European Journal of Neuroscience, Vol. 40, pp. 3120–3127, 2014
doi:10.1111/ejn.12647
DISORDERS OF THE NERVOUS SYSTEM
Effect of amiloride on endoplasmic reticulum stress response in the injured spinal cord of rats Masahiro Kuroiwa,1 Masahiko Watanabe,1 Hiroyuki Katoh,1 Kaori Suyama,2 Daisuke Matsuyama,1 Takeshi Imai1 and Joji Mochida1 1
Department of Orthopaedic Surgery, Surgical Science, Tokai University School of Medicine, 143 Shimokasuya, Isehara, Kanagawa 259-1193, Japan 2 Department of Anatomy and Cellular Biology, Tokai University School of Medicine, Isehara, Kanagawa, Japan Keywords: apoptosis, glial cells, in vivo, rat, spinal cord injury
Abstract After traumatic spinal cord injury (SCI), endoplasmic reticulum (ER) stress exacerbates secondary injury, leading to expansion of demyelination and reduced remyelination due to oligodendrocyte precursor cell (OPC) apoptosis. Although recent studies have revealed that amiloride controls ER stress and leads to improvement in several neurological disorders including SCI, its mechanism is not completely understood. Here, we used a rat SCI model to assess the effects of amiloride on functional recovery, secondary damage expansion, ER stress-induced cell death and OPC survival. Hindlimb function in rats with spinal cord contusion significantly improved after amiloride administration. Amiloride significantly decreased the expression of the pro-apoptotic transcription factor CHOP in the injured spinal cord and significantly increased the expression of the ER chaperone GRP78, which protects cells against ER stress. In addition, amiloride treatment led to a significant decrease in ER stress-induced apoptosis and a significant increase of NG2-positive OPCs in the injured spinal cord. Furthermore, in vitro experiments performed to investigate the direct effect of amiloride on OPCs revealed that amiloride reduced CHOP expression in OPCs cultured under ER stress. These results suggest that amiloride controls ER stress in SCI and inhibits cellular apoptosis, contributing to OPC survival. The present study suggests that amiloride may be an effective treatment to reduce ER stress-induced cell death in the acute phase of SCI.
Introduction In traumatic spinal cord injury (SCI), primary injury refers to the physical tissue disruption that is caused by direct external forces. This is followed by a secondary injury in which biochemical and vascular factors (Tator & Fehlings, 1991; Watanabe et al., 1998; Lu et al., 2000; Park et al., 2004) cause delayed damage that expands through the spinal cord (Schwab & Bartholdi, 1996). One of the main factors contributing to secondary injury is the demyelination that occurs as a result of glial cell apoptosis (Crowe et al., 1997). Although oligodendrocyte precursor cells (OPCs) proliferate at the periphery of the injury epicenter after SCI, a majority of OPCs fail to differentiate into mature oligodendrocytes and are eliminated through apoptosis (Ishii et al., 2001; McTigue et al., 2001). As OPCs proliferate, survive and differentiate into mature oligodendrocytes and promote remyelination in multiple sclerosis chemical demyelination models (Chang et al., 2000; Watanabe et al., 2002), it is likely that the apoptosis of OPCs is a major factor in the failure of remyelination following SCI. Given that astrocytes proliferate without succumbing to apoptosis and participate in the formation of the glial scar after SCI (Mckeon et al., 1991; Baldwin et al., 1998;
Correspondence: Dr M. Watanabe, as above. E-mail: [email protected] Received 27 February 2014, revised 30 April 2014, accepted 2 May 2014
Fawcett & Asher, 1999; Eng et al., 2000), the significance of understanding the regulation of apoptosis is clear. To elucidate the mechanism underlying OPC apoptosis and the diverse reactions observed among different cell types after SCI, we focused on the endoplasmic reticulum (ER) stress pathway, which is one of many apoptotic pathways. When a cell is exposed to external stress factors, structurally abnormal, misfolded proteins (unfolded proteins) accumulate in the ER. This triggers an ER stress pathway that activates the C/EBP homologous transcription factor protein (CHOP), which is a proapoptotic factor that mediates programmed cell death. To protect cells against unfolded proteins, the ER chaperone glucose-regulated protein 78 (GRP78) mediates the refolding of unfolded proteins. It has been shown that the unfolded protein response (UPR), which is the cellular stress response against ER stress, is also involved in demyelination and the apoptosis of glial cells that leads to failure of remyelination in the secondary injury process following SCI (Penas et al., 2007; Ohri et al., 2011). Therefore, if ER stress can be controlled, it might be possible to reduce secondary injury and to induce remyelination via OPC survival. In this study, we used amiloride, a therapeutic agent that has been shown to have cytoprotective and ER stress-modulating effects in various models of neurological diseases. We administered amiloride to a rat SCI model and examined the effects of amiloride on secondary
© 2014 Federation of European Neuroscience Societies and John Wiley & Sons Ltd
Effect of amiloride in spinal cord injury 3121 injury expansion, ER stress-induced apoptosis, OPC survival and functional recovery. In addition, we examined the direct effect of amiloride on OPCs by adding amiloride to rat OPC cultures under ER stress and assessing its effects on cell activity.
Materials and methods Rat SCI model All experimental procedures were performed in accordance with the National Institute of Health Guidelines for the Care and Use of Laboratory Animals and were approved by the Animal Experimentation Committee of Tokai University School of Medicine. For these experiments, female Sprague–Dawley (SD) rats (age, 10 weeks, 280–320 g) were obtained from CLEA Japan, Inc. (Kanagawa, Japan). Surgical procedures were performed under aseptic conditions, and the rats were anesthetized with inhaled 4% isoflurane. Laminectomy of the 10th thoracic vertebra was performed to expose the dura mater. A spinal cord contusion model was created using an Infinite Horizon spinal cord impactor device (IH impactor; Precision Systems & Instrumentation, Lexington, KY, USA) with a force of 200 Kdyne (2 mN). To care for the bladder dysfunction that occurs after SCI, abdominal massages were performed twice a day to empty the bladder. The injured rats were divided into two groups: an amiloride group and a phosphate-buffered saline (PBS) control group (n = 6 per group). At 24 h after injury and every 24 h thereafter for 28 days, the amiloride group received intraperitoneal administration of 10 mg/kg amiloride (amiloride hydrochloride hydrate, catalog #A7410; Sigma-Aldrich, St. Louis, MO, USA) in accordance with a protocol used for a mouse multiple sclerosis model (Friese et al., 2007). The body weight of each rat was measured daily, and the drug dosage was calculated. The PBS group received intraperitoneal administration of PBS using the same protocol. Control animals were age-matched normal female SD rats (age, 10 weeks; 280–320 g) that received a sham surgery consisting of a laminectomy of the 10th thoracic vertebra. Evaluation of hindlimb function The effects of amiloride treatment on hindlimb motor function after SCI were assessed using the Basso, Beattie and Bresnahan locomotor rating scale (BBB scale), which is an open-field locomotor test for rats. Locomotor behavior was evaluated immediately before injury and each following day at approximately the same time for 28 days after injury. The mean score was calculated using the measurements derived by three observers through 5 min of careful observation. Each observer was blinded regarding the treatment used (n = 6 per group). The BBB scores were analysed by two-way repeated-measures analysis of variance (ANOVA) with Tukey’s post hoc multiple comparison test. Western blot At 3, 7 and 14 days after injury, a rat was anesthetized with inhaled 4% isoflurane and 5 mm of the injured spinal cord (2.5 mm rostral and caudal to the epicenter) was microscopically dissected. The excised spinal cord tissue was immediately washed in ice cold PBS, after which extracts were prepared using a Cell Lytic NuCLEAR extraction kit (Sigma-Aldrich). These extracts were subjected to electrophoresis on a 12.5% sodium dodecyl sulfate polyacrylamide gel; 20 lg of protein solution was applied to each gel lane. After electrophoresis, the proteins were electrotransferred onto nitrocellu-
lose membranes (Bio-Rad, Hercules, CA, USA). The membranes were blocked with 5% bovine serum albumin (BSA) in TBST (50 mM Tris, pH 7.6, 150 mM NaCl, 0.1% Tween-20), followed by incubation with anti-GRP78 (rabbit anti-HSPA5/GRP78, catalog #APO6149PU-N, 1 : 2000, catalog #BS1136, 1 : 200; ACRIS, Herford, Germany) and anti-CHOP (rabbit anti-GADD153, bioWORLD, Atlanta, GA, USA) antibodies overnight at 4 °C. The membranes were washed for 7 h in PBS containing 0.05% Tween20 and incubated for 60 min at 25 °C with horseradish peroxidase (HRP)-linked anti-rabbit IgG (catalog #NA9340; GRP78 1 : 5000, CHOP 1 : 1000; GE Healthcare, Amersham, UK). The proteins were labeled with Immobilon Western Chemiluminescent HRP and were quantified by densitometric scanning analysis of the films using MACINTOSH CS ANALYSER SOFTWARE (Atto, Tokyo, Japan). bActin was used as an internal control and was labeled with a mouse monoclonal anti-b-actin antibody (catalog #A5441, 1 : 1000; Sigma-Aldrich). GRP78 and CHOP expression levels were analysed with Mann–Whitney U test (n = 6 per group). Tissue processing for TUNEL staining and immunohistochemical staining Perfusion fixation was performed at 3, 7 and 14 days after injury using 2% paraformaldehyde (PFA) – 0.1 M phosphate buffer (PB) under inhaled 4% isoflurane anesthesia. After fixation, the spinal cord was excised and post-fixed in 2% PFA in 0.1 M PB for 2 days at 4 °C and dehydrated in a series of increasing sucrose concentrations (7, 15, 20%). Frozen spinal cord tissue blocks were embedded in Optimal Cutting Temperature (OCT) compound (Sakura Finetek, Tokyo, Japan), and 10-lm-thick tissue sections were prepared using a cryostat. The epicenter was defined as the width of the tip of the IH impactor: 2 mm. From this epicenter, spinal cord sections were dissected at 3-mm increments: at the epicenter, and at 4, 7, 10 and 13 mm rostral to the epicenter. TUNEL staining Using specimens obtained on days 3, 7 and 14 after injury, TUNEL staining was performed on sections from the epicenter and 4, 7, 10 and 13 mm rostral from the epicenter (n = 6 per group). Because cauda equina was encountered in the region caudal to the epicenter, we used specimens taken from the epicenter and rostral regions. The sections were washed three times (10 min each) with PBS and incubated in a permeabilization solution (0.1% Triton-X, 0.1% sodium citrate) for 2 min on ice. The sections were then subjected to TUNEL staining using the In Situ Cell Death Detection Kit TMR red (catalog #12156792910; Roche, Mannheim, Germany) and incubated at 37 °C for 60 min. As negative controls, sections were incubated in the label solution without the enzyme solution (terminal transferase) instead of the TUNEL reaction solution, which contains both solutions. The sections were washed twice with PBS, and nuclei were stained and mounted using Vectashield with DAPI H-1500 (Vector Laboratories, Burlingame, CA, USA). Images were taken with a Nikon DS-Ri1 (Nikon, Tokyo, Japan) camera using mono 10-bit lenses (4 9 objective NA=0.13, 20 9 objective NA=0.4) on an Olympus IX70 fluorescence microscope (Olympus, Tokyo, Japan) and analysed with Nikon NIS-elements Ver.3.1 software (Nikon). TUNEL-positive cells with clearly recognizable nuclei and cytoplasm and with co-localized TMR red- and DAPI-stained nuclei were recorded as positive. The numbers of TUNEL-positive cells in the dorsal funiculus were counted and analysed with two-way
© 2014 Federation of European Neuroscience Societies and John Wiley & Sons Ltd European Journal of Neuroscience, 40, 3120–3127
3122 M. Kuroiwa et al. repeated-measures ANOVA with Tukey’s post hoc multiple comparison test. The differences in the temporal and spatial expansion of cell death were compared between the two groups. Immunohistochemical staining For immunohistochemical staining, 10-lm-thick tissue sections taken 7 mm rostral to the epicenter of post-injury 3-, 7- and 14-day animals were used (n = 6 per group). The sections were washed three times (10 min each) with PBS and blocked for 60 min in PBS with 5% normal goat serum at 24 °C. The sections were washed again for 10 min and incubated overnight at 4 °C with anti-GRP78 (rabbit anti-HSPA5/GRP78, ACRIS, catalog #APO6149PU-N, 1 : 200), anti-NG2 (mouse anti-NG2, catalog #MAB5384, 1 : 50; a marker for OPCs; Merck Millipore, Billerica, MA, USA) and antiGFAP (mouse anti-GFAP, catalog #MAB360, 1 : 400; a marker for astrocytes; Millipore). The sections were washed again with PBS and incubated for 60 min at 24 °C in the dark with the respective fluorescent secondary antibodies (GRP78: Alexa Fluor594, anti-rabbit; Invitrogen, Carlsbad, CA, USA, catalog #A11012, 1 : 1000; NG2 and GFAP: Alexa Fluor488, anti-mouse, Invitrogen, catalog #A11029, 1 : 1000). Subsequently, nuclei were stained and mounted using Vectashield with DAPI H-1500 (Vector Laboratories). Spinal cord tissue from sham surgery rats was used as control. Images were taken with a Nikon DS-Ri1 camera using mono 10-bit lenses (40 9 objective NA=0.6) on an Olympus IX70 fluorescence microscope and analysed with Nikon NIS-elements Ver.3.1 software. To quantify the ratios of OPCs or astrocytes, we counted the number of cells positive for each cell marker and divided it by the total number of nuclei in the dorsal funiculus. To quantify GRP78 expression ratios in each cell type, the number of cells double positive for each cell marker and GRP78 was counted and was divided by the total number of cells positive for each cell marker in the dorsal funiculus. We determined the following: (i) percentages of GRP78-positive cells among all DAPI-positive cells; (ii) percentages of NG2-positive cells among all DAPI-positive cells; (iii) percentages of GFAP-positive cells among all DAPI-positive cells; (iv) percentages of GRP78positive cells among all NG2-positive cells; and (v) percentages of GRP78-positive cells among all GFAP-positive cells. Only those cells with clearly recognizable nuclei and cytoplasm that had cytoplasmic staining corresponding to each secondary antibody were scored as positive. Negative controls were incubated with secondary antibodies in the absence of primary antibodies. Data obtained through immunohistochemistry were analysed with Mann–Whitney U test. Cell culture and treatment of OPCs
pyruvate (0.11 mg/mL), and penicillin-streptomycin (10 IU/mL and 100 lg/mL, respectively)], and 0.1% BSA (all from Sigma-Aldrich). After 1–2 days, the cells were incubated with platelet-derived growth factor (PDGF; PeproTech, Rocky Hill, NJ, USA; 10 ng/mL) and were cultured for 2–3 days to allow for OPC differentiation. To evaluate the ER stress response, the OPCs were incubated with 3 lM tunicamycin (Sigma-Aldrich) and 20 lM amiloride (ami+tuni group) or 3 lM tunicamycin alone (tuni group) for 6 h (n = 4 per group). Control cells were cultured under the same conditions without tunicamycin or amiloride. Immunostaining to evaluate OPC activity Live cells were immunolabeled as described Bansal et al. (2003). Briefly, cells were blocked for non-specific absorption with HEPESbuffered Earl’s balanced salt solution (EBSS-HEPES) containing 3% normal goat serum (also used for diluting antibodies), and then double immunolabeled with oligodendrocyte lineage stage-specific markers [early progenitors, A2B5 (R and D Systems, Minneapolis, MN, USA); late progenitors, O4 (R and D Systems)]. After fixation with 4% PFA for 15 min, cells were permeabilized (0.3% Triton X100), blocked with 5% BSA/PBS, and stained for CHOP (rabbit anti-GADD153, bioWORLD, catalog #BS1136, 1 : 200). Cells were then labeled with the appropriate secondary antibodies [Alexa 488conjugated anti-mouse antibody; Alexa Fluor 594 (anti-rabbit, catalog #A11012, 1 : 1000; Invitrogen) and a nuclear label, Vectashield with DAPI H-1500 (Vector Laboratories)]. Cells were then mounted and evaluated using the Thermo Scientific Cellomics Array Scan (Thermo Fisher Scientific, Kanagawa, Japan). The average intensity of CHOP that overlapped with the nuclei in OPCs was measured from ten random fields per well and was analysed with Mann–Whitney U test. Statistical analysis Statistical significance was determined using PASW statistics 18 (IBM SPSS, Foster City, CA, USA). Mann–Whitney U tests were used to analyse GRP78 and CHOP expression levels obtained by Western blotting, the in vivo immunohistochemical measurements and the in vitro evaluation of OPCs. The BBB scores and TUNEL analysis were evaluated with a two-way repeated-measures ANOVA with Tukey’s post hoc multiple comparison test. Significant interactions were further analysed with a one-way ANOVA followed by Tukey’s test. Asterisks in figures indicate statistical significance (*P < 0.05, **P < 0.01).
Results
Enriched cultures of OPCs were prepared as described elsewhere (Armstrong, 1998; Guardiola-Diaz et al., 2012). Mixed primary cultures from neonatal (P1–2) rat telencephalon were shaken overnight in an orbital shaker. Dislodged OPCs (and some preprogenitors) were further purified by differential adhesion. Early progenitors were seeded on poly-L-lysine- (Sigma-Aldrich) coated four-well plates (1 9 105 cells per well for immunocytochemistry). The cells were first cultured for 3 h in Dulbecco’s modified Eagle’s medium (DMEM; Invitrogen) supplemented with 5% (v/v) fetal bovine serum (FBS) to allow for attachment, after which the cells were grown in serum-free N2 defined medium [DMEM supplemented with human transferrin (50 lL/mL), bovine pancreatic insulin (5 lL/mL), 3,3,5-triiodo-L-thyronine (10 ng/mL), sodium selenium (30 nM), D-biotin (10 ng/mL), hydrocortisone (10 nM), sodium
Amiloride improves functional recovery after SCI For both the amiloride and the PBS groups, the BBB scores dropped to 0 after injury and gradually improved thereafter. No significant differences were found between the two groups until day 11 after injury. However, at day 12, the amiloride group showed a significant improvement compared with the PBS group, and this difference was maintained for the duration of the study after day 15 (interaction between group and days post operation: F27.280 = 3.8, P < 0.01 (5.82 9 10 9), one-way ANOVA: F55.280 = 76.95, P < 0.01 (7.39 9 10 140), Tukey’s test: P < 0.05). The mean scores on the last observation day (day 28 after injury) were 15.0 1.23 for the amiloride group and 12.8 1.17 for the PBS group (Fig. 1).
© 2014 Federation of European Neuroscience Societies and John Wiley & Sons Ltd European Journal of Neuroscience, 40, 3120–3127
Effect of amiloride in spinal cord injury 3123
Fig. 1. Amiloride improves functional recovery after SCI. BBB scale scores were significantly higher in the amiloride group compared with the PBS group from day 15 and later. Asterisks indicate significant difference between amiloride group (Ami) and PBS group (PBS), two-way repeated measures ANOVA, followed by Tukey’s multiple comparison test (*P < 0.05, **P < 0.01, n = 6 per group, error bars are standard deviations).
Amiloride administration increases the expression of the anti-apoptotic ER chaperone GRP78 Western blot analyses revealed that GRP78 expression was highest on day 3 after injury in both groups but subsequently showed a declining trend. On day 3 after injury, GRP78 expression was significantly higher in the amiloride group than in the PBS group (P = 0.02). No significant difference was found between these groups on days 7 and 14 after injury (7 dpi: P = 0.42, 14 dpi: P = 0.63) (Fig. 2A). In contrast, CHOP expression gradually increased in both groups. Although no significant difference was found on day 3 (P = 0.94), CHOP expression was significantly lower in the amiloride group than in the PBS group on day 7 after injury (P = 0.04). No significant difference was found between these groups on day 14 after injury (P = 0.87) (Fig. 2B). Amiloride ameliorates apoptosis In both the amiloride and the PBS groups, the TUNEL assay revealed high rates of cell death on post-injury day 3 that was
focused in the lesion epicenter (Fig. 3A). The number of TUNELpositive cells at all locations (from epicenter to 13 mm rostral) decreased on post-injury day 7 compared with day 3, and the peak for apoptosis shifted to 7 mm from the epicenter, perhaps reflecting the expansion of cell death caused by secondary injury. A further decrease in the number of TUNEL-positive cells was found in all sections on day 14 after injury; only a limited number of cells were detected at the epicenter. When the two groups were compared, the number of TUNELpositive cells in the lesion epicenter was significantly lower in the amiloride group on post-injury day 3: 65.5 4.2 for the amiloride group vs. 111.8 9.6 for the PBS group [interactions between group and location: F4.50 = 1.88, P = 0.14, main effect of amiloride: F1.50 = 21.22, P < 0.01 (2.85 9 10 5), Tukey’s test: P < 0.01]. Although the differences did not reach statistical significance, the numbers of TUNEL-positive cells were constantly lower in the amiloride group in all sections (7, 10 and 13 mm from the epicenter). Similarly, at 7 and 14 days after injury, the numbers of TUNELpositive cells were lower in the amiloride group compared with the PBS group [7 dpi: interactions: F4.50 = 0.47, P = 0.76; main effect of amiloride: F1.50 = 9.18, P < 0.01 (3.87 9 10 3), Tukey’s test: P > 0.05; 14 dpi: interactions: F4.50 = 0.92, P = 0.45, main effect of amiloride: F1.50 = 0.11, P = 0.74] (Fig. 3B). These results demonstrate that amiloride treatment suppressed cell death in the injured spinal cord. Amiloride treatment spared OPCs from apoptosis Although the differences did not reach statistical significance (P > 0.05), immunohistochemistry showed that GRP78 expression in NG2-positive OPCs and GFAP-positive astrocytes was higher in the amiloride group. When these two cell types were further compared, GRP78 expression in the astrocytes tended to be higher than OPCs in both the amiloride and PBS groups, but no significant difference was observed between these groups (P > 0.05). Quantification of all cell types in the dorsal funiculus revealed that the ratio of NG2-positive OPCs gradually decreased over time in both groups with higher values in the amiloride group at all time points. In particular, on post-injury day 14, the ratio of OPCs was
A
B
Fig. 2. Western blotting was performed using anti-GRP78 (78 kDa), anti-CHOP (25 kDa) and anti-b actin (45 kDa) antibodies. (A) GRP78 expression gradually decreased in both groups. On day 3 after injury, GRP78 expression was significantly higher in the amiloride group than in the PBS group. (B) CHOP expression gradually increased in both groups. Although no significant difference was found on day 3, CHOP expression was significantly lower in the amiloride group than in the PBS group on day 7 after injury. Asterisks indicate significant difference between amiloride group and PBS group, Mann–Whitney U test (*P < 0.05, n = 6 per group, error bars are standard deviations). © 2014 Federation of European Neuroscience Societies and John Wiley & Sons Ltd European Journal of Neuroscience, 40, 3120–3127
3124 M. Kuroiwa et al. significantly higher in the amiloride group: 11.5 2.0% for the amiloride group vs. 8.4 0.7% for the PBS group (P = 0.01). These results indicate that OPCs were preferentially spared by amilA (a)
(b)
oride treatment. In contrast, while the ratio of GFAP-positive astrocytes gradually increased, there was no significant difference observed between the two groups (P > 0.05) (Fig. 4).
(c)
(d)
B
Fig. 3. (A) TUNEL staining of the lesion epicenter on day 3 after injury. High rates of cell death are observed in both the amiloride (a, c) and PBS groups (b, d), with the peak of positive cells located at the epicenter. Red = TUNEL, Blue = DAPI. Scale bars: (a, b) 500 lm, (c, d) 200 lm. (B) Significantly fewer TUNEL-positive cells were observed in the amiloride group compared with the PBS group at the lesion epicenter on day 3. Asterisks indicate significant difference between amiloride group (Ami) and PBS group (PBS), two-way repeated measures ANOVA, followed by Tukey’s multiple comparison test (**P < 0.01, n = 6 per group, error bars are standard deviations). Although the differences did not reach statistical significance, the numbers of TUNEL-positive cells at other time points were also lower in the amiloride group than in the PBS group.
A
C
B
Fig. 4. (A) Double immunostaining of the dorsal funiculus from the amiloride group for GRP78 (red) and NG2 (green) on sections taken 7 mm rostal from the epicenter of post injury day 7. Blue: DAPI. Scale bars 50 lm. (B) Double immunostaining on a similar section for GRP78 (red) and GFAP (green). Blue: DAPI. Scale bars 50 lm. GRP78 immunoreactivity is localized in the cytoplasm. (C) Quantification of each cell type. NG2-positive OPCs decreased over time in both groups. Significantly more NG2-positive cells were observed in the amiloride group than in the PBS group on day 14 after injury. Asterisks indicate significant difference between amiloride group and PBS group, Mann–Whitney U test (*P < 0.05, n = 6 per group, error bars are standard deviations). In contrast, GFAP-positive astrocytes tended to increase over time in both groups, but the differences did not reach significant levels. GRP78 expression in NG2- and GFAP-positive cells was higher in the amiloride group than in the PBS group, although no significant differences were observed between these groups. © 2014 Federation of European Neuroscience Societies and John Wiley & Sons Ltd European Journal of Neuroscience, 40, 3120–3127
Effect of amiloride in spinal cord injury 3125 A
B
Fig. 5. (A) Double immunostaining of cultured OPCs from the ami+tuni group. Red: CHOP, Green: O4, Blue: DAPI. Scale bars 100 lm. (B) The average intensity of CHOP that overlapped with the nuclei in OPCs was significantly lower in the ami+tuni group compared with the tuni group. The expression of the proapoptotic factor CHOP was lower in cultured OPCs treated with amiloride, demonstrating the protective effect of amiloride against ER stress. Asterisks indicate significant difference between the ami+tuni group and the tuni group, Mann–Whitney U test (*P < 0.05, n = 4 per group, error bars are standard deviations).
In vitro confirmation of the protective effect of amiloride Cultured OPCs were treated with tunicamycin, an ER stress inducer that increases a cell’s risk for apoptosis. The average intensity of the pro-apoptotic factor CHOP in the ami+tuni group was significantly lower than that of the tuni group (P = 0.02). Cell activity analysis performed on cultured OPCs with the Thermo Scientific Cellomics Array Scan revealed the protective effect of amiloride against ER stress (Fig. 5).
Discussion Amiloride treatment decreased cell death in the injured spinal cord, improved survival of OPCs and brought about improved motor function. Expression levels of GRP78, which protects cells against ER stress, increased after administration of amiloride, while the levels of the pro-apoptotic factor CHOP decreased. These results showed that amiloride affects ER stress and inhibits cell death in the injured spinal cord. ER stress in SCI After SCI, external stress such as excitatory amino acids and electrolyte imbalance cause structurally abnormal unfolded proteins to accumulate in the ER (Schr€oder & Kaufman, 2005; Boyce & Yuan, 2006; Larner et al., 2006), triggering the ER stress pathway that leads to cellular apoptosis. The accumulation of unfolded proteins in the ER induces the dissociation of the ER chaperone GRP78 from three ER transmembrane effector proteins: activating transcription factor 6 (ATF6), inositol requiring kinase 1 (IRE1) and RNA-activated protein kinase-like ER resident kinase (PERK) (Bertolotti et al., 2000; Shen et al., 2002; Ron & Walter, 2007; Schr€ oder, 2008). GRP78 facilitates the refolding of unfolded proteins and
exhibits cytoprotective effects against ER stress, and also activates ATF6, IRE1 and PERK. Activated ATF6 is proteolytically cleaved at the Golgi apparatus and promotes transcription of ER chaperones at the nucleus (Haze et al., 1999). Similarly, activated IRE1 promotes the transcription of ER chaperones by splicing X-box binding protein 1 (XBP1) mRNA to induce XBP1 protein synthesis (Yoshida et al., 2001; Ron & Walter, 2007). PERK inhibits protein synthesis via phosphorylation of the a-subunit of eukaryotic initiation factor-2 (eIF2a) (Harding et al., 1999, 2000). Activated eIF2a induces the translation of activating transcription factor 4 (ATF4), which promotes GRP78 gene transcription (Schr€ oder & Kaufman, 2005) and concurrently induces the expression of CHOP, a proapoptotic transcription factor that is a crucial mediator of ER stressinduced apoptosis (Zinszner et al., 1998; Oyadomari & Mori, 2004; Woehlbier & Hetz, 2011). Thus, the PERK-eIF2a pathway elicits two opposing effects: a cytoprotective effect by inhibiting mRNA translation and inducing GRP78 transcription, and a cytotoxic effect via CHOP expression. Although it has been reported that UPR is observed in SCI (Penas et al., 2007; Ohri et al., 2011), the detailed mechanisms involved remain unclear. To date, our research efforts have sought to elucidate the mechanisms by which UPR tailors specific responses to ER stress. We introduced the GRP78 gene into the rat C6 glioma cell line and added ER stress-inducing tunicamycin to these cultures and quantified Annexin V-positive apoptotic cells using fluorescence-activated cell sorting (FACS). The numbers of apoptotic cells were significantly decreased in the group overexpressing GRP78, confirming its protective effects on glial cells against ER stress (Suyama et al., 2011). Immunohistochemistry of the injured spinal cord of rats was conducted to evaluate cell-specific responses to ER stress following SCI. GRP78 expression was significantly lower in OPCs and significantly higher in astrocytes compared with
© 2014 Federation of European Neuroscience Societies and John Wiley & Sons Ltd European Journal of Neuroscience, 40, 3120–3127
3126 M. Kuroiwa et al. other cell types in the dorsal funiculus, indicating that ER stress tolerance was lower in OPCs than in other cell types (Matsuyama et al., 2014). Therefore, if apoptosis could be inhibited by controlling the ER stress response, then it might be possible to increase OPC survival, achieve remyelination and improve functional recovery. Based on the above results, we here used amiloride, a therapeutic agent that has been shown to have cytoprotective and ER stressmodulating effects in various models of neurological diseases, including SCI (Friese et al., 2007; Arias et al., 2008; Durham-Lee et al., 2011). Amiloride can affect ER stress in SCI Amiloride is a potassium-sparing diuretic approved by the Food and Drug Administration (FDA) to treat hypertension in the United States. In recent years, its neuroprotective effects against SCI (Durham-Lee et al., 2011) and neurodegenerative diseases, such as Parkinson’s disease (Arias et al., 2008) and multiple sclerosis (Friese et al., 2007), have been reported. Although the relationship between amiloride and ER stress has not been sufficiently investigated, Hosoi et al. (2010) reported that 20 lM amiloride modulated ER stress and significantly inhibited apoptosis in mouse primary cultured glial cells under tunicamycininduced ER stress. Amiloride increased eIF2a phosphorylation and, contrary to its normal behavior, decreased CHOP expression in a dose-dependent manner. The dual action of activated eIF2a makes interpretation of these results complicated, but they suggest the possibility that amiloride administered at an appropriate dose may be protective against ER stress-induced apoptosis via CHOP inhibition. In fact, Hosoi et al. (2010) reported that amiloride at 200 lM, unlike the result at 20 lM, resulted in an increase of tunicamycin-induced apoptosis by inhibiting the IRE1/XBP1/GRP78 signaling pathway. Therefore, amiloride’s effect on cell death seems be dependent on the dose used, but a careful in vivo examination of the dose– response relationship will be required. Here, we examined the effects of amiloride in a rat SCI model at a dosage of 10 mg/kg/day, in accordance with a mouse multiple sclerosis protocol that showed the neuroprotective effects of amiloride (Friese et al., 2007). We found that cell death in the injured spinal cord was significantly inhibited by amiloride, with increased GRP78 expression on post-injury day 3 and decreased CHOP expression on post-injury day 7, confirming our previous study in which GRP78 increased in the acute phase of SCI, and CHOP increased later in the sub-acute phase (Matsuyama et al., 2014). The in vitro effects of amiloride were examined at a concentration of 20 lM, based on the protocol of Hosoi et al. (2010) in which suppression of apoptosis was observed. Our results showed that the addition of 20 lM amiloride decreased CHOP expression in OPCs cultured under tunicamycin-induced ER stress, confirming the cytoprotective effects of amiloride against ER stress. Although the effects of amiloride on ER stress require further investigation, our results suggest that amiloride ameliorates ER stress and inhibits cell death in the injured spinal cord. Neuroprotective effects of amiloride in SCI Numerous studies have reported the neuroprotective effects of amiloride in various neurological disease models. In an in vitro model of brain ischemia under acidic conditions (pH 6.0), cytotoxicity was inhibited by treating mouse cortical neurons with amiloride (Xiong et al., 2004). Amiloride administration reduced demyelination and axonal degeneration in a multiple sclerosis mouse model (Friese
et al., 2007; Vergo et al., 2011) and suppressed neuronal degeneration in the substantia nigra in a Parkinson’s disease mouse model (Arias et al., 2008). Among the few studies that investigated the effects of amiloride in an SCI model, one report showed that amiloride had neuroprotective effects in an in vitro model of SCI (Agrawal & Fehlings, 1996). Another study reported improvements in hindlimb function and increased myelin oligodendrocyte glycoprotein in a rat SCI model (Durham-Lee et al., 2011). Similarly, in our current study, significant improvements in hindlimb function were observed after amiloride administration in a rat SCI model, possibly brought about by the reduced cell death revealed by TUNEL assays. Interestingly, the number of NG2-positive OPCs in the dorsal funiculus decreased over time in both of our experimental groups, but the NG2-positive cells were preferentially spared in the amiloride group compared with the PBS group. Furthermore, our in vitro results showed that amiloride decreased the expression of the proapoptic factor CHOP in OPCs cultured under ER stress. Based on these findings, we suggest that amiloride contributes to the survival of OPCs and may play a role in remyelination after SCI.
Conclusion Our results suggest that amiloride ameliorates ER stress in the injured spinal cord and improves hindlimb function after SCI by inhibiting cell death, leading to OPC survival. We consider amiloride to be a potential therapeutic agent for treating spinal cord injuries by targeting ER stress pathways.
Acknowledgements We thank our staff and Dr. Chisa Okada at the Teaching and Research Support Center, Tokai University, for their advice and technical assistance. All animal experiments were conducted in accordance with the protocol approved by the Animal Experimentation Committee at our institution. This work was supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology, Japan (21591907).
Abbreviations ATF, activating transcription factor; BBB scale, the Basso, Beattie, Bresnahan locomotor rating scale; BSA, bovine serum albumin; CHOP, C/EBP homologous transcription factor protein; DMEM, Dulbecco’s modified Eagle’s medium; eIF2a, a-subunit of eukaryotic initiation factor-2; ER, endoplasmic reticulum; GRP78, glucose-regulated protein 78; IRE1, inositol requiring kinase 1; OPC, oligodendrocyte precursor cell; PERK, RNA-activated protein kinase-like ER resident kinase; SCI, spinal cord injury; UPR, unfolded protein response; XBP1, X-box binding protein 1.
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doi:10.1111/ejn.12647
DISORDERS OF THE NERVOUS SYSTEM
Effect of amiloride on endoplasmic reticulum stress response in the injured spinal cord of rats Masahiro Kuroiwa,1 Masahiko Watanabe,1 Hiroyuki Katoh,1 Kaori Suyama,2 Daisuke Matsuyama,1 Takeshi Imai1 and Joji Mochida1 1
Department of Orthopaedic Surgery, Surgical Science, Tokai University School of Medicine, 143 Shimokasuya, Isehara, Kanagawa 259-1193, Japan 2 Department of Anatomy and Cellular Biology, Tokai University School of Medicine, Isehara, Kanagawa, Japan Keywords: apoptosis, glial cells, in vivo, rat, spinal cord injury
Abstract After traumatic spinal cord injury (SCI), endoplasmic reticulum (ER) stress exacerbates secondary injury, leading to expansion of demyelination and reduced remyelination due to oligodendrocyte precursor cell (OPC) apoptosis. Although recent studies have revealed that amiloride controls ER stress and leads to improvement in several neurological disorders including SCI, its mechanism is not completely understood. Here, we used a rat SCI model to assess the effects of amiloride on functional recovery, secondary damage expansion, ER stress-induced cell death and OPC survival. Hindlimb function in rats with spinal cord contusion significantly improved after amiloride administration. Amiloride significantly decreased the expression of the pro-apoptotic transcription factor CHOP in the injured spinal cord and significantly increased the expression of the ER chaperone GRP78, which protects cells against ER stress. In addition, amiloride treatment led to a significant decrease in ER stress-induced apoptosis and a significant increase of NG2-positive OPCs in the injured spinal cord. Furthermore, in vitro experiments performed to investigate the direct effect of amiloride on OPCs revealed that amiloride reduced CHOP expression in OPCs cultured under ER stress. These results suggest that amiloride controls ER stress in SCI and inhibits cellular apoptosis, contributing to OPC survival. The present study suggests that amiloride may be an effective treatment to reduce ER stress-induced cell death in the acute phase of SCI.
Introduction In traumatic spinal cord injury (SCI), primary injury refers to the physical tissue disruption that is caused by direct external forces. This is followed by a secondary injury in which biochemical and vascular factors (Tator & Fehlings, 1991; Watanabe et al., 1998; Lu et al., 2000; Park et al., 2004) cause delayed damage that expands through the spinal cord (Schwab & Bartholdi, 1996). One of the main factors contributing to secondary injury is the demyelination that occurs as a result of glial cell apoptosis (Crowe et al., 1997). Although oligodendrocyte precursor cells (OPCs) proliferate at the periphery of the injury epicenter after SCI, a majority of OPCs fail to differentiate into mature oligodendrocytes and are eliminated through apoptosis (Ishii et al., 2001; McTigue et al., 2001). As OPCs proliferate, survive and differentiate into mature oligodendrocytes and promote remyelination in multiple sclerosis chemical demyelination models (Chang et al., 2000; Watanabe et al., 2002), it is likely that the apoptosis of OPCs is a major factor in the failure of remyelination following SCI. Given that astrocytes proliferate without succumbing to apoptosis and participate in the formation of the glial scar after SCI (Mckeon et al., 1991; Baldwin et al., 1998;
Correspondence: Dr M. Watanabe, as above. E-mail: [email protected] Received 27 February 2014, revised 30 April 2014, accepted 2 May 2014
Fawcett & Asher, 1999; Eng et al., 2000), the significance of understanding the regulation of apoptosis is clear. To elucidate the mechanism underlying OPC apoptosis and the diverse reactions observed among different cell types after SCI, we focused on the endoplasmic reticulum (ER) stress pathway, which is one of many apoptotic pathways. When a cell is exposed to external stress factors, structurally abnormal, misfolded proteins (unfolded proteins) accumulate in the ER. This triggers an ER stress pathway that activates the C/EBP homologous transcription factor protein (CHOP), which is a proapoptotic factor that mediates programmed cell death. To protect cells against unfolded proteins, the ER chaperone glucose-regulated protein 78 (GRP78) mediates the refolding of unfolded proteins. It has been shown that the unfolded protein response (UPR), which is the cellular stress response against ER stress, is also involved in demyelination and the apoptosis of glial cells that leads to failure of remyelination in the secondary injury process following SCI (Penas et al., 2007; Ohri et al., 2011). Therefore, if ER stress can be controlled, it might be possible to reduce secondary injury and to induce remyelination via OPC survival. In this study, we used amiloride, a therapeutic agent that has been shown to have cytoprotective and ER stress-modulating effects in various models of neurological diseases. We administered amiloride to a rat SCI model and examined the effects of amiloride on secondary
© 2014 Federation of European Neuroscience Societies and John Wiley & Sons Ltd
Effect of amiloride in spinal cord injury 3121 injury expansion, ER stress-induced apoptosis, OPC survival and functional recovery. In addition, we examined the direct effect of amiloride on OPCs by adding amiloride to rat OPC cultures under ER stress and assessing its effects on cell activity.
Materials and methods Rat SCI model All experimental procedures were performed in accordance with the National Institute of Health Guidelines for the Care and Use of Laboratory Animals and were approved by the Animal Experimentation Committee of Tokai University School of Medicine. For these experiments, female Sprague–Dawley (SD) rats (age, 10 weeks, 280–320 g) were obtained from CLEA Japan, Inc. (Kanagawa, Japan). Surgical procedures were performed under aseptic conditions, and the rats were anesthetized with inhaled 4% isoflurane. Laminectomy of the 10th thoracic vertebra was performed to expose the dura mater. A spinal cord contusion model was created using an Infinite Horizon spinal cord impactor device (IH impactor; Precision Systems & Instrumentation, Lexington, KY, USA) with a force of 200 Kdyne (2 mN). To care for the bladder dysfunction that occurs after SCI, abdominal massages were performed twice a day to empty the bladder. The injured rats were divided into two groups: an amiloride group and a phosphate-buffered saline (PBS) control group (n = 6 per group). At 24 h after injury and every 24 h thereafter for 28 days, the amiloride group received intraperitoneal administration of 10 mg/kg amiloride (amiloride hydrochloride hydrate, catalog #A7410; Sigma-Aldrich, St. Louis, MO, USA) in accordance with a protocol used for a mouse multiple sclerosis model (Friese et al., 2007). The body weight of each rat was measured daily, and the drug dosage was calculated. The PBS group received intraperitoneal administration of PBS using the same protocol. Control animals were age-matched normal female SD rats (age, 10 weeks; 280–320 g) that received a sham surgery consisting of a laminectomy of the 10th thoracic vertebra. Evaluation of hindlimb function The effects of amiloride treatment on hindlimb motor function after SCI were assessed using the Basso, Beattie and Bresnahan locomotor rating scale (BBB scale), which is an open-field locomotor test for rats. Locomotor behavior was evaluated immediately before injury and each following day at approximately the same time for 28 days after injury. The mean score was calculated using the measurements derived by three observers through 5 min of careful observation. Each observer was blinded regarding the treatment used (n = 6 per group). The BBB scores were analysed by two-way repeated-measures analysis of variance (ANOVA) with Tukey’s post hoc multiple comparison test. Western blot At 3, 7 and 14 days after injury, a rat was anesthetized with inhaled 4% isoflurane and 5 mm of the injured spinal cord (2.5 mm rostral and caudal to the epicenter) was microscopically dissected. The excised spinal cord tissue was immediately washed in ice cold PBS, after which extracts were prepared using a Cell Lytic NuCLEAR extraction kit (Sigma-Aldrich). These extracts were subjected to electrophoresis on a 12.5% sodium dodecyl sulfate polyacrylamide gel; 20 lg of protein solution was applied to each gel lane. After electrophoresis, the proteins were electrotransferred onto nitrocellu-
lose membranes (Bio-Rad, Hercules, CA, USA). The membranes were blocked with 5% bovine serum albumin (BSA) in TBST (50 mM Tris, pH 7.6, 150 mM NaCl, 0.1% Tween-20), followed by incubation with anti-GRP78 (rabbit anti-HSPA5/GRP78, catalog #APO6149PU-N, 1 : 2000, catalog #BS1136, 1 : 200; ACRIS, Herford, Germany) and anti-CHOP (rabbit anti-GADD153, bioWORLD, Atlanta, GA, USA) antibodies overnight at 4 °C. The membranes were washed for 7 h in PBS containing 0.05% Tween20 and incubated for 60 min at 25 °C with horseradish peroxidase (HRP)-linked anti-rabbit IgG (catalog #NA9340; GRP78 1 : 5000, CHOP 1 : 1000; GE Healthcare, Amersham, UK). The proteins were labeled with Immobilon Western Chemiluminescent HRP and were quantified by densitometric scanning analysis of the films using MACINTOSH CS ANALYSER SOFTWARE (Atto, Tokyo, Japan). bActin was used as an internal control and was labeled with a mouse monoclonal anti-b-actin antibody (catalog #A5441, 1 : 1000; Sigma-Aldrich). GRP78 and CHOP expression levels were analysed with Mann–Whitney U test (n = 6 per group). Tissue processing for TUNEL staining and immunohistochemical staining Perfusion fixation was performed at 3, 7 and 14 days after injury using 2% paraformaldehyde (PFA) – 0.1 M phosphate buffer (PB) under inhaled 4% isoflurane anesthesia. After fixation, the spinal cord was excised and post-fixed in 2% PFA in 0.1 M PB for 2 days at 4 °C and dehydrated in a series of increasing sucrose concentrations (7, 15, 20%). Frozen spinal cord tissue blocks were embedded in Optimal Cutting Temperature (OCT) compound (Sakura Finetek, Tokyo, Japan), and 10-lm-thick tissue sections were prepared using a cryostat. The epicenter was defined as the width of the tip of the IH impactor: 2 mm. From this epicenter, spinal cord sections were dissected at 3-mm increments: at the epicenter, and at 4, 7, 10 and 13 mm rostral to the epicenter. TUNEL staining Using specimens obtained on days 3, 7 and 14 after injury, TUNEL staining was performed on sections from the epicenter and 4, 7, 10 and 13 mm rostral from the epicenter (n = 6 per group). Because cauda equina was encountered in the region caudal to the epicenter, we used specimens taken from the epicenter and rostral regions. The sections were washed three times (10 min each) with PBS and incubated in a permeabilization solution (0.1% Triton-X, 0.1% sodium citrate) for 2 min on ice. The sections were then subjected to TUNEL staining using the In Situ Cell Death Detection Kit TMR red (catalog #12156792910; Roche, Mannheim, Germany) and incubated at 37 °C for 60 min. As negative controls, sections were incubated in the label solution without the enzyme solution (terminal transferase) instead of the TUNEL reaction solution, which contains both solutions. The sections were washed twice with PBS, and nuclei were stained and mounted using Vectashield with DAPI H-1500 (Vector Laboratories, Burlingame, CA, USA). Images were taken with a Nikon DS-Ri1 (Nikon, Tokyo, Japan) camera using mono 10-bit lenses (4 9 objective NA=0.13, 20 9 objective NA=0.4) on an Olympus IX70 fluorescence microscope (Olympus, Tokyo, Japan) and analysed with Nikon NIS-elements Ver.3.1 software (Nikon). TUNEL-positive cells with clearly recognizable nuclei and cytoplasm and with co-localized TMR red- and DAPI-stained nuclei were recorded as positive. The numbers of TUNEL-positive cells in the dorsal funiculus were counted and analysed with two-way
© 2014 Federation of European Neuroscience Societies and John Wiley & Sons Ltd European Journal of Neuroscience, 40, 3120–3127
3122 M. Kuroiwa et al. repeated-measures ANOVA with Tukey’s post hoc multiple comparison test. The differences in the temporal and spatial expansion of cell death were compared between the two groups. Immunohistochemical staining For immunohistochemical staining, 10-lm-thick tissue sections taken 7 mm rostral to the epicenter of post-injury 3-, 7- and 14-day animals were used (n = 6 per group). The sections were washed three times (10 min each) with PBS and blocked for 60 min in PBS with 5% normal goat serum at 24 °C. The sections were washed again for 10 min and incubated overnight at 4 °C with anti-GRP78 (rabbit anti-HSPA5/GRP78, ACRIS, catalog #APO6149PU-N, 1 : 200), anti-NG2 (mouse anti-NG2, catalog #MAB5384, 1 : 50; a marker for OPCs; Merck Millipore, Billerica, MA, USA) and antiGFAP (mouse anti-GFAP, catalog #MAB360, 1 : 400; a marker for astrocytes; Millipore). The sections were washed again with PBS and incubated for 60 min at 24 °C in the dark with the respective fluorescent secondary antibodies (GRP78: Alexa Fluor594, anti-rabbit; Invitrogen, Carlsbad, CA, USA, catalog #A11012, 1 : 1000; NG2 and GFAP: Alexa Fluor488, anti-mouse, Invitrogen, catalog #A11029, 1 : 1000). Subsequently, nuclei were stained and mounted using Vectashield with DAPI H-1500 (Vector Laboratories). Spinal cord tissue from sham surgery rats was used as control. Images were taken with a Nikon DS-Ri1 camera using mono 10-bit lenses (40 9 objective NA=0.6) on an Olympus IX70 fluorescence microscope and analysed with Nikon NIS-elements Ver.3.1 software. To quantify the ratios of OPCs or astrocytes, we counted the number of cells positive for each cell marker and divided it by the total number of nuclei in the dorsal funiculus. To quantify GRP78 expression ratios in each cell type, the number of cells double positive for each cell marker and GRP78 was counted and was divided by the total number of cells positive for each cell marker in the dorsal funiculus. We determined the following: (i) percentages of GRP78-positive cells among all DAPI-positive cells; (ii) percentages of NG2-positive cells among all DAPI-positive cells; (iii) percentages of GFAP-positive cells among all DAPI-positive cells; (iv) percentages of GRP78positive cells among all NG2-positive cells; and (v) percentages of GRP78-positive cells among all GFAP-positive cells. Only those cells with clearly recognizable nuclei and cytoplasm that had cytoplasmic staining corresponding to each secondary antibody were scored as positive. Negative controls were incubated with secondary antibodies in the absence of primary antibodies. Data obtained through immunohistochemistry were analysed with Mann–Whitney U test. Cell culture and treatment of OPCs
pyruvate (0.11 mg/mL), and penicillin-streptomycin (10 IU/mL and 100 lg/mL, respectively)], and 0.1% BSA (all from Sigma-Aldrich). After 1–2 days, the cells were incubated with platelet-derived growth factor (PDGF; PeproTech, Rocky Hill, NJ, USA; 10 ng/mL) and were cultured for 2–3 days to allow for OPC differentiation. To evaluate the ER stress response, the OPCs were incubated with 3 lM tunicamycin (Sigma-Aldrich) and 20 lM amiloride (ami+tuni group) or 3 lM tunicamycin alone (tuni group) for 6 h (n = 4 per group). Control cells were cultured under the same conditions without tunicamycin or amiloride. Immunostaining to evaluate OPC activity Live cells were immunolabeled as described Bansal et al. (2003). Briefly, cells were blocked for non-specific absorption with HEPESbuffered Earl’s balanced salt solution (EBSS-HEPES) containing 3% normal goat serum (also used for diluting antibodies), and then double immunolabeled with oligodendrocyte lineage stage-specific markers [early progenitors, A2B5 (R and D Systems, Minneapolis, MN, USA); late progenitors, O4 (R and D Systems)]. After fixation with 4% PFA for 15 min, cells were permeabilized (0.3% Triton X100), blocked with 5% BSA/PBS, and stained for CHOP (rabbit anti-GADD153, bioWORLD, catalog #BS1136, 1 : 200). Cells were then labeled with the appropriate secondary antibodies [Alexa 488conjugated anti-mouse antibody; Alexa Fluor 594 (anti-rabbit, catalog #A11012, 1 : 1000; Invitrogen) and a nuclear label, Vectashield with DAPI H-1500 (Vector Laboratories)]. Cells were then mounted and evaluated using the Thermo Scientific Cellomics Array Scan (Thermo Fisher Scientific, Kanagawa, Japan). The average intensity of CHOP that overlapped with the nuclei in OPCs was measured from ten random fields per well and was analysed with Mann–Whitney U test. Statistical analysis Statistical significance was determined using PASW statistics 18 (IBM SPSS, Foster City, CA, USA). Mann–Whitney U tests were used to analyse GRP78 and CHOP expression levels obtained by Western blotting, the in vivo immunohistochemical measurements and the in vitro evaluation of OPCs. The BBB scores and TUNEL analysis were evaluated with a two-way repeated-measures ANOVA with Tukey’s post hoc multiple comparison test. Significant interactions were further analysed with a one-way ANOVA followed by Tukey’s test. Asterisks in figures indicate statistical significance (*P < 0.05, **P < 0.01).
Results
Enriched cultures of OPCs were prepared as described elsewhere (Armstrong, 1998; Guardiola-Diaz et al., 2012). Mixed primary cultures from neonatal (P1–2) rat telencephalon were shaken overnight in an orbital shaker. Dislodged OPCs (and some preprogenitors) were further purified by differential adhesion. Early progenitors were seeded on poly-L-lysine- (Sigma-Aldrich) coated four-well plates (1 9 105 cells per well for immunocytochemistry). The cells were first cultured for 3 h in Dulbecco’s modified Eagle’s medium (DMEM; Invitrogen) supplemented with 5% (v/v) fetal bovine serum (FBS) to allow for attachment, after which the cells were grown in serum-free N2 defined medium [DMEM supplemented with human transferrin (50 lL/mL), bovine pancreatic insulin (5 lL/mL), 3,3,5-triiodo-L-thyronine (10 ng/mL), sodium selenium (30 nM), D-biotin (10 ng/mL), hydrocortisone (10 nM), sodium
Amiloride improves functional recovery after SCI For both the amiloride and the PBS groups, the BBB scores dropped to 0 after injury and gradually improved thereafter. No significant differences were found between the two groups until day 11 after injury. However, at day 12, the amiloride group showed a significant improvement compared with the PBS group, and this difference was maintained for the duration of the study after day 15 (interaction between group and days post operation: F27.280 = 3.8, P < 0.01 (5.82 9 10 9), one-way ANOVA: F55.280 = 76.95, P < 0.01 (7.39 9 10 140), Tukey’s test: P < 0.05). The mean scores on the last observation day (day 28 after injury) were 15.0 1.23 for the amiloride group and 12.8 1.17 for the PBS group (Fig. 1).
© 2014 Federation of European Neuroscience Societies and John Wiley & Sons Ltd European Journal of Neuroscience, 40, 3120–3127
Effect of amiloride in spinal cord injury 3123
Fig. 1. Amiloride improves functional recovery after SCI. BBB scale scores were significantly higher in the amiloride group compared with the PBS group from day 15 and later. Asterisks indicate significant difference between amiloride group (Ami) and PBS group (PBS), two-way repeated measures ANOVA, followed by Tukey’s multiple comparison test (*P < 0.05, **P < 0.01, n = 6 per group, error bars are standard deviations).
Amiloride administration increases the expression of the anti-apoptotic ER chaperone GRP78 Western blot analyses revealed that GRP78 expression was highest on day 3 after injury in both groups but subsequently showed a declining trend. On day 3 after injury, GRP78 expression was significantly higher in the amiloride group than in the PBS group (P = 0.02). No significant difference was found between these groups on days 7 and 14 after injury (7 dpi: P = 0.42, 14 dpi: P = 0.63) (Fig. 2A). In contrast, CHOP expression gradually increased in both groups. Although no significant difference was found on day 3 (P = 0.94), CHOP expression was significantly lower in the amiloride group than in the PBS group on day 7 after injury (P = 0.04). No significant difference was found between these groups on day 14 after injury (P = 0.87) (Fig. 2B). Amiloride ameliorates apoptosis In both the amiloride and the PBS groups, the TUNEL assay revealed high rates of cell death on post-injury day 3 that was
focused in the lesion epicenter (Fig. 3A). The number of TUNELpositive cells at all locations (from epicenter to 13 mm rostral) decreased on post-injury day 7 compared with day 3, and the peak for apoptosis shifted to 7 mm from the epicenter, perhaps reflecting the expansion of cell death caused by secondary injury. A further decrease in the number of TUNEL-positive cells was found in all sections on day 14 after injury; only a limited number of cells were detected at the epicenter. When the two groups were compared, the number of TUNELpositive cells in the lesion epicenter was significantly lower in the amiloride group on post-injury day 3: 65.5 4.2 for the amiloride group vs. 111.8 9.6 for the PBS group [interactions between group and location: F4.50 = 1.88, P = 0.14, main effect of amiloride: F1.50 = 21.22, P < 0.01 (2.85 9 10 5), Tukey’s test: P < 0.01]. Although the differences did not reach statistical significance, the numbers of TUNEL-positive cells were constantly lower in the amiloride group in all sections (7, 10 and 13 mm from the epicenter). Similarly, at 7 and 14 days after injury, the numbers of TUNELpositive cells were lower in the amiloride group compared with the PBS group [7 dpi: interactions: F4.50 = 0.47, P = 0.76; main effect of amiloride: F1.50 = 9.18, P < 0.01 (3.87 9 10 3), Tukey’s test: P > 0.05; 14 dpi: interactions: F4.50 = 0.92, P = 0.45, main effect of amiloride: F1.50 = 0.11, P = 0.74] (Fig. 3B). These results demonstrate that amiloride treatment suppressed cell death in the injured spinal cord. Amiloride treatment spared OPCs from apoptosis Although the differences did not reach statistical significance (P > 0.05), immunohistochemistry showed that GRP78 expression in NG2-positive OPCs and GFAP-positive astrocytes was higher in the amiloride group. When these two cell types were further compared, GRP78 expression in the astrocytes tended to be higher than OPCs in both the amiloride and PBS groups, but no significant difference was observed between these groups (P > 0.05). Quantification of all cell types in the dorsal funiculus revealed that the ratio of NG2-positive OPCs gradually decreased over time in both groups with higher values in the amiloride group at all time points. In particular, on post-injury day 14, the ratio of OPCs was
A
B
Fig. 2. Western blotting was performed using anti-GRP78 (78 kDa), anti-CHOP (25 kDa) and anti-b actin (45 kDa) antibodies. (A) GRP78 expression gradually decreased in both groups. On day 3 after injury, GRP78 expression was significantly higher in the amiloride group than in the PBS group. (B) CHOP expression gradually increased in both groups. Although no significant difference was found on day 3, CHOP expression was significantly lower in the amiloride group than in the PBS group on day 7 after injury. Asterisks indicate significant difference between amiloride group and PBS group, Mann–Whitney U test (*P < 0.05, n = 6 per group, error bars are standard deviations). © 2014 Federation of European Neuroscience Societies and John Wiley & Sons Ltd European Journal of Neuroscience, 40, 3120–3127
3124 M. Kuroiwa et al. significantly higher in the amiloride group: 11.5 2.0% for the amiloride group vs. 8.4 0.7% for the PBS group (P = 0.01). These results indicate that OPCs were preferentially spared by amilA (a)
(b)
oride treatment. In contrast, while the ratio of GFAP-positive astrocytes gradually increased, there was no significant difference observed between the two groups (P > 0.05) (Fig. 4).
(c)
(d)
B
Fig. 3. (A) TUNEL staining of the lesion epicenter on day 3 after injury. High rates of cell death are observed in both the amiloride (a, c) and PBS groups (b, d), with the peak of positive cells located at the epicenter. Red = TUNEL, Blue = DAPI. Scale bars: (a, b) 500 lm, (c, d) 200 lm. (B) Significantly fewer TUNEL-positive cells were observed in the amiloride group compared with the PBS group at the lesion epicenter on day 3. Asterisks indicate significant difference between amiloride group (Ami) and PBS group (PBS), two-way repeated measures ANOVA, followed by Tukey’s multiple comparison test (**P < 0.01, n = 6 per group, error bars are standard deviations). Although the differences did not reach statistical significance, the numbers of TUNEL-positive cells at other time points were also lower in the amiloride group than in the PBS group.
A
C
B
Fig. 4. (A) Double immunostaining of the dorsal funiculus from the amiloride group for GRP78 (red) and NG2 (green) on sections taken 7 mm rostal from the epicenter of post injury day 7. Blue: DAPI. Scale bars 50 lm. (B) Double immunostaining on a similar section for GRP78 (red) and GFAP (green). Blue: DAPI. Scale bars 50 lm. GRP78 immunoreactivity is localized in the cytoplasm. (C) Quantification of each cell type. NG2-positive OPCs decreased over time in both groups. Significantly more NG2-positive cells were observed in the amiloride group than in the PBS group on day 14 after injury. Asterisks indicate significant difference between amiloride group and PBS group, Mann–Whitney U test (*P < 0.05, n = 6 per group, error bars are standard deviations). In contrast, GFAP-positive astrocytes tended to increase over time in both groups, but the differences did not reach significant levels. GRP78 expression in NG2- and GFAP-positive cells was higher in the amiloride group than in the PBS group, although no significant differences were observed between these groups. © 2014 Federation of European Neuroscience Societies and John Wiley & Sons Ltd European Journal of Neuroscience, 40, 3120–3127
Effect of amiloride in spinal cord injury 3125 A
B
Fig. 5. (A) Double immunostaining of cultured OPCs from the ami+tuni group. Red: CHOP, Green: O4, Blue: DAPI. Scale bars 100 lm. (B) The average intensity of CHOP that overlapped with the nuclei in OPCs was significantly lower in the ami+tuni group compared with the tuni group. The expression of the proapoptotic factor CHOP was lower in cultured OPCs treated with amiloride, demonstrating the protective effect of amiloride against ER stress. Asterisks indicate significant difference between the ami+tuni group and the tuni group, Mann–Whitney U test (*P < 0.05, n = 4 per group, error bars are standard deviations).
In vitro confirmation of the protective effect of amiloride Cultured OPCs were treated with tunicamycin, an ER stress inducer that increases a cell’s risk for apoptosis. The average intensity of the pro-apoptotic factor CHOP in the ami+tuni group was significantly lower than that of the tuni group (P = 0.02). Cell activity analysis performed on cultured OPCs with the Thermo Scientific Cellomics Array Scan revealed the protective effect of amiloride against ER stress (Fig. 5).
Discussion Amiloride treatment decreased cell death in the injured spinal cord, improved survival of OPCs and brought about improved motor function. Expression levels of GRP78, which protects cells against ER stress, increased after administration of amiloride, while the levels of the pro-apoptotic factor CHOP decreased. These results showed that amiloride affects ER stress and inhibits cell death in the injured spinal cord. ER stress in SCI After SCI, external stress such as excitatory amino acids and electrolyte imbalance cause structurally abnormal unfolded proteins to accumulate in the ER (Schr€oder & Kaufman, 2005; Boyce & Yuan, 2006; Larner et al., 2006), triggering the ER stress pathway that leads to cellular apoptosis. The accumulation of unfolded proteins in the ER induces the dissociation of the ER chaperone GRP78 from three ER transmembrane effector proteins: activating transcription factor 6 (ATF6), inositol requiring kinase 1 (IRE1) and RNA-activated protein kinase-like ER resident kinase (PERK) (Bertolotti et al., 2000; Shen et al., 2002; Ron & Walter, 2007; Schr€ oder, 2008). GRP78 facilitates the refolding of unfolded proteins and
exhibits cytoprotective effects against ER stress, and also activates ATF6, IRE1 and PERK. Activated ATF6 is proteolytically cleaved at the Golgi apparatus and promotes transcription of ER chaperones at the nucleus (Haze et al., 1999). Similarly, activated IRE1 promotes the transcription of ER chaperones by splicing X-box binding protein 1 (XBP1) mRNA to induce XBP1 protein synthesis (Yoshida et al., 2001; Ron & Walter, 2007). PERK inhibits protein synthesis via phosphorylation of the a-subunit of eukaryotic initiation factor-2 (eIF2a) (Harding et al., 1999, 2000). Activated eIF2a induces the translation of activating transcription factor 4 (ATF4), which promotes GRP78 gene transcription (Schr€ oder & Kaufman, 2005) and concurrently induces the expression of CHOP, a proapoptotic transcription factor that is a crucial mediator of ER stressinduced apoptosis (Zinszner et al., 1998; Oyadomari & Mori, 2004; Woehlbier & Hetz, 2011). Thus, the PERK-eIF2a pathway elicits two opposing effects: a cytoprotective effect by inhibiting mRNA translation and inducing GRP78 transcription, and a cytotoxic effect via CHOP expression. Although it has been reported that UPR is observed in SCI (Penas et al., 2007; Ohri et al., 2011), the detailed mechanisms involved remain unclear. To date, our research efforts have sought to elucidate the mechanisms by which UPR tailors specific responses to ER stress. We introduced the GRP78 gene into the rat C6 glioma cell line and added ER stress-inducing tunicamycin to these cultures and quantified Annexin V-positive apoptotic cells using fluorescence-activated cell sorting (FACS). The numbers of apoptotic cells were significantly decreased in the group overexpressing GRP78, confirming its protective effects on glial cells against ER stress (Suyama et al., 2011). Immunohistochemistry of the injured spinal cord of rats was conducted to evaluate cell-specific responses to ER stress following SCI. GRP78 expression was significantly lower in OPCs and significantly higher in astrocytes compared with
© 2014 Federation of European Neuroscience Societies and John Wiley & Sons Ltd European Journal of Neuroscience, 40, 3120–3127
3126 M. Kuroiwa et al. other cell types in the dorsal funiculus, indicating that ER stress tolerance was lower in OPCs than in other cell types (Matsuyama et al., 2014). Therefore, if apoptosis could be inhibited by controlling the ER stress response, then it might be possible to increase OPC survival, achieve remyelination and improve functional recovery. Based on the above results, we here used amiloride, a therapeutic agent that has been shown to have cytoprotective and ER stressmodulating effects in various models of neurological diseases, including SCI (Friese et al., 2007; Arias et al., 2008; Durham-Lee et al., 2011). Amiloride can affect ER stress in SCI Amiloride is a potassium-sparing diuretic approved by the Food and Drug Administration (FDA) to treat hypertension in the United States. In recent years, its neuroprotective effects against SCI (Durham-Lee et al., 2011) and neurodegenerative diseases, such as Parkinson’s disease (Arias et al., 2008) and multiple sclerosis (Friese et al., 2007), have been reported. Although the relationship between amiloride and ER stress has not been sufficiently investigated, Hosoi et al. (2010) reported that 20 lM amiloride modulated ER stress and significantly inhibited apoptosis in mouse primary cultured glial cells under tunicamycininduced ER stress. Amiloride increased eIF2a phosphorylation and, contrary to its normal behavior, decreased CHOP expression in a dose-dependent manner. The dual action of activated eIF2a makes interpretation of these results complicated, but they suggest the possibility that amiloride administered at an appropriate dose may be protective against ER stress-induced apoptosis via CHOP inhibition. In fact, Hosoi et al. (2010) reported that amiloride at 200 lM, unlike the result at 20 lM, resulted in an increase of tunicamycin-induced apoptosis by inhibiting the IRE1/XBP1/GRP78 signaling pathway. Therefore, amiloride’s effect on cell death seems be dependent on the dose used, but a careful in vivo examination of the dose– response relationship will be required. Here, we examined the effects of amiloride in a rat SCI model at a dosage of 10 mg/kg/day, in accordance with a mouse multiple sclerosis protocol that showed the neuroprotective effects of amiloride (Friese et al., 2007). We found that cell death in the injured spinal cord was significantly inhibited by amiloride, with increased GRP78 expression on post-injury day 3 and decreased CHOP expression on post-injury day 7, confirming our previous study in which GRP78 increased in the acute phase of SCI, and CHOP increased later in the sub-acute phase (Matsuyama et al., 2014). The in vitro effects of amiloride were examined at a concentration of 20 lM, based on the protocol of Hosoi et al. (2010) in which suppression of apoptosis was observed. Our results showed that the addition of 20 lM amiloride decreased CHOP expression in OPCs cultured under tunicamycin-induced ER stress, confirming the cytoprotective effects of amiloride against ER stress. Although the effects of amiloride on ER stress require further investigation, our results suggest that amiloride ameliorates ER stress and inhibits cell death in the injured spinal cord. Neuroprotective effects of amiloride in SCI Numerous studies have reported the neuroprotective effects of amiloride in various neurological disease models. In an in vitro model of brain ischemia under acidic conditions (pH 6.0), cytotoxicity was inhibited by treating mouse cortical neurons with amiloride (Xiong et al., 2004). Amiloride administration reduced demyelination and axonal degeneration in a multiple sclerosis mouse model (Friese
et al., 2007; Vergo et al., 2011) and suppressed neuronal degeneration in the substantia nigra in a Parkinson’s disease mouse model (Arias et al., 2008). Among the few studies that investigated the effects of amiloride in an SCI model, one report showed that amiloride had neuroprotective effects in an in vitro model of SCI (Agrawal & Fehlings, 1996). Another study reported improvements in hindlimb function and increased myelin oligodendrocyte glycoprotein in a rat SCI model (Durham-Lee et al., 2011). Similarly, in our current study, significant improvements in hindlimb function were observed after amiloride administration in a rat SCI model, possibly brought about by the reduced cell death revealed by TUNEL assays. Interestingly, the number of NG2-positive OPCs in the dorsal funiculus decreased over time in both of our experimental groups, but the NG2-positive cells were preferentially spared in the amiloride group compared with the PBS group. Furthermore, our in vitro results showed that amiloride decreased the expression of the proapoptic factor CHOP in OPCs cultured under ER stress. Based on these findings, we suggest that amiloride contributes to the survival of OPCs and may play a role in remyelination after SCI.
Conclusion Our results suggest that amiloride ameliorates ER stress in the injured spinal cord and improves hindlimb function after SCI by inhibiting cell death, leading to OPC survival. We consider amiloride to be a potential therapeutic agent for treating spinal cord injuries by targeting ER stress pathways.
Acknowledgements We thank our staff and Dr. Chisa Okada at the Teaching and Research Support Center, Tokai University, for their advice and technical assistance. All animal experiments were conducted in accordance with the protocol approved by the Animal Experimentation Committee at our institution. This work was supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology, Japan (21591907).
Abbreviations ATF, activating transcription factor; BBB scale, the Basso, Beattie, Bresnahan locomotor rating scale; BSA, bovine serum albumin; CHOP, C/EBP homologous transcription factor protein; DMEM, Dulbecco’s modified Eagle’s medium; eIF2a, a-subunit of eukaryotic initiation factor-2; ER, endoplasmic reticulum; GRP78, glucose-regulated protein 78; IRE1, inositol requiring kinase 1; OPC, oligodendrocyte precursor cell; PERK, RNA-activated protein kinase-like ER resident kinase; SCI, spinal cord injury; UPR, unfolded protein response; XBP1, X-box binding protein 1.
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