Expression of DNA methyltransferases in adult dorsal root ganglia is cell-type specific and up regulated in a rodent model of neuropathic pain

Abstract

Neuropathic pain is associated with hyperexcitability and intrinsic firing of dorsal root ganglia (DRG) neurons. These phenotypical changes can be long lasting, potentially spanning the entire life of animal models, and depend on altered expression of numerous proteins, including many ion channels. Yet, how DRGs maintain long-term changes in protein expression in neuropathic conditions remains unclear. DNA methylation is a well-known mechanism of epigenetic control of gene expression and is achieved by the action of three enzymes: DNA methyltransferase (DNMT) 1, 3a, and 3b, which have been studied primarily during development. We first performed immunohistochemical analysis to assess whether these enzymes are expressed in adult rat DRGs (L4-5) and found that DNMT1 is expressed in both glia and neurons, DNMT3a is preferentially expressed in glia and DNMT3b is preferentially expressed in neurons. A rat model of neuropathic pain was then used to determine whether nerve injury may induce epigenetic changes in DRGs at multiple time points after pain onset. Real-time RT PCR analysis revealed robust and time-dependent changes in DNMT transcript expression in ipsilateral DRGs from spared nerve injury (SNI) but not sham rats. Interestingly, DNMT3b transcript showed a robust upregulation that appeared already 1 week after surgery and persisted at 4 weeks (our endpoint); in contrast, DNMT1 and DNMT3a transcripts showed only moderate upregulation that was transient and did not appear until the second week. We suggest that DNMT regulation in adult DRGs may be a contributor to the pain phenotype and merits further study.

Keywords: DNA methylation; DNMT expression; SNI model; dorsal root ganglia; gene expression; neuropathic pain.

FIGURE 1

DNA methyltransferase expression in DRG is cell-type specific. 20-micron L4 and L5 DRG sections from control rats aged 5–7 weeks were immuno-labeled with DNMT antibodies as well as incubated with the nuclear labeling dye 4’,6-diamidino-2-phenylindole (DAPI). (A–C) Show robust and ubiquitous labeling of DNMT1 in nuclei of DRG neurons. Notably, there is little expression of DNMT1 detected in the satellite cells surrounding DRG neurons. In addition, widespread overlap of DAPI and DNMT1 was detected in axons, demonstrating ubiquitous expression in Schwann cells (D–F). (G–I) In contrast to DNMT1, DNMT3a was not detected in neuronal nuclei but rather in satellite cells surrounding DRG neurons. (J–L) DNMT3a also showed widespread overlap with DAPI in nuclei of Schwann cells. (M–O) DNMT3b was detected in nuclei of all DRG neurons. (P–R) Notably, DNMT3b staining did not reveal overlap with DAPI in DRG axons, suggesting DNMT3b is not expressed in glia. Scale bar: 20 μM.

FIGURE 2

Relative abundance of DNMT transcripts in adult rat DRGs. (A) PCR products run on a 1.8% agarose gel demonstrate a single amplification product for each gene of interest. DNMT3b and GAPDH were run together as this assay involved multiplexing DNMT3b and GAPDH in the same PCR well, thus the two bands in one lane. (B) Quantitative RT-PCR was first used to determine the relative abundance of DNMTs from five naive rats at 1 month of age. All DNMT gene expression is normalized to GAPDH. Note the higher expression levels of DNMT1 and DNMT3a while DNMT3b is lower, consistent with immunocytochemistry data (Figure 1). Note the plot is on a logarithmic scale.

FIGURE 3

Von Frey measurements in SNI and sham rats. The spared nerve injury (SNI) model of neuropathic pain was used. The left or ipsilateral nerve was injured while the right or contralateral nerve was intact. Tactile sensitivity of the spared region of the operated paws was measured from the withdrawal responses to mechanical stimulation with von Frey filaments. 50% response thresholds were calculated according to Chaplan et al. (1994). Data are shown by dividing the contralateral/right paw over the ipsilateral/left paw, at 1, 2, and 4 weeks post SNI or sham surgery. All animals that underwent SNI surgery developed significant tactile allodynia in the ipsilateral/left paw that persisted until they were sacrificed, while sham rats showed no changes, *p < 0.01. n = 8 SNI and sham for 1 week, 4 for 2 weeks, and 6 for 4 weeks. The left foot von Frey thresholds for sham-operated and SNI rats, respectively, were 3.93 ± 0.58 g and 0.55 ± 0.41 g at 1 week, 5.16 ± 0.49 g and 0.64 ± 0.16 g at 2 weeks, and 7.93 ± 0.77 g and 0.97 ± 0.44 g at 4 weeks after surgery.

FIGURE 4

Nerve injury induces a robust and long lasting increase in DNMT3b expression. Gene expression is normalized to GAPDH. All SNI data are normalized to sham controls. (A) At 1 week post nerve injury, qRT-PCR experiments demonstrate a significant, almost fourfold increase in DNMT3b transcript in the DRG ipsilateral to the injured nerve. DRGs contralateral to nerve injury showed no significant changes, *p < 0.001. n = 8 SNI and 8 sham. (B) At 2 weeks post nerve injury, qRT-PCR experiments demonstrate a significant increase in DNMT1 (38%), DNMT3a (58%), and DNMT3b (twofold) transcripts in the DRGs ipsilateral to the injured nerve. DRGs contralateral to nerve injury showed no changes, *p < 0.05. n = 4 SNI and 4 sham animals. (C) At 4 weeks post nerve injury, qRT-PCR experiments demonstrate a significant, approximately 40% increase in DNMT3b transcripts in the DRGs ipsilateral to the injured nerve. DRGs contralateral to nerve injury showed no changes. For DRGs ipsilateral to injury in SNI rats, DNMT1 showed a trend toward being increased by 27% with p = 0.09 while DNMT3a also showed a slight trend of being increased by 42% with p = 0.22, *p < 0.05. n = 6 SNI and 6 sham.

FIGURE 5

DNMT3b transcript expression remains exclusively neuronal after peripheral nerve injury. Immunohistochemisry was performed on sections obtained from DRG ipsilateral to the peripheral injury in two SNI rats 1 week post-surgery. (A,B) are two examples of images obtained by staining the DRG sections with DNMT3b (green) antibody and DAPI (blue). Note that, similar to the naïve tissue, DNMT3b expression is ubiquitous in neurons, but absent from glia.