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Keywords:

  • Bcl-2 expression;
  • CREB;
  • neurotrophin-3;
  • oligodendrocyte progenitors

Abstract

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Isolation and culture of OLG progenitors
  5. Western blot analysis
  6. Detection of apoptosis by TUNEL labeling
  7. Inhibition of CREB protein expression by antisense treatment
  8. Luciferase reporter gene assays
  9. Electrophoretic mobility shift assay (EMSA)
  10. Nuclear extract preparation
  11. Electrophoretic mobility shift assay (EMSA)
  12. Statistical Analysis
  13. Results
  14. Treatment of oligodendrocyte progenitors with NT-3 results in decreased caspase-3 activation and PARP degradation accompanied by inhibition of DNA-fragmentation
  15. Treatment of OLG progenitors with NT-3 up-regulates Bcl-2 levels by a process that requires CREB expression
  16. NT-3 stimulates Bcl-2 gene promoter activity by a process that involves a putative CREB binding site
  17. The CREB protein present in freshly isolated OLG progenitors binds to the Bcl-2 promoter CRE.
  18. Discussion
  19. Acknowledgements
  20. References

Our previous results suggested that the transcription factor CREB mediates the actions of neuroligands and growth factor signals that coupled to different signaling pathways may play different roles along oligodendrocyte (OLG) development. We showed before that CREB phosphorylation in OLG progenitors is up-regulated by neurotrophin-3 (NT-3); and moreover CREB is required for NT-3 to stimulate the proliferation of these cells. We now show that treatment of OLG progenitors with NT-3 is also accompanied by an increase in the levels of the anti-apoptotic protein Bcl-2. Interestingly, the presence of a putative CREB binding site (CRE) in the Bcl-2 gene raised the possibility that CREB could also be involved in regulating Bcl-2 expression in the OLGs. Supporting this hypothesis, the NT-3 dependent increase in Bcl-2 levels is abolished by inhibition of CREB expression. In addition, transient transfection experiments using various regions of the Bcl-2 promoter and mutation of the CRE site indicate a direct role of CREB in regulating Bcl-2 gene activity in response to NT-3. Furthermore, protein-DNA binding assays show that the CREB protein from freshly isolated OLGs indeed binds to the Bcl-2 promoter CRE. Together with our previous results, these observations suggest that CREB may play an important role in linking proliferation and survival pathways in the OLG progenitors.

Abbreviations used
BDNF

brain-derived neurotrophic factor

CDM

chemically defined medium

CRE

Ca2+/cAMP-response element

CREB

cAMP-response element binding protein

EMSA

electrophoretic mobility shift assay

FGF

fibroblast growth factor

IL-3

interleukin-3

MBP

myelin basic protein

MS

multiple sclerosis

NGF

nerve growth factor

NT-3

neurotrophin-3

OLG

oligodendrocyte

PARP

poly(ADP-ribose)polymerase

PDGF

platelet derived growth factor

TNF

tumor necrosis factor

TUNEL

terminal deoxynucleotidyl transferase dUTP nick-end labeling

Neurotrophins are a family of structurally related homodimeric proteins which include nerve growth factor (NGF), brain derived growth factor (BDNF), neurotrophin-3 (NT-3), and neurotrophin-4/5. These molecules specifically bind to members of the Trk family of receptor tyrosine kinases (Barbacid 1994) inducing a variety of biological responses in the nervous system that include neuronal survival and development (Levi-Montalcini 1987; Ghosh et al. 1994; Fagan et al. 1996; Kavanaugh et al. 2000), neurite outgrowth (Segal et al. 1995), microglial chemotaxis (Gilad and Gilad 1995), proliferation of neuroblasts (Confort et al. 1991; Frade et al. 1999) and astroglial behavior (Hutton and Perez-Polo 1995). Neurotrophins, in particular NGF and NT-3, are also known to affect the proliferation and survival of oligodendrocytes (OLGs), the cells responsible for synthesizing and maintaining the myelin membrane that facilitates the saltatory conduction of nerve impulses in the central nervous system (CNS) (Barres et al. 1993; Barres et al. 1994; Casaccia-Bonnefil et al. 1996; Cohen et al. 1996; Kumar et al. 1998; Kahn et al. 1999). During CNS development, cells of the OLG lineage undergo multiple stages of differentiation prior to maturing into myelin producing post-mitotic OLGs (Small et al. 1987; LeVine and Goldman 1988; Reynolds and Wilkin 1988). As part of this developmental program, more than half of the OLG progenitors are eliminated by apoptosis (Barres et al. 1992a,b; Raff et al. 1993; Barres and Raff 1994; Raff et al. 1994). Survival and proliferation of OLGs are both determined by the presence or absence of growth factors and neurotrophins as well as the ability of these cells to form neuron–glial interactions (Casaccia-Bonnefil 2000). However, the molecular mechanisms involved in these processes are not clearly understood. In this regard, we have previously shown that developing OLGs express elevated levels of the transcription factor CREB (cyclic AMP response element binding protein), suggesting a regulatory function of this protein in a developmental window that precedes the peak of myelination (Sato-Bigbee and Yu 1993; Sato-Bigbee et al. 1994). CREB is a member of the leucine zipper family of transcription factors and binds to the consensus sequence known as Ca2+/cyclic AMP response element (CRE, TGACGTCA) (Montminy et al. 1990; Sheng et al. 1991). CREB capacity to activate transcription is regulated by phosphorylation at ser133 (Gonzalez and Montminy 1989). We have shown before that different neuroligands and signal transduction pathways stimulate CREB phosphorylation at specific stages of OLG differentiation, suggesting that this protein could play different roles at specific stages of OLG development (Sato-Bigbee et al. 1999). In support of this hypothesis, we found that in committed OLGs, CREB plays a crucial role in the cyclic AMP-mediated stimulation of myelin basic protein (MBP) expression (Sato-Bigbee and DeVries 1996; Afshari et al. 2001). On the other hand, at an earlier stage when the cells are immature OLG progenitors, CREB is a crucial player in the regulation of cell proliferation in response to NT-3 (Johnson et al. 2000). As indicated before, in addition to stimulating proliferation, NT-3 is also known to promote OLG survival (Cohen et al. 1996; Kumar et al. 1998). However, the molecular mechanisms involved in this dual action of NT-3 in the OLGs are poorly understood. In the present study we decided to investigate whether CREB could also be involved in the NT-3 dependent mechanisms leading to the survival of OLG progenitors. We focused on the possibility that NT-3 and CREB could regulate the expression of the anti-apoptotic protein Bcl-2. A member of the BH3-domain family of proteins, Bcl-2 was first suggested to play a crucial role in protecting lymphoma cells from IL-3 deprivation-induced cell death (Pegoraro et al. 1984; Vaux et al. 1988; Vaux et al. 1992). Consequent studies have shown that Bcl-2 interacts with other members of the Bcl-2 family and modulates mitochondrial membrane permeability thereby regulating the release of apoptogenic factors from the intermembrane space into the cytoplasm (Green 2000). Bcl-2 expression has been associated with protecting OLGs from different cell-death inducers (Burgmaier et al. 2000; FitzGerald et al. 2003) as well as with increased remyelinating capacity of OLGs in multiple sclerosis (MS) lesions (Kuhlmann et al. 1999). Interestingly, studies on Bcl-2 gene regulation in other cell types have identified a critical CREB binding region in the Bcl-2 gene promoter (Ji et al. 1996; Wilson et al. 1996; Pugazhenthi et al. 1999; Pugazhenthi et al. 2000; Freeland et al. 2001; Heckman et al. 2002; Pugazhenthi et al. 2003). These observations prompted us to investigate the possible existence of a CREB-mediated mechanism that could regulate Bcl-2 expression in the OLG progenitors. The present results demonstrate for the first time that treatment of OLG progenitors with NT-3 not only results in a significant up-regulation of Bcl-2 levels but also that CREB plays a crucial role in this stimulation.

Isolation and culture of OLG progenitors

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Isolation and culture of OLG progenitors
  5. Western blot analysis
  6. Detection of apoptosis by TUNEL labeling
  7. Inhibition of CREB protein expression by antisense treatment
  8. Luciferase reporter gene assays
  9. Electrophoretic mobility shift assay (EMSA)
  10. Nuclear extract preparation
  11. Electrophoretic mobility shift assay (EMSA)
  12. Statistical Analysis
  13. Results
  14. Treatment of oligodendrocyte progenitors with NT-3 results in decreased caspase-3 activation and PARP degradation accompanied by inhibition of DNA-fragmentation
  15. Treatment of OLG progenitors with NT-3 up-regulates Bcl-2 levels by a process that requires CREB expression
  16. NT-3 stimulates Bcl-2 gene promoter activity by a process that involves a putative CREB binding site
  17. The CREB protein present in freshly isolated OLG progenitors binds to the Bcl-2 promoter CRE.
  18. Discussion
  19. Acknowledgements
  20. References

OLG progenitors were isolated from 2 to 3 day-old Sprague–Dawley rat brain by using a Percoll (Sigma, St Louis, MO, USA) gradient and differential adhesion, as we previously reported (Sato-Bigbee et al. 1999; Johnson et al. 2000). Immediately after isolation, the cells were plated on 24-well plates coated with reduced growth factor-Matrigel (Becton Dickinson, Franklin Lakes, NJ, USA) and maintained overnight in chemically defined medium (CDM) [Dulbecco's modified Eagle medium (DMEM)/Ham-F12 medium (1 : 1) (Invitrogen, Grand Island, NY, USA) supplemented with 1 mg/mL bovine serum albumin, 50 µg/mL transferrin, 5 µg/mL insulin, 30 nm sodium selenite, 0.11 mg/mL sodium pyruvate, 10 nm biotin, 20 nm progesterone, 15 nm triiodothyronine (T3) and 100 µm putrescine (Sigma)]. Cultures prepared from these cells are OLG progenitors that are either bipolar or possess several simple processes and are labeled with the A2B5/O4 antibodies. Astroglial contamination of these cultures, as assessed by GFAP staining, was less than 5%. The identity of these cells as OLG progenitors is further supported by the observation that when maintained for 5–7 days in medium containing T3, a hormone that stimulates OLG differentiation (Matthieu et al. 1975), the majority of these cells first become multipolar and O4/O1 positive, and then MBP positive mature OLGs (Sato-Bigbee et al. 1999). For each set of experiments, the purity of the cultures was assessed by immunocytochemical staining with the antibodies mentioned above. In studies investigating the effects of NT-3, NT-3 (PeproTech Inc., Rocky Hill, NJ, USA) was used at a concentration of 50 ng/mL.

Western blot analysis

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Isolation and culture of OLG progenitors
  5. Western blot analysis
  6. Detection of apoptosis by TUNEL labeling
  7. Inhibition of CREB protein expression by antisense treatment
  8. Luciferase reporter gene assays
  9. Electrophoretic mobility shift assay (EMSA)
  10. Nuclear extract preparation
  11. Electrophoretic mobility shift assay (EMSA)
  12. Statistical Analysis
  13. Results
  14. Treatment of oligodendrocyte progenitors with NT-3 results in decreased caspase-3 activation and PARP degradation accompanied by inhibition of DNA-fragmentation
  15. Treatment of OLG progenitors with NT-3 up-regulates Bcl-2 levels by a process that requires CREB expression
  16. NT-3 stimulates Bcl-2 gene promoter activity by a process that involves a putative CREB binding site
  17. The CREB protein present in freshly isolated OLG progenitors binds to the Bcl-2 promoter CRE.
  18. Discussion
  19. Acknowledgements
  20. References

Cell lysates were prepared by solubilization of the cultures in 70 µL of 60 mm Tris-HCl buffer (pH 6.8) containing 10% glycerol, 2% sodium dodecylsulfate (SDS) and 5%β-mercaptoethanol. Equal amounts of protein sample were subjected to SDS–polyacrylamide gel electrophoresis (PAGE) in 10% acrylamide gels and the proteins were electrotransferred to nitrocellulose membranes. The membranes were then subjected to immunoblot analysis as previously reported (Johnson et al. 2000; Afshari et al. 2001), using the following antibodies: anti-CREB (dil. 1 : 1000; Santa Cruz Biotech., Santa Cruz, CA, USA), anti-phosphorylated CREB (dil. 1 : 1000; Upstate Biotech. Inc., USA), anti-Bcl-2 (dil. 1 : 1000; Santa Cruz Biotech., Lake Placid, NY, USA), anti-caspase-3 (dil. 1 : 1000; Cell Signaling Tech., Beverly, MA, USA), anti-PARP (dil. 1 : 1000; Cell Signaling Tech.), and anti-beta-actin (dil. 1 : 2000; Sigma). The immunoreactive bands were detected by chemiluminescent reaction with Super Signal West Dura reagent (Pierce, Rockford, Il, USA). The relative amount of immunoreactive protein in each band was determined by scanning densitometric analysis of the X-ray films.

Detection of apoptosis by TUNEL labeling

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Isolation and culture of OLG progenitors
  5. Western blot analysis
  6. Detection of apoptosis by TUNEL labeling
  7. Inhibition of CREB protein expression by antisense treatment
  8. Luciferase reporter gene assays
  9. Electrophoretic mobility shift assay (EMSA)
  10. Nuclear extract preparation
  11. Electrophoretic mobility shift assay (EMSA)
  12. Statistical Analysis
  13. Results
  14. Treatment of oligodendrocyte progenitors with NT-3 results in decreased caspase-3 activation and PARP degradation accompanied by inhibition of DNA-fragmentation
  15. Treatment of OLG progenitors with NT-3 up-regulates Bcl-2 levels by a process that requires CREB expression
  16. NT-3 stimulates Bcl-2 gene promoter activity by a process that involves a putative CREB binding site
  17. The CREB protein present in freshly isolated OLG progenitors binds to the Bcl-2 promoter CRE.
  18. Discussion
  19. Acknowledgements
  20. References

OLG progenitors were isolated as indicated above and after one day in culture in CDM, the medium was replaced by DMEM/HAMF12 medium alone or DMEM/F12 medium supplemented with 50 ng/mL NT-3. After overnight incubation, the cells were fixed with 4% paraformaldehyde for 1 h at room temperature. DNA fragmentation was then assessed by terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay using an In Situ cell death detection kit (Roche Diagnostics Corp., IN, USA), following the manufacturer's recommendations. Fluorescein-labeled nucleotides incorporated at-3′-OH ends were detected by incubation with horseradish peroxidase-conjugated antifluorescein antibody followed by detection of peroxidase activity with diaminobenzidine (DAB) (metal enhanced DAB substrate kit, Roche Diagnostics Corp.) and microscopic examination.

Inhibition of CREB protein expression by antisense treatment

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Isolation and culture of OLG progenitors
  5. Western blot analysis
  6. Detection of apoptosis by TUNEL labeling
  7. Inhibition of CREB protein expression by antisense treatment
  8. Luciferase reporter gene assays
  9. Electrophoretic mobility shift assay (EMSA)
  10. Nuclear extract preparation
  11. Electrophoretic mobility shift assay (EMSA)
  12. Statistical Analysis
  13. Results
  14. Treatment of oligodendrocyte progenitors with NT-3 results in decreased caspase-3 activation and PARP degradation accompanied by inhibition of DNA-fragmentation
  15. Treatment of OLG progenitors with NT-3 up-regulates Bcl-2 levels by a process that requires CREB expression
  16. NT-3 stimulates Bcl-2 gene promoter activity by a process that involves a putative CREB binding site
  17. The CREB protein present in freshly isolated OLG progenitors binds to the Bcl-2 promoter CRE.
  18. Discussion
  19. Acknowledgements
  20. References

CREB protein synthesis was inhibited using an oligodeoxynucleotide (ODN) corresponding to the CREB sequence (Gonzalez and Montminy 1989) in the antisense orientation spanning the initiation codon to nucleotide 20 (5′-GCTCCAGAGTCCATGGTCAT-3′), as we previously reported (Johnson et al. 2000; Afshari et al. 2001). Control cultures were treated in a similar manner with the corresponding sense (5′-ATGACCATGGACTCTGGAGC-3′) ODN. Transfections were carried out using Lipofectamine Plus transfection reagent (Invitrogen). Sense or antisense ODNs (1 µg/well) were incubated for 15 min with 5 µL Plus reagent followed by incubation for 15 min with 1.25 µL Lipofectamine. Cells were then incubated with the oligonucleotide mixture for 5 h followed by 12–14 h in CDM. The effectiveness of inhibition of CREB protein expression after transfection was assessed by western blot analysis using anti-total CREB antibody as described above.

Luciferase reporter gene assays

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Isolation and culture of OLG progenitors
  5. Western blot analysis
  6. Detection of apoptosis by TUNEL labeling
  7. Inhibition of CREB protein expression by antisense treatment
  8. Luciferase reporter gene assays
  9. Electrophoretic mobility shift assay (EMSA)
  10. Nuclear extract preparation
  11. Electrophoretic mobility shift assay (EMSA)
  12. Statistical Analysis
  13. Results
  14. Treatment of oligodendrocyte progenitors with NT-3 results in decreased caspase-3 activation and PARP degradation accompanied by inhibition of DNA-fragmentation
  15. Treatment of OLG progenitors with NT-3 up-regulates Bcl-2 levels by a process that requires CREB expression
  16. NT-3 stimulates Bcl-2 gene promoter activity by a process that involves a putative CREB binding site
  17. The CREB protein present in freshly isolated OLG progenitors binds to the Bcl-2 promoter CRE.
  18. Discussion
  19. Acknowledgements
  20. References

The reporter constructs, containing various regions of the Bcl-2 promoter originated by progressive 5′ truncations of the Bcl-2 promoter sequence by restriction digestion, linked to a luciferase reporter gene have been described previously (Wilson et al. 1996; Liu et al. 1999) (see Fig. 6a). Numbering of the Bcl-2 promoter sequence is relative to the translation start site. For transfection, cultures plated in 24 well plates and containing equal cell numbers (60% confluent) were incubated with 0.5 µg/well of the different luciferase reporter genes in the presence of 2.5 µL GeneJammer reagent (Stratagene, La Jolla, CA, USA), following the manufacturer's recommendations. After 3 h, the transfection medium was replaced by CDM. Following overnight incubation, the transfected cells were incubated for 6 h in DMEM/F12 in the presence or absence of 50 ng/mL NT-3. At the end of the incubation, the luciferase activity in the samples was determined using the Luciferase Assay System (Promega, Madison, WI, USA). For each plasmid, the luciferase activity in the presence of NT-3 was normalized to the values corresponding to the controls in medium alone and by using the beta-actin levels in each of the cultures as determined by western blot analysis of the cell extracts.

image

Figure 6. NT-3 stimulates the Bcl-2 promoter activity by a process that requires an intact CREB binding site. OLG progenitors were transfected with the luciferase reporter plasmids shown in (a), using GeneJammer reagent (Stratagene). On the following day, cells were incubated for 6 h in the presence or absence of 50 ng/mL NT-3. (b) The luciferase activity in the cell lysates was determined and the results expressed as percentage of the corresponding controls in the absence of NT-3. The results are the mean ± standard error from 3 to 6 independent experiments done in duplicates. LB124 and LB334, controls vs. cells + NT-3, p < 0.01. Closed bars, medium alone; open bars, medium + NT-3.

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Nuclear extracts were prepared as we previously reported (Afshari et al. 2002). Freshly isolated OLG progenitors (7 × 106 cells) were resuspended in 1 mL 10 mm HEPES (pH 7.9), 10 mm KCl, 1 mm dithiothreitol (DTT), 2 mm EDTA, 0.6% (v/v) NP-40, and 10 µL protease inhibitor cocktail (Sigma-Aldrich). After 15 min at 4°C, the suspension was centrifuged for 30 s at 10 000 ×g, and the nuclear pellet was resuspended and incubated for 15 min in 500 µL 20 mm HEPES (pH 7.9), 0.4 m NaCl, 1 mm DTT, 2 mm EDTA and 5 µL protease inhibitors. The nuclear extract was collected after centrifugation at 11 000 ×g for 5 min.

EMSAs were carried out as we previously reported (Afshari et al. 2002), with minor modifications. The binding reaction mixture (final volume 25 µL) contained 10 mm Tris (pH 7.6), 100 mm NaCl, 1 mm EDTA, 1 mm DTT, 4% (v/v) glycerol, 1 µg poly(dI-dC), 5 µg nuclear protein and 0.4 ng 32P-end labeled 17mer double stranded oligonucleotide corresponding to the promoter region of the Bcl-2 promoter containing either the wild-type or a mutated form of the CRE (CREB binding site). Specificity of binding with each 32P-end labeled probe was determined by competition with a 50-fold excess unlabeled oligonucleotide containing the consensus CRE sequence. Samples were then subjected to non-denaturing electrophoresis in 5% polyacrylamide gels at 100 V in 25 mm Tris, 25 mm boric acid, 0.5 mm EDTA (pH 8.4). The protein/DNA complexes were detected by autoradiography of the dried gels. For identification of the nuclear protein responsible for binding the CRE, prior to EMSA, the nuclear extracts were incubated for 30 min in the presence of 1 µg of polyclonal antibody directed against CREB (Upstate Biotech.).

Treatment of oligodendrocyte progenitors with NT-3 results in decreased caspase-3 activation and PARP degradation accompanied by inhibition of DNA-fragmentation

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Isolation and culture of OLG progenitors
  5. Western blot analysis
  6. Detection of apoptosis by TUNEL labeling
  7. Inhibition of CREB protein expression by antisense treatment
  8. Luciferase reporter gene assays
  9. Electrophoretic mobility shift assay (EMSA)
  10. Nuclear extract preparation
  11. Electrophoretic mobility shift assay (EMSA)
  12. Statistical Analysis
  13. Results
  14. Treatment of oligodendrocyte progenitors with NT-3 results in decreased caspase-3 activation and PARP degradation accompanied by inhibition of DNA-fragmentation
  15. Treatment of OLG progenitors with NT-3 up-regulates Bcl-2 levels by a process that requires CREB expression
  16. NT-3 stimulates Bcl-2 gene promoter activity by a process that involves a putative CREB binding site
  17. The CREB protein present in freshly isolated OLG progenitors binds to the Bcl-2 promoter CRE.
  18. Discussion
  19. Acknowledgements
  20. References

As described before, results from different laboratories have shown that NT-3 stimulates OLG survival. The results depicted in Fig. 1 indicate that this protective effect of NT-3 is also observed in our cultures of OLG progenitors. In these experiments we have investigated the effect of NT-3 on caspase-3 activation (Fig. 1a and b). Caspases, the final executioners of apoptotic cell death, are synthesized as inactive pro-caspases which upon apoptotic stimuli such as the lack of growth factors, are activated by proteolytic cleavage (Earnshaw et al. 1999). In this case, caspase-3 activation was detected by western blot analysis with an antibody that recognizes both, the inactive 32 kDa intact pro-caspase and the active 18 kDa cleaved forms of caspase-3. Figure 1a indicates that overnight incubation of OLGs in medium lacking growth factors and insulin clearly results in the presence of cleaved caspase-3, however, this caspase-3 cleavage is blocked by supplementing the medium with NT-3. Thus, OLG progenitors treated with NT-3 exhibit a higher procaspase-3/cleaved caspase-3 ratio than control cells (bar graph, Fig. 1b). Moreover, as shown in Fig. 1(c), NT-3 also inhibits the degradation of nuclear poly(ADP-ribose)polymerase (PARP). This enzyme is involved in DNA repair in response to stress and plays a crucial role in cell survival (Simbulan-Rosenthal et al. 1999). PARP is a major substrate of caspase-3 and cleavage of the 116 kDa intact form originates an 89 kDa fragment that is used as a marker of apoptosis. The results indicated that this 89 kDa fragment is easily detected after overnight incubation of the OLGs in DMEM/F12 medium alone but its levels are drastically decreased when the medium is supplemented with NT-3. The protective effect of NT-3 is also observed after TUNEL labeling to detect DNA fragmentation. As shown in Fig. 2a and b, control cultures incubated in medium alone show a significant proportion of TUNEL-positive OLG progenitors. However, a much lower number of TUNEL-positive cells is observed in the cultures treated with NT-3 (Fig. 2c and d).

image

Figure 1. (a, b) NT-3 prevents pro-caspase 3 cleavage. OLG progenitors were isolated and cultured in chemically defined medium (CDM) as indicated in Methods. On the following day, the cells were incubated for 18 h in DMEM/F12 alone or DMEM/F12 with 50 ng/mL NT-3. (a) Caspase-3 cleavage was studied by western blotting with an antibody recognizing both the inactive pro-caspase and the active cleaved form of caspase-3. The picture shows a representative experiment. (b) The bar graph depicts the mean ± SEM from 3 independent experiments and indicates the pro-caspase/cleaved caspase-3 ratio in the cells. Control vs. NT-3, p = 0.03. (c) NT-3 inhibits PARP degradation. OLG progenitors were incubated for 18 h in DMEM/F12 alone or DMEM/F12 with 50 ng/mL NT-3. PARP cleavage was investigated by western blot analysis with an antibody that reacts with both the 116 kDa intact PARP and its 89 kDa degradation product. The picture shows a representative western blot, similar results were obtained in 3 independent experiments. Each lane of the gels was loaded with 10 µg of protein.

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image

Figure 2. NT-3 inhibits DNA fragmentation in OLG progenitors. OLG progenitors were isolated as indicated under ‘Methods’ and after one day in culture in CDM, the cells were incubated overnight in DMEM/HAMF12 medium alone (panels a and b) or DMEM/F12 medium supplemented with 50 ng/mL NT-3 (panels c and d). Cells were then fixed with 4% paraformaldehyde and DNA fragmentation was then assessed by terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay as indicated in Methods.

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Altogether, these results indicate that NT-3 prevents apoptotic cell death in the OLG progenitors.

Treatment of OLG progenitors with NT-3 up-regulates Bcl-2 levels by a process that requires CREB expression

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Isolation and culture of OLG progenitors
  5. Western blot analysis
  6. Detection of apoptosis by TUNEL labeling
  7. Inhibition of CREB protein expression by antisense treatment
  8. Luciferase reporter gene assays
  9. Electrophoretic mobility shift assay (EMSA)
  10. Nuclear extract preparation
  11. Electrophoretic mobility shift assay (EMSA)
  12. Statistical Analysis
  13. Results
  14. Treatment of oligodendrocyte progenitors with NT-3 results in decreased caspase-3 activation and PARP degradation accompanied by inhibition of DNA-fragmentation
  15. Treatment of OLG progenitors with NT-3 up-regulates Bcl-2 levels by a process that requires CREB expression
  16. NT-3 stimulates Bcl-2 gene promoter activity by a process that involves a putative CREB binding site
  17. The CREB protein present in freshly isolated OLG progenitors binds to the Bcl-2 promoter CRE.
  18. Discussion
  19. Acknowledgements
  20. References

Interestingly, treatment of the OLG progenitors with NT-3 is accompanied by a time-dependent increase in the expression of the anti-apoptotic protein Bcl-2. As depicted in Fig. 3, western blot analysis indicated that incubation of OLG progenitors with NT-3 for a 3–6-h period results in a significant increase in Bcl-2 protein levels.

image

Figure 3. Treatment of OLG progenitors with NT-3 results in increased expression of the anti-apoptotic protein Bcl-2. After one day in culture, OLG progenitors were incubated for different times in the presence or absence of 50 ng/mL NT-3. Bcl-2 expression in the cells was determined by western blot analysis. Each lane of the gel was loaded with 5 µg of protein. The figure shows a representative western blot. The bar graph corresponds to the results obtained after densitometric scanning of the autoradiographs. The results are the average ± SEM from 4 to 5 independent experiments done in duplicates. 0 vs. 3 and 6 h, p < 0.001.

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This observation raised the possibility that this stimulation could result from Bcl-2 gene activation by the transcription factor CREB. Two main reasons supported this possibility. Firstly, the Bcl-2 promoter contains a non concensus CREB binding site (CRE) which in other cell types has been shown to play an important role in regulating Bcl-2 expression (Ji et al. 1996; Wilson et al. 1996; Pugazhenthi et al. 1999; Pugazhenthi et al. 2000; Pugazhenthi et al. 2003; Freeland et al. 2001; Heckman et al. 2002). Secondly, we have shown before (Johnson et al. 2000) that NT-3, causes a dramatic stimulation of CREB phosphorylation in OLG progenitors (Fig. 4a). Moreover, Fig. 4(b) shows that this stimulation of CREB phosphorylation is in fact accompanied by increased CREB capacity to activate transcription. In these experiments, cells were transfected with a control luciferase gene vector containing a classic promoter element (TATA box) (control plasmid) or an inducible reporter vector that contains the luciferase gene driven by a TATA box plus a synthetic promoter containing four copies of the consensus CREB binding site (CRE plasmid). The results indicate that incubation of the CRE-plasmid containing cells with NT-3 results in a 3.5-fold increase in luciferase expression. On the other hand, this stimulation by NT-3 is not observed in the cells transfected with the plasmid control lacking CREB binding sites. Therefore, NT-3 not only stimulates CREB phosphorylation in the OLG progenitors but this action also correlates with increased CREB capacity to activate transcription.

image

Figure 4. The stimulation of CREB phosphorylation by NT-3 is accompanied by increased CREB capacity to activate transcription. (a) OLG progenitors were isolated from 2-day-old rat brain and after 1 day in culture, the cells were incubated for various times in the presence of 50 ng/mL NT-3. Phosphorylated (p-CREB) levels were detected by western blot analysis with anti-p-CREB antibody. Total CREB was detected with an antibody that reacts with both CREB and p-CREB. Each lane of the gels was loaded with 10 µg of protein. The figure shows a representative western blot. The bar graph shows the p-CREB levels determined by scanning densitometric analysis of the films. The results are the means ± SEM from 5 to 6 independent experiments done in duplicates. *p < 0.001. (b) OLG progenitors were transfected with a luciferase gene control plasmid (Control Plasmid) or a luciferase gene driven by a promoter containing 4 copies of the CREB binding site (CRE Plasmid), by using GeneJammer transfection reagent. The cells were then incubated for 6 h in the presence (closed bars) or absence (open bars) of 50 ng/mL NT-3. The luciferase activity in the samples was determined and expressed a percentage of the controls in the absence of NT-3 (open bars). The results are the average of 3 independent experiments done in duplicates. CRE plasmid control cells vs. CRE plasmid cells with NT-3, p < 0.04.

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Thus, we next investigated the possible role of CREB in mediating the stimulation of Bcl-2 expression by NT-3. For this, CREB expression in the OLG progenitors was blocked by treating the cells with an antisense oligonucleotide directed against CREB mRNA.

We have previously used this technique to successfully block CREB expression at different stages of OLG development (Sato-Bigbee and DeVries 1996; Johnson et al. 2000; Afshari et al. 2001). As shown in Fig. 5(a), treatment of the OLG progenitors with CREB antisense effectively reduces CREB protein levels when compared to the controls treated with CREB sense oligonucleotide. Figure 5(b) shows that NT-3 is able to up-regulate Bcl-2 levels in the control cells that were transfected with CREB sense oligonucleotide. However, this stimulation is not observed in the cultures in which CREB expression has been inhibited with the antisense construct.

image

Figure 5. NT-3 increases Bcl-2 expression in the oligodendrocyte progenitors by a process that requires CREB. OLG progenitors were isolated from 2 to 3 day-old rat brain and after 1 day in culture, CREB expression was blocked by using an antisense oligonucleotide. Control cultures were treated with the respective sense construct. (a) Representative western blot corresponding to Total CREB levels at the end of the oligonucleotide (antisense or sense) treatment. Following the oligonucleotide treatment, the cells were incubated for 6 h in the presence or absence of 50 ng/mL NT-3. (b) Representative western blot depicting Bcl-2 expression after oligonucleotide treatment followed by incubation in the presence or absence of NT-3. The bar graph, obtained after densitometric scanning of the films, shows the average relative Bcl-2 level values from 3 independent experiments done in duplicates. The value for each sample was corrected by the corresponding values of actin levels. Sense vs. sense + NT-3, p = 0.022; sense vs. antisense + NT-3, NS.

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These results therefore suggested that NT-3 increases Bcl-2 levels in the OLG progenitors by a mechanism that requires CREB.

NT-3 stimulates Bcl-2 gene promoter activity by a process that involves a putative CREB binding site

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Isolation and culture of OLG progenitors
  5. Western blot analysis
  6. Detection of apoptosis by TUNEL labeling
  7. Inhibition of CREB protein expression by antisense treatment
  8. Luciferase reporter gene assays
  9. Electrophoretic mobility shift assay (EMSA)
  10. Nuclear extract preparation
  11. Electrophoretic mobility shift assay (EMSA)
  12. Statistical Analysis
  13. Results
  14. Treatment of oligodendrocyte progenitors with NT-3 results in decreased caspase-3 activation and PARP degradation accompanied by inhibition of DNA-fragmentation
  15. Treatment of OLG progenitors with NT-3 up-regulates Bcl-2 levels by a process that requires CREB expression
  16. NT-3 stimulates Bcl-2 gene promoter activity by a process that involves a putative CREB binding site
  17. The CREB protein present in freshly isolated OLG progenitors binds to the Bcl-2 promoter CRE.
  18. Discussion
  19. Acknowledgements
  20. References

To further investigate the mechanism by which NT-3 and CREB could regulate Bcl-2 expression in the OLGs we determined: (1) the effect of NT-3 on Bcl-2 gene promoter activity and (2) the role that the CRE site could play in that regulation. For this purpose, the cells were transiently transfected with different plasmids containing various regions of the Bcl-2 promoter linked to a luciferase reporter gene (Fig. 6a). The Bcl-2 promoter sequences studied were 5′-truncations of the P1 promoter that initiates transcription at − 1280 and contains a putative CREB binding site. These promoter sequences have been previously used to demonstrate the role of CREB in regulating Bcl-2 expression in other cell types (Ji et al. 1996; Wilson et al. 1996; Liu et al. 1999; Pugazhenthi et al. 1999, 2000, 2003; Freeland et al. 2001; Zhang et al. 2002; ).

As shown in Fig. 6(b), the full length construct LB124 (−3934 to −1280) was significantly induced by NT-3, supporting the role of this neurotrophin in regulating Bcl-2 expression through activation of the Bcl-2 gene. Sequential deletion analysis of the promoter indicated that plasmid LB334 (−1644 to −1280) was inducible by NT-3 to a similar extent to that for the full length construct. However, a lack of induction was observed in the cells transfected with plasmids LB375 (−1526 to −1280) and LB360 (−1337 to −1280) which lack the putative CRE site. More importantly, a total lack of response to NT-3 was also observed with plasmid LB595 (−1644 to −1280) which expands the same length as plasmid LB334 but in which the wild-type Bcl-2 CRE (5′-TGACGTTA-3′) has been mutated (mutated CRE, 5′-GGCCTTTA-3′). Altogether, these results indicate that NT-3 has a direct effect on Bcl-2 promoter activation and this stimulation indeed requires the presence of an intact CRE site.

The CREB protein present in freshly isolated OLG progenitors binds to the Bcl-2 promoter CRE.

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Isolation and culture of OLG progenitors
  5. Western blot analysis
  6. Detection of apoptosis by TUNEL labeling
  7. Inhibition of CREB protein expression by antisense treatment
  8. Luciferase reporter gene assays
  9. Electrophoretic mobility shift assay (EMSA)
  10. Nuclear extract preparation
  11. Electrophoretic mobility shift assay (EMSA)
  12. Statistical Analysis
  13. Results
  14. Treatment of oligodendrocyte progenitors with NT-3 results in decreased caspase-3 activation and PARP degradation accompanied by inhibition of DNA-fragmentation
  15. Treatment of OLG progenitors with NT-3 up-regulates Bcl-2 levels by a process that requires CREB expression
  16. NT-3 stimulates Bcl-2 gene promoter activity by a process that involves a putative CREB binding site
  17. The CREB protein present in freshly isolated OLG progenitors binds to the Bcl-2 promoter CRE.
  18. Discussion
  19. Acknowledgements
  20. References

The following experiments indicated that the CREB protein present in OLG progenitors freshly isolated from rat brain actually binds to the Bcl-2 promoter CRE site. Figure 7(b), shows an electrophoretic mobility shift assay (EMSA) in which nuclear extracts from these cells were incubated with double stranded oligonucleotide probes corresponding to the Bcl-2 CRE region containing either the wild-type Bcl-2 CRE or a mutated form of this site (Fig. 7a). As shown in lanes 2 and 4, incubation of the nuclear extracts with the wild-type probe results in the formation of a prominent protein-DNA complex. However, this complex is not detected when the nuclear extracts are incubated with the probe containing the mutated CRE (lanes 6 and 8). The specificity of binding to the Bcl-2 CRE is supported by the lack of complex formation in the presence of a 50-fold excess unlabeled oligonucleotide (lane 3).

image

Figure 7. Freshly isolated OLG progenitors contain a nuclear protein that binds to the putative CREB binding site in the Bcl-2 gene promoter region. OLG progenitors were isolated from 2 to 3 day-old rat brain and immediately used to prepare nuclear extracts as indicated in Methods. (a) oligonucleotide sequences. (b) Electrophoretic mobility shift assays (EMSA) were carried by incubation of nuclear extract (5 µg protein) with 32P-labeled double stranded oligonucleotides corresponding to the Bcl-2 promoter region containing the wild-type or a mutated form of the putative Bcl-2 CREB binding site. Specificity of protein binding to the Bcl-2 CREB binding site was analyzed by competition with a 50-fold excess of unlabeled probe containing the consensus CREB binding sequence (CRE). Lanes 1: wild-type probe alone; lanes 2 and 4: nuclear extracts + wild-type probe; lane 3: nuclear extract + wild-type probe + 50X consensus CRE; lane 5: mutant probe alone; lanes 6 and 8: nuclear extract + mutant probe; lane 7: nuclear extract + mutant probe + 50X consensus CRE. (c) EMSAs were carried out using nuclear extract from freshly isolated OLG progenitors (5 µg protein) and a 32P-labeled double stranded oligonucleotide corresponding to the Bcl-2 promoter region containing the putative CREB binding site. Notice that formation of the protein-DNA complex is abolished by pre-incubation of the nuclear extract with anti-CREB antibody (lanes 4 and 5). Lane1: wild-type probe alone; lane 2: nuclear extracts + wild-type probe; lane 3: nuclear extract + wild-type probe + 50X consensus CRE; lanes 4 and 5: nuclear extract + wild-type probe + anti-CREB antibody.

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The identity of the protein binding to the Bcl-2 CRE as CREB is further supported by the results of EMSAs carried out in the presence of anti-CREB antibody. As shown in Fig. 7(c), preincubation of the OLG nuclear extracts with anti-CREB antibody (lanes 4 and 5) completely disrupts the formation of the protein-DNA complex that is observed when the extracts are incubated with the probe alone (lane 2).

Altogether, our present observations implicate NT-3 and CREB in directly regulating Bcl-2 gene expression in the OLG progenitors.

Discussion

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Isolation and culture of OLG progenitors
  5. Western blot analysis
  6. Detection of apoptosis by TUNEL labeling
  7. Inhibition of CREB protein expression by antisense treatment
  8. Luciferase reporter gene assays
  9. Electrophoretic mobility shift assay (EMSA)
  10. Nuclear extract preparation
  11. Electrophoretic mobility shift assay (EMSA)
  12. Statistical Analysis
  13. Results
  14. Treatment of oligodendrocyte progenitors with NT-3 results in decreased caspase-3 activation and PARP degradation accompanied by inhibition of DNA-fragmentation
  15. Treatment of OLG progenitors with NT-3 up-regulates Bcl-2 levels by a process that requires CREB expression
  16. NT-3 stimulates Bcl-2 gene promoter activity by a process that involves a putative CREB binding site
  17. The CREB protein present in freshly isolated OLG progenitors binds to the Bcl-2 promoter CRE.
  18. Discussion
  19. Acknowledgements
  20. References

Several lines of evidence have shown that Bcl-2 levels in the nervous system are highly regulated by neurotrophins, in particular NGF and BDNF (Allsopp et al. 1995; Riccio et al. 1999). The present studies demonstrate for the first time that Bcl-2 protein levels, as well as the activity of the Bcl-2 gene promoter in OLG progenitors can be up-regulated by another neurotrophin, NT-3. Moreover, this stimulation involves CREB, a transcription factor which we have previously shown to play a crucial role in the NT-3 dependent stimulation of OLG proliferation (Johnson et al. 2000).

Results from different laboratories have shown that NT-3 is able to stimulate OLG survival and proliferation, both in vitro and in vivo. Developing OLGs express the NT-3 receptor TrkC (Cohen et al. 1996; Kumar and de Vellis 1996; Kahn et al. 1999) and NT-3 by itself, in the absence of either PDGF or bFGF, is known to induce both survival and proliferation of cultured OLG progenitors (Barres et al. 1992b; Cohen et al. 1996; Johnson et al. 2000). In support of a role of NT-3 and TrkC in OLG development in vivo, the number of OLGs in the optic nerve was shown to be significantly reduced by delivery of neutralizing antibodies against NT-3 (Barres et al. 1994), and mutant mice lacking NT-3 and TrkC expression have decreased brain and spinal cord OLG numbers (Kahn et al. 1999). Moreover, several lines of evidence suggest that NT-3 could also exert a beneficial effect under pathological conditions affecting myelination. NT-3 has been shown to diminish the susceptibility of OLGs to TNF-alpha and AMPA-glutamate receptor-mediated excitotoxicity (Kavanaugh et al. 2000). Although an indirect effect mediated by neurons could not be eliminated, in vivo studies in which NT-3 was administered to adult rats after chemically induced brain demyelination (Jean et al. 2003) and experiments in which fibroblasts engineered to produce NT-3 were transplanted into rat contused spinal cord (McTigue et al. 1998) showed that this neurotrophin may also play an important role in regulating OLG numbers and myelin regeneration after injury. Moreover, recent studies have indicated that NT-3 not only affect rodent cells but is also able to stimulate the proliferation of human OLG progenitors (Wilson et al. 2003). However, the molecular mechanisms underlying the actions of NT-3 in OLGs are poorly understood.

Treatment of OLGs with NT-3 is known to result in activation of Akt (Ness et al. 2002) and the mitogen-activated protein kinases ERK1/2 (Cohen et al. 1996; Kumar et al. 1998; Johnson et al. 2000), kinases that play important roles in mediating survival signals from growth factors and cytokines (Marshall 1995; Franke et al. 1997; Kolch 2000; Tang et al. 2000). In this regard, we have previously found that both an ERK as well as a PKC activity are involved in the rapid stimulation of CREB phosphorylation that is observed upon treatment of OLG progenitors with NT-3 (Johnson et al. 2000). Importantly, CREB phosphorylation in response to activation of mitogen-activated protein kinases and Akt has been shown to play an important role in the survival of other cell types (Du and Montminy 1998; Bonni et al. 1999). Interestingly, immunocytochemical studies (Tanaka et al. 2001) indicated increased CREB phosphorylation together with elevated Bcl-2 expression in OLGs in the corpus callosum following recirculation after focal cerebral ischemia in rats, suggesting that CREB activation may be closely associated with survival of OLGs and maintenance of myelination following cerebral ischemia.

The present studies demonstrate that CREB plays in effect a crucial function in mediating the up-regulation in the expression of the anti-apoptotic protein Bcl-2 that is observed following treatment of OLG progenitors with NT-3. This is supported by the observation that NT-3 capacity to increase Bcl-2 levels in the OLGs progenitors is lost when CREB expression in these cells is inhibited by treatment with CREB antisense oligonucleotide. In addition, the promoter transient transfection experiments demonstrated that the region of the Bcl-2 promoter which is responsive to NT-3 stimulation is the one containing a putative CREB binding site and furthermore, mutation of this site, abolishes the response to NT-3. Altogether, these observations are consistent with the idea that CREB plays a direct role in stimulating Bcl-2 gene expression in OLG progenitors. This possibility is further supported by the DNA binding experiments demonstrating that the CREB protein from freshly isolated OLG progenitors is indeed able to directly bind to the non-consensus CRE sequence present in the Bcl-2 promoter.

The identification of molecular mechanisms regulating Bcl-2 expression in the OLGs is particularly important as several lines of evidence indicate that manipulation of Bcl-2 levels can alter the capacity of these cells to overcome cell death. Down-regulation of Bcl-2 expression has been suggested as a major cause of the increased susceptibility to the TNF-alpha induced apoptosis that is observed in the spinal cord OLGs of rats infected with HTLV-I virus (Jiang 2000) and a rapid decline in Bcl-2 mRNA expression is known to accompany the oligodendroglial apoptotic cell death that results from growth factor deprivation (Soane et al. 1999). On the other hand, increased Bcl-2 levels have been associated with rescue from TNF-alpha induced cell death in the OLG cell line OLN-93 (Burgmaier et al. 2000), as well as protection of immature OLGs from cell death induced by glucose oxidase, ceramide and brefeldin A (FitzGerald et al. 2003). More importantly (Kuhlmann et al. 1999) correlated Bcl-2 expression in OLGs in multiple sclerosis (MS) lesions with the stage of demyelinating activity and disease course. These authors found that the highest proportion of Bcl-2 positive OLGs was observed in a subgroup of patients with relapsing-remitting disease course of MS and indicated a direct correlation between the presence of Bcl-2 positive OLGs and remyelination.

Thus, it is possible to hypothesize that mechanisms that increase Bcl-2 expression could protect OLGs from apoptotic cell death by altering the proportion between pro- and anti-apoptotic proteins.

Studies from different laboratories indicated that the expression of pro- and anti-apoptotic proteins in OLGs is subjected to developmental regulation. A recent study from Itoh et al. (2003) analyzing the mRNA expression of different Bcl-2 related proteins indicated that as OLGs mature, mRNA levels corresponding to the anti-apoptotic proteins Bcl-xL and Mcl-1 show a significant increase that is accompanied by a dramatic reduction in Bcl-2 mRNA expression. mRNA levels for the pro-apoptotic protein Bax were shown to be lower in mature OLGs than in the progenitor cells (Madison and Pfeiffer 1996). When analyzed at the protein level, maturation of the OLGs was shown to be accompanied by increased (Khorchid et al. 2002; Itoh et al. 2003) or unchanged Bcl-xL and Bcl-2 expression (Osterhout et al. 2002). On the other hand, levels of the pro-apoptotic Bax were reported to be either relatively constant (Itoh et al. 2003), decreased (Khorchid et al. 2002) or increased (Osterhout et al. 2002) along OLG maturation. Such apparent discrepancies between results from different laboratories may result from different culture conditions and/or variability in the precise timing of differentiation at which the cells were analyzed and could therefore reflect the existence of very sensitive mechanisms regulating the expression of both pro- and anti-apoptotic proteins.

Thus, changes in the ratio between pro- and anti-apoptotic proteins may be crucial in determining the sensitivity of OLGs to apoptosis at different developmental stages. In addition, several lines of evidence suggest that different mechanisms may be involved in protecting the OLGs from different cell death-inducing agents. For example, results from Khorchid et al. (2002) demonstrated that OLG progenitors are more susceptible to apoptotic cell death induced by catecholamines than differentiated OLGs. Similarly, immature OLGs appear to be more sensitive than mature OLGs to both oxidative stress caused by glutathione depletion (Back et al. 1998) and to ischemic injury induced cell death (Fern and Moller 2000). In contrast, activation of p75 receptor in response to NGF results in apoptotic cell death of mature OLGs but has no effect on the OLG progenitors (Casaccia-Bonnefil et al. 1996).

Thus, it would be important to consider that the variation in susceptibility to apoptosis of OLGs at different maturational stages and to various insults may result from a balance between pro- and anti-apoptotic components of the cells as well from the different mechanisms by which those insults induce apoptotic cell death. Nevertheless, our present results indicating a role of CREB in the regulation of Bcl-2 expression in the OLGs, suggest that treatments that trigger CREB activation may have a protective effect against certain apoptotic insults to these cells.

As described above, previous results from this laboratory indicated that CREB is also involved in the mechanisms leading to the NT-3 dependent stimulation of OLG proliferation (Johnson et al. 2000), an observation that suggests that in the OLGs, CREB could also target genes encoding cell cycle related proteins. We are currently investigating the possibility that CREB could also regulate the oligodendroglial expression of the cyclins A and D1. Both cyclins are crucial players in controlling eukaryotic cell proliferation and are encoded by genes that contain putative CREB binding sites (Herber et al. 1994; Desdouets et al. 1995).

In summary, our previous results indicating the involvement of CREB in OLG proliferation (Johnson et al. 2000) together with the present report indicating that CREB plays a pivotal role in the NT-3-dependent up-regulation of Bcl-2 levels, support the idea that this transcription factor may play a crucial function in ‘linking’ proliferation and survival pathways in the OLGs. A better understanding of these mechanisms should be of fundamental importance in the development of treatments to replace lost OLGs and to stimulate myelin regeneration after demyelinating lesions of the CNS.

References

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Isolation and culture of OLG progenitors
  5. Western blot analysis
  6. Detection of apoptosis by TUNEL labeling
  7. Inhibition of CREB protein expression by antisense treatment
  8. Luciferase reporter gene assays
  9. Electrophoretic mobility shift assay (EMSA)
  10. Nuclear extract preparation
  11. Electrophoretic mobility shift assay (EMSA)
  12. Statistical Analysis
  13. Results
  14. Treatment of oligodendrocyte progenitors with NT-3 results in decreased caspase-3 activation and PARP degradation accompanied by inhibition of DNA-fragmentation
  15. Treatment of OLG progenitors with NT-3 up-regulates Bcl-2 levels by a process that requires CREB expression
  16. NT-3 stimulates Bcl-2 gene promoter activity by a process that involves a putative CREB binding site
  17. The CREB protein present in freshly isolated OLG progenitors binds to the Bcl-2 promoter CRE.
  18. Discussion
  19. Acknowledgements
  20. References
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