Abnormal DNA methylation in the lumbar spinal cord following chronic constriction injury in rats

doi:10.1016/j.neulet.2015.10.048

Neuroscience Letters

Volume 610, 1 January 2016, Pages 1-5

https://doi.org/10.1016/j.neulet.2015.10.048 Get rights and content

Highlights

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The role of abnormal DNA methylation in the lumbar spinal cord in neuropathic pain was explored in this study.
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DNMT3a, DNMT3b and MeCP2 expression increased, MBD2 expression decreased, and DNMT1, MBD1 and MBD3 expression hardly changed in the lumbar spinal cord in CCI rats on day 14 after surgery.
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GAD 67 expression decreased, and GAD 1 promoter methylation increased in the lumbar spinal cord in CCI rats on day 14 after surgery.

Abstract

Pathogenesis of neuropathic pain is complex and not clearly understood. Glutamate decarboxylase 67 (GAD 67) is a key synthetic enzyme for the main inhibitory transmitter gamma-aminobutyric acid (GABA), and diminishes in the spinal dorsal horn in rats following chronic constriction injury (CCI). GAD 67 is coded by gene GAD 1. DNA methylation can regulate the expression of GAD 67 by regulating the methylation of GAD 1 promoter in the psychotic brain. DNA methylation is primarily mediated by DNA methyltransferases (DNMTs) and methyl-DNA binding domain proteins (MBDs). In this study, in order to discover whether DNA methylation regulates GAD 67 expression in the spinal cord in CCI rats and is involved in neuropathic pain, we examined mRNA levels of DNMTs, MBDs and GAD 67 with real-time reverse transcriptase-polymerase chain reaction (qRT-PCR), and methylation of GAD 1 promoter with Pyromark CpG Assays in the lumbar spinal cord in CCI rats on day 14 after surgery. Our results showed that DNMT3a, DNMT3b and methyl-CpG binding protein 2 (MeCP2) expression increased, MBD2 expression decreased, and DNMT1, MBD1 and MBD3 expression hardly changed in the lumbar spinal cord in CCI rats on day 14 after surgery. GAD 67 expression decreased, and methylation of GAD 1 promoter increased in the lumbar spinal cord in CCI rats on day 14 after surgery. These results indicate that decreased GAD 67 may be associated with increased GAD 1 promoter methylation, which may be mediated by DNMT3a, DNMT3b, MeCP2 and MBD2 in CCI rats. These indicate that abnormal DNA methylation may be highly involved in CCI-induced neuropathic pain.

Introduction

Neuropathic pain is a chronic pain, which is caused by primary or secondary damage or dysfunction of the nervous system, and is characterized by hyperalgesia, allodynia and spontaneous pain [18]. The pathogenesis of neuropathic pain is complex and not clearly understood. The transcription and expression level of pain-associated genes are highly involved in the generation and maintenance of neuropathic pain [25]. Decreased gamma-aminobutyric acid (GABA)-ergic inhibition in the spinal dorsal horn plays an important role in neuropathic pain [4], [28]. Glutamate decarboxylase 67 (GAD 67) is a key synthetic enzyme for the main inhibitory transmitter GABA, and diminishes in the spinal dorsal horn in rats following chronic constriction injury (CCI) [19], [29].

Epigenetic mechanisms that induce heritable changes in gene expression without causing alterations in the DNA sequence can regulate the transcription and expression of pro- or antinociceptive genes [5], [8], and may play an important role in neuropathic pain [3], [27]. DNA methylation is one of the earliest and most characteristic epigenetic mechanisms in mammals, and is a major contributor to the stability of gene expression [12]. DNA methylation is catalyzed by DNA methyltransferases (DNMTs), which transfer a methyl group from S-adenosyl-l-methionine to cytosine bases of cytosine–phosphate–guanine (CpG) in DNA [9]. The CpG dinucleotides tend to cluster in regions called CpG islands, defined as regions of more than 200 bases with a G + C content of at least 50% and a ratio of observed to statistically expected CpG frequencies of at least 0.6 [23]. The methylation of CpG islands in the gene promoter plays an important role in regulating gene expression. Increased methylation of CpG islands in the gene promoter can downregulate gene expression, and decreased methylation of CpG islands in the gene promoter can upregulate gene expression [26]. There are two functionally different classes of DNMTs—maintenance methyltransferases DNMT1 and de novo methyltransferases DNMT3a and DNMT3b [16]. Methylated DNA relies on interfering with transcription factors directly and mainly via methyl-CpG-binding proteins (MBDs) indirectly to inhibit gene transcription [2], [20]. Methyl-CpG binding protein 2 (MeCP2) and MBD 1–3 are the most studied MBDs in the central nervous system (CNS) [16].

GAD 67 is encoded by gene GAD 1. Recent evidences indicate that DNA methylation can regulate the expression of GAD 67 by regulating the methylation of GAD 1 promoter in the psychotic brain [6], [10]. And there is a CpG island in rat GAD 1 promoter by scanning the rat GAD 1 promoter region (GenBank accession number AF110132) and recent rat GAD 1 sequence from UCSC Genome Browser on Rat Nov. 2004 (Baylor 3.4/rn4) Assembly. In this study, in order to discover whether DNA methylation regulates GAD 67 expression in the spinal cord in CCI rats and is involved in neuropathic pain, we examined mRNA levels of DNMTs, MBDs and GAD 67, and methylation of GAD 1 promoter in the lumbar spinal cord in CCI rats on day 14 after surgery.

Section snippets

Animal surgeries and neuropathic pain measurements

This study was performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. The Animal Care Committee of the Fujian Provincial Cancer Hospital in China approved all the procedures. Sixteen male Sprague-Dawley (SD) rats, weighing 220–250 g, provided by the animal experiment center of Fujian Medical University were divided into CCI rats (n = 8) and sham operation rats (n = 8). Under deep anesthesia induced by intraperitoneal injection of 10% chloral

Changes in behavior and neuropathic pain measurements

CCI rats suffered from neuropathic pain showing various signs of protecting postures and moving disorders such like foot and toe closing together, dorsiflexion, eversion and marked limp. Sham operation rats showed no such signs. MWT and TWL had similar changes (Fig. 1). There was no significant difference in MWT and TWL among all the rats before surgery (P > 0.05). MWT and TWL of sham operation rats showed no significant change compared with that of before operation (P > 0.05). The MWT and TWL of

Discussion

Pain-associated gene' transcription and expression, on which DNA methylation may exert a major effect, are highly relevant to the genesis of neuropathic pain. DNA methylation is mediated by DNMTs and MBDs. DNMT1 is the primary maintenance methyltransferase, which associates with hemi-methylated CpG during DNA replication and preserves methylation patterns in daughter cells [24]. DNMT3a and DNMT3b are the primary de novo methyltransferases, which target unmethylated CpG and establish new

Conclusions

In this study, GAD 67 expression decreased, GAD 1 promoter methylation increased, DNMT3a, DNMT3b and MeCP2 expression increased, and MBD2 expression decreased in the lumbar spinal cord in CCI rats on day 14 after surgery. These results indicate that decreased GAD 67 expression may be associated with increased GAD 1 promoter methylation, which may be mediated by DNMT3a, DNMT3b, MeCP2 and MBD2 in CCI rats. These indicate that abnormal DNA methylation may be highly involved in CCI-induced

Acknowledgements

This study was supported by National Natural Science Foundation of China (81400921) and Natural Science Foundation of Fujian Province of China (2015J05144).

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Citation Excerpt :
This is referred to as the infant lesion effect (Goldberger, 1986;Robinson & Goldberger, 1986) a phenomenon of which the mechanisms are not fully understood, though they are thought to be due to plasticity intrinsic to the spinal cord and not due to regrowth of axons over the lesion site (Cummings & Stelzner, 1988; Tillakaratne et al., 2010; Weber & Stelzner, 1980). Existing studies of epigenetic mechanisms in the spinal cord are sparse and mainly focus on pain (Jiang et al., 2017; Zhao et al., 2017), disease (Martin & Wong, 2013; Miranpuri et al., 2017), or injury (Mahar & Cavalli, 2018; Wang et al., 2011, 2016; Wang, Li, Li, Guan, & Deng, 2017; Weng et al., 2017, 2018) in adulthood. Given that these studies suggest a functional role for the spinal epigenome, and that there is some evidence of epigenetic regulation in the developing spinal cord (Akizu, Estarás, Guerrero, Martí, & Martínez-Balbás, 2010; Chestnut et al., 2011; Moyon et al., 2016; Ocklenburg et al., 2017; Ye et al., 2009), it is reasonable to hypothesize that the epigenome of the developing spinal cord contributes to its unique plasticity and behavioral outcomes.
Although the importance of epigenetic mechanisms in behavioral development has been gaining attention in recent years, research has largely focused on the brain. To our knowledge, no studies to date have investigated epigenetic changes in the developing spinal cord to determine the dynamic manner in which the spinal epigenome may respond to environmental input during behavioral development. Animal studies demonstrate that spinal cord plasticity is heightened during early development, is somewhat preserved following neonatal transection, and that spinal injured animals are responsive to sensory feedback. Because epigenetic alterations have been implicated in brain plasticity and are highly responsive to experience, these alterations are promising candidates for molecular substrates of spinal plasticity as well. Thus, the current study investigated behavioral changes in the development of weight-bearing locomotion and epigenetic modifications in the spinal cord of infant rats following a neonatal low-thoracic spinal transection or sham surgery on postnatal day (P)1. Specifically, global levels of methylation and methylation status of the brain-derived neurotrophic factor (Bdnf) gene, a neurotrophin heavily involved in both CNS and behavioral plasticity, particularly in development, were examined in lumbar tissue harvested on P10 from sham and spinal-transected subjects. Behavioral results demonstrate that compared to shams, spinal-transected subjects exhibit significantly reduced partial-weight bearing hindlimb activity. Molecular data demonstrate group differences in global lumbar methylation levels as well as exon-specific group differences in Bdnf methylation. This study represents an initial step toward understanding the relationship between epigenetic mechanisms and plasticity associated with spinal cord and locomotor development.
Hyperbaric oxygenation alleviates chronic constriction injury (CCI)-induced neuropathic pain and inhibits GABAergic neuron apoptosis in the spinal cord
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The release of Cytc and activation of caspase-3 have been observed in CCI-induced neuropathic pain [39]. In nerve-injury-induced neuropathic pain, the synthesis of GAD67 and GAD65 proteins and mRNA was observed being down-regulated, which causes the hypofunction of GABAergic transmission [9,30,34,40–42]. Increasing the GAD67 level in GABAergic neurons by gene transfer technique alleviates neuropathic pain [41,43].
Dysfunction of GABAergic inhibitory controls contributes to the development of neuropathic pain. We examined our hypotheses that (1) chronic constriction injury (CCI)-induced neuropathic pain is associated with increased spinal GABAergic neuron apoptosis, and (2) hyperbaric oxygen therapy (HBO) alleviates CCI-induced neuropathic pain by inhibiting GABAergic neuron apoptosis.
Male rats were randomized into 3 groups: CCI, CCI+HBO and the control group (SHAM). Mechanical allodynia was tested daily following CCI procedure. HBO rats were treated at 2.4 atmospheres absolute (ATA) for 60 min once per day. The rats were euthanized and the spinal cord harvested on day 8 and 14 post-CCI. Detection of GABAergic cells and apoptosis was performed. The percentages of double positive stained cells (NeuN/GABA), cleaved caspase-3 or Cytochrome C in total GABAergic cells or in total NeuN positive cells were calculated.
HBO significantly alleviated mechanical allodynia. CCI-induced neuropathic pain was associated with significantly increased spinal apoptotic GABA-positive neurons. HBO considerably decreased these spinal apoptotic cells. Cytochrome-C-positive neurons and cleaved caspase-3-positive neurons were also significantly higher in CCI rats. HBO significantly decreased these positive cells. Caspase-3 mRNA was also significantly higher in CCI rats. HBO reduced mRNA expression of caspase-3.
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Increased apoptotic GABAergic neurons induced by activation of key proteins of mitochondrial apoptotic pathways in the dorsal horn of the spinal cord is critical in CCI-induced neuropathic pain. The inhibitory role of HBO in GABAergic neuron apoptosis suppresses ongoing neuropathic pain.
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Research paperAbnormal DNA methylation in the lumbar spinal cord following chronic constriction injury in rats

Highlights

Abstract

Introduction

Section snippets

Animal surgeries and neuropathic pain measurements

Changes in behavior and neuropathic pain measurements

Discussion

Conclusions

Acknowledgements

Pain

Trends Neurosci.

Neuropharmacology

Eur. J. Pain

Prog. Mol. Biol. Transl. Sci.

Neurosci. Lett.

Mol. Ther.

Prog. Neurobiol.

Neuropharmacology

Brain Res. Rev.

Cell

J. Biol. Chem.

Neuromodulation

Neurosci. Res.

Neurosci. Lett.

Research paper
Abnormal DNA methylation in the lumbar spinal cord following chronic constriction injury in rats