J Mol Neurosci DOI 10.1007/s12031-014-0422-4
Cannabinoid Receptor CB1 Is Involved in Nicotine-Induced Protection Against Aβ1–42 Neurotoxicity in HT22 Cells Mingchun Wu & Ji Jia & Chong Lei & Ling Ji & Xiaodan Chen & Hanfei Sang & Lize Xiong
Received: 4 August 2014 / Accepted: 8 September 2014 # Springer Science+Business Media New York 2014
Abstract Emerging evidences suggest that nicotine exerts a neuroprotective effect on Alzheimer’s disease (AD), yet the precise mechanism is not fully elucidated. Here, HT22 cells were exposed to amyloid beta protein fragment (Aβ)1–42 to mimic the pathological process of neuron in AD. We hypothesized that cannabinoid receptor CB1 is involved in the nicotine-induced neuroprotection against Aβ1–42 injury in HT22 cells. CB1 expression in HT22 cells was investigated by immunocytochemistry and Western blot. The injury of HT22 cells was evaluated by cellular morphology, cell viability, and lactate dehydrogenase (LDH) release. The apoptosis of HT22 cells was assessed by flow cytometry and expressions of Bcl-2 and Bax. The results demonstrated that nicotine markedly upregulated CB1 expression, increased cell viability, ameliorated cellular morphology, decreased LDH release, and reduced the apoptotic rate of HT22 cells exposed to Aβ1– 42 for 24 h, while the blockade of CB1 or the inhibition of protein kinase C (PKC) partially reversed the neuroprotection. Furthermore, the blockade of CB1 reversed nicotine-induced PKC activation in HT22 cells exposed to Aβ1–42. These results suggest that CB1 is involved in the nicotine-induced neuroprotection against Aβ1–42 neurotoxicity, and the neuroprotection may be dependent on the activation of PKC. Keywords Nicotine . Alzheimer’s disease . Cannabinoid receptor CB1 . Amyloid β . HT22 cells Mingchun Wu Ji Jia and Chong Lei contributed equally to this work. M. Wu : J. Jia : C. Lei : X. Chen : H. Sang (*) : L. Xiong (*) Department of Anesthesiology, Xijing Hospital, The Fourth Military Medical University, Xi’an 710032, China e-mail: [email protected] e-mail: [email protected] L. Ji Department of Anesthesiology, Tangdu Hospital, The Fourth Military Medical University, Xi’an 710038, China
Abbreviations Aβ Amyloid beta protein fragment AD Alzheimer’s disease ANOVA Analysis of variance BSA Bovine serum albumin CB1 Cannabinoid receptor CB1 CB2 Cannabinoid receptor CB2 DAPI 4′,6-Diamidino-2-phenylindole DMEM Dulbecco’s modified Eagle’s medium DMSO Dimethyl sulfoxide EA Electroacupuncture LDH Lactate dehydrogenase MTT 3-(4,5-Dimethyl-2-thiazolyl)-2,5-dipheny l-2-tetrazolium bromide PBS Phosphate-buffered saline NMDA N-methyl-D-aspartate PKC Protein kinase C
Introduction Some epidemiological evidences indicated that the incidence of Alzheimer’s disease (AD) is lower in smokers than nonsmokers (Lee 1994; Morens et al. 1995). Nicotine is one of the main constituents of tobacco, which can protect neuronal cells against the insults of AD (Yu et al. 2011). Moreover, exposure to nicotine for a short period ameliorated the cognitive performance in AD patients (Ciobica et al. 2012). These findings explained, to some extent, the phenomenon that smokers have a lower incidence of AD. However, the mechanism of nicotine-induced neuroprotection against AD is not fully elucidated. It was reported that cannabinoid receptor CB1 mediated nicotine-induced place preference in rats (Hashemizadeh et al. 2014) and that rimonabant, a selective CB1 antagonist, is an efficacious treatment for nicotine dependence (Le Foll et al. 2008). We also found that CB1 was involved in the nicotine-
J Mol Neurosci
induced neuroprotection against transient brain ischemic injury in rats (Chen et al. 2013a). Yet, no obvious evidence has been found to prove that CB1 could mediate nicotine-induced neuroprotection in AD. Amyloid beta protein fragment (Aβ) is a main biomarker of AD. The levels of soluble Aβ were found to be increased in brains from AD patients and correlated highly with disease severity (Bateman et al. 2012), and overaccumulation of Aβ in brains could result in the injury of neuronal cells (Tan et al. 2013). Thus, reducing the toxicity of Aβ could be a therapeutic target for AD. Aβ1–42 plays a more important role than the other peptides in Aβ family during the development of AD (Ashley et al. 2006). HT22 cell, a mouse hippocampal neuronal cell line, was used widely to investigate the pathophysiological mechanism of AD (Shim and Kwon 2012). Therefore, we took HT22 cells exposed to Aβ1–42 as the cell injury model to mimic pathogenesis of AD. Since we have found that protein kinase C (PKC) was involved in the electroacupuncture (EA)-induced neuroprotection against brain ischemia through CB1 (Wang et al. 2011), some other researchers reported that PKC inhibitor could reverse nicotine-induced neuroprotection against N-methyl- D -
aspartate (NMDA) in hippocampal slices (Ferchmin et al. 2005). However, whether PKC mediates nicotine-induced neuroprotection against Aβ has not been investigated. In this study, we hypothesized that CB1 may be involved in nicotine-induced neuroprotection against Aβ1–42 neurotoxicity in HT22 cells, and the CB1-mediated neuroprotection may be dependent on the activation of PKC.
Fig. 1 Experimental protocol diagram. a The HT22 cells were divided into four groups. Control cells were cultured in drug-free medium, and the other three groups were exposed to 1, 5, and 10 μM Aβ1–42 for 24 h, respectively. MTT assay was taken to measure the cell injury degree. b The HT22 cells were divided into six groups; except for the control and Aβ1–42-only groups, the other four groups were incubated with 5 μM Aβ1–42 plus different concentrations of nicotine (Nico) for 24 h. MTT assay was used to determine the cell viability. c The HT22 cells were divided into five groups, including control, Aβ1–42, Nico+Aβ1–42,
Nico+AM251+Aβ1–42, and CB1 antagonist AM251-only groups. After the incubation for 24 h, MTT assay, LDH release, flow cytometry, and Western blot were used to determine the nicotine-induced protection. d Then, the cells were divided into five groups, including control, Aβ1–42, PKC inhibitor chelerythrine (Che)+Aβ1–42, Nico+Aβ1–42, and Nico+ Che+Aβ1–42 groups. After the incubation, cell morphology, MTT assay, and LDH release were taken to evaluate the role of PKC in nicotineinduced protection
Materials and Methods Materials The HT22 cell line was obtained as a gift from Xuzhou Medical College (Xuzhou, China). The primary anti-CB1 antibody (rabbit anti-mouse) was purchased from Abcam Ltd. (Cambridge, UK), with bovine serum albumin (BSA), and the Cy3-labeled secondary antibody (Goat Anti-Rabbit) was purchased from Beijing Cowin Bioscience Co., Ltd. (Beijing, China). The nicotine, Aβ1–42, Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-
J Mol Neurosci
tetrazolium bromide (MTT), and dimethyl sulfoxide (DMSO) were purchased from Sigma-Aldrich (St. Louis, MO, USA). The 4′,6-diamidino-2-phenylindole (DAPI) was obtained from Beyotime (Nantong, China). The lactate dehydrogenase (LDH) reagent kit was purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). AM251 and chelerythrine were purchased from Tocris Bioscience (Bristol, UK). Cell Culture HT22 cells were cultured in DMEM with 10 % FBS (v/v), 100 U/ml penicillin, and 100 μg/ml streptomycin at 37 °C in a humidified atmosphere containing 5 % CO2 and 95 % air. The medium was replaced every 2 days. When cell density reached roughly 70–80 %, cells were exposed to the indicated drugs for 24 h. Then, some evaluations were performed. Experimental Protocols To find a suitable Aβ1–42 concentration, the cells were divided into four groups (Fig. 1a). The control cells were cultured in normal medium, and the other three groups were exposed to different concentrations of Aβ1–42 for 24 h. Then, MTT assay was taken to determine the cell injury degree. To find a more effective concentration of nicotine, the cells were divided into six groups, including control, Aβ1–42-only, and other four groups treated with 5 μM Aβ1–42 plus different concentrations of nicotine (Fig. 1b). After an incubation of 24 h, MTT assay was performed to evaluate the cell injury degree. The HT22 cells were divided into five groups (Fig. 1c), including control, Aβ1–42, nicotine (Nico)+Aβ1–42, Nico+ AM251+Aβ1–42, and AM251 (CB1 antagonist) groups. After the incubation, MTT assay, LDH release, flow cytometry, and Western blot were taken to investigate the nicotine-induced protection and the potential mechanism. Then, the cells were divided into five groups (Fig. 1d), including control, Aβ1–42, PKC inhibitor chelerythrine (Che)+Aβ1–42, Nico+Aβ1–42, and Nico+Che+Aβ1–42 groups. After the incubation, cell morphology, MTT assay, and LDH release were taken to evaluate the role of PKC in nicotine-induced protection.
Immunocytochemistry HT22 cells were seeded into five confocal microscopy special dishes at a density of 2×104 cells/dish. After 24 h, cells were incubated with various drugs for 24 h, and then the cells were fixed with 4 % paraformaldehyde solution for 1 h and blocked with 50 mg BSA/ml in phosphate-buffered saline (PBS) for 30 min. Cells were incubated with primary anti-CB1 antibody (1:50) overnight at 4 °C before incubation with Cy3-labeled secondary antibody (1:200) for 1 h at room temperature. Two hundred microliters of DAPI staining solution was added into each dish for 5 min, and then, the dishes were washed three times with PBS. CB1 expression was assessed using a confocal microscope (FV10i, Olympus, Japan) (excitation = 550 nm, emission=570 nm). LDH Measurement HT22 cells were plated at a density of 2×104 cells/well into a 24-well plate. After treatments with drugs, the supernatant of each well was removed to measure the level of LDH, which was measured as we previously described (Gou et al. 2011), based on measurement of the activity of LDH released from injured cells into the supernatant.
Cell Viability HT22 cells were plated at a density of 1×104 cells/well in 96well plates. After the treatments, cell viability was evaluated by the MTT assay. Briefly, MTT solution (20 μl, 5 mg/ml) was added into each well, and after 4-h incubation at 37 °C, the supernatant of each well was carefully removed and 150 μl DMSO was added into each well to solubilize the formazan product. The plate was then shaken for 10 min to ensure that the formazan had completely dissolved. Absorbance at 490 nm was evaluated using a spectrophotometer (TECAN, CH).
Fig. 2 Nicotine restored the cell viability of HT22 cells exposed to Aβ1– 42. a HT22 cells were exposed to 1–10 μM Aβ1–42 for 24 h. b The HT22 cells were treated with 1–500 μM nicotine in the presence of 5 μM Aβ1– 42 for 24 h. The cell viability was assessed by MTT assay. Results are expressed as means±SD (n=6). *P
Cannabinoid Receptor CB1 Is Involved in Nicotine-Induced Protection Against Aβ1–42 Neurotoxicity in HT22 Cells Mingchun Wu & Ji Jia & Chong Lei & Ling Ji & Xiaodan Chen & Hanfei Sang & Lize Xiong
Received: 4 August 2014 / Accepted: 8 September 2014 # Springer Science+Business Media New York 2014
Abstract Emerging evidences suggest that nicotine exerts a neuroprotective effect on Alzheimer’s disease (AD), yet the precise mechanism is not fully elucidated. Here, HT22 cells were exposed to amyloid beta protein fragment (Aβ)1–42 to mimic the pathological process of neuron in AD. We hypothesized that cannabinoid receptor CB1 is involved in the nicotine-induced neuroprotection against Aβ1–42 injury in HT22 cells. CB1 expression in HT22 cells was investigated by immunocytochemistry and Western blot. The injury of HT22 cells was evaluated by cellular morphology, cell viability, and lactate dehydrogenase (LDH) release. The apoptosis of HT22 cells was assessed by flow cytometry and expressions of Bcl-2 and Bax. The results demonstrated that nicotine markedly upregulated CB1 expression, increased cell viability, ameliorated cellular morphology, decreased LDH release, and reduced the apoptotic rate of HT22 cells exposed to Aβ1– 42 for 24 h, while the blockade of CB1 or the inhibition of protein kinase C (PKC) partially reversed the neuroprotection. Furthermore, the blockade of CB1 reversed nicotine-induced PKC activation in HT22 cells exposed to Aβ1–42. These results suggest that CB1 is involved in the nicotine-induced neuroprotection against Aβ1–42 neurotoxicity, and the neuroprotection may be dependent on the activation of PKC. Keywords Nicotine . Alzheimer’s disease . Cannabinoid receptor CB1 . Amyloid β . HT22 cells Mingchun Wu Ji Jia and Chong Lei contributed equally to this work. M. Wu : J. Jia : C. Lei : X. Chen : H. Sang (*) : L. Xiong (*) Department of Anesthesiology, Xijing Hospital, The Fourth Military Medical University, Xi’an 710032, China e-mail: [email protected] e-mail: [email protected] L. Ji Department of Anesthesiology, Tangdu Hospital, The Fourth Military Medical University, Xi’an 710038, China
Abbreviations Aβ Amyloid beta protein fragment AD Alzheimer’s disease ANOVA Analysis of variance BSA Bovine serum albumin CB1 Cannabinoid receptor CB1 CB2 Cannabinoid receptor CB2 DAPI 4′,6-Diamidino-2-phenylindole DMEM Dulbecco’s modified Eagle’s medium DMSO Dimethyl sulfoxide EA Electroacupuncture LDH Lactate dehydrogenase MTT 3-(4,5-Dimethyl-2-thiazolyl)-2,5-dipheny l-2-tetrazolium bromide PBS Phosphate-buffered saline NMDA N-methyl-D-aspartate PKC Protein kinase C
Introduction Some epidemiological evidences indicated that the incidence of Alzheimer’s disease (AD) is lower in smokers than nonsmokers (Lee 1994; Morens et al. 1995). Nicotine is one of the main constituents of tobacco, which can protect neuronal cells against the insults of AD (Yu et al. 2011). Moreover, exposure to nicotine for a short period ameliorated the cognitive performance in AD patients (Ciobica et al. 2012). These findings explained, to some extent, the phenomenon that smokers have a lower incidence of AD. However, the mechanism of nicotine-induced neuroprotection against AD is not fully elucidated. It was reported that cannabinoid receptor CB1 mediated nicotine-induced place preference in rats (Hashemizadeh et al. 2014) and that rimonabant, a selective CB1 antagonist, is an efficacious treatment for nicotine dependence (Le Foll et al. 2008). We also found that CB1 was involved in the nicotine-
J Mol Neurosci
induced neuroprotection against transient brain ischemic injury in rats (Chen et al. 2013a). Yet, no obvious evidence has been found to prove that CB1 could mediate nicotine-induced neuroprotection in AD. Amyloid beta protein fragment (Aβ) is a main biomarker of AD. The levels of soluble Aβ were found to be increased in brains from AD patients and correlated highly with disease severity (Bateman et al. 2012), and overaccumulation of Aβ in brains could result in the injury of neuronal cells (Tan et al. 2013). Thus, reducing the toxicity of Aβ could be a therapeutic target for AD. Aβ1–42 plays a more important role than the other peptides in Aβ family during the development of AD (Ashley et al. 2006). HT22 cell, a mouse hippocampal neuronal cell line, was used widely to investigate the pathophysiological mechanism of AD (Shim and Kwon 2012). Therefore, we took HT22 cells exposed to Aβ1–42 as the cell injury model to mimic pathogenesis of AD. Since we have found that protein kinase C (PKC) was involved in the electroacupuncture (EA)-induced neuroprotection against brain ischemia through CB1 (Wang et al. 2011), some other researchers reported that PKC inhibitor could reverse nicotine-induced neuroprotection against N-methyl- D -
aspartate (NMDA) in hippocampal slices (Ferchmin et al. 2005). However, whether PKC mediates nicotine-induced neuroprotection against Aβ has not been investigated. In this study, we hypothesized that CB1 may be involved in nicotine-induced neuroprotection against Aβ1–42 neurotoxicity in HT22 cells, and the CB1-mediated neuroprotection may be dependent on the activation of PKC.
Fig. 1 Experimental protocol diagram. a The HT22 cells were divided into four groups. Control cells were cultured in drug-free medium, and the other three groups were exposed to 1, 5, and 10 μM Aβ1–42 for 24 h, respectively. MTT assay was taken to measure the cell injury degree. b The HT22 cells were divided into six groups; except for the control and Aβ1–42-only groups, the other four groups were incubated with 5 μM Aβ1–42 plus different concentrations of nicotine (Nico) for 24 h. MTT assay was used to determine the cell viability. c The HT22 cells were divided into five groups, including control, Aβ1–42, Nico+Aβ1–42,
Nico+AM251+Aβ1–42, and CB1 antagonist AM251-only groups. After the incubation for 24 h, MTT assay, LDH release, flow cytometry, and Western blot were used to determine the nicotine-induced protection. d Then, the cells were divided into five groups, including control, Aβ1–42, PKC inhibitor chelerythrine (Che)+Aβ1–42, Nico+Aβ1–42, and Nico+ Che+Aβ1–42 groups. After the incubation, cell morphology, MTT assay, and LDH release were taken to evaluate the role of PKC in nicotineinduced protection
Materials and Methods Materials The HT22 cell line was obtained as a gift from Xuzhou Medical College (Xuzhou, China). The primary anti-CB1 antibody (rabbit anti-mouse) was purchased from Abcam Ltd. (Cambridge, UK), with bovine serum albumin (BSA), and the Cy3-labeled secondary antibody (Goat Anti-Rabbit) was purchased from Beijing Cowin Bioscience Co., Ltd. (Beijing, China). The nicotine, Aβ1–42, Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-
J Mol Neurosci
tetrazolium bromide (MTT), and dimethyl sulfoxide (DMSO) were purchased from Sigma-Aldrich (St. Louis, MO, USA). The 4′,6-diamidino-2-phenylindole (DAPI) was obtained from Beyotime (Nantong, China). The lactate dehydrogenase (LDH) reagent kit was purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). AM251 and chelerythrine were purchased from Tocris Bioscience (Bristol, UK). Cell Culture HT22 cells were cultured in DMEM with 10 % FBS (v/v), 100 U/ml penicillin, and 100 μg/ml streptomycin at 37 °C in a humidified atmosphere containing 5 % CO2 and 95 % air. The medium was replaced every 2 days. When cell density reached roughly 70–80 %, cells were exposed to the indicated drugs for 24 h. Then, some evaluations were performed. Experimental Protocols To find a suitable Aβ1–42 concentration, the cells were divided into four groups (Fig. 1a). The control cells were cultured in normal medium, and the other three groups were exposed to different concentrations of Aβ1–42 for 24 h. Then, MTT assay was taken to determine the cell injury degree. To find a more effective concentration of nicotine, the cells were divided into six groups, including control, Aβ1–42-only, and other four groups treated with 5 μM Aβ1–42 plus different concentrations of nicotine (Fig. 1b). After an incubation of 24 h, MTT assay was performed to evaluate the cell injury degree. The HT22 cells were divided into five groups (Fig. 1c), including control, Aβ1–42, nicotine (Nico)+Aβ1–42, Nico+ AM251+Aβ1–42, and AM251 (CB1 antagonist) groups. After the incubation, MTT assay, LDH release, flow cytometry, and Western blot were taken to investigate the nicotine-induced protection and the potential mechanism. Then, the cells were divided into five groups (Fig. 1d), including control, Aβ1–42, PKC inhibitor chelerythrine (Che)+Aβ1–42, Nico+Aβ1–42, and Nico+Che+Aβ1–42 groups. After the incubation, cell morphology, MTT assay, and LDH release were taken to evaluate the role of PKC in nicotine-induced protection.
Immunocytochemistry HT22 cells were seeded into five confocal microscopy special dishes at a density of 2×104 cells/dish. After 24 h, cells were incubated with various drugs for 24 h, and then the cells were fixed with 4 % paraformaldehyde solution for 1 h and blocked with 50 mg BSA/ml in phosphate-buffered saline (PBS) for 30 min. Cells were incubated with primary anti-CB1 antibody (1:50) overnight at 4 °C before incubation with Cy3-labeled secondary antibody (1:200) for 1 h at room temperature. Two hundred microliters of DAPI staining solution was added into each dish for 5 min, and then, the dishes were washed three times with PBS. CB1 expression was assessed using a confocal microscope (FV10i, Olympus, Japan) (excitation = 550 nm, emission=570 nm). LDH Measurement HT22 cells were plated at a density of 2×104 cells/well into a 24-well plate. After treatments with drugs, the supernatant of each well was removed to measure the level of LDH, which was measured as we previously described (Gou et al. 2011), based on measurement of the activity of LDH released from injured cells into the supernatant.
Cell Viability HT22 cells were plated at a density of 1×104 cells/well in 96well plates. After the treatments, cell viability was evaluated by the MTT assay. Briefly, MTT solution (20 μl, 5 mg/ml) was added into each well, and after 4-h incubation at 37 °C, the supernatant of each well was carefully removed and 150 μl DMSO was added into each well to solubilize the formazan product. The plate was then shaken for 10 min to ensure that the formazan had completely dissolved. Absorbance at 490 nm was evaluated using a spectrophotometer (TECAN, CH).
Fig. 2 Nicotine restored the cell viability of HT22 cells exposed to Aβ1– 42. a HT22 cells were exposed to 1–10 μM Aβ1–42 for 24 h. b The HT22 cells were treated with 1–500 μM nicotine in the presence of 5 μM Aβ1– 42 for 24 h. The cell viability was assessed by MTT assay. Results are expressed as means±SD (n=6). *P
