Immobilization stress induces XBP1 splicing in the mouse brain
Toru Hosoi, Hitomi Kimura, Yosuke Yamawaki, Kohei Mori, Koichiro Ozawa
a Department of Pharmacotherapy, Graduate School of Biomedical and Health Sciences, Hiroshima University, 1-2-3 Kasumi, Minami-ku, Hiroshima 734- 8551, Japan
b Department of Cellular and Molecular Pharmacology, Institute of Biomedical and Health Sciences, Hiroshima University, 1-2-3 Kasumi, Minami-ku, Hiroshima 734-8553, Japan
A B S T R A C T
Cells activate the unfolded protein response (UPR) to cope with endoplasmic reticulum (ER) stress. In the present study, we investigated the possible involvement of psychological stress on UPR induction in the mouse brain. When mice were exposed to immobilization stress for 8 h, XBP1 mRNA splicing was significantly induced in the hippocampus, cortex, hypothalamus, cerebellum, and brain stem. On the other hand, we did not observe any increase in XBP1 splicing in the liver, suggesting that this effect is specific to the brain. Stress-induced XBP1 splicing was attenuated 2 days after immobilization stress. We did not observe increases in any other UPR genes, such as CHOP or GRP78, in mouse brains after immobilization stress. These findings indicate an important specific role of XBP1 in response to psy- chological stress in the mouse brain.
1. Introduction
The endoplasmic reticulum (ER) is an organelle involved in maintaining protein homeostasis. However, stress exposure leads to an accumulation of unfolded proteins in the ER, causing ER stress [1e3]. To cope with ER stress upon accumulation of unfolded/ aggregated proteins, cells activate the unfolded protein response (UPR) [4]. There are three major pathways of the UPR: the inositol- requiring enzyme-1 (IRE1) pathway, the double stranded RNA- activated protein kinase (PKR)-like ER kinase (PERK) pathway, and the activating transcription factor 6 (ATF6)-mediated path- ways. UPR activation increases X-box binding protein 1 (XBP1) splicing, C/EBP-homologous protein (CHOP), and glucose regulated protein 78 (GRP78). Accumulating evidence indicates the possible involvement of ER stress in the development of neurodegenerative diseases, including Alzheimer’s disease, Parkinson’s disease, and brain ischemia [5e7]. The pathophysiological mechanisms under- lying the development of mental disorders are currently unknown, but it has been speculated that genetic as well as environmental factors contribute to the pathogenesis of disease. In the present study, we hypothesized possibility that psychological stress would affect ER stress in the brain. We explored a possible link between psychological stress and ER stress, and found that immobilization stress activated XBP1 splicing, an ER stress-response factor, in the mouse brain.
2. Materials and methods
2.1. Animals
Adult male C57BL/6 Cr Slc mice were obtained from SLC (Hamamatsu, Japan). Mice were maintained in our animal facility at 22e24 ◦C under a constant day-night rhythm, and given food and water ad libitum. All animal experiments were carried out in accordance with the NIH Guidelines for the Care and Use of Labo- ratory Animals, and approved by the Animal Care and Use Com- mittee at Hiroshima University.
2.2. Immobilization stress
On the day of the experiment, the four limbs of the mice were corded and bonded to the board, so that the mice were lying on their backs for 8 h from 9 a.m. to 5 p.m. Food and water were not available during the experiment. For unstressed control mice, the four limbs of the mice were corded, but not bonded to the board. Food and water were also not available for control mice during the experiment.
2.3. Tissue isolation
Mice were killed by cervical spine fracture dislocation, and the brain and liver were quickly removed. The tissue were washed with ice cold PBS and the hippocampus, cortex, hypothalamus, cere- bellum, and brain stem were dissected on an ice-cold plate. Then, the samples were snap-frozen in liquid nitrogen and stored at —80 ◦C until use.
2.4. Gene expression analysis
Total RNA was isolated using TRI reagent (Sigma-Aldrich, St. Louis, MO, USA). Reverse transcriptase-polymerase chain reaction (RT-PCR) was performed as previously described [8]. cDNA was synthesized from 2 mg of total RNA by reverse transcription using 25 U of Superscript™ Reverse Transcriptase III (Invitrogen) and 0.25 mg of Oligo(dt)12e18 primer (Invitrogen) in a 20-ml reaction mixture containing First-Strand Buffer (Invitrogen), 1 mM dNTP mix, 10 mM DTT, and 20 U of RNaseOUT Recombinant Ribonuclease Inhibitor (Invitrogen). Total RNA and the Oligo (dt) 12e18 primer were pre-incubated at 70 ◦C for 10 min prior to reverse transcrip- tion. After incubation for 1.5 h at 46 ◦C, the reaction was terminated by incubating samples for 15 min at 70 ◦C. For PCR amplification, 1.2 ml of cDNA was added to 10.8 ml of a reaction mix containing 0.2 mM of each primer, 0.2 mM dNTP mix, 0.6 U Taq polymerase (PCR for XBP1 was performed using Phusion hot start, Thermo Fisher Scientific. Other PCR reactions were performed using Expand High FidelityPLUS PCR System, Roche Diagnostics), and reaction buffer. PCR was performed in a DNA Thermal Cycler (MJ Research, PTC-220). The following primer sequences were used: XBP1; up- stream, 50ecct tgt ggt tga gaa cca gg-30, and downstream, 50-cta gag gct tgg tgt ata-30, GRP78; upstream, 50ectg ggt aca ttt gat ctg act gg-30, and downstream, 50-gca tcc tgg tgg ctt tcc agc cat tc-30, CHOP; upstream, 50eccc tgc ctt tca cct tgg-30, and downstream, 50-ccg ctc gtt ctc ctg ctc-30, GAPDH; upstream, 50-aaa ccc atc acc atc ttc cag-30 and downstream, 50-agg ggc cat cca cag tct tct-3’. The PCR products (10 ml) were resolved by electrophoresis in an 8% polyacrylamide gel in TBE buffer. The gel was stained with ethidium bromide and photographed under ultraviolet light. The density of each band was measured using Image J 1.37v (Wayne Rasband, NIH) software.
2.5. Statistics
Results are expressed as the mean ± S.E. Statistical analyses were performed using Student’s t-test.
3. Results
3.1. Immobilization stress induced XBP1 splicing in the brain but not the liver
Upon ER stress, ER stress sensor protein, IRE1, is activated and induces XBP1 splicing [1,9,10]. To analyze whether psychological stress affects the UPR, we investigated the effect of immobilization stress on XBP1 splicing in mice. Eight hours after immobilization stress, we analyzed the levels of XBP1 splicing in the hippocampus, cortex, hypothalamus, cerebellum, and brain stem of mice brains. We observed that XBP1 splicing was increased after immobilization stress in these brain regions (Fig. 1CeE). To analyze whether the XBP1 splicing is increased peripherally, we analyzed the effect of immobilization stress on XBP1 splicing in the liver. As shown in Fig. 1F, we did not observe any increase in XBP1 splicing in the liver 8 h after immobilization stress. These findings suggest that psy- chological stress increases XBP1 splicing specifically in the brain.
3.2. Immobilization stress-induced XBP1 splicing was normalized 2 days after stress
Next, we investigated whether immobilization stress-induced XBP1 splicing is sustained after stress exposure. Mice were exposed to immobilization stress from 9 a.m. to 5 p.m., and the level of XBP1 splicing in the brain 48 h after the stress exposure was analyzed. As shown in Fig. 2, we did not observe any increase in XBP1 splicing in the cortex and hypothalamus regions of the brain. These findings suggest that immobilization stress-induced XBP1 splicing is normalized to basal levels 48 h after stress exposure.
3.3. Immobilization stress may not increase GRP78 or CHOP levels in the brain
ER stress-induced activation of stress sensor proteins, such as IRE1, PERK, and ATF6, increases UPR genes, such as GRP78 and CHOP [3,11,12]. Therefore, we next analyzed whether immobiliza- tion stress increased expression of GRP78 and CHOP. Eight hours after stress exposure, expression levels of GRP78 and CHOP in mice brains were analyzed. As shown in Fig. 3, we did not detect any increase in GRP78 and CHOP levels after stress exposure. We next exposed mice to immobilization stress for 8 h from 9 a.m. to 5 p.m. and analyzed GRP78 and CHOP levels in the brain 48 h after the
4. Discussion
Environmental factors may contribute to the progression of mental disorders. However, the underlying mechanisms that un- derlie this process are currently unknown. In the present study, we found that XBP1, an ER stress-induced UPR gene, may be involved in the psychological stress response in the central nervous system (CNS).
When cells are exposed to stress, which causes accumulation of unfolded proteins, ER stress is induced and IRE1, PERK, and ATF6 branches of the UPR are activated [1,3]. XBP1 is an ER stress- induced UPR component, which is spliced when ER stress sensor protein IRE1 is activated [10]. In the present study, we found immobilization stress induced XBP1 splicing in several brain re- gions, including the hippocampus, cortex, hypothalamus, cere- bellum, and brain stem. Importantly, we did not observe XBP1 splicing in stress-exposed mice livers, indicating that immobiliza- tion stress-induced XBP1 splicing may be specific to the brain. We also did not observe GRP78 or CHOP induction in the stressed mice brains. These findings suggest that immobilization stress does not induce global responses to ER stress. Instead, it may specifically induce the IRE1-XBP1 arm of the UPR. These results raise the possibility that immobilization stress may not cause accumulation of unfolded proteins in the ER, as, if unfolded proteins were accu- mulated in the ER, all branches of the UPR would be activated. Then, what are the mechanisms underlying immobilization stress- induced XBP1 splicing? Increasing evidence suggests that the UPR is involved in regulating physiological responses [13,14]. Therefore, it is possible that immobilization stress activates physiological re- sponses, which specifically induce XBP1 splicing in the brain. XBP1 was reported to regulate lipid homeostasis in the liver [15]. In neurons, XBP1 has been shown to promote axon regeneration [16]. Furthermore, BiP Inducer X controls dopaminergic neuronal survival in a Parkinson’s disease model [17,18] and prevents amyloid b-induced neuronal cell death in an Alzheimer’s disease model [19]. Therefore, these findings suggest that the induction of XBP1 splicing in the brain may have neuro-protective functions, which may protect living organisms after exposure to environment stressors. Overall, our findings suggest that XBP1 is a key regulator of psychological stress, and may provide useful information for the development of therapeutics targeting mental disorders.