Imipramine protects mouse hippocampus against tunicamycin-induced cell death

European Journal of Pharmacology 696 (2012) 83–88

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European Journal of Pharmacology journal homepage: www.elsevier.com/locate/ejphar

Neuropharmacology and analgesia

Imipramine protects mouse hippocampus against tunicamycin-induced cell death Yoko Ono 1, Masamitsu Shimazawa 1, Mitsue Ishisaka, Atsushi Oyagi, Kazuhiro Tsuruma, Hideaki Hara n Molecular Pharmacology, Department of Biofunctional Evaluation, Gifu Pharmaceutical University, 1-25-4 Daigaku-nishi, Gifu 501-1196, Japan

a r t i c l e i n f o

abstract

Article history: Received 23 June 2012 Received in revised form 11 September 2012 Accepted 22 September 2012 Available online 3 October 2012

Endoplasmic reticulum (ER) stress is implicated in various diseases. Recently, some reports have suggested that the sigma-1 receptor may play a role in ER stress, and many antidepressants have a high affinity for the sigma-1 receptor. In the present study, we focused on imipramine, a widely used antidepressant, and investigated whether it might protect against the neuronal cell death induced by tunicamycin, an ER stress inducer. In mouse cultured hippocampal HT22 cells, imipramine inhibited cell death and caspase-3 activation induced by tunicamycin, although it did not alter the elevated expressions of 78 kDa glucose-regulated protein (GRP78) and C/EBP-homologous protein (CHOP). Interestingly, in such cells application of imipramine normalized the expression of the sigma-1 receptor, which was decreased by treatment with tunicamycin alone. Additionally, NE-100, a selective sigma-1 receptor antagonist, abolished the protective effect of imipramine against such tunicamycininduced cell death. Imipramine inhibited the reduction of mitochondrial membrane potential induced by tunicamycin, and NE-100 blocked this modulating effect of imipramine. Furthermore, in anesthetized mice intracerebroventricular administration of tunicamycin decreased the number of neuronal cells in the hippocampus, particularly in the CA1 and dentate gyrus (DG) areas, and 7 days’ imipramine treatment (10 mg/kg/day; i.p.) significantly suppressed these reductions in CA1 and DG. These findings suggest that imipramine protects against ER stress-induced hippocampal neuronal cell death both in vitro and in vivo. Such protection may be partly due to the sigma-1 receptor. & 2012 Elsevier B.V. All rights reserved.

Keywords: Endoplasmic reticulum stress Hippocampus Imipramine Sigma-1 receptor Tunicamycin

1. Introduction Endoplasmic reticulum (ER) stress is involved in such neurodegenerative diseases as Parkinson’s disease, Alzheimer’s disease, and cerebral ischemia (Arduino et al., 2009; Lindholm et al., 2006; Morimoto et al., 2007; Oida et al., 2008; Oono et al., 2004). Additionally, various psychiatric diseases are also associated with ER stress (Kakiuchi et al., 2003). The ER regulates protein synthesis, protein folding and trafficking, and intracellular calcium levels (Rao et al., 2004). Accumulation of misfolded proteins in the ER can result in ER stress, which activates the cellular unfolded protein response (UPR), leading to a reduced quantity of unfolded proteins. The UPR results in attenuation of protein synthesis via ER chaperone proteins such as glucose-regulated protein 78 (GRP78) and GRP 94. Long-term activation of UPR, however, induces C/EBP homologous protein (CHOP), and promotes cell death (Malhotra and Kaufman, 2007; Miyake et al., 2000; Oyadomari and Mori, 2004; Scheuner and Kaufman, 2008).

n

Corresponding author. Tel./fax: þ 81 58 230 8126. E-mail address: [email protected] (H. Hara). 1 Contributed equally.

0014-2999/$ - see front matter & 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.ejphar.2012.09.037

ER stress also results in cell death by causing a disturbance of Ca2 þ homeostasis in mitochondria, leading to activation of caspase-3 (Leem and Koh, 2012). The sigma-1 receptor, which plays a role in the control of Ca2 þ release (Monnet, 2005), is an ER protein that resides specifically at the ER-mitochondrion interface (termed the mitochondriaassociated ER membrane). Upon ER Ca2 þ depletion or via ligand stimulation, the sigma-1 receptor binds to inositol-1, 4, 5-trisphosphate (IP3) receptors to enhance Ca2 þ signaling from the ER into the mitochondria (Hayashi and Su, 2007). Ligands have been reported to exert neuroprotective effects via the sigma-1 receptor in both the brain (Katnik et al., 2006; Maurice and Su, 2009) and the retina (Martin et al., 2004). Recently, it has been suggested that this receptor may have a protective function against ER stress (Ha et al., 2011). Interestingly, many antidepressants—such as fluvoxamine, a selective serotonin-reuptake inhibitor, and imipramine, a tricyclic antidepressant—have a high affinity for the sigma-1 receptor (Narita et al., 1996). Moreover, imipramine potentiates nerve growth factor-induced neurite outgrowth through the sigma-1 receptor (Takebayashi et al., 2002). Hence, imipramine could reduce ER stress through the sigma-1 receptor. For the present study, we hypothesized that imipramine dose reduce both ER stress and the subsequent development of neurodegeneration, and we therefore investigated its

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effects on ER stress-induced damage in the hippocampus both in vitro and in vivo.

2. Materials and methods 2.1. Cell cultures Mouse hippocampal HT22 cells were kindly gifted by Yoko Hirata Ph.D. (Gifu University, Japan). Cells were maintained in Dulbecco’s modified Eagle’s medium (D-MEM; Nacalai tesque, Kyoto, Japan) containing 10% fetal bovine serum (FBS), 100 units/ ml penicillin (Meiji Seika Kaisha Ltd.,Tokyo, Japan), and 100 mg/ml streptomycin (Meiji Seika) in a humidified atmosphere containing 5% CO2 at 37 1C. Cells were passaged by trypsinization every 2 or 3 days, and maintained in a 10 cm dish (BD Biosciences, Franklin Lakes, NJ, USA). 2.2. Cell death assay HT22 cells were seeded at 3  103 cells per well into 96-well plates (BD Biosciences), then incubated for 24 h at 37 1C in a humidified atomosphere containing 5% CO2. The entire medium was then replaced with fresh medium containing 1% FBS, and imipramine hydrochloride (0.1–10 mM; Wako Pure Chemical Industries, Osaka, Japan) or vehicle, and pretreated for 1 h, followed by the addition of 50 ng/ml tunicamycin (Wako) or vehicle. Imipramine and tunicamycin were dissolved in phosphate-buffered saline (PBS; pH 7.4) containing 1% dimethyl sulfoxide (DMSO) as vehicle. To investigate the involvement of the sigma-1 receptor, NE-100 (0.1 mM; Santa Cruz, CA, USA), a sigma-1 receptor antagonist, was added at the same time as imipramine. Nuclear staining assays were carried out after a further 24 h of incubation. At the end of the culture period, the Hoechst 33342 (lex ¼360 nm, lem 4490 nm, Molecular Probes, Eugene, OR) and propidium iodide ( PI; lex ¼535 nm, lem 4617 nm, Molecular Probes) dyes were added to the culture medium (8 mM and 1.5 mM, respectively) for 15 min (Dive et al., 1992). Images were collected via an inverted epifluorescence microscope (IX70; Olympus. Co., Tokyo, Japan). The number of cells per condition was counted in a blind manner by a single observer (Y.O.) with the aid of image-processing software (Image-J, version 1.33f; National Institutes of Health, Bethesda, MD, USA). Cell mortality was quantified by expressing the number of PI-positive cells as a percentage of the number of Hoechst 33342-positive cells. 2.3. Western blot analysis At the end of the culture period, HT22 cells were washed with PBS twice in order to remove dead cells. Then, the HT22 cells were lysed using a cell-lysis buffer (RIPA buffer: 50 mM Tris HCl, 150 mM NaCl, 0.5% sodium deoxycholate, 0.1% SDS, and 1% Igepal CA-630) together with protease inhibitor (Sigma-Aldrich, St. Louis, MO, USA) and phosphatase inhibitor cocktails (Sigma-Aldrich). The lysate was centrifuged at 12,000  rpm for 10 min, and the supernatant was used for this study. Assays to determine protein concentrations were performed by comparison with a known concentration of bovine serum albumin using a BCA protein assay kit (Pierce Biotechnology, Rockford, IL, USA). A mixture of equal parts of an aliquot of protein and sample buffer with 20% 2-mercaptoethanol (Wako) was subjected to 5–20% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Wako). The separated protein was then transferred onto a polyvinylidene difluoride membrane (Immobilon-P; Millipore Corporation, Bedford, MA, USA). For western blotting, the following primary antibodies were used: mouse anti-GRP78 antibody (Santa Cruz), mouse anti-CHOP (GADD153) antibody (Santa Cruz), rabbit anti-sigma-1 receptor (OPRS1) antibody (Abcam, Cambridge, UK),

and mouse anti-b-actin monoclonal antibody (Sigma-Aldrich). The secondary antibody was goat anti-mouse HRP-conjugated and goat anti-rabbit HRP-conjugated (PIERCE Biotechnology, Inc. MA, USA). The immunoreactive bands were visualized using a chemiluminescent substrate (ImmunoStars LD; Wako). The band intensity was measured using an imaging analyzer (LAS-4000; Fuji Film, Tokyo, Japan). 2.4. Caspase-3 assay To measure caspase-3 activity, a CaspACETM Assay System (Promega Co., Madison, WI, USA) was used. HT22 cells were seeded at 4  106 cells per 10 cm dish, then incubated for 24 h at 37 1C. After treatment with tunicamycin in the presence or absence of 10 mM of imipramine for 24 h, cells were collected by trypsinization and suspended in cell lysis buffer. Cell lysates were mixed in CaspACETM Assay substrates and incubated for 1 h at 37 1C. The fluorescence was measured using SkanIt RE for Varioskan Flash 2.4 (Thermo Fisher Scientific, Waltham, MA, USA) with excitation/emission wavelengths of 360/460 nm. 2.5. Mitochondrial membrane potential assay To evaluate effects on mitochondrial membrane potential, a JC-1 Mitochondrial Membrane Potential Assay Kit (Cayman Chemical Company, Ann Arbor, MI, USA) was used according to the manufacturer’s protocol. Images were collected using a fluorescence microscope (Keyence, Osaka, Japan), which detects unhealthy or apoptotic cells with JC-1 monomers (excitation/ emission¼ 480/510 nm) and healthy cells with JC-1 J-aggregates (excitation/emission ¼540/605 nm). The total number of cells was counted and the percentage of JC-1 monomers-positive cells was calculated as a measure of the number of cells with a low mitochondria membrane potential. Hoechst 33342 (8 mM) was used at the same time to detect all cells, and the total number of cells per condition was counted. The number of cells per condition was counted in a blind manner by a single observer (Y.O.) with the aid of image-processing software (Image-J). 2.6. Animals Male 9-week-old ddY mice (Japan SLC, Hamamatsu, Japan) were used for the following experiments. They were housed at 2571 1C under a 12 h light–dark cycle (lights on from 8:00 to 20:00) and had ad libitum access to food and water. All procedures relating to animal care and treatment conformed to the animal care guidelines of the Animal Experiment Committee of Gifu Pharmaceutical University. All efforts were made to minimize both suffering and the number of animal used. 2.7. Intracerebroventricular (i.c.v.) administration Anesthesia was induced with 3.0% isoflurane (Escain; Mylan, Canonsburg, PA, USA) and maintained with 1.5% isoflurane in 70% N2O and 30% O2 via an animal general anesthesia machine (Soft Lander; Sin-ei Industry Co., Ltd., Saitama, Japan). The coordinates used for placement of the microliter syringe (Hamilton, Reno, NV, USA) were 0.5 mm posterior to bregma, 1.0 mm lateral, and 2.0 mm below the skull surface at the point of entry. Under anesthesia, mice were slowly (over 1.5 min) injected i.c.v. with PBS containing 10% DMSO or tunicamycin (0.1 mg/ml; Wako) in a volume of 2 ml (n ¼4–6). The syringe was left in place for 1 min following the injection. Mice were allowed to recover for a minimum of 7 days before perfusion fixation.

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Tunicamycin Imipramine

2.8. Drug treatment Imipramine hydrochloride was dissolved in saline and injected intraperitoneally (i.p.) at 10 mg/kg/day for 7 days, the first injection being given 30 min before the i.c.v. administration with PBS containing 10% DMSO or tunicamycin (0.1 mg/ml; Wako) in a volume of 2 ml.

Control

10

3

Vehicle

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2.9. Cresyl violet staining

PI

PI positive cells (% of total cell number)

Seven days after the i.c.v. administration of PBS containing 10% DMSO or tunicamycin, mice were anesthetized with sodium pentobarbital (50 mg/kg) and perfused with PBS (pH 7.4) until the outflow became clear. The perfusate was then immediately changed to 0.1 M phosphate buffer (PB; pH 7.4) containing 4% paraformaldehyde (Wako) for 10 min. Brains were removed and postfixed in the same fixative for 24 h at 4 1C. The fixed specimens were dehydrated through a graded series of ethanol and xylene, and finally embedded in paraffin. For cresyl violet staining, paraffin-embedded specimens were cut at 5 mm thickness, and the sections were mounted on microslide glass (Matsunami Glass Ind. Ltd, Osaka, Japan), and then deparaffinized. Next, they were stained in cresyl violet for 1 min, dehydrated for 10 min using absolute ethanol, cleared in xylene for another 10 min, and covered with EUKITT (O. Kindler, Germany). Images were collected using a light microscope (Keyence), and the neuronal cells were counted in a blind manner by a single observer (Y.O.).

50 ## 40

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2.10. Statistical analysis Data are presented as the means7S.E.M. Statistical comparisons were made using a two-tailed t-test or one-way ANOVA followed by Dunnett’s test, Po0.05 being considered to indicate statistical significance.

3. Results 3.1. Imipramine protects against tunicamycin-induced cell death in hippocampal HT22 cells. We evaluated the effect of imipramine against tunicamycininduced cell death by the use of dual-staining with Hoechst 33342 and PI. Hoechst 33342 stains all cells (living and dead cells), whereas PI stains only dead cells (Fig. 1A). Compared with the vehicle control, tunicamycin (50 ng/ml) significantly increased the number of dead cells. Pretreatment with imipramine at 0.1–10 mM protected HT22 cells against tunicamycin-induced cell death in a concentrationdependent manner, the effect being significant at 0.3–10 mM (Fig. 1B). 3.2. Effects of imipramine on expressions of ER stress-related proteins and caspase-3 activation and expressions of sigma-1 receptor in HT22 cells. To clarify the mechanism underlying the above protective effects of imipramine against tunicamycin-induced cell death, we first investigated the expressions of GRP78 and CHOP by Western blot. Tunicamycin, at 50 ng/ml, significantly upregulated both GRP78 and CHOP expressions (versus control), but 10 mM imipramine had no effects on the elevated expression levels of GRP78 and CHOP induced by tunicamycin (Fig. 2A and B). Next, we measured caspase-3 activation using the CaspASETM Assay System. At 50 ng/ml, tunicamycin significantly increased caspase-3 activation, and imipramine at 10 mM significantly reduced this elevated level (Fig. 2C). Sigma-1-receptor expression is altered by ER stress

Fig. 1. Imipramine protects HT22 cells against endoplasmic reticulum (ER) stressinduced cell death. (A) Representative fluorescence microscopy showing nuclear staining for Hoechst 33342 (blue) and propidium iodide (PI) (red) in HT22 cells. Cells were pretreated with vehicle or with various concentrations of imipramine for 1 h, followed by tunicamycin (50 ng/ml) or vehicle for 24 h. (B) The number of cells displaying PI or Hoechst 33342 fluorescence was counted, and the cell death rate was expressed as the percentage of PI-positive cells to Hoechst 33342positive cells. ]], Po 0.01 versus control (Student’s t-test). *, P o 0.05; **, Po 0.01 versus tunicamycin-treated (vehicle) group (Dunnett’s test). Each column and bar represent mean 7 S.E.M. (n¼ 6). Scale bar ¼ 50 mm. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

(Hayashi and Su, 2007). Therefore, we investigated whether treatment with tunicamycin and/or imipramine might alter this receptor’s expression in HT22 cells. Sigma-1 receptor protein expression was significantly decreased under ER stress lasting for 24 h, and imipramine significantly normalized it (Fig. 2D).

3.3. Involvement of the sigma-1 receptor in the protective effect of imipramine against ER stress-induced cell death. Next, we examined whether the sigma-1 receptor is involved in the protective effect of imipramine. Representative photographs of staining with Hoechst 33342 and PI are shown in Fig. 3A. At 0.1 mM, NE-100, a sigma-1-receptor antagonist, inhibited the protective effect of imipramine against tunicamycininduced cell death (Fig. 3B). We also investigated whether imipramine might inhibit the reduction of the mitochondrial membrane potential mediated via this receptor. Healthy cells were detected with JC-1 J-aggregates (red) and unhealthy or apoptotic cells were detected with JC-1 monomers (green) (Fig. 3C). Imipramine significantly reduced the number of cells exhibiting evidence of a tunicamycin-induced reduction of the mitochondrial membrane potential, and NE-100 significantly inhibited this effect of imipramine (Fig. 3D).

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Tunicamycin Control Vehicle

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Fig. 2. Effects of imipramine on expressions of endoplasmic reticulum (ER) stress-related proteins and activation of caspase-3 and sigma-1 receptor in HT22 cells. HT22 cells were pretreated with vehicle or with imipramine (10 mM) for 1 h, and then treated with tunicamycin (50 ng/ml) or vehicle for 24 h. Proteins were analyzed by Western blot using antibodies specific to (A) GRP78 (n¼ 6), (B) CHOP (n¼ 6), (D) sigma-1 receptor (n ¼10 or 11) or b-actin. Protein levels were quantified by densitometry and normalized to the level of b-actin (n¼ 6). (C) Caspase-3 activity was measured at 24 h after tunicamycin-treatment (n¼ 3). ]], Po 0.01 versus control (Student’s t-test). *, Po 0.05 versus tunicamycin-treated (vehicle) group (Student’s t-test). Each column and bar represent mean 7S.E.M.

3.4. Effects of imipramine against tunicamycin-induced cell damage in mouse hippocampus. To investigate the effects of tunicamycin and imipramine in vivo, we artificially induced ER stress in the mouse brain by injecting tunicamycin at 0.2 mg (i.c.v.). Histological analysis based on cresyl violet staining (for representative photographs, see Fig. 4A) revealed that tunicamycin decreased the numbers of neuronal cells in the CA1 and dentate gyrus (DG) areas (versus control), but not that in the CA3 area. Seven days’ treatment with imipramine (10 mg/kg/day; i.p.) significantly suppressed the tunicamycin-induced loss of neuronal cells in the CA1 and DG areas (Fig. 4B).

4. Discussion The purpose of present study was to investigate the effect of imipramine, if any, against ER stress. Our findings provide evidence that imipramine protects against ER stress-induced cell death in the hippocampus both in vitro and in vivo.

Here, we demonstrated that in mouse hippocampal HT22 cells, imipramine significantly protected against tunicamycin-induced cell death and caspase-3 activation, without affecting the elevated expression levels of GRP78 and CHOP induced by tunicamycin treatment. These results suggest that the protective effects of imipramine are mediated by inhibition of the mitochondriamediated caspase signaling pathway, not by inhibition of the UPR-CHOP pathway. Many antidepressants are sigma-1-receptor agonists with a high affinity for the sigma-1 receptor (Hayashi and Su, 2004), and the selective serotonin-reuptake inhibitor fluvoxamine exerts several effects via the sigma-1 receptor. For example, fluvoxamine potentiates nerve-growth factor (NGF)induced neurite outgrowth in PC 12 cells (Takebayashi et al., 2004), and also ameliorates myocardial hypertrophy and dysfunction in mice (Tagashira et al., 2010) by stimulating the sigma-1 receptor. Imipramine blocks both the serotonin transporter (Ki ¼1.41 nM) and the norepinephrine transporter (Ki ¼37 nM) (Getachew et al., 2010), and has a high affinity for the sigma-1 receptor (Ki ¼343 nM). In the present study, imipramine at 300 nM or more protected against the neuronal cell death induced by tunicamycin in vitro. The concentrations of

Y. Ono et al. / European Journal of Pharmacology 696 (2012) 83–88

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Fig. 4. Effects of imipramine against tunicamycin-induced cell damage in the hippocampus. Mice were injected intracerebroventricularly (i.c.v.) with vehicle or with tunicamycin (0.2 mg). For long-term treatment, imipramine hydrochloride was injected intraperitoneally (i.p.) at 10 mg/kg/day for 7 days. (A) Representative cresyl violet staining of hippocampal CA1, CA3, and DG areas. (B) The numbers of neuronal cells in the CA1, CA3, and DG areas were counted. ]], Po 0.01 versus control group (Student’s t-test). **, P o0.01 versus tunicamycin-treated (vehicle) group (Student’s t-test). Each column and bar represent mean 7 S.E.M. (n¼ 4–6). Scale bars; a, e, i¼300 mm, others¼50 mm.

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Fig. 3. Involvement of the sigma-1 receptor in the protective effect of imipramine against endoplasmic reticulum (ER) stress-induced cell death. (A) Representative fluorescence microscopy showing nuclear staining for Hoechst 33342 (blue) and propidium iodide (PI) (red) in HT22 cells. Cells were pretreated with tunicamycin (50 ng/ml) and/or with imipramine (10 mM) in the presence or absence of the sigma-1- receptor antagonist NE-100 (0.1 mM). (B) The number of cells displaying PI or Hoechst 33342 fluorescence was counted, and the cell death rate was expressed as the percentage of PI-positive cells to Hoechst 33342-positive cells. (C) Representative images showing JC-1 stained cells. Healthy cells with mainly JC-1 J-aggregates are detected as red cells, and unhealthy or apoptotic cells with mainly JC-1 monomers are detected as green. (D) Comparison of mitochondrial membrane potential (JC-1 monomers/total cell ratio) between groups. The number of cells was counted and expressed as a percentage of total cell number. ]] , Po 0.01 versus control (Welch’s t-test). **, P o0.01 versus tunicamycin-treated (vehicle) group (Student’s t-test). y, Po 0.05, yy, Po 0.01 versus tunicamycin and imipramine-treated group (Student’s t-test). Each column and bar represent mean 7 S.E.M. (n ¼6). Scale bar ¼ 50 mm. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

imipramine used in the present study were in accord with its affinity for the sigma-1 receptor. Therefore, we focused on the effects of imipramine mediated via the sigma-1 receptor. In HT22 cells, tunicamycin decreased the expression of sigma-1 receptor protein, and imipramine at 10 mM inhibited this downregulation of the sigma-1 receptor. Tagashira et al. (2010) have reported that fluvoxamine attenuates the downregulation of the sigma-1

receptor that is induced by transverse aortic constriction, suggesting that sigma-1 receptor agonists have stabilization or upregulation effects on the sigma-1 receptor. Thus, imipramine, like fluvoxamine, may be able to stabilize or upregulate the sigma-1 receptor. Although NE-100, a selective sigma-1-receptor antagonist, significantly inhibited the protective effect of imipramine against the tunicamycin-induced cell death, NE-100 could not block the effect of imipramine completely. From this result, it is considered a possibility that other receptors/transporters may be involved in the protective effect of imipramine. Some reports show that serotonin transporter (SERT) and norepinephrine transporter (NET) functions may be involved in ER stress. Briefly, ER stress attenuates SERT function (Nobukuni et al., 2009) and norepinephrine causes NET dysfunction and induces secondary ER stress (Mao et al., 2005). Since imipramine affects on SERT, NET, and other receptors/transporters, it may also show the protective effect via those receptors/ transporters. Therefore, it is cannot rule out the possible involvement of other receptors/transporters. However, in the present study, imipramine reduced the number of HT22 cells displaying evidence of a tunicamycin-induced depression of the mitochondrial membrane potential, and NE-100, completely attenuated this effect of imipramine. This result suggests that imipramine may stimulate the sigma-1 receptor to increase Ca2 þ influx into mitochondria, and thereby rescue the mitochondria from dysfunction Depression of the mitochondrial membrane potential is followed by a release of cytochrome c, caspase activation, and apoptosis (Zhu et al., 2000). Hence, imipramine may inhibit caspase-3 activation through its action on the sigma-1 receptor, and thereby protect against tunicamycin-induced cell death in HT22 cells. To determine whether the protective effect of imipramine against ER stress-induced cell-damage occurs in vivo, we looked

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for evidence of such an effect of imipramine against cell-damage induced by i.c.v. injection of tunicamycin. CA1, but not CA3, is highly vulnerable to ischemic injury (Kirino, 2000), and so the CA1 area may have a general vulnerability to various stresses. It has been reported that in hippocampal slices, the CA1 and DG areas are more vulnerable than the CA3 area to tunicamycin-induced neuronal damage (Kosuge et al., 2008). Our results suggest that the CA1 and DG areas are more sensitive to ER stress-induced damage, too, and that imipramine may protect hippocampal neuronal cells in vivo, as well as in vitro. Although it remains to be determined whether and/or how the sigma-1 receptor is involved in vivo, it is an intriguing possibility that long-term imipramine treatment might enhance sigma-1-receptor function and thereby protect neuronal cells against ER stress-induced cell damage. In conclusion, imipramine protects against ER stress-induced neuronal cell death partly by rescuing sigma-1 receptor protein levels from downregulation and by increasing stimulation of the sigma-1 receptor. Our findings provide evidence of a novel effect of imipramine against tunicamycin-induced neuronal damage via a modulating influence over the sigma-1 receptor. We suggest that imipramine warrants further investigation as a potential drug for diseases in which neuronal damage is induced by the ER stress.

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