Abstract

Post-traumatic stress disorder (PTSD) is closely related to the exposure to traumatic events and results in the structural and functional changes of hippocampus. Human basic helix-loop-helix family member e40 (BHLHE40) was reported to be implicated with neuron maturity and neuronal differentiation. The present study aimed to reveal the role of BHLHE40 on single-prolonged stress (SPS) model of PTSD in mice. The morris water maze test, open field test and contextual fear test were conducted to assess memory deficits, anxiety-like behaviors, and freezing of mice. Western blot was performed to identify proteins and reveal their levels in hippocampal tissues. We found that mice receiving SPS exhibited increased anxiety-like behaviors, memory deficits, and prolonged freezing time. The protein levels of BHLHE40 were downregulated in the hippocampal tissues of SPS mice. SPS reduced the protein levels of glutamate receptors, while overexpression of BHLHE40 promoted glutamate receptor protein levels in SPS mice. Moreover, BHLHE40 overexpression activated the PI3K/AKT pathway. BHLHE40 overexpression ameliorated the SPS-induced PTSD-like behavioral deficits. Overall, BHLHE40 promotes glutamate receptor protein levels to ameliorate PTSD-like behaviors with the involvement of the PI3K/AKT pathway. This novel discovery may provide a potential target for the improvement of PTSD.

Key words
BHLHE40; Post-traumatic stress disorder; PI3K/AKT pathway; single-prolonged stress

INTRODUCTION

Post-traumatic stress disorder (PTSD), a complicated mental disease, is closely related to the exposure to traumatic events (Bisson et al. 2015BISSON JI, COSGROVE S, LEWIS C & ROBERT NP. 2015. Post-traumatic stress disorder. Bmj 351:.h6161 doi:10.1136/bmj.h6161.). The occurrence of PTSD is about 10-11% worldwide (Kirkpatrick & Heller 2014KIRKPATRICK HA & HELLER GM. 2014. Post-traumatic stress disorder: theory and treatment update. Int J Psychiatry Med 47: 337-346. doi:10.2190/PM.47.4.h.). In addition to war, vital diseases or accidental injury contribute to the initiation of PTSD (Turcek 2017TURCEK M. 2017. Post-Traumatic Stress Disorder. N Engl J Med 377: 1796. doi:10.1056/NEJMc1709522.). The typical symptoms of PTSD are intense fear, anxiety, hyperarousal or emotional numbing when re-experiencing trauma-related stimuli (Yehuda 2002YEHUDA R. 2002. Post-traumatic stress disorder. N Engl J Med 346: 108-114. doi:10.1056/NEJMra012941.). A previous study suggested that these dysregulations of emotion related to fear, anxiety or stress are based on a persistent, aberrant adaptation of neurobiological systems in response to traumatic events (Bisson 2007BISSON JI. 2007. Post-traumatic stress disorder. Bmj 334: 789-793. doi:10.1136/bmj.39162.538553.80.). Furthermore, the dysfunction of neurons in hippocampus is closely associated with PTSD (Kaplan et al. 2018KAPLAN GB, LEITE-MORRIS KA, WANG L, RUMBIKA KK, HEINRICHS SC, ZENG X, WU L, ARENA DT & TENG YD. 2018. Pathophysiological Bases of Comorbidity: Traumatic Brain Injury and Post-Traumatic Stress Disorder. J Neurotrauma 35: 210-225. doi:10.1089/neu.2016.4953., Liu et al. 2018bLIU R, TANG A, WANG X, CHEN X, ZHAO L, XIAO Z & SHEN S. 2018b. Inhibition of lncRNA NEAT1 suppresses the inflammatory response in IBD by modulating the intestinal epithelial barrier and by exosome-mediated polarization of macrophages. Int J Mol Med 42: 2903-2913. doi:10.3892/ijmm.2018.3829., Lucassen et al. 2014LUCASSEN PJ, PRUESSNER J, SOUSA N, ALMEIDA OF, VAN DAM AM, RAJKOWSKA G, SWAAB DF & CZÉH B. 2014. Neuropathology of stress. Acta Neuropathol 127: 109-135. doi:10.1007/s00401-013-1223-5., Shin et al. 2006SHIN LM, RAUCH SL & PITMAN RK. 2006. Amygdala, medial prefrontal cortex, and hippocampal function in PTSD. Ann N Y Acad Sci 1071: 67-79. doi:10.1196/annals.1364.007.). Over the past decades, great progress has been achieved on the potential mechanisms of PTSD. However, the exact pathology of PTSD remains unclear.

Human basic helix-loop-helix family member e40 (BHLHE40), a member of basic helix-loop-helix transcription factors, is also called differentiated embryonic chondrocyte gene 1 (DEC1), enhancer of split and hairy related protein 2 (SHARP2, rat), or stimulated with retinoic acid 13 (STRA13, mouse) (Boudjelal et al. 1997BOUDJELAL M, TANEJA R, MATSUBARA S, BOUILLET P, DOLLE P & CHAMBON P. 1997. Overexpression of Stra13, a novel retinoic acid-inducible gene of the basic helix-loop-helix family, inhibits mesodermal and promotes neuronal differentiation of P19 cells. Genes Dev 11: 2052-2065. doi:10.1101/gad.11.16.2052., Rossner et al. 1997ROSSNER MJ, DORR J, GASS P, SCHWAB MH & NAVE KA. 1997. SHARPs: mammalian enhancer-of-split- and hairy-related proteins coupled to neuronal stimulation. Mol Cell Neurosci 9: 460-475. doi:10.1006/mcne.1997.0640., Shen et al. 1997SHEN M, KAWAMOTO T, YAN W, NAKAMASU K, TAMAGAMI M, KOYANO Y, NOSHIRO M & KATO Y. 1997. Molecular characterization of the novel basic helix-loop-helix protein DEC1 expressed in differentiated human embryo chondrocytes. Biochem Biophys Res Commun 236: 294-298. doi:10.1006/bbrc.1997.6960.). A previous study showed that BHLHE40 is widely expressed in various tissues and involved in the regulation of cell proliferation, differentiation, apoptosis, biological rhythm, and lipid metabolism (Kato et al. 2014KATO Y, KAWAMOTO T, FUJIMOTO K & NOSHIRO M. 2014. DEC1/STRA13/SHARP2 and DEC2/SHARP1 coordinate physiological processes, including circadian rhythms in response to environmental stimuli. Curr Top Dev Biol 110: 339-372. doi:10.1016/b978-0-12-405943-6.00010-5., Li et al. 2016LI XM ET AL. 2016. Dec1 expression predicts prognosis and the response to temozolomide chemotherapy in patients with glioma. Mol Med Rep 14: 5626-5636. doi:10.3892/mmr.2016.5921.). BHLHE40 has anti-apoptotic potential by increasing survivin protein expression (Li et al. 2006LI Y, XIE M, YANG J, YANG D, DENG R, WAN Y & YAN B. 2006. The expression of antiapoptotic protein survivin is transcriptionally upregulated by DEC1 primarily through multiple sp1 binding sites in the proximal promoter. Oncogene 25: 3296-3306. doi:10.1038/sj.onc.1209363.). BHLHE40 overexpression selectively inactivates procaspase 3, 7, 9 to antagonize apoptosis induced by serum starvation (Li et al. 2002LI Y, ZHANG H, XIE M, HU M, GE S, YANG D, WAN Y & YAN B. 2002. Abundant expression of Dec1/stra13/sharp2 in colon carcinoma: its antagonizing role in serum deprivation-induced apoptosis and selective inhibition of procaspase activation. Biochem J 367: 413-422. doi:10.1042/bj20020514.). In addition, knockdown of endogenous BHLHE40 reverses the promotive effect of TGF-β on cell survival in breast cancer (Ehata et al. 2007EHATA S, HANYU A, HAYASHI M, ABURATANI H, KATO Y, FUJIME M, SAITOH M, MIYAZAWA K, IMAMURA T & MIYAZONO K. 2007. Transforming growth factor-beta promotes survival of mammary carcinoma cells through induction of antiapoptotic transcription factor DEC1. Cancer Res 67:.9694-9703. doi:10.1158/0008-5472.Can-07-1522.). More importantly, BHLHE40 was reported to be implicated with neuron maturity and neuronal differentiation. SHARP1 and SHARP2 (BHLHE40) are associated with synaptic plasticity in the subregions of central nervous system (Rossner et al. 1997ROSSNER MJ, DORR J, GASS P, SCHWAB MH & NAVE KA. 1997. SHARPs: mammalian enhancer-of-split- and hairy-related proteins coupled to neuronal stimulation. Mol Cell Neurosci 9: 460-475. doi:10.1006/mcne.1997.0640.). Additionally, STRA13 (BHLHE40) overexpression improves neuronal differentiation (Boudjelal et al. 1997BOUDJELAL M, TANEJA R, MATSUBARA S, BOUILLET P, DOLLE P & CHAMBON P. 1997. Overexpression of Stra13, a novel retinoic acid-inducible gene of the basic helix-loop-helix family, inhibits mesodermal and promotes neuronal differentiation of P19 cells. Genes Dev 11: 2052-2065. doi:10.1101/gad.11.16.2052.). Recently, the downregulation of DEC1 (BHLHE40) was reported to promote neurotoxicity and aggravate neurological deficit induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine in a mice model of Parkinson’s disease (Zhu et al. 2017bZHU Z, WANG YW, GE DH, LU M, LIU W, XIONG J, HU G, LI XP & YANG J. 2017b. Downregulation of DEC1 contributes to the neurotoxicity induced by MPP(+) by suppressing PI3K/Akt/GSK3beta pathway. CNS Neurosci Ther 23: 736-747. doi:10.1111/cns.12717.).

Phosphatidylinositol 3-kinase/protein kinase B (PI3K/AKT) signaling pathway, a highly conserved pathway, regulates multiple signaling cascades and plays an important role in neuronal survival (Kim et al. 2011KIM SN, KIM ST, DOO AR, PARK JY, MOON W, CHAE Y, YIN CS, LEE H & PARK HJ. 2011. Phosphatidylinositol 3-kinase/Akt signaling pathway mediates acupuncture-induced dopaminergic neuron protection and motor function improvement in a mouse model of Parkinson’s disease. Int J Neurosci 121: 562-569. doi:10.3109/00207454.2011.591515., Zhu et al. 2017aZHU Y, TANG Q, WANG G & HAN R. 2017a. Tanshinone IIA Protects Hippocampal Neuronal Cells from Reactive Oxygen Species Through Changes in Autophagy and Activation of Phosphatidylinositol 3-Kinase, Protein Kinas B, and Mechanistic Target of Rapamycin Pathways. Curr Neurovasc Res 14: 132-140. doi:10.2174/1567202614666170306105315.). Additionally, the PI3K/AKT signaling pathway is inactivated in hippocampal tissues of PTSD rat models (Zhang et al. 2020ZHANG J, XUE R, LI YF, ZHANG YZ & WEI HW. 2020. Anxiolytic-like effects of treadmill exercise on an animal model of post-traumatic stress disorder and its mechanism. J Sports Med Phys Fitness 60: 172-179. doi:10.23736/s0022-4707.20.10120-8.). The Akt signaling pathway can ameliorate PTSD symptoms by promoting synaptic plasticity and glutamate transmission (Liu et al. 2018aLIU G ET AL. 2018a. rTMS Ameliorates PTSD Symptoms in Rats by Enhancing Glutamate Transmission and Synaptic Plasticity in the ACC via the PTEN/Akt Signalling Pathway. Mol Neurobiol 55: 3946-3958. doi:10.1007/s12035-017-0602-7.). Glutamate receptors, the predominant excitatory neurotransmitter receptors in the mammalian brain, determine the synaptic transmission efficiency (Barnes et al. 2020BARNES JR, MUKHERJEE B, ROGERS BC, NAFAR F, GOSSE M & PARSONS MP. 2020. The Relationship Between Glutamate Dynamics and Activity-Dependent Synaptic Plasticity. J Neurosci 40:.2793-2807. doi:10.1523/jneurosci.1655-19.2020., Zarebidaki et al. 2020ZAREBIDAKI F, CAMACHO M, BROCKMANN MM, TRIMBUCH T, HERMAN MA & ROSENMUND C. 2020. Disentangling the Roles of RIM and Munc13 in Synaptic Vesicle Localization and Neurotransmission. J Neurosci 40 :9372-9385. doi:10.1523/jneurosci.1922-20.2020.), and play crucial roles in learning and memory behaviors (Krugers et al. 2010KRUGERS HJ, HOOGENRAAD CC & GROC L. 2010. Stress hormones and AMPA receptor trafficking in synaptic plasticity and memory. Nat Rev Neurosci 11: 675-681. doi:10.1038/nrn2913., Van den Oever et al. 2008VAN DEN OEVER MC, GORIOUNOVA NA, LI KW, VAN DER SCHORS RC, BINNEKADE R, SCHOFFELMEER AN, MANSVELDER HD, SMIT AB, SPIJKER S & DE VRIES TJ. 2008. Prefrontal cortex AMPA receptor plasticity is crucial for cue-induced relapse to heroin-seeking. Nat Neurosci 11: 1053-1058. doi:10.1038/nn.2165.). Increasing evidence relates abnormalities in the glutamatergic system to stress response and PTSD (Pitman et al. 2012PITMAN RK, RASMUSSON AM, KOENEN KC, SHIN LM, ORR SP, GILBERTSON MW, MILAD MR & LIBERZON I. 2012. Biological studies of post-traumatic stress disorder. Nat Rev Neurosci 13: 769-787. doi:10.1038/nrn3339., Popoli et al. 2011POPOLI M, YAN Z, MCEWEN BS & SANACORA G. 2011. The stressed synapse: the impact of stress and glucocorticoids on glutamate transmission. Nat Rev Neurosci 13: 22-37. doi:10.1038/nrn3138.). When re-experiencing trauma-related stimuli, PTSD subjects have an injured glutamate system and fail to maintain adequate glutamate transmission, which causes heightened over-attention, stress reactions and fear levels (Yang et al. 2015YANG ZY, QUAN H, PENG ZL, ZHONG Y, TAN ZJ & GONG QY. 2015. Proton magnetic resonance spectroscopy revealed differences in the glutamate + glutamine/creatine ratio of the anterior cingulate cortex between healthy and pediatric post-traumatic stress disorder patients diagnosed after 2008 Wenchuan earthquake. Psychiatry Clin Neurosci 69: 782-790. doi:10.1111/pcn.12332.).

In our study, we investigated the role of BHLHE40 in single-prolonged stress (SPS) mice model of PTSD. Protein levels of glutamate receptors in hippocampal tissues of mice were detected, and the effects of BHLHE40 on glutamate receptors were explored. Furthermore, the regulatory effects of BHLHE40 on the PI3K/AKT signaling pathway were identified. This novel discovery may provide a potential target for the improvement of PTSD.

MATERIALS AND METHODS

Animals and ethics statement

Adult male C57BL/6J mice (7-9 weeks) were purchased from Vital River Co. Ltd. (Beijing, China) and were housed in a temperature-controlled room (24 ± 1°C) under 12-hour light/dark cycles (6:00 AM to 6:00 PM) with ad libitum access to food and water for 1-week for acclimatization. All experimental procedures were approved by the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals and the Animal Care and Use Committee of Mental Health Center of Xinjiang in China.

The single prolonged stress (SPS) operation

SPS mice models were established as previously described (Azevedo et al. 2020AZEVEDO H, FERREIRA M, MASCARELLO A, OSTEN P & GUIMARãES CRW. 2020. Brain-wide mapping of c-fos expression in the single prolonged stress model and the effects of pretreatment with ACH-000029 or prazosin. Neurobiol Stress 13: 100226. doi:10.1016/j.ynstr.2020.100226., Song et al. 2018SONG D, GE Y, CHEN Z, SHANG C, GUO Y, ZHAO T, LI Y, WU N, SONG R & LI J. 2018. Role of dopamine D3 receptor in alleviating behavioural deficits in animal models of post-traumatic stress disorder. Prog Neuropsychopharmacol Biol Psychiatry 84: 190-200. doi:10.1016/j.pnpbp.2018.03.001., Wang et al. 2012WANG H, ZUO D, HE B, QIAO F, ZHAO M & WU Y. 2012. Conditioned fear stress combined with single-prolonged stress: a new PTSD mouse model. Neurosci Res 73: 142-152. doi:10.1016/j.neures.2012.03.003., Yu et al. 2013YU H, WATT H, KESAVAN C & MOHAN S. 2013. The negative impact of single prolonged stress (SPS) on bone development in mice. Stress 16: 564-570. doi:10.3109/10253890.2013.806908.). In brief, the mice were restrained for 2 h in a BD Falcon© 50-mL conical tube with a screw-on top and air holes located 1/2 cm apart. Next, the mice were put in an 18-cm diameter, 50-cm height clear acrylic cylinder individually, and the mice were forced to swim in cylinder filled with water (22-24°C) to 2/3 of the height of the cylinder for 20 min. After a 15-min recuperation period, mice were exposed to diethyl ether till they lost consciousness. Next, the mice were immediately placed in their cages and left undisturbed for 7 days. The mice in sham group were kept in cages adjacent to the SPS mice during the treatment without food and water. The treatment schedule and behavioral test design is shown in Figure 1.

Figure 1
The design of the current study. To investigate whether SPS mice develops PTSD-like behaviors, the mice were randomly divided into sham (n = 18) and SPS (n = 36) groups. Open field test, MWM and contextual fear test were conducted on mice on day 9, 10 and 11, separately, after SPS induction. On day 12, some of the mice (n = 9 in sham group; n = 9 in SPS group) were sacrificed, and the hippocampal tissues were collected to detect the BHLHE40 protein levels. To investigate the functions of BHLHE40 on PTSD-like behaviors of mice, the remaining SPS mice were divided into 4 groups: sham (n = 9), SPS (n = 9), SPS+AAV-NC (n = 9), SPS+AAV-BHLHE40 (n = 9). Some of the mice were injected with AAV-NC and AAV-BHLHE40 on day 12. After injection of AAV for 28, 29, 30 days, open field test, MWM and contextual fear test were conducted, respectively. After injection of AAV for 30 days (42 days after SPS induction), mice were sacrificed, and the hippocampal tissues were collected.

Adeno-associated virus (AAV) injection

AAV (serotype 2, characterized by poor-immunogenicity; Vigene Biosciences, shanghai, China) containing the coding sequences (provided in Supplementary Material) of BHLHE40 or empty AAV vectors (pAV-MeCP2-GFP; 4272 bp) as the negative controls were injected into mice. First, mice were intraperitoneally injected with a mixture of ketamine (7.0 mg/mL) and xylazine (0.44 mg/mL) dissolved in 0.9% saline for anesthesia. With the head of each mouse fixed in a stereotactic apparatus, 0.5 µL of AAVs (10¹² v.g/mL) were bilaterally microinjected to the bilateral hippocampal CA1 region (2.0 mm anteroposterior from bregma; ± 1.5 mm mediolateral; 1.70 mm dorsoventral). Four weeks after AAV injection, mice were used for behavioral tests. After behavioral tests, the rats were sacrificed, and the hippocampal tissues were collected for western blot analysis.

Morris water maze (MWM) test

The MWM test was conducted to measure the memorizing and spatial learning abilities of mice according to a previous study (Broussard et al. 2018BROUSSARD JI ET AL. 2018. Repeated mild traumatic brain injury produces neuroinflammation, anxiety-like behaviour and impaired spatial memory in mice. Brain Inj 32: 113-122 doi:10.1080/02699052.2017.1380228.). The test includes a platform trial and a probe trial and was conducted in a standing pool (diameter: 120 cm; height: 40 cm) filled with water (depth: 30 cm; temperature: 22-24°C). Before the first trial, the visible escape platform was placed 1 cm above the water surface. For platform trial, mice were placed in a random point with their noses facing the wall, and the escape latency (the time that mice reached the platform) of mice was automatically recorded by a video-tracking system. If the mice were unable to find the hidden platform in 2 minutes, they would be helped to stand on the platform for 20 s. After the last acquisition trial, the probe test was performed. For probe trial, the platform was removed, and mice were placed at a new position with their noses facing the wall to swim at random. The total distance of mice spent in the target quadrant in 120 s was recorded and calculated.

Open field test

The open field test was used to evaluate anxiety-like behaviors of mice as previously described (Feng et al. 2020FENG DY, GUO BL, LIU GH, XU K, YANG J, TAO K, HUANG J, WANG LY, WANG W & WU SX. 2020. Nerve growth factor against PTSD symptoms: Preventing the impaired hippocampal cytoarchitectures. Prog Neurobiol 184: 101721. doi:10.1016/j.pneurobio.2019.101721., Pi et al. 2019PI H ET AL. 2019. AKT inhibition-mediated dephosphorylation of TFE3 promotes overactive autophagy independent of MTORC1 in cadmium-exposed bone mesenchymal stem cells. Autophagy 15: 565-582. doi:10.1080/15548627.2018.1531198.). In the present study, mice were put in the center of a cubic chamber (36 × 36 × 36 cm). The mice were permitted to move for 5 min under dim white light. The time and distance of mice that spent in central area were recorded and analyzed by an automatic analyzing system (Intelligent Recognition & Communications Biotech Co. Ltd, Suzhou). The inner surface was cleaned with 70% ethanol between adjacent sessions.

The contextual fear test

The contextual fear test was conducted as previously described (Feng et al. 2020FENG DY, GUO BL, LIU GH, XU K, YANG J, TAO K, HUANG J, WANG LY, WANG W & WU SX. 2020. Nerve growth factor against PTSD symptoms: Preventing the impaired hippocampal cytoarchitectures. Prog Neurobiol 184: 101721. doi:10.1016/j.pneurobio.2019.101721.). In brief, mice were placed in a contextual chamber (242 × 242 × 300 mm operant chambers) for an acclimation period (180 s). Then, mice were treated with a pure tone (28 s, 1 kHz, 90 dB) and co-terminated with a foot shock (2 s, 0.8 mA) using a stainless-steel grid floor (Med Associates Inc, USA) three times. Afterwards, mice were kept in the chamber for another 2 min. After 24 h of fear conditioning training, mice were replaced in the contextual chamber for 5 min without tone or foot shock exposure, and the freezing time of mice was automatically recorded using a FreezeScan software (CleverSys Inc). The freezing response upon re-exposure to the shock context is a measure of conditioned associative fear memory reflecting the response to trauma-related cues as a symptom of PTSD (Siegmund & Wotjak 2007SIEGMUND A & WOTJAK CT. 2007. A mouse model of posttraumatic stress disorder that distinguishes between conditioned and sensitised fear. J Psychiatr Res 41: 848-860. doi:10.1016/j.jpsychires.2006.07.017.). Freezing was defined as the absence of any movement except for respiration.

Western blot

The mice were killed respectively at 12 or 42 days after SPS. The brains of mice were quickly resected, and the hippocampal tissues of mice were isolated and immediately maintained at -80°C. The total proteins from mice hippocampus were extracted (Beyotime Biotechnology, China). Then, the protein samples (50 μg/lane) from each group were subjected to 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Later, protein samples were transferred onto a polyvinylidene fluoride membrane, and were then blocked with 5% defatted milk for 2 h. Afterwards, the membrane was incubated with Glutamate Receptor 1 antibody (1:1000, ab31232), Glutamate Receptor 2 antibody (1:1000, ab206293), Glutamate Receptor 3 antibody (1:1000, ab40845), Glutamate Receptor 4 antibody (1:1000, ab115322), BHLHE40 antibody (1:1000, ab23797), PI3Kp110α antibody (1:1000, ab40776), phosphorylated AKT (ser473) antibody (1:1000, ab8932), AKT antibody (1:1000, ab8805) and GAPDH antibody (1:1000, ab9485) at 4°C overnight, and then incubated with secondary antibodies at room temperature for 2 h. The antibodies were purchased from Abcam (Cambridge, UK). Then, blots were placed to the autoradiography by ECL reagents after washing in Tris buffered saline with Tween. The protein bands were visualized by the Gel Image Analysis System and quantified by ImageJ software (National Institute of Health, USA).

Statistical Analysis

The data were analyzed by SPSS 19.0 software and shown as the means ± standard deviation. The unpaired student’s t test was employed to compare the differences between the groups. One-way analysis of variance (ANOVA) followed by Tukey’s post hoc test was applied for the comparison of more than two groups. p value less than 0.05 had statistical significance.

RESULT

Successful establishment of SPS model mice and the downregulation of BHLHE40 protein levels in hippocampal tissues of SPS mice

As shown in Fig. 2a (t(16)=10.804, p<0.001), the escape latency of mice in visible platform trial was prolonged by treatment of SPS. The total distance that mice spent in target quadrant was shorter in SPS group compared with that in sham group in the probe trial (t(16)=3.64, p=0.002, Fig. 2b). In addition, according to the results of the open field test, compared to sham operated mice, SPS mice traveled shorter central distance and spent less central time (2c: t(16)=19.955, p<0.001; 2d: t(16)=12.363, p<0.001, Fig. 2c-d). Moreover, mice exhibit increased fear response to SPS in contextual fear test. As illustrated in Fig. 2e (t(16)=12.913, p<0.001), the freezing time of mice in SPS group was longer than those in sham group in the contextual fear conditioning paradigms. All these data indicated that SPS model was successfully established and can be used for following assays. At last, we measured the protein levels of BHLHE40 in hippocampal tissues of mice and found that the BHLHE40 protein levels were lower in SPS group than that in sham group (t(16)=10.927, p<0.001, Fig. 2f).

Figure 2
Successful establishment of SPS model mice and the downregulation of BHLHE40 protein levels in hippocampal tissues of SPS mice. (a) The escape latency that the mice (n=9/group) of the sham/SPS group spent in the visible platform trial on day 10. This experiment was conducted 3 times. (b) Total distance that the mice (n=9/group) of the sham/SPS group spent in target quadrant in the probe trial on day 10. This experiment was conducted once. (c-d) The distance and time that sham and SPS mice (n=9/group) spent in the central area in open field test on day 9. This experiment was conducted once. (e) Fear conditioning test of contextual fear memory assessment in sham and SPS groups (n=9/group) on day 11. This experiment was conducted once. (f) The protein levels of BHLHE40 in hippocampal tissues of SPS or sham mice (n=9/group) were determined by western blot. This experiment was conducted 3 times. Student’s t test was applied for data analysis in Figure 2. **p< 0.01, ***p< 0.001.

BHLHE40 overexpression promoted glutamate receptor protein levels and activated the PI3K/AKT signaling pathway

To begin with, we significantly overexpressed BHLHE40 protein levels by injection of AAV-BHLHE40 in mice (F(3,32)=832.201, p<0.001, Fig. 3a). Effects of BHLHE40 on glutamate receptors were assessed. According to the western blot, the protein levels of glutamate receptors including glutamate receptor 1, glutamate receptor 2, glutamate receptor 3 and glutamate receptor 4 were reduced by SPS, and such results were partially reversed by BHLHE40 overexpression (GluR1: F(3,32)=139.966, p<0.001; GluR2: F(3,32)=89.293, p<0.001; GluR3: F(3,32)=42.95, p<0.001; GluR4: F(3,32)=138.745, p<0.001, Fig. 3b). Moreover, we evaluated the protein levels of PI3Kp110α, phosphorylated AKT (ser473) and total AKT. The results demonstrated that the decreased protein levels of PI3Kp110α and phosphorylated AKT (ser473) induced by SPS were rescued by overexpression of BHLHE40 (Pi3kp110α: F(3,32)=65.276, p<0.001; p-AKT/AKT: F(3,32)=194.665, p<0.001, Fig. 3c).

Figure 3
BHLHE40 overexpression promoted protein levels of glutamate receptors and activated the PI3K/AKT signaling pathway. (a) The overexpression efficacy of BHLHE40 in mice of sham, SPS, SPS+AAV-NC, SPS+AAV-BHLHE40 groups (n=9/group) was evaluated by western blot analysis. This experiment was conducted 3 times. (b-c) The protein levels of glutamate receptors, PI3Kp110α, phosphorylated AKT (ser473) and total AKT in mice of sham, SPS, SPS+AAV-NC, SPS+AAV-BHLHE40 groups (n=9/group) were examined by western blot analysis. This experiment was conducted 3 times. One-way ANOVA was applied for data analysis in Figure 3. ***p< 0.001.

BHLHE40 overexpression mitigated PTSD-like behaviors of mice

Next, we explored the effects of BHLHE40 on animal PTSD-like behaviors. First, the prolonged escape latency (F(3,32)=87.766, p<0.001) and the shortened total distance (F(3,32)=14.553, p<0.001) of mice spent in target quadrant induced by SPS were rescued by BHLHE40 overexpression (Fig. 4a-b), In addition, the open field test revealed that the decreased central distance (F(3,32)=163.421, p<0.001) and time (F(3,32)=172.643, p<0.001) spent by SPS mice were neutralized by the injection of AAV-BHLHE40 (Fig. 4c-d). Finally, the contextual fear test showed that the increase of freezing time resulting from SPS was partially rescued by BHLHE40 overexpression (F(3,32)=122.241, p<0.001, Fig. 4e).

Figure 4
BHLHE40 overexpression mitigated PTSD-like behaviors of mice. (a) The escape latency of mice of sham, SPS, SPS+AAV-NC, SPS+AAV-BHLHE40 groups (n=9/group) in the visible platform trial on day 41. This experiment was conducted 3 times. (b) Total distance that mice of sham, SPS, SPS+AAV-NC, SPS+AAV-BHLHE40 groups (n=9/group) spent in target quadrant in the probe trial on day 41. This experiment was conducted once. (c-d) The distance and time that mice of sham, SPS, SPS+AAV-NC, SPS+AAV-BHLHE40 groups (n=9/group) spent in the central area in open field test on day 40. This experiment was conducted once. (e) Fear conditioning test of contextual fear memory assessment in mice of sham, SPS, SPS+AAV-NC, SPS+AAV-BHLHE40 groups (n=9/group) on day 42. This experiment was conducted once. One-way ANOVA was applied for data analysis in Figure 4. ***p< 0.001.

DISCUSSION

As a complex and debilitating neuropathology, PTSD has brought considerable troubles to life quality of the patients (Series 2019SERIES P. 2019. Post-traumatic stress disorder as a disorder of prediction. Nat Neurosci 22: 334-336. doi:10.1038/s41593-019-0345-z.). PTSD has an overlap of comorbidity and symptoms with other disorders such as fear, anxiety, and suicidal ideation. To date, acupuncture, psychoanalytic therapy, psychotherapy, and medical treatment have been widely used to improve PTSD (Buhmann & Andersen 2017BUHMANN CB & ANDERSEN HS. 2017. Diagnosing and treating post-traumatic stress disorder. Ugeskr Laeger 179: V12160914., Oh et al. 2018OH JY, KIM YK, KIM SN, LEE B, JANG JH, KWON S & PARK HJ. 2018. Acupuncture modulates stress response by the mTOR signaling pathway in a rat post-traumatic stress disorder model. Sci Rep 8: 11864. doi:10.1038/s41598-018-30337-5.). Additionally, selective serotonin reuptake inhibitors (SSRIs) including sertraline, paroxetine and fluoxetine were reported to be effective for PTSD treatment (Akiki & Abdallah 2018AKIKI TJ & ABDALLAH CG. 2018. Are There Effective Psychopharmacologic Treatments for PTSD? J Clin Psychiatry 80: 18ac12473. doi:10.4088/JCP.18ac12473., Bushnell et al. 2018BUSHNELL GA, COMPTON SN, DUSETZINA SB, GAYNES BN, BROOKHART MA, WALKUP JT, RYNN MA & STÜRMER T. 2018. Treating Pediatric Anxiety: Initial Use of SSRIs and Other Antianxiety Prescription Medications. J Clin Psychiatry 79: 16m11415. doi:10.4088/JCP.16m11415.). However, the increased prescription of SSRIs may induce suicide, especially for adolescents (Locher et al. 2017LOCHER C, KOECHLIN H, ZION SR, WERNER C, PINE DS, KIRSCH I, KESSLER RC & KOSSOWSKY J. 2017. Efficacy and Safety of Selective Serotonin Reuptake Inhibitors, Serotonin-Norepinephrine Reuptake Inhibitors, and Placebo for Common Psychiatric Disorders Among Children and Adolescents: A Systematic Review and Meta-analysis. JAMA Psychiatry 74: 1011-1020. doi:10.1001/jamapsychiatry.2017.2432.). Thus, to identify novel biomarkers for PTSD is necessary.

To explore the development of PTSD, previous studies have constructed a variety of models including the restraint stress model, the inescapable shock model, the predator-stress model, the SPS model and the social defeat stress model to mimic PTSD (Aspesi & Pinna 2019ASPESI D & PINNA G. 2019. Animal models of post-traumatic stress disorder and novel treatment targets. Behav Pharmacol 30: 130-150. doi:10.1097/fbp.0000000000000467). In our study, we established SPS mice models to explore the role of BHLHE40 in PTSD. According to previous studies (Aspesi & Pinna 2019ASPESI D & PINNA G. 2019. Animal models of post-traumatic stress disorder and novel treatment targets. Behav Pharmacol 30: 130-150. doi:10.1097/fbp.0000000000000467, Flandreau & Toth 2018FLANDREAU EI & TOTH M. 2018. Animal Models of PTSD: A Critical Review. Curr Top Behav Neurosci 38: 47-68. doi:10.1007/7854_2016_65.), SPS can imitate the physiological challenges of PTSD. The enhancement of hypothalamic-pituitary-adrenal (HPA) negative feedback may contribute to a rise of glucocorticoid receptor (GR) expression in hippocampus 7 days after the exposure to stress. Furthermore, other research revealed that SPS operation contributed to behavioral abnormalities to imitate PTSD symptoms like hyperarousal or contextual freezing. In our study, the SPS caused increased anxiety levels, acoustic startle responses and contextual freezing time. Hippocampus, a region involved in memory, learning and contextual fear extinction, has been confirmed to be correlated with the progression of PTSD (Coburn 2018COBURN D. 2018. Using MR to View PTSD’s Effect on the Amygdala and Hippocampus. Radiol Technol 89: 501-504.). Additionally, the structural or functional alterations of hippocampus have been observed in the pathophysiology of PTSD (Wingenfeld & Wolf 2014WINGENFELD K & WOLF OT. 2014. Stress, memory, and the hippocampus. Front Neurol Neurosci 34: 109-120. doi:10.1159/000356423.). We detected BHLHE40 protein levels in hippocampal tissues and found that BHLHE40 protein levels were lower in SPS group than sham group.

The glutamate receptor system consists of three subfamilies, two ligand-gated ion channels (ionotropic receptors), the N-methyl-d-aspartate receptor (NMDAR) and the α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid/kainate receptors (AMPAR/KAR), and one metabotropic receptor (Hollmann & Heinemann 1994HOLLMANN M & HEINEMANN S. 1994. Cloned glutamate receptors. Annu Rev Neurosci 17: 31-108. doi:10.1146/annurev.ne.17.030194.000335.) q. Metabotropic glutamate receptors are classified into eight subtypes (mGluR1-8), which are subdivided into group I-III based on sequence homology and intracellular G-protein coupling activity (Niswender & Conn 2010NISWENDER CM & CONN PJ. 2010. Metabotropic glutamate receptors: physiology, pharmacology, and disease. Annu Rev Pharmacol Toxicol 50: 295-322. doi:10.1146/annurev.pharmtox.011008.145533.). Group I is characterized by mGluR1 and mGluR5 and causes stimulation of Phospholipase C beta and activation of protein kinase C. Group II, composed of mGluR2 and mGluR3, and group III consisting of mGluR4 and mGluR6-8, are responsible for the inhibition of adenylyl cyclase, which potentiates the PI3K/Akt signaling pathway (Iacovelli et al. 2002IACOVELLI L, BRUNO V, SALVATORE L, MELCHIORRI D, GRADINI R, CARICASOLE A, BARLETTA E, DE BLASI A & NICOLETTI F. 2002. Native group-III metabotropic glutamate receptors are coupled to the mitogen-activated protein kinase/phosphatidylinositol-3-kinase pathways. J Neurochem 82: 216-223. doi:10.1046/j.1471-4159.2002.00929.x.). The metabotropic GluRs in the central nervous system mediate the postsynaptic neuronal response to glutamate, modulating both NMDAR and AMPAR activities, as well as cellular proliferation, growth, migration, survival, and calcium-mediated cellular homeostasis (Pin & Duvoisin 1995PIN JP & DUVOISIN R. 1995. The metabotropic glutamate receptors: structure and functions. Neuropharmacology 34: 1-26. doi:10.1016/0028-3908(94)00129-g.)Montpellier, were decreased in hippocampal tissues of sham mice and were increased by BHLHE40 overexpression. BHLHE40 has been reported to be a crucial repressor of IL-10 (Huynh et al. 2018HUYNH JP ET AL. 2018. Bhlhe40 is an essential repressor of IL-10 during Mycobacterium tuberculosis infection. J Exp Med 215: 1823-1838. doi:10.1084/jem.20171704.), and IL-10 restores glutamate receptor-controlled Ca 2⁺-pathway in brain circuits (Turovskaya et al. 2020TUROVSKAYA MV, EPIFANOVA EA, TARABYKIN VS, BABAEV AA & TUROVSKY EA. 2020. Interleukin-10 restores glutamate receptor-mediated Ca(2+)-signaling in brain circuits under loss of Sip1 transcription factor. Int J Neurosci 6: 1-12. doi:10.1080/00207454.2020.1803305.). BHLHE40 can increase the expression of CXCL12 (Teng et al. 2020TENG YS ET AL. 2020. Upexpression of BHLHE40 in gastric epithelial cells increases CXCL12 production through interaction with p-STAT3 in Helicobacter pylori-associated gastritis. Faseb J 34: 1169-1181. doi:10.1096/fj.201900464RR.), and CXCL12 facilitates glutamate synaptic activity at serotonin neurons in the rat dorsal raphe nucleus (Heinisch & Kirby 2010HEINISCH S & KIRBY LG. 2010. SDF-1alpha/CXCL12 enhances GABA and glutamate synaptic activity at serotonin neurons in the rat dorsal raphe nucleus. Neuropharmacology 58:.501-514. doi:10.1016/j.neuropharm.2009.08.022.). Our study revealed that BHLHE40 overexpression increased expression of metabotropic glutamate receptors to reverse the increase of anxiety levels and contextual freezing time resulting from SPS in mice.

Moreover, BHLHE40 can activate the PI3K/AKT signaling pathway by promoting the phosphorylation processes of PI3K and AKT in neurons (Zhu et al. 2017bZHU Z, WANG YW, GE DH, LU M, LIU W, XIONG J, HU G, LI XP & YANG J. 2017b. Downregulation of DEC1 contributes to the neurotoxicity induced by MPP(+) by suppressing PI3K/Akt/GSK3beta pathway. CNS Neurosci Ther 23: 736-747. doi:10.1111/cns.12717.). Additionally, repetitive transcranial magnetic stimulation regulates the PTEN/Akt signaling pathway to ameliorate PTSD symptoms by promoting synaptic plasticity and glutamate transmission in the anterior cingulate cortex of rats (Liu et al. 2018aLIU G ET AL. 2018a. rTMS Ameliorates PTSD Symptoms in Rats by Enhancing Glutamate Transmission and Synaptic Plasticity in the ACC via the PTEN/Akt Signalling Pathway. Mol Neurobiol 55: 3946-3958. doi:10.1007/s12035-017-0602-7.). Metabotropic glutamate receptors can activate the PI3K/Akt signaling pathway (Willard & Koochekpour 2013WILLARD SS & KOOCHEKPOUR S. 2013. Glutamate, glutamate receptors, and downstream signaling pathways. Int J Biol Sci 9: 948-959. doi:10.7150/ijbs.642.6). Therefore, we hypothesized that BHLHE40 activates the PI3K/AKT signaling pathway in hippocampus to mitigate PTSD-like behaviors. The present study revealed that protein levels of PI3Kp110α, phosphorylated AKT were lower in hippocampal tissues of SPS mice than sham mice, while BHLHE40 overexpression increased PI3Kp110α and phosphorylated AKT protein levels.

However, our study has limitations. First, the expression status and biological roles of BHLHE40 in neurons were not explored. Second, the effects of the PI3K/AKT signaling pathway on PTSD behaviors were not evaluated. At last, the detailed molecular mechanisms of BHLHE40 downregulation in PTSD deserved further exploration.

In conclusion, BHLHE40 alleviates PTSD-like behaviors with the involvement of the PI3K/AKT signaling pathway in a glutamate receptor-dependent manner in mice. This novel discovery may provide a potential target for the improvement of PTSD.

ACKNOWLEDGMENTS

The authors sincerely appreciate all members participated in this study.

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