CpH methylome analysis in human cortical neurons identifies novel gene pathways and drug targets for opioid use disorder

Abstract

IntroductionDNA methylation (DNAm), an epigenetic mechanism, has been associated with opioid use disorder (OUD) in preclinical and human studies. However, most of the studies have focused on DNAm at CpG sites. DNAm at non-CpG sites (mCpHs, where H indicates A, T, or C) has been recently shown to have a role in gene regulation and to be highly abundant in neurons. However, its role in OUD is unknown. This work aims to evaluate mCpHs in the human postmortem orbital frontal cortex (OFC) in the context of OUD.MethodsA total of 38 Postmortem OFC samples were obtained from the VA Brain Bank (OUD = 12; Control = 26). mCpHs were assessed using reduced representation oxidative bisulfite sequencing in neuronal nuclei. Differential analysis was performed using the “methylkit” R package. Age, ancestry, postmortem interval, PTSD, and smoking status were included as covariates. Significant mCpHs were set at q-value < 0.05. Gene Ontology (GO) and KEGG enrichment analyses were performed for the annotated genes of all differential mCpH loci using String, ShinyGO, and amiGO software. Further, all annotated genes were analyzed using the Drug gene interaction database (DGIdb).ResultsA total of 2,352 differentially methylated genome-wide significant mCpHs were identified in OUD, mapping to 2,081 genes. GO analysis of genes with differential mCpH loci showed enrichment for nervous system development (p-value = 2.32E-19). KEGG enrichment analysis identified axon guidance and glutamatergic synapse (FDR 9E-4–2.1E-2). Drug interaction analysis found 3,420 interactions between the annotated genes and drugs, identifying interactions with 15 opioid-related drugs, including lofexidine and tizanidine, both previously used for the treatment of OUD-related symptoms.ConclusionOur findings suggest a role of mCpHs for OUD in cortical neurons and reveal important biological pathways and drug targets associated with the disorder.

Neuronal-specific methylome and hydroxymethylome analysis reveal significant loci associated with alcohol use disorder

“…Another limitation is that all samples are male, limiting to identify the effect of sex in our results. The analyses were only carried out on CpGs sites and it would be important to conduct a study on non-CpGs sites, because of the role of 5mC at non-CpGs on neuropsychiatric diseases from our group and others ( Jang et al, 2017 ; Nagamatsu et al, 2022 ). Further, since the technologies to assess 5mC and 5hmC in our study and Clark et al, 2022 are different, we were only able to evaluate the convergence between the two studies at the gene-level, not at the CpG level, which limits the interpretation in gene regulatory processes of the differential epigenetic marks identified.…”

“…Neuronal nuclei isolation was performed using fluorescence-activated nuclei sorting (FANS), described by Rompala and Nagamatsu et al (2022) ( Rompala et al, 2022 ), obtaining 0.5–1 M NeuN + nuclei for DNA extraction. Sorted nuclei were centrifuged at 1,500 × g for 15 min at 4°C to obtain a pellet.…”

Single nuclei transcriptomics in human and non-human primate striatum implicates neuronal DNA damage and proinflammatory signaling in opioid use disorder

“…Efforts to develop new treatment strategies and to bolster avenues of existing treatments for opioid addiction require a deeper understanding of the changes that occur in the human brain with chronic opioid use. To date, few studies have investigated the cellular and molecular changes in the human brain associated with OUD [2][3][4][5][6][7] . Recently, we reported alterations in neuroinflammation, among other pathways, in postmortem brain regions from subjects with OUD 2,5 .…”

Circadian rhythm disruptions associated with opioid use disorder in the synaptic proteomes of the human dorsolateral prefrontal cortex and nucleus accumbens

“…Understanding the impact of long-term opioid use and overdose on the human brain is critical for developing new interventions, therapeutics, and for evaluating current OUD treatments. In-depth investigations into the cellular and molecular changes in human postmortem brain may provide valuable insights into the biological mechanisms associated with OUD and lead to new therapeutic strategies for treating opioid addiction [2][3][4][5][6][7][8].…”

“…The dorsolateral prefrontal cortex (DLPFC) and nucleus accumbens (NAc) are key brain regions involved in cognitive, mood, and reward alterations in OUD [9][10][11]. Recent human postmortem brain studies from us [2,3] and others [4,6,12,13] reported a potential interplay between proinflammatory and synaptic remodeling in DLPFC and NAc in OUD. Moreover, we recently implicated circadian rhythm regulation of neuroinflammatory, neurovascular, and neurotransmission (e.g., dopaminergic, GABAergic, and opioidergic signaling) in DLPFC and NAc of OUD subjects [2,3].…”