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LanCL1 attenuates ischemia-induced oxidative stress by Sirt3-mediated preservation of mitochondrial function
Brain Research Bulletin 142 (2018) 216–223
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Research report
LanCL1 attenuates ischemia-induced oxidative stress by Sirt3-mediated preservation of mitochondrial function
T
Zhen Xieb, Bing-Qing Caob, Tao Wangb, Qi Leib, Tao Kangb, Chao-Yuan Gea, Wen-Jie Gaoa, ⁎ Hao Huia, a b
Department of Spine Surgery, Honghui Hospital, Xi’an Jiaotong University, No. 555 Youyi East Road, Xi’an, Shaanxi, 710054, China Department 2 of Neurology, Shaanxi Provincial People’s Hospital, Beilin Distinct, Xi’an, Shaanxi, 710068, China
A R T I C LE I N FO
A B S T R A C T
Keywords: LanCL1 Oxygen and glucose deprivation Oxidative stress Sirt3 PGC-1α
Lanthionine synthetase C-like protein 1 (LanCL1) is homologous to prokaryotic lanthionine cyclases, and has been shown to have novel functions in neuronal redox homeostasis. A recent study showed that LanCL1 expression was developmental and activity-dependent regulated, and LanCL1 transgene protected neurons against oxidative stress. In the present study, the potential protective effects of LanCL1 against ischemia was investigated in an in vitro model mimicked by oxygen and glucose deprivation (OGD) in neuronal HT22 cells. We found that OGD exposure induced a temporal increase and persistent decreases in the expression of LanCL1 at both mRNA and protein levels. Overexpression of LanCL1 by lentivirus (LV-LanCL1) transfection preserved cell viability, reduced lactate dehydrogenase (LDH) release and attenuated apoptosis after OGD. These protective effects were accompanied by decreased protein radical formation, lipid peroxidation and mitochondrial dysfunction. In addition, LanCL1 significantly stimulated mitochondrial enzyme activities and SOD2 deacetylation in a Sirt3-dependent manner. The results of western blot analysis showed that LanCL1-induced activation of Sirt3 was dependent on Akt-PGC-1α pathway. Knockdown of PGC-1α expression using small interfering RNA (siRNA) or blocking Akt activation using specific antagonist partially prevented the protective effects of LanCL1 in HT22 cells. Taken together, our results show that LanCL1 protects against OGD through activating the Akt-PGC-1αSirt3 pathway, and may have potential therapeutic value for ischemic stroke.
1. Introduction Despite the decrease in mortality rate (approximately 37%) in the past three decades, stroke is still a leading cause of disability and death worldwide (Lee et al., 2017; Peisker et al., 2017). In the United States, a stroke attack happens every 40 s, and a stroke-associated death occurs every 4 min, making it the second-leading cause of death behind ischemic heart disease (Benjamin et al., 2017). Thus far, many neuroprotective drugs and treatments have failed the clinical translation from experimental models to stroke patients. Tissue plasminogen activator is the only drug approved by the FDA for acute ischemic stroke treatment, but only 4–5% of all stroke patients can benefit from it (Docagne et al., 2015). The lanthionine synthetase C-like protein 1 (LanCL1), originally known as p40/GPR69 A, is one member of the LanC-like protein family, which are homologous to prokaryotic lanthionine synthetase component C (lanthionine cyclases) (Bauer et al., 2000). Prokaryotic lanthionine cyclases act in concert with dehydratases to facilitate
⁎
intramolecular conjugation of cysteine to serine or threonine residues, yielding potent “lantibiotics”, which are not present in mammals (Bierbaum and Sahl, 2009; Chatterjee et al., 2005). Three genes coding for the LanCL proteins are present in the human genome, named LanCL1, LanCL2 and LanCL3 (He et al., 2017). LanCL1 was previously identified as a novel binding partner for the redox regulatory, reduced glutathione (GSH), but has been demonstrated to have no enzymatic activity (Chung et al., 2007; Mayer et al., 2001; Zhang et al., 2009). The LanCL1 protein is highly expressed in the brain, spinal cord and testis, and increased levels of LanCL1 were found in the spinal cord of a mouse model of amyotrophic lateral sclerosis (ALS) (Chung et al., 2007). Recently, LanCL1 was shown to be developmentally regulated by neuronal activity, and confer antioxidant activity that is required for neuronal survival (Huang et al., 2014). However, the primary function of LanCL1 in ischemic stroke has not been determined. In this study, the role of LanCL1 in neuronal ischemia was investigated using an in vitro model mimicked by oxygen and glucose deprivation (OGD) in hippocampal HT22 cells. We also elucidated the potential molecular mechanisms
Corresponding author at: Department of Spine Surgery, Honghui Hospital, Xi’an Jiaotong University, No. 555 Youyi East Road, Xi’an, Shaanxi, 710054, China. E-mail address: [email protected] (H. Hui).
https://doi.org/10.1016/j.brainresbull.2018.07.017 Received 29 April 2018; Received in revised form 3 July 2018; Accepted 4 July 2018 Available online 31 July 2018 0361-9230/ © 2018 Elsevier Inc. All rights reserved.
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2. Materials and methods
secondary antibodies for 1 h at room temperature. Coverslips were mounted in mounting medium and visualized using a fluorescence microscope.
2.1. Cell culture
2.8. Quantification of lipid peroxidation
HT22 cells were purchased from the Institute of Biochemistry and Cell Biology, and cultured in Dulbecco’s modified Eagle’s medium (DMEM) with 10% fetal bovine serum at 37 °C.
Lipid peroxidation was determined by measuring MDA and 4-HNE levels using ELLAS kits (R&D Systems, Minneapolis, MN, USA) according to user’s manual.
2.2. OGD model
2.9. Measurement of ATP synthesis
Ischemia was induced by OGD method in vitro. Briefly, culture medium was replaced by glucose-free DMEM, and the cells were placed into a humidified chamber containing 5% CO2, 95% N2 at 37 °C. After 2, 4 or 6 h challenge, cells were returned to normal medium and normoxic conditions. Cells cultured under normal conditions during the experimental period served as the control.
HT22 cells were subjected to fission and centrifuged at 12 000 g for 5 min. In 24-well plates, 100 μl of each supernatant was mixed with 100 μl ATP working dilution. Luminance was measured using a monochromator microplate reader. The ATP release levels were expressed as a percentage of the luminescence levels in the control cells.
with focus on mitochondrial function and the Sirtuin3 (Sirt3) pathway.
2.10. Measurement of mitochondria swelling 2.3. Cell viability assay Mitochondria swelling was measured following a previously published protocol (Yu et al., 2013). The swelling of mitochondria was monitored by a decrease in absorbance at 540 nm in the presence of CaCl2 (200 μM).
WST-1 colorimetric assay was used to determine the viability of HT22 cells. Cells were seeded in 96-well plates and exposed to OGD. Ten microliters of WST-1 solution was added into each well, and the cells were incubated for another 4 h. Absorbance of each sample was measured at 450 nm with a reference wavelength of 600 nm. The results were expressed as the percentage of control values.
2.11. Mitochondrial enzyme activity The enzyme activities of IDH2, SOD2 and GSH-Px in the mitochondria were determined by commercial assay kits purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, Jiangsu, China), according to the manufacturer’s instructions.
2.4. Lactate dehydrogenase (LDH) release assay The cytotoxicity was determined by measuring the LDH activity in culture medium using a kit according to the manufacturer’s instructions (Pierce, IL, USA). The absorbance values were measured at 490 nm and 650 nm using a microplate reader. The results were expressed as the fold of control values.
2.12. RNA interference The specific siRNA targeted PGC-1α (sc-72151) or Sirt3 (sc-61556), and control siRNA (sc-37007) were purchased from Santa Cruz. The above siRNA molecules were transfected with Lipofectamine 2000 for 48 h before OGD.
2.5. Lentivirus construction and transfection The coding sequence of LanCL1 was amplified by RT-PCR with the following sequences: forward, 5′−CCT TCA GGT GAA CCA AGG AA-3′; reverse, 5′-AGA TCA CGT CAG CAC ACT GC-3′. The PCR fragments and the pGC-FU plasmid were digested with Age I and then ligated with T4 DNA ligase to produce pGC-FU-LanCL1. To generate the recombinant Lentivirus LV-LanCL1, 293 T cells were co-transfected with a pGC-FU plasmid (20 μg) with cDNA encoding LanCL1, pHelper1.0 plasmid (15 μg) and pHelper 2.0 plasmid (10 μg) using Lipofectamine 2000 (100 μl). After 48 h, the supernatant was harvested, and the viral titer was calculated by transducing 293 T cells.
2.13. Quantitative real-time PCR Total RNA was extracted from tissues using TRizol reagent. RNA was subjected to reverse transcription with reverse transcriptase as the manufacturer’s instructions (Fermentas). Quantitative real-time PCR was performed using the Bio-Rad iQ5 gradient real-time PCR system, and the relative gene expression was normalized to internal control as GAPDH. 2.14. Western blot analysis
2.6. TUNEL staining Standard Western blotting procedures were performed with the following antibodies: anti-LanCL1 antibody (1:500, ab229257, Abcam), anti-Sirt3 antibody (1:600, ab86671, Abcam), anti-SOD2 antibody (1:1000, ab13533, Abcam), anti-ac-SOD2 antibody (1:500, ab137037, Abcam), anti-Akt antibody (1:800, #4685, Cell Signaling), anti-p-Akt antibody (1:500, #4060, Cell signaling), anti- PGC-1α antibody (1:800, #2178, Cell signaling), and anti-β-actin antibody (1:1000, #4970, Cell signaling). The Image J software was used to quantify the optical density of each band.
Apoptotic cell death in HT22 cells was detected using the terminal deoxynucleotidy TUNEL staining. After various treatments, cells seeded on slides were washed with PBS, fixed with formaldehyde and permeabilized with 0.2% Tri-ton X-100. Then cells were incubated with 1.5 μM fluorescein isothiocyanate-coupled dUTP and TdT for 1 h in dark. The nucleus was stained with DAPI, and the slides were examined by fluorescence microscopy. 2.7. Immunocytochemistry (ICC)
2.15. Statistical analysis HT22 cells were fixed with formaldehyde and permeabilized with 0.2% Tri-ton X-100. Following two PBS rinses, the cells were incubated with blocking solution for 20 min and incubated with the primary DMPO antibody at 4 °C overnight. The cells were then rinsed with PBS and incubated with fluorescein isothiocyanate (FITC) labelled
Statistical analysis was performed using the GraphPad Prism 6 software. Statistical evaluation of the data was performed by one-way analysis of variance (ANOVA) followed by Newman–Keuls post-test. The differences were considered statistically significant when the value 217
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Fig. 1. Dynamic changes of LanCL1 expression after ischemia in HT22 cells. HT22 cells were exposed to OGD insults of different durations (2, 4 or 6 h) followed by further 24 h reperfusion. The cell viability was measured by WST-1 assay (A), and cytotoxicity was determined by LDH release assay (B). HT22 cells were exposed to 4 h OGD and 24 h reperfusion. The levels of LanCL1 mRNA was determined by RT-PCR (C), and the expression of LanCL1 protein was detected by western blot at different time points (D). Values are the mean ± SEM from five experiments. *p < 0.05 vs. Control.
of p < 0.05.
(Fig. 2E).
3. Results
3.3. LanCL1 inhibits ischemia-induced oxidative stress and mitochondrial dysfunction
3.1. Dynamic changes of LanCL1 expression after ischemia in HT22 cells HT22 cells were transfected with LV-LanCL1 or LV-control for 48 h and exposed to OGD. Immunofluorescence staining was used to detect cytosolic levels of DMPO, a marker of protein radical formation (Fig. 3A). The OGD-induced increase in DMPO fluorescence intensity was significantly reduced by LanCL1 overexpression (Fig. 3B). We also found that LV-LanCL1 transfection inhibited the OGD-induced lipid peroxidation, which was measured by MDA and 4-HNE (Fig. 3C). To further detect mitochondrial dysfunction, we isolated and purified mitochondria from HT22 cells in each group. As shown in Fig. 3D, LanCL1 overexpression preserved ATP generation after OGD. In addition, we measured mitochondrial swelling, which was induced by adding 200 μM Ca2+ into the isolated mitochondria (Fig. 3E). The results showed that OGD-induced mitochondrial swelling (decrease of absorbance) was partially prevented by LV-LanCL1 transfection.
To mimic brain ischemia in vitro, HT22 cells were exposed to OGD of different durations (2, 4 or 6 h) followed by 24 h reperfusion. OGD treatment resulted in significant decrease in cell viability (Fig. 1A) and increase in LDH release (Fig. 1B). Exposure of HT22 cells to 4 h OGD caused nearly half of the cells to die, so 4 h of OGD was used in following experiments. The results of RT-PCR showed that LanCL1 mRNA levels rapidly increased within 3 h after OGD, but significantly decreased from 6 h to 48 h (Fig. 1C). As shown in Fig. 1D, LanCL1 protein levels markedly increased at 3 h, but decreased at 12, 24 and 48 h after OGD treatment. 3.2. LanCL1 attenuates ischemia-induced cytotoxicity and apoptosis To overexpress LanCL1 in vitro, HT22 cells was transfected with LVLanCL1 or LV-control for 48 h, and western blot assay showed that LVLanCL1 transfection led to a significant increase in LanCL1 protein levels (Fig. 2A). The results of cell viability (Fig. 2B) and LDH release (Fig. 2C) showed that LV-LanCL1 transfection had no toxic effects on HT22 cells. However, LV-LanCL1-transfected cell are more resistant to OGD, as evidenced by increased cell viability (Fig. 2B) and decreased LDH release (Fig. 2C) compared to LV-control group. We also detected apoptosis in HT22 cells by TUNEL staining (Fig. 2D), and overexpression of LanCL1 significantly reduced OGD-induced apoptosis
3.4. LanCL1 stimulates Sirt3-mediated mitochondrial enzyme activities and SOD2 deacetylation To determine the effect of LanCL1 on endogenous antioxidant system, the enzymatic activities of IDH2, GSH-Px and SOD2 were measured. As shown in Fig. 4A–C, OGD treatment markedly reduced enzymatic activities of IDH2, GSH-Px and SOD2, whereas LanCL1 overexpression significantly enhanced these enzymatic activities. The results of western blot showed that LV-LanCL1 transfection suppressed 218
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Fig. 2. LanCL1 attenuates ischemia-induced cytotoxicity and apoptosis. HT22 cells were transfected with LV-LanCL1 or LV-control for 48 h, and the expression of LanCL1 was detected by western blot (A). HT22 cells were transfected with LV-LanCL1 or LV-control for 48 h and exposed to OGD. The cell viability was measured by WST-1 assay (B), and cytotoxicity was determined by LDH release assay (C). The apoptotic cell death was detected by TUNEL staining (D), and the apoptotic rate was calculated (E). Scale bars, 50 μm. Values are the mean ± SEM from five experiments. #p < 0.05 vs. Control. *p < 0.05 vs. LV-control.
Fig. 3. LanCL1 inhibits ischemia-induced oxidative stress and mitochondrial dysfunction. HT22 cells were transfected with LV-LanCL1 or LV-control for 48 h and exposed to OGD. The expression of DMPO was detected by immunofluorescence staining (A and B). Lipid peroxidation was determined by measuring MDA and 4HNE levels (C), and ATP generation was measured in isolated mitochondria (D). Mitochondrial swelling was examined by monitoring the absorbance at 540 nm induced by 200 μM Ca2+ (E), and the baseline absorbance was measured without Ca2+. Scale bars, 50 μm. Values are the mean ± SEM from five experiments. #p < 0.05 vs. Control. *p < 0.05 vs. LV-control. 219
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Fig. 4. LanCL1 stimulates Sirt3-mediated mitochondrial enzyme activities and SOD2 deacetylation. HT22 cells were transfected with LV-LanCL1 or LV-control for 48 h with transfection of Si-control or Si-Sirt3 and exposed to OGD. The enzyme activities of IDH2 (A), GSH-Px (B) and SOD2 (C) were determined using the corresponding assay kits, and the expression of Sirt3, ac-SOD2 and SOD2 was detected by western blot (D and E). Values are the mean ± SEM from five experiments. # p < 0.05 vs. Control. *p < 0.05 vs. OGD. &p < 0.05 vs. Si-control.
LanCL1 reduces OGD-induced cytotoxicity and apoptosis; (b) this protection is further supported by inhibition of oxidative stress and mitochondrial dysfunction; (c) LanCL1 stimulates Sirt3 protein expression and its downstream enzyme activity; and (e) mechanistically, the LanCL1-induced protection is partially dependent on the Akt-PGC-1αSirt3 pathway (Fig. 6). Experiments in postnatal rodents and primary cultured neurons showed that the expression of LanCL1 protein in brain was developmentally regulated and paralleled the formation of synapses and spontaneous neuronal activity (Huang et al., 2014). LanCL1 could be induced not only by several neurotrophic factors, including insulin growth factor 1 (IGF-1) and brain-derived neurotrophic factor (BDNF), but also by many neurotoxic agents, such as glutamate and hydrogen peroxide (H2O2). In our in vitro model, the expression of LanCL1 mRNA and protein were measured up to 48 h after OGD. A rapid induction of LanCL1, both at mRNA and protein levels, was observed within 3 h after OGD, which was followed by reduced expression from 6 to 48 h. These temporal increases in mRNA and protein levels are similar to the pattern of typical neuronal immediate early genes (Brakeman et al., 1997; Chen et al., 2017). A previous study using SOD1G93 A transgenic mice model showed that the expression of LanCL1 was increased at presymptomatic stages of ALS (Chung et al., 2007). These dynamic changes of LanCL1 mRNA and protein expression suggest a key role in neurological disorders. In the first few hours after ischemic stroke, many protective signaling cascades, such as antioxidant enzymes, mitochondrial biogenesis and unfolded protein response (UPR), are activated to prevent neuronal cell death (Prentice et al., 2015). If the ischemic stress is so severe that it cannot be overcome, these pro-survival cascades would be inhibited by several other detrimental pathways. Thus, we speculated that LanCL1 might represent one of this kind of endogenous protective mechanisms. We designed a lentivirus expressing exogenous LanCL1, and significant increase in LanCL1 protein expression was observed
the OGD-induced acetylation of SOD2 (Fig. 4D and E). In addition, based on the findings that LanCL1 overexpression increased the expression of Sirt3 protein both with and without OGD exposure (Fig. 4D), the related mechanisms were further investigated via Sirt3 knockdown using specific targeted siRNA (Si-Sirt3). The effects of LVLanCL1 on mitochondrial enzyme activities and SOD2 acetylation were all partially abolished by Sirt3 knockdown. 3.5. Involvement of Sirt3 signaling in LanCL1-induced protection against ischemia In consistent with the increased expression of Sirt3 after LanCL1 overexpression, increased expression of p-Akt and PGC-1α proteins was also observed (Fig. 5A and B), indicating the potential involvement of Akt-PGC-1α-Sirt3 pathway. To further confirm these findings, LY294002 was pretreated to inhibit Akt activity, and PGC-1α targeted siRNA (Si-PGC-1α) was used to knockdown PGC-1α in vitro. The results of western blot showed that LanCL1-induced increase in Sirt3 expression was partially ablated by both LY294002 and Si-PGC-1α transfection (Fig. 5C). In addition, the results of cell viability (Fig. 5D), LDH release (Fig. 5E) and TUNEL staining (Fig. 5F) indicated that LY294002 and PGC-1α knockdown partially reversed the protection induced by LanCL1 overexpression against OGD. 4. Discussion Because of its high expression in organs separated by blood-tissue barriers, such as brain and testis, LanCL1 was previously considered to play a role in the immune surveillance of these organs (Mayer et al., 2001). Recently, LanCL1 was found to exert a novel function in neuronal redox homeostasis (Zhong et al., 2012). Our present study provides evidence that LanCL1 protects against ischemia-induced oxidative stress in neuronal HT22 cells. We found that (a) overexpression of 220
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Fig. 5. Involvement of Sirt3 signaling in LanCL1-induced protection against ischemia. HT22 cells were transfected with LV-LanCL1 in the presence or absence of OGD. The expression of p-Akt, Akt and PGC-1α was detected by western blot (A and B). HT22 cells were transfected with LV-LanCL1 for 48 h with transfection of Sicontrol or Si-Sirt3, or treated with LY294002 in the presence of OGD. The expression of Sirt3 was detected by western blot (C). The cell viability was measured by WST-1 assay (D), and cytotoxicity was determined by LDH release assay (E). The apoptotic cell death was detected by TUNEL staining (F). Values are the mean ± SEM from five experiments. #p < 0.05 vs. Control. *p < 0.05 vs. OGD. &p < 0.05 vs. LV-LanCL1. $p < 0.05 vs. Si-control.
antioxidant enzymes (Giordano et al., 2014; Molenaar et al., 2014). Interestingly, a previous study using LanCL1 targeted siRNA and peptide showed that knockdown of LanCL1 occluded cystathionine β-synthase activation and exerted protective effects in cortical neurons (Zhong et al., 2012). This contradiction might be explained by several factors, such as the differences in cell types, toxic agents used in the experiments and different injury degrees. In consistent with our data, a recent paper showed that LanCL1 transgene could protect neurons from ROS (Huang et al., 2014), indicating the neuron-specific protective role of LanCL1 protein against oxidative stress. Sirt3 is a NAD+-dependent protein deacetylase that is exclusively localized and active in mitochondria. Due to its role in regulating mitochondrial enzyme activities and energy metabolism processes, Sirt3 is well known as a ROS scavenger and anti-apoptotic factor (Ansari et al., 2017). Many recent studies have demonstrated that overexpression of Sirt3 could prevent neuronal derangements in both in vitro and in vivo experimental models of aging and neurological disorders (Anamika et al., 2017; Su et al., 2017). In addition, activation of Sirt3 was shown to mediate the beneficial effects of various protective agents against stroke (Chen et al., 2018; Liu et al., 2017; Su et al., 2017). In the present study, the results of western blot showed that LanCL1 overexpression significantly increased Sirt3 expression after OGD. Sirt3 could directly deacetylate IDH2 and regulate mitochondrial redox status (Yu et al., 2012). Previous studies showed that knockdown of Sirt3 is associated with worsen neurological function due to the declined activity of SOD2 during cerebral ischemia (Anamika et al., 2017; Wang et al., 2015). Thus, we also detected the activities of Sirt3 associated antioxidant enzymes, and enhanced activities of IDH2, GSH-Px and SOD2 were observed after LV-LanCL1 transfection. SOD2 is a major downstream
Fig. 6. A proposed diagram tying together the observations involved in the LanCL1-induced neuroprotection against OGD.
after transfection in HT22 cells. LV-LanCL1 transfected HT22 cells exerted potent tolerance to OGD insult, which was accompanied by reduced oxidative stress and preserved mitochondrial function. Although LanCL1 is not a member of GST superfamily, its catalytic activity resembles that of GST proteins (Sheehan et al., 2001; Zhang et al., 2009). Our results showed that LanCL1 overexpression significantly promotes the enzymatic activities of IDH2, SOD2 and GSH-Px, three important 221
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signal of Sirt3-mediated mitochondrial O2.− reduction and deacetylation of SOD2 by Sirt3 regulates SOD2 enzymatic activity (Tao et al., 2010). Here, both increased ac-SOD2 expression and SOD2 activity were observed in LV-LanCL1-transfected HT22 cells. In addition, these changes in enzymatic activities was partially prevented by Sirt3 knockdown, indicating the involvement of Sirt3 signaling in LanCL1induced protection in our in vitro model. PGC-1α is a transcription co-activator that plays a key role in regulating mitochondrial biogenesis and energy metabolism in multiple organs, including the brain (Schilling and Kelly, 2011; Shoag and Arany, 2010). Previous studies in muscle cells, hepatocytes and neurons found that PGC-1α stimulates Sirt3 expression by binding to the Sirt3 promoter region (Giralt et al., 2011; Park et al., 2012). Silencing of PGC-1α reduced the expression of Sirt3, and overexpression of Sirt3 stimulated PGC-1α and its target UCP1, causing a decrease of ROS in a positive feedback (Kong et al., 2010). In this study, our results using PGC-1α specific siRNA showed that LanCL1-induced regulation in Sirt3 and protection against OGD were both dependent on PGC-1α. There are many protein kinases acting as the upstream factors in regulating PGC1α, such as oestrogen related receptor α (EERα) (Zhang et al., 2011), adenosine monophosphate dependent protein kinase (AMPK) (Wan et al., 2014) and protein kinase B (Akt) (Eddy and Storey, 2003). Recently, a study demonstrated that the Akt-PGC-1α axis contribute to melatonin-induced upregulation of Sirt3 and protection against hepatotoxicity (Song et al., 2017). Similar results were also observed in our neuronal injury model. We found that the Akt antagonist LY294002 partially prevented the Sirt3 expression and protection, as evidenced by cell viability, LDH release and apoptosis, in LV-LanCL1-transfected cells after OGD exposure. All these data suggest that the protective effects of LanCL1 are mediated by the Akt-PGC-1α-Sirt3 pathway.
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Melatoninmediated upregulation of Sirt3 attenuates sodium fluoride-induced hepatotoxicity by activating the MT1-PI3K/AKT-PGC-1alpha signaling pathway. Free Radic. Biol. Med. 112, 616–630. Su, J., Liu, J., Yan, X.Y., Zhang, Y., Zhang, J.J., Zhang, L.C., Sun, L.K., 2017. Cytoprotective effect of the UCP2-SIRT3 signaling pathway by decreasing mitochondrial oxidative stress on cerebral ischemia-reperfusion injury. Int. J. Mol. Sci. 18. Tao, R., Coleman, M.C., Pennington, J.D., Ozden, O., Park, S.H., Jiang, H., Kim, H.S., Flynn, C.R., Hill, S., Hayes McDonald, W., Olivier, A.K., Spitz, D.R., Gius, D., 2010. Sirt3-mediated deacetylation of evolutionarily conserved lysine 122 regulates MnSOD activity in response to stress. Mol. Cell 40, 893–904. Wan, Z., Root-McCaig, J., Castellani, L., Kemp, B.E., Steinberg, G.R., Wright, D.C., 2014. Evidence for the role of AMPK in regulating PGC-1 alpha expression and mitochondrial proteins in mouse epididymal adipose tissue. Obesity (Silver Spring) 22, 730–738. Wang, Q., Li, L., Li, C.Y., Pei, Z., Zhou, M., Li, N., 2015. SIRT3 protects cells from hypoxia via PGC-1alpha- and MnSOD-dependent pathways. Neuroscience 286, 109–121. Yu, W., Dittenhafer-Reed, K.E., Denu, J.M., 2012. SIRT3 protein deacetylates isocitrate dehydrogenase 2 (IDH2) and regulates mitochondrial redox status. J. Biol. Chem. 287, 14078–14086. Yu, Z., Liu, N., Li, Y., Xu, J., Wang, X., 2013. Neuroglobin overexpression inhibits oxygen-
5. Conclusions In conclusion, we found that LanCL1 facilitates the expression and activity of Sirt3 through activating the Akt-PGC-1α signaling pathway, thereby protecting HT22 cells against ischemia in vitro. These results many provide valuable clues in the research for new therapeutic targets that can be applied to clinical application for the treatment of ischemic stroke. Conflicts of interest The authors have no conflicts of interest relevant to this article. Acknowledgement This work was financially supported by the National Nature Science Foundation of China (No.81601898). References Anamika, Khanna, A., Acharjee, P., Acharjee, A., Trigun, S.K., 2017. MitochondrialSIRT3 and neurodegenerative brain disorders. J. Chem. Neuroanat. https://doi.org/10. 1016/j.jchemneu.2017.11.009. Ansari, A., Rahman, M.S., Saha, S.K., Saikot, F.K., Deep, A., Kim, K.H., 2017. Function of the SIRT3 mitochondrial deacetylase in cellular physiology, cancer, and neurodegenerative disease. Aging Cell 16, 4–16. Bauer, H., Mayer, H., Marchler-Bauer, A., Salzer, U., Prohaska, R., 2000. Characterization of p40/GPR69A as a peripheral membrane protein related to the lantibiotic synthetase component C. Biochem. Biophys. Res. Commun. 275, 69–74. Benjamin, E.J., Blaha, M.J., Chiuve, S.E., Cushman, M., Das, S.R., Deo, R., de Ferranti, S.D., Floyd, J., Fornage, M., Gillespie, C., Isasi, C.R., Jimenez, M.C., Jordan, L.C., Judd, S.E., Lackland, D., Lichtman, J.H., Lisabeth, L., Liu, S., Longenecker, C.T., Mackey, R.H., Matsushita, K., Mozaffarian, D., Mussolino, M.E., Nasir, K., Neumar, R.W., Palaniappan, L., Pandey, D.K., Thiagarajan, R.R., Reeves, M.J., Ritchey, M., Rodriguez, C.J., Roth, G.A., Rosamond, W.D., Sasson, C., Towfighi, A., Tsao, C.W., Turner, M.B., Virani, S.S., Voeks, J.H., Willey, J.Z., Wilkins, J.T., Wu, J.H., Alger, H.M., Wong, S.S., Muntner, P., 2017. Heart Disease and Stroke Statistics-2017 update: a report from the American Heart Association. Circulation 135, e146–e603. Bierbaum, G., Sahl, H.G., 2009. Lantibiotics: mode of action, biosynthesis and
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Baicalin increases VEGF expression and angiogenesis by activating the ERR{alpha}/ PGC-1{alpha} pathway. Cardiovasc. Res. 89, 426–435. Zhong, W.X., Wang, Y.B., Peng, L., Ge, X.Z., Zhang, J., Liu, S.S., Zhang, X.N., Xu, Z.H., Chen, Z., Luo, J.H., 2012. Lanthionine synthetase C-like protein 1 interacts with and inhibits cystathionine beta-synthase: a target for neuronal antioxidant defense. J. Biol. Chem. 287, 34189–34201.
glucose deprivation-induced mitochondrial permeability transition pore opening in primary cultured mouse cortical neurons. Neurobiol. Dis. 56, 95–103. Zhang, W., Wang, L., Liu, Y., Xu, J., Zhu, G., Cang, H., Li, X., Bartlam, M., Hensley, K., Li, G., Rao, Z., Zhang, X.C., 2009. Structure of human lanthionine synthetase C-like protein 1 and its interaction with Eps8 and glutathione. Genes Dev. 23, 1387–1392. Zhang, K., Lu, J., Mori, T., Smith-Powell, L., Synold, T.W., Chen, S., Wen, W., 2011.
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Research report
LanCL1 attenuates ischemia-induced oxidative stress by Sirt3-mediated preservation of mitochondrial function
T
Zhen Xieb, Bing-Qing Caob, Tao Wangb, Qi Leib, Tao Kangb, Chao-Yuan Gea, Wen-Jie Gaoa, ⁎ Hao Huia, a b
Department of Spine Surgery, Honghui Hospital, Xi’an Jiaotong University, No. 555 Youyi East Road, Xi’an, Shaanxi, 710054, China Department 2 of Neurology, Shaanxi Provincial People’s Hospital, Beilin Distinct, Xi’an, Shaanxi, 710068, China
A R T I C LE I N FO
A B S T R A C T
Keywords: LanCL1 Oxygen and glucose deprivation Oxidative stress Sirt3 PGC-1α
Lanthionine synthetase C-like protein 1 (LanCL1) is homologous to prokaryotic lanthionine cyclases, and has been shown to have novel functions in neuronal redox homeostasis. A recent study showed that LanCL1 expression was developmental and activity-dependent regulated, and LanCL1 transgene protected neurons against oxidative stress. In the present study, the potential protective effects of LanCL1 against ischemia was investigated in an in vitro model mimicked by oxygen and glucose deprivation (OGD) in neuronal HT22 cells. We found that OGD exposure induced a temporal increase and persistent decreases in the expression of LanCL1 at both mRNA and protein levels. Overexpression of LanCL1 by lentivirus (LV-LanCL1) transfection preserved cell viability, reduced lactate dehydrogenase (LDH) release and attenuated apoptosis after OGD. These protective effects were accompanied by decreased protein radical formation, lipid peroxidation and mitochondrial dysfunction. In addition, LanCL1 significantly stimulated mitochondrial enzyme activities and SOD2 deacetylation in a Sirt3-dependent manner. The results of western blot analysis showed that LanCL1-induced activation of Sirt3 was dependent on Akt-PGC-1α pathway. Knockdown of PGC-1α expression using small interfering RNA (siRNA) or blocking Akt activation using specific antagonist partially prevented the protective effects of LanCL1 in HT22 cells. Taken together, our results show that LanCL1 protects against OGD through activating the Akt-PGC-1αSirt3 pathway, and may have potential therapeutic value for ischemic stroke.
1. Introduction Despite the decrease in mortality rate (approximately 37%) in the past three decades, stroke is still a leading cause of disability and death worldwide (Lee et al., 2017; Peisker et al., 2017). In the United States, a stroke attack happens every 40 s, and a stroke-associated death occurs every 4 min, making it the second-leading cause of death behind ischemic heart disease (Benjamin et al., 2017). Thus far, many neuroprotective drugs and treatments have failed the clinical translation from experimental models to stroke patients. Tissue plasminogen activator is the only drug approved by the FDA for acute ischemic stroke treatment, but only 4–5% of all stroke patients can benefit from it (Docagne et al., 2015). The lanthionine synthetase C-like protein 1 (LanCL1), originally known as p40/GPR69 A, is one member of the LanC-like protein family, which are homologous to prokaryotic lanthionine synthetase component C (lanthionine cyclases) (Bauer et al., 2000). Prokaryotic lanthionine cyclases act in concert with dehydratases to facilitate
⁎
intramolecular conjugation of cysteine to serine or threonine residues, yielding potent “lantibiotics”, which are not present in mammals (Bierbaum and Sahl, 2009; Chatterjee et al., 2005). Three genes coding for the LanCL proteins are present in the human genome, named LanCL1, LanCL2 and LanCL3 (He et al., 2017). LanCL1 was previously identified as a novel binding partner for the redox regulatory, reduced glutathione (GSH), but has been demonstrated to have no enzymatic activity (Chung et al., 2007; Mayer et al., 2001; Zhang et al., 2009). The LanCL1 protein is highly expressed in the brain, spinal cord and testis, and increased levels of LanCL1 were found in the spinal cord of a mouse model of amyotrophic lateral sclerosis (ALS) (Chung et al., 2007). Recently, LanCL1 was shown to be developmentally regulated by neuronal activity, and confer antioxidant activity that is required for neuronal survival (Huang et al., 2014). However, the primary function of LanCL1 in ischemic stroke has not been determined. In this study, the role of LanCL1 in neuronal ischemia was investigated using an in vitro model mimicked by oxygen and glucose deprivation (OGD) in hippocampal HT22 cells. We also elucidated the potential molecular mechanisms
Corresponding author at: Department of Spine Surgery, Honghui Hospital, Xi’an Jiaotong University, No. 555 Youyi East Road, Xi’an, Shaanxi, 710054, China. E-mail address: [email protected] (H. Hui).
https://doi.org/10.1016/j.brainresbull.2018.07.017 Received 29 April 2018; Received in revised form 3 July 2018; Accepted 4 July 2018 Available online 31 July 2018 0361-9230/ © 2018 Elsevier Inc. All rights reserved.
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2. Materials and methods
secondary antibodies for 1 h at room temperature. Coverslips were mounted in mounting medium and visualized using a fluorescence microscope.
2.1. Cell culture
2.8. Quantification of lipid peroxidation
HT22 cells were purchased from the Institute of Biochemistry and Cell Biology, and cultured in Dulbecco’s modified Eagle’s medium (DMEM) with 10% fetal bovine serum at 37 °C.
Lipid peroxidation was determined by measuring MDA and 4-HNE levels using ELLAS kits (R&D Systems, Minneapolis, MN, USA) according to user’s manual.
2.2. OGD model
2.9. Measurement of ATP synthesis
Ischemia was induced by OGD method in vitro. Briefly, culture medium was replaced by glucose-free DMEM, and the cells were placed into a humidified chamber containing 5% CO2, 95% N2 at 37 °C. After 2, 4 or 6 h challenge, cells were returned to normal medium and normoxic conditions. Cells cultured under normal conditions during the experimental period served as the control.
HT22 cells were subjected to fission and centrifuged at 12 000 g for 5 min. In 24-well plates, 100 μl of each supernatant was mixed with 100 μl ATP working dilution. Luminance was measured using a monochromator microplate reader. The ATP release levels were expressed as a percentage of the luminescence levels in the control cells.
with focus on mitochondrial function and the Sirtuin3 (Sirt3) pathway.
2.10. Measurement of mitochondria swelling 2.3. Cell viability assay Mitochondria swelling was measured following a previously published protocol (Yu et al., 2013). The swelling of mitochondria was monitored by a decrease in absorbance at 540 nm in the presence of CaCl2 (200 μM).
WST-1 colorimetric assay was used to determine the viability of HT22 cells. Cells were seeded in 96-well plates and exposed to OGD. Ten microliters of WST-1 solution was added into each well, and the cells were incubated for another 4 h. Absorbance of each sample was measured at 450 nm with a reference wavelength of 600 nm. The results were expressed as the percentage of control values.
2.11. Mitochondrial enzyme activity The enzyme activities of IDH2, SOD2 and GSH-Px in the mitochondria were determined by commercial assay kits purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, Jiangsu, China), according to the manufacturer’s instructions.
2.4. Lactate dehydrogenase (LDH) release assay The cytotoxicity was determined by measuring the LDH activity in culture medium using a kit according to the manufacturer’s instructions (Pierce, IL, USA). The absorbance values were measured at 490 nm and 650 nm using a microplate reader. The results were expressed as the fold of control values.
2.12. RNA interference The specific siRNA targeted PGC-1α (sc-72151) or Sirt3 (sc-61556), and control siRNA (sc-37007) were purchased from Santa Cruz. The above siRNA molecules were transfected with Lipofectamine 2000 for 48 h before OGD.
2.5. Lentivirus construction and transfection The coding sequence of LanCL1 was amplified by RT-PCR with the following sequences: forward, 5′−CCT TCA GGT GAA CCA AGG AA-3′; reverse, 5′-AGA TCA CGT CAG CAC ACT GC-3′. The PCR fragments and the pGC-FU plasmid were digested with Age I and then ligated with T4 DNA ligase to produce pGC-FU-LanCL1. To generate the recombinant Lentivirus LV-LanCL1, 293 T cells were co-transfected with a pGC-FU plasmid (20 μg) with cDNA encoding LanCL1, pHelper1.0 plasmid (15 μg) and pHelper 2.0 plasmid (10 μg) using Lipofectamine 2000 (100 μl). After 48 h, the supernatant was harvested, and the viral titer was calculated by transducing 293 T cells.
2.13. Quantitative real-time PCR Total RNA was extracted from tissues using TRizol reagent. RNA was subjected to reverse transcription with reverse transcriptase as the manufacturer’s instructions (Fermentas). Quantitative real-time PCR was performed using the Bio-Rad iQ5 gradient real-time PCR system, and the relative gene expression was normalized to internal control as GAPDH. 2.14. Western blot analysis
2.6. TUNEL staining Standard Western blotting procedures were performed with the following antibodies: anti-LanCL1 antibody (1:500, ab229257, Abcam), anti-Sirt3 antibody (1:600, ab86671, Abcam), anti-SOD2 antibody (1:1000, ab13533, Abcam), anti-ac-SOD2 antibody (1:500, ab137037, Abcam), anti-Akt antibody (1:800, #4685, Cell Signaling), anti-p-Akt antibody (1:500, #4060, Cell signaling), anti- PGC-1α antibody (1:800, #2178, Cell signaling), and anti-β-actin antibody (1:1000, #4970, Cell signaling). The Image J software was used to quantify the optical density of each band.
Apoptotic cell death in HT22 cells was detected using the terminal deoxynucleotidy TUNEL staining. After various treatments, cells seeded on slides were washed with PBS, fixed with formaldehyde and permeabilized with 0.2% Tri-ton X-100. Then cells were incubated with 1.5 μM fluorescein isothiocyanate-coupled dUTP and TdT for 1 h in dark. The nucleus was stained with DAPI, and the slides were examined by fluorescence microscopy. 2.7. Immunocytochemistry (ICC)
2.15. Statistical analysis HT22 cells were fixed with formaldehyde and permeabilized with 0.2% Tri-ton X-100. Following two PBS rinses, the cells were incubated with blocking solution for 20 min and incubated with the primary DMPO antibody at 4 °C overnight. The cells were then rinsed with PBS and incubated with fluorescein isothiocyanate (FITC) labelled
Statistical analysis was performed using the GraphPad Prism 6 software. Statistical evaluation of the data was performed by one-way analysis of variance (ANOVA) followed by Newman–Keuls post-test. The differences were considered statistically significant when the value 217
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Fig. 1. Dynamic changes of LanCL1 expression after ischemia in HT22 cells. HT22 cells were exposed to OGD insults of different durations (2, 4 or 6 h) followed by further 24 h reperfusion. The cell viability was measured by WST-1 assay (A), and cytotoxicity was determined by LDH release assay (B). HT22 cells were exposed to 4 h OGD and 24 h reperfusion. The levels of LanCL1 mRNA was determined by RT-PCR (C), and the expression of LanCL1 protein was detected by western blot at different time points (D). Values are the mean ± SEM from five experiments. *p < 0.05 vs. Control.
of p < 0.05.
(Fig. 2E).
3. Results
3.3. LanCL1 inhibits ischemia-induced oxidative stress and mitochondrial dysfunction
3.1. Dynamic changes of LanCL1 expression after ischemia in HT22 cells HT22 cells were transfected with LV-LanCL1 or LV-control for 48 h and exposed to OGD. Immunofluorescence staining was used to detect cytosolic levels of DMPO, a marker of protein radical formation (Fig. 3A). The OGD-induced increase in DMPO fluorescence intensity was significantly reduced by LanCL1 overexpression (Fig. 3B). We also found that LV-LanCL1 transfection inhibited the OGD-induced lipid peroxidation, which was measured by MDA and 4-HNE (Fig. 3C). To further detect mitochondrial dysfunction, we isolated and purified mitochondria from HT22 cells in each group. As shown in Fig. 3D, LanCL1 overexpression preserved ATP generation after OGD. In addition, we measured mitochondrial swelling, which was induced by adding 200 μM Ca2+ into the isolated mitochondria (Fig. 3E). The results showed that OGD-induced mitochondrial swelling (decrease of absorbance) was partially prevented by LV-LanCL1 transfection.
To mimic brain ischemia in vitro, HT22 cells were exposed to OGD of different durations (2, 4 or 6 h) followed by 24 h reperfusion. OGD treatment resulted in significant decrease in cell viability (Fig. 1A) and increase in LDH release (Fig. 1B). Exposure of HT22 cells to 4 h OGD caused nearly half of the cells to die, so 4 h of OGD was used in following experiments. The results of RT-PCR showed that LanCL1 mRNA levels rapidly increased within 3 h after OGD, but significantly decreased from 6 h to 48 h (Fig. 1C). As shown in Fig. 1D, LanCL1 protein levels markedly increased at 3 h, but decreased at 12, 24 and 48 h after OGD treatment. 3.2. LanCL1 attenuates ischemia-induced cytotoxicity and apoptosis To overexpress LanCL1 in vitro, HT22 cells was transfected with LVLanCL1 or LV-control for 48 h, and western blot assay showed that LVLanCL1 transfection led to a significant increase in LanCL1 protein levels (Fig. 2A). The results of cell viability (Fig. 2B) and LDH release (Fig. 2C) showed that LV-LanCL1 transfection had no toxic effects on HT22 cells. However, LV-LanCL1-transfected cell are more resistant to OGD, as evidenced by increased cell viability (Fig. 2B) and decreased LDH release (Fig. 2C) compared to LV-control group. We also detected apoptosis in HT22 cells by TUNEL staining (Fig. 2D), and overexpression of LanCL1 significantly reduced OGD-induced apoptosis
3.4. LanCL1 stimulates Sirt3-mediated mitochondrial enzyme activities and SOD2 deacetylation To determine the effect of LanCL1 on endogenous antioxidant system, the enzymatic activities of IDH2, GSH-Px and SOD2 were measured. As shown in Fig. 4A–C, OGD treatment markedly reduced enzymatic activities of IDH2, GSH-Px and SOD2, whereas LanCL1 overexpression significantly enhanced these enzymatic activities. The results of western blot showed that LV-LanCL1 transfection suppressed 218
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Fig. 2. LanCL1 attenuates ischemia-induced cytotoxicity and apoptosis. HT22 cells were transfected with LV-LanCL1 or LV-control for 48 h, and the expression of LanCL1 was detected by western blot (A). HT22 cells were transfected with LV-LanCL1 or LV-control for 48 h and exposed to OGD. The cell viability was measured by WST-1 assay (B), and cytotoxicity was determined by LDH release assay (C). The apoptotic cell death was detected by TUNEL staining (D), and the apoptotic rate was calculated (E). Scale bars, 50 μm. Values are the mean ± SEM from five experiments. #p < 0.05 vs. Control. *p < 0.05 vs. LV-control.
Fig. 3. LanCL1 inhibits ischemia-induced oxidative stress and mitochondrial dysfunction. HT22 cells were transfected with LV-LanCL1 or LV-control for 48 h and exposed to OGD. The expression of DMPO was detected by immunofluorescence staining (A and B). Lipid peroxidation was determined by measuring MDA and 4HNE levels (C), and ATP generation was measured in isolated mitochondria (D). Mitochondrial swelling was examined by monitoring the absorbance at 540 nm induced by 200 μM Ca2+ (E), and the baseline absorbance was measured without Ca2+. Scale bars, 50 μm. Values are the mean ± SEM from five experiments. #p < 0.05 vs. Control. *p < 0.05 vs. LV-control. 219
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Fig. 4. LanCL1 stimulates Sirt3-mediated mitochondrial enzyme activities and SOD2 deacetylation. HT22 cells were transfected with LV-LanCL1 or LV-control for 48 h with transfection of Si-control or Si-Sirt3 and exposed to OGD. The enzyme activities of IDH2 (A), GSH-Px (B) and SOD2 (C) were determined using the corresponding assay kits, and the expression of Sirt3, ac-SOD2 and SOD2 was detected by western blot (D and E). Values are the mean ± SEM from five experiments. # p < 0.05 vs. Control. *p < 0.05 vs. OGD. &p < 0.05 vs. Si-control.
LanCL1 reduces OGD-induced cytotoxicity and apoptosis; (b) this protection is further supported by inhibition of oxidative stress and mitochondrial dysfunction; (c) LanCL1 stimulates Sirt3 protein expression and its downstream enzyme activity; and (e) mechanistically, the LanCL1-induced protection is partially dependent on the Akt-PGC-1αSirt3 pathway (Fig. 6). Experiments in postnatal rodents and primary cultured neurons showed that the expression of LanCL1 protein in brain was developmentally regulated and paralleled the formation of synapses and spontaneous neuronal activity (Huang et al., 2014). LanCL1 could be induced not only by several neurotrophic factors, including insulin growth factor 1 (IGF-1) and brain-derived neurotrophic factor (BDNF), but also by many neurotoxic agents, such as glutamate and hydrogen peroxide (H2O2). In our in vitro model, the expression of LanCL1 mRNA and protein were measured up to 48 h after OGD. A rapid induction of LanCL1, both at mRNA and protein levels, was observed within 3 h after OGD, which was followed by reduced expression from 6 to 48 h. These temporal increases in mRNA and protein levels are similar to the pattern of typical neuronal immediate early genes (Brakeman et al., 1997; Chen et al., 2017). A previous study using SOD1G93 A transgenic mice model showed that the expression of LanCL1 was increased at presymptomatic stages of ALS (Chung et al., 2007). These dynamic changes of LanCL1 mRNA and protein expression suggest a key role in neurological disorders. In the first few hours after ischemic stroke, many protective signaling cascades, such as antioxidant enzymes, mitochondrial biogenesis and unfolded protein response (UPR), are activated to prevent neuronal cell death (Prentice et al., 2015). If the ischemic stress is so severe that it cannot be overcome, these pro-survival cascades would be inhibited by several other detrimental pathways. Thus, we speculated that LanCL1 might represent one of this kind of endogenous protective mechanisms. We designed a lentivirus expressing exogenous LanCL1, and significant increase in LanCL1 protein expression was observed
the OGD-induced acetylation of SOD2 (Fig. 4D and E). In addition, based on the findings that LanCL1 overexpression increased the expression of Sirt3 protein both with and without OGD exposure (Fig. 4D), the related mechanisms were further investigated via Sirt3 knockdown using specific targeted siRNA (Si-Sirt3). The effects of LVLanCL1 on mitochondrial enzyme activities and SOD2 acetylation were all partially abolished by Sirt3 knockdown. 3.5. Involvement of Sirt3 signaling in LanCL1-induced protection against ischemia In consistent with the increased expression of Sirt3 after LanCL1 overexpression, increased expression of p-Akt and PGC-1α proteins was also observed (Fig. 5A and B), indicating the potential involvement of Akt-PGC-1α-Sirt3 pathway. To further confirm these findings, LY294002 was pretreated to inhibit Akt activity, and PGC-1α targeted siRNA (Si-PGC-1α) was used to knockdown PGC-1α in vitro. The results of western blot showed that LanCL1-induced increase in Sirt3 expression was partially ablated by both LY294002 and Si-PGC-1α transfection (Fig. 5C). In addition, the results of cell viability (Fig. 5D), LDH release (Fig. 5E) and TUNEL staining (Fig. 5F) indicated that LY294002 and PGC-1α knockdown partially reversed the protection induced by LanCL1 overexpression against OGD. 4. Discussion Because of its high expression in organs separated by blood-tissue barriers, such as brain and testis, LanCL1 was previously considered to play a role in the immune surveillance of these organs (Mayer et al., 2001). Recently, LanCL1 was found to exert a novel function in neuronal redox homeostasis (Zhong et al., 2012). Our present study provides evidence that LanCL1 protects against ischemia-induced oxidative stress in neuronal HT22 cells. We found that (a) overexpression of 220
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Fig. 5. Involvement of Sirt3 signaling in LanCL1-induced protection against ischemia. HT22 cells were transfected with LV-LanCL1 in the presence or absence of OGD. The expression of p-Akt, Akt and PGC-1α was detected by western blot (A and B). HT22 cells were transfected with LV-LanCL1 for 48 h with transfection of Sicontrol or Si-Sirt3, or treated with LY294002 in the presence of OGD. The expression of Sirt3 was detected by western blot (C). The cell viability was measured by WST-1 assay (D), and cytotoxicity was determined by LDH release assay (E). The apoptotic cell death was detected by TUNEL staining (F). Values are the mean ± SEM from five experiments. #p < 0.05 vs. Control. *p < 0.05 vs. OGD. &p < 0.05 vs. LV-LanCL1. $p < 0.05 vs. Si-control.
antioxidant enzymes (Giordano et al., 2014; Molenaar et al., 2014). Interestingly, a previous study using LanCL1 targeted siRNA and peptide showed that knockdown of LanCL1 occluded cystathionine β-synthase activation and exerted protective effects in cortical neurons (Zhong et al., 2012). This contradiction might be explained by several factors, such as the differences in cell types, toxic agents used in the experiments and different injury degrees. In consistent with our data, a recent paper showed that LanCL1 transgene could protect neurons from ROS (Huang et al., 2014), indicating the neuron-specific protective role of LanCL1 protein against oxidative stress. Sirt3 is a NAD+-dependent protein deacetylase that is exclusively localized and active in mitochondria. Due to its role in regulating mitochondrial enzyme activities and energy metabolism processes, Sirt3 is well known as a ROS scavenger and anti-apoptotic factor (Ansari et al., 2017). Many recent studies have demonstrated that overexpression of Sirt3 could prevent neuronal derangements in both in vitro and in vivo experimental models of aging and neurological disorders (Anamika et al., 2017; Su et al., 2017). In addition, activation of Sirt3 was shown to mediate the beneficial effects of various protective agents against stroke (Chen et al., 2018; Liu et al., 2017; Su et al., 2017). In the present study, the results of western blot showed that LanCL1 overexpression significantly increased Sirt3 expression after OGD. Sirt3 could directly deacetylate IDH2 and regulate mitochondrial redox status (Yu et al., 2012). Previous studies showed that knockdown of Sirt3 is associated with worsen neurological function due to the declined activity of SOD2 during cerebral ischemia (Anamika et al., 2017; Wang et al., 2015). Thus, we also detected the activities of Sirt3 associated antioxidant enzymes, and enhanced activities of IDH2, GSH-Px and SOD2 were observed after LV-LanCL1 transfection. SOD2 is a major downstream
Fig. 6. A proposed diagram tying together the observations involved in the LanCL1-induced neuroprotection against OGD.
after transfection in HT22 cells. LV-LanCL1 transfected HT22 cells exerted potent tolerance to OGD insult, which was accompanied by reduced oxidative stress and preserved mitochondrial function. Although LanCL1 is not a member of GST superfamily, its catalytic activity resembles that of GST proteins (Sheehan et al., 2001; Zhang et al., 2009). Our results showed that LanCL1 overexpression significantly promotes the enzymatic activities of IDH2, SOD2 and GSH-Px, three important 221
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signal of Sirt3-mediated mitochondrial O2.− reduction and deacetylation of SOD2 by Sirt3 regulates SOD2 enzymatic activity (Tao et al., 2010). Here, both increased ac-SOD2 expression and SOD2 activity were observed in LV-LanCL1-transfected HT22 cells. In addition, these changes in enzymatic activities was partially prevented by Sirt3 knockdown, indicating the involvement of Sirt3 signaling in LanCL1induced protection in our in vitro model. PGC-1α is a transcription co-activator that plays a key role in regulating mitochondrial biogenesis and energy metabolism in multiple organs, including the brain (Schilling and Kelly, 2011; Shoag and Arany, 2010). Previous studies in muscle cells, hepatocytes and neurons found that PGC-1α stimulates Sirt3 expression by binding to the Sirt3 promoter region (Giralt et al., 2011; Park et al., 2012). Silencing of PGC-1α reduced the expression of Sirt3, and overexpression of Sirt3 stimulated PGC-1α and its target UCP1, causing a decrease of ROS in a positive feedback (Kong et al., 2010). In this study, our results using PGC-1α specific siRNA showed that LanCL1-induced regulation in Sirt3 and protection against OGD were both dependent on PGC-1α. There are many protein kinases acting as the upstream factors in regulating PGC1α, such as oestrogen related receptor α (EERα) (Zhang et al., 2011), adenosine monophosphate dependent protein kinase (AMPK) (Wan et al., 2014) and protein kinase B (Akt) (Eddy and Storey, 2003). Recently, a study demonstrated that the Akt-PGC-1α axis contribute to melatonin-induced upregulation of Sirt3 and protection against hepatotoxicity (Song et al., 2017). Similar results were also observed in our neuronal injury model. We found that the Akt antagonist LY294002 partially prevented the Sirt3 expression and protection, as evidenced by cell viability, LDH release and apoptosis, in LV-LanCL1-transfected cells after OGD exposure. All these data suggest that the protective effects of LanCL1 are mediated by the Akt-PGC-1α-Sirt3 pathway.
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