- Email: [email protected]
Monoacylglycerol lipase inhibitor protects primary cultured neurons against homocysteine-induced impairments in rat caudate nucleus through COX-2 signaling
LFS-14329; No of Pages 8 Life Sciences xxx (2015) xxx–xxx
Contents lists available at ScienceDirect
Life Sciences journal homepage: www.elsevier.com/locate/lifescie
Monoacylglycerol lipase inhibitor protects primary cultured neurons against homocysteine-induced impairments in rat caudate nucleus through COX-2 signaling Manman Dong, Yongli Lu, Ziliang Zou, Hongwei Yang ⁎ Department of Physiology and Pathophysiology, College of Medical Sciences, China Three Gorges University, 443002 Yichang, Hubei, PR China
a r t i c l e
i n f o
Article history: Received 15 December 2014 Received in revised form 1 March 2015 Accepted 11 March 2015 Available online xxxx Keywords: Endocannabinoid Monoacylglycerol lipase Alzheimer's disease Neuroinflammation Caudate nucleus Cyclooxygenase-2
a b s t r a c t Aims: URB602 is a selective inhibitor of monoacylglycerol lipase (MAGL), a serine hydrolase involved in the biological deactivation of the endocannabinoid 2-arachidonoyl glycerol (2-AG). It has been described that URB602 significantly enhances depolarization-induced increases in 2-AG. A high level of homocysteine (Hcy) is a modifiable risk factor for developing Alzheimer's disease (AD). The aim of this study was to investigate the protective effects of URB602 on Hcy-induced impairments underlying its cellular and molecular mechanism in primary cultured caudate nucleus (CN) neurons. Main methods: The expressions of cyclooxygenase-2 (COX-2), ERK1/2, NF-κB and IκB-α as well as cleaved caspase3 and p-Bcl-2 in Hcy-, URB602 or SR1 (a selective inhibitor of CB1 receptor)-treated primary cultured neurons in CN were measured by immunoblotting technique and neurotoxicity assays were performed by using Hoechst staining. Key findings: The MAGL inhibitor URB602 exerted a neuroprotective effect on Hcy-induced impairment through suppression of cyclooxygenase-2 (COX-2) elevation and ERK1/2 and NF-κB phosphorylation as well as suppressions of IκB-α degradation in a CB1 receptor-dependent way. Moreover, anti-neuronal impairments of URB602 were mediated by modulating down-regulation of cleaved caspase-3 expression and up-regulation of p-Bcl-2 expression in a CB1 receptor-dependent manner in primary cultured CN neurons. Significance: These data suggest that the MAGL inhibitor is a promising therapeutic target for some neurodegenerative disorders, such as AD, via the COX-2 signaling pathway. © 2015 Elsevier Inc. All rights reserved.
1. Introduction Epidemiological and clinical studies have revealed that elevated total homocysteine (Hcy) level is a modifiable risk factor for developing Alzheimer's disease (AD) [8,15,26,31,32,35]. Hcy, an amino acid, is produced in the methylation cycle of protein metabolism requiring folate, vitamins B12 and B6 as cofactors [8,27]. The major neuropathological features of AD are amyloid-β peptide aggregation and tau hyperphosphorylation in the hippocampus and cortex, along with brain atrophy and cognitive decline [17]. An elevated concentration of Hcy is associated with a serious decrease of volume in the hippocampus in a group of old outpatients [16]. Several potential mechanisms underlying the deleterious effect of Hcy in the brain have been proposed. These included the impact on cerebrovascular pathology [20], direct neurotoxic effects [24] or the malignant influence on amyloid-β peptide generation and tau hyperphosphorylation [23], but the cellular and molecular mechanisms for Hcy in AD are not fully understood. Moreover, ⁎ Corresponding author at: Department of Physiology and Pathophysiology, College of Medical Sciences, China Three Gorges University, 8 University Road, 443002 Yichang, Hubei, PR China. Tel.: +86 717 6397620; fax: +86 717 6397328. E-mail address: [email protected] (H. Yang).
there are currently no effective medications to lower the concentration of Hcy and alleviate Hcy-induced brain disorders. The caudate nucleus (CN), a cluster of nerve cells that is part of the striatum, has been deemed to have little relationship with AD. However, neuropathological studies suggested that atrophy of the CN plays a vital role in AD progression, because the cortico–striato–thalamic loops are involved in cognitive processes, particularly, in attention and executive function, as well as in movement disorders [7,13]. Moreover, a significant reduction in the caudate volume of AD patients was compared to the normal volunteers at a presymptomatic stage, while no significant hippocampal volume loss was evident presymptomatically [19,30]. Therefore the change in the CN may be an important factor in the search for AD biomarkers in the brain. In the last decade, the endocannabinoid system (eCBs), comprised of cannabinoid receptors (CB1 and CB2), endocannabinoids and their interrelated biosynthesis or degradation enzymes, has been recognized to be important in controlling many physiological and pathological processes and has become an area of interest for novel drug development [11,12,14,25]. 2-Arachidonoyl glycerol (2-AG), the main endocannabinoid, is synthesized on demand and rapidly inactivated by enzymatic hydrolysis, therefore limiting their potential protective effects [5]. A comprehensive profile of brain serine hydrolases revealed
http://dx.doi.org/10.1016/j.lfs.2015.03.006 0024-3205/© 2015 Elsevier Inc. All rights reserved.
Please cite this article as: M. Dong, et al., Monoacylglycerol lipase inhibitor protects primary cultured neurons against homocysteine-induced impairments in rat caudate nucleus through COX-2 signaling, Life Sci (2015), http://dx.doi.org/10.1016/j.lfs.2015.03.006
2
M. Dong et al. / Life Sciences xxx (2015) xxx–xxx
that about 85% of total 2-AG in mouse brain is hydrolyzed by monoacylglycerol lipase (MAGL) [6]. Recently, Chen et al. reported that the application of the MAGL inhibitor or deletion gene of MAGL suppresses neuroinflammation and prevents neurodegeneration, which is beneficial for decelerating AD progress, on account of the marked and sustained elevations in 2-AG levels [11]. URB602, a selective inhibitor of MAGL's activity, known to decrease 2-AG's metabolism and enhance 2-AG-mediated signaling in neurons, exerts neuroprotective effects, such as inhibition of neurotrophic cytokine cyclooxygenase-2 (COX-2), nuclear factor-κB (NF-κB) and mitogen-activated protein kinase (MAPK) family [37], but little is known about whether suppression of MAGL has the ability to protect neurons against Hcy harmful insults in the CN neuron. In the present study, we found that the MAGL inhibitor could protect primary cultured neurons against Hcy-induced impairments in rat CN through COX-2 signaling. 2. Materials and methods 2.1. Materials Neurobasal-A, B27, 0.25% trypsin–EDTA solution, fetal bovine serum, and Dulbecco's Modified Eagle's Medium (DMEM) dry powder were obtained from Gibco BRL (Grand Island, NY, USA). Homocysteine, AM630, poly-D-lysine, L-glutamine, L-glutamate, and cytosine arabinoside were purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). Antibodies for detecting nuclear factor-kappaB (NF-κB), phospho-p38 mitogen activated protein kinase (p-p38MAPK), extracellular signal-regulated kinase (ERK1/2), and cleaved caspase-3 were purchased from Beyotime Institute of Biotechnology (Haimen, China). Cyclooxygenase-2 (COX-2) polyclonal antibody, URB602, and Rimonabant (SR141716, SR1) were purchased from Cayman Chemical (Ann Arbor, MI, USA). 2.2. Cell culture In this study, subject care complied with the China Three Gorges University Guide for the Care and Use of Laboratory Animals, and all protocols were approved by the Institutional Animal Care and Use Committee of the China Three Gorges University. The methods for obtaining primary cultured CN neurons have been described in detail elsewhere [14,25]. Briefly, primary CN cultures were obtained from neonatal Sprague–Dawley rats (within 24 h) which were provided by the Experimental Animal Research Center of Hubei Province. The caudate nuclei were taken out and washed several times in a cold (4 °C) DMEM solution. After the caudate nuclei were cut to small pieces (1 mm × 1 mm × 1 mm) with spring scissors, they were incubated into 5 mL 0.25% trypsin–EDTA sterile solution and incubated at 37 °C for 10–15 min with 5% CO 2. Then, they were triturated with a flamed and siliconized Pasteur pipette to separate cells gently. Subsequently, they were placed in a centrifuge tube with 5–10 mL DMEM solution containing 10% fetal bovine serum and centrifuged one time for 8 min at 800 rpm/min. Cells were spun down and resuspended in neurobasalA/B27 medium (Gibco) supplemented with 1 mM L-glutamine, penicillin/streptomycin, and 25 μM glutamate. Cells (0.5 × 106–1 × 106/mL) were loaded into poly-D-lysine-coated 35-mm culture dishes for Western blot analysis. After 5 days in culture, the cells were fed with the medium supplemented with 2.5 μg/mL cytosine arabinoside to proliferation of non-neuronal cells. The culture medium was changed every 3 days with the same medium without glutamate until use. The cells were used in experiments between 7 and 12 days in culture. 2.3. Western blot analysis The CN neurons were harvested after 7 days of Hcy or URB602 or vehicle treatment. The cells were washed with ice-cold PBS and lysed with the solubilization buffer containing a Radio Immunoprecipitation Assay (RIPA) lysis buffer and phenylmethanesulfonyl fluoride (PMSF)
(100:1) (Beyotime, Haimen, China). After sonication, the samples were centrifuged for 10 min at 12000 g. The protein concentration of the supernatant was determined by BCA (bicinchoninic acid) assay, and the loading quantity of the protein sample was chosen to be 50 μg. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis was done by loading equal amounts of proteins per lane. Proteins were separated on SDS-12% polyacrylamide gel and transferred to PVDF membranes by wet transfer. The membranes were blocked with 5% non-fat milk and incubated with primary antibodies at 4 °C overnight, and the primary antibodies (all the primary antibodies diluted 1:1,000) including Rabbit anti-COX-2 polyclonal antibodies (Cayman, Ann Arbor, MI, USA), Rabbit anti-cleaved caspase-3, Rabbit anti-NFκB-65 or anti-p-NF-κB65 antibodies, Rabbit anti-IκB-α, Rabbit antiBcl-2 or anti-p-Bcl-2, Mouse anti-ERK1/2 or anti-p-ERK1/2, Mouse anti-p38 or anti-p-38MAPK (Beyotime, Haimen, China). The blot was washed for four times with TBST (20 mM Tris–HCl, 150 mM NaCl, 0.05% Tween 20, pH 7.4) for a total of 30 min, and incubated with the corresponding secondary antibody (goat anti-rabbit/mouse 1:2000, Beyotime, Haimen, China), at room temperature for 2 h and washed for four times with TBST. Proteins were detected by ECL Plus (Beyotime, Haimen, China) with films according to the manufacturer's instructions. Quantitation of the proteins was done by densitometric analysis using ImageJ 1.46i software. Band densities were normalized to the total amount of protein loaded in each well as determined by mouse anti-βactin (1:4000, Beyotime, Haimen, China). 2.4. Hoechst 33258 staining analysis The primary CN neurons were seeded in 35 mm plates as described in the Cell culture section. On the 7th day the CN neurons were treated with Hcy or URB602 or vehicle treatment by 12 h. Then the neurons were fixed with 4% paraformaldehyde (Beyotime, Haimen, China) for 10 min, and washed with ice-cold PBS twice. Then the cells were incubated in a Hoechst 33258 (Beyotime, Haimen, China) staining solution (final concentration: 10 μg/mL) for 5 min at 37 °C. The cells were analyzed with a fluorescence microscopic system (100× magnification, excitation wavelength 350 nm, emission wavelength 460 nm). Stained condensed chromatin appeared small and bright blue, whereas stained normal nuclei were large and light blue. The proportion of nuclei with condensed chromatin was calculated to reflect the level of injury. Five randomly chosen fields were analyzed for each group. The rate of apoptotic cells was equal to the ratio of apoptotic cells to the number of total cells. 2.5. Data analysis All values are presented as mean ± SEM unless stated otherwise. The significance of inter-group differences was evaluated by one-way analyses of variance (ANOVA: least-significant difference test for post-hoc comparisons). Differences were considered significant when P b 0.05. 3. Results 3.1. URB602 suppresses excessive COX-2 expression in response to Hcy in the cultured CN neurons through the CB1 receptor, not the CB2 receptor URB602, a selective inhibitor of MAGL activity, is known to elevate the levels of endogenous 2-AG and enhance 2-AG-mediated signaling in neurons, without affecting the level of arachidonoyl ethanolamide (anandamide, AEA), another endocannabinoid ligand [37]. As a key player in inflammation, COX-2 is markedly elevated by a variety of stimulation of inflammation [12,25,41]. Hcy serves as an inducer of proinflammatory response to induce neural injury. To determine whether URB602 is capable of inhibiting elevation of Hcy-induced COX-2 expression, the CN neurons were cultured with Hcy (100 μM) and URB602
Please cite this article as: M. Dong, et al., Monoacylglycerol lipase inhibitor protects primary cultured neurons against homocysteine-induced impairments in rat caudate nucleus through COX-2 signaling, Life Sci (2015), http://dx.doi.org/10.1016/j.lfs.2015.03.006
M. Dong et al. / Life Sciences xxx (2015) xxx–xxx
(10 μM) for 12 h and COX-2 protein expression was detected by Western blot. As shown in Fig. 1A & B, under the induction of Hcy, the level of COX-2 proteins increased significantly compared with the vehicle control (n = 3, P b 0.01, versus vehicle control). Meanwhile, this elevation was suppressed in the presence of URB602 (n = 3, P b 0.01, versus Hcy), suggesting that URB602 is able to suppress Hcy-induced COX-2 elevation in the CN neurons. Elevated 2-AG by URB602 is an endogenous agonist for the CB1 receptor and the CB1 receptor is expressed mainly in the nervous system [34]. To ascertain whether the URB602-induced inhibition of COX-2 is mediated through the CB1 receptor in the CN neurons, we used SR141716 (SR1, a selective CB1 antagonist) and AM630 (a selective CB2 antagonist) to detect it. As seen in Fig. 1A & B, SR1 inhibits the URB602-produced suppression of COX-2 (n = 3, P b 0.01, versus URB602 and Hcy), while AM630 has no effect on URB602-produced suppression of COX-2 expression elevation (n = 3, P b 0.01, versus URB602 and Hcy). And URB602-produced suppression of COX-2 is not completely inhibited by SR1 (n = 3, P b 0.01, versus Hcy). These findings indicate that CB1R-, but not CB2R-dependent suppressions of COX-2 expression may partially be involved in URB602-induced neuroprotection against Hcy. To exclude the non-specific effect of drugs on COX-2 expression, we employed URB602, SR1 and AM630 to examine it. As shown in Fig. 1C & D, URB602, SR1 and AM630 alone did not affect the COX-2 expression.
3
3.2. URB602 inhibits the phosphorylation of NF-κB and degradation of IκB-α in response to Hcy in the primary CN neurons via the CB1 receptor A preceding study showed that the NF-κB signaling pathway was involved in the lipopolysaccharide (LPS)-induced or Hcy-induced neuroinflammation in the hippocampus and CN [14,25,40,41]. To determine whether this signaling pathway is involved in the URB602 suppression of COX-2 induced by Hcy in the CN neurons, we detected the phosphorylation level of NF-κB in the CN neurons. As shown in Fig. 2A & B, Western blot showed that the expression of pho-NF-κB (p-NF-κB) was significantly enhanced after the CN neurons were cultured with Hcy (100 μM, 12 h) (n = 3, P b 0.01, versus vehicle control). However, the Hcy-induced enhancement of p-NF-κB expression was significantly suppressed when co-incubation with URB602 (10 μM, 12 h) (n = 3, P b 0.01, versus Hcy). Furthermore, when the CN neurons were co-treated with Hcy, URB602 and SR1 (10 μM, 12 h), this action of URB602 was significantly alleviated (n = 3, P b 0.01, versus URB602 and Hcy), not completely alleviated (n = 3, P b 0.05, versus Hcy). The data clearly demonstrates that CB1-dependent suppression of NF-κB phosphorylation may be partially involved in URB602-induced neuroprotection against Hcy in the primary CN neurons. NF-κB dimer is a kind of protein and is sequestered in the cytoplasm by IκB-α. Upon stimulation IκB-α is rapidly degraded [38]. Meanwhile,
Fig. 1. URB602 inhibits the elevation of Hcy-induced COX-2 expression via CB1R-, but not CB2R-dependent way in the primary CN neurons. (A) A representative autoradiograph of COX-2 expression in the CN neurons in vehicle control, Hcy (100 μM), URB (10 μM) + Hcy, SR1 (10 μM) + URB + Hcy and AM630 (5 μM) + URB + Hcy. The CN neurons in culture were treated with Hcy in the absence and presence of URB or SR1 or AM630 for 12 h. (B) Quantitative values of protein expressions under different treatments. (C) A representative autoradiograph of COX-2 expression in the CN neurons in vehicle control, URB (10 μM), SR1 (10 μM) and AM630 (5 μM) in culture for 12 h. (D) Quantitative values of COX-2 expressions under different treatments. Values are expressed as mean ± SEM of three independent experiments. In all blots, staining for β-actin was used as a loading control. The level of COX-2 expression obtained in each experimental condition was calculated as a fold of the control. **P b 0.01, compared with the vehicle control; ##P b 0.01, compared with Hcy; ++ P b 0.01, compared with URB + Hcy.
Please cite this article as: M. Dong, et al., Monoacylglycerol lipase inhibitor protects primary cultured neurons against homocysteine-induced impairments in rat caudate nucleus through COX-2 signaling, Life Sci (2015), http://dx.doi.org/10.1016/j.lfs.2015.03.006
4
M. Dong et al. / Life Sciences xxx (2015) xxx–xxx
Fig. 2. URB602 reduces Hcy-induced NF-κB phosphorylation and degradation of IκB-α through a CB1R-dependent manner in the primary CN neurons. (A) A representative autoradiograph of NF-κB phosphorylation in the CN neurons in vehicle control, Hcy (100 μM), URB (10 μM) + Hcy, and SR1 (10 μM) + URB + Hcy. The CN neurons in culture were treated with Hcy in the absence and presence of URB or SR1 for 12 h. (B) Quantitative values of protein expressions under different treatments. Values are expressed as mean ± SEM of three independent experiments. (C) A representative autoradiograph of degradation of IκB-α in the CN neurons in vehicle control, Hcy (100 μM), URB (10 μM) + Hcy, and SR1 (10 μM) + URB + Hcy. The CN neurons in culture were treated with Hcy in the absence and presence of URB or SR1 for 12 h. (D) Quantitative values of protein expressions under different treatments. Values are expressed as mean ± SEM of three independent experiments. **P b 0.01, compared with the vehicle control; ##P b 0.01, compared with Hcy; ++ P b 0.01, compared with URB + Hcy.
as shown in Fig. 2C & D, Western blot showed that Hcy induced the degradation of IκB-α (n = 3, P b 0.01, versus vehicle control), and this degradation was significantly suppressed by co-incubation with URB602 (10 μM) (Fig. 2C & D, n = 3, P b 0.01, versus Hcy). Furthermore, when the CN neurons were co-treated with Hcy, URB602 and SR1 (10 μM, 12 h), this action of URB602 was significantly alleviated (n = 3, P b 0.01, versus URB602 and Hcy), not completely alleviated (n = 3, P b 0.05, versus Hcy). These findings suggest that CB1-dependent suppression of IκB-α degradation may be partially involved in URB602-induced neuroprotection against Hcy in the primary CN neurons.
3.3. URB602 prevents the phosphorylation of ERK1/2 in response to Hcy in the cultured CN neurons through the CB1 receptor In the hippocampus and CN, mitogen-activated protein kinase (MAPK) ERK1/2 is also known to be involved in the antiinflammatory properties of eCBs [12,14,25,40,41]. Therefore, we tested the level of phospho-ERK1/2 (p-ERK1/2) by Western blot. As shown in Fig. 3, treatment with Hcy (100 μM, 12 h) induced an increase in pERK1/2 expression in the CN neurons (n = 3, P b 0.01, versus vehicle control). The Hcy-induced elevation of p-ERK1/2 expression was significantly reduced in the presence of URB602 (10 μM, 12 h) (n = 3, P b 0.01, versus Hcy). Furthermore, this reduction was reversed by SR1 (10 μM, 12 h) (n = 3, P b 0.01, versus URB602 and Hcy), but not
completely reversed (n = 3, P b 0.01, versus Hcy). These results suggest that CB1-dependent suppression of ERK1/2 phosphorylation may partially be implicated in URB602-induced neuroprotection against Hcy in the CN neurons.
3.4. URB602 reduces down-regulated phosphorylation of p38MAPK in response to Hcy in the cultured CN neurons through the CB1 receptor The p38MAPK signal pathway is one of the important branches of the MAPK pathway [12,14,25,40,41]. To explore whether the p38MAPK signal pathway is involved in the effects of URB602 in the CN neurons, we treated the CN neurons with Hcy in the absence and presence of URB602 or SR1. The level of phospho-p38MAPK (p-p38MAPK) was measured by Western blot analysis. As seen in Fig. 4, treatment with Hcy (100 μM, 12 h) significantly decreased the expression of p-p38MAPK proteins in the CN neurons (n = 3, P b 0.01, versus vehicle control). This reduction was significantly enhanced in the presence of URB602 (10 μM, 12 h) (n = 3, P b 0.01, versus Hcy). Furthermore, the URB602 inducedreduction of p-p38MAPK expression was inhibited by SR1 (10 μM, 12 h) (n = 3, P b 0.01, versus URB602 and Hcy), but not completely inhibited (n = 3, P b 0.01, versus Hcy). These findings indicated that the URB602 modulating down-regulation of p-p38MAPK expression in response to Hcy was partially mediated through CB1 receptor in the cultured CN neurons.
Please cite this article as: M. Dong, et al., Monoacylglycerol lipase inhibitor protects primary cultured neurons against homocysteine-induced impairments in rat caudate nucleus through COX-2 signaling, Life Sci (2015), http://dx.doi.org/10.1016/j.lfs.2015.03.006
M. Dong et al. / Life Sciences xxx (2015) xxx–xxx
Fig. 3. URB602 suppresses Hcy-induced ERK1/2 phosphorylation in a CB1R-dependent way in the primary CN neurons. (A) A representative autoradiograph of ERK1/2 phosphorylation in the CN neurons in vehicle control, Hcy (100 μM), URB (10 μM) + Hcy, and SR1 (10 μM) + URB + Hcy. The CN neurons in culture were treated with Hcy in the absence and presence of URB or SR1 for 12 h. (B) Quantitative values of protein expressions under different treatments. Values are expressed as mean ± SEM of three independent experiments. **P b 0.01, compared with the vehicle control; ##P b 0.01, compared with Hcy; ++ P b 0.01, compared with URB + Hcy.
3.5. URB602 inhibits cleaved caspase-3 activation and increases p-Bcl-2 expression induced by Hcy in the cultured CN neurons via the CB1 receptor Caspase-3 is involved in the regulation of cell apoptosis [14,25]. To examine whether suppression of MAGL could prevent the CN neurons from apoptosis induced by Hcy, we directly add URB602 to the CN neurons. The exposure of Hcy (100 μM, 12 h) to the CN neurons showed a significant up-regulation of cleaved caspase-3 expression (Fig. 5A & B, n = 3, P b 0.01, versus vehicle control). The Hcy-induced elevation of cleaved caspase-3 expression was significantly reduced in the presence of URB602 (10 μM, 12 h) (n = 3, P b 0.01, versus Hcy). Furthermore, this reduction was reversed by SR1 (10 μM, 12 h) (n = 3, P b 0.01, versus URB602 and Hcy), but not completely inhibited (n = 3, P b 0.01, versus Hcy). These results suggested that the URB602 modulating down-regulation of cleaved caspase-3 expression in response to Hcy was partially mediated through the CB1 receptor in the cultured CN neurons. The Bcl-2 protein family is endowed with an anti-apoptotic effect, which is involved in the progression of AD [1]. To verify whether inhibition of MAGL activity could prevent the CN neurons by means of elevating the level of the anti-apoptotic protein p-Bcl-2 reduced by Hcy, we directly add URB602 to the CN neurons. The exposure of Hcy (100 μM, 12 h) to the CN neurons showed a significant down-regulation of p-Bcl-2 expression (Fig. 5C & D, n = 3, P b 0.01, versus vehicle control). The Hcy-induced diminution of p-Bcl-2 expression was significantly
5
Fig. 4. URB602 attenuates Hcy-induced p38MAPK expression through a CB1R-dependent way in the primary CN neurons. (A) A representative autoradiograph of p38MAPK phosphorylation in the CN neurons in vehicle control, Hcy (100 μM), URB (10 μM) + Hcy, and SR1 (10 μM) + URB + Hcy. The CN neurons in culture were treated with Hcy in the absence and presence of URB or SR1 for 12 h. (B) Quantitative values of protein expressions under different treatments. Values are expressed as mean ± SEM of three independent experiments. **P b 0.01, compared with the vehicle control; ##P b 0.01, compared with Hcy; ++ P b 0.01, compared with URB + Hcy.
elevated in the presence of URB602 (10 μM, 12 h) (n = 3, P b 0.01, versus Hcy). Furthermore, this elevation was reversed by SR1 (10 μM, 12 h) (n = 3, P b 0.01, versus URB602 and Hcy), but not completely inhibited (n = 3, P b 0.01, versus Hcy). These results suggested that URB602 modulated the up-regulation of p-Bcl-2 expression in response to Hcy partially via a CB1 receptor-dependent manner in the cultured CN neurons.
3.6. URB602 inhibits the cellular apoptosis induced by Hcy using Hoechst staining in the cultured CN neurons through the CB1 receptor To determine whether suppression of MAGL could prevent the CN neurons from apoptosis induced by Hcy using Hoechst staining, we directly add URB602 to the CN neurons. As shown in Fig. 6A & B, the exposure of Hcy (100 μM, 12 h) to the CN neurons showed a significant elevation of apoptotic neurons (n = 3, P b 0.01, versus vehicle control). The Hcy-induced elevation of apoptotic neurons was significantly reduced in the presence of URB602 (10 μM, 12 h) (n = 3, P b 0.01, versus Hcy). Furthermore, this reduction was inhibited by SR1 (10 μM, 12 h) (n = 3, P b 0.01, versus URB602 and Hcy), but not completely inhibited (n = 3, P b 0.01, versus Hcy). These findings suggested that URB602 inhibition of cellular apoptosis induced by Hcy using Hoechst staining was partially mediated through the CB1 receptor in the cultured CN neurons.
Please cite this article as: M. Dong, et al., Monoacylglycerol lipase inhibitor protects primary cultured neurons against homocysteine-induced impairments in rat caudate nucleus through COX-2 signaling, Life Sci (2015), http://dx.doi.org/10.1016/j.lfs.2015.03.006
6
M. Dong et al. / Life Sciences xxx (2015) xxx–xxx
Fig. 5. URB602 suppresses the elevation of cleaved caspase-3 expression and increases p-Bcl-2 expression induced by Hcy via CB1R-dependent way in the primary CN neurons. (A) A representative autoradiograph of cleaved caspase-3 in the CN neurons in vehicle control, Hcy (100 μM), URB (10 μM) + Hcy, and SR1 (10 μM) + URB + Hcy. The CN neurons in culture were treated with Hcy in the absence and presence of URB or SR1 for 12 h. (B) Quantitative values of cleaved caspase-3 expressions under different treatments. Values are expressed as mean ± SEM of three independent experiments. (C) A representative autoradiograph of p-Bcl-2 in the CN neurons in vehicle control, Hcy (100 μM), URB (10 μM) + Hcy, and SR1 (10 μM) + URB + Hcy. The CN neurons in culture were treated with Hcy in the absence and presence of URB or SR1 for 12 h. (D) Quantitative values of p-Bcl-2 expressions under different treatments. Values are expressed as mean ± SEM of three independent experiments. **P b 0.01, compared with the vehicle control; **P b 0.01, compared with the vehicle control, ##P b 0.01, compared with Hcy; ++ P b 0.01, compared with URB + Hcy.
4. Discussion In this study, we found that pretreatment with the MAGL inhibitor URB602 has protective effects against Hcy-induced neurotoxicity to the CN neurons, through suppression of COX-2 elevation and ERK1/2 and NF-κB phosphorylation in a CB1 receptor-dependent way. Moreover, URB602-mediated down-regulation of cleaved caspase-3 expression and up-regulation level of p-Bcl-2 participated in defending from Hcy-induced CN neuron apoptosis. Various lines of research have implicated neuroinflammation as an important participant in AD pathophysiology [42]. Most recent studies show that MAGL inactivation suppresses the expression of neuroinflammation factors COX-2, NF-κB and PGE-2, through CB1 receptor-mediated mechanisms, suggesting that MAGL is a promising therapeutic target for preventing and treating neurodegeneration diseases such as AD [9,11]. Our experiment found that the MAGL inhibitor URB602 exerts a protective response to Hcy harmfulness via the CB1 receptors and not the CB2 receptors in the CN neurons since the protective effects of URB602 were blocked or attenuated by SR141716, a selective CB1 receptor antagonist, but not by AM630, a selective CB2 receptor antagonist. It's consistent with our recent studies that directly adding 2AG protected the CN neurons from lipopolysaccharide (LPS) and Hcy toxicity, which binds to CB1 receptors [14,25]. The reason may be that the CB1 receptor is largely expressed in the brain, particularly in the striatum, the hippocampus and the amygdaloid nucleus [34]. However,
the extent of CB2 receptor expression and the precise distribution of CB2 receptors in the nervous system are still debated [3]. COX-2 is a medium of inflammation, through prostaglandin E2 glycerol (PGE2-G) ester inducing NF-κB, caspase-3, ERK1/2, p38MAPK and other signaling activation, which has been implicated in the pathogenesis of neurodegenerative diseases like AD [40]. Hcy induced COX-2 expression in macrophages, hepatic cell, as well as in the primary cultured CN neurons, which was hazardous to the function of cells [14,22, 38]. Previous findings showed that the direct application of 2-AG or elevation of endogenous 2-AG by URB602 protects the hippocampal and CN neurons through suppression of cyclooxygenase-2 (COX-2) expression, which is provoked by pro-inflammatory IL-1β and LPS, excitotoxic glutamate and kainic acid and β-amyloid, via a CB1 receptor-dependent NFκB signaling pathway [12,25,41]. In our study, indirectly increasing the level of 2-AG by URB602 and binding to CB1 receptors exert an inhibitory action on COX-2 elevation induced by Hcy insults in the CN neurons. The result is consistent with our previous finding that adding pure 2-AG solution could protect the CN neurons from Hcy assault by decreasing the expression of COX-2 [14]. In our study, we observed that the CN neurons in culture treated with Hcy significantly elevated phosphorylation of NF-κB and degradation of IκB-α. The transcription factor NF-κB plays a critical role in the regulating the induction and resolution of inflammation and is involved in the brain proinflammatory process of AD [10,33]. Once IκB-α degrades, the released NF-κB translocates to the nucleus, which is the onset
Please cite this article as: M. Dong, et al., Monoacylglycerol lipase inhibitor protects primary cultured neurons against homocysteine-induced impairments in rat caudate nucleus through COX-2 signaling, Life Sci (2015), http://dx.doi.org/10.1016/j.lfs.2015.03.006
M. Dong et al. / Life Sciences xxx (2015) xxx–xxx
Fig. 6. URB602 protects the CN neurons from Hcy insult using Hoechst staining. (A) Representative Hoechst staining images of the CN neurons in vehicle control, Hcy (100 μM), URB (10 μM) + Hcy, and SR1 (10 μM) + URB + Hcy. The CN neurons in culture were treated with Hcy in the absence and presence of URB or SR1 for 12 h. (B) Percentages of apoptotic neurons under different treatments. Values are expressed as mean ± SEM of three independent experiments. **P b 0.01, compared with the vehicle control; ##P b 0.01, compared with Hcy; ++ P b 0.01, compared with URB + Hcy.
of inflammatory responses by regulating the expression of proinflammatory genes such as iNOS, COX-2, and TNF-α [21]. Suppression of the NF-κB signaling pathway can ameliorate neurotoxicity [18]. In our finding, the elevation of NF-κB was inhibited or eliminated by URB602, suggesting that 2-AG-produced neuroprotective effects are mediated via a CB1R-dependent suppression of NF-κB phosphorylation. This is consistent with previous observations where we demonstrated that 2-AG protected the CN neurons from LPS and Hcy through the CB1R-dependent suppression of NF-κB phosphorylation [14,25]. Mitogen-activated protein kinase (MAPK) signaling pathways, which regulate cellular activities such as differentiation, neuronal apoptosis and survival, have been implicated in the development of many neurodegenerative diseases such as AD [36]. The two main MAPK signaling pathways, the extracellular signal-regulated kinase (ERK) and p38MAPK, are involved in Hcy-induced primary cultured cortical neuronal death through excessive activation of the N-methyl-D-aspartic acid receptor [28]. Hyperphosphorylation of ERK1/2 and p38MAPK molecules ultimately activates the transcription factor NF-κB and production
7
of COX-2 [4]. Our findings demonstrated that Hcy down-regulated, but not up-regulated, the phosphorylation of p38MAPK, and URB602 enhanced this down-regulation effect. This change of p38MAPK signaling was identical with our recent study in which Hcy or LPS downregulated the p38MAPK pathways and 2-AG enhanced this effect further [14,25]. This suggests that the p38MAPK signaling pathway is not involved in Hcy or LPS-induced COX-2 and NF-κB expression in the CN neurons. Nonetheless, in the hippocampus LPS induced the phosphorylation of p38MAPK. The discrepancy in the phosphorylation of p38MAPK may be ascribed to the regional specificity of the CN and hippocampus [41]. In any case, in our study the enhancement of URB602 on Hcy-induced p38MAPK down-regulation demonstrated that URB602 can inhibit phosphorylation of p38MAPK in the CN neurons. However, the difference is that Hcy induced ERK1/2 phosphorylation, which is suppressed by URB602 through a CB1 receptor dependent way in this study. In the cultured primary cortical neurons, activation of the ERK1/ 2 pathway plays a critical role in mediating Hcy dependent neuronal cell death [29]. These results were different from Yan et al.'s [39] study, they found that Hcy decreased ERK1/2 protein phosphorylation and neural stem cell proliferation, but it did not induce cell death or apoptosis. The cause of the diversity may be concerned with the fact that the primary CN and cortical neuron do not posess the ability to proliferation while neural stem cell does. Our recent study also revealed that 2-AG binding to CB1 receptor inhibited LPS and Hcy-induced ERK1/2 phosphorylation [14,25]. In the present study, our findings also showed that the expression of apoptosis regulatory proteins, cleaved caspase-3 and p-Bcl-2 was altered by Hcy, while URB602 counteracted this alteration in a CB1receptor dependent way. A previous finding implicated that Hcy induces neuronal apoptosis by other mechanisms such as excitoxicity, oxidative and metabolic insults in the hippocampal neurons [2]. In our paper the expression of pro-apoptotic caspase-3 activation and antiapoptosis p-Bcl-2 inhibition participated in the apoptosis of neurons by Hcy. Previous studies reported that Hcy did not induce apoptosis in neural progenitor cells and neuronal stem cells [39]. The reason about the diversity may be that we use the primary CN neurons while they chose neural stem cells, while in cultured primary cortical neurons, activation of the ERK1/2 pathway plays a critical role in mediating Hcydependent neuronal cell death [29]. Interestingly, the initial activation of p38MAPK is downstream of, and dependent on activation of ERK1/2 in primary cortical neuronal cultures [28]. Our discovery revealed that the exposure of URB602 alleviated Hcy-induced CN neuronal death, which was achieved by indirectly activating CB1R through increasing 2-AG levels. 5. Conclusion Our results demonstrate that URB602 exerted potent antiinflammatory effects on Hcy-stimulated CN neurons. The COX-2, NF-κB and ERK1/2 MAPK signaling pathways were largely involved in URB602-mediated modulation of inflammatory mediators via a CB1-receptor-dependent way. Due to the promising potential of MAGL to regulate Hcy mediated neuroinflammatory events, URB602 could be further explored for its beneficial role in suppressing neuroinflammation. Hence, our findings may provide new insight for the development of therapeutics from suppression of MAGL activity to action against inflammation in neuroinflammatory diseases, such as AD. Conflict of interest statement The authors declare no conflict of interest.
Acknowledgments This work was supported by the National Natural Science Foundation of China (No. 30970930).
Please cite this article as: M. Dong, et al., Monoacylglycerol lipase inhibitor protects primary cultured neurons against homocysteine-induced impairments in rat caudate nucleus through COX-2 signaling, Life Sci (2015), http://dx.doi.org/10.1016/j.lfs.2015.03.006
8
M. Dong et al. / Life Sciences xxx (2015) xxx–xxx
References [1] J.A. Araya, A.E. Ramírez, D. Figueroa-Aroca, G.J. Sotes, C. Pérez, J. Becerra, et al., Modulation of neuronal nicotinic receptor by quinolizidine alkaloids causes neuroprotection on a cellular Alzheimer model, J. Alzheimers Dis. 42 (1) (2014) 143-55e. [2] A. Ataie, R. Ataee, M. Shadifar, S. Shahabi, S.M. Aghajanpour, Y. Hosseinpour, Interaction of memantine with homocysteine on the apoptosis in the rat hippocampus cells, Int. J. Mol. Cell Med. 1 (3) (2012) 145–152. [3] B.K. Atwood, A. Straiker, K. Mackie, CB2 cannabinoid receptors inhibit synaptic transmission when expressed in cultured autaptic neurons, Neuropharmacology 63 (4) (2012) 514–523. [4] Y.P. Bai, Y.H. Liu, J. Chen, T. Song, Y. You, Z.Y. Tang, et al., Rosiglitazone attenuates NF-kappaB-dependent ICAM-1 and TNF-alpha production caused by homocysteine via inhibiting ERK1/2/p38MAPK activation, Biochem. Biophys. Res. Commun. 360 (1) (2007) 20–26. [5] T. Bisogno, F. Howell, G. Williams, A. Minassi, M.G. Cascio, A. Ligresti, et al., Cloning of the first sn1-DAG lipases points to the spatial and temporal regulation of endocannabinoid signaling in the brain, J. Cell Biol. 163 (3) (2003) 463–468. [6] J.L. Blankman, G.M. Simon, B.F. Cravatt, A comprehensive profile of brain enzymes that hydrolyze the endocannabinoid 2-arachidonoylglycerol, Chem. Biol. 14 (12) (2007) 1347–1356. [7] A. Brück, R. Portin, A. Lindell, A. Laihinen, J. Bergman, M. Haaparanta, et al., Positron emission tomography shows that impaired frontal lobe functioning in Parkinson's disease is related to dopaminergic hypofunction in the caudate nucleus, Neurosci. Lett. 311 (2) (2001) 81–84. [8] F. Cacciapuoti, Lowering homocysteine levels with folic acid and B-vitamins do not reduce early atherosclerosis, but could interfere with cognitive decline and Alzheimer's disease, J. Thromb. Thrombolysis 36 (3) (2013) 258–262. [9] S. Carloni, D. Alonso-Alconada, S. Girelli, A. Duranti, A. Tontini, D. Piomelli, et al., Pretreatment with the monoacylglycerol lipase inhibitor URB602 protects from the long-term consequences of neonatal hypoxic–ischemic brain injury in rats, Pediatr. Res. 72 (4) (2012) 400–406. [10] J. Chen, Y. Zhou, S. Mueller-Steiner, L.F. Chen, H. Kwon, S. Yi, et al., SIRT1 protects against microglia-dependent amyloid-beta toxicity through inhibiting NF-kappaB signaling, J. Biol. Chem. 280 (48) (2005) 40364–40374. [11] R. Chen, J. Zhang, Y. Wu, D. Wang, G. Feng, Y.P. Tang, et al., Monoacylglycerol lipase is a therapeutic target for Alzheimer's disease, Cell Rep. 2 (5) (2012) 1329–1339. [12] X. Chen, J. Zhang, C. Chen, Endocannabinoid 2-arachidonoylglycerol protects neurons against β-amyloid insults, Neuroscience 178 (2011) 159–168. [13] B. Deweer, S. Lehéricy, B. Pillon, M. Baulac, J. Chiras, C. Marsault, et al., Memory disorders in probable Alzheimer's disease: the role of hippocampal atrophy as shown with MRI, J. Neurol. Neurosurg. Psychiatry 58 (5) (1999) 590–597. [14] M. Dong, Y. Lu, Y. Zha, et al., Endocannabinoid 2-arachidonylglycerol protects primary cultured neurons against homocysteine-induced impairments in rat caudate nucleus through CB1 receptor, J. Mol. Neurosci. 55 (2) (2015) 500–508. [15] B.E. Dwyer, A.K. Raina, G. Perry, M.A. Smith, Homocysteine and Alzheimer's disease: a modifiable risk? Free Radic. Biol. Med. 36 (11) (2004) 1471–1475. [16] M. Gallucci, A. Zanardo, M. Bendini, F. Di Paola, P. Boldrini, E. Grossi, Serum folate, homocysteine, brain atrophy, and auto-CM system: the Treviso Dementia (TREDEM) study, J. Alzheimers Dis. 38 (3) (2014) 581–587. [17] H. Guo, X. Song, M.H. Schmidt, R. Vandorpe, Z. Yang, E. LeBlanc, J. Zhang, et al., Evaluation of whole brain health in aging and Alzheimer’s disease: a standard procedure for scoring an MRI-based brain atrophy and lesion index, J. Alzheimers Dis. 42 (2) (2014) 691–703. [18] M. Hüll, B. Müksch, R.S. Akundi, A. Waschbisch, J.J. Hoozemans, Amyloid beta peptide (25–35) activates protein kinase C leading to cyclooxygenase-2 induction and prostaglandin E2 release in primary midbrain astrocytes, Neurochemistry 48 (8) (2006) 663–672. [19] S. Jiji, K.A. Smitha, A.K. Gupta, V.P. Pillai, R.S. Jayasree, Segmentation and volumetric analysis of the caudate nucleus in Alzheimer's disease, Eur. J. Radiol. 82 (9) (2013) 1525–1530. [20] P.K. Kamat, A. Kalani, S. Givvimani, P.B. Sathnur, S.C. Tyagi, N. Tyagi, Hydrogen sulfide attenuates neurodegeneration and neurovascular dysfunction induced
[21] [22]
[23]
[24]
[25]
[26] [27]
[28]
[29]
[30]
[31] [32]
[33] [34]
[35] [36]
[37]
[38]
[39]
[40] [41] [42]
by intracerebral-administered homocysteine in mice, Neuroscience 252 (2013) 302–319. T. Krakauer, Molecular therapeutic targets in inflammation: cyclooxygenase and NF-kappaB, Curr. Drug Targets Inflamm. Allergy 3 (3) (2004) 317–324. Y.S. Lee, S.J. Lee, K.W. Seo, J.U. Bae, S.Y. Park, C.D. Kim, Homocysteine induces COX-2 expression in macrophages through ROS generated by NMDA receptor–calcium signaling pathways, Free Radic. Res. 47 (5) (2013) 422–431. J.G. Li, J. Chu, C. Barrero, S. Merali, D. Praticò, Homocysteine exacerbates β-amyloid pathology, tau pathology, and cognitive deficit in a mouse model of Alzheimer disease with plaques and tangles, Ann. Neurol. 75 (6) (2014) 851–863. M.H. Li, J.P. Tang, P. Zhang, X. Li, C.Y. Wang, H.J. Wei, et al., Disturbance of endogenous hydrogen sulfide generation and endoplasmic reticulum stress in hippocampus are involved in homocysteine-induced defect in learning and memory of rats, Behav. Brain Res. 262 (2014) 35–41. Y. Lu, F. Peng, M. Dong, H. Yang, Endocannabinoid 2-arachidonylglycerol protects primary cultured neurons against LPS-induced impairments in rat caudate nucleus, J. Mol. Neurosci. 54 (1) (2014) 49–58. J.W. Miller, Homocysteine and Alzheimer's disease, Nutr. Rev. 57 (4) (1999) 126–129. M.S. Morris, J. Selhub, P.F. Jacques, Vitamin B-12 and folate status in relation to decline in scores on the mini-mental state examination in the Framingham heart study, J. Am. Geriatr. Soc. 60 (8) (2012) 1457–1464. R. Poddar, S. Paul, Novel crosstalk between ERK MAPK and p38 MAPK leads to homocysteine–NMDA receptor-mediated neuronal cell death, J. Neurochem. 124 (4) (2013) 558–570. R. Poddar, S. Paul, Homocysteine–NMDA receptor-mediated activation of extracellular signal-regulated kinase leads to neuronal cell death, J. Neurochem. 110 (3) (2009) 1095–1106. N.S. Ryan, S. Keihaninejad, T.J. Shakespeare, M. Lehmann, S.J. Crutch, I.B. Malone, et al., Magnetic resonance imaging evidence for presymptomatic change in thalamus and caudate in familial Alzheimer's disease, Brain 136 (2013) 1399–1414. R.J. Schulz, Homocysteine is a biomarker for cognitive dysfunction in the elderly, Curr. Opin Clin. Nutr. Metab. Care 10 (6) (2007) 718–723. S. Seshadri, A. Beiser, J. Selhub, P.F. Jacques, I.H. Rosenberg, R.B. D'Agostino, et al., Plasma homocysteine as a risk factor for dementia and Alzheimer's disease, N. Engl. J. Med. 346 (7) (2002) 476–483. L. Steinman, Nuanced roles of cytokines in three major human brain disorders, J. Clin. Invest. 118 (11) (2008) 3557–3563. T. Sugiura, S. Kishimoto, S. Oka, M. Gokoh, Biochemistry, pharmacology and physiology of 2-arachidonoylglycerol, an endogenous cannabinoid receptor ligand, Prog. Lipid Res. 45 (2006) 405–446. F. Van Dam, W.A. Van Gool, Hyperhomocysteinemia and Alzheimer's disease: a systematic review, Arch. Gerontol. Geriatr. 48 (3) (2009) 425–430. S. Wang, C. Zhang, X. Sheng, X. Zhang, B. Wang, G. Zhang, Peripheral expression of MAPK pathways in Alzheimer's and Parkinson's diseases, J. Clin. Neurosci. 21 (5) (2014) 810–814. J. Wiskerke, C. Irimia, B.F. Cravatt, T.J. De Vries, A.N. Schoffelmeer, T. Pattij, et al., Characterization of the effects of reuptake and hydrolysis inhibition on interstitial endocannabinoid levels in the brain: an in vivo microdialysis study, ACS Chem. Neurosci. 3 (5) (2012) 407–417. N. Wu, Y.L. Siow, K. O, Induction of hepatic cyclooxygenase-2 by hyperhomocysteinemia via nuclear factor-kappaB activation, Am. J. Physiol. Regul. Integr. Comp. Physiol. 297 (4) (2009) R1086–R1094. H. Yan, X. Zhang, S. Luo, H. Liu, X. Wang, Y. Gao, et al., Effects of homocysteine on ERK signaling and cell proliferation in fetal neural stem cells in vitro, Cell Biochem. Biophys. 66 (1) (2013) 131–137. H. Yang, C. Chen, Cyclooxygenase-2 in synaptic signaling, Curr. Pharm. Des. 14 (14) (2008) 1443–1451. J. Zhang, C. Chen, Endocannabinoid 2-arachidonoylglycerol protects neurons by limiting COX-2 elevation, J. Biol. Chem. 283 (33) (2008) 22601–22611. E.R. Zimmer, A. Leuzy, A.L. Benedet, J. Breitner, S. Gauthier, P. Rosa-Neto, Tracking neuroinflammation in Alzheimer's disease: the role of positron emission tomography imaging, J. Neuroinflammation 11 (2014) 120.
Please cite this article as: M. Dong, et al., Monoacylglycerol lipase inhibitor protects primary cultured neurons against homocysteine-induced impairments in rat caudate nucleus through COX-2 signaling, Life Sci (2015), http://dx.doi.org/10.1016/j.lfs.2015.03.006
Contents lists available at ScienceDirect
Life Sciences journal homepage: www.elsevier.com/locate/lifescie
Monoacylglycerol lipase inhibitor protects primary cultured neurons against homocysteine-induced impairments in rat caudate nucleus through COX-2 signaling Manman Dong, Yongli Lu, Ziliang Zou, Hongwei Yang ⁎ Department of Physiology and Pathophysiology, College of Medical Sciences, China Three Gorges University, 443002 Yichang, Hubei, PR China
a r t i c l e
i n f o
Article history: Received 15 December 2014 Received in revised form 1 March 2015 Accepted 11 March 2015 Available online xxxx Keywords: Endocannabinoid Monoacylglycerol lipase Alzheimer's disease Neuroinflammation Caudate nucleus Cyclooxygenase-2
a b s t r a c t Aims: URB602 is a selective inhibitor of monoacylglycerol lipase (MAGL), a serine hydrolase involved in the biological deactivation of the endocannabinoid 2-arachidonoyl glycerol (2-AG). It has been described that URB602 significantly enhances depolarization-induced increases in 2-AG. A high level of homocysteine (Hcy) is a modifiable risk factor for developing Alzheimer's disease (AD). The aim of this study was to investigate the protective effects of URB602 on Hcy-induced impairments underlying its cellular and molecular mechanism in primary cultured caudate nucleus (CN) neurons. Main methods: The expressions of cyclooxygenase-2 (COX-2), ERK1/2, NF-κB and IκB-α as well as cleaved caspase3 and p-Bcl-2 in Hcy-, URB602 or SR1 (a selective inhibitor of CB1 receptor)-treated primary cultured neurons in CN were measured by immunoblotting technique and neurotoxicity assays were performed by using Hoechst staining. Key findings: The MAGL inhibitor URB602 exerted a neuroprotective effect on Hcy-induced impairment through suppression of cyclooxygenase-2 (COX-2) elevation and ERK1/2 and NF-κB phosphorylation as well as suppressions of IκB-α degradation in a CB1 receptor-dependent way. Moreover, anti-neuronal impairments of URB602 were mediated by modulating down-regulation of cleaved caspase-3 expression and up-regulation of p-Bcl-2 expression in a CB1 receptor-dependent manner in primary cultured CN neurons. Significance: These data suggest that the MAGL inhibitor is a promising therapeutic target for some neurodegenerative disorders, such as AD, via the COX-2 signaling pathway. © 2015 Elsevier Inc. All rights reserved.
1. Introduction Epidemiological and clinical studies have revealed that elevated total homocysteine (Hcy) level is a modifiable risk factor for developing Alzheimer's disease (AD) [8,15,26,31,32,35]. Hcy, an amino acid, is produced in the methylation cycle of protein metabolism requiring folate, vitamins B12 and B6 as cofactors [8,27]. The major neuropathological features of AD are amyloid-β peptide aggregation and tau hyperphosphorylation in the hippocampus and cortex, along with brain atrophy and cognitive decline [17]. An elevated concentration of Hcy is associated with a serious decrease of volume in the hippocampus in a group of old outpatients [16]. Several potential mechanisms underlying the deleterious effect of Hcy in the brain have been proposed. These included the impact on cerebrovascular pathology [20], direct neurotoxic effects [24] or the malignant influence on amyloid-β peptide generation and tau hyperphosphorylation [23], but the cellular and molecular mechanisms for Hcy in AD are not fully understood. Moreover, ⁎ Corresponding author at: Department of Physiology and Pathophysiology, College of Medical Sciences, China Three Gorges University, 8 University Road, 443002 Yichang, Hubei, PR China. Tel.: +86 717 6397620; fax: +86 717 6397328. E-mail address: [email protected] (H. Yang).
there are currently no effective medications to lower the concentration of Hcy and alleviate Hcy-induced brain disorders. The caudate nucleus (CN), a cluster of nerve cells that is part of the striatum, has been deemed to have little relationship with AD. However, neuropathological studies suggested that atrophy of the CN plays a vital role in AD progression, because the cortico–striato–thalamic loops are involved in cognitive processes, particularly, in attention and executive function, as well as in movement disorders [7,13]. Moreover, a significant reduction in the caudate volume of AD patients was compared to the normal volunteers at a presymptomatic stage, while no significant hippocampal volume loss was evident presymptomatically [19,30]. Therefore the change in the CN may be an important factor in the search for AD biomarkers in the brain. In the last decade, the endocannabinoid system (eCBs), comprised of cannabinoid receptors (CB1 and CB2), endocannabinoids and their interrelated biosynthesis or degradation enzymes, has been recognized to be important in controlling many physiological and pathological processes and has become an area of interest for novel drug development [11,12,14,25]. 2-Arachidonoyl glycerol (2-AG), the main endocannabinoid, is synthesized on demand and rapidly inactivated by enzymatic hydrolysis, therefore limiting their potential protective effects [5]. A comprehensive profile of brain serine hydrolases revealed
http://dx.doi.org/10.1016/j.lfs.2015.03.006 0024-3205/© 2015 Elsevier Inc. All rights reserved.
Please cite this article as: M. Dong, et al., Monoacylglycerol lipase inhibitor protects primary cultured neurons against homocysteine-induced impairments in rat caudate nucleus through COX-2 signaling, Life Sci (2015), http://dx.doi.org/10.1016/j.lfs.2015.03.006
2
M. Dong et al. / Life Sciences xxx (2015) xxx–xxx
that about 85% of total 2-AG in mouse brain is hydrolyzed by monoacylglycerol lipase (MAGL) [6]. Recently, Chen et al. reported that the application of the MAGL inhibitor or deletion gene of MAGL suppresses neuroinflammation and prevents neurodegeneration, which is beneficial for decelerating AD progress, on account of the marked and sustained elevations in 2-AG levels [11]. URB602, a selective inhibitor of MAGL's activity, known to decrease 2-AG's metabolism and enhance 2-AG-mediated signaling in neurons, exerts neuroprotective effects, such as inhibition of neurotrophic cytokine cyclooxygenase-2 (COX-2), nuclear factor-κB (NF-κB) and mitogen-activated protein kinase (MAPK) family [37], but little is known about whether suppression of MAGL has the ability to protect neurons against Hcy harmful insults in the CN neuron. In the present study, we found that the MAGL inhibitor could protect primary cultured neurons against Hcy-induced impairments in rat CN through COX-2 signaling. 2. Materials and methods 2.1. Materials Neurobasal-A, B27, 0.25% trypsin–EDTA solution, fetal bovine serum, and Dulbecco's Modified Eagle's Medium (DMEM) dry powder were obtained from Gibco BRL (Grand Island, NY, USA). Homocysteine, AM630, poly-D-lysine, L-glutamine, L-glutamate, and cytosine arabinoside were purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). Antibodies for detecting nuclear factor-kappaB (NF-κB), phospho-p38 mitogen activated protein kinase (p-p38MAPK), extracellular signal-regulated kinase (ERK1/2), and cleaved caspase-3 were purchased from Beyotime Institute of Biotechnology (Haimen, China). Cyclooxygenase-2 (COX-2) polyclonal antibody, URB602, and Rimonabant (SR141716, SR1) were purchased from Cayman Chemical (Ann Arbor, MI, USA). 2.2. Cell culture In this study, subject care complied with the China Three Gorges University Guide for the Care and Use of Laboratory Animals, and all protocols were approved by the Institutional Animal Care and Use Committee of the China Three Gorges University. The methods for obtaining primary cultured CN neurons have been described in detail elsewhere [14,25]. Briefly, primary CN cultures were obtained from neonatal Sprague–Dawley rats (within 24 h) which were provided by the Experimental Animal Research Center of Hubei Province. The caudate nuclei were taken out and washed several times in a cold (4 °C) DMEM solution. After the caudate nuclei were cut to small pieces (1 mm × 1 mm × 1 mm) with spring scissors, they were incubated into 5 mL 0.25% trypsin–EDTA sterile solution and incubated at 37 °C for 10–15 min with 5% CO 2. Then, they were triturated with a flamed and siliconized Pasteur pipette to separate cells gently. Subsequently, they were placed in a centrifuge tube with 5–10 mL DMEM solution containing 10% fetal bovine serum and centrifuged one time for 8 min at 800 rpm/min. Cells were spun down and resuspended in neurobasalA/B27 medium (Gibco) supplemented with 1 mM L-glutamine, penicillin/streptomycin, and 25 μM glutamate. Cells (0.5 × 106–1 × 106/mL) were loaded into poly-D-lysine-coated 35-mm culture dishes for Western blot analysis. After 5 days in culture, the cells were fed with the medium supplemented with 2.5 μg/mL cytosine arabinoside to proliferation of non-neuronal cells. The culture medium was changed every 3 days with the same medium without glutamate until use. The cells were used in experiments between 7 and 12 days in culture. 2.3. Western blot analysis The CN neurons were harvested after 7 days of Hcy or URB602 or vehicle treatment. The cells were washed with ice-cold PBS and lysed with the solubilization buffer containing a Radio Immunoprecipitation Assay (RIPA) lysis buffer and phenylmethanesulfonyl fluoride (PMSF)
(100:1) (Beyotime, Haimen, China). After sonication, the samples were centrifuged for 10 min at 12000 g. The protein concentration of the supernatant was determined by BCA (bicinchoninic acid) assay, and the loading quantity of the protein sample was chosen to be 50 μg. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis was done by loading equal amounts of proteins per lane. Proteins were separated on SDS-12% polyacrylamide gel and transferred to PVDF membranes by wet transfer. The membranes were blocked with 5% non-fat milk and incubated with primary antibodies at 4 °C overnight, and the primary antibodies (all the primary antibodies diluted 1:1,000) including Rabbit anti-COX-2 polyclonal antibodies (Cayman, Ann Arbor, MI, USA), Rabbit anti-cleaved caspase-3, Rabbit anti-NFκB-65 or anti-p-NF-κB65 antibodies, Rabbit anti-IκB-α, Rabbit antiBcl-2 or anti-p-Bcl-2, Mouse anti-ERK1/2 or anti-p-ERK1/2, Mouse anti-p38 or anti-p-38MAPK (Beyotime, Haimen, China). The blot was washed for four times with TBST (20 mM Tris–HCl, 150 mM NaCl, 0.05% Tween 20, pH 7.4) for a total of 30 min, and incubated with the corresponding secondary antibody (goat anti-rabbit/mouse 1:2000, Beyotime, Haimen, China), at room temperature for 2 h and washed for four times with TBST. Proteins were detected by ECL Plus (Beyotime, Haimen, China) with films according to the manufacturer's instructions. Quantitation of the proteins was done by densitometric analysis using ImageJ 1.46i software. Band densities were normalized to the total amount of protein loaded in each well as determined by mouse anti-βactin (1:4000, Beyotime, Haimen, China). 2.4. Hoechst 33258 staining analysis The primary CN neurons were seeded in 35 mm plates as described in the Cell culture section. On the 7th day the CN neurons were treated with Hcy or URB602 or vehicle treatment by 12 h. Then the neurons were fixed with 4% paraformaldehyde (Beyotime, Haimen, China) for 10 min, and washed with ice-cold PBS twice. Then the cells were incubated in a Hoechst 33258 (Beyotime, Haimen, China) staining solution (final concentration: 10 μg/mL) for 5 min at 37 °C. The cells were analyzed with a fluorescence microscopic system (100× magnification, excitation wavelength 350 nm, emission wavelength 460 nm). Stained condensed chromatin appeared small and bright blue, whereas stained normal nuclei were large and light blue. The proportion of nuclei with condensed chromatin was calculated to reflect the level of injury. Five randomly chosen fields were analyzed for each group. The rate of apoptotic cells was equal to the ratio of apoptotic cells to the number of total cells. 2.5. Data analysis All values are presented as mean ± SEM unless stated otherwise. The significance of inter-group differences was evaluated by one-way analyses of variance (ANOVA: least-significant difference test for post-hoc comparisons). Differences were considered significant when P b 0.05. 3. Results 3.1. URB602 suppresses excessive COX-2 expression in response to Hcy in the cultured CN neurons through the CB1 receptor, not the CB2 receptor URB602, a selective inhibitor of MAGL activity, is known to elevate the levels of endogenous 2-AG and enhance 2-AG-mediated signaling in neurons, without affecting the level of arachidonoyl ethanolamide (anandamide, AEA), another endocannabinoid ligand [37]. As a key player in inflammation, COX-2 is markedly elevated by a variety of stimulation of inflammation [12,25,41]. Hcy serves as an inducer of proinflammatory response to induce neural injury. To determine whether URB602 is capable of inhibiting elevation of Hcy-induced COX-2 expression, the CN neurons were cultured with Hcy (100 μM) and URB602
Please cite this article as: M. Dong, et al., Monoacylglycerol lipase inhibitor protects primary cultured neurons against homocysteine-induced impairments in rat caudate nucleus through COX-2 signaling, Life Sci (2015), http://dx.doi.org/10.1016/j.lfs.2015.03.006
M. Dong et al. / Life Sciences xxx (2015) xxx–xxx
(10 μM) for 12 h and COX-2 protein expression was detected by Western blot. As shown in Fig. 1A & B, under the induction of Hcy, the level of COX-2 proteins increased significantly compared with the vehicle control (n = 3, P b 0.01, versus vehicle control). Meanwhile, this elevation was suppressed in the presence of URB602 (n = 3, P b 0.01, versus Hcy), suggesting that URB602 is able to suppress Hcy-induced COX-2 elevation in the CN neurons. Elevated 2-AG by URB602 is an endogenous agonist for the CB1 receptor and the CB1 receptor is expressed mainly in the nervous system [34]. To ascertain whether the URB602-induced inhibition of COX-2 is mediated through the CB1 receptor in the CN neurons, we used SR141716 (SR1, a selective CB1 antagonist) and AM630 (a selective CB2 antagonist) to detect it. As seen in Fig. 1A & B, SR1 inhibits the URB602-produced suppression of COX-2 (n = 3, P b 0.01, versus URB602 and Hcy), while AM630 has no effect on URB602-produced suppression of COX-2 expression elevation (n = 3, P b 0.01, versus URB602 and Hcy). And URB602-produced suppression of COX-2 is not completely inhibited by SR1 (n = 3, P b 0.01, versus Hcy). These findings indicate that CB1R-, but not CB2R-dependent suppressions of COX-2 expression may partially be involved in URB602-induced neuroprotection against Hcy. To exclude the non-specific effect of drugs on COX-2 expression, we employed URB602, SR1 and AM630 to examine it. As shown in Fig. 1C & D, URB602, SR1 and AM630 alone did not affect the COX-2 expression.
3
3.2. URB602 inhibits the phosphorylation of NF-κB and degradation of IκB-α in response to Hcy in the primary CN neurons via the CB1 receptor A preceding study showed that the NF-κB signaling pathway was involved in the lipopolysaccharide (LPS)-induced or Hcy-induced neuroinflammation in the hippocampus and CN [14,25,40,41]. To determine whether this signaling pathway is involved in the URB602 suppression of COX-2 induced by Hcy in the CN neurons, we detected the phosphorylation level of NF-κB in the CN neurons. As shown in Fig. 2A & B, Western blot showed that the expression of pho-NF-κB (p-NF-κB) was significantly enhanced after the CN neurons were cultured with Hcy (100 μM, 12 h) (n = 3, P b 0.01, versus vehicle control). However, the Hcy-induced enhancement of p-NF-κB expression was significantly suppressed when co-incubation with URB602 (10 μM, 12 h) (n = 3, P b 0.01, versus Hcy). Furthermore, when the CN neurons were co-treated with Hcy, URB602 and SR1 (10 μM, 12 h), this action of URB602 was significantly alleviated (n = 3, P b 0.01, versus URB602 and Hcy), not completely alleviated (n = 3, P b 0.05, versus Hcy). The data clearly demonstrates that CB1-dependent suppression of NF-κB phosphorylation may be partially involved in URB602-induced neuroprotection against Hcy in the primary CN neurons. NF-κB dimer is a kind of protein and is sequestered in the cytoplasm by IκB-α. Upon stimulation IκB-α is rapidly degraded [38]. Meanwhile,
Fig. 1. URB602 inhibits the elevation of Hcy-induced COX-2 expression via CB1R-, but not CB2R-dependent way in the primary CN neurons. (A) A representative autoradiograph of COX-2 expression in the CN neurons in vehicle control, Hcy (100 μM), URB (10 μM) + Hcy, SR1 (10 μM) + URB + Hcy and AM630 (5 μM) + URB + Hcy. The CN neurons in culture were treated with Hcy in the absence and presence of URB or SR1 or AM630 for 12 h. (B) Quantitative values of protein expressions under different treatments. (C) A representative autoradiograph of COX-2 expression in the CN neurons in vehicle control, URB (10 μM), SR1 (10 μM) and AM630 (5 μM) in culture for 12 h. (D) Quantitative values of COX-2 expressions under different treatments. Values are expressed as mean ± SEM of three independent experiments. In all blots, staining for β-actin was used as a loading control. The level of COX-2 expression obtained in each experimental condition was calculated as a fold of the control. **P b 0.01, compared with the vehicle control; ##P b 0.01, compared with Hcy; ++ P b 0.01, compared with URB + Hcy.
Please cite this article as: M. Dong, et al., Monoacylglycerol lipase inhibitor protects primary cultured neurons against homocysteine-induced impairments in rat caudate nucleus through COX-2 signaling, Life Sci (2015), http://dx.doi.org/10.1016/j.lfs.2015.03.006
4
M. Dong et al. / Life Sciences xxx (2015) xxx–xxx
Fig. 2. URB602 reduces Hcy-induced NF-κB phosphorylation and degradation of IκB-α through a CB1R-dependent manner in the primary CN neurons. (A) A representative autoradiograph of NF-κB phosphorylation in the CN neurons in vehicle control, Hcy (100 μM), URB (10 μM) + Hcy, and SR1 (10 μM) + URB + Hcy. The CN neurons in culture were treated with Hcy in the absence and presence of URB or SR1 for 12 h. (B) Quantitative values of protein expressions under different treatments. Values are expressed as mean ± SEM of three independent experiments. (C) A representative autoradiograph of degradation of IκB-α in the CN neurons in vehicle control, Hcy (100 μM), URB (10 μM) + Hcy, and SR1 (10 μM) + URB + Hcy. The CN neurons in culture were treated with Hcy in the absence and presence of URB or SR1 for 12 h. (D) Quantitative values of protein expressions under different treatments. Values are expressed as mean ± SEM of three independent experiments. **P b 0.01, compared with the vehicle control; ##P b 0.01, compared with Hcy; ++ P b 0.01, compared with URB + Hcy.
as shown in Fig. 2C & D, Western blot showed that Hcy induced the degradation of IκB-α (n = 3, P b 0.01, versus vehicle control), and this degradation was significantly suppressed by co-incubation with URB602 (10 μM) (Fig. 2C & D, n = 3, P b 0.01, versus Hcy). Furthermore, when the CN neurons were co-treated with Hcy, URB602 and SR1 (10 μM, 12 h), this action of URB602 was significantly alleviated (n = 3, P b 0.01, versus URB602 and Hcy), not completely alleviated (n = 3, P b 0.05, versus Hcy). These findings suggest that CB1-dependent suppression of IκB-α degradation may be partially involved in URB602-induced neuroprotection against Hcy in the primary CN neurons.
3.3. URB602 prevents the phosphorylation of ERK1/2 in response to Hcy in the cultured CN neurons through the CB1 receptor In the hippocampus and CN, mitogen-activated protein kinase (MAPK) ERK1/2 is also known to be involved in the antiinflammatory properties of eCBs [12,14,25,40,41]. Therefore, we tested the level of phospho-ERK1/2 (p-ERK1/2) by Western blot. As shown in Fig. 3, treatment with Hcy (100 μM, 12 h) induced an increase in pERK1/2 expression in the CN neurons (n = 3, P b 0.01, versus vehicle control). The Hcy-induced elevation of p-ERK1/2 expression was significantly reduced in the presence of URB602 (10 μM, 12 h) (n = 3, P b 0.01, versus Hcy). Furthermore, this reduction was reversed by SR1 (10 μM, 12 h) (n = 3, P b 0.01, versus URB602 and Hcy), but not
completely reversed (n = 3, P b 0.01, versus Hcy). These results suggest that CB1-dependent suppression of ERK1/2 phosphorylation may partially be implicated in URB602-induced neuroprotection against Hcy in the CN neurons.
3.4. URB602 reduces down-regulated phosphorylation of p38MAPK in response to Hcy in the cultured CN neurons through the CB1 receptor The p38MAPK signal pathway is one of the important branches of the MAPK pathway [12,14,25,40,41]. To explore whether the p38MAPK signal pathway is involved in the effects of URB602 in the CN neurons, we treated the CN neurons with Hcy in the absence and presence of URB602 or SR1. The level of phospho-p38MAPK (p-p38MAPK) was measured by Western blot analysis. As seen in Fig. 4, treatment with Hcy (100 μM, 12 h) significantly decreased the expression of p-p38MAPK proteins in the CN neurons (n = 3, P b 0.01, versus vehicle control). This reduction was significantly enhanced in the presence of URB602 (10 μM, 12 h) (n = 3, P b 0.01, versus Hcy). Furthermore, the URB602 inducedreduction of p-p38MAPK expression was inhibited by SR1 (10 μM, 12 h) (n = 3, P b 0.01, versus URB602 and Hcy), but not completely inhibited (n = 3, P b 0.01, versus Hcy). These findings indicated that the URB602 modulating down-regulation of p-p38MAPK expression in response to Hcy was partially mediated through CB1 receptor in the cultured CN neurons.
Please cite this article as: M. Dong, et al., Monoacylglycerol lipase inhibitor protects primary cultured neurons against homocysteine-induced impairments in rat caudate nucleus through COX-2 signaling, Life Sci (2015), http://dx.doi.org/10.1016/j.lfs.2015.03.006
M. Dong et al. / Life Sciences xxx (2015) xxx–xxx
Fig. 3. URB602 suppresses Hcy-induced ERK1/2 phosphorylation in a CB1R-dependent way in the primary CN neurons. (A) A representative autoradiograph of ERK1/2 phosphorylation in the CN neurons in vehicle control, Hcy (100 μM), URB (10 μM) + Hcy, and SR1 (10 μM) + URB + Hcy. The CN neurons in culture were treated with Hcy in the absence and presence of URB or SR1 for 12 h. (B) Quantitative values of protein expressions under different treatments. Values are expressed as mean ± SEM of three independent experiments. **P b 0.01, compared with the vehicle control; ##P b 0.01, compared with Hcy; ++ P b 0.01, compared with URB + Hcy.
3.5. URB602 inhibits cleaved caspase-3 activation and increases p-Bcl-2 expression induced by Hcy in the cultured CN neurons via the CB1 receptor Caspase-3 is involved in the regulation of cell apoptosis [14,25]. To examine whether suppression of MAGL could prevent the CN neurons from apoptosis induced by Hcy, we directly add URB602 to the CN neurons. The exposure of Hcy (100 μM, 12 h) to the CN neurons showed a significant up-regulation of cleaved caspase-3 expression (Fig. 5A & B, n = 3, P b 0.01, versus vehicle control). The Hcy-induced elevation of cleaved caspase-3 expression was significantly reduced in the presence of URB602 (10 μM, 12 h) (n = 3, P b 0.01, versus Hcy). Furthermore, this reduction was reversed by SR1 (10 μM, 12 h) (n = 3, P b 0.01, versus URB602 and Hcy), but not completely inhibited (n = 3, P b 0.01, versus Hcy). These results suggested that the URB602 modulating down-regulation of cleaved caspase-3 expression in response to Hcy was partially mediated through the CB1 receptor in the cultured CN neurons. The Bcl-2 protein family is endowed with an anti-apoptotic effect, which is involved in the progression of AD [1]. To verify whether inhibition of MAGL activity could prevent the CN neurons by means of elevating the level of the anti-apoptotic protein p-Bcl-2 reduced by Hcy, we directly add URB602 to the CN neurons. The exposure of Hcy (100 μM, 12 h) to the CN neurons showed a significant down-regulation of p-Bcl-2 expression (Fig. 5C & D, n = 3, P b 0.01, versus vehicle control). The Hcy-induced diminution of p-Bcl-2 expression was significantly
5
Fig. 4. URB602 attenuates Hcy-induced p38MAPK expression through a CB1R-dependent way in the primary CN neurons. (A) A representative autoradiograph of p38MAPK phosphorylation in the CN neurons in vehicle control, Hcy (100 μM), URB (10 μM) + Hcy, and SR1 (10 μM) + URB + Hcy. The CN neurons in culture were treated with Hcy in the absence and presence of URB or SR1 for 12 h. (B) Quantitative values of protein expressions under different treatments. Values are expressed as mean ± SEM of three independent experiments. **P b 0.01, compared with the vehicle control; ##P b 0.01, compared with Hcy; ++ P b 0.01, compared with URB + Hcy.
elevated in the presence of URB602 (10 μM, 12 h) (n = 3, P b 0.01, versus Hcy). Furthermore, this elevation was reversed by SR1 (10 μM, 12 h) (n = 3, P b 0.01, versus URB602 and Hcy), but not completely inhibited (n = 3, P b 0.01, versus Hcy). These results suggested that URB602 modulated the up-regulation of p-Bcl-2 expression in response to Hcy partially via a CB1 receptor-dependent manner in the cultured CN neurons.
3.6. URB602 inhibits the cellular apoptosis induced by Hcy using Hoechst staining in the cultured CN neurons through the CB1 receptor To determine whether suppression of MAGL could prevent the CN neurons from apoptosis induced by Hcy using Hoechst staining, we directly add URB602 to the CN neurons. As shown in Fig. 6A & B, the exposure of Hcy (100 μM, 12 h) to the CN neurons showed a significant elevation of apoptotic neurons (n = 3, P b 0.01, versus vehicle control). The Hcy-induced elevation of apoptotic neurons was significantly reduced in the presence of URB602 (10 μM, 12 h) (n = 3, P b 0.01, versus Hcy). Furthermore, this reduction was inhibited by SR1 (10 μM, 12 h) (n = 3, P b 0.01, versus URB602 and Hcy), but not completely inhibited (n = 3, P b 0.01, versus Hcy). These findings suggested that URB602 inhibition of cellular apoptosis induced by Hcy using Hoechst staining was partially mediated through the CB1 receptor in the cultured CN neurons.
Please cite this article as: M. Dong, et al., Monoacylglycerol lipase inhibitor protects primary cultured neurons against homocysteine-induced impairments in rat caudate nucleus through COX-2 signaling, Life Sci (2015), http://dx.doi.org/10.1016/j.lfs.2015.03.006
6
M. Dong et al. / Life Sciences xxx (2015) xxx–xxx
Fig. 5. URB602 suppresses the elevation of cleaved caspase-3 expression and increases p-Bcl-2 expression induced by Hcy via CB1R-dependent way in the primary CN neurons. (A) A representative autoradiograph of cleaved caspase-3 in the CN neurons in vehicle control, Hcy (100 μM), URB (10 μM) + Hcy, and SR1 (10 μM) + URB + Hcy. The CN neurons in culture were treated with Hcy in the absence and presence of URB or SR1 for 12 h. (B) Quantitative values of cleaved caspase-3 expressions under different treatments. Values are expressed as mean ± SEM of three independent experiments. (C) A representative autoradiograph of p-Bcl-2 in the CN neurons in vehicle control, Hcy (100 μM), URB (10 μM) + Hcy, and SR1 (10 μM) + URB + Hcy. The CN neurons in culture were treated with Hcy in the absence and presence of URB or SR1 for 12 h. (D) Quantitative values of p-Bcl-2 expressions under different treatments. Values are expressed as mean ± SEM of three independent experiments. **P b 0.01, compared with the vehicle control; **P b 0.01, compared with the vehicle control, ##P b 0.01, compared with Hcy; ++ P b 0.01, compared with URB + Hcy.
4. Discussion In this study, we found that pretreatment with the MAGL inhibitor URB602 has protective effects against Hcy-induced neurotoxicity to the CN neurons, through suppression of COX-2 elevation and ERK1/2 and NF-κB phosphorylation in a CB1 receptor-dependent way. Moreover, URB602-mediated down-regulation of cleaved caspase-3 expression and up-regulation level of p-Bcl-2 participated in defending from Hcy-induced CN neuron apoptosis. Various lines of research have implicated neuroinflammation as an important participant in AD pathophysiology [42]. Most recent studies show that MAGL inactivation suppresses the expression of neuroinflammation factors COX-2, NF-κB and PGE-2, through CB1 receptor-mediated mechanisms, suggesting that MAGL is a promising therapeutic target for preventing and treating neurodegeneration diseases such as AD [9,11]. Our experiment found that the MAGL inhibitor URB602 exerts a protective response to Hcy harmfulness via the CB1 receptors and not the CB2 receptors in the CN neurons since the protective effects of URB602 were blocked or attenuated by SR141716, a selective CB1 receptor antagonist, but not by AM630, a selective CB2 receptor antagonist. It's consistent with our recent studies that directly adding 2AG protected the CN neurons from lipopolysaccharide (LPS) and Hcy toxicity, which binds to CB1 receptors [14,25]. The reason may be that the CB1 receptor is largely expressed in the brain, particularly in the striatum, the hippocampus and the amygdaloid nucleus [34]. However,
the extent of CB2 receptor expression and the precise distribution of CB2 receptors in the nervous system are still debated [3]. COX-2 is a medium of inflammation, through prostaglandin E2 glycerol (PGE2-G) ester inducing NF-κB, caspase-3, ERK1/2, p38MAPK and other signaling activation, which has been implicated in the pathogenesis of neurodegenerative diseases like AD [40]. Hcy induced COX-2 expression in macrophages, hepatic cell, as well as in the primary cultured CN neurons, which was hazardous to the function of cells [14,22, 38]. Previous findings showed that the direct application of 2-AG or elevation of endogenous 2-AG by URB602 protects the hippocampal and CN neurons through suppression of cyclooxygenase-2 (COX-2) expression, which is provoked by pro-inflammatory IL-1β and LPS, excitotoxic glutamate and kainic acid and β-amyloid, via a CB1 receptor-dependent NFκB signaling pathway [12,25,41]. In our study, indirectly increasing the level of 2-AG by URB602 and binding to CB1 receptors exert an inhibitory action on COX-2 elevation induced by Hcy insults in the CN neurons. The result is consistent with our previous finding that adding pure 2-AG solution could protect the CN neurons from Hcy assault by decreasing the expression of COX-2 [14]. In our study, we observed that the CN neurons in culture treated with Hcy significantly elevated phosphorylation of NF-κB and degradation of IκB-α. The transcription factor NF-κB plays a critical role in the regulating the induction and resolution of inflammation and is involved in the brain proinflammatory process of AD [10,33]. Once IκB-α degrades, the released NF-κB translocates to the nucleus, which is the onset
Please cite this article as: M. Dong, et al., Monoacylglycerol lipase inhibitor protects primary cultured neurons against homocysteine-induced impairments in rat caudate nucleus through COX-2 signaling, Life Sci (2015), http://dx.doi.org/10.1016/j.lfs.2015.03.006
M. Dong et al. / Life Sciences xxx (2015) xxx–xxx
Fig. 6. URB602 protects the CN neurons from Hcy insult using Hoechst staining. (A) Representative Hoechst staining images of the CN neurons in vehicle control, Hcy (100 μM), URB (10 μM) + Hcy, and SR1 (10 μM) + URB + Hcy. The CN neurons in culture were treated with Hcy in the absence and presence of URB or SR1 for 12 h. (B) Percentages of apoptotic neurons under different treatments. Values are expressed as mean ± SEM of three independent experiments. **P b 0.01, compared with the vehicle control; ##P b 0.01, compared with Hcy; ++ P b 0.01, compared with URB + Hcy.
of inflammatory responses by regulating the expression of proinflammatory genes such as iNOS, COX-2, and TNF-α [21]. Suppression of the NF-κB signaling pathway can ameliorate neurotoxicity [18]. In our finding, the elevation of NF-κB was inhibited or eliminated by URB602, suggesting that 2-AG-produced neuroprotective effects are mediated via a CB1R-dependent suppression of NF-κB phosphorylation. This is consistent with previous observations where we demonstrated that 2-AG protected the CN neurons from LPS and Hcy through the CB1R-dependent suppression of NF-κB phosphorylation [14,25]. Mitogen-activated protein kinase (MAPK) signaling pathways, which regulate cellular activities such as differentiation, neuronal apoptosis and survival, have been implicated in the development of many neurodegenerative diseases such as AD [36]. The two main MAPK signaling pathways, the extracellular signal-regulated kinase (ERK) and p38MAPK, are involved in Hcy-induced primary cultured cortical neuronal death through excessive activation of the N-methyl-D-aspartic acid receptor [28]. Hyperphosphorylation of ERK1/2 and p38MAPK molecules ultimately activates the transcription factor NF-κB and production
7
of COX-2 [4]. Our findings demonstrated that Hcy down-regulated, but not up-regulated, the phosphorylation of p38MAPK, and URB602 enhanced this down-regulation effect. This change of p38MAPK signaling was identical with our recent study in which Hcy or LPS downregulated the p38MAPK pathways and 2-AG enhanced this effect further [14,25]. This suggests that the p38MAPK signaling pathway is not involved in Hcy or LPS-induced COX-2 and NF-κB expression in the CN neurons. Nonetheless, in the hippocampus LPS induced the phosphorylation of p38MAPK. The discrepancy in the phosphorylation of p38MAPK may be ascribed to the regional specificity of the CN and hippocampus [41]. In any case, in our study the enhancement of URB602 on Hcy-induced p38MAPK down-regulation demonstrated that URB602 can inhibit phosphorylation of p38MAPK in the CN neurons. However, the difference is that Hcy induced ERK1/2 phosphorylation, which is suppressed by URB602 through a CB1 receptor dependent way in this study. In the cultured primary cortical neurons, activation of the ERK1/ 2 pathway plays a critical role in mediating Hcy dependent neuronal cell death [29]. These results were different from Yan et al.'s [39] study, they found that Hcy decreased ERK1/2 protein phosphorylation and neural stem cell proliferation, but it did not induce cell death or apoptosis. The cause of the diversity may be concerned with the fact that the primary CN and cortical neuron do not posess the ability to proliferation while neural stem cell does. Our recent study also revealed that 2-AG binding to CB1 receptor inhibited LPS and Hcy-induced ERK1/2 phosphorylation [14,25]. In the present study, our findings also showed that the expression of apoptosis regulatory proteins, cleaved caspase-3 and p-Bcl-2 was altered by Hcy, while URB602 counteracted this alteration in a CB1receptor dependent way. A previous finding implicated that Hcy induces neuronal apoptosis by other mechanisms such as excitoxicity, oxidative and metabolic insults in the hippocampal neurons [2]. In our paper the expression of pro-apoptotic caspase-3 activation and antiapoptosis p-Bcl-2 inhibition participated in the apoptosis of neurons by Hcy. Previous studies reported that Hcy did not induce apoptosis in neural progenitor cells and neuronal stem cells [39]. The reason about the diversity may be that we use the primary CN neurons while they chose neural stem cells, while in cultured primary cortical neurons, activation of the ERK1/2 pathway plays a critical role in mediating Hcydependent neuronal cell death [29]. Interestingly, the initial activation of p38MAPK is downstream of, and dependent on activation of ERK1/2 in primary cortical neuronal cultures [28]. Our discovery revealed that the exposure of URB602 alleviated Hcy-induced CN neuronal death, which was achieved by indirectly activating CB1R through increasing 2-AG levels. 5. Conclusion Our results demonstrate that URB602 exerted potent antiinflammatory effects on Hcy-stimulated CN neurons. The COX-2, NF-κB and ERK1/2 MAPK signaling pathways were largely involved in URB602-mediated modulation of inflammatory mediators via a CB1-receptor-dependent way. Due to the promising potential of MAGL to regulate Hcy mediated neuroinflammatory events, URB602 could be further explored for its beneficial role in suppressing neuroinflammation. Hence, our findings may provide new insight for the development of therapeutics from suppression of MAGL activity to action against inflammation in neuroinflammatory diseases, such as AD. Conflict of interest statement The authors declare no conflict of interest.
Acknowledgments This work was supported by the National Natural Science Foundation of China (No. 30970930).
Please cite this article as: M. Dong, et al., Monoacylglycerol lipase inhibitor protects primary cultured neurons against homocysteine-induced impairments in rat caudate nucleus through COX-2 signaling, Life Sci (2015), http://dx.doi.org/10.1016/j.lfs.2015.03.006
8
M. Dong et al. / Life Sciences xxx (2015) xxx–xxx
References [1] J.A. Araya, A.E. Ramírez, D. Figueroa-Aroca, G.J. Sotes, C. Pérez, J. Becerra, et al., Modulation of neuronal nicotinic receptor by quinolizidine alkaloids causes neuroprotection on a cellular Alzheimer model, J. Alzheimers Dis. 42 (1) (2014) 143-55e. [2] A. Ataie, R. Ataee, M. Shadifar, S. Shahabi, S.M. Aghajanpour, Y. Hosseinpour, Interaction of memantine with homocysteine on the apoptosis in the rat hippocampus cells, Int. J. Mol. Cell Med. 1 (3) (2012) 145–152. [3] B.K. Atwood, A. Straiker, K. Mackie, CB2 cannabinoid receptors inhibit synaptic transmission when expressed in cultured autaptic neurons, Neuropharmacology 63 (4) (2012) 514–523. [4] Y.P. Bai, Y.H. Liu, J. Chen, T. Song, Y. You, Z.Y. Tang, et al., Rosiglitazone attenuates NF-kappaB-dependent ICAM-1 and TNF-alpha production caused by homocysteine via inhibiting ERK1/2/p38MAPK activation, Biochem. Biophys. Res. Commun. 360 (1) (2007) 20–26. [5] T. Bisogno, F. Howell, G. Williams, A. Minassi, M.G. Cascio, A. Ligresti, et al., Cloning of the first sn1-DAG lipases points to the spatial and temporal regulation of endocannabinoid signaling in the brain, J. Cell Biol. 163 (3) (2003) 463–468. [6] J.L. Blankman, G.M. Simon, B.F. Cravatt, A comprehensive profile of brain enzymes that hydrolyze the endocannabinoid 2-arachidonoylglycerol, Chem. Biol. 14 (12) (2007) 1347–1356. [7] A. Brück, R. Portin, A. Lindell, A. Laihinen, J. Bergman, M. Haaparanta, et al., Positron emission tomography shows that impaired frontal lobe functioning in Parkinson's disease is related to dopaminergic hypofunction in the caudate nucleus, Neurosci. Lett. 311 (2) (2001) 81–84. [8] F. Cacciapuoti, Lowering homocysteine levels with folic acid and B-vitamins do not reduce early atherosclerosis, but could interfere with cognitive decline and Alzheimer's disease, J. Thromb. Thrombolysis 36 (3) (2013) 258–262. [9] S. Carloni, D. Alonso-Alconada, S. Girelli, A. Duranti, A. Tontini, D. Piomelli, et al., Pretreatment with the monoacylglycerol lipase inhibitor URB602 protects from the long-term consequences of neonatal hypoxic–ischemic brain injury in rats, Pediatr. Res. 72 (4) (2012) 400–406. [10] J. Chen, Y. Zhou, S. Mueller-Steiner, L.F. Chen, H. Kwon, S. Yi, et al., SIRT1 protects against microglia-dependent amyloid-beta toxicity through inhibiting NF-kappaB signaling, J. Biol. Chem. 280 (48) (2005) 40364–40374. [11] R. Chen, J. Zhang, Y. Wu, D. Wang, G. Feng, Y.P. Tang, et al., Monoacylglycerol lipase is a therapeutic target for Alzheimer's disease, Cell Rep. 2 (5) (2012) 1329–1339. [12] X. Chen, J. Zhang, C. Chen, Endocannabinoid 2-arachidonoylglycerol protects neurons against β-amyloid insults, Neuroscience 178 (2011) 159–168. [13] B. Deweer, S. Lehéricy, B. Pillon, M. Baulac, J. Chiras, C. Marsault, et al., Memory disorders in probable Alzheimer's disease: the role of hippocampal atrophy as shown with MRI, J. Neurol. Neurosurg. Psychiatry 58 (5) (1999) 590–597. [14] M. Dong, Y. Lu, Y. Zha, et al., Endocannabinoid 2-arachidonylglycerol protects primary cultured neurons against homocysteine-induced impairments in rat caudate nucleus through CB1 receptor, J. Mol. Neurosci. 55 (2) (2015) 500–508. [15] B.E. Dwyer, A.K. Raina, G. Perry, M.A. Smith, Homocysteine and Alzheimer's disease: a modifiable risk? Free Radic. Biol. Med. 36 (11) (2004) 1471–1475. [16] M. Gallucci, A. Zanardo, M. Bendini, F. Di Paola, P. Boldrini, E. Grossi, Serum folate, homocysteine, brain atrophy, and auto-CM system: the Treviso Dementia (TREDEM) study, J. Alzheimers Dis. 38 (3) (2014) 581–587. [17] H. Guo, X. Song, M.H. Schmidt, R. Vandorpe, Z. Yang, E. LeBlanc, J. Zhang, et al., Evaluation of whole brain health in aging and Alzheimer’s disease: a standard procedure for scoring an MRI-based brain atrophy and lesion index, J. Alzheimers Dis. 42 (2) (2014) 691–703. [18] M. Hüll, B. Müksch, R.S. Akundi, A. Waschbisch, J.J. Hoozemans, Amyloid beta peptide (25–35) activates protein kinase C leading to cyclooxygenase-2 induction and prostaglandin E2 release in primary midbrain astrocytes, Neurochemistry 48 (8) (2006) 663–672. [19] S. Jiji, K.A. Smitha, A.K. Gupta, V.P. Pillai, R.S. Jayasree, Segmentation and volumetric analysis of the caudate nucleus in Alzheimer's disease, Eur. J. Radiol. 82 (9) (2013) 1525–1530. [20] P.K. Kamat, A. Kalani, S. Givvimani, P.B. Sathnur, S.C. Tyagi, N. Tyagi, Hydrogen sulfide attenuates neurodegeneration and neurovascular dysfunction induced
[21] [22]
[23]
[24]
[25]
[26] [27]
[28]
[29]
[30]
[31] [32]
[33] [34]
[35] [36]
[37]
[38]
[39]
[40] [41] [42]
by intracerebral-administered homocysteine in mice, Neuroscience 252 (2013) 302–319. T. Krakauer, Molecular therapeutic targets in inflammation: cyclooxygenase and NF-kappaB, Curr. Drug Targets Inflamm. Allergy 3 (3) (2004) 317–324. Y.S. Lee, S.J. Lee, K.W. Seo, J.U. Bae, S.Y. Park, C.D. Kim, Homocysteine induces COX-2 expression in macrophages through ROS generated by NMDA receptor–calcium signaling pathways, Free Radic. Res. 47 (5) (2013) 422–431. J.G. Li, J. Chu, C. Barrero, S. Merali, D. Praticò, Homocysteine exacerbates β-amyloid pathology, tau pathology, and cognitive deficit in a mouse model of Alzheimer disease with plaques and tangles, Ann. Neurol. 75 (6) (2014) 851–863. M.H. Li, J.P. Tang, P. Zhang, X. Li, C.Y. Wang, H.J. Wei, et al., Disturbance of endogenous hydrogen sulfide generation and endoplasmic reticulum stress in hippocampus are involved in homocysteine-induced defect in learning and memory of rats, Behav. Brain Res. 262 (2014) 35–41. Y. Lu, F. Peng, M. Dong, H. Yang, Endocannabinoid 2-arachidonylglycerol protects primary cultured neurons against LPS-induced impairments in rat caudate nucleus, J. Mol. Neurosci. 54 (1) (2014) 49–58. J.W. Miller, Homocysteine and Alzheimer's disease, Nutr. Rev. 57 (4) (1999) 126–129. M.S. Morris, J. Selhub, P.F. Jacques, Vitamin B-12 and folate status in relation to decline in scores on the mini-mental state examination in the Framingham heart study, J. Am. Geriatr. Soc. 60 (8) (2012) 1457–1464. R. Poddar, S. Paul, Novel crosstalk between ERK MAPK and p38 MAPK leads to homocysteine–NMDA receptor-mediated neuronal cell death, J. Neurochem. 124 (4) (2013) 558–570. R. Poddar, S. Paul, Homocysteine–NMDA receptor-mediated activation of extracellular signal-regulated kinase leads to neuronal cell death, J. Neurochem. 110 (3) (2009) 1095–1106. N.S. Ryan, S. Keihaninejad, T.J. Shakespeare, M. Lehmann, S.J. Crutch, I.B. Malone, et al., Magnetic resonance imaging evidence for presymptomatic change in thalamus and caudate in familial Alzheimer's disease, Brain 136 (2013) 1399–1414. R.J. Schulz, Homocysteine is a biomarker for cognitive dysfunction in the elderly, Curr. Opin Clin. Nutr. Metab. Care 10 (6) (2007) 718–723. S. Seshadri, A. Beiser, J. Selhub, P.F. Jacques, I.H. Rosenberg, R.B. D'Agostino, et al., Plasma homocysteine as a risk factor for dementia and Alzheimer's disease, N. Engl. J. Med. 346 (7) (2002) 476–483. L. Steinman, Nuanced roles of cytokines in three major human brain disorders, J. Clin. Invest. 118 (11) (2008) 3557–3563. T. Sugiura, S. Kishimoto, S. Oka, M. Gokoh, Biochemistry, pharmacology and physiology of 2-arachidonoylglycerol, an endogenous cannabinoid receptor ligand, Prog. Lipid Res. 45 (2006) 405–446. F. Van Dam, W.A. Van Gool, Hyperhomocysteinemia and Alzheimer's disease: a systematic review, Arch. Gerontol. Geriatr. 48 (3) (2009) 425–430. S. Wang, C. Zhang, X. Sheng, X. Zhang, B. Wang, G. Zhang, Peripheral expression of MAPK pathways in Alzheimer's and Parkinson's diseases, J. Clin. Neurosci. 21 (5) (2014) 810–814. J. Wiskerke, C. Irimia, B.F. Cravatt, T.J. De Vries, A.N. Schoffelmeer, T. Pattij, et al., Characterization of the effects of reuptake and hydrolysis inhibition on interstitial endocannabinoid levels in the brain: an in vivo microdialysis study, ACS Chem. Neurosci. 3 (5) (2012) 407–417. N. Wu, Y.L. Siow, K. O, Induction of hepatic cyclooxygenase-2 by hyperhomocysteinemia via nuclear factor-kappaB activation, Am. J. Physiol. Regul. Integr. Comp. Physiol. 297 (4) (2009) R1086–R1094. H. Yan, X. Zhang, S. Luo, H. Liu, X. Wang, Y. Gao, et al., Effects of homocysteine on ERK signaling and cell proliferation in fetal neural stem cells in vitro, Cell Biochem. Biophys. 66 (1) (2013) 131–137. H. Yang, C. Chen, Cyclooxygenase-2 in synaptic signaling, Curr. Pharm. Des. 14 (14) (2008) 1443–1451. J. Zhang, C. Chen, Endocannabinoid 2-arachidonoylglycerol protects neurons by limiting COX-2 elevation, J. Biol. Chem. 283 (33) (2008) 22601–22611. E.R. Zimmer, A. Leuzy, A.L. Benedet, J. Breitner, S. Gauthier, P. Rosa-Neto, Tracking neuroinflammation in Alzheimer's disease: the role of positron emission tomography imaging, J. Neuroinflammation 11 (2014) 120.
Please cite this article as: M. Dong, et al., Monoacylglycerol lipase inhibitor protects primary cultured neurons against homocysteine-induced impairments in rat caudate nucleus through COX-2 signaling, Life Sci (2015), http://dx.doi.org/10.1016/j.lfs.2015.03.006