Metab Brain Dis DOI 10.1007/s11011-014-9622-4
RESEARCH ARTICLE
Induction of DJ-1 protects neuronal cells from isoflurane induced neurotoxicity Wenjie Liu & Qulian Guo & Xiaoling Hu & Liangyu Peng & Bin Zhou
Received: 17 July 2014 / Accepted: 1 October 2014 # Springer Science+Business Media New York 2014
Abstract Oxidative stress, mitochondrial dysfunction and neuronal apoptosis are thought to be major contributors of Isoflurane toxicity. However, the underlying mechanisms remain largely to be determined. DJ-1, a protein that is involved in the response to various kinds of stress, has shown its neuroprotective effects. Whether DJ-1 has a neuroprotective effect against isoflurane-induced neurotoxocity is still unknown. In this study, we found that expression of DJ-1 is elevated in response to isoflurane treatment in human SHSY5Y neuroblastoma cells. In order to clarify whether DJ-1 plays a potential role in isoflurane neurotoxicity or as a compensatory response for survival, we investigated the effects of DJ-1 silencing in isoflurane neurotoxicity. Our findings indicate that knockdown of DJ-1 promotes isoflurane-induced oxidative stress and mitochondrial dysfunction. Importantly, DJ-1 silencing was found to exacerbate isoflurane- induced apoptosis through modulation of mitochondria-dependent apoptosis pathways, thereby suggesting that induction of DJ-1 in response to isoflurane might act as a compensatory response for cell survival.
Keywords Anesthetic . Isoflurane . DJ-1 . Mitochondria . Oxidative stress . Apoptosis
W. Liu : Q. Guo (*) Department of Anesthesiology, Xiangya Hospital, Central South University, 87 Xiangya Road, 410008 Changsha, Hunan, China e-mail: [email protected] W. Liu : X. Hu : L. Peng : B. Zhou Department of Anesthesiology, The First affiliated Hospital of University of South China, Hengyang 421001, China
Introduction Anesthetic and anticonvulsant drugs have been reported to cause widespread apoptotic neurodegeneration in the developing brains and persistent cognitive impairment in animals. Isoflurane is one of the widely used inhalation anesthetics, which has been reported to impair the cognitive function in the rodent models (Rammes et al. 2009). On one hand, the use of isoflurane causes structural and functional changes in the central nervous system, thereby leads to the cognitive impairments. On the other hand, exposure to isoflurane leads to the exacerbation of oxidative stress (Shen et al. 2014). Thirdly, isoflurane has also been shown to induce neuroapoptosis in both developing animal brains and primary neuronal cells by activating the caspase-3 and changing the ratio of the Bcl/Bax in the hippocampus of the aged rats (Kong et al. 2013). Increased caspase-3 activity has been associated with impairment in the synaptic plasticity and consequent deficits in the cognitive function. In this regard, oxidative stress, dysfunction of mitochondria and apoptosis may contribute to isoflurane anesthesia- induced cognitive deficits in the brains. DJ-1, a product of DJ-1/PARK7 gene, is a small 20-kDa protein. It is highly conserved across diverse species (Bandyopadhyay and Cookson 2004). As a matter of fact, DJ-1 was originally identified as an oncogene protein. DJ-1 has multifunctional features, however, the most striking and consistent findings about DJ-1 are its involvement in the response to various kinds of stress (Cookson 2010). Multiple lines of evidence have shown that DJ-1 is capable to protect cells against stress through multiple pathways including gene transcription regulation, protein stabilization, signal transduction and reactive oxygen species (ROS) elimination (Nagakubo et al. 1997). Notably, it has been found that oxidative stress induces rapid re-localization of DJ-1 to mitochondria (Junn et al. 2009). Consistently, a recent study has reported that wild-type DJ-1 translocates to mitochondria and
Metab Brain Dis
binds to Bcl-XL in response to UVB irradiation and inhibits Bcl-XL rapid degradation and mitochondrial apoptosis pathway induced by UVB irradiation (Ren et al. 2011), suggesting that mitochondria could be a site of neuroprotective action for DJ-1. Interestingly, DJ-1 has been involved in familial early onset PD as its deletion mutants or point mutations (Moore et al. 2005). Wild-type DJ-1 but not DJ-1 mutant shows its capability of protecting cells from cytotoxicity caused by oxidative stress. In contrast, in vivo and in vitro studies have displayed that DJ-1 deficiency caused increased cellular vulnerability to oxidative insults (Kim et al. 2005). However, whether DJ-1 has a neuroprotective effect against isofluraneinduced neurotoxocity is still unknown.
Materials and methods Cell cultures, treatment, and transfections Human SH-SY5Y neuroblastoma cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) containing 10 % fetal bovine serum, 1 % penicillin and streptomycin at 37 °C in a humidified atmosphere of 5 % CO2. Cells were exposed to 1, 2, or 3 % isoflurane for 36 h in one chamber as described previously (Johnsen and Murphy 2011). The siRNA sequence targeting both DJ-1 and a random sequence of annealed oligonucleotides was transfected into SH-SY5Y cells using Lipofectamine 2,000 reagent (Invitrogen) in accordance with the manufacturer’s instructions. Reactive oxygen species (ROS) assay ROS levels were measured using 2′, 7′-dichlorofluorescein diacetate (Sigma, USA) according to the method described previously (Sheng et al. 2009). Briefly, after indicated infection and treatment, cells were washed for 3 times with PBS followed by pretreated with 100 μM 2′, 7′-dichlorofluorescein diacetate for 30 min at 37 °C in darkness. Then 2′, 7′Dichlorofluorescein diacetate was removed, and 2′, 7′Dichlorofluorescein fluorescence signal was recorded by a fluorescence microscope. Mitochondrial membrane potential (MMP) determination Fluorescent probes for monitoring mitochondrial membrane potential are frequently used for assessing mitochondrial function. Here, the fluorescence dye tetramethylrhodamine methyl ester (TMRM) was used to assay MMP according to the manual instructions. Briefly, cells were loaded with 10 nmol/ L TMRM (Invitrogen, USA) for 60 min at 37 °C in darkness. Then the dye TMRM was removed and the cells were washed for three times with PBS. The level of MMP was indexed by
the average fluorescence intensity recorded using a fluorescence microscope. Western blot analysis Cell lysates were prepared by using cell lysis buffer (Cell signaling, USA). 20 μg of total protein extract in each group was subject to 10 % SDS-PAGE and was electrotransfered onto Immobilon-P Membrane (Millipore, USA) according to the method described previously (Zhou et al. 2008) Transferred membranes were blocked in TBS containing 10 % non-fat dry milk and 0.5 % Tween-20 for 2 h at room temperature (RT), followed by probed with the indicated primary antibodies overnight at 4 °C and with the secondary antibodies for 2 h at RT. Immunoreactive bands were visualized by Immobilon Western Chemiluminescent HRP Substrate (Millipore, USA). Cytochrome C assay After indicated treatment, cells were incubated with ice-cold cytosolic extraction buffer (250 mM sucrose, 20 mM Hepes pH 7.4, 10 mM KCl, 1 mM EGTA, 1 mM EDTA, 1 mM MgCl2, 1 mM dithiothreitol, 1 mM phenylmethylsulphonyl fluoride, 1 mM benzamidine, 1 mM pepstatin A, 10 mg/mL leupeptin, and 2 mg/mL aprotonin) for 30 min on ice. Cells were collected and a glass of Dounce homogenizer was then used to homogenize cell suspensions with a tight-fitting pestle. Then samples were centrifuged at 2,500 rpm for 10 min at 4 °C, and the supernatant was collected and used for another centrifugation at 13,000 rpm for 30 min at 4 °C. The yielded cytosolic extract was used for western blot analysis to determine the levels of cytochrome C levels in the cytosol. Mitochondrial cytochrome C oxidase subunit IV (COX 4) was used as a negative control. Lactate dehydrogenase (LDH) release determination Release of lactate dehydrogenase (LDH) from damaged cells was assayed by using a commercial kit according to the manual instructions. Absorbance captured at 490 nm was used to quantify the amount of LDH. The percentage of cell death was characterized by the ratio of LDH activity in the supernatant to the total LDH activity outlined by the manufacturer’s instructions. Determination of adenosine triphosphate (ATP) levels via bioluminescence assay The level of ATP in SH-SY5Y cell lines was assayed by an adenosine triphosphate (ATP) bioluminescence assay kit following the manufacturer’s instructions. Briefly, after indicated treatment and transfection, cells were lysed and centrifuged at
Metab Brain Dis
10,000×g for 10 min at 4 °C. 100 μl of supernatant and luciferase reagent were mixed together in darkness, which catalyzed the light production from ATP and luciferin. Signals were recorded by a microplate luminometer to reflect ATP concentration. 4-hydroxy-2-nonenal (4-HNE) immunofluorescence staining The levels of 4-HNE were determined by the immunofluorescence staining method. Briefly, cells were fixed in 4 % paraformaldehyde for 10 min at room tempreture (RT) followed by permeabilization with 0.4 % Triton X-100 on ice for 15 min. Then cells were blocked with 5 % bovine serum albumin and Fig. 1 Isoflurane treatment increased expression of DJ-1 in SH-SY5Y cells. SHSY5Y cells were stimulated with isoflurane at various concentrations (1, 2, 3 %) for 36 h. a mRNA levels of DJ-1 at various concentrations were determined by real time PCR. b Protein levels of DJ-1 at various concentrations were determined by western blot analysis (*p
RESEARCH ARTICLE
Induction of DJ-1 protects neuronal cells from isoflurane induced neurotoxicity Wenjie Liu & Qulian Guo & Xiaoling Hu & Liangyu Peng & Bin Zhou
Received: 17 July 2014 / Accepted: 1 October 2014 # Springer Science+Business Media New York 2014
Abstract Oxidative stress, mitochondrial dysfunction and neuronal apoptosis are thought to be major contributors of Isoflurane toxicity. However, the underlying mechanisms remain largely to be determined. DJ-1, a protein that is involved in the response to various kinds of stress, has shown its neuroprotective effects. Whether DJ-1 has a neuroprotective effect against isoflurane-induced neurotoxocity is still unknown. In this study, we found that expression of DJ-1 is elevated in response to isoflurane treatment in human SHSY5Y neuroblastoma cells. In order to clarify whether DJ-1 plays a potential role in isoflurane neurotoxicity or as a compensatory response for survival, we investigated the effects of DJ-1 silencing in isoflurane neurotoxicity. Our findings indicate that knockdown of DJ-1 promotes isoflurane-induced oxidative stress and mitochondrial dysfunction. Importantly, DJ-1 silencing was found to exacerbate isoflurane- induced apoptosis through modulation of mitochondria-dependent apoptosis pathways, thereby suggesting that induction of DJ-1 in response to isoflurane might act as a compensatory response for cell survival.
Keywords Anesthetic . Isoflurane . DJ-1 . Mitochondria . Oxidative stress . Apoptosis
W. Liu : Q. Guo (*) Department of Anesthesiology, Xiangya Hospital, Central South University, 87 Xiangya Road, 410008 Changsha, Hunan, China e-mail: [email protected] W. Liu : X. Hu : L. Peng : B. Zhou Department of Anesthesiology, The First affiliated Hospital of University of South China, Hengyang 421001, China
Introduction Anesthetic and anticonvulsant drugs have been reported to cause widespread apoptotic neurodegeneration in the developing brains and persistent cognitive impairment in animals. Isoflurane is one of the widely used inhalation anesthetics, which has been reported to impair the cognitive function in the rodent models (Rammes et al. 2009). On one hand, the use of isoflurane causes structural and functional changes in the central nervous system, thereby leads to the cognitive impairments. On the other hand, exposure to isoflurane leads to the exacerbation of oxidative stress (Shen et al. 2014). Thirdly, isoflurane has also been shown to induce neuroapoptosis in both developing animal brains and primary neuronal cells by activating the caspase-3 and changing the ratio of the Bcl/Bax in the hippocampus of the aged rats (Kong et al. 2013). Increased caspase-3 activity has been associated with impairment in the synaptic plasticity and consequent deficits in the cognitive function. In this regard, oxidative stress, dysfunction of mitochondria and apoptosis may contribute to isoflurane anesthesia- induced cognitive deficits in the brains. DJ-1, a product of DJ-1/PARK7 gene, is a small 20-kDa protein. It is highly conserved across diverse species (Bandyopadhyay and Cookson 2004). As a matter of fact, DJ-1 was originally identified as an oncogene protein. DJ-1 has multifunctional features, however, the most striking and consistent findings about DJ-1 are its involvement in the response to various kinds of stress (Cookson 2010). Multiple lines of evidence have shown that DJ-1 is capable to protect cells against stress through multiple pathways including gene transcription regulation, protein stabilization, signal transduction and reactive oxygen species (ROS) elimination (Nagakubo et al. 1997). Notably, it has been found that oxidative stress induces rapid re-localization of DJ-1 to mitochondria (Junn et al. 2009). Consistently, a recent study has reported that wild-type DJ-1 translocates to mitochondria and
Metab Brain Dis
binds to Bcl-XL in response to UVB irradiation and inhibits Bcl-XL rapid degradation and mitochondrial apoptosis pathway induced by UVB irradiation (Ren et al. 2011), suggesting that mitochondria could be a site of neuroprotective action for DJ-1. Interestingly, DJ-1 has been involved in familial early onset PD as its deletion mutants or point mutations (Moore et al. 2005). Wild-type DJ-1 but not DJ-1 mutant shows its capability of protecting cells from cytotoxicity caused by oxidative stress. In contrast, in vivo and in vitro studies have displayed that DJ-1 deficiency caused increased cellular vulnerability to oxidative insults (Kim et al. 2005). However, whether DJ-1 has a neuroprotective effect against isofluraneinduced neurotoxocity is still unknown.
Materials and methods Cell cultures, treatment, and transfections Human SH-SY5Y neuroblastoma cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) containing 10 % fetal bovine serum, 1 % penicillin and streptomycin at 37 °C in a humidified atmosphere of 5 % CO2. Cells were exposed to 1, 2, or 3 % isoflurane for 36 h in one chamber as described previously (Johnsen and Murphy 2011). The siRNA sequence targeting both DJ-1 and a random sequence of annealed oligonucleotides was transfected into SH-SY5Y cells using Lipofectamine 2,000 reagent (Invitrogen) in accordance with the manufacturer’s instructions. Reactive oxygen species (ROS) assay ROS levels were measured using 2′, 7′-dichlorofluorescein diacetate (Sigma, USA) according to the method described previously (Sheng et al. 2009). Briefly, after indicated infection and treatment, cells were washed for 3 times with PBS followed by pretreated with 100 μM 2′, 7′-dichlorofluorescein diacetate for 30 min at 37 °C in darkness. Then 2′, 7′Dichlorofluorescein diacetate was removed, and 2′, 7′Dichlorofluorescein fluorescence signal was recorded by a fluorescence microscope. Mitochondrial membrane potential (MMP) determination Fluorescent probes for monitoring mitochondrial membrane potential are frequently used for assessing mitochondrial function. Here, the fluorescence dye tetramethylrhodamine methyl ester (TMRM) was used to assay MMP according to the manual instructions. Briefly, cells were loaded with 10 nmol/ L TMRM (Invitrogen, USA) for 60 min at 37 °C in darkness. Then the dye TMRM was removed and the cells were washed for three times with PBS. The level of MMP was indexed by
the average fluorescence intensity recorded using a fluorescence microscope. Western blot analysis Cell lysates were prepared by using cell lysis buffer (Cell signaling, USA). 20 μg of total protein extract in each group was subject to 10 % SDS-PAGE and was electrotransfered onto Immobilon-P Membrane (Millipore, USA) according to the method described previously (Zhou et al. 2008) Transferred membranes were blocked in TBS containing 10 % non-fat dry milk and 0.5 % Tween-20 for 2 h at room temperature (RT), followed by probed with the indicated primary antibodies overnight at 4 °C and with the secondary antibodies for 2 h at RT. Immunoreactive bands were visualized by Immobilon Western Chemiluminescent HRP Substrate (Millipore, USA). Cytochrome C assay After indicated treatment, cells were incubated with ice-cold cytosolic extraction buffer (250 mM sucrose, 20 mM Hepes pH 7.4, 10 mM KCl, 1 mM EGTA, 1 mM EDTA, 1 mM MgCl2, 1 mM dithiothreitol, 1 mM phenylmethylsulphonyl fluoride, 1 mM benzamidine, 1 mM pepstatin A, 10 mg/mL leupeptin, and 2 mg/mL aprotonin) for 30 min on ice. Cells were collected and a glass of Dounce homogenizer was then used to homogenize cell suspensions with a tight-fitting pestle. Then samples were centrifuged at 2,500 rpm for 10 min at 4 °C, and the supernatant was collected and used for another centrifugation at 13,000 rpm for 30 min at 4 °C. The yielded cytosolic extract was used for western blot analysis to determine the levels of cytochrome C levels in the cytosol. Mitochondrial cytochrome C oxidase subunit IV (COX 4) was used as a negative control. Lactate dehydrogenase (LDH) release determination Release of lactate dehydrogenase (LDH) from damaged cells was assayed by using a commercial kit according to the manual instructions. Absorbance captured at 490 nm was used to quantify the amount of LDH. The percentage of cell death was characterized by the ratio of LDH activity in the supernatant to the total LDH activity outlined by the manufacturer’s instructions. Determination of adenosine triphosphate (ATP) levels via bioluminescence assay The level of ATP in SH-SY5Y cell lines was assayed by an adenosine triphosphate (ATP) bioluminescence assay kit following the manufacturer’s instructions. Briefly, after indicated treatment and transfection, cells were lysed and centrifuged at
Metab Brain Dis
10,000×g for 10 min at 4 °C. 100 μl of supernatant and luciferase reagent were mixed together in darkness, which catalyzed the light production from ATP and luciferin. Signals were recorded by a microplate luminometer to reflect ATP concentration. 4-hydroxy-2-nonenal (4-HNE) immunofluorescence staining The levels of 4-HNE were determined by the immunofluorescence staining method. Briefly, cells were fixed in 4 % paraformaldehyde for 10 min at room tempreture (RT) followed by permeabilization with 0.4 % Triton X-100 on ice for 15 min. Then cells were blocked with 5 % bovine serum albumin and Fig. 1 Isoflurane treatment increased expression of DJ-1 in SH-SY5Y cells. SHSY5Y cells were stimulated with isoflurane at various concentrations (1, 2, 3 %) for 36 h. a mRNA levels of DJ-1 at various concentrations were determined by real time PCR. b Protein levels of DJ-1 at various concentrations were determined by western blot analysis (*p
