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Decreased cpg15 augments oxidative stress in sleep deprived mouse brain
Biochemical and Biophysical Research Communications xxx (xxxx) xxx
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Decreased cpg15 augments oxidative stress in sleep deprived mouse brain Cheng-Jing Li a, Jun-Jie Li a, Yi Jiang a, Ya-Wei Mu a, De-Xin Lu a, Zi-Yao Xiao b, Han-Yang Jiang a, Jing-Jing Zhao c, Xian-Hua Chen a, * a Department of Translational Neuroscience, Jing’ an District Centre Hospital of Shanghai, State Key Laboratory of Medical Neurobiology and MOE Frontiers Center for Brain Science, Institutes of Brain Science, Fudan University, Shanghai, China b Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou, China c Center of Clinical Research, The Affiliated Wuxi People’s Hospital of Nanjing Medical University, Wuxi, 214023, PR China
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Article history: Received 9 November 2019 Accepted 20 November 2019 Available online xxx
Sleep deprivation (SD) has detrimental effects on the physiological function of the brain. However, the underlying mechanism remains elusive. In the present study, we investigated the expression of candidate plasticity-related gene 15 (cpg15), a neurotrophic gene, and its potential role in SD using a REM-SD mouse model. Immunofluorescent and Western blot analysis revealed that the expression of cpg15 protein decreased in the hippocampus, ventral group of the dorsal thalamus (VENT), and somatosensory area of cerebral cortex (SSP) after 24e72 h of REM-SD, and the oxidative stress in these brain regions was increased in parallel, as indicated by the ratio of glutathione (GSH) to its oxidative product (GSSG). Overexpression of cpg15 in thalamus, hippocampus, and cerebral cortex mediated by AAV reduced the oxidative stress in these regions, indicating that the decrease of cpg15 might be a cause that augments oxidative stress in the sleep deprived mouse brain. Collectively, the results imply that cpg15 may play a protective function in the SD-subjected mouse brain via an anti-oxidative function. To our knowledge, this is the first time to provide evidences in the role of cpg15 against SD-induced oxidative stress in the brain. © 2019 Elsevier Inc. All rights reserved.
Keywords: Sleep deprivation cpg15 Oxidative stress
1. Introduction Sleep deprivation (SD) or insufficient sleep which is caused by a variety of reasons including improper lifestyles and psychosocial stress is a widespread and serious problem in present-day society [1,2]. Accumulated evidences have indicated that SD may have detrimental effects on the physiological function of the brain, such as cognitive and mood, and is related to the increasing risk of a variety of neurodegenerative diseases, including Alzheimer’s disease, Parkinson’s diseases, depression and anxiety [3,4]. However, how sleep is linked to maintain normal brain functions, and how SD increase susceptibility of brain to insult and disease is largely unclear. Therefore, revealing the mechanism under the SD-induced disturbance in the structure and function of brain will be
* Corresponding author. State Key Laboratory of Medical Neurobiology, Institutes of Brain Science, Fudan University, 138 Yixueyuan Road, Shanghai, 200032, PR China. E-mail address: [email protected] (X.-H. Chen).
beneficial to exploring strategies to intervene in SD-induced brain injury. Neurotrophic factors are a family of secreted proteins that have multiple functions in mediating the neural development, survival and maintaining homeostasis of nervous system [5e7]. Evidences have indicated that neurotrophic factors, such as the brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF) and neurotrophin-3(NT-3), play important roles in the regulation of sleep homeostasis and sleep-related synaptic plasticity, and misregulations of the expression of these neurotrophic factors have causal link with the SD-induced neuropsychiatric disorders such as depression and anxiety [8e11]. Mechanism studies reveal that neurotrophic factors regulate sleep homeostasis in multiple aspects including affecting neuronal oxidative stress, regulating synaptic plasticity of neurons and modulating metabolic signals [12e14]. Accordingly, studying the role of neurotrophic factors in the SDinduced brain dysfunction is of interest for the development of the intervene strategies against the SD-induced brain injury. Candidate plasticity-related gene 15 (cpg15, also named
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Please cite this article as: C.-J. Li et al., Decreased cpg15 augments oxidative stress in sleep deprived mouse brain, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.11.132
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Abbreviations SD cpg15 REM-SD VENT SSP GSH GSSG BDNF NGF NT-3
sleep deprivation candidate plasticity-related gene 15 rapid-eye movement sleep deprivation ventral group of the dorsal thalamus somatosensory area of cerebral cortex glutathione oxidized glutathione brain-derived neurotrophic factor nerve growth factor neurotrophin-3
neuritin)is a small, highly conserved GPI-anchored neurotrophic gene expressed specifically in neurons. It has been shown that cpg15 protein plays roles in promoting neurites outgrowth [15], synaptic formation or maturation [16,17], and neuronal survival [18] during the brain development, and has protective function in alleviating the injury and enhancing the repair of neurons induced by a variety of stimulations such as cerebral ischemia, neurotrauma and depression [19e21]. However, whether cpg15 is involved in the regulation of sleep homeostasis, or whether it plays a role in SDinduced neural injury in the brain remains unknown. In the present study, the regulation of cpg15 expression and its potential role in the SD-subjected brain was investigated using the REM SD mouse model. The results showed that, the expression of cpg15 decreased significantly in the brain regions of hippocampus, ventral group of the dorsal thalamus (VENT), and somatosensory area of cerebral cortex (SSP) after 24e72 h of SD. In parallel, the ratio of GSH/GSSG, a biomarker of oxidative stress, decreased significantly in these brain regions after 72 h of SD. In addition, supplement of cpg15 via AAV mediated cpg15 over-expression in the thalamus and hippocampus reversed the change of GSH/GSSG ratio induced by SD, indicating the anti-oxidative function of cpg15 in the SD mouse brain. To our knowledge, this is the first study to provide evidence in the regulation of cpg15 expression level and its role in the SD-induced oxidative stress in the brain. The results may provide a potential intervening target for the SD-induced brain injury. 2. Materials and methods 2.1. Experimental animals and treatments Adult male C57BL/6 mice were provided by Slacc as Laboratory Animal Center of Chinese Academy of Sciences and Shanghai Model Organisms, housed under conditions of constant temperature (25 C) and humidity, a 12 h light-dark cycle, and were used to induce REM sleep deprivation (REM-SD). All animal experiments and surgical procedures were approved by the Institutional Animal Care and Fudan University Shanghai Medical College Committee (IACUC Animal Project 20170223e092), and were used in strict accordance with the recommendations in the Guide for the care and use of laboratory animals of the National Institutes of Health. All surgery was performed under chloral hydrate anesthesia, and all efforts were made to minimize suffering.
with slight modification. The device is a 38 32.5 18 cm (L W H) tank, with 9 platforms, each 3 cm in diameter and 2 cm in height, the distances between the adjacent platforms are 3 cm.The mice were placed in the platforms 24 h ahead of time for adapting to the device, and then the device was filled with clean water until the water surface was 1 cm below the platforms. For the control group, mice were subjected to the same procedure except that the platform is 8 cm in diameter (in order that the mice were able to sleep on the platforms). The devices were placed on a heating blanket to maintain a water temperature of 25 C. Food and water were made available ad libitum through a grid placed on top of the water tank. The light is illuminated according to the 12 h light/12 h dark rhythm (lighting from 8 a.m. to 8 p.m.). Analysis was performed immediately after sleep deprivation in mice. 5e6 mice per group were used in the experiment. 2.3. Construction of recombinant pAAV plasmid and AAV virus for cpg15 overexpression For over-expression of secretary HA-tagged cpg15 protein in cells, the HA coding sequence was inserted in frame in the cDNA fragments encoding the full-length mouse cpg15 protein (NM_153529.2,Mus musculus neuritin 1-Nrn1, 188-616 nt) between the upstream signal sequence and the core domain sequence. The obtained coding sequence of HA-tagged mouse cpg15 was then inserted into the pAAV plasmid to construct the recombinant pAAV plasmid. The plasmid contains the GFP coding sequence upstream and is used to express the HA-tagged cpg15 protein as well as the upstream GFP in the cultured cells. The same pAAV plasmid containing only the GFP coding sequence but not the HA-cpg15 coding sequence was used as the control. The recombinant AAV2/9 virus and its control virus were packaged using the pAAV-HA-cpg15 plasmids and its control (pAAV-Ctrl), respectively, by Heyuan Biotechnology (Shanghai) Co., Ltd. 2.4. Stereotaxic injection of mouse thalamus with the recombinant AAV In vivo injections of the recombinant AAV-HA-cpg15 were performed on the two -month old C57 adult male mice according to the previous description [23]. In detail, 0.8 mL of virus with the titer of 1.6 1013 v. g./mL was stereotactically injected into the ventral group of the dorsal thalamus (1.57, ±1.4, þ3.9) at 0.08 mL/min. Equal amount of the AAV-Ctrl was also injected as the control group. 21 days after the virus injection, REM sleep deprivation was performed on the mice when the AAV-mediated HA-cpg15 expression in mouse brain reaches a high efficiency. 2.5. Cell culture and transfection COS-1 cells were cultured in DMEM supplemented with 10% fetal bovine serum at 37 C with 5% CO2 in a humidified incubator. Transfections of pAAV-HA-cpg15 plasmid (pAAV-Ctrl as the control) were conducted using Lipofectamin®2000 (Invitrogen) following the supplier’s protocol. Briefly, cells were transfected with 0.5 mg of plasmid DNA per well of 24-well plate (60e80% confluent cells), in the presence of 2 mL of Lipofectamin 2000 Reagent. 24e48 h after transfection, cells were harvested for further investigation.
2.2. Mice REM sleep deprivation (REM-SD)
2.6. Fluorescence immunolabeling of mouse brain sections
Two-month-old male mice were subjected to REM-SD for 24 h or 72 h using a self-made multiple-platform water environment sleep deprivation device according to the previous description [22],
For fluorescence immunolabeling, REM sleep-deprived and the control mice were anesthetized with chloral hydrate and perfused with PBS at a rate of 5 mL/min for 10min.The brains were then
Please cite this article as: C.-J. Li et al., Decreased cpg15 augments oxidative stress in sleep deprived mouse brain, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.11.132
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dissected and fixed in 4% PFA at 4 C for 24 h, followed by dipping in 20% and 30% sucrose solution at 4 C successively until the tissues were sunk. Coronal sections were prepared in a thickness of 20 mm with a freezing microtome (Leica) when the brain tissues were completely frozen in the Optimum Cutting Temperature (OCT) compound at 20 C. Sections were then post-fixed with 4% PFA for 15 min, incubated with blocking solution containing 0.1% Triton and 10% horse serum in PBS at 37 C for 1 h, then incubated with primary antibodies overnight at 4 C. The sections were then incubated with the corresponding fluorescence-labeled secondary antibodies for 1 h at 37 C and stained with DAPI (Prod#D1306,Invitrogen, 1:100) and mounted on glass slides and covered slipped using fluoromount medium (Sigma). The negative controls received the same procedures except that the primary antibodies were omitted and no unspecific staining was observed. The immunofluorescent staining signals were observed under a fluorescent microscope or a confocal laser-scanning microscopy (TCS SP8, Leica). Anti-cpg15Antibody (Prod#AF283, R&D, 1:200), rabbit anti-HA (Prod #3724, CST, 1:1000), rabbit anti-NeuN (Prod#177487,Abcam, 1:500) were used as the primary antibodies. Donkey anti-goat IgG-Alexa Fluor 488(Prod#A11055,Invitrogen, 1:1000) and donkey anti-rabbit IgG-Alexa Fluor 594(Prod#A21207,Invitrogen, 1:1000) were used as the second antibodies.
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2.8. Western blot For western blot analysis, brain tissues or cells were lysed in RIPA buffer containing a complete protease inhibitor cocktail (Roche). 30e40 mg of each sample were separated in a 15% SDS-PAGE and transferred to PVDF membrane. The membranes were incubated with the primary antibody and then the corresponding second antibody. Goat anti-cpg15 antibody (Prod#AF283, R&D, 1:300), rabbit anti-HA (Prod #3724, CST, 1:1000) were used as the primary antibodies. Goat anti-rabbit IgG-IRDye 680CW(Prod#926e68021,LI-COR ODYSSEY, 1:10000), goat anti-mouse IgG-IRDye 680CW (Prod#926e32220,LI-COR ODYSSEY, 1:10000), and rabbit anti-goat IgG-IRDye800CW(Prod#926e32214,LI-COR ODYSSEY, 1:10000) were used as the second antibodies. Signals were detected with an odyssey scanner. The protein levels were quantified by densitometry analysis using Quantity One 4.5.2 software (Bio-Rad, USA). 2.9. Measurement of the glutathione concentration The concentration of the GSH and GSSG were measured using the GSH/GSSG quantitation kit (Prod #G263, Tongren Chemical) following the supplier’s instructions. 2.10. Statistical analysis
2.7. Immunocytochemistry For cell immunofluorescence, cells were cultured on the slides coated with poly-L-lysine, then fixed with 4% PFA for 30 min. After being incubated in the blocking buffer, the cell slides were incubated with the primary antibody and then a corresponding fluorescenceconjugated secondary antibody, followed by DAPI staining.
All data are expressed as mean ± standard error (mean ± S. E. M.) and analyses were performed using Prism software. The comparison between the two sets of data was performed using the ttest of independent samples; the comparison between the three sets of data was performed using one-way ANOVA. Statistical significance was defined as *P < 0.05, **P < 0.01, ***P < 0.001.
Fig. 1. Expression of cpg15 was decreased significantly in mouse hippocampus, thalamus and cerebral cortex after 72 h of sleep deprivation. A to F: Double immunofluorescent staining of cpg15 and NeuN in CA3 region of hippocampus (A and D), ventral group of the dorsal thalamus (VENT, B and E), and somatosensory area of cerebral cortex (SSP, C and F) in the mouse brain after 24 (SD24h) and 72 h (SD72h) of sleep deprivation. D, E, F are statistical data of relative fluorescent intensities of cpg15 staining in A, B, and C, respectively. G to L: Western blot analysis of cpg15 in mouse hippocampus (G), thalamus (I) and cerebral cortex (K) after 72 h of sleep deprivation (SD 72h). H, J and L are statistical data of G, I and K respectively. Non-sleep deprived mice were used as the control (Ctrl). DAPI was used to stain the nucleus. Bar equals to 50 mmb-actin was used as the loading control. n ¼ 3 for each group. *P < 0.05, **P < 0.01, ***P < 0.001.
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3. Results 3.1. The expression of cpg15 protein decreased in the brain regions of hippocampus, thalamus and cerebral cortex after 72 h of REM-SD To investigate whether cpg15 is involved in SD-induced neuronal injury or disturb in brain function, expression of cpg15 was analyzed in the mouse brain regions after REM sleep deprivation for 24e72 h by immunofluorescent and western blot analysis. The results showed that the expression of cpg15 protein decreased in the brain regions of hippocampus, VENT and SSP after 24e72 h of REM sleep deprivation, compared with the non-SD control mice, the decrease was significant for 72 h of REM sleep deprivation (Fig. 1).
3.2. The oxidative stress increased in the brain regions of hippocampus, VENT, and SSP after 72 h of REM-SD It has been reported that several kinds of sleep deprivation induce oxidative stress in the brain [24], we further want to reveal whether the regulation of cpg15 expression after REM sleep deprivation was related to the SD-induced oxidative stress in the brain. The concentrations of the reduced and oxidative glutathione were analyzed to determine the oxidative status in these brain regions after REM-SD for 72 h. The results indicated that, the ratio of the concentration of glutathione to oxidative glutathione (GSH/ GSSG) decreased obviously in the hippocampus, thalamus and cerebral cortex after REM-SD for 72 h, compared with the non-SD control mice (Fig. 2). The results indicated that 72 h of REM-SD causes obviously oxidative stress in these brain regions.
Fig. 2. Concentration of GSSG, GSH and ratio of GSH/GSSG in the hippocampus, thalamus and cerebral cortex after 72 h of sleep deprivation. The concentration of glutathione (GSH), oxidized glutathione (GSSG) and ratio of GSH/GSSG in the hippocampus (A), thalamus (B) and cerebral cortex (C) of SD-subjected mice (SD 72h) compared with the non-SD control (Ctrl) were analyzed. Each column is the Mean ± SEM, n ¼ 3. *P < 0.05, **P < 0.01,***P < 0.001.
Please cite this article as: C.-J. Li et al., Decreased cpg15 augments oxidative stress in sleep deprived mouse brain, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.11.132
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3.3. cpg15 over-expression alleviated the REM-SD-induced oxidative stress in the hippocampus, thalamus and cerebral cortex The parallel occurrence of decrease in the cpg15 expression and increase in the oxidative stress implies that there might be any correlation between them. In order to investigate whether cpg15 is involved in the SD-induced oxidative stress, recombinant AAV were constructed for over-expressing HA-tagged cpg15 proteins in the SD-subjected mice brain. The efficient expression of HA-cpg15 was first confirmed in cultured COS-1 cells transfected with of the pAAV-HA-cpg15 plasmid (Fig. 3), then the recombinant AAV-HA-
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cpg15 virus were injected into the VENT region of mouse brain, and 21 days later, the mice were subjected to 72 h of SD, followed by the analysis of the expression of HA-cpg15 and the concentration of GSH and GSSG in the brain regions. The results showed that the AAV-mediated HA-cpg15 fusion protein was expressed extensively in the hippocampus and cerebral cortex as well as in the thalamus (Fig. 3), and the ratio of GSH/GSSG increased significantly (Fig. 4) in the hippocampus, thalamus, and cerebral cortex of the mice, compared with these injected with the control AAV. These results indicated that supplement of cpg15 expression alleviated the oxidative stress in these brain regions in mice after 72 h of REM-
Fig. 3. Recombinant AAV-mediated HA-cpg15 protein overexpression in mouse hippocampus and thalamus. A. Confirmation of overexpression of pAAV-HA-cpg15 plasmid in culture cells. Immunofluorescent analysis of HA and cpg15 expression in COS-1 cells transfected with pAAV-HA-cpg15 plasmid or the control (pAAV-Ctrl). Bar equals to 50 mm. B. Immunofluorescent analysis of HA in sections of mouse brain injected with AAV-HA-cpg15 virus or the control virus (AAV-Ctrl). GFP was expressed in both AAV-HA-cpg15 and AAVCtrl virus. Bar equals to 50 mm.
Please cite this article as: C.-J. Li et al., Decreased cpg15 augments oxidative stress in sleep deprived mouse brain, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.11.132
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Fig. 4. cpg15 overexpression alleviates the oxidative stress induced by sleep deprivation in mouse hippocampus and thalamus. The concentration of GSH, GSSG and the ratio of GSH/GSSG in hippocampus (A), thalamus (B) and cerebral cortex (C) in SD-subjected mice injected with the AAV-HA-cpg15 virus and the AAV-Control virus were analyzed. Each column is the Mean ± SEM, n ¼ 3. **P < 0.01,***P < 0.001.
sleep deprivation. 4. Discussion Nowadays, sleep deprivation (SD) is commonplace worldwide and becomes a public health epidemic. Accumulating evidence indicates that SD causes wide-ranging deleterious effects such as impaired brain function and increased risk of neurodegenerative diseases [25,26]. Neurotrophins are reportedly involved in the SDinduced injury of brain function [27,28]. cpg15 is known to be an activity-dependent neurotrophins and play important role in regulating brain function by modulating nervous system plasticity such as promoting neurites outgrowth, synaptic formation or maturation, and neuronal survival [15e17,29]. However, it remains unknown whether cpg15 is involved in SD-induced brain injury. The question was addressed in the present study. Neurotrophins reportedly play important roles in hippocampaldependent learning and memory. Increasing studies reveal that
neurotrophins are susceptible to be misregulated after SD. For example, previous work showed that SD down-regulated the expression of BDNF in hippocampus [30]. ELISA analysis revealed that the level of NGF decreased in hippocampus after REM-SD [31]. Consistent with these results, we demonstrated that cpg15 expression was down-regulated in hippocampus after REM-SD. Whether decreased cpg15 in hippocampus was associated with impaired hippocampal-dependent learning and memory after SD needs further investigation. Thalamus is a critical brain region involved in sleep-wake regulation [32]. In our study, the down-regulation of cpg15 in thalamus was observed after REM-SD. Our study implies that cpg15 may be involved in SD-induced alteration in the structure and function in thalamus. In addition, somatosensory area of cerebral cortex (SSP) is a brain region in which the activities of the neurons are susceptible to the sleep-wake status [33,34]. The downregulation of cpg15 in SSP suggests that cpg15 might be involved in the somatosensory function after SD. This result is consistent
Please cite this article as: C.-J. Li et al., Decreased cpg15 augments oxidative stress in sleep deprived mouse brain, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.11.132
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with the neuronal activity-dependent expressional regulation property of cpg15 [17,35,36] and the previous report that cpg15 protein distributed in the sensory regions of the Xenopus brain [37]. Whether and how decreased cpg15 in SSP was associated with the alteration of somatosensory function after SD needs further investigation. Oxidative stress is one of the main causes that induce the injury of brain after SD. Although there are a minority of contradictory results reported, majority of evidences showed that decrease in the content of GSH, a scavenger of ROS, after SD, as well as the antioxidative enzymes SOD2 and COX2, which is consistent with our results that the ratio of GSH/GSSG decreased significantly after 72 h SD [24,38,39]. Our study further supports that SD aggravates oxidative stress in the brain. Large number of evidences indicated that neurotrophins may play protective roles via anti-oxidative function in the brain. For example, NGF, BDNF and NT-5 reportedly protect against MPPþ toxicity by attenuating oxidative stress in neonatal animals [40]. In order to investigate whether cpg15 also involved in the oxidative stress in SD-mouse, recombinant AAV was injected into the thalamus of mouse for supplement of the exogenous cpg15, by confirming that the AAV has diffused and spread widely in the hippocampus and cerebral cortex as well as in the injected site thalamus after 21 days from AAV injection, we found that the oxidative stress in these brain regions were alleviated significantly, indicating that cpg15 can provide protective anti-oxidative function in the SD-subjected brain. The result is consistent with the report that cpg15 protected Schwann cells from oxidative stress-induced apoptosis [41]. How the decreased cpg15 expression relates to the aggravation of oxidative stress in the SD-subjected brain need further investigation. 5. Conclusions The present work showed that cpg15 was involved in the regulation of oxidative stress in mouse brain after SD and might ameliorate the brain function via alleviating the neuronal injury in the affected brain regions. To our knowledge, this is the first time to provide evidences in the role of cpg15 against SD-induced oxidative stress in the brain. The results provide a potential intervening strategy for the SD-induced structural and functional injuries in the brain, and for the neurodegenerative diseases with sleep disorder, such as anxiety, depression, and Parkinson’s diseases. Acknowledgments This work was supported by grants from the National Foundation of Natural Sciences of China (grant numbers 31771112, 31571037, 81901259), Shanghai Municipal Science and Technology Major Project (grant number 2018SHZDZX01) and ZJLab. Natural Science Foundation of Jiangsu Province (BK20180168), Key Project of Scientific Developing Fund of Nanjing Medical University (20l7NJMUZD125). Transparency document Transparency document related to this article can be found online at https://doi.org/10.1016/j.bbrc.2019.11.132. References [1] R.E. Roberts, C.R. Roberts, Y. Xing, Restricted sleep among adolescents: prevalence, incidence, persistence, and associated factors, Behav. Sleep Med. 9 (2011) 18e30. [2] S.M. Rajaratnam, J. Arendt, Health in a 24-h society, Lancet 358 (2001) 999e1005.
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Decreased cpg15 augments oxidative stress in sleep deprived mouse brain Cheng-Jing Li a, Jun-Jie Li a, Yi Jiang a, Ya-Wei Mu a, De-Xin Lu a, Zi-Yao Xiao b, Han-Yang Jiang a, Jing-Jing Zhao c, Xian-Hua Chen a, * a Department of Translational Neuroscience, Jing’ an District Centre Hospital of Shanghai, State Key Laboratory of Medical Neurobiology and MOE Frontiers Center for Brain Science, Institutes of Brain Science, Fudan University, Shanghai, China b Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou, China c Center of Clinical Research, The Affiliated Wuxi People’s Hospital of Nanjing Medical University, Wuxi, 214023, PR China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 9 November 2019 Accepted 20 November 2019 Available online xxx
Sleep deprivation (SD) has detrimental effects on the physiological function of the brain. However, the underlying mechanism remains elusive. In the present study, we investigated the expression of candidate plasticity-related gene 15 (cpg15), a neurotrophic gene, and its potential role in SD using a REM-SD mouse model. Immunofluorescent and Western blot analysis revealed that the expression of cpg15 protein decreased in the hippocampus, ventral group of the dorsal thalamus (VENT), and somatosensory area of cerebral cortex (SSP) after 24e72 h of REM-SD, and the oxidative stress in these brain regions was increased in parallel, as indicated by the ratio of glutathione (GSH) to its oxidative product (GSSG). Overexpression of cpg15 in thalamus, hippocampus, and cerebral cortex mediated by AAV reduced the oxidative stress in these regions, indicating that the decrease of cpg15 might be a cause that augments oxidative stress in the sleep deprived mouse brain. Collectively, the results imply that cpg15 may play a protective function in the SD-subjected mouse brain via an anti-oxidative function. To our knowledge, this is the first time to provide evidences in the role of cpg15 against SD-induced oxidative stress in the brain. © 2019 Elsevier Inc. All rights reserved.
Keywords: Sleep deprivation cpg15 Oxidative stress
1. Introduction Sleep deprivation (SD) or insufficient sleep which is caused by a variety of reasons including improper lifestyles and psychosocial stress is a widespread and serious problem in present-day society [1,2]. Accumulated evidences have indicated that SD may have detrimental effects on the physiological function of the brain, such as cognitive and mood, and is related to the increasing risk of a variety of neurodegenerative diseases, including Alzheimer’s disease, Parkinson’s diseases, depression and anxiety [3,4]. However, how sleep is linked to maintain normal brain functions, and how SD increase susceptibility of brain to insult and disease is largely unclear. Therefore, revealing the mechanism under the SD-induced disturbance in the structure and function of brain will be
* Corresponding author. State Key Laboratory of Medical Neurobiology, Institutes of Brain Science, Fudan University, 138 Yixueyuan Road, Shanghai, 200032, PR China. E-mail address: [email protected] (X.-H. Chen).
beneficial to exploring strategies to intervene in SD-induced brain injury. Neurotrophic factors are a family of secreted proteins that have multiple functions in mediating the neural development, survival and maintaining homeostasis of nervous system [5e7]. Evidences have indicated that neurotrophic factors, such as the brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF) and neurotrophin-3(NT-3), play important roles in the regulation of sleep homeostasis and sleep-related synaptic plasticity, and misregulations of the expression of these neurotrophic factors have causal link with the SD-induced neuropsychiatric disorders such as depression and anxiety [8e11]. Mechanism studies reveal that neurotrophic factors regulate sleep homeostasis in multiple aspects including affecting neuronal oxidative stress, regulating synaptic plasticity of neurons and modulating metabolic signals [12e14]. Accordingly, studying the role of neurotrophic factors in the SDinduced brain dysfunction is of interest for the development of the intervene strategies against the SD-induced brain injury. Candidate plasticity-related gene 15 (cpg15, also named
https://doi.org/10.1016/j.bbrc.2019.11.132 0006-291X/© 2019 Elsevier Inc. All rights reserved.
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Abbreviations SD cpg15 REM-SD VENT SSP GSH GSSG BDNF NGF NT-3
sleep deprivation candidate plasticity-related gene 15 rapid-eye movement sleep deprivation ventral group of the dorsal thalamus somatosensory area of cerebral cortex glutathione oxidized glutathione brain-derived neurotrophic factor nerve growth factor neurotrophin-3
neuritin)is a small, highly conserved GPI-anchored neurotrophic gene expressed specifically in neurons. It has been shown that cpg15 protein plays roles in promoting neurites outgrowth [15], synaptic formation or maturation [16,17], and neuronal survival [18] during the brain development, and has protective function in alleviating the injury and enhancing the repair of neurons induced by a variety of stimulations such as cerebral ischemia, neurotrauma and depression [19e21]. However, whether cpg15 is involved in the regulation of sleep homeostasis, or whether it plays a role in SDinduced neural injury in the brain remains unknown. In the present study, the regulation of cpg15 expression and its potential role in the SD-subjected brain was investigated using the REM SD mouse model. The results showed that, the expression of cpg15 decreased significantly in the brain regions of hippocampus, ventral group of the dorsal thalamus (VENT), and somatosensory area of cerebral cortex (SSP) after 24e72 h of SD. In parallel, the ratio of GSH/GSSG, a biomarker of oxidative stress, decreased significantly in these brain regions after 72 h of SD. In addition, supplement of cpg15 via AAV mediated cpg15 over-expression in the thalamus and hippocampus reversed the change of GSH/GSSG ratio induced by SD, indicating the anti-oxidative function of cpg15 in the SD mouse brain. To our knowledge, this is the first study to provide evidence in the regulation of cpg15 expression level and its role in the SD-induced oxidative stress in the brain. The results may provide a potential intervening target for the SD-induced brain injury. 2. Materials and methods 2.1. Experimental animals and treatments Adult male C57BL/6 mice were provided by Slacc as Laboratory Animal Center of Chinese Academy of Sciences and Shanghai Model Organisms, housed under conditions of constant temperature (25 C) and humidity, a 12 h light-dark cycle, and were used to induce REM sleep deprivation (REM-SD). All animal experiments and surgical procedures were approved by the Institutional Animal Care and Fudan University Shanghai Medical College Committee (IACUC Animal Project 20170223e092), and were used in strict accordance with the recommendations in the Guide for the care and use of laboratory animals of the National Institutes of Health. All surgery was performed under chloral hydrate anesthesia, and all efforts were made to minimize suffering.
with slight modification. The device is a 38 32.5 18 cm (L W H) tank, with 9 platforms, each 3 cm in diameter and 2 cm in height, the distances between the adjacent platforms are 3 cm.The mice were placed in the platforms 24 h ahead of time for adapting to the device, and then the device was filled with clean water until the water surface was 1 cm below the platforms. For the control group, mice were subjected to the same procedure except that the platform is 8 cm in diameter (in order that the mice were able to sleep on the platforms). The devices were placed on a heating blanket to maintain a water temperature of 25 C. Food and water were made available ad libitum through a grid placed on top of the water tank. The light is illuminated according to the 12 h light/12 h dark rhythm (lighting from 8 a.m. to 8 p.m.). Analysis was performed immediately after sleep deprivation in mice. 5e6 mice per group were used in the experiment. 2.3. Construction of recombinant pAAV plasmid and AAV virus for cpg15 overexpression For over-expression of secretary HA-tagged cpg15 protein in cells, the HA coding sequence was inserted in frame in the cDNA fragments encoding the full-length mouse cpg15 protein (NM_153529.2,Mus musculus neuritin 1-Nrn1, 188-616 nt) between the upstream signal sequence and the core domain sequence. The obtained coding sequence of HA-tagged mouse cpg15 was then inserted into the pAAV plasmid to construct the recombinant pAAV plasmid. The plasmid contains the GFP coding sequence upstream and is used to express the HA-tagged cpg15 protein as well as the upstream GFP in the cultured cells. The same pAAV plasmid containing only the GFP coding sequence but not the HA-cpg15 coding sequence was used as the control. The recombinant AAV2/9 virus and its control virus were packaged using the pAAV-HA-cpg15 plasmids and its control (pAAV-Ctrl), respectively, by Heyuan Biotechnology (Shanghai) Co., Ltd. 2.4. Stereotaxic injection of mouse thalamus with the recombinant AAV In vivo injections of the recombinant AAV-HA-cpg15 were performed on the two -month old C57 adult male mice according to the previous description [23]. In detail, 0.8 mL of virus with the titer of 1.6 1013 v. g./mL was stereotactically injected into the ventral group of the dorsal thalamus (1.57, ±1.4, þ3.9) at 0.08 mL/min. Equal amount of the AAV-Ctrl was also injected as the control group. 21 days after the virus injection, REM sleep deprivation was performed on the mice when the AAV-mediated HA-cpg15 expression in mouse brain reaches a high efficiency. 2.5. Cell culture and transfection COS-1 cells were cultured in DMEM supplemented with 10% fetal bovine serum at 37 C with 5% CO2 in a humidified incubator. Transfections of pAAV-HA-cpg15 plasmid (pAAV-Ctrl as the control) were conducted using Lipofectamin®2000 (Invitrogen) following the supplier’s protocol. Briefly, cells were transfected with 0.5 mg of plasmid DNA per well of 24-well plate (60e80% confluent cells), in the presence of 2 mL of Lipofectamin 2000 Reagent. 24e48 h after transfection, cells were harvested for further investigation.
2.2. Mice REM sleep deprivation (REM-SD)
2.6. Fluorescence immunolabeling of mouse brain sections
Two-month-old male mice were subjected to REM-SD for 24 h or 72 h using a self-made multiple-platform water environment sleep deprivation device according to the previous description [22],
For fluorescence immunolabeling, REM sleep-deprived and the control mice were anesthetized with chloral hydrate and perfused with PBS at a rate of 5 mL/min for 10min.The brains were then
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dissected and fixed in 4% PFA at 4 C for 24 h, followed by dipping in 20% and 30% sucrose solution at 4 C successively until the tissues were sunk. Coronal sections were prepared in a thickness of 20 mm with a freezing microtome (Leica) when the brain tissues were completely frozen in the Optimum Cutting Temperature (OCT) compound at 20 C. Sections were then post-fixed with 4% PFA for 15 min, incubated with blocking solution containing 0.1% Triton and 10% horse serum in PBS at 37 C for 1 h, then incubated with primary antibodies overnight at 4 C. The sections were then incubated with the corresponding fluorescence-labeled secondary antibodies for 1 h at 37 C and stained with DAPI (Prod#D1306,Invitrogen, 1:100) and mounted on glass slides and covered slipped using fluoromount medium (Sigma). The negative controls received the same procedures except that the primary antibodies were omitted and no unspecific staining was observed. The immunofluorescent staining signals were observed under a fluorescent microscope or a confocal laser-scanning microscopy (TCS SP8, Leica). Anti-cpg15Antibody (Prod#AF283, R&D, 1:200), rabbit anti-HA (Prod #3724, CST, 1:1000), rabbit anti-NeuN (Prod#177487,Abcam, 1:500) were used as the primary antibodies. Donkey anti-goat IgG-Alexa Fluor 488(Prod#A11055,Invitrogen, 1:1000) and donkey anti-rabbit IgG-Alexa Fluor 594(Prod#A21207,Invitrogen, 1:1000) were used as the second antibodies.
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2.8. Western blot For western blot analysis, brain tissues or cells were lysed in RIPA buffer containing a complete protease inhibitor cocktail (Roche). 30e40 mg of each sample were separated in a 15% SDS-PAGE and transferred to PVDF membrane. The membranes were incubated with the primary antibody and then the corresponding second antibody. Goat anti-cpg15 antibody (Prod#AF283, R&D, 1:300), rabbit anti-HA (Prod #3724, CST, 1:1000) were used as the primary antibodies. Goat anti-rabbit IgG-IRDye 680CW(Prod#926e68021,LI-COR ODYSSEY, 1:10000), goat anti-mouse IgG-IRDye 680CW (Prod#926e32220,LI-COR ODYSSEY, 1:10000), and rabbit anti-goat IgG-IRDye800CW(Prod#926e32214,LI-COR ODYSSEY, 1:10000) were used as the second antibodies. Signals were detected with an odyssey scanner. The protein levels were quantified by densitometry analysis using Quantity One 4.5.2 software (Bio-Rad, USA). 2.9. Measurement of the glutathione concentration The concentration of the GSH and GSSG were measured using the GSH/GSSG quantitation kit (Prod #G263, Tongren Chemical) following the supplier’s instructions. 2.10. Statistical analysis
2.7. Immunocytochemistry For cell immunofluorescence, cells were cultured on the slides coated with poly-L-lysine, then fixed with 4% PFA for 30 min. After being incubated in the blocking buffer, the cell slides were incubated with the primary antibody and then a corresponding fluorescenceconjugated secondary antibody, followed by DAPI staining.
All data are expressed as mean ± standard error (mean ± S. E. M.) and analyses were performed using Prism software. The comparison between the two sets of data was performed using the ttest of independent samples; the comparison between the three sets of data was performed using one-way ANOVA. Statistical significance was defined as *P < 0.05, **P < 0.01, ***P < 0.001.
Fig. 1. Expression of cpg15 was decreased significantly in mouse hippocampus, thalamus and cerebral cortex after 72 h of sleep deprivation. A to F: Double immunofluorescent staining of cpg15 and NeuN in CA3 region of hippocampus (A and D), ventral group of the dorsal thalamus (VENT, B and E), and somatosensory area of cerebral cortex (SSP, C and F) in the mouse brain after 24 (SD24h) and 72 h (SD72h) of sleep deprivation. D, E, F are statistical data of relative fluorescent intensities of cpg15 staining in A, B, and C, respectively. G to L: Western blot analysis of cpg15 in mouse hippocampus (G), thalamus (I) and cerebral cortex (K) after 72 h of sleep deprivation (SD 72h). H, J and L are statistical data of G, I and K respectively. Non-sleep deprived mice were used as the control (Ctrl). DAPI was used to stain the nucleus. Bar equals to 50 mmb-actin was used as the loading control. n ¼ 3 for each group. *P < 0.05, **P < 0.01, ***P < 0.001.
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3. Results 3.1. The expression of cpg15 protein decreased in the brain regions of hippocampus, thalamus and cerebral cortex after 72 h of REM-SD To investigate whether cpg15 is involved in SD-induced neuronal injury or disturb in brain function, expression of cpg15 was analyzed in the mouse brain regions after REM sleep deprivation for 24e72 h by immunofluorescent and western blot analysis. The results showed that the expression of cpg15 protein decreased in the brain regions of hippocampus, VENT and SSP after 24e72 h of REM sleep deprivation, compared with the non-SD control mice, the decrease was significant for 72 h of REM sleep deprivation (Fig. 1).
3.2. The oxidative stress increased in the brain regions of hippocampus, VENT, and SSP after 72 h of REM-SD It has been reported that several kinds of sleep deprivation induce oxidative stress in the brain [24], we further want to reveal whether the regulation of cpg15 expression after REM sleep deprivation was related to the SD-induced oxidative stress in the brain. The concentrations of the reduced and oxidative glutathione were analyzed to determine the oxidative status in these brain regions after REM-SD for 72 h. The results indicated that, the ratio of the concentration of glutathione to oxidative glutathione (GSH/ GSSG) decreased obviously in the hippocampus, thalamus and cerebral cortex after REM-SD for 72 h, compared with the non-SD control mice (Fig. 2). The results indicated that 72 h of REM-SD causes obviously oxidative stress in these brain regions.
Fig. 2. Concentration of GSSG, GSH and ratio of GSH/GSSG in the hippocampus, thalamus and cerebral cortex after 72 h of sleep deprivation. The concentration of glutathione (GSH), oxidized glutathione (GSSG) and ratio of GSH/GSSG in the hippocampus (A), thalamus (B) and cerebral cortex (C) of SD-subjected mice (SD 72h) compared with the non-SD control (Ctrl) were analyzed. Each column is the Mean ± SEM, n ¼ 3. *P < 0.05, **P < 0.01,***P < 0.001.
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3.3. cpg15 over-expression alleviated the REM-SD-induced oxidative stress in the hippocampus, thalamus and cerebral cortex The parallel occurrence of decrease in the cpg15 expression and increase in the oxidative stress implies that there might be any correlation between them. In order to investigate whether cpg15 is involved in the SD-induced oxidative stress, recombinant AAV were constructed for over-expressing HA-tagged cpg15 proteins in the SD-subjected mice brain. The efficient expression of HA-cpg15 was first confirmed in cultured COS-1 cells transfected with of the pAAV-HA-cpg15 plasmid (Fig. 3), then the recombinant AAV-HA-
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cpg15 virus were injected into the VENT region of mouse brain, and 21 days later, the mice were subjected to 72 h of SD, followed by the analysis of the expression of HA-cpg15 and the concentration of GSH and GSSG in the brain regions. The results showed that the AAV-mediated HA-cpg15 fusion protein was expressed extensively in the hippocampus and cerebral cortex as well as in the thalamus (Fig. 3), and the ratio of GSH/GSSG increased significantly (Fig. 4) in the hippocampus, thalamus, and cerebral cortex of the mice, compared with these injected with the control AAV. These results indicated that supplement of cpg15 expression alleviated the oxidative stress in these brain regions in mice after 72 h of REM-
Fig. 3. Recombinant AAV-mediated HA-cpg15 protein overexpression in mouse hippocampus and thalamus. A. Confirmation of overexpression of pAAV-HA-cpg15 plasmid in culture cells. Immunofluorescent analysis of HA and cpg15 expression in COS-1 cells transfected with pAAV-HA-cpg15 plasmid or the control (pAAV-Ctrl). Bar equals to 50 mm. B. Immunofluorescent analysis of HA in sections of mouse brain injected with AAV-HA-cpg15 virus or the control virus (AAV-Ctrl). GFP was expressed in both AAV-HA-cpg15 and AAVCtrl virus. Bar equals to 50 mm.
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Fig. 4. cpg15 overexpression alleviates the oxidative stress induced by sleep deprivation in mouse hippocampus and thalamus. The concentration of GSH, GSSG and the ratio of GSH/GSSG in hippocampus (A), thalamus (B) and cerebral cortex (C) in SD-subjected mice injected with the AAV-HA-cpg15 virus and the AAV-Control virus were analyzed. Each column is the Mean ± SEM, n ¼ 3. **P < 0.01,***P < 0.001.
sleep deprivation. 4. Discussion Nowadays, sleep deprivation (SD) is commonplace worldwide and becomes a public health epidemic. Accumulating evidence indicates that SD causes wide-ranging deleterious effects such as impaired brain function and increased risk of neurodegenerative diseases [25,26]. Neurotrophins are reportedly involved in the SDinduced injury of brain function [27,28]. cpg15 is known to be an activity-dependent neurotrophins and play important role in regulating brain function by modulating nervous system plasticity such as promoting neurites outgrowth, synaptic formation or maturation, and neuronal survival [15e17,29]. However, it remains unknown whether cpg15 is involved in SD-induced brain injury. The question was addressed in the present study. Neurotrophins reportedly play important roles in hippocampaldependent learning and memory. Increasing studies reveal that
neurotrophins are susceptible to be misregulated after SD. For example, previous work showed that SD down-regulated the expression of BDNF in hippocampus [30]. ELISA analysis revealed that the level of NGF decreased in hippocampus after REM-SD [31]. Consistent with these results, we demonstrated that cpg15 expression was down-regulated in hippocampus after REM-SD. Whether decreased cpg15 in hippocampus was associated with impaired hippocampal-dependent learning and memory after SD needs further investigation. Thalamus is a critical brain region involved in sleep-wake regulation [32]. In our study, the down-regulation of cpg15 in thalamus was observed after REM-SD. Our study implies that cpg15 may be involved in SD-induced alteration in the structure and function in thalamus. In addition, somatosensory area of cerebral cortex (SSP) is a brain region in which the activities of the neurons are susceptible to the sleep-wake status [33,34]. The downregulation of cpg15 in SSP suggests that cpg15 might be involved in the somatosensory function after SD. This result is consistent
Please cite this article as: C.-J. Li et al., Decreased cpg15 augments oxidative stress in sleep deprived mouse brain, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.11.132
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with the neuronal activity-dependent expressional regulation property of cpg15 [17,35,36] and the previous report that cpg15 protein distributed in the sensory regions of the Xenopus brain [37]. Whether and how decreased cpg15 in SSP was associated with the alteration of somatosensory function after SD needs further investigation. Oxidative stress is one of the main causes that induce the injury of brain after SD. Although there are a minority of contradictory results reported, majority of evidences showed that decrease in the content of GSH, a scavenger of ROS, after SD, as well as the antioxidative enzymes SOD2 and COX2, which is consistent with our results that the ratio of GSH/GSSG decreased significantly after 72 h SD [24,38,39]. Our study further supports that SD aggravates oxidative stress in the brain. Large number of evidences indicated that neurotrophins may play protective roles via anti-oxidative function in the brain. For example, NGF, BDNF and NT-5 reportedly protect against MPPþ toxicity by attenuating oxidative stress in neonatal animals [40]. In order to investigate whether cpg15 also involved in the oxidative stress in SD-mouse, recombinant AAV was injected into the thalamus of mouse for supplement of the exogenous cpg15, by confirming that the AAV has diffused and spread widely in the hippocampus and cerebral cortex as well as in the injected site thalamus after 21 days from AAV injection, we found that the oxidative stress in these brain regions were alleviated significantly, indicating that cpg15 can provide protective anti-oxidative function in the SD-subjected brain. The result is consistent with the report that cpg15 protected Schwann cells from oxidative stress-induced apoptosis [41]. How the decreased cpg15 expression relates to the aggravation of oxidative stress in the SD-subjected brain need further investigation. 5. Conclusions The present work showed that cpg15 was involved in the regulation of oxidative stress in mouse brain after SD and might ameliorate the brain function via alleviating the neuronal injury in the affected brain regions. To our knowledge, this is the first time to provide evidences in the role of cpg15 against SD-induced oxidative stress in the brain. The results provide a potential intervening strategy for the SD-induced structural and functional injuries in the brain, and for the neurodegenerative diseases with sleep disorder, such as anxiety, depression, and Parkinson’s diseases. Acknowledgments This work was supported by grants from the National Foundation of Natural Sciences of China (grant numbers 31771112, 31571037, 81901259), Shanghai Municipal Science and Technology Major Project (grant number 2018SHZDZX01) and ZJLab. Natural Science Foundation of Jiangsu Province (BK20180168), Key Project of Scientific Developing Fund of Nanjing Medical University (20l7NJMUZD125). Transparency document Transparency document related to this article can be found online at https://doi.org/10.1016/j.bbrc.2019.11.132. References [1] R.E. Roberts, C.R. Roberts, Y. Xing, Restricted sleep among adolescents: prevalence, incidence, persistence, and associated factors, Behav. Sleep Med. 9 (2011) 18e30. [2] S.M. Rajaratnam, J. Arendt, Health in a 24-h society, Lancet 358 (2001) 999e1005.
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