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The expression of spinal methyl-CpG-binding protein 2, DNA methyltransferases and histone deacetylases is modulated in persistent pain states.

Tochiki KK, Cunningham J, Hunt SP, Géranton SM - Mol Pain (2012)

Bottom Line: Recently, we have found that MeCP2 activity had a crucial role in the pattern of gene expression seen in the superficial dorsal horn rapidly after injection of Complete Freund's Adjuvant (CFA) in the rat ankle joint.However, there were no significant changes in the expression of the MeCP2 targets that we had previously shown are regulated in the early time points following CFA injection in the ankle joint.Our results strongly suggest that changes in chromatin compaction, regulated by the binding of MeCP2 complexes to methylated DNA, are involved in the modulation of gene expression in the superficial dorsal horn and dorsal root ganglia during the maintenance of persistent pain states.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Cell and Developmental Biology, University College London, London WC1E 6BT, UK.

ABSTRACT

Background: DNA CpG methylation is carried out by DNA methyltransferases and induces chromatin remodeling and gene silencing through a transcription repressor complex comprising the methyl-CpG-binding protein 2 (MeCP2) and a subset of histone deacetylases. Recently, we have found that MeCP2 activity had a crucial role in the pattern of gene expression seen in the superficial dorsal horn rapidly after injection of Complete Freund's Adjuvant (CFA) in the rat ankle joint. The aim of the present study was to analyse the changes in expression of MeCP2, DNA methyltransferases and a subset of histone deacetylases in the superficial dorsal horn during the maintenance phase of persistent pain states. In this process, the cell specific expression of MeCP2 was also investigated.

Results: Using immunohistochemistry, we found that neurones, oligodendrocytes and astrocytes expressed MeCP2. Microglia, oligodendrocyte precursor cells and Schwann cells never showed any positive stain for MeCP2. Quantitative analyses showed that MeCP2 expression was increased in the superficial dorsal horn 7 days following CFA injection in the ankle joint but decreased 7 days following spared nerve injury. Overall, the expression of DNA methyltransferases and a subset of histone deacetylases followed the same pattern of expression. However, there were no significant changes in the expression of the MeCP2 targets that we had previously shown are regulated in the early time points following CFA injection in the ankle joint. Finally, the expression of MeCP2 was also down regulated in damaged dorsal root ganglion neurones following spared nerve injury.

Conclusion: Our results strongly suggest that changes in chromatin compaction, regulated by the binding of MeCP2 complexes to methylated DNA, are involved in the modulation of gene expression in the superficial dorsal horn and dorsal root ganglia during the maintenance of persistent pain states.

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MeCP2 expression is reduced within damaged DRG neurons following SNI surgery. A1 to A3, Images of DRG sections 7 days following SNI surgery. Colocalization of MeCP2 (green; Millipore antibody) and ATF3 (red) in DRG neurones. Arrow indicates neurones expressing both MeCP2 and ATF3. Scale bar, 20 μm. A4, Measure of MeCP2 expression in ATF3 positive neurons using confocal microscopy. MeCP2 expression was quantified by measuring staining intensity and normalized to MeCP2 expression in ATF3 negative cells within each sub-group (small and large fibers; cell body diameter < 25 μm and > 25 μm). B, Confocal images of rat dorsal root ganglion sections. Expression of MeCP2 (green; Millipore antibody), S100 (red) and DAPI, a nuclear marker (blue). Pictures show single focal plane. Scale bars, 50 μm.
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Figure 4: MeCP2 expression is reduced within damaged DRG neurons following SNI surgery. A1 to A3, Images of DRG sections 7 days following SNI surgery. Colocalization of MeCP2 (green; Millipore antibody) and ATF3 (red) in DRG neurones. Arrow indicates neurones expressing both MeCP2 and ATF3. Scale bar, 20 μm. A4, Measure of MeCP2 expression in ATF3 positive neurons using confocal microscopy. MeCP2 expression was quantified by measuring staining intensity and normalized to MeCP2 expression in ATF3 negative cells within each sub-group (small and large fibers; cell body diameter < 25 μm and > 25 μm). B, Confocal images of rat dorsal root ganglion sections. Expression of MeCP2 (green; Millipore antibody), S100 (red) and DAPI, a nuclear marker (blue). Pictures show single focal plane. Scale bars, 50 μm.

Mentions: Since peripheral nerve damage (SNI) was shown to reduce MeCP2 expression in the dorsal horn, we wanted to know what the effect of nerve transection would be on MeCP2 neuronal levels in DRGs. Our hypothesis was that MeCP2 would be decreased in injured neurones allowing for up-regulation of gene expression required for regeneration. MeCP2 expression was analysed in DRG sections stained for ATF3, a marker for damaged neurones, 7 days following SNI surgery (Figure 1, 2, 3). We measured MeCP2 expression in 4 to 5 DRG sections per animal, 1 to 9 cells per section in 4 animals. We found that MeCP2 expression in both small and large fibers was decreased in ATF3 positive neurones when compared with ATF3 negative neurones (100 ± 10.1 vs 79.4 ± 8.5 in small fibers, P < 0.05, and 100 ± 8.4 vs 87.6 ± 7.4 in large fibers, P < 0.01; Figure 4A4). Finally, since MeCP2 was expressed in subsets of glial cells in the superficial dorsal horn, we analysed MeCP2 expression in Schwann cells and found that Schwann cells (S100 positive cells) did not express MeCP2 (Figure 4B).


The expression of spinal methyl-CpG-binding protein 2, DNA methyltransferases and histone deacetylases is modulated in persistent pain states.

Tochiki KK, Cunningham J, Hunt SP, Géranton SM - Mol Pain (2012)

MeCP2 expression is reduced within damaged DRG neurons following SNI surgery. A1 to A3, Images of DRG sections 7 days following SNI surgery. Colocalization of MeCP2 (green; Millipore antibody) and ATF3 (red) in DRG neurones. Arrow indicates neurones expressing both MeCP2 and ATF3. Scale bar, 20 μm. A4, Measure of MeCP2 expression in ATF3 positive neurons using confocal microscopy. MeCP2 expression was quantified by measuring staining intensity and normalized to MeCP2 expression in ATF3 negative cells within each sub-group (small and large fibers; cell body diameter < 25 μm and > 25 μm). B, Confocal images of rat dorsal root ganglion sections. Expression of MeCP2 (green; Millipore antibody), S100 (red) and DAPI, a nuclear marker (blue). Pictures show single focal plane. Scale bars, 50 μm.
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Figure 4: MeCP2 expression is reduced within damaged DRG neurons following SNI surgery. A1 to A3, Images of DRG sections 7 days following SNI surgery. Colocalization of MeCP2 (green; Millipore antibody) and ATF3 (red) in DRG neurones. Arrow indicates neurones expressing both MeCP2 and ATF3. Scale bar, 20 μm. A4, Measure of MeCP2 expression in ATF3 positive neurons using confocal microscopy. MeCP2 expression was quantified by measuring staining intensity and normalized to MeCP2 expression in ATF3 negative cells within each sub-group (small and large fibers; cell body diameter < 25 μm and > 25 μm). B, Confocal images of rat dorsal root ganglion sections. Expression of MeCP2 (green; Millipore antibody), S100 (red) and DAPI, a nuclear marker (blue). Pictures show single focal plane. Scale bars, 50 μm.
Mentions: Since peripheral nerve damage (SNI) was shown to reduce MeCP2 expression in the dorsal horn, we wanted to know what the effect of nerve transection would be on MeCP2 neuronal levels in DRGs. Our hypothesis was that MeCP2 would be decreased in injured neurones allowing for up-regulation of gene expression required for regeneration. MeCP2 expression was analysed in DRG sections stained for ATF3, a marker for damaged neurones, 7 days following SNI surgery (Figure 1, 2, 3). We measured MeCP2 expression in 4 to 5 DRG sections per animal, 1 to 9 cells per section in 4 animals. We found that MeCP2 expression in both small and large fibers was decreased in ATF3 positive neurones when compared with ATF3 negative neurones (100 ± 10.1 vs 79.4 ± 8.5 in small fibers, P < 0.05, and 100 ± 8.4 vs 87.6 ± 7.4 in large fibers, P < 0.01; Figure 4A4). Finally, since MeCP2 was expressed in subsets of glial cells in the superficial dorsal horn, we analysed MeCP2 expression in Schwann cells and found that Schwann cells (S100 positive cells) did not express MeCP2 (Figure 4B).

Bottom Line: Recently, we have found that MeCP2 activity had a crucial role in the pattern of gene expression seen in the superficial dorsal horn rapidly after injection of Complete Freund's Adjuvant (CFA) in the rat ankle joint.However, there were no significant changes in the expression of the MeCP2 targets that we had previously shown are regulated in the early time points following CFA injection in the ankle joint.Our results strongly suggest that changes in chromatin compaction, regulated by the binding of MeCP2 complexes to methylated DNA, are involved in the modulation of gene expression in the superficial dorsal horn and dorsal root ganglia during the maintenance of persistent pain states.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Cell and Developmental Biology, University College London, London WC1E 6BT, UK.

ABSTRACT

Background: DNA CpG methylation is carried out by DNA methyltransferases and induces chromatin remodeling and gene silencing through a transcription repressor complex comprising the methyl-CpG-binding protein 2 (MeCP2) and a subset of histone deacetylases. Recently, we have found that MeCP2 activity had a crucial role in the pattern of gene expression seen in the superficial dorsal horn rapidly after injection of Complete Freund's Adjuvant (CFA) in the rat ankle joint. The aim of the present study was to analyse the changes in expression of MeCP2, DNA methyltransferases and a subset of histone deacetylases in the superficial dorsal horn during the maintenance phase of persistent pain states. In this process, the cell specific expression of MeCP2 was also investigated.

Results: Using immunohistochemistry, we found that neurones, oligodendrocytes and astrocytes expressed MeCP2. Microglia, oligodendrocyte precursor cells and Schwann cells never showed any positive stain for MeCP2. Quantitative analyses showed that MeCP2 expression was increased in the superficial dorsal horn 7 days following CFA injection in the ankle joint but decreased 7 days following spared nerve injury. Overall, the expression of DNA methyltransferases and a subset of histone deacetylases followed the same pattern of expression. However, there were no significant changes in the expression of the MeCP2 targets that we had previously shown are regulated in the early time points following CFA injection in the ankle joint. Finally, the expression of MeCP2 was also down regulated in damaged dorsal root ganglion neurones following spared nerve injury.

Conclusion: Our results strongly suggest that changes in chromatin compaction, regulated by the binding of MeCP2 complexes to methylated DNA, are involved in the modulation of gene expression in the superficial dorsal horn and dorsal root ganglia during the maintenance of persistent pain states.

Show MeSH
Related in: MedlinePlus