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

Pollema-Mays SL, Centeno MV, Apkarian AV, Martina M - Front Cell Neurosci (2014)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology, Northwestern University Feinberg School of Medicine Chicago, IL, USA.

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.

No MeSH data available.


Related in: MedlinePlus

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.
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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.

Mentions: As no data are available concerning DNMT expression in rat DRG, we next quantified the relative abundance of DNMTs in L4 and L5 DRGs from naive rats. One month old rats were used to perform qRT-PCR measurements of DNMT transcript. qRT-PCR data was efficiency corrected, normalized to the GAPDH reference gene, and expression levels compared. PCR products were first run on a gel to demonstrate primer accuracy and confirmed a single band for each reaction (Figure 2A). DNMT1 and DNMT3a had much higher transcript levels than DNMT3b (Figure 2B). This is consistent with immunocytochemistry data showing that DNMT1 and DNMT3a staining was present in both neurons and glia, while staining for DNMT3b was restricted to the neuronal population (Figure 1). These experiments confirmed that DNMTs are expressed in rat DRG at the level of both mRNA and protein.


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

Pollema-Mays SL, Centeno MV, Apkarian AV, Martina M - Front Cell Neurosci (2014)

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.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4126486&req=5

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.
Mentions: As no data are available concerning DNMT expression in rat DRG, we next quantified the relative abundance of DNMTs in L4 and L5 DRGs from naive rats. One month old rats were used to perform qRT-PCR measurements of DNMT transcript. qRT-PCR data was efficiency corrected, normalized to the GAPDH reference gene, and expression levels compared. PCR products were first run on a gel to demonstrate primer accuracy and confirmed a single band for each reaction (Figure 2A). DNMT1 and DNMT3a had much higher transcript levels than DNMT3b (Figure 2B). This is consistent with immunocytochemistry data showing that DNMT1 and DNMT3a staining was present in both neurons and glia, while staining for DNMT3b was restricted to the neuronal population (Figure 1). These experiments confirmed that DNMTs are expressed in rat DRG at the level of both mRNA and protein.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology, Northwestern University Feinberg School of Medicine Chicago, IL, USA.

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.

No MeSH data available.


Related in: MedlinePlus