Matthew J. Girgenti

Genomic studies have improved our understanding of posttraumatic stress disorder (PTSD) biology. Epigenetic differences in DNA methylation can reflect environmental influences, which are critical in PTSD etiology, and may additionally differentiate between PTSD cases and controls or distinguish severity of PTSD symptoms. We conducted epigenome-wide association studies (EWAS) of PTSD in 22,141 subjects from the United States V.A. Million Veteran Program (MVP), using the PTSD Checklist (PCL-C), including 17,674 European ancestry (EUR), 3,430 African ancestry (AFR), and 1,037 admixed American descent (AMR) individuals. We evaluated the 17-item PCL-C and its symptom subdomains (re-experiencing, avoidance, and hyperarousal) and used our previously-reported algorithmically-defined PTSD diagnoses validated against clinical records. There were 9,413 total cases of Lifetime PTSD and 27,257 controls, with 5,184 cases and 19,382 controls in EUR, 3,095 cases and 6,240 controls in AFR, and 1,134 cases and 1,635 controls in AMR. We identified a total of 272 epigenome-wide-significant associated CpG sites across all PTSD traits, or 28 after adjusting for smoking status. We replicated 5 out of 11 CpG sites reported in a prior (Psychiatric Genomics Consortium) PTSD EWAS. There was significant enrichment of genes downregulated in somatostatin interneurons and of genes expressed in GABAergic interneurons. Regression analyses showed that PTSD case status was significantly correlated with accelerated DNA methylation aging. Contrary to previous PTSD EWAS findings, smoking status stratification suggested that the association between PTSD and methylation of the AHRR gene may be confounded by smoking status. This is the largest-scale PTSD EWAS in multiple ancestries and the first large PTSD EWAS on symptom severity. We replicate findings from previous EWAS and greatly extend knowledge of the epigenetics of PTSD.

Cannabidiol (CBD) has shown therapeutic potential for post-traumatic stress disorder (PTSD) by reducing spontaneous recovery (SR), yet the underlying mechanisms remain unclear. We examined the effects of CBD on SR in male and female mice and found greater behavioral efficacy in males. To uncover the molecular basis of these effects, we performed transcriptomic profiling of the prelimbic (PL) and infralimbic (IL) subregions of the medial prefrontal cortex (mPFC). We identified distinct and overlapping SR-associated gene sets in each subregion, and CBD shifted the expression of a significant subset of these genes toward non-stressed control levels, including genes involved in retrograde endocannabinoid signaling. Cross-species comparisons with human PTSD transcriptomic datasets revealed conserved gene alterations within each subregion, suggesting shared molecular signatures between mouse SR and human PTSD. These findings highlight region-specific and conserved pathways through which CBD may exert therapeutic effects and provide mechanistic insight to advance CBD-based interventions for PTSD-associated spontaneous recovery.

Regulation of gene expression is a highly coordinated process in both the healthy and pathological brain with unique patterns across a multitude of cell types. Here we present a multi-omic single nucleus study of ~175,000 nuclei from 50 donors with alcohol use disorder (AUD) and control donors without AUD, profiling cell type specific gene expression and chromatin accessibility in the human central amygdala. We identify all major CNS cell types and neuronal subtypes and find inhibitory neurons are particularly affected by AUD. We find high numbers of differentially expressed genes (DEGs) including GABRA2 , GRM8 , and NCAM1 and show significant enrichment for AUD risk genes within these DEGs. We identified 51,431 cell type-specific, disease associated candidate cis -regulatory elements including an interneuron-associated set of chromatin loops at the AUD risk gene CALN1 . Transcription factor footprinting identified Kruppel-like factors upstream of AUD GWAS genes and DEGs. Finally, we also perform cell type-specific fine mapping for AUD GWAS to prioritize variants within functional genomic elements.

Histone deacetylases (HDACs), typically known for regulating gene expression, also play a major role in protein regulation outside of histone modification. Emerging evidence suggests the HDACs may be novel pharmacologic targets in complex disorders such as posttraumatic stress disorder (PTSD). Histone deacetylase 6 (HDAC6) regulates microtubule function and plays a role in stress-related cortisol signaling in serotonergic regions of the brain by maintaining the nuclear translocation of glucocorticoid receptors. Here, we report results of a translational positron emission tomography brain imaging study using a novel HDAC6-selective radiotracer, [¹⁸F]Bavarostat. In humans, we demonstrate significantly lower availability of HDAC6 in the amygdala of individuals with PTSD compared to non-trauma exposed controls. These proof-of-concept human findings are supported by rodent findings of reduced HDAC6 availability both in case-control groups and within-subject longitudinal analysis using a single prolonged stress model. Together, our translational findings demonstrate a potential role for HDAC6 in the pathophysiology of PTSD.

Chronic pain affects one-fifth of American adults, contributing significant public health burden. Chronic pain can be further understood through investigating brain gene expression, potentially informing on brain regions, cell types, and gene pathways. We tested for differentially expressed genes (DEGs) in chronic pain, migraine, lifetime fentanyl and oxymorphone use, and with chronic pain genetic risk in 4 brain regions (dorsal anterior cingulate cortex [dACC], dorsolateral prefrontal cortex [DLPFC], medial amygdala [MeA], and basolateral amygdala [BLA]) and imputed cell type expression data from 304 deeply phenotyped postmortem donors, potentially highlighting variation relevant to factors such as predisposition to chronic pain development, mechanisms of chronic pain development and persistence, and indirect effects of chronic pain and associated treatment or medication, and substance use. We also investigated sex differences in chronic pain differential gene expression. At the brain region level, we identified 2 chronic pain DEGs: B4GALT2 and VEGFB in dACC. At the cell level, we found more than 2000 chronic pain cell-type DEGs, significantly enriched in microglia of the basolateral amygdala. The findings were enriched for mouse microglia pain genes, and for hypoxia and immune response pathways. Small amounts of cross-trait DEG overlap in migraine and chronic pain highlighted medial amygdala cells, and in chronic pain and oxymorphone use suggested the amygdala as a key region. Chronic pain differential gene expression was not significantly different between men and women. Overall, chronic pain-associated gene expression is heterogeneous across region and cell type, is largely distinct from that in pain-related factors and migraine, and our results highlight BLA microglia as a key brain cell type in chronic pain.

Post-traumatic stress disorder (PTSD) is a polygenic disorder occurring after extreme trauma exposure. Recent studies have begun to detail the molecular biology of PTSD. However, given the array of PTSD-perturbed molecular pathways identified so far¹, it is implausible that a single cell type is responsible. Here we profile the molecular responses in over two million nuclei from the dorsolateral prefrontal cortex of 111 human brains, collected post-mortem from individuals with and without PTSD and major depressive disorder. We identify neuronal and non-neuronal cell-type clusters, gene expression changes and transcriptional regulators, and map the epigenomic regulome of PTSD in a cell-type-specific manner. Our analysis revealed PTSD-associated gene alterations in inhibitory neurons, endothelial cells and microglia and uncovered genes and pathways associated with glucocorticoid signalling, GABAergic transmission and neuroinflammation. We further validated these findings using cell-type-specific spatial transcriptomics, confirming disruption of key genes such as SST and FKBP5. By integrating genetic, transcriptomic and epigenetic data, we uncovered the regulatory mechanisms of credible variants that disrupt PTSD genes, including ELFN1, MAD1L1 and KCNIP4, in a cell-type-specific context. Together, these findings provide a comprehensive characterization of the cell-specific molecular regulatory mechanisms that underlie the persisting effects of traumatic stress response on the human prefrontal cortex.

Background: Post-traumatic stress disorder (PTSD) is a common and disabling psychiatric disorder. PTSD involves multiple brain regions and is often comorbid with other psychiatric disorders, such as major depressive disorder (MDD). Recent genome-wide association studies (GWASs) have identified many PTSD risk loci and transcriptomics studies of postmortem brain have found differentially expressed genes associated with PTSD cases. In this study, we integrated genome-wide measures across modalities to identify convergent molecular effects in the PTSD brain. Methods: We performed tandem mass spectrometry (MS/MS) on a large cohort of donors (N = 66) in two prefrontal cortical areas, dorsolateral prefrontal cortex (DLPFC), and subgenual prefrontal cortex (sgPFC). We also coupled the proteomics data with transcriptomics and microRNA (miRNA) profiling from RNA-seq and small-RNA sequencing, respectively for the same cohort. Additionally, we utilized published GWAS results of multiple psychiatric disorders for integrative analysis. Results: We found differentially expressed proteins and co-expression protein modules disrupted by PTSD. Integrative analysis with transcriptomics and miRNA data from the same cohort pointed to hsa-mir-589 as a regulatory miRNA responsible for dysregulation of neuronal protein networks for PTSD, including the gamma-aminobutyric acid (GABA) vesicular transporter, SLC32A1. In addition, we identified significant enrichment of risk genes for other psychiatric disorders, such as autism spectrum disorder (ASD) and major depressive disorder (MDD) within PTSD protein co-expression modules, suggesting shared molecular pathology. Conclusions: We integrated genome-wide measures of mRNA and miRNA expression and proteomics profiling from PTSD, MDD, and control (CON) brains to identify convergent and divergent molecular processes across genomic modalities. We substantially expand the number of differentially expressed genes and proteins in PTSD and identify downregulation of GABAergic processes in the PTSD proteome. This provides a novel framework for future studies integrating proteomic profiling with transcriptomics and non-coding RNAs in the human brain studies.

There is growing interest to investigate classic psychedelics and ketamine as therapeutics for mental illnesses. Previous studies have demonstrated that one dose of psilocybin or ketamine leads to persisting neural and behavioral changes. The durability of these effects suggests that there are likely alterations in gene expression at the transcriptional level. In this study, we performed single-nucleus RNA sequencing of the dorsal medial frontal cortex of male and female mice. Samples were collected at 1, 2, 4, 24, or 72 hours after psilocybin or ketamine administration and from control animals. At baseline, major subtypes of excitatory and GABAergic neurons selectively express particular serotonin receptor transcripts. The psilocybin-evoked differentially expressed genes in excitatory neurons are involved in synaptic plasticity, distinct from genes enriched in GABAergic neurons, which contribute to mitochondrial function and cellular metabolism, and non-neuronal glial cells. The effect of psilocybin on gene expression is time-dependent, including an early phase at 1 hour followed by a late phase at 72 hours of transcriptional response after administration, and differs from the changes following ketamine administration, which peaks at 2 – 4 hours. Collectively, the results provide a resource for understanding the cell type-specific and time-dependent changes in gene expression induced by psilocybin and ketamine in the mouse medial frontal cortex, which may underpin the drug’s long-term effects on neural circuits and behavior.