DNA methyltransferase DNMT3a contributes to neuropathic pain by repressing Kcna2 in primary afferent neurons

Abstract

Nerve injury induces changes in gene transcription in dorsal root ganglion (DRG) neurons, which may contribute to nerve injury-induced neuropathic pain. DNA methylation represses gene expression. Here, we report that peripheral nerve injury increases expression of the DNA methyltransferase DNMT3a in the injured DRG neurons via the activation of the transcription factor octamer transcription factor 1. Blocking this increase prevents nerve injury-induced methylation of the voltage-dependent potassium (Kv) channel subunit Kcna2 promoter region and rescues Kcna2 expression in the injured DRG and attenuates neuropathic pain. Conversely, in the absence of nerve injury, mimicking this increase reduces the Kcna2 promoter activity, diminishes Kcna2 expression, decreases Kv current, increases excitability in DRG neurons and leads to spinal cord central sensitization and neuropathic pain symptoms. These findings suggest that DNMT3a may contribute to neuropathic pain by repressing Kcna2 expression in the DRG.

Figure 1. Distribution of DNMT3a protein in lumbar dorsal root ganglia of naive rats.

(a,b) DNMT3a is co-expressed exclusively with NeuN in cellular nuclei (a) and undetected in cellular nuclei (labelled by 4′, 6-diamidino-2-phenylindole (DAPI)) of glutamine synthetase (GS)-labelled cells (b). (c) Distribution of DNMT3a-positive somata: large, 30.2%; medium, 48.2%; small, 21.6%. (d–f) DNMT3a-positive neurons were labelled by calcitonin gene-related peptide (CGRP; d), isolectin B4 (IB4; e) or neurofilament-200 (NF200; f). n=5 rats. Scale bars, 50 μm.

Figure 2. Nerve injury-induced increases in Dnmt3a mRNA and DNMT3a protein levels in the injured DRG.

(a) DNMT3a and DNMT3b protein expression in the ipsilateral L5 DRG after SNL or sham surgery. n=6 rats/time point. One-way ANOVA followed by post hoc Tukey test, F_time (3, 15)=8.09 for DNMT3a and F_time (3, 15)=0.07 for DNMT3b. *P<0.05 versus the corresponding control group (0 day). Full-length blots are presented in Supplementary Fig. 6. (b,c) Neurons labelled by DNMT3a and NeuN in the ipsilateral L5 DRG on days 3 (c) and 7 (b,c) after SNL or sham surgery. n=5 rats/time point/group. **P<0.01 versus the corresponding sham group by two-tailed unpaired Student's t-test. Scale bar: 50 μm. (d) Dnmt3a mRNA expression in the ipsilateral L5 DRG on days 0, 3, and 7 after SNL or sham surgery. n=6 rats/time point/group. Two-way ANOVA followed by post hoc Tukey test, F_time (2, 48)=7.4. *P<0.05 versus the corresponding control group (0 day), (e,f) DNMT3a and DNMT3b proteins (e) and their mRNAs (f) in the ipsilateral L4 and L5 DRG on day 7 after CCI or sham surgery. n=6 rats/group. *P<0.05 or **P<0.01 versus the corresponding sham group by two-tailed unpaired Student's t-test. Full-length blots are presented in Supplementary Fig. 6.

Figure 3. OCT1-triggered Dnmt3a gene transcription following peripheral nerve injury in rat injured DRG.

(a) The Dnmt3a promotor fragment immunoprecipitated by rabbit anti-OCT1 antibody in the ipsilateral L5 DRG on day 7 post-SNL or sham surgery. Input, total purified fragments. M, ladder marker. n=3 repeats. *P<0.05 versus the sham group by two-tailed unpaired Student's t-test. (b) OCT1 expression in the ipsilateral L5 DRG after SNL or sham surgery. n=6 rats/time point/group. Two-way ANOVA followed by post hoc Tukey test, F_time (2, 17)=33.0. **P<0.01 versus the corresponding control group (0 day). Full-length blots are presented in Supplementary Fig. 6. (c) Dnmt3a gene promotor activity in HEK-293 T cells transfected with the vectors and/or siRNAs as shown. Ctl: control empty pGL3-Basic. n=3 repeats per treatment. One-way ANOVA followed by post hoc Tukey test, F_group (7, 23)=4,501.1. **P<0.01 versus the pGL3-Dnmt3a vector (Dnmt3a) alone. (d,e) Levels of OCT1 and DNMT3a proteins in PC12 cells transfected with the vectors as shown. Ctl: control pHpa-trs-GFP. Oct1: pHpa-trs-Oct1. siRNA: Oct1 siRNA. NC: negative control siRNA. N=5 repeats per treatment. One-way ANOVA followed by post hoc Tukey test, F_group (5, 17)=13.8 for OCT1 and F_group (5, 17)=20.9 for DNMT3a. **P<0.01 versus control GFP-treated group. ^#P<0.05, ^##P<0.01 versus the Oct1-treated group. Full-length blots are presented in Supplementary Fig. 6. (f) Levels of Oct1 mRNA and Dnmt3a mRNA in rat lumbar DRG cultured neurons transduced with AAV5-GFP (GFP) or AAV5-Oct1 (Oct1). n=3 repeats per treatment. One-way ANOVA followed by post hoc Tukey test, F_group (2, 8)=14.7 for Oct1 mRNA and F_group (2, 8)=10.2 for Dnmt3a mRNA. *P<0.05 or **P<0.01 versus the corresponding naive condition. (g) Co-expression of Oct1 mRNA with Dnmt3a mRNA in individual small, medium, and large neurons from the rat lumbar DRG. n=3 repeats.

Figure 4. Blocking DRG DNMT3a increase attenuates neuropathic pain development in rats.

Scram: AAV5-scrambled Dnmt3a shRNA. shRNA: AAV5-Dnmt3a shRNA. (a) Levels of Dnmt3a and Dnmt3b mRNAs in the ipsilateral and contralateral L5 DRG on day 7 post-SNL or sham surgery from the treated groups as shown. n=6 rats per group. One-way ANOVA followed by post hoc Tukey test, F_group (3, 15)=7.25 for Dnmt3a mRNA and F_group (3, 15)=0.29 for Dnmt3b mRNA. **P<0.01 versus the corresponding scram plus sham group. (b) Levels of DNMT3a and DNMT3b proteins in the ipsilateral L5 DRG on day 7 post-SNL or sham surgery from the treated groups as shown. n=6 rats per group. One-way ANOVA followed by post hoc Tukey test, F_group (3, 11)=46.6 for DNMT3a protein and F_group (3, 11)=0.54 for DNMT3b protein. **P<0.01 versus the corresponding scram plus sham group. ^##P<0.01 versus the corresponding scram plus SNL group. Full-length blots are presented in Supplementary Fig. 6. (c–e) The effect of microinjection of AAV5-Dnmt3a shRNA, AAV5-scrambled Dnmt3a shRNA, or PBS into the ipsilateral L5 DRG on paw withdrawal responses to mechanical (c), thermal (d) and cold (e) stimuli on the ipsilateral side at days shown before or after SNL or sham surgery in rats. n=5 rats per group. Two-way ANOVA followed by post hoc Tukey test, F_group (3, 124)=52.2 for (c), F_group (3, 124)=81.3 for (d) and F_group (3, 124)=611.1 for (e). *P<0.05 or **P<0.01 versus the corresponding PBS plus SNL group. (f) The effect of microinjection of AAV5-Dnmt3a shRNA or AAV5-scrambled Dnmt3a shRNA into the ipsilateral L5 DRG on the duration of time spent in saline- or lidocaine-paired chambers on day 7 post-SNL or sham surgery. Difference scores=post-conditioning time (post)—pre-conditioning time (pre) spent in the lidocaine-paired chamber. n=5 rats/group. One-way ANOVA followed by post hoc Tukey test, F_group (3, 19)=6.3. *P<0.01 versus the scram plus sham group. ^#P<0.01 versus the scram plus SNL group.

Figure 5. DRG DNMT3a knockdown attenuates neuropathic pain development in mice.

Cre: AAV5-Cre. GFP: AAV5-GFP. (a) Levels of Dnmt3a and Dnmt3b mRNAs in the ipsilateral L4 DRG on day 7 post-SNL or sham surgery from the Dnmt3a^fl/fl mice injected with PBS, AAV5-Cre or AAV5-GFP. n=12 mice per group. One-way ANOVA followed by post hoc Tukey test, F_group (4, 16)=5.45 for Dnmt3a mRNA and F_group (4, 16)=0.15 for Dnmt3b mRNA. **P<0.01 versus the corresponding GFP plus sham group. ^##P<0.01 versus the corresponding PBS plus SNL group. (b) Levels of DNMT3a and DNMT3b proteins in the ipsilateral L4 DRG on day 7 post-SNL or sham surgery from the Dnmt3a^fl/fl mice injected with AAV5-Cre or AAV5-GFP. n=12 mice per group. One-way ANOVA followed by post hoc Tukey test, F_group (2, 8)=10.5 for DNMT3a protein and F_group (2, 8)=0.05 for DNMT3b protein. *P<0.05 versus the corresponding GFP plus sham group. #P<0.05 versus the corresponding GFP plus SNL group. Full-length blots are presented in Supplementary Fig. 6. (c–e) The effect of microinjection of AAV5-Cre or AAV5-GFP into the ipsilateral L4 DRG of Dnmt3a^fl/fl mice on paw withdrawal responses to mechanical (c), thermal (d) and cold (e) stimuli on the ipsilateral side at days shown before or after SNL or sham surgery. n=8 mice per group. Two-way ANOVA followed by post hoc Tukey test, F_group (3, 129)=238.9 for (c), F_group (3, 129)=357.9 for (d) and F_group (3, 129)=367.9 for (e). *P<0.05 versus the corresponding GFP plus SNL group.

Figure 6. DRG Dnmt3a overexpression produces neuropathic pain symptoms in both rats and mice.

GFP: AAV5-GFP in a–e or HSV-GFP in f–j. DNMT3a: AAV5-Dnmt3a in a–e or HSV-Dnmt3a in f–j. (a) Amounts of Dnmt3a, Dnmt1, and Dnmt3b mRNAs in the ipsilateral and contralateral L4/5 DRG 8 weeks after viral microinjection into the ipsilateral L4/5 DRG in rats. n=3 rats per group. **P<0.01 versus the corresponding GFP group by two-tailed unpaired Student's t-test. (b) Levels of DNMT3a and DNMT1 proteins in the ipsilateral L4/5 DRG in naive rats and 8 weeks after viral microinjection into the ipsilateral L4/5 DRG in rats. n=3 rats/group. One-way ANOVA followed by post hoc Tukey test, F_group (2, 8)=40.7 for DNMT3a and F_group (2, 8)=1.21 for DNMT1. **P<0.01 versus naive rats. Full-length blots are presented in Supplementary Fig. 6. (c–e) Paw withdrawal responses to mechanical (c), thermal (d) and cold (e) stimuli on the ipsilateral (ipsi) and contralateral (contral) sides at time points as shown after viral microinjection into the ipsilateral L4/5 DRG in rats. n=10 rats per group. Two-way ANOVA followed by post hoc Tukey test, F_group (3, 119)=206.9 for (c), F_group (3, 119)=69.1 for (d), and F_group (1, 59)=178.0 for (e). **P<0.01 versus the corresponding GFP group on the ipsilateral side. (f–i) Ipsilateral paw withdrawal responses to mechanical (f,g), thermal (h) and cold (i) stimuli at time points as shown after viral microinjection into the ipsilateral L3/4 DRG in mice. n=10 mice per group. Two-way ANOVA followed by post hoc Tukey test, F_group (1, 23)=88.2 for (f), F_group (1, 23)=28.4 for (g), F_group (1, 23)=25.2 for (h) and F_group (1, 23)=37.8 for (i). *P<0.05 or **P<0.01 versus the corresponding GFP group. (j) Levels of DNMT3a, p-ERK1/2, ERK1/2 and GFAP in the ipsilateral L3/4 dorsal horn 6 days after viral microinjection into the ipsilateral L3/4 DRG in mice. n=12 mice per group. *P<0.05 versus the corresponding HSV-GFP group by two-tailed unpaired Student's t-test. Full-length blots are presented in Supplementary Fig. 6.

Figure 7. Blocking DRG DNMT3a increase rescues Kcna2 expression in the injured DRG of rats or mice post-SNL.

(a,b) Levels of Kcna1, Kcna2, and Kcna4 mRNAs in the ipsilateral (Ipsi) or contralateral (Contral) L5 DRG (a) and levels of Kcna1 and Kcna2 proteins in the ipsilateral L5 DRG (b) from the AAV5-Dnmt3a shRNA (shRNA)- or AAV5-Dnmt3a scrambled shRNA (scram)-injected rats on day 7 post-SNL or sham surgery. n=6 rats/group. One-way ANOVA followed by post hoc Tukey test, F_group (3, 15)=18.9 for Kcna1 mRNA, F_group (3, 15)=128.6 for Kcna2 mRNA, F_group (3, 15)=38.9 for Kcna4 mRNA, F_group (2, 8)=0.02 for Kcna1 protein, and F_group (2, 8)=12.2 for Kcna2 protein. *P<0.05 or **P<0.01 versus the corresponding sham plus scram group. ^#P<0.05 or ^##P<0.01 versus the corresponding SNL plus scram group. Full-length blots are presented in Supplementary Fig. 6. (c,d) Levels of Kcna1, Kcna2 and Kcna4 mRNAs in the ipsilateral (Ipsi) or contralateral (Contral) L4 DRG (c) and levels of Kcna2 and Kcna4 proteins in the ipsilateral L4 DRG (d) from the AAV5-Cre (Cre)- or AAV5-GFP (GFP)-injected Dnmt3a^fl/fl mice on day 7 post-SNL or sham surgery. n=12 mice/group. One-way ANOVA followed by post hoc Tukey test, F_group (4, 16)=9.68 for Kcna1 mRNA, F_group (4, 16)=36.9 for Kcna2 mRNA, F_group (4, 16)=6.40 for Kcna4 mRNA, F_group (2, 8)=13.4 for Kcna2 protein, and F_group (2, 8)=0.10 for Kcna4 protein. *P<0.05 or **P<0.01 versus the corresponding sham plus GFP group. ^##P<0.01 versus the corresponding SNL plus GFP group. Full-length blots are presented in Supplementary Fig. 6.

Figure 8. DRG DNMT3a overexpression represses Kcna2 expression.

(a,b) Levels of Kcna1, Kcna2, and Kcna4 mRNAs on the ipsilateral (Ipsi) or contralateral (Contral) sides (a) and levels of Kcna2 and Kcna4 proteins on the ipsilateral side (b) 8 weeks after microinjection of AAV5-Dnmt3a (DNMT3a) or AAV5-GFP (GFP) into the unilateral L4/5 DRG. n=6 rats/group. *P<0.05 or **P<0.01 versus the corresponding GFP group by two-tailed unpaired Student's t-test. Full-length blots are presented in Supplementary Fig. 6. (c) Co-localization of DNMT3a with Kcna2 in rat L5 DRG neurons. Scale bar, 40 μm. (d) Amounts of Dnmt3a, Dnmt1, Dnmt3b, Kcna1, Kcna2 and Kcna4 mRNAs in Dnmt3a^fl/fl mouse DRG cultured neurons transduced with PBS or virus as indicated. Inset: GFP-labelled neurons. n=3 repeats per group. One-way ANOVA (relative level versus. group) followed by post hoc Tukey test, F_group (5, 17)=136.9 for Dnmt3a, F_group (5, 17)=0.85 for Dnmt1, F_group (5, 17)=0.67 for Dnmt3b, F_group (5, 17)=0.56 for Kcna1, F_group (5, 17)=38.1 for Kcna2, and F_group (5, 17)=1.52 for Kcna4. **P<0.01 versus the corresponding PBS group. ^##P<0.01 versus the corresponding AAV5-Cre-treated group.

Figure 9. DNMT3a is required for the nerve injury-induced increase in Kcna2 DNA methylation in rat injured DRG.

(a) Two regions (R1, −663/−389; R2, −491/−199), but not other regions (R3, −237/+42; R4, +20/+278; R5, +185/+464; R6: +411/+649; R7: +680/+928), from the Kcna2 gene were immunoprecipitated by rabbit anti-DNMT3a (not by rabbit normal serum) in rat lumbar DRG. Input, total purified fragments. M, ladder marker. n=3 repeats. (b) DNMT3a binding to R1 and R2 regions within the Dnmt3a gene in the ipsilateral L5 DRG of rats on day 7 post-SNL or sham surgery. Left: the binding of Dnmt3a to the R1 region of Kcna2 gene. Right: the quantitative analysis of the binding. n=3 repeats (3 rats/repeat)/group. **P<0.01 versus the corresponding sham group by two-tailed unpaired Student's t-test. (c,d) The increases in the levels of DNA methylation at −457, −444, −440, and −374 CpG sites by bisulfite clone-sequencing assay (c) and at −482, −457, and −444 CpG sites by bisulfite pyro-sequencing assay (d) from −540 to 500 bp sites of Kcna2 gene consisted of 65 CpG sites in the ipsilateral L5 DRG of rats on day 7 post-SNL. n=3 repeats (6 rats/repeat)/group. **P<0.01 versus the corresponding sham group by two-tailed unpaired Student's t-test. (e) The percentages of methylation at −457 and −444 CpG sites of the Kcna2 gene in the ipsilateral L5 DRG on day 7 post-SNL or sham surgery from five groups as indicated. GFP: AAV5-GFP. scram: AAV5-Dnmt3a scrambled shRNA. shRNA: AAV5-Dnmt3a shRNA. Left: Representation of a single cloned allele per group at the -457 CpG site. Right: Statistical analysis at −457 and −444 CpG sites. n=3 repeats (3 rats/repeat)/group. One-way ANOVA (methylation versus. group) followed by post hoc Tukey test, F_group (3, 19)=15.8 for −457 site and F_group (3, 19)=6.4 for −440 site. *P<0.05 versus the corresponding PBS plus sham group. ^#P<0.05 versus the corresponding GFP plus SNL group.

Figure 10. Dnmt3a overexpression reduces total Kv current and increases the excitability in the injected DRG neurons 5–8 weeks after microinjection of AAV5-GFP (Control) or AAV5-Dnmt3a (DNMT3a) into the unilateral L4 and L5 DRG of rats.

(a) Representative traces of total Kv currents before or after bath perfusion of 100 nM maurotoxin (MTX) in the large DRG neurons. (b) I–V curves before or after 100 nM MTX treatment in the large DRG neurons from control (n=17 neurons from 7 rats) and DNMT3a-treated (n=25 neurons from 9 rats) groups. One-way ANOVA (current density versus. group) followed by post hoc Tukey test, F_group (1, 13)=145.1. *P<0.05, **P<0.01 versus the DNMT3a-treated group. (c) At +50 mV, reduction in total Kv current in the large DRG neurons after MTX treatment was greater in the control group than in the DNMT3a-treated group. *P<0.05 versus control group by two-tailed unpaired Student's t-test. (d,e) Resting membrane potential (d) and current threshold for pulses (I_threshold, e). n=25 large, 25 medium and 16 small neurons from control group (8 rats). n=22 large, 24 medium and 16 small neurons from the DNMT3a-treated group (10 rats). *P<0.05, **P<0.01 versus the corresponding control group by two-tailed unpaired Student's t-test. (f) Representative traces of evoked action potentials in DRG neurons. (g–i) Numbers of evoked action potentials from control and DNMT3a-treated groups after application of different currents as indicated. Numbers of the recorded cells are the same as in d. Two-way ANOVA (effect versus. group × stimulation interaction) followed by post hoc Tukey test, F_group (1, 13)=98.3 for large, F_group (1, 13)=91.4 for medium and F_group (1, 13)=134.9 for small. *P<0.05, **P<0.01 versus the same stimulation intensity in the control group.