SEARCH

SEARCH BY CITATION

Keywords:

  • axon;
  • inflammation;
  • mitogen-activated protein kinase;
  • neurone;
  • neuroprotection;
  • trophic

Abstract

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Neuronal cell culture
  5. Preparation of oligodendrocyte conditioned medium
  6. Assays for cell survival and cell morphology
  7. Immunocytochemistry
  8. Morphological analysis using immunocytochemistry
  9. Analysis of axons in culture
  10. Immunoblotting for cell signalling proteins
  11. Statistical analysis
  12. Results
  13. The nitric oxide donor DETANONOate causes cortical neuronal death in a dose-dependent manner
  14. Oligodendrocyte conditioned medium and insulin-like growth factor type-1 increases neuronal survival in neurones exposed to DETANONOate
  15. The nitric oxide donor DETANONOate causes axonal loss independent of neuronal survival, and oligodendocyte conditioned medium, insulin-like growth factor type-1 and glial cell line-derived neurotrophic factor improve axonal survival
  16. Attenuation of nitric oxide mediated axon destruction requires mitogen-activated protein kinase/extracellular signal-regulated kinase signalling
  17. P38 pathways are activated in neurones exposed to DETANONOate and both oligodendrocyte conditioned medium and insulin-like growth factor reduce levels of p38 activation
  18. Discussion
  19. Nitric oxide causes neuronal and axonal destruction
  20. Attenuation of neuronal death
  21. Attenuation of axonal destruction
  22. Relevance to inflammatory diseases of the central nervous system
  23. Acknowledgement
  24. References

Inflammation in the central nervous system occurs in diseases such as multiple sclerosis and leads to axon dysfunction and destruction. Both in vitro and in vivo observations have suggested an important role for nitric oxide (NO) in mediating inflammatory axonopathy. The purposes of this study were to model inflammatory axonopathy in vitro and identify modulators of the process. Rat cortical neurones were cultured and exposed to an NO-donor plus potential protective factors. Cultures were then assessed for neuronal survival, axon survival and markers of intracellular signalling pathways. The NO-donor produced dose-dependent neuronal loss and a large degree of axon destruction. Oligodendrocyte conditioned medium (OCM) and insulin-like growth factor type-1 (IGF-1), but not glial cell line-derived neurotrophic factor (GDNF), improved survival of neurones exposed to NO donors. In addition p38 MAP kinase was activated by NO exposure and inhibition of p38 signalling led to neuronal and axonal survival effects. OCM and IGF-1 (but not GDNF) reduced p38 activation in NO-exposed cortical neurones. OCM, IGF-1 and GDNF improved axon survival in cultures exposed to NO, a process dependent on mitogen-activated protein kinase/extracellular signal-related kinase signalling. This study emphasizes that different mechanisms may underlie neuronal/axonal destructive processes, and suggests that trophic factors may modulate NO-mediated neurone/axon destruction via specific pathways.

Abbreviations used
DETANONOate

(Z)-1-[2-(2-aminoethyl)-N-(2-ammonioethyl)amino]daizen-1-ium-1,2-diolate

DIV

day in vitro

Erk

extracellular signal-related kinase

GDNF

glial cell line-derived neurotrophic factor

IGF-1

insulin-like growth factor type-1

MAP

kinase, mitogen-activated protein kinase

NO

nitric oxide

OCM

oligodendrocyte conditioned medium

OPC

oligodendrocyte precursor cell

Inflammation in the central nervous system occurs in a variety of diseases and may be associated with neuronal, axonal and glial destruction (Trapp et al. 1998). Both in experimentally induced encephalomyelitis and multiple sclerosis, inflammatory cells, including activated T-lymphocytes and microglia, accumulate in discrete lesions where they cause widespread cellular damage (Kornek and Lassmann 2003). Mechanisms of cell death within inflammatory lesions rely heavily upon release of cytokines and other cytotoxic agents (Bauer et al. 2001). Of the many substances that have been shown to be toxic to neurones and axons, nitric oxide (NO) appears to be of particular importance. NO serves many physiological functions within the nervous system, including roles in synaptic plasticity, long-term potentiation and neurotransmitter release, but at sites of inflammation high concentrations of NO are thought to mediate cell death (Smith and Lassmann 2002). Several studies have shown that microglial-derived NO is neurotoxic in vitro (Bal-Price and Brown 2001; Golde et al. 2002). Mechanisms underlying this neurotoxicity are not entirely clear, but NO activates a number of intracellular signalling pathways, including mitogen-activated protein kinases (MAP kinases) and may ultimately lead to neuronal death via inhibition of mitochondrial respiration (Ghatan et al. 2000; Brown and Borutaite 2002). Recent in vivo work has shown that NO may also mediate activity dependent axon destruction (Smith et al. 2001). Studies of acute inflammatory lesions of multiple sclerosis have revealed the involvement of NO (Bagasra et al. 1995).

Inflammatory cells not only release toxic compounds, but also provide neuroprotective factors at sites of tissue injury (Kerschensteiner et al. 2003). Indeed, the balance between these two opposing activities may determine the outcome of tissue damage, with trophic factors modulating the deleterious effect of molecules such as NO (Matsuzaki et al. 1999). As glial cells are another potent source of neurotrophic factors, the local environment of the central nervous system may also influence the effects of neurotoxic substances (Komoly et al. 1992). We have previously shown that oligodendrocyte-derived insulin-like growth factor (IGF)-1 and glial cell line-derived neurotrophic factor (GDNF) increase neuronal survival and axonal length in trophically deprived neuronal cultures, respectively (Wilkins et al. 2001, 2003). The close and reciprocal relationship between neurones and oligodendrocytes makes it important also to consider how oligodendrocyte-derived factors may influence axonal survival during inflammation.

Here we show that a NO donor causes neuronal death in cultured cortical neurones and that oligodendrocyte-conditioned medium (OCM) and IGF-1 improve neuronal survival under these conditions. The NO donor also causes axonal loss within cultures independent of whole neuronal survival. Again, OCM, GDNF and IGF-1 attenuate this process, to different extents, acting though MAP kinase/extracellular signal-regulated kinase (Erk) signalling pathways. Furthermore, exposure of neurones to NO activates p38 MAP kinase signalling. Its inhibition improves neuronal and axonal survival in NO-exposed neurones; and both OCM and IGF-1 inhibit p38 signalling. Thus, this study emphasizes the general importance of trophic factors in protection against NO-mediated inflammatory injury of neurones and axons. More specifically, we distinguish components of the MAP kinase signalling pathway that differentially affect axons and whole neurones.

Neuronal cell culture

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Neuronal cell culture
  5. Preparation of oligodendrocyte conditioned medium
  6. Assays for cell survival and cell morphology
  7. Immunocytochemistry
  8. Morphological analysis using immunocytochemistry
  9. Analysis of axons in culture
  10. Immunoblotting for cell signalling proteins
  11. Statistical analysis
  12. Results
  13. The nitric oxide donor DETANONOate causes cortical neuronal death in a dose-dependent manner
  14. Oligodendrocyte conditioned medium and insulin-like growth factor type-1 increases neuronal survival in neurones exposed to DETANONOate
  15. The nitric oxide donor DETANONOate causes axonal loss independent of neuronal survival, and oligodendocyte conditioned medium, insulin-like growth factor type-1 and glial cell line-derived neurotrophic factor improve axonal survival
  16. Attenuation of nitric oxide mediated axon destruction requires mitogen-activated protein kinase/extracellular signal-regulated kinase signalling
  17. P38 pathways are activated in neurones exposed to DETANONOate and both oligodendrocyte conditioned medium and insulin-like growth factor reduce levels of p38 activation
  18. Discussion
  19. Nitric oxide causes neuronal and axonal destruction
  20. Attenuation of neuronal death
  21. Attenuation of axonal destruction
  22. Relevance to inflammatory diseases of the central nervous system
  23. Acknowledgement
  24. References

Neuronal cultures were prepared from cortices of E16 rat embryos as previously described (Wilkins et al. 2001). Following enzymatic dissociation, cells were plated onto poly-l-lysine coated 13-mm coverslips and cultured in Dulbecco's modified Eagle's medium supplemented with 2% B27 (Gibco, Paisley, UK). After 6 days in vitro (DIV), > 95% of cells were positive for the neuronal marker β-tubulin. At this point, cultures were exposed to experimental conditions. The base medium for all experiments was ‘minimal’, which consisted of Dulbecco's modified Eagle's medium supplemented with insulin-free Sato (containing 100 µg/mL bovine serum albumin, 100 µg/mL transferrin, 0.06 µg/mL progesterone, 16 µg/mL putrescine, 0.04 µg/mL selenite, 0.04 µg/mL thyroxine, 0.04 µg/mL triiodothryonine).

Recombinant IGF-1 was obtained from Boehringer (Bracknell, Berkshire, UK) and recombinant GDNF from Chemicon (Chandler's Ford, Hampshire, UK). The pathway inhibitors SB202190 and PD98059 were obtained from Calbiochem (San Diego, CA, USA), and SB203580 from Sigma (Gillingham, Dorset, UK). A stock solution (50 mm in 10 mm NaOH) of (Z)-1-[2-(2-aminoethyl)-N-(2-ammonioethyl)amino]daizen-1-ium-1,2-diolate (DETANONOate; Alexis Biochemicals, Nottingham, UK) was prepared immediately before use. In order to confirm NO generation in cultures, a modified Greiss reaction (Sigma, UK) was performed on culture supernatants after 24 h of DETANONOate exposure.

Preparation of oligodendrocyte conditioned medium

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Neuronal cell culture
  5. Preparation of oligodendrocyte conditioned medium
  6. Assays for cell survival and cell morphology
  7. Immunocytochemistry
  8. Morphological analysis using immunocytochemistry
  9. Analysis of axons in culture
  10. Immunoblotting for cell signalling proteins
  11. Statistical analysis
  12. Results
  13. The nitric oxide donor DETANONOate causes cortical neuronal death in a dose-dependent manner
  14. Oligodendrocyte conditioned medium and insulin-like growth factor type-1 increases neuronal survival in neurones exposed to DETANONOate
  15. The nitric oxide donor DETANONOate causes axonal loss independent of neuronal survival, and oligodendocyte conditioned medium, insulin-like growth factor type-1 and glial cell line-derived neurotrophic factor improve axonal survival
  16. Attenuation of nitric oxide mediated axon destruction requires mitogen-activated protein kinase/extracellular signal-regulated kinase signalling
  17. P38 pathways are activated in neurones exposed to DETANONOate and both oligodendrocyte conditioned medium and insulin-like growth factor reduce levels of p38 activation
  18. Discussion
  19. Nitric oxide causes neuronal and axonal destruction
  20. Attenuation of neuronal death
  21. Attenuation of axonal destruction
  22. Relevance to inflammatory diseases of the central nervous system
  23. Acknowledgement
  24. References

Mixed glial cell cultures were prepared following the protocol of McCarthy and de Vellis 1980). Briefly, forebrains of newborn rat pups were removed and the meninges stripped before mechanical and enzymatic dissociation. The resulting cell suspension was plated onto poly-l-lysine coated 75-cm2 tissue culture flasks. Culture medium (Dulbecco's modified Eagle's medium/10% fetal calf serum) was changed at 24 h and twice weekly thereafter until the cells reached confluence after 10–12 days. At this stage, a loosely adherent superficial layer of cells that represents oligodendrocyte precursor cells (OPCs) and microglia, lying on a confluent astrocyte layer, was isolated by two-stage differential adhesion. Microglia were removed by vigorous shaking of the cultures at 240 rpm, followed by a change to fresh medium. Top-dwelling OPCs were dislodged by overnight shaking of the cultures at 160 rpm and the supernatant placed into uncoated tissue culture flasks for 30 min to allow adherence of any residual microglia. The loosely adherent OPCs were dislodged by gentle manual shaking. The final supernatant from these shaken cultures contained approximately 85–90% OPCs. Cells were expanded in B104 conditioned medium to further enrich and expand the oligodendrocyte cultures (Canoll et al. 1996). Enriched OPCs were plated at high density (2 × 106 per poly-l-lysine coated 6-well plates) in serum-free Dulbecco's modified Eagle's medium supplemented with 2% B27 (Gibco, UK). After 4 DIV, cultures were washed and used to condition ‘minimal’ medium for 24 h. At this stage the cells were predominantly differentiated oligodendrocytes [12.6 ± 7.8% of cells were A2B5 positive, 78.8 ± 3.7% were galactocerebroside (GalC) positive and 4.3 ± 3.2% were glial fibrillary acidic protein (GFAP) positive].

Immunocytochemistry was used to identify cell phenotypes. Cells were stained after fixation with 4% paraformaldehyde. Primary antibodies against intracellular markers were used after treatment of fixed cultures with 100% methanol at −20°C for 10 min. These were β-tubulin III (Sigma, UK) 1 : 400 and SMI312 (phosphorylated neurofilaments) 1 : 1000 (Sternberger Monoclonals, Lutherville, MD, USA). Secondary antibodies coupled to fluorochromes FITC, TRITC or AMCA were used to visualize primary antibody staining. Hoechst 33258 (bisbenzamide 1 : 5000) was used (10 min at room temperature) for nuclear identification.

Neurones were identified by β-tubulin expression. In addition, nuclear staining (Hoechst) of cells enabled a morphological assessment of apoptosis. Neuronal survival was assessed using counts of live β-tubulin-positive stained cells, taking five random fields per culture and at least three cultures per experiment. In all cases, control cultures grown throughout the experimental period in Dulbecco's modified Eagle's medium/2% B27 were also analysed and values for experimental conditions divided by this value, in order to standardize results between experiments.

The antibody SMI312 (Sternberger Monoclonals, USA) labels phosphorylated neurofilaments and can be used to distinguish axons from dendrites (Lee et al. 1987). Cultures were stained for SMI312 following fixation and viewed under fluorescent microscopy with digital images (× 40) of three random fields within each culture (at least three cultures per condition per experiment). The images were analysed using Scion imaging software (Scion Corp., Frederick, MD, USA). Lengths of processes staining for SMI312 per field were obtained, together with the number of live cells per field. The ratio of SMI312 length to cell number therefore gave an index of phosphorylated neurofilament length per live cell. Again control cultures grown throughout the experimental period in Dulbecco's modified Eagle's medium/2% B27 were also analysed and values for experimental conditions divided by this value, in order to standardize results between experiments.

Immunoblotting for cell signalling proteins

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Neuronal cell culture
  5. Preparation of oligodendrocyte conditioned medium
  6. Assays for cell survival and cell morphology
  7. Immunocytochemistry
  8. Morphological analysis using immunocytochemistry
  9. Analysis of axons in culture
  10. Immunoblotting for cell signalling proteins
  11. Statistical analysis
  12. Results
  13. The nitric oxide donor DETANONOate causes cortical neuronal death in a dose-dependent manner
  14. Oligodendrocyte conditioned medium and insulin-like growth factor type-1 increases neuronal survival in neurones exposed to DETANONOate
  15. The nitric oxide donor DETANONOate causes axonal loss independent of neuronal survival, and oligodendocyte conditioned medium, insulin-like growth factor type-1 and glial cell line-derived neurotrophic factor improve axonal survival
  16. Attenuation of nitric oxide mediated axon destruction requires mitogen-activated protein kinase/extracellular signal-regulated kinase signalling
  17. P38 pathways are activated in neurones exposed to DETANONOate and both oligodendrocyte conditioned medium and insulin-like growth factor reduce levels of p38 activation
  18. Discussion
  19. Nitric oxide causes neuronal and axonal destruction
  20. Attenuation of neuronal death
  21. Attenuation of axonal destruction
  22. Relevance to inflammatory diseases of the central nervous system
  23. Acknowledgement
  24. References

Neurones were cultured at high density (2 × 106 per 6-well plate) before exposure to test conditions. After set time points, cells were lysed in 150 mm Tris-HCl, 8 m urea, 2.5% w/v sodium dodecyl sulfate, 20% v/v glycerol, 10% v/v 2-mercaptoethanol, 3% w/v dithiothreitol, 0.1% bromophenol blue, pH 6.8 and stored at −20°C until use. Continuous gradient sodium dodecyl sulfate–polyacrylamide gels (5–20%) were used and equal volumes of cell lysates [equivalent to equal amounts of total protein as judged by protein determination using the BCA protein assay kit (Pierce, Cheshire, UK)] run on each lane. After transfer to nitrocellulose membrane (Hybond C-super, Amersham Pharmacia Biotech, Chalfont St Giles, UK) and blocking in 4% w/v powdered milk, membranes were incubated overnight in primary antibody at 4°C (in Tris-buffered saline/5% bovine serum albumin). Antibodies used were phospho-p38 (Thr180/Tyr182; Chemicon, UK), p38 antibody, phospho-MAP kinase p42/p44 and non-phosphorylated MAP kinase p42/p44 (all 1 : 1000, New England Biolabs, Beverley, MA, USA). Immunoreactivity was visualized by secondary anti-rabbit horseradish peroxidase conjugated antibodies (Dako, UK) and enhanced chemoluminescence (Renaissance ECL reagent, NEN Life Science Products, Boston, MA, USA). Where necessary, membranes were stripped (Restore Western Blot stripping buffer, Pierce) and reblotted.

Statistical analysis

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Neuronal cell culture
  5. Preparation of oligodendrocyte conditioned medium
  6. Assays for cell survival and cell morphology
  7. Immunocytochemistry
  8. Morphological analysis using immunocytochemistry
  9. Analysis of axons in culture
  10. Immunoblotting for cell signalling proteins
  11. Statistical analysis
  12. Results
  13. The nitric oxide donor DETANONOate causes cortical neuronal death in a dose-dependent manner
  14. Oligodendrocyte conditioned medium and insulin-like growth factor type-1 increases neuronal survival in neurones exposed to DETANONOate
  15. The nitric oxide donor DETANONOate causes axonal loss independent of neuronal survival, and oligodendocyte conditioned medium, insulin-like growth factor type-1 and glial cell line-derived neurotrophic factor improve axonal survival
  16. Attenuation of nitric oxide mediated axon destruction requires mitogen-activated protein kinase/extracellular signal-regulated kinase signalling
  17. P38 pathways are activated in neurones exposed to DETANONOate and both oligodendrocyte conditioned medium and insulin-like growth factor reduce levels of p38 activation
  18. Discussion
  19. Nitric oxide causes neuronal and axonal destruction
  20. Attenuation of neuronal death
  21. Attenuation of axonal destruction
  22. Relevance to inflammatory diseases of the central nervous system
  23. Acknowledgement
  24. References

Statistical analysis on data expressed as a percentage of control was performed on arcsine transformed values. Counting data was analysed using non-parametric tests (Kruskal–Wallis with post hoc Dunn's testing between groups). Other data was analysed using one-way anova with post hoc testing for comparison of multiple sets (Neumann–Keuls). Values are expressed as the mean ± SEM from at least three independent experiments, unless otherwise stated.

The nitric oxide donor DETANONOate causes cortical neuronal death in a dose-dependent manner

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Neuronal cell culture
  5. Preparation of oligodendrocyte conditioned medium
  6. Assays for cell survival and cell morphology
  7. Immunocytochemistry
  8. Morphological analysis using immunocytochemistry
  9. Analysis of axons in culture
  10. Immunoblotting for cell signalling proteins
  11. Statistical analysis
  12. Results
  13. The nitric oxide donor DETANONOate causes cortical neuronal death in a dose-dependent manner
  14. Oligodendrocyte conditioned medium and insulin-like growth factor type-1 increases neuronal survival in neurones exposed to DETANONOate
  15. The nitric oxide donor DETANONOate causes axonal loss independent of neuronal survival, and oligodendocyte conditioned medium, insulin-like growth factor type-1 and glial cell line-derived neurotrophic factor improve axonal survival
  16. Attenuation of nitric oxide mediated axon destruction requires mitogen-activated protein kinase/extracellular signal-regulated kinase signalling
  17. P38 pathways are activated in neurones exposed to DETANONOate and both oligodendrocyte conditioned medium and insulin-like growth factor reduce levels of p38 activation
  18. Discussion
  19. Nitric oxide causes neuronal and axonal destruction
  20. Attenuation of neuronal death
  21. Attenuation of axonal destruction
  22. Relevance to inflammatory diseases of the central nervous system
  23. Acknowledgement
  24. References

Cortical neurones (1.4 × 103 cells/mm2) were maintained in B27 supplemented Dulbecco's modified Eagle's medium for 6 DIV before exposure to different concentrations of DETANONOate, a slow releasing NO donor. After a further 1 DIV, cultures were fixed and stained for β-tubulin and the nuclear marker bisbenzamide, and neuronal survival was determined. Neurones exposed to the DETANONOate showed decreased survival compared to control (Fig. 1a). Moreover, the phenomenon was dose dependent, with no survival occurring in cells exposed to 1 mm. In order to verify that the parent compound of DETANONOate was not toxic to neurones, cells were exposed to DETANONOate that had been activated 7 days prior to conditioning and that no longer possessed NO donor effects. Under these conditions, neurones showed no change in survival compared to control (data not shown).

image

Figure 1. (a) DETANONOate causes neuronal loss in cortical neuronal cultures. Effect of DETANONOate on numbers of live (β-tubulin positive) neurones. Cultures treated without DETANONOate were exposed to vehicle only (MIN). Controls were cultured in 2% B27 throughout and values expressed as a percentage of this control. Neurones were cultured for 6 DIV and exposed to test condition for 24 h (n = 5; **p < 0.01; ***p < 0.001). (b) Oligodendrocyte conditioned media (OCM) and insulin-like growth factor-1 (IGF-1) increase survival neurones exposed to DETANONOate. Effect of DETANONOate (0.1 mm; NO), DETANONOate plus OCM (NO/OCM), DETANONOate plus IGF-1 (50 ng/mL; NO/IGF), DETANONOate plus glial cell line-derived neurotrophic factor (10 ng/mL; NO/GDNF) and DETANONOate plus IGF-1 and GDNF (NO/I/G) on numbers of live (β-tubulin positive) neurones. Cultures treated without DETANONOate were exposed to vehicle only (MIN). Neurones were cultured for 6 DIV and exposed to test condition for 24 h (n = 6; **p < 0.01; ***p < 0.001).

Download figure to PowerPoint

Oligodendrocyte conditioned medium and insulin-like growth factor type-1 increases neuronal survival in neurones exposed to DETANONOate

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Neuronal cell culture
  5. Preparation of oligodendrocyte conditioned medium
  6. Assays for cell survival and cell morphology
  7. Immunocytochemistry
  8. Morphological analysis using immunocytochemistry
  9. Analysis of axons in culture
  10. Immunoblotting for cell signalling proteins
  11. Statistical analysis
  12. Results
  13. The nitric oxide donor DETANONOate causes cortical neuronal death in a dose-dependent manner
  14. Oligodendrocyte conditioned medium and insulin-like growth factor type-1 increases neuronal survival in neurones exposed to DETANONOate
  15. The nitric oxide donor DETANONOate causes axonal loss independent of neuronal survival, and oligodendocyte conditioned medium, insulin-like growth factor type-1 and glial cell line-derived neurotrophic factor improve axonal survival
  16. Attenuation of nitric oxide mediated axon destruction requires mitogen-activated protein kinase/extracellular signal-regulated kinase signalling
  17. P38 pathways are activated in neurones exposed to DETANONOate and both oligodendrocyte conditioned medium and insulin-like growth factor reduce levels of p38 activation
  18. Discussion
  19. Nitric oxide causes neuronal and axonal destruction
  20. Attenuation of neuronal death
  21. Attenuation of axonal destruction
  22. Relevance to inflammatory diseases of the central nervous system
  23. Acknowledgement
  24. References

In order to investigate factors that may be protective against NO-mediated neurotoxicity, neurones were exposed to NO donor in the presence of selected factors. DETANONOate exposed neurones were cultured in the presence of IGF-1 and GDNF, factors produced by oligodendrocytes and previously shown to provide trophic support for ‘trophically deprived’ neurones (Wilkins et al. 2001, 2003). Neurones were cultured as above for 6 DIV before exposure to DETANONOate (0.1 mm) plus each of the trophic factors described for a further 1 DIV. The addition of OCM and IGF-1 (50 ng/mL), but not GDNF (10 ng/mL), significantly improved neuronal survival (Fig. 1b).

The nitric oxide donor DETANONOate causes axonal loss independent of neuronal survival, and oligodendocyte conditioned medium, insulin-like growth factor type-1 and glial cell line-derived neurotrophic factor improve axonal survival

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Neuronal cell culture
  5. Preparation of oligodendrocyte conditioned medium
  6. Assays for cell survival and cell morphology
  7. Immunocytochemistry
  8. Morphological analysis using immunocytochemistry
  9. Analysis of axons in culture
  10. Immunoblotting for cell signalling proteins
  11. Statistical analysis
  12. Results
  13. The nitric oxide donor DETANONOate causes cortical neuronal death in a dose-dependent manner
  14. Oligodendrocyte conditioned medium and insulin-like growth factor type-1 increases neuronal survival in neurones exposed to DETANONOate
  15. The nitric oxide donor DETANONOate causes axonal loss independent of neuronal survival, and oligodendocyte conditioned medium, insulin-like growth factor type-1 and glial cell line-derived neurotrophic factor improve axonal survival
  16. Attenuation of nitric oxide mediated axon destruction requires mitogen-activated protein kinase/extracellular signal-regulated kinase signalling
  17. P38 pathways are activated in neurones exposed to DETANONOate and both oligodendrocyte conditioned medium and insulin-like growth factor reduce levels of p38 activation
  18. Discussion
  19. Nitric oxide causes neuronal and axonal destruction
  20. Attenuation of neuronal death
  21. Attenuation of axonal destruction
  22. Relevance to inflammatory diseases of the central nervous system
  23. Acknowledgement
  24. References

The axonal marker SMI312 was employed in order to assess influences on axonal survival in vitro (Lee et al. 1987). SMI312 labels highly phosphorylated neurofilament epitopes and is thus a useful axon marker. As DETANONOate causes significant neuronal death, an index of SMI312 length per live neurone was used in order to study axon specific effects. Cortical neurones (1.4 × 103 cells/mm2) were maintained in B27 supplemented medium for 6 DIV before exposure to DETANONOate and selected factors. After a further 1 DIV, cultures were fixed and stained for β-tubulin, SMI312 and the nuclear marker bisbenzamide. SMI312 lengths per live neurone were calculated for each condition. Exposure of cortical neurones to 0.1 mm DETANONOate for 24 h led to a significant reduction in axon length per neurone, implying a severe axon destructive effect induced by the NO donor (Figs 2a and b). This effect was attenuated by the addition of OCM, recombinant GDNF and IGF-1. As expected from previous work, the effect of GDNF was significantly greater than that of IGF-1.

image

Figure 2. (a) Trophic factors attenuate SMI312 length per cell reductions induced by DETANONOate. Effect of DETANONOate (0.1 mm; NO), DETANONOate plus medium conditioned oligodendrocytes (NO/OCM), DETANONOate plus insulin-like growth factor-1 (50 ng/mL; NO/IGF) and DETANONOate plus glial cell line-derived neurotrophic factor (10 ng/mL; NO/GDNF) on SMI312 length per live neurone. Cultures treated without DETANONOate were exposed to vehicle only (MIN). Controls were cultured in 2% B27 throughout and values expressed as a percentage of this control. Neurones were cultured for 6 DIV and exposed to test condition for 24 h (n = 5; **p < 0.01; ***p < 0.001). (b) Immunographs showing the effect of DETANONOate (0.1 mm; NO) and DETANONOate plus OCM (NO/OCM) on SMI312 (phosphorylated neurofilament; green) staining in neuronal cultures (6 DIV) exposed to test conditions for 24 h. Nuclei stained with Hoechst (blue).

Download figure to PowerPoint

Attenuation of nitric oxide mediated axon destruction requires mitogen-activated protein kinase/extracellular signal-regulated kinase signalling

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Neuronal cell culture
  5. Preparation of oligodendrocyte conditioned medium
  6. Assays for cell survival and cell morphology
  7. Immunocytochemistry
  8. Morphological analysis using immunocytochemistry
  9. Analysis of axons in culture
  10. Immunoblotting for cell signalling proteins
  11. Statistical analysis
  12. Results
  13. The nitric oxide donor DETANONOate causes cortical neuronal death in a dose-dependent manner
  14. Oligodendrocyte conditioned medium and insulin-like growth factor type-1 increases neuronal survival in neurones exposed to DETANONOate
  15. The nitric oxide donor DETANONOate causes axonal loss independent of neuronal survival, and oligodendocyte conditioned medium, insulin-like growth factor type-1 and glial cell line-derived neurotrophic factor improve axonal survival
  16. Attenuation of nitric oxide mediated axon destruction requires mitogen-activated protein kinase/extracellular signal-regulated kinase signalling
  17. P38 pathways are activated in neurones exposed to DETANONOate and both oligodendrocyte conditioned medium and insulin-like growth factor reduce levels of p38 activation
  18. Discussion
  19. Nitric oxide causes neuronal and axonal destruction
  20. Attenuation of neuronal death
  21. Attenuation of axonal destruction
  22. Relevance to inflammatory diseases of the central nervous system
  23. Acknowledgement
  24. References

MAP kinase/Erk signalling has been shown to be an important intracellular signalling pathway for the phosphorylation of neurofilaments (Veeranna et al. 1998; Li et al. 1999). Indeed, we have previously shown that under conditions of trophic deprivation, GDNF acts via Erk signalling to promote axonal survival in vitro (Wilkins et al. 2003). Therefore, investigation of this pathway was undertaken in neurones exposed to DETANONOate. Levels of p-Erk were initially low in NO-exposed neurones and reduced to undetectable levels over time (Figs 3a and b). Addition of OCM, GDNF and IGF-1 to neuronal cultures exposed to DETANONOate for 30 min led to significant increases in levels of phosphorylated Erk (Fig. 3b). Analysis of band intensity demonstrated that GDNF exposure yielded significantly greater levels of Erk phosphorylation compared to IGF-1 stimulation. Maximal Erk activation occurred early after exposure of cultures to growth factors, but trophic factor-induced Erk activation was detected at later time points (Fig. 3b). Furthermore, addition of the Erk pathway inhibitor PD98059 to neurones (cultured for 6 DIV) exposed to DETANONOate with OCM, GDNF or IGF-1 for a further 1 DIV significantly reduced SMI312 lengths per neurone (Fig. 3c), suggesting that the effect of these factors in protecting axons from NO requires signalling via MAP kinase/Erk pathways. Inhibition of MAP kinase/Erk pathways under the above conditions had no effect on whole neuronal survival (Fig. 3d).

image

Figure 3. (a) Extracellular signal-related kinase (Erk) activation in neurones exposed to DETANONOate. Mitogen-activated protein kinase (MAP kinase)/Erk 1/2 phosphorylation in neurones (cultured for 6 DIV) and exposed to 10% fetal calf serum (FCS; positive control) for 30 min or DETANONOate (0.1 mm) for times shown. Upper panel shows phospho-Erk 1/2 (p-Erk) immunoblot. Lower panel shows total-Erk 1/2 (T-Erk) immunoblot of same membrane. Representative blot of three independent experiments. (b) Trophic factors increase Erk activation within neuronal cultures exposed to DETANONOate. MAP kinase/Erk 1/2 phosphorylation in neurones (cultured for 6 DIV) and exposed to DETANONOate (0.1 mm) plus insulin-like growth factor type-1 (50 ng/mL; IGF), glial cell line-derived neurotrophic factor (10 ng/mL; GDNF) or oligodendrocyte conditioned medium (OCM) for 30 min (upper pair) or 4 h (lower pair). Upper panel shows phospho-Erk 1/2 (p-Erk) immunoblot. Lower panel shows total-Erk 1/2 (T-Erk) immunoblot of same membrane. Representative blot of five independent experiments. Bar chart shows analysis of band density of phosphorylated Erk2/total Erk2 in 30-min treated cultures, relative to no growth factor exposure (nil; n = 5, **p < 0.01). (c) PD98059 reduces axon effects of trophic factors. Effect of DETANONOate (0.1 mm; NO), DETANONOate plus GDNF (10 ng/mL; NO/GDNF), DETANONOate plus GDNF plus PD98059 (30 µm; NO/G/PD), DETANONOate plus IGF-1 (50 ng/mL; NO/IGF), DETANONOate plus IGF-1 plus PD98059 (NO/I/PD), DETANONOate plus OCM (NO/OCM) and DETANONOate plus OCM plus PD98059 (NO/OCM/PD) on SMI312 length per live neurone. Controls were cultured in 2% B27 throughout and values expressed as a percentage of this control. Neurones were cultured for 6 DIV and exposed to test condition for 24 h (n = 4; *p < 0.05; **p < 0.01). (d) PD98059 has no effect on neuronal survival. Effect of above conditions on numbers of live β-tubulin-positive cells per field. Neurones were cultured for 6 DIV and exposed to test condition for 24 h (n = 4).

Download figure to PowerPoint

P38 pathways are activated in neurones exposed to DETANONOate and both oligodendrocyte conditioned medium and insulin-like growth factor reduce levels of p38 activation

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Neuronal cell culture
  5. Preparation of oligodendrocyte conditioned medium
  6. Assays for cell survival and cell morphology
  7. Immunocytochemistry
  8. Morphological analysis using immunocytochemistry
  9. Analysis of axons in culture
  10. Immunoblotting for cell signalling proteins
  11. Statistical analysis
  12. Results
  13. The nitric oxide donor DETANONOate causes cortical neuronal death in a dose-dependent manner
  14. Oligodendrocyte conditioned medium and insulin-like growth factor type-1 increases neuronal survival in neurones exposed to DETANONOate
  15. The nitric oxide donor DETANONOate causes axonal loss independent of neuronal survival, and oligodendocyte conditioned medium, insulin-like growth factor type-1 and glial cell line-derived neurotrophic factor improve axonal survival
  16. Attenuation of nitric oxide mediated axon destruction requires mitogen-activated protein kinase/extracellular signal-regulated kinase signalling
  17. P38 pathways are activated in neurones exposed to DETANONOate and both oligodendrocyte conditioned medium and insulin-like growth factor reduce levels of p38 activation
  18. Discussion
  19. Nitric oxide causes neuronal and axonal destruction
  20. Attenuation of neuronal death
  21. Attenuation of axonal destruction
  22. Relevance to inflammatory diseases of the central nervous system
  23. Acknowledgement
  24. References

Several recent reports have highlighted the importance of p38 MAP kinase signalling in NO-mediated cell death in a variety of models (Ghatan et al. 2000; Bossy-Wetzel et al. 2004). Therefore, an assessment of p38 signalling and its importance to neurone/axon loss in the current system was undertaken. Neurones were cultured for 6 DIV before exposure to DETANONOate (0.1 mm). At specific time points, cells were lysed and immunoblotting performed for activated p38 (p-p38). p-p38 was detected at high levels after exposure to DETANONOate for 4 h, and this was maintained at 8 h (Fig. 4a). To assess the functional significance of p-38 activation, cortical neurones (1.4 × 103 cells/mm2) were maintained in B27 supplemented Dulbecco's modified Eagle's medium for 6 DIV before exposure to DETANONOate (0.1 mm) in the presence of the MAP kinase inhibitors SB203580 (20 µm), SB202190 (2 µm; both p38 pathway inhibitors), PD98059 (30 µm; Erk pathway inhibitor), or combinations of inhibitors for a further 1 DIV. p38 pathway inhibitors led to an increase in neuronal survival in NO-exposed neurones (Fig. 4b). No significant changes in neuronal survival were seen in NO-exposed neurones in the presence of PD98059. Cultures exposed to p38 inhbitors in the absence of DETANONOate showed no change in neuronal survival compared to control (data not shown). Neurones were then exposed to DETANONOate for 4 h in the presence of the trophic agents previously studied and an analysis of p38 pathway activation undertaken. Both IGF-1 and OCM, but not GDNF, led to significant decreases in p-p38 levels (Fig. 4c).

image

Figure 4. (a) DETANONOate exposure leads to activation of p38 pathways in cortical neurones over time. p38 phosphorylation in neurones (cultured for 6 DIV) and exposed to DETANONOate (0.1 mm) for times shown. Upper panel shows phospho-p38 (p-p38) immunoblot. Lower panel shows total-p38 (T-p38) immunoblot of same membrane. Representative blot of three independent experiments. (b) Inhibitors of p38 pathway improve survival of neurones exposed to DETANONOate. Effect of combinations of PD98059 (30 µm), SB203580 (20 µm) and SB202190 (2 µm) on numbers of live β-tubulin-positive cells per field. Controls were cultured in 2% B27 throughout and values expressed as a percentage of this control. Neurones were cultured for 6 DIV and exposed to DETANONOate plus inhibitors for 24 h (n = 5; *p < 0.05; ***p < 0.001). (c) Insulin-like growth factor type-1 (IGF-1) and oligodendrocyte conditioned medium (OCM) reduce activation of p38 pathway in neurones exposed to DETANONOate. p38 phosphorylation in neurones (cultured for 6 DIV) exposed to DETANONOate (0.1 mm) for 4 h plus IGF-1 (50 ng/mL), GDNF (10 ng/mL), OCM or SB203580 (20 µm). Upper panel shows phospho-p38 (p-p38) immunoblot. Lower panel shows total-p38 (T-p38) immunoblot of same membrane. Representative blot of four independent experiments.

Download figure to PowerPoint

Next, the importance of p-38 pathways to axonal length in NO-exposed neurones was assessed. Addition of SB203580 (20 µm) or SB202190 (2 µm) to NO-exposed neurones resulted in significant increases in SMI312 lengths per cell (Figs 5a and b). As expected from previous data, PD98059 had no significant axon protective effects (Figs 5a and b). These results suggest that p38 signalling may mediate both neurotoxic and axonotoxic effects of NO in the current system.

image

Figure 5. (a) Immunographs showing effect DETANONOate (0.1 mm; NO), DETANONOate plus SB203580 (20 µm; NO/SB) and DETANONOate plus PD98059 (30 µm; NO/PD) on SMI312 (phosphorylated neurofilament; green) staining in neuronal cultures (6 DIV) exposed to test conditions for 24 h. Nuclei stained with Hoechst (blue). (b) p38 inhibitors attenuate SMI312 length per cell reductions induced by DETANONOate. Effect of combinations of PD98059 (30 µm), SB203580 (20 µm) and SB202190 (2 µm) on SMI312 length per live neurone. Controls were cultured in 2% B27 throughout and values expressed as a percentage of this control. Neurones were cultured for 6 DIV and exposed to test condition for 24 h (n = 5; **p < 0.01; ***p < 0.001). (c) p38 inhibitors increase extracellular signal-regulated kinase (Erk) activation within neuronal cultures exposed to DETANONOate. Mitogen-activated protein kinase(MAP kinase)/Erk 1/2 phosphorylation in neurones (cultured for 6 DIV) and exposed to DETANONOate (0.1 mm) alone or plus SB203580 (20 µm) or SB202190 (2 µm) for 30 min. Upper panel shows phospho-Erk 1/2 (p-Erk) immunoblot. Lower panel shows total-Erk 1/2 (T-Erk) immunoblot of same membrane. Representative blot of three independent experiments.

Download figure to PowerPoint

In order further to examine the interactions of Erk and p38 pathways in terms of axon length, we measured axon length per cell in cultured neurones (6 DIV) exposed to DETANONOate (0.1 mm), p38 inhibitors and the Erk pathway inhibitor PD98059. Addition of PD98059 significantly attenuated p38 inhibitor-induced axon length per cell growth enhancement (Fig. 5b), again implying the need for Erk signalling in the protection of axons from NO-mediated damage. Furthermore, addition of the p38 inhibitors to neurones exposed to DETANONOate led to significant activation of Erk pathways (Fig. 5c), implying close interactions between these signalling pathways.

Discussion

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Neuronal cell culture
  5. Preparation of oligodendrocyte conditioned medium
  6. Assays for cell survival and cell morphology
  7. Immunocytochemistry
  8. Morphological analysis using immunocytochemistry
  9. Analysis of axons in culture
  10. Immunoblotting for cell signalling proteins
  11. Statistical analysis
  12. Results
  13. The nitric oxide donor DETANONOate causes cortical neuronal death in a dose-dependent manner
  14. Oligodendrocyte conditioned medium and insulin-like growth factor type-1 increases neuronal survival in neurones exposed to DETANONOate
  15. The nitric oxide donor DETANONOate causes axonal loss independent of neuronal survival, and oligodendocyte conditioned medium, insulin-like growth factor type-1 and glial cell line-derived neurotrophic factor improve axonal survival
  16. Attenuation of nitric oxide mediated axon destruction requires mitogen-activated protein kinase/extracellular signal-regulated kinase signalling
  17. P38 pathways are activated in neurones exposed to DETANONOate and both oligodendrocyte conditioned medium and insulin-like growth factor reduce levels of p38 activation
  18. Discussion
  19. Nitric oxide causes neuronal and axonal destruction
  20. Attenuation of neuronal death
  21. Attenuation of axonal destruction
  22. Relevance to inflammatory diseases of the central nervous system
  23. Acknowledgement
  24. References

In this study, we show that a slow-releasing NO donor causes neuronal loss and axonal destruction in vitro. Trophic factors attenuate these processes, acting through specific intracellular signalling pathways. Activation of MAP kinase/Erk pathway is important for attenuating NO-mediated axon loss but has little effect on neuronal survival. p38 MAP kinase activation occurs following exposure to NO and is important in death of neurones but may also interact with Erk pathways to influence axonal length. We show that trophic factors differentially activate these pathways with different consequential effects on neurones and axons. IGF-1 and medium conditioned by oligodendrocytes have beneficial effects on both neuronal survival and axon length in culture, and both activate Erk pathways whilst inhibiting p-38 signalling. In contrast, GDNF improves axonal length in NO-exposed cultures and strongly activates Erk signalling, but has no effect on neuronal survival and does not inhibit p-38 signalling (Fig. 6).

image

Figure 6. Proposed schema for neuronal/axonal protection mechanisms by oligodendrocyte-derived factors against acute nitric oxide-mediated damage. Erk, extracellular signal-related kinase; GDNF, glial cell line-derived neurotrophic factor; IGF, insulin-like growth factor; NO, nitric oxide.

Download figure to PowerPoint

Nitric oxide causes neuronal and axonal destruction

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Neuronal cell culture
  5. Preparation of oligodendrocyte conditioned medium
  6. Assays for cell survival and cell morphology
  7. Immunocytochemistry
  8. Morphological analysis using immunocytochemistry
  9. Analysis of axons in culture
  10. Immunoblotting for cell signalling proteins
  11. Statistical analysis
  12. Results
  13. The nitric oxide donor DETANONOate causes cortical neuronal death in a dose-dependent manner
  14. Oligodendrocyte conditioned medium and insulin-like growth factor type-1 increases neuronal survival in neurones exposed to DETANONOate
  15. The nitric oxide donor DETANONOate causes axonal loss independent of neuronal survival, and oligodendocyte conditioned medium, insulin-like growth factor type-1 and glial cell line-derived neurotrophic factor improve axonal survival
  16. Attenuation of nitric oxide mediated axon destruction requires mitogen-activated protein kinase/extracellular signal-regulated kinase signalling
  17. P38 pathways are activated in neurones exposed to DETANONOate and both oligodendrocyte conditioned medium and insulin-like growth factor reduce levels of p38 activation
  18. Discussion
  19. Nitric oxide causes neuronal and axonal destruction
  20. Attenuation of neuronal death
  21. Attenuation of axonal destruction
  22. Relevance to inflammatory diseases of the central nervous system
  23. Acknowledgement
  24. References

Although NO performs many physiological roles at low levels, it is associated with cytotoxicity and degeneration during central nervous system inflammation (Garthwaite et al. 2002). NO may have many actions during inflammation, but there is evidence from both in vitro and in vivo studies for a neurotoxic and axonotoxic role. NO can induce apoptosis in a variety of cultured peripheral and central neurones (Estevez et al. 1998; Wang et al. 2003). Mechanisms of neuronal cell death induced by NO are complex and involve the formation of reactive nitrogen species and, ultimately, inhibition of mitochondrial respiration (Brown and Borutaite 2002).

Mechanisms of NO-mediated axon destruction are less clear (Waxman 2003). NO has been shown to affect axons in experimental models, causing either transient conduction block or permanent damage dependent on methods of exposure (Redford et al. 1997; Smith and Lassmann 2002). Prolonged exposure of isolated optic nerve preparations to NO donors leads to permanent axonal damage (Garthwaite et al. 2002). The mechanism of this process to some extent appears dependent on calcium overload that follows a reduction in ATP levels in the face of continued sodium influx through voltage-gated channels. Indeed, modulation of sodium/calcium levels within axons may protect against NO-mediated axonopathy (Kapoor et al. 2003). Axonal influx of calcium due to ATP depletion would appear to be a late step in the cascade of NO signalling and little is known about signals upstream of this event.

The current study is consistent with previous in vitro and in vivo observations implying that axons are particularly sensitive to inflammatory mediators (Kornek et al. 2000). By using an index of axon length per neurone in cultures exposed to an NO donor, we found that axon length is reduced to approximately 30% of control levels, indicating that NO has more profound axonotoxic effects than its ability to induce neuronal cell death. Alternatively, axons in culture may show reduced stability and thus manifest earlier changes compared to nuclear condensation. Differential susceptibility of cellular components to toxins is a concept that has emerged from the study of neuronal populations and may be highly relevant when considering the degeneration of axons that may be topographically far removed from their cell body (Finn et al. 2000).

Attenuation of neuronal death

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Neuronal cell culture
  5. Preparation of oligodendrocyte conditioned medium
  6. Assays for cell survival and cell morphology
  7. Immunocytochemistry
  8. Morphological analysis using immunocytochemistry
  9. Analysis of axons in culture
  10. Immunoblotting for cell signalling proteins
  11. Statistical analysis
  12. Results
  13. The nitric oxide donor DETANONOate causes cortical neuronal death in a dose-dependent manner
  14. Oligodendrocyte conditioned medium and insulin-like growth factor type-1 increases neuronal survival in neurones exposed to DETANONOate
  15. The nitric oxide donor DETANONOate causes axonal loss independent of neuronal survival, and oligodendocyte conditioned medium, insulin-like growth factor type-1 and glial cell line-derived neurotrophic factor improve axonal survival
  16. Attenuation of nitric oxide mediated axon destruction requires mitogen-activated protein kinase/extracellular signal-regulated kinase signalling
  17. P38 pathways are activated in neurones exposed to DETANONOate and both oligodendrocyte conditioned medium and insulin-like growth factor reduce levels of p38 activation
  18. Discussion
  19. Nitric oxide causes neuronal and axonal destruction
  20. Attenuation of neuronal death
  21. Attenuation of axonal destruction
  22. Relevance to inflammatory diseases of the central nervous system
  23. Acknowledgement
  24. References

In this study we show that IGF-1 and OCM attenuate NO-mediated neuronal death. These ‘factors’ also attenuate neuronal death resulting from trophic deprivation, and OCM contains IGF-1 (Wilkins et al. 2001). The close relationship between oligodendrocytes and neurones suggests extensive cross-talk and raises the possibility that oligodendrocyte-derived factors may influence neuronal survival and phenotype. Indeed, oligodendrocyte-derived soluble factors induce sodium channel clustering, necessary for efficient saltatory conduction (Kaplan et al. 1997). Also, oligodendrocytes influence local accumulation and phosphorylation of neurofilaments during development, adding to axonal stability (Colello et al. 1994; Sanchez et al. 1996; Brady et al. 1999). Oligodendrocytes have been shown to produce a wide range of trophic factors in vitro, including IGF-1, NGF, BDNF and GDNF (Byravan et al. 1994; Strelau and Unsicker 1999; Wilkins et al. 2001; Wilkins et al. 2003). During inflammation, the role of oligodendrocyte-derived neuronal support is less clear, but it is logical that the local environment is key to determining neuronal survival.

Several reports have shown p38 MAP kinase activation during NO-mediated neuronal death (Lin et al. 2001; Ishikawa et al. 2003). We confirm that NO exposure leads to activation of p38 pathways in cortical neurones and show that inhibition of p38 signalling increases neuronal survival. Although p38 inhibitors also activate Erk signalling pathways, the latter has no apparent role in mediating or attenuating NO-mediated cell death. GDNF is a potent activator of Erk in the current system but does not improve neuronal survival. Furthermore, inhibition of Erk signalling does not influence neuronal survival under any condition used in this study (Creedon et al. 1996). Both OCM and IGF-1 inhibited NO-induced activation of p38 signalling, consistent with their effects on inhibition of neuronal cell death. Several in vitro studies have previously demonstrated the importance of IGF-1 on neuronal survival via a variety of mechanisms (Meyer-Franke et al. 1995; Lindholm et al. 1996; Dudek et al. 1997). Previous reports have demonstrated inhibition of p38 signalling by IGF-1 in cerebellar neurones undergoing apoptosis in response to low potassium, and in hippocampal neurones exposed to NO (Matsuzaki et al. 1999; Yamagishi et al. 2003). Together, these results point to a major role of the p38 signalling pathway in inducing neuronal death induced by NO (Ghatan et al. 2000; Ishikawa et al. 2003; Bossy-Wetzel et al. 2004).

Attenuation of axonal destruction

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Neuronal cell culture
  5. Preparation of oligodendrocyte conditioned medium
  6. Assays for cell survival and cell morphology
  7. Immunocytochemistry
  8. Morphological analysis using immunocytochemistry
  9. Analysis of axons in culture
  10. Immunoblotting for cell signalling proteins
  11. Statistical analysis
  12. Results
  13. The nitric oxide donor DETANONOate causes cortical neuronal death in a dose-dependent manner
  14. Oligodendrocyte conditioned medium and insulin-like growth factor type-1 increases neuronal survival in neurones exposed to DETANONOate
  15. The nitric oxide donor DETANONOate causes axonal loss independent of neuronal survival, and oligodendocyte conditioned medium, insulin-like growth factor type-1 and glial cell line-derived neurotrophic factor improve axonal survival
  16. Attenuation of nitric oxide mediated axon destruction requires mitogen-activated protein kinase/extracellular signal-regulated kinase signalling
  17. P38 pathways are activated in neurones exposed to DETANONOate and both oligodendrocyte conditioned medium and insulin-like growth factor reduce levels of p38 activation
  18. Discussion
  19. Nitric oxide causes neuronal and axonal destruction
  20. Attenuation of neuronal death
  21. Attenuation of axonal destruction
  22. Relevance to inflammatory diseases of the central nervous system
  23. Acknowledgement
  24. References

A defining feature of axons is the presence of highly phosphorylated neurofilaments within their cytoskeleton. Phosphorylation of neurofilaments leads to enhancement of axonal stability and an increase in axonal diameter (Carden et al. 1987; Sanchez et al. 1996). Dephosphorylation of neurofilaments within axons occurs in pathological states and leads to destabilization of the cytoskeleton and subsequent axon degeneration (Trapp et al. 1998). Therefore, mechanisms involved in maintenance of neurofilament phosphorylation are likely to be highly relevant to the treatment of axonopathies.

In the current study we used an index of phosphorylated neurofilament length per live neurone in order to study axon specific processes independent of whole neuronal survival. Although selective changes in neurofilament phosphorylation may not fully correlate with axonal length, SMI312 provides useful information concerning axonal stability. To varying degrees, IGF, GDNF and OCM attenuated the severe in vitro NO-mediated axon destruction. GDNF exerted a greater effect than IGF-1, and the improvement in axon lengths was inhibited by Erk inhibition. GDNF activated higher levels of the Erk pathway, suggesting a correlation with axon survival. Mechanisms by which neurofilaments become phosphorylated are complex, but a role for Erk signalling has previously been established (Veeranna et al. 1998; Li et al. 1999). Whether Erk signalling promotes general augmentation of neurite integrity or is specific to the axon is an important question and requires further study.

GDNF was originally identified as a potent survival factor for midbrain dopaminergic neurones but appears to have a role in trophic support of a wide variety of neuronal populations (Lin et al. 1993). GDNF may also be an important neuroprotective agent acting against a variety of pathological insults, including glutamate excitotoxicity (Nicole et al. 2001). Here, we argue that GDNF has specific axonal effects in keeping with trophism for cortical neurones (Wilkins et al. 2003). Indeed, delivery of GDNF to areas of axonal pathology leads to reduction in axonal degeneration and promotes axonal regeneration in a number of in vivo models (Wang et al. 2002; Cao et al. 2004).

p38 pathway inhibitors not only improve survival of neurones exposed to NO, but also attenuate NO-mediated axon loss within the cultures. This process was again dependent on Erk signalling. We therefore investigated the effect of p38 inhibition on Erk pathway activation and found that SB203580 and SB202190 exposure leads to greatly increased levels of p-Erk. This is consistent with a recent report that p38 activation in fibroblasts leads to dephosphorylation of Erk pathway components and, conversely, p38 inhibition leads to phosphorylation of Erk pathway components (Li et al. 2003). Inhibition of p38 with Erk pathway activation is a novel finding in neurones and links the two important MAP kinase signalling pathways. In addition, this finding is an important consideration when interpreting effects of p38 pathway inhibitors.

The above data therefore suggest a central role for Erk activation in protection of axons from NO. Furthermore, Erk pathway activation in this context can occur independently of p38 pathways (in the case of GDNF) or secondary to p38 inhibition (in the case of IGF-1).

Relevance to inflammatory diseases of the central nervous system

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Neuronal cell culture
  5. Preparation of oligodendrocyte conditioned medium
  6. Assays for cell survival and cell morphology
  7. Immunocytochemistry
  8. Morphological analysis using immunocytochemistry
  9. Analysis of axons in culture
  10. Immunoblotting for cell signalling proteins
  11. Statistical analysis
  12. Results
  13. The nitric oxide donor DETANONOate causes cortical neuronal death in a dose-dependent manner
  14. Oligodendrocyte conditioned medium and insulin-like growth factor type-1 increases neuronal survival in neurones exposed to DETANONOate
  15. The nitric oxide donor DETANONOate causes axonal loss independent of neuronal survival, and oligodendocyte conditioned medium, insulin-like growth factor type-1 and glial cell line-derived neurotrophic factor improve axonal survival
  16. Attenuation of nitric oxide mediated axon destruction requires mitogen-activated protein kinase/extracellular signal-regulated kinase signalling
  17. P38 pathways are activated in neurones exposed to DETANONOate and both oligodendrocyte conditioned medium and insulin-like growth factor reduce levels of p38 activation
  18. Discussion
  19. Nitric oxide causes neuronal and axonal destruction
  20. Attenuation of neuronal death
  21. Attenuation of axonal destruction
  22. Relevance to inflammatory diseases of the central nervous system
  23. Acknowledgement
  24. References

Inflammatory diseases of the central nervous system, such as multiple sclerosis, cause profound disability and are associated with significant axon pathology (Davie et al. 1995; Trapp et al. 1998). Mechanisms of axonopathy are not fully understood but inflammatory injury may be responsible for a large percentage of acute and chronic axonopathy (Kornek and Lassmann 2003). Several end-stage mechanisms for axon degeneration have been proposed including terminal energy depletion and influx of calcium ions (Waxman 2003). Following axonal destruction, regrowth and correct re-innervation of axons is usually incomplete. Protection against the effects of inflammatory mediators is therefore vital to prevent axon destruction.

We present here further mechanisms for NO-mediated neurone/axon degeneration and identify several factors that modulate the process. Further studies are warranted to extend these observations to more mature neurones and those derived from a variety of brain regions. In an age of burgeoning therapies for central nervous system disorders, trophic factor modulation of axonopathy in the context of inflammatory injury is an intriguing prospect.

References

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Neuronal cell culture
  5. Preparation of oligodendrocyte conditioned medium
  6. Assays for cell survival and cell morphology
  7. Immunocytochemistry
  8. Morphological analysis using immunocytochemistry
  9. Analysis of axons in culture
  10. Immunoblotting for cell signalling proteins
  11. Statistical analysis
  12. Results
  13. The nitric oxide donor DETANONOate causes cortical neuronal death in a dose-dependent manner
  14. Oligodendrocyte conditioned medium and insulin-like growth factor type-1 increases neuronal survival in neurones exposed to DETANONOate
  15. The nitric oxide donor DETANONOate causes axonal loss independent of neuronal survival, and oligodendocyte conditioned medium, insulin-like growth factor type-1 and glial cell line-derived neurotrophic factor improve axonal survival
  16. Attenuation of nitric oxide mediated axon destruction requires mitogen-activated protein kinase/extracellular signal-regulated kinase signalling
  17. P38 pathways are activated in neurones exposed to DETANONOate and both oligodendrocyte conditioned medium and insulin-like growth factor reduce levels of p38 activation
  18. Discussion
  19. Nitric oxide causes neuronal and axonal destruction
  20. Attenuation of neuronal death
  21. Attenuation of axonal destruction
  22. Relevance to inflammatory diseases of the central nervous system
  23. Acknowledgement
  24. References