NF-jB p65and phosphorylated p65 in nucleus of primary neurons, and block-ade of CysLT1R or NF-jB signaling prevented the nuclear transloca-tion of p65 and the consequent phosphorylation that is requiredfor transcriptional activity of NF-jB, suggesting that LTD4 stimu-lates NF-jB activation through CysLT1R in neurons.

BACE1 is a key rate-limiting enzyme that initiates Ab formation,and without b-secretase, Ab synthesis is either abolished or consid-erably reduced (Walter et al., 2001; Cai et al., 2001). It is considereda prime target for lowering Ab levels in the treatment and/or pre-vention of AD (Vassar, 2001). It was demonstrated that BACE1 pro-moter contains NF-jB binding domain, and NF-jB signalingregulates BACE1 expression and Ab production (Buggia-Prevotet al., 2008; Bourne et al., 2007; Guglielmotto et al., 2010; Jianget al., 2012). As expected, our data showed that CysLT1R-mediatedNF-jB signaling promoted enhancement of expression and activityof BACE1 in neurons. There is relatively few data concerning theregulation of the c-secretase activity by NF-jB. However, the lateststudies showed NF-jB-dependent regulation of c-secretase, pro-moting Ab production (Choi et al., 2012; Chami et al., 2012). Inpresent study, we also found NF-jB-dependent enhancement ofc-secretase activity in the neurons treated with LTD4. This effectwas inhibited by CysLT1R antagonist pranlukast or NF-jB inhibitorPDTC, which suggests that CysLT1R-mediated activation of NF-jBis involved in enhancement of c-secretase activity in LTD4-inducedAb production.

Overall, our study demonstrates for the first time the contribu-tions of LTD4 to Ab production in vitro. This refers to upregulationof b- or c-secretase resulting from activation of CysLT1R-mediatedNF-jB signaling. Thus, CysLT1R could be a novel potential target fordeveloping new drugs in the treatment of AD, and CysLT1R antag-onists such as pranlukast, zafirlukast and montelukast clinicallyused for the treatment of asthma might be benefit for AD.

Conflict of interest

The Author(s) declare(s) that they have no conflicts of interestto disclose.

Acknowledgements

This work was supported by the college graduate research andinnovation projects funded by Jiangsu Province, China.

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