Chronic pain: emerging evidence for the involvement of epigenetics

Abstract

Epigenetic processes, such as histone modifications and DNA methylation, have been associated with many neural functions including synaptic plasticity, learning, and memory. Here, we critically examine emerging evidence linking epigenetic mechanisms to the development or maintenance of chronic pain states. Although in its infancy, research in this area potentially unifies several pathophysiological processes underpinning abnormal pain processing and opens up a different avenue for the development of novel analgesics.

Copyright © 2012 Elsevier Inc. All rights reserved.

Figures

Figure 1. Schematic Representation of the Key Processes Thought to Underlie Chronic Pain States

(A) Changes in brain function: a network of cortical and subcortical areas is involved in processing nociceptive signals and the sensation of pain (among others: prefrontal cortex [PFC], sensory motor cortices [SI/SII], posterior parietal cortex [PPC], anterior cingulate cortex [ACC], insula [INS], thalamus [Th], periaqueductal gray [PAG], and rostral ventromedial medulla [RVM]). In chronic pain patients, many of these display profound changes in fMRI bold signal, interconnectivity, and top-down modulation of ascending spinal signals. (B) Abnormal amplification of pain signals in DRG and spinal cord neurons: sensory neurons display hyperexcitability as a result of altered neurotrophic support and extensive changes in the expression of relevant genes, most notably ion channels and nociceptors. Second-order cells exhibit central sensitization as a result of several processes including immune and glial cell recruitment in the CNS. (C) Peripheral inflammation and sensitization of nociceptors: tissue damage activates and recruits immune cells (e.g., mast cells, macrophages and neutrophils). These cells will release or stimulate the production of a variety of cytokines (e.g., IL-6, IL-1β, TNFα) and proinflammatory mediators (e.g., NGF and prostaglandins). This will activate or modulate the action of receptors on the sensory nerve terminals (e.g., the TrkA, cytokine, and prostaglandin receptors [EP/IP] are activated and Trp channels can be modulated). This process will result in sensitization of the nociceptive neuron.

Figure 2. Schematic Representation of Epigenetic Processes and General Chromatin Structure

(A) DNA methylation was traditionally considered mainly in the context of CpG islands and is known to have silencing functions (e.g., silencing of the inactive X chromosome). More recent evidence indicates that methylation can also occur in non-CpG context, may sometimes increase transcription, and is highly variable between tissues and individuals–particularly around CpG island shores (Irizarry et al., 2009; Lister et al., 2009). Another contentious point is the stability and reversibility of methyl marks in adult mammalian cells, since it is unclear whether and to what extent active demethylation occurs. Several putative mechanisms have now been proposed (Ma et al., 2009; Ito et al., 2011; He et al., 2011). (B) Histone variants and modifications: DNA is wrapped around a histone octamer consistent of a histone H3, H4 tetramer and two histone H2A, H2B dimers. The lysine residues of histones can be modified, e.g., by phosphorylation (P), acetylation (Ac) and methylation (Me), and this changes chromatin conformation (Bannister and Kouzarides, 2011). Different histone variants exist (e.g., H3.2) that have distinct posttranslational modification patterns, occur at different stages of neuronal development (Piña and Suau, 1987), and hence affect chromatin function. (C) Chromatin remodeling complexes: chromatin conformation can also be changed through protein complexes whose actions are fuelled by ATP hydrolysis (Hargreaves and Crabtree, 2011). (D) Nucleosomes are further condensed into chromatin fibers and packaged into chromosomes to fit inside the nucleus. The structure of chromatin and chromosomes is highly dynamic and varies with cell cycle.

Figure 3. Techniques Used to Probe Epigenetic Mechanisms

In ChIP, an antibody is used on sheared, crosslinked chromatin to select modifications of interest and determine gene-specific (qPCR) or genome-wide (NGS) enrichment. The latter is commonly referred to as “ChIP-seq.” Antibody availability and specificity is one of the main difficulties with this technique (Egelhofer et al., 2011). In addition, great care has to be taken to include appropriate controls: 10% input chromatin, total histone ChIP to control for nucleosome density and negative control primers for qPCR. DNA methylation can be probed with (1) affinity-based methods like MeDIP where an antibody against 5-methyl-cytosine (5mC) is used to pull down methylated regions of DNA. If followed by next generation sequencing (NGS), this is referred to as MeDIP-seq. Alternatively, there are sequence-specific methods (2), like reduced representation bisulfite sequencing (RRBS), which include bisulfite conversion. For genome-wide studies, DNA methylation methods have been compared (e.g., by Bock et al., 2010). RRBS was found to have the best resolution, accuracy, and robustness. However, it is important to bear in mind that sequence-specific methods cannot distinguish between 5mc and 5-hydroxymethyl-cytosine (5hmC). If not probed with 5hmC-specific antibodies using MeDIP, conversion of 5mC to 5hmC can thus be mistaken for the disappearance of methyl-sites (for a review on 5hmC and its potential functions, see Branco et al., 2012).

Figure 4. Schematic Illustrating One Possible Mechanism by which Epigenetic Processes Could Impact Development of Chronic Pain

(A) Acetylation at relevant nociceptive genes (Pain Gene, PG) could lead to a more open chromatin conformation through the negative charge of acetyl groups and the recruitment of transcription factor complexes (TF complex) containing bromodomain (bromo) readers. This would then lead to increased transcription of the genes in question, as is indeed observed in chronic pain states, where a large number of loci show abnormal upregulation (Lacroix-Fralish et al., 2011). (B) In contrast, in a nonpain state, HDACs may be present to deacetylate the nociceptive promoters thus leaving the region in a heterochromatic, silenced state.