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Keywords:

  • caspases;
  • cerebellar granule neurones;
  • mitogen-activated protein kinase;
  • neuronal apoptosis;
  • tetanus toxin;
  • tyrosine kinase receptor

Abstract

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Materials
  5. Cell cultures
  6. Immunoprecipitation
  7. Western blot analysis
  8. Determination of p21Ras-GTP
  9. MTT reduction assay
  10. DNA staining and fluorescence measurement
  11. Results
  12. Hc-TeTx fragment rescues mature CGN from apoptotic cell death
  13. Hc-TeTx induces phosphorylation of Trk receptor and p21Ras activation
  14. Hc-TeTx induces phosphatidylinositol 3-kinase (PI3K)/Akt signalling pathway
  15. Hc-TeTx induces mitogen-activated protein kinase (MAPK) signalling pathway
  16. Hc-TeTx prevents the proteolytic activation of pro-caspase-3
  17. Discussion
  18. Acknowledgements
  19. References

When cultured cerebellar granule neurones are transferred from a medium containing high extracellular potassium concentration ([K+]e) (25 mm) to one with lower [K+]e (5 mm), caspase-3 activity is induced and cells die apoptotically. In contrast, if cells in non-depolarizing conditions are treated with brain-derived neurotrophic factor (BDNF), caspase-3 activity, chromatin condensation and cell death are markedly diminished. In this study, we show that the C-terminal domain of the tetanus toxin heavy-chain (Hc-TeTx) is able to produce the same neuroprotective effect, as assessed by reduction of tetrazolium salts and by chromatin condensation. Hc-TeTx-conferred neuroprotection appears to depend on phosphatidylinositol 3-kinase (PI3K) and mitogen-activated protein kinase kinase, as is demonstrated by the selective inhibitors Wortmannin and PD98059, respectively. Hc-TeTx also induces phosphorylation of the tyrosine kinase BDNF receptor, activation of p21Ras in its GTP-bound form, and phosphorylation of the cascade including extracellular-signal-regulated kinases-1/2 (ERK-1/2), p90 ribosomal S6 kinase (p90rsk) and CREB (cAMP-response-element-binding protein). On the other hand, activation of the Akt pathway is also detected, as well as inhibition of the active form of caspase-3. These results point to an implication of both PI3K- and ERK-dependent pathways in the promotion of cerebellar granule cell survival by Hc-TeTx.

Abbreviations used
Akt/PKB

Akt/protein kinase B

BDNF

brain-derived neurotrophic factor

CGN

cerebellar granule neurones

CREB

cAMP-response-element-binding protein

DAPI

4′,6-diamino-2-phenylindole

ERK

extracellular signal-regulated kinase

GSK3β

glycogen synthase kinase 3β

GST

glutathione S-transferase

Hc-TeTx

C-terminal domain of the heavy-chain of tetanus toxin

[K+]e

extracellular potassium concentration

MAPK

mitogen-activated protein kinase

MAPKK

mitogen-activated protein kinase kinase

MTT

3(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide

NGF

nerve growth factor

PI3K

phosphatidylinositol 3-kinase

PKC

protein kinase C

PLCγ-1

phospholipase Cγ-1

p90rsk

p90 ribosomal S6 kinase

RBD

Ras-binding domain of Raf-1

TeTx

tetanus toxin

Trk

high-affinity tyrosine kinase receptor for neurotrophins

Tetanus toxin (TeTx), a close analogue of the larger family of botulinum neurotoxins, is a powerful neurotoxin acting on both central and peripheral nervous system by yet not completely understood modes of action (reviewed by Montecucco and Schiavo 1994). The toxin, produced by Clostridium tetani as a single polypeptide chain (150 kDa), is cleaved by an endogenous protease to release a 50-kDa amino-terminal L-chain that is responsible for toxicity. The L-chain remains attached to the rest of the polypeptide (the heavy chain or H-chain) by means of a disulphide bond. The H-chain can be cleaved with papain into two non-toxic fragments, consisting of a C-terminal (Hc-TeTx, 50 kDa) and an N-terminal (HN-TeTx 50 kDa) part. Each fragment is believed to have a distinct function: Hc-TeTx is responsible for binding to cell membrane of sensitive cells, whereas HN-TeTx promotes the translocation of the L-chain across the vesicular membrane (Halpern and Neale 1995; Lalli et al. 1999). Although the N-terminal of Hc-TeTx is similar in structure to many lectins, deletion mutagenesis studies suggest that the C-terminus is essential for ganglioside-binding activity in the cell (Halpern and Loftus 1993). As a Zn+-metalloprotease, TeTx appears to target synaptobrevin II, an essential protein for synaptic vesicles docking and neurotransmitter release (Schiavo et al. 1992). This selective proteolysis of synaptic proteins by clostridial neurotoxins accounts for the total block of neurotransmitter release in infected synapses, and appears to be the event responsible for motor symptoms in the tetanus and in the botulism (Pellizzari et al. 1999).

In previous reports we demonstrated a rapid translocation of protein kinase C (PKC) followed by down-regulation after intracerebral injection of TeTx (Aguilera and Yavin 1990). This rapid PKC translocation/down-regulation was detected in parallel with phosphatidyl inositol hydrolysis (Pelliccioni et al. 2001). Furthering these studies, we documented, in rat-brain synaptosomal preparations, a substantial phosphorylation of the nerve growth factor (NGF) high-affinity receptor (TrkA) and activation of other elements of a kinase cascade, including phospholipase Cγ-1 (PLCγ-1), classical and novel PKCs and extracellular signal-regulated kinases 1 and 2 (ERK-1/2), after short-time application of both TeTx or its Hc-TeTx fragment (Gil et al. 2001). On the other hand, a recent report in rat primary cortical cells (Gil et al. 2003) shows also the activation of the brain-derived neurotrophic factor (BDNF) high-affinity receptor (TrkB) and of the akt/PKB signalling pathway. Interestingly, this report also describes the phosphorylation of the transcription factor CREB (cAMP-response-element-binding protein) in Ser 133 after Hc-TeTx treatment, pointing to a putative enhancing effect on gene expression.

Since activation of Trk receptors, and subsequent activation of ERK and Akt cascades, has been described as responsible for trophic actions, such as enhancement of survival or neuritogenesis, in neuronal cells (Kaplan and Miller 2000), we addressed our efforts to study the hypothetical neurotrophic action of the Hc-TeTx fragment in cultured cerebellar granule neurones (CGN). In the present work we have further explored the involvement of Hc-TeTx on Trk-dependent signalling cascades in low extracellular potassium concentration ([K+]e) conditions, and a remarkable neuroprotective action of the fragment on the CGN is found. The molecular mechanism for this rescue appears to act via stimulation of signalling pathways typical to neurotrophic factors such as BDNF. Moreover, these studies also can help to better understand still unresolved issues concerning the molecular mechanisms of TeTx action, such as its extreme high potency at nanomolar concentration and its remarkable capacity of undergo retroaxonal transport along neurones (Lalli and Schiavo 2002).

Materials

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Materials
  5. Cell cultures
  6. Immunoprecipitation
  7. Western blot analysis
  8. Determination of p21Ras-GTP
  9. MTT reduction assay
  10. DNA staining and fluorescence measurement
  11. Results
  12. Hc-TeTx fragment rescues mature CGN from apoptotic cell death
  13. Hc-TeTx induces phosphorylation of Trk receptor and p21Ras activation
  14. Hc-TeTx induces phosphatidylinositol 3-kinase (PI3K)/Akt signalling pathway
  15. Hc-TeTx induces mitogen-activated protein kinase (MAPK) signalling pathway
  16. Hc-TeTx prevents the proteolytic activation of pro-caspase-3
  17. Discussion
  18. Acknowledgements
  19. References

Sprague-Dawley (OFA) rats were obtained from the Servei d'Estabulari of the Universitat Autònoma de Barcelona (Barcelona, Spain). Culture media, fetal calf serum, culture dishes and penicillin/streptomycin came from Pan Biotechnology GmbH, Fontlab (Barcelona, Spain). 4′,6-Diamino-2-phenylindole (DAPI) was from Vector Laboratories Inc. (Burlingame, CA, USA). Protein A-agarose beads were purchased from Boehringer-Mannheim GmbH (Mannheim, Germany). The specific antibody against phosphotyrosine (Clone PY20) was obtained from Zymed Laboratories Inc. (San Francisco, CA, USA). Anti-diphospho(Thr202-Tyr204)p44/p42 MAP kinase (ERK-1/2), anti-phospho(Ser380)p90 ribosomal S6 kinase (p90rsk), anti-phospho(Ser133)CREB, anti-phospho(Tyr674/675)TrkA, anti-phospho(Ser9)GSK3β (glycogen synthase kinase 3β), anti-phospho(Ser473)Akt, anti-ERK1/2, and anti-Akt (PKB) antibodies, as well as a polyclonal antibody against cleaved caspase-3 were from Cell Signalling Technology (Beverly, MA, USA). The rabbit polyclonal antibody against TrkB was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Monoclonal antibody against ERK-1, and anti-mouse and anti-rabbit secondary antibodies conjugated with horseradish peroxidase were from Transduction Laboratories (Lexington, KY, USA). Despite the anti-ERK-1 antibody being described as specific for this kinase, it also detects ERK-2, although with less potency. Monoclonal antibody against β-tubulin was from Sigma (St Louis, MO, USA). BDNF was supplied by Alomone Laboratories, Ltd. (Jerusalem, Israel). All other chemicals or reagents used were supplied by Sigma-Aldrich Química, SA (Madrid, Spain).

Cell cultures

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Materials
  5. Cell cultures
  6. Immunoprecipitation
  7. Western blot analysis
  8. Determination of p21Ras-GTP
  9. MTT reduction assay
  10. DNA staining and fluorescence measurement
  11. Results
  12. Hc-TeTx fragment rescues mature CGN from apoptotic cell death
  13. Hc-TeTx induces phosphorylation of Trk receptor and p21Ras activation
  14. Hc-TeTx induces phosphatidylinositol 3-kinase (PI3K)/Akt signalling pathway
  15. Hc-TeTx induces mitogen-activated protein kinase (MAPK) signalling pathway
  16. Hc-TeTx prevents the proteolytic activation of pro-caspase-3
  17. Discussion
  18. Acknowledgements
  19. References

Cerebellar granule neurones (CGN) cultures were prepared as described by Morán and Patel (1989) with slight modifications. Briefly, cell suspension from dissociated 7–8-day-old rat cerebella tissue was plated at a density of 1.3 × 106 cells/cm2 in plastic dishes previously coated with poly-l-lysine (5 µg/mL) or on glass coverslips (25 × 25 mm). Cells were grown regularly in basal Eagle's medium supplemented with 10% (v/v) heat-inactivated fetal calf serum, 20 mm KCl, 5 mm glucose, 2 mm glutamine, 50 U/mL penicillin and 50 µg/mL streptomycin at 37°C in a humidified atmosphere consisting of 5% CO2/95% air. This medium is referred to in the text as K25, since BME initially contains 5 mm KCl. Cytosine arabinofuranoside (10 µm) was added to the medium 24 h after seeding to reduce the number of non-neuronal cells to approximately 5% of the total. Cells were maintained for 6–8 days in vitro. For experiments, CGN were transferred to a serum-free medium without cytosine arabinofuranoside as described in the Results section, with either 5 mm or 25 mm KCl and a number of additives, including recombinant Hc-TeTx (0.1–100 nm), BDNF (25 ng/mL), PD98059 (50 µm) and/or Wortmannin (100 nm). Recombinant Hc-TeTx fragment was obtained as previously reported (Gil et al. 2003).

Immunoprecipitation

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Materials
  5. Cell cultures
  6. Immunoprecipitation
  7. Western blot analysis
  8. Determination of p21Ras-GTP
  9. MTT reduction assay
  10. DNA staining and fluorescence measurement
  11. Results
  12. Hc-TeTx fragment rescues mature CGN from apoptotic cell death
  13. Hc-TeTx induces phosphorylation of Trk receptor and p21Ras activation
  14. Hc-TeTx induces phosphatidylinositol 3-kinase (PI3K)/Akt signalling pathway
  15. Hc-TeTx induces mitogen-activated protein kinase (MAPK) signalling pathway
  16. Hc-TeTx prevents the proteolytic activation of pro-caspase-3
  17. Discussion
  18. Acknowledgements
  19. References

After treatment, CGN were rinsed and scraped from the wells with ice-cold phosphate-buffered saline (2.5 mm Na2HPO4, 6 mm NaH2PO4 and 0.14 mm NaCl, pH 7.4), collected by centrifugation, and the reaction medium was eliminated. Next, 0.3 mL of homogenization buffer (20 mm Tris-HCl, pH 7.5, 2 mm EDTA, 0.5 mm EGTA, 2 mm dithiothreitol, 10 µg/mL leupeptin, 25 µg/mL aprotinin, 2 mm phenylmethylsulphonyl fluoride and 1 mm Na3VO4) supplemented with 0.3% Triton X-100 was added and the cells were disrupted by sonication. For immunoprecipitation, 1 mg of total protein was incubated by gentle rocking at 4°C overnight in the presence of 4 µg of antibody. The immunocomplex was then trapped by adding 50 µL of washed Protein A-agarose bead slurry (25 µL of packed beads) previously incubated with 3% bovine serum albumin/phosphate-buffered saline to eliminate unspecific binding and gently rocked at 4°C for 3 h. The agarose beads were collected by centrifugation and the supernatant was drained off. The beads were washed three times with ice-cold phosphate-buffered saline, re-suspended in 200 µL of 2 × non-reducing Laemmli sample buffer (62.5 mm Tris-HCl, pH 6.8, 2% sodium dodecyl sulfate, 10% glycerol, 50 mm dithiothreitol, 0.01% bromophenol blue) and then boiled for 2 min.

Western blot analysis

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Materials
  5. Cell cultures
  6. Immunoprecipitation
  7. Western blot analysis
  8. Determination of p21Ras-GTP
  9. MTT reduction assay
  10. DNA staining and fluorescence measurement
  11. Results
  12. Hc-TeTx fragment rescues mature CGN from apoptotic cell death
  13. Hc-TeTx induces phosphorylation of Trk receptor and p21Ras activation
  14. Hc-TeTx induces phosphatidylinositol 3-kinase (PI3K)/Akt signalling pathway
  15. Hc-TeTx induces mitogen-activated protein kinase (MAPK) signalling pathway
  16. Hc-TeTx prevents the proteolytic activation of pro-caspase-3
  17. Discussion
  18. Acknowledgements
  19. References

Cells were washed twice with ice-cold phosphate-buffered saline prior to scraping and lysis. In the case of cleaved caspase-3, cell lysates were obtained by adding Chaps Cell Extract Buffer (50 mm Pipes/NaOH pH 6.5, 2 mm EDTA, 0.1% Chaps Cell Extract Buffer, 5 mm dithiothreitol, 20 µg/mL Leupeptin, 10 µg/mL aprotinin, and 1 mm phenylmethylsulphonyl fluoride). After a brief centrifugation, the lysate supernatants were used for immunoblotting. An aliquot of the cell lysate was mixed with loading sample buffer and boiled for 5 min. Equal amounts of protein (20–50 µg) were resolved on sodium dodecyl sulfate–polyacrylamide gel electrophoresis. After electrophoresis, the separated proteins were transferred to a nitrocellulose membrane at 75 V for 2 h. The blotting buffer used contained 48 mm Tris, 39 mm glycine and 20% methanol (pH 8.3). The membrane filters were blocked for 2 h with 5% non-fat dry milk in Tris-buffered saline (20 mm Tris base, 0.15 mm NaCl pH 7.6), which was supplemented with 0.1% Tween 20, and subsequently incubated overnight with the corresponding antibody diluted to 1 : 1000 in the same blocking buffer. Membrane filters were washed in Tris-buffered saline/Tween 20 (3 × 15 min) and incubated for 2 h with a secondary antibody conjugated with horseradish peroxidase diluted to 1 : 5000. This was followed by three washes (15 min/wash) in blocking buffer. Several washes with Tris-buffered saline/Tween 20 were performed after all of the steps. The western blots were developed using Super Signal West Pico Chemiluminescent Substrate from Pierce (Rockford, IL, USA) and exposed to ECL film membranes (Amersham Pharmacia Biotech, Buckinghamshire, UK). Computer-assisted analysis of bands was performed with a Bio-Rad GS700 system (Hercules, CA, USA), and data processed with a Bio-Rad Molecular Analyst image program using a DELL workstation. Repeated scans were taken for film non-linearity corrections. Equal loading of samples was checked by stripping the blots and re-testing with an antibody against total ERK or Akt. The membranes were stripped by incubation for 30 min at 65°C in stripping buffer (Tris-HCl 62.5 mm, pH 6.7, 2% sodium dodecyl sulfate, 1.5 mmβ-mercaptoethanol). The blots were incubated with Tris-buffered saline buffer for 1 h before incubation with primary antibody.

Determination of p21Ras-GTP

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Materials
  5. Cell cultures
  6. Immunoprecipitation
  7. Western blot analysis
  8. Determination of p21Ras-GTP
  9. MTT reduction assay
  10. DNA staining and fluorescence measurement
  11. Results
  12. Hc-TeTx fragment rescues mature CGN from apoptotic cell death
  13. Hc-TeTx induces phosphorylation of Trk receptor and p21Ras activation
  14. Hc-TeTx induces phosphatidylinositol 3-kinase (PI3K)/Akt signalling pathway
  15. Hc-TeTx induces mitogen-activated protein kinase (MAPK) signalling pathway
  16. Hc-TeTx prevents the proteolytic activation of pro-caspase-3
  17. Discussion
  18. Acknowledgements
  19. References

Granule cells were serum-deprived for 2 h before treatment. After treatment, cells were lysed at 4°C in a lysis buffer containing 20 mm HEPES, pH 7.5, 0.1 m NaCl, 1% Triton X-100, 10 mm EGTA, 40 mmβ-glycerophosphate, 20 mm MgCl2, 1 mm Na3VO4, 1 mm dithiothreitol, 10 µg/mL aprotinin, 10 µg/mL leupeptin, 1 mm phenylmethylsulphonyl fluoride, and 10% glycerol. Lysates were incubated for 30 min with 10 µg of a glutathione S-transferase (GST) fusion protein including the Ras-binding domain of Raf-1 (RBD) previously bound to glutathione-Sepharose beads (Upstate Biotechnology Inc., Lake Placid, NY, USA), followed by three washes with lysis buffer. Total p21Ras levels in the cell lysates and GTP-bound form of Ras associated with GST-RBD were quantified by western blot analysis using a monoclonal anti-p21Ras antibody (Oncogene Science, San Diego, CA, USA).

MTT reduction assay

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Materials
  5. Cell cultures
  6. Immunoprecipitation
  7. Western blot analysis
  8. Determination of p21Ras-GTP
  9. MTT reduction assay
  10. DNA staining and fluorescence measurement
  11. Results
  12. Hc-TeTx fragment rescues mature CGN from apoptotic cell death
  13. Hc-TeTx induces phosphorylation of Trk receptor and p21Ras activation
  14. Hc-TeTx induces phosphatidylinositol 3-kinase (PI3K)/Akt signalling pathway
  15. Hc-TeTx induces mitogen-activated protein kinase (MAPK) signalling pathway
  16. Hc-TeTx prevents the proteolytic activation of pro-caspase-3
  17. Discussion
  18. Acknowledgements
  19. References

Mitochondrial activity was estimated by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) reduction assay after 24 h following maintenance of cells in 5 mm (K5) or 25 mm (K25) KCl. MTT (0.2 mg/mL) was added to cells and after 45 min at 37°C medium was discarded and 100% dimethyl sulfoxide was added to the dishes. After incubation for 15 min at room temperature in darkness, the amount of formazan blue formed after MTT reduction was quantified spectroscopically at 560 nm excitation wavelengths using a Multiskan RC (Labsystems, Helsinki, Finland) microplate reader. As previously shown (Balàzs et al. 1990), there is a very good correlation between the capacity of cultures to form formazan blue, protein and DNA content, and the proportion of neurones that appeared intact by phase-contrast microscopy.

DNA staining and fluorescence measurement

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Materials
  5. Cell cultures
  6. Immunoprecipitation
  7. Western blot analysis
  8. Determination of p21Ras-GTP
  9. MTT reduction assay
  10. DNA staining and fluorescence measurement
  11. Results
  12. Hc-TeTx fragment rescues mature CGN from apoptotic cell death
  13. Hc-TeTx induces phosphorylation of Trk receptor and p21Ras activation
  14. Hc-TeTx induces phosphatidylinositol 3-kinase (PI3K)/Akt signalling pathway
  15. Hc-TeTx induces mitogen-activated protein kinase (MAPK) signalling pathway
  16. Hc-TeTx prevents the proteolytic activation of pro-caspase-3
  17. Discussion
  18. Acknowledgements
  19. References

After different treatments, CGN, cultured in 6-well dishes, were rapidly washed with cold Tris-buffered saline (Tris 0.05 m, NaCl 0.15 m, pH 7.4) and fixed for 10 min in cold 4% paraformaldehyde. After fixation, cells were washed twice with ice-cold Tris-buffered saline, and a drop of mounting medium with DAPI fromVectashield (Burlingame, CA, USA) was added prior to placing the coverslip. Preparations were excited at 360 nm and absorbance recorded at 460 nm using a fluorescence microscope (Leica DMRB) equipped with a Q500MC QuantiMed program. Apoptotic cells were characterized by the presence of condensed and fragmented nuclei, as opposed to diffuse staining observed in non-apoptotic cells. Data shown were obtained from two plates per condition from two to three separate cell preparations.

Hc-TeTx fragment rescues mature CGN from apoptotic cell death

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Materials
  5. Cell cultures
  6. Immunoprecipitation
  7. Western blot analysis
  8. Determination of p21Ras-GTP
  9. MTT reduction assay
  10. DNA staining and fluorescence measurement
  11. Results
  12. Hc-TeTx fragment rescues mature CGN from apoptotic cell death
  13. Hc-TeTx induces phosphorylation of Trk receptor and p21Ras activation
  14. Hc-TeTx induces phosphatidylinositol 3-kinase (PI3K)/Akt signalling pathway
  15. Hc-TeTx induces mitogen-activated protein kinase (MAPK) signalling pathway
  16. Hc-TeTx prevents the proteolytic activation of pro-caspase-3
  17. Discussion
  18. Acknowledgements
  19. References

Our previous findings showing effects due to the whole tetanus toxin or to its Hc-TeTx fragment on signalling (Gil et al. 2001, 2003; Pelliccioni et al. 2001) warranted further investigation on the possible effects on cellular physiology exerted by the Hc fragment, as well as on the pathways responsible for these effects. Formation of MTT product is a well-established method to assess cellular viability. As expected, switching the granular cells to low potassium (K5) medium resulted in a 44.2% ± 7.8% reduction of MTT conversion (Fig. 1a), indicative of cell damage. A modest but significant increase in MTT conversion (59.5% ± 10.3%) was observed when cells were pre-incubated for 30 min with 1 nm Hc-TeTx, prior to potassium withdrawal. This concentration is by far lower than most pharmacological agents used in similar types of studies. In the presence of 10 nm Hc-TeTx, the toxic effect due to potassium withdrawal could be markedly reduced (85.2% ± 7.6%). On the other hand, pre-incubation with 100 nm Hc-TeTx also shows an increase (75.3% ± 9.7%), this being lower than that produced by 10 nm Hc-TeTx. The neuroprotective effect of Hc-TeTx was also apparent, but also lower, when Hc-TeTx was applied either simultaneously (57.5% ± 4%) or after 30 min (56.9% ± 4.2%) following low [K+]e stress (Fig. 1b). The more potent survival effect observed when CGN are pre-treated for 30 min with Hc fragment is probably due to the necessity that the Hc-TeTx-activated survival pathway was activated before cells go into K5 in order to be fully effective. In experiments using full-length TeTx (10 nm, 30 min pre-treatment before changing to low K), a protection of 60% was obtained (results not shown).

image

Figure 1. Effects of the Hc-TeTx fragment on the viability of cultured cerebellar granule neurones switched to a low [K+]e medium (5 mm, K5). (a) Cells were grown for 7 days in vitro in 25 mm KCl (K25) medium. Hc-TeTx (1 nm to 100 nm) was added 30 min before switching the medium from K25 to K5, and maintained for 24 h. After this time, cell survival was estimated by MTT assay. **p < 0.01 and *p < 0.05 when compared with neurones switched to K5 medium; #p < 0.05 and ##p < 0.01 when compared with the corresponding control groups, using one-way anova, followed by Dunnett's post-hoc test for each Hc-TeTx concentration. (b) In the same conditions as (a), the Hc-TeTx fragment (10 nm) was added 30 min before, at the same time or 30 min after potassium deprivation. Results are presented as mean ± SEM of five to 10 independent experiments presented as a percentage of control cultures. (c) Hc-TeTx avoids nuclear condensation due to potassium withdrawal. CGN were cultured in basal Eagle's medium supplemented with high [K+]e (25 mm KCl) and 10% FCS. After 7 days in vitro, the cultures were switched to serum-free basal Eagle's medium normally containing 5 mm KCl, supplemented with 20 mm KCl (K25), no additives (K5) or 10 nm Hc-TeTx (K5 + Hc-TeTx). The images show fluorescent imaging of cells by DAPI staining at 24 h after treatment. Note an increased number of cells with nuclear condensation and fragmentation typical of apoptosis in K5 compared with culture switched to K25, and culture incubated with Hc-TeTx in K5. Arrows indicate cells with condensed chromatin. The histogram represents the differences of three experiments, each one of one register up to 10 fields.

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Further evidence for the changes in cell viability and the rescue phenomenon observed in Hc-TeTx-treated cultures, was obtained by nuclear staining with DAPI and fluorescence measurements. As shown in the graphic of Fig 1(c), 16.5% of the cells transferred to the low [K+]e medium showed, after 24 h, nuclear fragmentation, a typical phenomenon associated with apoptotic cell death. In contrast, in the presence of Hc-TeTx, only about 10.7% of the cells showed signs of apoptosis. The nuclear condensation in Hc-TeTx pre-treated cells in K25 was not altered with respect to control cells. These values are in good agreement with the effect of low [K+]e induced cell damage shown by others (D'Mello et al. 1993; Valencia and Morán 2001). It should be noted that the differences in cell viability values as estimated by the MTT assay or by DAPI might not be alike, since the state of cell damage as measured by the mitochondria activity may precede the state of apoptosis (Valencia and Morán 2001; Ho et al. 2002).

Hc-TeTx induces phosphorylation of Trk receptor and p21Ras activation

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Materials
  5. Cell cultures
  6. Immunoprecipitation
  7. Western blot analysis
  8. Determination of p21Ras-GTP
  9. MTT reduction assay
  10. DNA staining and fluorescence measurement
  11. Results
  12. Hc-TeTx fragment rescues mature CGN from apoptotic cell death
  13. Hc-TeTx induces phosphorylation of Trk receptor and p21Ras activation
  14. Hc-TeTx induces phosphatidylinositol 3-kinase (PI3K)/Akt signalling pathway
  15. Hc-TeTx induces mitogen-activated protein kinase (MAPK) signalling pathway
  16. Hc-TeTx prevents the proteolytic activation of pro-caspase-3
  17. Discussion
  18. Acknowledgements
  19. References

Western blot analysis using an antibody specific for Trk receptors dually phosphorylated in tyrosines 674 and 675 showed that Trk receptors are phosphorylated in K25 conditions, but not in K5 medium (Fig. 2a). On the other hand, 10 nm Hc-TeTx under K5 induce a transient and rapid phosphorylation of the Trk receptor (Fig. 2a), being evident at 10 min (71% respect to the BDNF signal, as determined by film densitometry), but subsequently decreasing. The induction of phosphorylation by BDNF was almost as effective as the induction achieved by K25 (83%). It has been reported that the antibody used for these experiments can detect in addition to phosphorylated TrkA, also phosphorylated TrkB and TrkC, since the epitope is common to all three receptors. Since it has been shown that CGN cells are devoid of TrkA receptors (Nonomura et al. 1996), it would appear that the antibody reacts with TrkB or TrkC receptors. Therefore, we pursued an immunoprecipitation experiment using anti-phosphotyrosine antibody (clone PY20) and subsequent western blotting with anti-TrkB antibody. The results shown in Fig. 2(b) indicate that the phospho-TrkB protein was undetectable when cells were transferred to low [K+]e medium alone but was significantly elevated in the presence of either Hc-TeTx (10 nm, 10 min or 30 min) or BDNF (25 ng/mL, 10 min). The total levels of TrkB (probed directly with anti-TrkB antibody) do not change significantly in any of these treatments, even in the CGN cells cultured in the high [K+]e medium (data not shown). In the same kind of experiments the activation of p21Ras, i.e. the presence of p21Ras-GTP bound form, was assessed. Being consistent with the detected activation of Trk receptors by Hc-TeTx, an increase in the p21Ras-GTP form is found due to Hc-TeTx after 10 min incubation in absence of depolarization, as well as due to BDNF (25 ng/mL, 10 min) (Fig. 2c).

image

Figure 2. Hc-TeTx induces Trk receptor phosphorylation in tyrosines 674 and 675, and activation of p21Ras. (a) CGN were grown for 7 days in vitro in a 25 mm KCl medium, then the cells were switched, in the K5 lanes, to a 5 mm KCl serum-free medium for 1 h and incubated with the Hc-TeTx fragment (10 nm) for 0, 10, 30 or 60 min. In the K25 lane, serum-free medium was supplemented with 25 mm potassium. As positive control, BDNF (25 ng/mL) was added for 10 min. Total lysates were separated in a 7.5% sodium dodecyl sulfate–polyacrylamide gel electrophoresis and subjected to immunoblotting with the anti-Trk phosphorylated in residues Tyr674/675 or against anti-TrkB. (b) Induction of Tyr-phosphorylation of TrkB by the Hc-TeTx fragment or by BDNF. Cells were grown for 7 days in vitro in 25 mm KCl, then the cells were switched, in the K5 lanes, to a 5 mm KCl serum-free medium for 1 h, and incubated with Hc-TeTx (10 nm)for 10, 30 or 60 min, or with BDNF (25 ng/mL) for 10 min. In the K25 lane, serum-free medium was supplemented with 25 mm potassium. After the respective treatment, the cells were lysated and 0.5 mg of protein were mmunoprecipitated using anti-phosphotyrosine antibody (clone PY20). The immunoprecipitated proteins were subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis, transferred to nitrocellulose filters, and probed with anti-TrkB antibody. Image shown is representative of three independent experiments. (c) Hc-TeTx-induced p21Ras activation in potassium deprivation. CGN were deprived of serum and potassium for 2 h in the K5 lanes, and then incubated with or without Hc-TeTx (10 nm) or BDNF (25 ng/mL) for 10 min. In K25 lanes, 25 mm potassium was present throughout all the experiment. Then, cell lysates were incubated for 30 min with 10 µg of a GST-RBD fusion protein, previously bound to glutathione-Sepharose beads. Subsequently, beads were washed three times with lysis buffer and Ras precipitated was analysed by western blot using a monoclonal anti-p21Ras antibody (indicated as Ras-GTP). In order to assess the initial amount of total Ras protein, aliquots of the lysates were taken and analysed by immunoblotting (indicated as Ras-total).

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Hc-TeTx induces phosphatidylinositol 3-kinase (PI3K)/Akt signalling pathway

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Materials
  5. Cell cultures
  6. Immunoprecipitation
  7. Western blot analysis
  8. Determination of p21Ras-GTP
  9. MTT reduction assay
  10. DNA staining and fluorescence measurement
  11. Results
  12. Hc-TeTx fragment rescues mature CGN from apoptotic cell death
  13. Hc-TeTx induces phosphorylation of Trk receptor and p21Ras activation
  14. Hc-TeTx induces phosphatidylinositol 3-kinase (PI3K)/Akt signalling pathway
  15. Hc-TeTx induces mitogen-activated protein kinase (MAPK) signalling pathway
  16. Hc-TeTx prevents the proteolytic activation of pro-caspase-3
  17. Discussion
  18. Acknowledgements
  19. References

Since it is well established that induction of the PI3K/Akt pathway is associated with cell survival under stress (Yuan and Yankner 2000), Akt phosphorylation, one of the key components of the inositol triphosphate cascade, was examined. As illustrated in Fig. 3(a), a substantial phosphorylation of Akt in Ser 473 takes place in response to increasing concentrations of Hc-TeTx fragment. Quantitative analysis of Akt phosphorylation was performed in cells treated with increasing concentrations of Hc-TeTx (10−10−10-7 m) for 15 min, after switching the cells to the K5 medium for 1 h. The signal intensities of the Akt phosphorylation of different experiments were determined by band densitometry analysis and the ratio of the band intensities was expressed as a percentage of values found in K5-treated cells (Fig. 3a). Since the highest Akt phosphorylation was found at 10 nm Hc-TeTx concentration, cells were treated with or without Hc-TeTx (10 nm) for 10, 30 or 60 min (Fig. 3b) in order to observe time-dependence of the phosphorylation. The signal intensities due to Akt phosphorylation in Ser 473 were determined by densitometric analysis and the ratio of band intensity was expressed as a percentage of values from naive cells. The Akt phosphorylation in low [K+]e (K5) conditions increases due to Hc-TeTx when compared to the non-treated cells in K5. The level of phosphorylation with Hc-TeTx in K5 was similar to the Akt phosphorylation found in high [K+]e (K25) conditions (Fig. 3b) at short-time. As can be seen, Hc-TeTx was more effective at short-time application. Western blots with anti-Akt antibody were performed to determine the protein levels, demonstrating that the total Akt protein was not modified under the three conditions assayed (K25, K5 and K5 +Hc-TeTx) in the three times determined. Similar results, using an antibody specific for GSK3β phosphorylated in Ser 9, were found in the phosphorylation of GSK3β, an Akt substrate (Fig. 4c), showing that Akt activity is enhanced due to Hc-TeTx treatment. This result is in good correlation with the results observed in Fig. 4(b).

image

Figure 3. Hc-TeTx fragment activates Akt in CGN. (a) Concentration-dependence of Hc-TeTx-induced Akt phosphorylation in Ser473. Cells were switched to K5 medium for 1 h and then treated for 15 min with increasing concentrations of Hc-TeTx (0–100 nm). Western blots were performed with anti-phospho(Ser473)Akt antibody or with anti-Akt antibody. Graphics shows densitometric determination of phospho-Akt levels. The ratio of the band intensity is expressed as a percentage of control. (b) Akt phosphorylation was detected using 10 nm Hc-TeTx at the indicated times in low [K+]e (K5) conditions compared with the non-treated cells in K5 and in high [K+]e K25 conditions. The signal intensities from western blot analysis (50 µg protein per lane) were determined by densitometry. The ratio of band intensity was expressed as a percentage of values from naive cells. The analysis was performed in three independent experiments with similar results. Values are the mean ± SEM (upper). Western blots of anti-Akt antibody (lower) were performed for determining the protein levels. (c) In the same experiments, the levels of phosphorylation of GSK3 (an Akt substrate) in the Ser9 residue were determined, using anti-phospho-GSK3 and anti-GSK3 antibodies.

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image

Figure 4. Phosphorylation of ERK1 and ERK2 members of the mitogen-activated protein kinases (MAPK) family in CGN cells after treatment with Hc-TeTx. Cells were grown for 7 days in vitro in 25 mm potassium and deprived for 1 h of serum and potassium in K5 lanes, and only of serum in K25 lanes. Then CGN were switched to serum-free BME containing either 25 mm or 5 mm KCl in the presence of increasing concentrations of Hc-TeTx (10–9 m to 10–7 m) for 15 min (a) or in the presence of 10 nm HcTeTx for the time indicated (b). Immunoblottings were performed with an antibody that recognizes the dually phosphorylated form of ERK-1 and ERK-2. Equal loading was demonstrated by reprobing the membranes with an antibody recognizing total ERK-1/2.

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Hc-TeTx induces mitogen-activated protein kinase (MAPK) signalling pathway

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Materials
  5. Cell cultures
  6. Immunoprecipitation
  7. Western blot analysis
  8. Determination of p21Ras-GTP
  9. MTT reduction assay
  10. DNA staining and fluorescence measurement
  11. Results
  12. Hc-TeTx fragment rescues mature CGN from apoptotic cell death
  13. Hc-TeTx induces phosphorylation of Trk receptor and p21Ras activation
  14. Hc-TeTx induces phosphatidylinositol 3-kinase (PI3K)/Akt signalling pathway
  15. Hc-TeTx induces mitogen-activated protein kinase (MAPK) signalling pathway
  16. Hc-TeTx prevents the proteolytic activation of pro-caspase-3
  17. Discussion
  18. Acknowledgements
  19. References

Phosphorylation of the MAPK family is another important yet ambiguous event in cellular signalling associated with survival, growth regulation and cell differentiation (Gunn-Moore et al. 1997). As illustrated in Fig. 4, ERK-1/2 remained phosphorylated at basal levels when cells were deprived of growth factors for 1 h and in a medium containing either 5 or 25 mm KCl (Fig. 4, lanes C). Addition for 15 min of various concentrations of Hc-TeTx caused an increase in ERK1 and in ERK 2 dually phosphorylated in Thr 202 and in Tyr 204 (Fig. 4a). The highest level of phosphorylation was obtained at 10 nm Hc-TeTx fragment. In time-course experiments, the maximum stimulation was obtained after 15 min, and thereafter a gradual decrease was noted (Fig. 4b). As can be observed in Figs 4(a) and (b), the basal phosphorylation in K25 medium is higher than in K5 medium (an average of 18% K5 with respect to K25), this being a result commonly described (e.g. Rosen et al. 1994). To further validate the Hc-TeTx-mediated stimulation of MAPK and PI3K/Akt signalling cascades, PD98059, a MEK inhibitor, and Wortmannin, a PI3K inhibitor, were used. In addition, the effect of Hc-TeTx on phosphorylation of p90rsk and of CREB, both members of the ERK pathway, were also examined using anti-phospho(Ser380)p90rsk, anti-phospho(Ser133)CREB antibodies. As shown in Fig. 5, addition of 10 nm Hc-TeTx for 15 min in low [K+]e medium enhanced p90rsk and CREB phosphorylation (Fig. 5a). Treatment with 50 µm PD98059 under these conditions blocked completely ERK1/2 phosphorylation and also reduced significantly p90rsk and CREB phosphorylation. Addition of 100 nm Wortmannin was less effective than PD98059 in blocking phosphorylation due to Hc-TeTx of ERK1/2, p90rsk and CREB. On the other hand, Hc-TeTx-induced cell survival was suppressed by pharmacological inhibition of MEK and PI3K (Fig. 5b). Compared to the 82% of cellular viability, with respect to the K25 control, after toxin addition under K5 medium, both PD98059 and Wortmannin partially blocked (64% and 59%, respectively) the Hc-TeTx-dependent rescue of the CGN cells.

image

Figure 5. Involvement of PI3K and MAPK pathways on cell survival. (a) Hc-TeTx-induced phosphorylation of ERK-1/2, p90rsk and CREB in a MAPK-dependent and PI3K-independent manner was demonstrated using PD98059 (a MEK inhibitor) and Wortmannin (a PI3K inhibitor), respectively. Cerebellar granule neurone cultures were grown for 7 days in vitro in 25 mm KCl, then the cells were deprived of serum and potassium for 1 h and then treated or not treated with Hc-TeTx (10 nm) for 15 min in low [K+]e. Cultures were also treated with PD98059 (50 µm), Wortmannin (100 nm), or the vehicle dimethyl sulfoxide (0.1%). Western blots were carried out by using anti-diphospho(Thr202-Tyr204)p44/p42 MAPK, anti-phospho(Ser380)p90rsk, anti-phospho(Ser133)CREB and anti-ERK-1/2 antibodies. (b) Hc-TeTx-induced cell survival suppressed by pharmacological inhibition of MEK and PI3K. CGN grown for 7 days in vitro were deprived of serum for 2 h and pre-incubated with Wortmannin (100 nm), PD98059 (50 µm), or the vehicle dimethyl sulfoxide (0.1%) for 30 min. Cultures were then switched to high (K25) and low potassium (K5) with or without Hc-TeTx (10 nm). After 24 h of incubation, cell viability was determined by MTT assay. Error bars represent the mean ± SEM (n = 3). The MEK and PI3K inhibitors significantly reduced Hc-TeTx enhancement of neuronal survival.

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Hc-TeTx prevents the proteolytic activation of pro-caspase-3

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Materials
  5. Cell cultures
  6. Immunoprecipitation
  7. Western blot analysis
  8. Determination of p21Ras-GTP
  9. MTT reduction assay
  10. DNA staining and fluorescence measurement
  11. Results
  12. Hc-TeTx fragment rescues mature CGN from apoptotic cell death
  13. Hc-TeTx induces phosphorylation of Trk receptor and p21Ras activation
  14. Hc-TeTx induces phosphatidylinositol 3-kinase (PI3K)/Akt signalling pathway
  15. Hc-TeTx induces mitogen-activated protein kinase (MAPK) signalling pathway
  16. Hc-TeTx prevents the proteolytic activation of pro-caspase-3
  17. Discussion
  18. Acknowledgements
  19. References

Since survival effects are activated following Hc-TeTx addition, experiments addressed to study the appearance of cleavage products of caspase-3 were performed. Time-course experiments on the effect of [K+]e deprivation on proteolytic activation of pro-caspase-3 in CGN were carried out (Fig. 6a) and the results were obtained by western blot using antibody against cleaved caspase-3. The results of the analysis of intensities of the bands show a clear and progressive increase in cleaved caspase-3, from a practically non-existing cleaved caspase-3 to a maximal amount observed after 24 h without potassium depolarization. When potassium-deprived cells are incubated in the presence of increasing concentrations of Hc-TeTx, a protection against caspase-3 cleavage is observed, with the maximal inhibition of cleavage at 10 nm Hc-TeTx (Fig. 6b). On the other hand, PD98059 inhibited the effect of the Hc fragment on cleaved-caspase-3 appearance by approximately 40%, and Wortmannin inhibited the effect by approximately 80% (results not shown). These results point to a shared responsibility of both pathways in the inhibition due to Hc fragment of caspase-3 cleavage, having the Akt pathway the main role in this inhibition. This dependence of caspase-3 cleavage on both pathways is in agreement with the results observed in Fig. 5(b).

image

Figure 6. (a) Time-course of the effect of [K+]e deprivation on proteolytic activation of pro-caspase-3 in cerebellar granule neurones (CGN). CGN were grown for 7 days in vitro in a 25 mm KCl medium (Control), the cultures were then transferred to a 5 mm KCl medium for a period of time ranging from 0 h to 24 h. Western blots (30 µg of protein/lane) were revealed using antibody against cleaved caspase-3. The results of the analysis of intensities of the bands were quantified by densitometry and represented as mean ± SEM of three independent experiments. (b) Concentration–response of Hc-TeTx on proteolytic activation of pro-caspase-3 in low [K+]e. CGN were switched to high or low [K+]ε with or without a 30 min Hc-TeTx-pre-treatment at different concentrations (1 nm, 10 nm or 100 nm) and the lysates were obtained at 24 h after treatment. Western blots (30 µg of protein/lane) were revealed using antibody against cleaved pro-caspase-3. The results of the analysis of intensities of the bands were quantified by densitometry and represented as mean ± SEM of three independent experiments. In order to show the equal loading between lanes β-tubulin was probed.

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Discussion

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Materials
  5. Cell cultures
  6. Immunoprecipitation
  7. Western blot analysis
  8. Determination of p21Ras-GTP
  9. MTT reduction assay
  10. DNA staining and fluorescence measurement
  11. Results
  12. Hc-TeTx fragment rescues mature CGN from apoptotic cell death
  13. Hc-TeTx induces phosphorylation of Trk receptor and p21Ras activation
  14. Hc-TeTx induces phosphatidylinositol 3-kinase (PI3K)/Akt signalling pathway
  15. Hc-TeTx induces mitogen-activated protein kinase (MAPK) signalling pathway
  16. Hc-TeTx prevents the proteolytic activation of pro-caspase-3
  17. Discussion
  18. Acknowledgements
  19. References

In the present study, we set the objective to document the molecular consequences of Hc-TeTx fragment interaction with CGN when stress conditions through low extracellular [K+] are applied on the cells. The overall rationale for this study has been the possibility that TeTx acts on neuronal targets by eliciting a number of signalling cascades that may share similar properties to the action of growth factors. From the present data it would appear that the Hc-TeTx shows neuroprotective properties, these being presumably the result of the direct or indirect interaction of the toxin fragment with the TrkB receptor. On the other hand, and taking into account that TrkB is already activated in K25 (Hc-TeTx pre-treatment conditions, see Figs 2a and b), Hc-TeTx activates, most probably, additional signalling besides those activated in high potassium, because the Hc-TeTx exerts a positive role in the presence of K25, which is not seen in K25 alone. The action of Hc-TeTx on the TrkB receptor leads to the activation of at least three major signalling pathways, PI3K/Akt, MEK/MAPK and PLCγ/PKC, in a similar way to BDNF (Nakagawara et al. 1994; Nonomura et al. 1996). Previous reports demonstrated that BDNF and NT-4/5, and NT-3 to a small extent, but not NGF, significantly protect differentiated CGN cells from low K+-induced cell death (Kubo et al. 1995), as well as from stress-mediated apoptotic death (Skaper et al. 1998). Furthermore, it has been demonstrated that PI3K has a crucial role in the prevention of low K+-induced apoptosis of CGN (Shimoke et al. 1997). In accordance with this observation, it has been demonstrated that CGN cultures express TrkB, and to a lesser extent TrkC, but not TrkA, under depolarizing conditions (Nonomura et al. 1996). The survival effects carried out by neurotrophins is similar to that produced by depolarization with a high [K+]e medium, and may be due to the influx of calcium into cells through voltage-activated channels (Gallo et al. 1987). In addition, tyrosine phosphorylation of TrkB receptors by neurotrophins, NGF and NT-3 in CGN cells was by far higher than TrkC and TrkA receptors. In any case, a demonstration of the interaction of Hc-TeTx with some component of the neurotrophins receptor machinery is lacking, and would shed light over how TeTx can undergo retroaxonal transport inside neurones. On the other hand, it has been established that TeTx interacts, through its Hc fragment, with the neurone membrane in specific microdomains known as ‘lipid rafts’ (Herreros et al. 2001). These are dynamic assemblies of cholesterol and sphingolipids that form in the cellular membranes, being highly enriched in gangliosides (Pike 2003). There is a general agreement that TeTx shows the highest affinity for the polysialogangliosides of the G1b series (Lalli et al. 1999), and these gangliosides are known to activate Trk receptors and ERK phosphorylation in rat brain slices (Duchemin et al. 2002). Thus, lipid rafts could act as a platform in which a receptor complex constituted by TeTx, ganglioside and Trk could be formed.

Another novel finding described in the present work is the enhancement by Hc-TeTx treatment of p21Ras in GTP-bound form, i.e. the activated form that leads to downstream signalling (White et al. 1995). These observations are in agreement with the relationship found between the amounts of GTP bound to p21Ras and the CGN survival induced by BDNF after potassium withdrawal (Zirrgiebel et al. 1995). The p21Ras protein has been described as an essential link in the inhibition of death pathways (Nobes et al. 1996), possibly due to the ability of p21Ras to promote signalling through the PI3K and ERK pathways simultaneously but non-redundantly, inhibiting two independent mechanisms of apoptosis, as has been described in rat sympathetic neurones (Xue et al. 2000). Interestingly, the same result was found after interleukin-3 deprivation in the Ba/F3 cell line, a mouse interleukin-3-dependent cell line (Kinoshita et al. 1997), pointing to the possibility of this dual manner of cell protection as a more general mechanism. Our finding, in experiments using PD98059 or Wortmannin, in which both pathways are responsible for Hc-TeTx-induced survival is in agreement with this view of PI3K and ERK as simultaneous and non-redundant survival pathways. Both these pathways can act over another prominent effect of potassium withdrawal as well as of serum deprivation in CGN, i.e. the appearance of the cleaved caspase-3 subunit, preceding the period of peak neuronal death (Cryns and Yuan 1998). Inhibition of caspase-3 cleavage participates in the survival effects elicited by several factors, such as lithium (Mora et al. 2001) or LIGA20 (Marks et al. 1998). Since caspases participate in many apoptotic events, such as DNA fragmentation, chromatin condensation, membrane blebbing, and disassembly into apoptotic bodies (Thornberry and Lazebnik 1998), inhibition of caspase-3 cleavage observed in the present work is a good clue about the interference by Hc-TeTx of pathways leading to programmed cell death. The inhibition of the 17 kDa caspase-3 fragment reaches the maximum at 10 nm Hc-TeTx, being partially reverted at 100 nm. This kind of pattern is also seen in the survival potentiation, in the ERK-1/2 activation at K5, and, to a lesser extent, in the Akt phosphorylation in Ser 473. One possible explanation for these results could be the acceleration, at high Hc-TeTx concentrations, of the deactivation rate of the Hc-TeTx-induced pathways, as is seen in the case of PKC when activated by TeTx (Gil et al. 1998).

Actually, the action of bacterial toxins on survival pathways is a common feature described in the literature, a lot of them causing apoptosis. Modulation of the host cell-death pathway may be used to abrogate key immune cells that could limit the infection or, alternatively, to facilitate the proliferation of intracellular pathogens (Weinrauch and Zychlinsky 1999). In this way, anthrax lethal factor, also a Zn2+-metalloprotease as is TeTx, causes cell death, as well as host death, by means of cleavage and inactivation of mitogen-activated protein kinase kinase (MAPKK), being, together with TeTx, the only protein toxin described as acting on the MAPK pathway (Chopra et al. 2003). Thus, whilst anthrax toxin causes cell death by MAPKK inactivation, Hc-TeTx would act in the reverse way, inducing cell survival by means of MAPKK activation through a signalling cascade coming from the plasma membrane. If the interpretation to the induction of apoptosis by a bacterial toxin is easy to obtain, i.e. elimination of key defence cells or to provide a safe haven for bacterial proliferation, the interpretation to the host cell survival enhancement performed by a toxin, Hc-TeTx in this case, is difficult to present. Would the retroaxonal transport, so important in the TeTx action, be enhanced in the context of the general activated metabolism in the cell survival conditions? On the other hand, since the impairment of exocytosis has been described to participate in neurological diseases in which apoptosis is present (Ferrer 2002), could this promotion of cell survival be a mechanism of compensation for the apoptosis that could be promoted by the inhibition of an essential function for neurones, such as exocytosis? In any case, the role of clostridial neurotoxins and derived molecules as therapeutical tools for the treatment of disorders of neuromuscular basis, or even of tumoral processes, seems to increase constantly (Rossetto et al. 2001). Now, the promotion of neuronal survival can be added to the list of possible therapeutical uses. Thus, clostridial neurotoxins seem to be not so bad at the end.

References

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Materials
  5. Cell cultures
  6. Immunoprecipitation
  7. Western blot analysis
  8. Determination of p21Ras-GTP
  9. MTT reduction assay
  10. DNA staining and fluorescence measurement
  11. Results
  12. Hc-TeTx fragment rescues mature CGN from apoptotic cell death
  13. Hc-TeTx induces phosphorylation of Trk receptor and p21Ras activation
  14. Hc-TeTx induces phosphatidylinositol 3-kinase (PI3K)/Akt signalling pathway
  15. Hc-TeTx induces mitogen-activated protein kinase (MAPK) signalling pathway
  16. Hc-TeTx prevents the proteolytic activation of pro-caspase-3
  17. Discussion
  18. Acknowledgements
  19. References
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