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Keywords:

  • CRE decoy oligonucleotide;
  • cyclic AMP responsive element binding protein;
  • gerbil;
  • hippocampus;
  • ischemic tolerance

Summary

  1. Top of page
  2. Summary
  3. Materials and methods
  4. Induction of forebrain ischemia
  5. Western blotting
  6. Immunohistochemistry
  7. Transfection of CRE decoy oligonucleotide into NG108-15 cells
  8. Intracerebroventricular CRE decoy oligonucleotide administration
  9. Electrophoretic mobility shift assay
  10. Statistical evaluation
  11. Results
  12. Changes in phosphorylation levels of CREB-Ser133 after ischemic insults
  13. Effect of CRE decoy oligonucleotide on NG108-15 cells
  14. Preliminary experiments
  15. Effect of CRE decoy oligonucleotide on CREB DNA-binding activity and ischemic tolerance
  16. Discussion
  17. References

Ischemic tolerance is well known as a neuroprotective effect of pre-conditioning ischemia against delayed neuronal death, however, the mechanism or mechanisms underlying this effect are not fully understood. We investigated the relationship between CREB and ischemic tolerance in gerbil hippocampal CA1 neurons using CRE decoy oligonucleotide. Sublethal ischemia led to an increase in the level of CREB phosphorylation in CA1 regions while lethal ischemia did not. Experiments with NG108-15 cells showed that adding CRE decoy oligonucleotide to culture media significantly inhibited the cell growth rate. The administration of CRE decoy oligonucleotide into gerbil cerebral ventricle decreased CREB-DNA binding activity to 38% of the control. Pre-treatment with CRE decoy oligonucleotide 24 h before the induction of ischemic tolerance decreased CA1 neuronal cell survival to 21% of the control. The present findings suggest that a CREB-mediated transcription system is necessary for the induction of ischemic tolerance.

Abbreviations used
ABC

avidin-biotin complex

BDNF

brain-derived neurotrophic factor

CBP

CREB binding protein

CREB

cyclic AMP responsive element binding protein

HRP

horseradish peroxidase

PBS

phosphate-buffered saline

PMSF

phenylmethylsulfonyl

SDS–PAGE

sodium dodecyl sulfate – polyacrylamide gel electrophoresis

Sublethal pre-conditioning ischemia avoids delayed neuronal cell death in the gerbil hippocampal CA1 region after severe ischemic insult. The mechanisms underlying this phenomenon, known as ischemic tolerance (Kirino et al. 1991), are not fully understood. Mild intracellular Ca2+ elevation through Ca2+ channels (Nakata et al. 1992) during the sublethal ischemia-reperfusion period appears to be required for the induction of ischemic tolerance. Sublethal, but not lethal, ischemic insults lead to rapid recovery of protein synthesis (Nakagomi et al. 1993). Brain-derived neurotrophic factor (BDNF) (Kokaia et al. 1995) and heat-shock protein 70 (Aoki et al. 1993) and Bcl-2 family members (Shimazaki et al. 1994; Zhang and Wang 1999) were expressed and showed neuroprotective effects after transient cerebral ischemia. These data suggest a role for gene expression triggered by mild intracellular Ca2+ elevation after sublethal ischemia. Therefore, transcriptional factors mediating between intracellular signals and these neuroprotective proteins are attracting attention as the key to identifying the mechanism(s) underlying ischemic tolerance.

Cyclic AMP responsive element binding protein (CREB) is a transcription factor with good responsiveness to intracellular Ca2+ influx via various protein kinases. The Ser133 residue of CREB is the target of these kinases and its phosphorylation is required for CREB activation. Activated CREB and its transcriptional co-activator CREB-binding protein (CBP) combine to regulate a variety of genes in response to intracellular signals. In hippocampal neurons, CREB plays a key role in long-term potentiation (LTP) (Schulz et al. 1999). In cerebellar granule cells, CREB activation inhibited potassium-induced apoptosis (See et al. 2001). CREB knockout mice revealed a dramatic increase in apoptotic cell death during periods of neurotrophin-dependent development of sensory neurons (Lonze et al. 2002). In adult rats, persistent phosphorylation of CREB was seen during the post-ischemic phase in hippocampal dentate granule cells that are resistant to transient cerebral ischemia (Hu et al. 1999). CREB regulates BDNF expression by Ca2+ influx signals (Tao et al. 1998) and Bcl-2 expression induced by Akt stimulation (Pugazhenthi et al. 2000) in vitro. We reported that sublethal ischemia induced Akt activation and that inhibition of Akt activation blocked the induction of ischemic tolerance (Yano et al. 2001).

These results suggest that sublethal ischemia may activate a neuroprotective signalling pathway that renders neuronal cells ischemia tolerant. During the induction of ischemic tolerance, a putative CREB-mediated transcription system may play a critical role in neuronal survival via the expression of various neuroprotective proteins. Our study was designed to investigate the role of CREB activity in the induction of ischemic tolerance in gerbil hippocampal neurons.

Induction of forebrain ischemia

  1. Top of page
  2. Summary
  3. Materials and methods
  4. Induction of forebrain ischemia
  5. Western blotting
  6. Immunohistochemistry
  7. Transfection of CRE decoy oligonucleotide into NG108-15 cells
  8. Intracerebroventricular CRE decoy oligonucleotide administration
  9. Electrophoretic mobility shift assay
  10. Statistical evaluation
  11. Results
  12. Changes in phosphorylation levels of CREB-Ser133 after ischemic insults
  13. Effect of CRE decoy oligonucleotide on NG108-15 cells
  14. Preliminary experiments
  15. Effect of CRE decoy oligonucleotide on CREB DNA-binding activity and ischemic tolerance
  16. Discussion
  17. References

Male mongolian gerbils (Seac Yoshitomi Ltd, Fukuoka, Japan) weighing 60–70 g were used. The experimental protocols were approved by the Committee on Animal Experiments of Kumamoto University School of Medicine. Gerbils were anaesthetized with a mixture of 2.5% halothane and nitrous oxide/oxygen (7 : 3). Both carotid arteries were exposed by a neck incision, occluded for 2 or 5 min with microaneurysmal clips, and visually inspected to ensure reflow after clip removal. Sham controls were operated but not made ischemic. Using a heat-lamp, the rectal temperature of all animals was maintained at 37–38°C throughout the operation. After they awoke from anaesthesia, the gerbils were returned to their cages in a room with a constant temperature of 22°C; food and water were available ad libitum. In our ischemic tolerance experiments, the gerbils were first subjected to 2-min, and 3 days later to 5-min, ischemia; 7 days after the second ischemia induction they were decapitated and their brains were removed.

Western blotting

  1. Top of page
  2. Summary
  3. Materials and methods
  4. Induction of forebrain ischemia
  5. Western blotting
  6. Immunohistochemistry
  7. Transfection of CRE decoy oligonucleotide into NG108-15 cells
  8. Intracerebroventricular CRE decoy oligonucleotide administration
  9. Electrophoretic mobility shift assay
  10. Statistical evaluation
  11. Results
  12. Changes in phosphorylation levels of CREB-Ser133 after ischemic insults
  13. Effect of CRE decoy oligonucleotide on NG108-15 cells
  14. Preliminary experiments
  15. Effect of CRE decoy oligonucleotide on CREB DNA-binding activity and ischemic tolerance
  16. Discussion
  17. References

The gerbils were killed by decapitation immediately after reperfusion or at the indicated times thereafter. Their brains were removed, rinsed once with cold phosphate-buffered saline (PBS), and dissected with a scalpel on a chilled plate in PBS. The hippocampi were removed, cross-sectioned into about six pieces under a binocular microscope, and further cut into subfields CA1, CA3, and the dentate gyrus. Individual tissue samples were stored in a test tube and kept at − 80°C until use. Frozen tissues were homogenized by hand and sonicated in a Bioruptor (COSMOBIO) at 0°C in 0.2 mL of homogenization buffer containing 50 mmol/L Tris-HCl (pH 7.5), 0.5% Triton X-100, 4 mmol/L EGTA, 10 mmol/L EDTA, 0.5 mol/L NaCl, 1 mmol/L Na3VO4, 30 mmol/L sodium pyrophosphate, 50 mmol/L NaF, 50 µg/mL leupeptin, 25 μg/mL pepstatin A, 0.5 mmol/L phenylmethylsulfonyl (PMSF), 10 μg/mL aprotinin, and 1 mmol/L dithiothreitol (DTT). Insoluble materials were removed by 15-min centrifugation at 20 000 g. The protein content in each supernatant fraction was determined using Bradford's solution and samples containing equivalent amounts of protein were applied to 12% acrylamide denaturing gels (SDS–PAGE) (Laemmli 1970). After electrophoresis, proteins were transferred to nitrocellulose membranes. Blotting membranes were incubated with 5% non-fat milk in TBST (10 mmol/L Tris [pH 7.5], 150 mmol/L NaCl, 0.05% Tween-20) at room temperature and then overnight at 4°C with a 1 : 1000 dilution of anti-phospho-Ser133 antibody or 1 : 1000 anti-CREB antibody (Cell Signaling Technology, Beverly, MA, USA) in 5% non-fat milk in TBST. After several washes with TBST, the membranes were incubated for 1 h with horseradish peroxidase (HRP)-conjugated anti-rabbit antibody (Vector Laboratories, Burlingame, CA, USA) diluted 1 : 5000. The membranes were then processed with enhanced chemiluminescence (ECL) western blotting detection reagents (Amersham Pharmacia Biotech, Piscataway, NJ, USA). The images were scanned and analyzed semi-quantitatively using the NIH image software (public domain software developed at the NIH and available at http://rsb.info.nih.gov/nih-image).

Immunohistochemistry

  1. Top of page
  2. Summary
  3. Materials and methods
  4. Induction of forebrain ischemia
  5. Western blotting
  6. Immunohistochemistry
  7. Transfection of CRE decoy oligonucleotide into NG108-15 cells
  8. Intracerebroventricular CRE decoy oligonucleotide administration
  9. Electrophoretic mobility shift assay
  10. Statistical evaluation
  11. Results
  12. Changes in phosphorylation levels of CREB-Ser133 after ischemic insults
  13. Effect of CRE decoy oligonucleotide on NG108-15 cells
  14. Preliminary experiments
  15. Effect of CRE decoy oligonucleotide on CREB DNA-binding activity and ischemic tolerance
  16. Discussion
  17. References

Anaesthetized gerbils (2.5% halothane and nitrous oxide/oxygen, 7 : 3), were perfused with 100 mL of ice-cold PBS containing 30 mmol/L sodium pyrophosphate, 50 mmol/L NaF, and 1 mmol/L Na3VO4, followed by 100 mL of 4% paraformaldehyde in 0.1 mol/L phosphate buffer containing 30 mmol/L sodium pyrophosphate, 50 mmol/L NaF, and 1 mmol/L Na3VO4 to prevent dephosphorylation. Their brains were removed and post fixed overnight at 4°C in the same fixative solution. Coronal brain sections (30 μm in thickness) at the level of the hippocampus were prepared using a vibratome. The sections were then floated in 0.1 m PB containing 0.01% Triton-X100 (30 min) and PBS with 3% bovine serum albumin (BSA) (blocking solution, 1 h); and incubated overnight with polyclonal anti-phospho-Ser133 antibody (diluted 1 : 100) (Cell Signaling Technology) in blocking solution. The sections were then labelled for 2 h with fluorescein isothiocyanate (FITC)-labelled anti-rabbit IgG (Vector) in PBS (diluted 1 : 200). After several washes with PBS, they were mounted on glass slides with coverslips and analyzed using a confocal laser microscope (Fluoview, Olympus, Tokyo, Japan).

Transfection of CRE decoy oligonucleotide into NG108-15 cells

  1. Top of page
  2. Summary
  3. Materials and methods
  4. Induction of forebrain ischemia
  5. Western blotting
  6. Immunohistochemistry
  7. Transfection of CRE decoy oligonucleotide into NG108-15 cells
  8. Intracerebroventricular CRE decoy oligonucleotide administration
  9. Electrophoretic mobility shift assay
  10. Statistical evaluation
  11. Results
  12. Changes in phosphorylation levels of CREB-Ser133 after ischemic insults
  13. Effect of CRE decoy oligonucleotide on NG108-15 cells
  14. Preliminary experiments
  15. Effect of CRE decoy oligonucleotide on CREB DNA-binding activity and ischemic tolerance
  16. Discussion
  17. References

For pre-treatment with CRE decoy oligonucleotide, we used a modified method of Park et al. (2001). CRE decoy oligonucleotide and control oligonucleotides were labelled by FITC (Nisshinbo, Chiba, Japan). Their sequences were as follows: 24-mer CRE decoy oligonucleotide, 5′-TGACGTCATGACGTCATGACGTCA-3′; 24-mer nonsense-sequence control, 5′- CTAGCTAGCTAGCTAGCTAGCTAG-3′. To increase the delivery of oligonucleotide into the cell, we used cationic lipid N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium methylsulfate (DOTAP; Boehringer-Mannheim, Mannheim, Germany). NG108-15 cells (1.0 × 105) were plated in 35-mm dishes in high-glucose DMEM (Gibco, Rockville, MD, USA) containing 5% foetal calf serum (FCS) at 37°C and, 48 h later, the medium was exchanged for new growth medium containing the above oligonucleotides and DOTAP.

For immunohistochemical analysis of CRE decoy oligonucleotide, cells were washed twice with PBS after 24-h incubation. To stain the nuclei, cells were incubated for 15 min with 1 μg/mL propidium iodide. After several washes, the distribution of FITC-labelled oligonucleotides and propidium iodide was analyzed under a confocal laser microscope. For cell viability assay, the trypan blue exclusion test was used. After incubation for the indicated periods in growth medium with CRE decoy oligonucleotide, the cells were dyed with trypan blue and the percentage of cells lacking trypan blue staining was calculated from the total number of cells.

Intracerebroventricular CRE decoy oligonucleotide administration

  1. Top of page
  2. Summary
  3. Materials and methods
  4. Induction of forebrain ischemia
  5. Western blotting
  6. Immunohistochemistry
  7. Transfection of CRE decoy oligonucleotide into NG108-15 cells
  8. Intracerebroventricular CRE decoy oligonucleotide administration
  9. Electrophoretic mobility shift assay
  10. Statistical evaluation
  11. Results
  12. Changes in phosphorylation levels of CREB-Ser133 after ischemic insults
  13. Effect of CRE decoy oligonucleotide on NG108-15 cells
  14. Preliminary experiments
  15. Effect of CRE decoy oligonucleotide on CREB DNA-binding activity and ischemic tolerance
  16. Discussion
  17. References

Anaesthetized gerbils (2.5% halothane and nitrous oxide/oxygen, 7 : 3) were placed on a stereotactic frame, the scalp was incised, and a burr hole was drilled on the right parietal skull at 2 mm posterior and 3.5 mm lateral to the bregma. A Hamilton syringe was inserted into the brain at a depth of 3 mm from the bone surface. To mark the desired needle insertion point, hematoxylin was first injected and allowed to spread in the ventricle. The success of the intracerebroventricular injection was confirmed by observing cerebrospinal fluid reflux through the burr hole after each injection, and by noting the presence of injection scars on fixed brain slices. A mixture (5 μL) consisting of 10 μmol/L oligonucleotide dissolved in DOTAP solution was continuously infused for 10 min and the infusion needle was kept in place for 10 additional minutes to allow diffusion into the ventricle. Pre-conditioning 2-min ischemia was induced 24 h later; the second 5-min ischemia was induced 3 days after the pre-conditioning ischemic episode.

For immunohistochemical examination of hippocampal CRE decoy oligonucleotide distribution, gerbil brains were removed 24 h after injection. CRE decoy- and control oligonucleotides were labelled with digoxigenin (Nisshinbo) and 30-μm thick slices were incubated overnight with biotin-conjugated anti-digoxigenin antibody (diluted 1 : 20) (ABCUM, Cambridge, UK) in blocking solution. After several washes with PBS, the sections were incubated for 1 h with FITC-labelled avidin (diluted 1 : 100) (Vector) in blocking solution, washed several times with PBS, mounted on glass slides with coverslips, and analyzed under a confocal laser microscope.

Following intracerebroventricular CRE decoy oligonucleotide administration, gerbils were reperfused for 10 days after 2-min ischemia (Group 1, n = 6) or reperfused for 7 days following 5-min ischemia and pre-conditioning ischemia (Group 2, n = 7). Group 3 (n = 6) was pre-treated with control oligonucleotide and reperfused for 7 days following 5-min ischemia and pre-conditioning ischemia. The gerbils were anaesthetized and perfusion-fixed with 4% paraformaldehyde as described above. Hippocampal serial sections (30 μm) were stained with cresyl violet for the counting of viable neurons under a light microscope. To be considered viable, neurons had to contain cells with normal morphological properties and round cresyl violet-stained nuclei. Viable neurons in the pyramidal cell layer of hippocampal CA1 regions were counted on video-captured computer images at the same magnification (× 400). For each animal, four sections were studied.

Electrophoretic mobility shift assay

  1. Top of page
  2. Summary
  3. Materials and methods
  4. Induction of forebrain ischemia
  5. Western blotting
  6. Immunohistochemistry
  7. Transfection of CRE decoy oligonucleotide into NG108-15 cells
  8. Intracerebroventricular CRE decoy oligonucleotide administration
  9. Electrophoretic mobility shift assay
  10. Statistical evaluation
  11. Results
  12. Changes in phosphorylation levels of CREB-Ser133 after ischemic insults
  13. Effect of CRE decoy oligonucleotide on NG108-15 cells
  14. Preliminary experiments
  15. Effect of CRE decoy oligonucleotide on CREB DNA-binding activity and ischemic tolerance
  16. Discussion
  17. References

Double-stranded oligonucleotide containing the CRE consensus sequence (5′-AGAGATTGCCTGACGTCAGAGAGCTAG-3′) (Promega, Madison, WI, USA) was end-labelled with [γ-32P]ATP (3000 Ci/mmol; Amersham Pharmacia Biotech, Piscataway, NJ, USA) and T4 polynucleotide kinase, and purified by centrifugation. To prepare nuclear extracts, frozen tissues were homogenized by hand at 0°C in 0.2 mL of the first homogenization buffer containing 10 mmol/L HEPES-KOH (pH 7.9), 10 mmol/L KCl, 1.5 mmol/L MgCl2, 1 mmol/L Na3VO4, 30 mmol/L sodium pyrophosphate, 50 mmol/L NaF, 50 μg/mL leupeptin, 25 μg/mL pepstatin A, 0.5 mmol/L PMSF, 10 μg/mL aprotinin, and 1 mmol/L DTT. The homogenates were then centrifuged for 10 s and the supernatant was restored as a cytosol fraction. The pellets were resuspended in 50 μL of the second homogenization buffer containing 20 mmol/L HEPES-KOH (pH 7.9), 25% glycerol, 0.5 m NaCl, 1.5 mmol/L MgCl2, 0.2 mmol/L EDTA, 1 mmol/L Na3VO4, 30 mmol/L sodium pyrophosphate, 50 mmol/L NaF, 50 μg/mL leupeptin, 25 μg/mL pepstatin A, 0.5 mmol/L PMSF, 10 μg/mL aprotinin, and 1 mmol/L DTT. The suspensions were kept on ice for 30 min and then the supernatants were removed by 15-min centrifugation at 20 000 g. DNA binding was performed in 23 μL of reaction buffer containing a 10-μg aliquot of extracted nuclear protein, 10 mmol/L HEPES (pH 7.8), 50 mmol/L KCl, 1 mmol/L EDTA, 5 mmol/L DTT, 0.7 mmol/L PMSF, 2 µg/μL aprotinin, 2 µg/μL pepstatin A, 2 μg/μL leupeptin, 1 mmol/L sodium orthovanadate, and 10% glycerol. After 30-min incubation on ice, 4 × 104 cpm of labelled probe was added to the reaction, and the total 25 μL reaction mixture was incubated for 30 min at 37°C. Protein-DNA complexes were resolved by electrophoresis (30 min at 30 mA) through 4% polyacrylamide gels in buffer containing 0.245% TBE buffer (24.5 mmol/L Tris base, 24.5 mmol/L boric acid, and 1 mmol/L EDTA). The gels were dried and visualized autoradiographically. The specificity of the identified CRE-binding proteins was determined by adding unlabelled competitor or an appropriate amount of anti-CREB antibody to 10 μg of nuclear extract before incubation with the binding mixture containing buffer and labelled probe. As an unlabelled non-specific competitor, we used transcription factor NF-kappa B (NFkB) oligonucleotide (5′-AGTTGAGGGGACTTTCCCAGGC-3′) (Promega). To confirm that the effect of CRE decoy pre-treatment was specific for CREB-DNA binding activity, we employed SP-1 as the negative control oligonucleotide (5′-ATTCGATCGGGGCGGGGCGAGC-3′) (Promega).

Changes in phosphorylation levels of CREB-Ser133 after ischemic insults

  1. Top of page
  2. Summary
  3. Materials and methods
  4. Induction of forebrain ischemia
  5. Western blotting
  6. Immunohistochemistry
  7. Transfection of CRE decoy oligonucleotide into NG108-15 cells
  8. Intracerebroventricular CRE decoy oligonucleotide administration
  9. Electrophoretic mobility shift assay
  10. Statistical evaluation
  11. Results
  12. Changes in phosphorylation levels of CREB-Ser133 after ischemic insults
  13. Effect of CRE decoy oligonucleotide on NG108-15 cells
  14. Preliminary experiments
  15. Effect of CRE decoy oligonucleotide on CREB DNA-binding activity and ischemic tolerance
  16. Discussion
  17. References

To evaluate CREB activity following ischemic insults, we used western blots with anti-phospho-Ser133 antibody to assay the phosphorylation level of CREB at Ser133, which is required for CREB activation. After 5-min ischemia and 12-h reperfusion, CREB phosphorylation was decreased to less than 20% of the control and never recovered to the control level in CA1 subfields. In contrast, 2-min ischemia led to a comparatively steep increase in CREB-Ser133 for about half a day; during the next few days there was a slow return to the basal phosphorylation level. CREB-Ser133 phosphorylation increased to 165.7% of the sham controls (p < 0.05, n = 8) at 6 h of reperfusion, to 223.2% (p < 0.05, n = 6) at 12 h of reperfusion, and then gradually decreased to 148.2% (p > 0.05, n = 9) after 1 day of reperfusion. It returned to the pre-ischemia level observed in sham-operated animals after 3 days of reperfusion. The CREB protein levels remained unchanged after both 2- and 5-min ischemia (Fig. 1a). In the dentate gyrus, CREB-Ser133 phosphorylation remained at the pre-ischemia level for 3 days after 2-min ischemia; after 5-min ischemia it gradually rose for 4 days (Fig. 1b).

image

Figure 1. (a) Western blot analysis of CREB Ser133 phosphorylation and quantitative analysis of relative phospho-Ser133 levels after sublethal or lethal ischemia in hippocampal CA1 subfields. Extracts were obtained at the indicated times from sham-operated gerbils, and from gerbils exposed to 2- or 5-min ischemia. For western blots, anti-phospho-Ser133 antibody or anti-CREB antibody was used in the 2- and 5-min ischemia groups. Data are the mean ± SD (n = 5 per time point). *p < 0.05 denotes a significant difference between sham- and post-ischemic groups obtained by analysis of variance followed by Fisher's post hoc test. (b) Western blot analysis of CREB Ser133 phosphorylation and quantitative analysis of relative phospho-Ser133 levels after sublethal or lethal ischemia in the hippocampal dentate gyrus region. Extracts were obtained at the indicated times from sham-operated gerbils, and from gerbils exposed to 2- or 5-min ischemia. For western blot, anti-phospho-Ser133 antibody or anti-CREB antibody was used in 2- and 5-min ischemia groups. Data are the mean ± SD (n = 5 per time point).

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Immunohistochemical analysis of CREB-Ser133 phosphorylation using antibody against phosphor-Ser133 revealed that, in sham-operated gerbils, immunoreactivity was present mainly in the neurons of the dentate granule cell subfield; there was weak immunoreactivity in the CA1 pyramidal cell subfield (Figs 2a, e and i). In gerbils exposed to 2-min ischemia followed by 12 h reperfusion, immunoreactivity was mildly increased in CA1 pyramidal cells; there was no apparent change in the dentate granule cells (Figs 2b. f and j). In the dentate gyrus of 5-min ischemia models, there was no obvious change in immunoreactivity until 4 days after ischemia (Figs 2k and l). However, immunoreactivity in their CA1 neurons was severely decreased at 12 h of reperfusion (Fig. 2g) and had disappeared at 4 days of reperfusion (Fig. 2h).

image

Figure 2. Immunohistochemical analysis of CREB phospho-Ser133 expression in whole hippocampus (upper), CA (middle) and dentate gyrus (lower). Coronal slices (30 μm) from gerbils subjected to sham operation (a, e, i) or to 2-min (b, f, j) or 5-min (c, g, k) followed by 12-h reperfusion or 4-day reperfusion (d, h, l) were immunostained with anti-phospho-Ser133 antibody and FITC-conjugated secondary antibody.

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Both western blotting and immunohistochemical analysis indicated that in the dentate gyrus, where neurons can be expected to survive ischemia, the level of CREB phosphorylation was sustained during the post-ischemic phase. In CA1 pyramidal neurons which are able to acquire ischemic tolerance after sublethal ischemia, CREB phosphorylation rose transiently and then maintained the basal level during the post-ischemic phase. These results led us to posit that CREB activation is necessary for ischemic tolerance induction in the CA1 region.

Preliminary experiments

  1. Top of page
  2. Summary
  3. Materials and methods
  4. Induction of forebrain ischemia
  5. Western blotting
  6. Immunohistochemistry
  7. Transfection of CRE decoy oligonucleotide into NG108-15 cells
  8. Intracerebroventricular CRE decoy oligonucleotide administration
  9. Electrophoretic mobility shift assay
  10. Statistical evaluation
  11. Results
  12. Changes in phosphorylation levels of CREB-Ser133 after ischemic insults
  13. Effect of CRE decoy oligonucleotide on NG108-15 cells
  14. Preliminary experiments
  15. Effect of CRE decoy oligonucleotide on CREB DNA-binding activity and ischemic tolerance
  16. Discussion
  17. References

After determining the effective concentration of CRE decoy ologonucleotide for transfection into NG108-15 cells by immunohistochemical methods, we examined the inhibitory effect of CRE decoy oligonucleotide treatment on the survival of NG108-15 cells. The cells were incubated for 24 h with CRE decoy oligonucleotide and the distribution of FITC-labelled CRE decoy oligonucleotide was examined under a confocal laser microscope. As shown in Fig. 3(a), CRE decoy oligonucleotide transfection was dose dependent. Next, we examined the effect of CRE decoy oligonucleotide on cell viability and on cell proliferation. After 6-, 12-, and 24-h incubation with 150 nm CRE decoy oligonucleotide, the viability of NG108-15 cells was decreased significantly (Fig. 3b (i)). The total number of cells did not increase during the first 12 h of incubation (Fig. 3b(ii)).

image

Figure 3. (a) Immunohistochemical examination of CRE decoy oligonucleotide transfection into NG108-15 cells. After 24-h incubation with CRE decoy oligonucleotide, the distribution of FITC-labelled CRE decoy oligonucleotide was detected under a confocal laser microscope. The number of propidium iodide-positive cells was decreased by CRE decoy oligonucleotide treatment. Transfection of CRE decoy oligonucleotide was dose dependent. (b) The effect of CRE decoy oligonucleotide on NG108-15 cells treated with control oligonucleotide or CRE decoy oligonucleotide (150 nm) for the indicated times. (i) Cell viability (%). (ii) Total number of cells. *p < 0.05 denotes a significant difference between the controls and CRE decoy oligonucleotide-treated gerbils at the indicated times (Fisher's test).

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Effect of CRE decoy oligonucleotide on CREB DNA-binding activity and ischemic tolerance

  1. Top of page
  2. Summary
  3. Materials and methods
  4. Induction of forebrain ischemia
  5. Western blotting
  6. Immunohistochemistry
  7. Transfection of CRE decoy oligonucleotide into NG108-15 cells
  8. Intracerebroventricular CRE decoy oligonucleotide administration
  9. Electrophoretic mobility shift assay
  10. Statistical evaluation
  11. Results
  12. Changes in phosphorylation levels of CREB-Ser133 after ischemic insults
  13. Effect of CRE decoy oligonucleotide on NG108-15 cells
  14. Preliminary experiments
  15. Effect of CRE decoy oligonucleotide on CREB DNA-binding activity and ischemic tolerance
  16. Discussion
  17. References

To investigate the effect of CREB activity on the induction of ischemic tolerance, CRE decoy oligonucleotide or control oligonucleotide (5 μL of 10 μmol/L) was infused into the right cerebral ventricle of gerbils 24 h before 2-min ischemia. To check the distribution of infused CRE decoy oligonucleotide, gerbils were perfusion-fixed 24 h after treatment and brain slices were examined. The distribution of DIG-labelled CRE decoy oligonucleotide was detected by the avidin-biotin complex method using anti-DIG antibody. As shown in Fig. 4, hippocampal CA1 and CA3 pyramidal cells were immunoreactive; there was only slight immunoreactivity in dentate gyrus granule cells.

image

Figure 4. Immunohistochemical analysis of transfection of CRE decoy oligonucleotide. Coronal slices subjected to intracerebroventricular administration of CRE decoy oligonucleotides were immunostained firstly with biotin conjugated anti-digoxigenin, secondly with FITC labelled avidin. Representative images obtained CA1 area (× 200), CA3 (× 200), dentate gyrus (× 200).

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Next, we examined the inhibitory effect of CRE decoy oligonucleotide on CREB-DNA binding. Nuclear extracts, prepared from the hippocampus of CRE decoy oligonucleotide-treated and -untreated gerbils exposed to 2-h ischemia and 12-h reperfusion, were subjected to electrophoretic mobility shift assay. As shown in Fig. 5(a), there was a single shifted band, confirmed by competitor experiments to be indicative of CRE-specific binding. As the addition of anti-CREB antibody resulted in supershift, we knew that the band contained CREB protein (Fig. 5a). CREB-DNA binding activity was reduced to 38% of the control (p < 0.05, n = 6) in CRE decoy oligonucleotide-treated gerbils; SP-1 binding activity was not affected by CRE decoy oligonucleotide treatment (Fig. 5b). We concluded that the intracerebroventricular administration of CRE decoy oligonucleotide inhibited CREB-DNA binding activity in gerbil hippocampus.

image

Figure 5. Electrophoretic mobility shift assay (EMSA) showing the effect of intracerebroventricular administration of CRE decoy oligonucleotide on CREB-DNA binding activity. Nuclear extracts were prepared from the hippocampus of gerbils subjected to 2-min ischemia followed by 12-h reperfusion. (1) EMSA in the absence or presence of unlabelled competitors or anti-CREB antibody. Lane 1, neither competitors nor antibody; lane 2, unlabelled CRE oligonucleotide; lane 3, unlabelled non-specific competitor; lane 4, anti-CREB antibody. The arrow indicates shifted CRE-protein complex. The triangle indicates the supershift band. (2) EMSA showing the effect of intracerebroventricular administration of CRE decoy oligonucleotide. The probes used are indicated. CRE, CRE oligonucleotide; SP-1, SP-oligonucleotide. (3) Quantitative analysis of the effect of pre-treatment with control oligo or CRE decoy oligonucleotide on CREB-DNA binding activity. *p < 0.05 denotes a significant difference between the two groups (Fisher's test).

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To investigate the effect of CRE decoy oligonucleotide on the induction of ischemic tolerance, gerbils received 10 μmol/L CRE decoy oligonucleotide or control oligonucleotide 24 h before the induction of forebrain ischemia. To assess ischemic tolerance, we used histological methods to examine CA1 neurons from gerbils that were pre-conditioned with 2-min ischemia, exposed to 5-min ischemia, and then reperfused for 7 days. The non-specific effect of control oligonucleotide on ischemic tolerance was assayed by counting surviving cells. There was no significant difference with respect to the viability of cells in CA1 pyramidal neurons between control oligonucleotide-untreated and -treated gerbils (Figs 6a i, ii, iv and v). Next, we examined the effect of CRE decoy oligonucleotide pre-treatment in gerbils exposed only to 2-min pre-conditioning ischemia. Neurons in the CA1 area survived for at least 10 days after reperfusion in CRE decoy oligonucleotide-pre-treated animals (Figs 6a iii and v).

image

Figure 6. (a) Effect of pre-treatment with CRE decoy oligonucleotide. Coronal slices were stained with cresyl violet and inspected under a light microscope. Representative histological sections of hippocampus (i–iii) and hippocampal CA1 subfields (v–vii) from CRE decoy oligonucleotide untreated gerbils exposed to 2-min pre-conditioning ischemia, 5-min ischemia and 7-day reperfusion (i, iv); gerbils pre-treated with control oligonucleotide and then exposed to 2- and 5-min ischemia followed by 7-day reperfusion (ii, v); gerbils CRE decoy oligonucleotide pre-treated and exposed to 2-min ischemia followed by 10-day reperfusion (iii, vii). (b) Effect of pre-treatment with CRE decoy oligonucleotide on ischemic tolerance. Coronal slices were stained with cresyl violet and inspected under a light microscope. Representative histological sections of hippocampus (i–v) and hippocampal CA1 subfields (vi–x) from gerbils exposed to 2-min pre-conditioning ischemia, 5-min ischemia and 7-day reperfusion. (c) Effect of pre-treatment with CRE decoy oligonucleotide on the induction of ischemic tolerance. Quantitative analysis of the relative survival of CA1 neurons in gerbils exposed to 2- and 5-min ischemia and 7-day reperfusion without oligonucleotide pre-treatment, with control oligonucleotide pre-treatment and with CRE decoy oligonucleotide pre-treatment. Surviving CA1 neurons were counted in video-captured images on a computer at identical magnification (× 400). The average number of neurons in the CA1 area of gerbils not pre-treated with oligonucleotides exposed to 2- and 5-min ischemia, followed by 7-day reperfusion, was 216.3 ± 4.8 mm. * p < 0.05 denotes a significant difference between the controls and gerbils pre-treated with CRE decoy oligonucleotide (Fisher's test).

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CRE decoy oligonucleotide pre-treatment affected the induction of ischemic tolerance in gerbils exposed to pre-conditioning (2-min) ischemia: in five of six animals studied, there was ischemic tolerance inhibition (Figs 6b i–v), in four the inhibition was complete (Figs 6b vii-x) and in the remaining gerbil it was mild (Fig. 6b vi). As shown in Fig. 6(c), significantly fewer cells survived in the CRE decoy oligonucleotide pre-treated group than the group pre-treated with control oligonucleotide (20.9 + 13.2% vs. 95.9 ± 5.8%, p < 0.05). Our results indicated that CREB-DNA binding played a critical role in the induction of ischemic tolerance and in cell survival.

Discussion

  1. Top of page
  2. Summary
  3. Materials and methods
  4. Induction of forebrain ischemia
  5. Western blotting
  6. Immunohistochemistry
  7. Transfection of CRE decoy oligonucleotide into NG108-15 cells
  8. Intracerebroventricular CRE decoy oligonucleotide administration
  9. Electrophoretic mobility shift assay
  10. Statistical evaluation
  11. Results
  12. Changes in phosphorylation levels of CREB-Ser133 after ischemic insults
  13. Effect of CRE decoy oligonucleotide on NG108-15 cells
  14. Preliminary experiments
  15. Effect of CRE decoy oligonucleotide on CREB DNA-binding activity and ischemic tolerance
  16. Discussion
  17. References

Ischemic tolerance is reportedly associated with sublethal Ca2+ elevation through NMDA receptor or voltage-dependent Ca2+ channels (Shimazaki et al. 1994). Sublethal ischemic insults are followed by rapid recovery of protein synthesis while lethal insults are not (Nakagomi et al. 1993). Transcriptional factors that mediate between intracellular Ca2+ signal(s) and neuroprotective proteins may hold the key for understanding the mechanisms underlying ischemic tolerance. Transcription factor CREB is highly responsive to intracellular Ca2+ influx. Various protein kinases involving Akt, CaM-KIV, and extracellular signal-regulated kinase (ERK) may contribute to CREB phosphorylation in response to Ca2+ influx (Impey et al. 1998; Dolmetsch et al. 2001; Perkinton et al. 2002).

Previous studies have shown that CREB plays a key role in cell survival. For example, CREB inhibited potassium-induced apoptosis of cerebellar granule cells (See et al. 2001). In rat hippocampal dentate granule cells resistant to transient cerebral ischemia, persistent phosphorylation of CREB was observed during the post-ischemic phase (Hu et al. 1999). We also found that CREB phosphorylation was maintained at the pre-ischemia level in surviving, but not in dying, cells. In CA1 neuronal cells exposed to sublethal ischemia, CREB phosphorylation was not only maintained at the pre-ischemia level, but was transiently increased for several hours. Phosphorylated-CREB overexpressing cells were resistant to apoptotic stimulation (Walton et al. 1999). During the acquisition of long-term potentiation, Schulz et al. (1999) observed a bi-phasic peak in the phosphorylation of CREB in rat hippocampal neurons. Based on these and our results, we posit that a transient increase in CREB activation after pre-conditioning (2-min) ischemia plays a key role in the induction of ischemic tolerance in gerbil CA1 neurons. As documented in Figs 5 and 6, CRE decoy oligonucleotide pre-treatment decreased CREB-DNA binding activity to 38% of the control and resulted in the failure of neurons to acquire ischemic tolerance. We concluded that activation of CREB-mediated transcription system and its down-stream target were necessary for introduction of ischemic tolerance.

In cultured tumour cells, CRE decoy oligonucleotide penetrates into cells and, by binding to CREB, competes with CRE enhancers for transcription factors (Park et al. 1999). However, upon immunohistochemical analysis, the CRE decoy oligonucleotide distribution pattern varied with different cell lines. In our study, CRE decoy oligonucleotide was immunohistochemically recognized in the extranuclear space. It is possible that the 24-h interval between CRE decoy oligonucleotide administration and our immunohistochemical analysis was too short for the oligo to penetrate into the cell nucleus. Alternatively, a CRE decoy oligonucleotide-CREB complex may exist in the extranuclear space. Western blot analysis using nuclear extracts showed that CRE decoy oligonucleotide pre-treatment tended to decrease the level of CREB protein (data not shown), suggesting that the molecule can competitively bind CREB away from the native enhancer sequence.

With respect to oligonucleotide pre-treatment, we found that exposure to the control oligonucleotide did not affect the acquisition of ischemic tolerance. Therefore, we think that the non-specific effect of the oligonucleotide was negligible. CRE decoy oligonucleotide pre-treatment did not reduce SP-1-DNA binding activity. Although the toxic effects of CRE decoy oligonucleotide pre-treatment remain unknown, we posit that the specific effect of CRE decoy oligonucleotide pre-treatment was within the limits of the CREB-mediating transcription signalling system. In our model, CRE decoy oligonucleotide pre-treatment before sublethal ischemia induction resulted in delayed neuronal death after lethal ischemia and had no effect on neuronal death in the absence of exposure to lethal ischemia. These observations suggest that the CREB transcription system may contribute to the generation of protective proteins that are not necessary for cell viability under normal conditions but are required to protect against lethal ischemia. In order to understand the mechanism(s) underlying the acquisition of ischemic tolerance, the key contributing protein(s) involved in the CREB transcription system must be identified.

As pre-treatment with BDNF enhanced the neuroprotective effect of pre-conditioning (Kawahara et al. 1997) and intracerebroventricular infusion of BDNF prevented delayed neuronal death in rats (Kiprianova et al. 1999), BDNF appears to have a role in ischemic tolerance. Glial cell line-derived neurotrophic factor (GDNF) (Woodbury et al. 1998) and anti-apoptotic protein Bcl-2 are known CREB target genes. Bcl-2 immunoreactivity increased after the induction of sublethal ischemic insult in gerbils (Shimazaki et al. 1994).

We demonstrated the transient increase and preservation of CREB phosphorylation after sublethal ischemic insult in gerbils. Pre-treatment with CRE decoy oligonucleotide prevented CREB-DNA binding and resulted in the failure of gerbil CA1 pyramidal neurons to acquire ischemic tolerance. Studies are underway in our laboratory to study the relevant proteins and to identify CREB target genes that bear on ischemic tolerance.

References

  1. Top of page
  2. Summary
  3. Materials and methods
  4. Induction of forebrain ischemia
  5. Western blotting
  6. Immunohistochemistry
  7. Transfection of CRE decoy oligonucleotide into NG108-15 cells
  8. Intracerebroventricular CRE decoy oligonucleotide administration
  9. Electrophoretic mobility shift assay
  10. Statistical evaluation
  11. Results
  12. Changes in phosphorylation levels of CREB-Ser133 after ischemic insults
  13. Effect of CRE decoy oligonucleotide on NG108-15 cells
  14. Preliminary experiments
  15. Effect of CRE decoy oligonucleotide on CREB DNA-binding activity and ischemic tolerance
  16. Discussion
  17. References
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