The Role of Epigenomic Regulatory Pathways in the Gut-Brain Axis and Visceral Hyperalgesia.

The gut-brain axis (GBA) is broadly accepted to describe the bidirectional circuit that links the gastrointestinal tract with the central nervous system (CNS). Interest in the GBA has grown dramatically over past two decades along with advances in our understanding of the importance of the axis in the pathophysiology of numerous common clinical disorders including mood disorders, neurodegenerative disease, diabetes mellitus, non-alcohol fatty liver disease (NAFLD) and enhanced abdominal pain (visceral hyperalgesia). Paralleling the growing interest in the GBA, there have been seminal developments in our understanding of how environmental factors such as psychological stress and other extrinsic factors alter gene expression, primarily via epigenomic regulatory mechanisms. This process has been driven by advances in next-generation multi-omics methods and bioinformatics. Recent reviews address various components of GBA, but the role of epigenomic regulatory pathways in chronic stress-associated visceral hyperalgesia in relevant regions of the GBA including the amygdala, spinal cord, primary afferent (nociceptive) neurons, and the intestinal barrier has not been addressed. Rapidly developing evidence suggests that intestinal epithelial barrier dysfunction and microbial dysbiosis play a potentially significant role in chronic stress-associated visceral hyperalgesia in nociceptive neurons innervating the lower intestine via downregulation in intestinal epithelial cell tight junction protein expression and increase in paracellular permeability. These observations support an important role for the regulatory epigenome in the development of future diagnostics and therapeutic interventions in clinical disorders affecting the GBA.

Druggable Transcriptional Networks in the Human Neurogenic Epigenome.

Chromosome conformation capture methods have revealed the dynamics of genome architecture which is spatially organized into topologically associated domains, with gene regulation mediated by enhancer-promoter pairs in chromatin space. New evidence shows that endogenous hormones and several xenobiotics act within circumscribed topological domains of the spatial genome, impacting subsets of the chromatin contacts of enhancer-gene promoter pairs in cis and trans Results from the National Institutes of Health-funded PsychENCODE project and the study of chromatin remodeling complexes have converged to provide a clearer understanding of the organization of the neurogenic epigenome in humans. Neuropsychiatric diseases, including schizophrenia, bipolar spectrum disorder, autism spectrum disorder, attention deficit hyperactivity disorder, and other neuropsychiatric disorders are significantly associated with mutations in neurogenic transcriptional networks. In this review, we have reanalyzed the results from publications of the PsychENCODE Consortium using pharmacoinformatics network analysis to better understand druggable targets that control neurogenic transcriptional networks. We found that valproic acid and other psychotropic drugs directly alter these networks, including chromatin remodeling complexes, transcription factors, and other epigenetic modifiers. We envision a new generation of CNS therapeutics targeted at neurogenic transcriptional control networks, including druggable parts of chromatin remodeling complexes and master transcription factor-controlled pharmacogenomic networks. This may provide a route to the modification of interconnected gene pathways impacted by disease in patients with neuropsychiatric and neurodegenerative disorders. Direct and indirect therapeutic strategies to modify the master regulators of neurogenic transcriptional control networks may ultimately help extend the life span of CNS neurons impacted by disease.

Improvement of Blood-Brain Barrier Integrity in Traumatic Brain Injury and Hemorrhagic Shock Following Treatment With Valproic Acid and Fresh Frozen Plasma.

OBJECTIVE: Combined traumatic brain injury and hemorrhagic shock are highly lethal. Following injuries, the integrity of the blood-brain barrier can be impaired, contributing to secondary brain insults. The status of the blood-brain barrier represents a potential factor impacting long-term neurologic outcomes in combined injuries. Treatment strategies involving plasma-based resuscitation and valproic acid therapy have shown efficacy in this setting. We hypothesize that a component of this beneficial effect is related to blood-brain barrier preservation. DESIGN: Following controlled traumatic brain injury, hemorrhagic shock, various resuscitation and treatment strategies were evaluated for their association with blood-brain barrier integrity. Analysis of gene expression profiles was performed using Porcine Gene ST 1.1 microarray. Pathway analysis was completed using network analysis tools (Gene Ontology, Ingenuity Pathway Analysis, and Parametric Gene Set Enrichment Analysis). SUBJECTS: Female Yorkshire swine were subjected to controlled traumatic brain injury and 2 hours of hemorrhagic shock (40% blood volume, mean arterial pressure 30-35 mmHg). INTERVENTIONS: Subjects were resuscitated with 1) normal saline, 2) fresh frozen plasma, 3) hetastarch, 4) fresh frozen plasma + valproic acid, or 5) hetastarch + valproic acid (n = 5 per group). After 6 hours of observation, brains were harvested for evaluation. MEASUREMENTS AND MAIN RESULTS: Immunofluoroscopic evaluation of the traumatic brain injury site revealed significantly increased expression of tight-junction associated proteins (zona occludin-1, claudin-5) following combination therapy (fresh frozen plasma + valproic acid and hetastarch + valproic acid). The extracellular matrix protein laminin was found to have significantly improved expression with combination therapies. Pathway analysis indicated that valproic acid significantly modulated pathways involved in endothelial barrier function and cell signaling. CONCLUSIONS: Resuscitation with fresh frozen plasma results in improved expression of proteins essential for blood-brain barrier integrity. The addition of valproic acid provides significant improvement to these protein expression profiles. This is likely secondary to activation of key pathways related to endothelial functions.

Rapid valproic acid-induced modulation of the traumatic proteome in a porcine model of traumatic brain injury and hemorrhagic shock.

BACKGROUND: Histone deacetylase inhibitors such as valproic acid (VPA) improve survival in lethal models of hemorrhagic shock and polytrauma. Although VPA is known to modulate transcription, its ability to reduce mortality within minutes of administration suggests involvement of a rapid, posttranslational mechanism. We hypothesized that VPA treatment would cause proteomic changes within minutes of treatment including quantitative and/or posttranslational differences in structural and/or effector proteins. MATERIALS AND METHODS: We used a porcine model of traumatic brain injury (computer-controlled cortical impact, 12 mm depth) and hemorrhagic shock (40% hemorrhage). Animals were kept in shock for 2 h and randomized to two groups (n = 3): normal saline (volume = 3:1 hemorrhage volume) or normal saline + VPA (150 mg/kg, single dose). Peripheral blood mononuclear cells were collected at baseline, postshock, and postresuscitation. Intracellular protein profiles were assessed using 1 dimensional gel electrophoresis, liquid chromatography, mass spectrometry, and analyzed with Ingenuity Pathway Analysis software. RESULTS: Animals treated with VPA demonstrated significant proteomic changes. Quantitative differences were found in over 200 proteins including effector, regulatory, and structural proteins in critical cell signaling pathways. Posttranslational modification analysis demonstrated differential VPA-induced acetylation of lysine residues in histone and nonhistone proteins. Pathway analysis correlated these changes with significant increases in numerous prosurvival and cytoskeletal intracellular pathways, including Rho GTPase signaling (P = 1.66E-11), integrin signaling (P = 4.19E-21), and a decrease in Rho guanosine nucleotide dissociation inhibitor signaling (P = 4.83E-12). CONCLUSIONS: In a porcine model of severe injuries, a single dose of VPA is associated with protective changes in the proteome that are measurable within minutes of treatment.

Mining the topography and dynamics of the 4D Nucleome to identify novel CNS drug pathways.

The pharmacoepigenome can be defined as the active, noncoding province of the genome including canonical spatial and temporal regulatory mechanisms of gene regulation that respond to xenobiotic stimuli. Many psychotropic drugs that have been in clinical use for decades have ill-defined mechanisms of action that are beginning to be resolved as we understand the transcriptional hierarchy and dynamics of the nucleus. In this review, we describe spatial, temporal and biomechanical mechanisms mediated by psychotropic medications. Focus is placed on a bioinformatics pipeline that can be used both for detection of pharmacoepigenomic variants that discretize drug response and adverse events to improve pharmacogenomic testing, and for the discovery of novel CNS therapeutics. This approach integrates the functional topology and dynamics of the transcriptional hierarchy of the pharmacoepigenome, gene variant-driven identification of pharmacogenomic regulatory domains, and mesoscale mapping for the discovery of novel CNS pharmacodynamic pathways in human brain. Examples of the application of this pipeline are provided, including the discovery of valproic acid (VPA) mediated transcriptional reprogramming of neuronal cell fate following injury, and mapping of a CNS pathway glutamatergic pathway for the mood stabilizer lithium. These examples in regulatory pharmacoepigenomics illustrate how ongoing research using the 4D nucleome provides a foundation to further insight into previously unrecognized psychotropic drug pharmacodynamic pathways in the human CNS.

Network Reconstruction Reveals that Valproic Acid Activates Neurogenic Transcriptional Programs in Adult Brain Following Traumatic Injury.

OBJECTIVES: To determine the mechanism of action of valproic acid (VPA) in the adult central nervous system (CNS) following traumatic brain injury (TBI) and hemorrhagic shock (HS). METHODS: Data were analyzed from different sources, including experiments in a porcine model, data from postmortem human brain, published studies, public and commercial databases. RESULTS: The transcriptional program in the CNS following TBI, HS, and VPA treatment includes activation of regulatory pathways that enhance neurogenesis and suppress gliogenesis. Genes which encode the transcription factors (TFs) that specify neuronal cell fate, including MEF2D, MYT1L, NEUROD1, PAX6 and TBR1, and their target genes, are induced by VPA. VPA represses genes responsible for oligodendrogenesis, maintenance of white matter, T-cell activation, angiogenesis, and endothelial cell proliferation, adhesion and chemotaxis. NEUROD1 has regulatory interactions with 38% of the genes regulated by VPA in a swine model of TBI and HS in adult brain. Hi-C spatial mapping of a VPA pharmacogenomic SNP in the GRIN2B gene shows it is part of a transcriptional hub that contacts 12 genes that mediate chromatin-mediated neurogenesis and neuroplasticity. CONCLUSIONS: Following TBI and HS, this study shows that VPA administration acts in the adult brain through differential activation of TFs responsible for neurogenesis, genes responsible for neuroplasticity, and repression of TFs that specify oligodendrocyte cell fate, endothelial cell chemotaxis and angiogenesis. Short title: Mechanism of action of valproic acid in traumatic brain injury.

Glucocorticoid receptor-mediated Nr1d1 chromatin circadian misalignment in stress-induced irritable bowel syndrome.

Stress-elevated glucocorticoids cause circadian disturbances and gut-brain axis (GBA) disorders, including irritable bowel syndrome (IBS). We hypothesized that the glucocorticoid receptor (GR/NR3C1) might cause chromatin circadian misalignment in the colon epithelium. We observed significantly decreased core circadian gene Nr1d1 in water avoidance stressed (WAS) BALB/c colon epithelium, like in IBS patients. WAS decreased GR binding at the Nr1d1 promoter E-box (enhancer box), and GR could suppress Nr1d1 via this site. Stress also altered GR binding at the E-box sites along the Ikzf3-Nr1d1 chromatin and remodeled circadian chromatin 3D structures, including Ikzf3-Nr1d1 super-enhancer, Dbp, and Npas2. Intestinal deletion of Nr3c1 specifically abolished these stress-induced transcriptional alternations relevant to IBS phenotypes in BALB/c mice. GR mediated Ikzf3-Nr1d1 chromatin disease related circadian misalignment in stress-induced IBS animal model. This animal model dataset suggests that regulatory SNPs of human IKZF3-NR1D1 transcription through conserved chromatin looping have translational potential based on the GR-mediated circadian-stress crosstalk.

Chronic psychological stress alters gene expression in rat colon epithelial cells promoting chromatin remodeling, barrier dysfunction and inflammation.

Chronic stress is commonly associated with enhanced abdominal pain (visceral hypersensitivity), but the cellular mechanisms underlying how chronic stress induces visceral hypersensitivity are poorly understood. In this study, we examined changes in gene expression in colon epithelial cells from a rat model using RNA-sequencing to examine stress-induced changes to the transcriptome. Following chronic stress, the most significantly up-regulated genes included Atg16l1, Coq10b, Dcaf13, Nat2, Ptbp2, Rras2, Spink4 and down-regulated genes including Abat, Cited2, Cnnm2, Dab2ip, Plekhm1, Scd2, and Tab2. The primary altered biological processes revealed by network enrichment analysis were inflammation/immune response, tissue morphogenesis and development, and nucleosome/chromatin assembly. The most significantly down-regulated process was the digestive system development/function, whereas the most significantly up-regulated processes were inflammatory response, organismal injury, and chromatin remodeling mediated by H3K9 methylation. Furthermore, a subpopulation of stressed rats demonstrated very significantly altered gene expression and transcript isoforms, enriched for the differential expression of genes involved in the inflammatory response, including upregulation of cytokine and chemokine receptor gene expression coupled with downregulation of epithelial adherens and tight junction mRNAs. In summary, these findings support that chronic stress is associated with increased levels of cytokines and chemokines, their downstream signaling pathways coupled to dysregulation of intestinal cell development and function. Epigenetic regulation of chromatin remodeling likely plays a prominent role in this process. Results also suggest that super enhancers play a primary role in chronic stress-associated intestinal barrier dysfunction.

Valproic acid-induced changes of 4D nuclear morphology in astrocyte cells.

Histone deacetylase inhibitors, such as valproic acid (VPA), have important clinical therapeutic and cellular reprogramming applications. They induce chromatin reorganization that is associated with altered cellular morphology. However, there is a lack of comprehensive characterization of VPA-induced changes of nuclear size and shape. Here, we quantify 3D nuclear morphology of primary human astrocyte cells treated with VPA over time (hence, 4D). We compared volumetric and surface-based representations and identified seven features that jointly discriminate between normal and treated cells with 85% accuracy on day 7. From day 3, treated nuclei were more elongated and flattened and then continued to morphologically diverge from controls over time, becoming larger and more irregular. On day 7, most of the size and shape descriptors demonstrated significant differences between treated and untreated cells, including a 24% increase in volume and 6% reduction in extent (shape regularity) for treated nuclei. Overall, we show that 4D morphometry can capture how chromatin reorganization modulates the size and shape of the nucleus over time. These nuclear structural alterations may serve as a biomarker for histone (de-)acetylation events and provide insights into mechanisms of astrocytes-to-neurons reprogramming.

Early treatment with exosomes following traumatic brain injury and hemorrhagic shock in a swine model promotes transcriptional changes associated with neuroprotection.

BACKGROUND: We have shown that administration of mesenchymal stem cell-derived exosomes (single dose given within 1 hour) in models of traumatic brain injury (TBI) and hemorrhagic shock is neuroprotective. The precise mechanisms responsible for the neuroprotection are not fully understood. This study was designed to investigate the transcriptomic changes in the brain that are associated with this treatment strategy. METHODS: Yorkshire swine (40-45 kg) were subjected to a severe TBI (12-mm cortical impact) and hemorrhagic shock (40% estimated total blood volume). One hour into shock, animals were randomized (n = 5/cohort) to receive either lactated Ringer's (LR; 5 mL) or exosomes suspended in LR (LR + EXO; 1 × 10 exosome particles in 5 mL LR). Animals then underwent additional shock (1 hour) followed by normal saline resuscitation. After 6 hours of observation, brain swelling (% increase compared with the uninjured side) and lesion size (mm) were assessed. Periinjured brain tissue was processed for RNA sequencing, analyzed with high through-put RNA sequencing data analysis, and results compared between control and experimental groups. RESULTS: Exosome treatment significantly increased (p < 0.005) gene expression associated with neurogenesis, neuronal development, synaptogenesis, and neuroplasticity. It also significantly reduced (p < 0.005) genes associated with stroke, neuroinflammation, neuroepithelial cell proliferation, and nonneuronal cell proliferation contributing to reactive gliosis. Exosome treatment also significantly increased (p < 0.005) the genes that are associated with stability of blood-brain barrier. CONCLUSIONS: Administration of a single dose of exosomes induces transcriptomic changes suggestive of neuroprotection. Their use as a treatment for TBI is promising and requires further investigation for human translation.

Early single-dose treatment with exosomes provides neuroprotection and improves blood-brain barrier integrity in swine model of traumatic brain injury and hemorrhagic shock.

BACKGROUND: Administration of human mesenchymal stem cell (MSC)-derived exosomes can enhance neurorestoration in models of traumatic brain injury (TBI) and hemorrhagic shock (HS). The impact of early treatment with MSC-derived exosomes on brain injury in a large animal model remains unknown. We sought to evaluate the impact of early single-dose exosome treatment on brain swelling and lesion size, blood-based cerebral biomarkers, and blood-brain barrier (BBB) integrity. METHODS: Female Yorkshire swine were subjected to a severe TBI (12-mm cortical impact) and HS (40% estimated total blood volume). One hour into shock, animals were randomized (n = 5/cohort) to receive either lactated Ringer's (LR; 5 mL) or LR + exosomes (1 × 10 exosome particles in 5 mL LR). Animals then underwent additional shock (1 hour) followed by normal saline resuscitation. After 6 hours of observation, brain swelling (% increase compared with the uninjured side) and lesion size (mm) were assessed. Cerebral hemodynamics and blood-based biomarkers of brain injury were compared. Immunofluorescence and RNA sequencing with differential gene expression and pathway analysis were used to assess the integrity of the perilesion BBB. RESULTS: Exosome-treated animals had significantly less (p < 0.05) brain swelling and smaller lesion size. They also had significantly decreased (p < 0.05) intracranial pressures and increased cerebral perfusion pressures. Exosome-treated animals had significantly decreased (p < 0.05) albumin extravasation and significantly higher (p < 0.05) laminin, claudin-5, and zonula occludens 1 levels. Differential gene expression and pathway analysis confirmed these findings. Serum glial fibrillary acidic protein levels were also significantly lower (p < 0.05) in the exosome-treated cohort at the end of the experiment. CONCLUSION: In a large animal model of TBI and HS, early treatment with a single dose of MSC-derived exosomes significantly attenuates brain swelling and lesion size, decreases levels of blood-based cerebral biomarkers, and improves BBB integrity.

A similarity-based approach to leverage multi-cohort medical data on the diagnosis and prognosis of Alzheimer's disease.

Motivation: Heterogeneous diseases such as Alzheimer's disease (AD) manifest a variety of phenotypes among populations. Early diagnosis and effective treatment offer cost benefits. Many studies on biochemical and imaging markers have shown potential promise in improving diagnosis, yet establishing quantitative diagnostic criteria for ancillary tests remains challenging. Results: We have developed a similarity-based approach that matches individuals to subjects with similar conditions. We modeled the disease with a Gaussian process, and tested the method in the Alzheimer's Disease Big Data DREAM Challenge. Ranked the highest among submitted methods, our diagnostic model predicted cognitive impairment scores in an independent dataset test with a correlation score of 0.573. It differentiated AD patients from control subjects with an area under the receiver operating curve of 0.920. Without knowing longitudinal information about subjects, the model predicted patients who are vulnerable to conversion from mild-cognitive impairment to AD through the similarity network. This diagnostic framework can be applied to other diseases with clinical heterogeneity, such as Parkinson's disease.

The pharmacoepigenomics informatics pipeline defines a pathway of novel and known warfarin pharmacogenomics variants.

AIM: 'Pharmacoepigenomics' methods informed by omics datasets and pre-existing knowledge have yielded discoveries in neuropsychiatric pharmacogenomics. Now we evaluate the generality of these methods by discovering an extended warfarin pharmacogenomics pathway. MATERIALS & METHODS: We developed the pharmacoepigenomics informatics pipeline, a scalable multi-omics variant screening pipeline for pharmacogenomics, and conducted an experiment in the genomics of warfarin. RESULTS: We discovered known and novel pharmacogenomics variants and genes, both coding and regulatory, for warfarin response, including adverse events. Such genes and variants cluster in a warfarin response pathway consolidating known and novel warfarin response variants and genes. CONCLUSION: These results can inform a new warfarin test. The pharmacoepigenomics informatics pipeline may be able to discover new pharmacogenomics markers in other drug-disease systems.

Transcriptomic changes following valproic acid treatment promote neurogenesis and minimize secondary brain injury.

BACKGROUND: Early treatment with valproic acid (VPA) has demonstrated benefit in preclinical models of traumatic brain injury, including smaller brain lesion size, decreased edema, reduced neurologic disability, and faster recovery. Mechanisms underlying these favorable outcomes are not fully understood. We hypothesized that VPA treatment would upregulate genes involved in cell survival and proliferation and downregulate those associated with cell death and the inflammatory response. METHODS: Ten female swine were subjected to a protocol of traumatic brain injury and hemorrhagic shock. They were assigned to two groups (n = 5): normal saline (NS; 3× volume of shed blood), or NS + VPA (150 mg/kg). Following 6 hours of observation, brain tissue was harvested to evaluate lesion size and edema. Brain tissue was processed for RNA sequencing. Gene set enrichment and pathway analysis was performed to determine the differential gene expression patterns following injury. RESULTS: Animals treated with VPA were noted to have a 46% reduction in brain lesion size and a 57% reduction in ipsilateral brain edema. Valproic acid significantly upregulated genes involved in morphology of the nervous system, neuronal development and neuron quantity. The VPA treatment downregulated pathways related to apoptosis, glial cell proliferation, and neuroepithelial cell differentiation. Ingenuity Pathway Analysis identified VPA as the top upstream regulator of activated transcription, supporting it as a direct cause of these transcriptional changes. Master transcriptional regulator NEUROD1 was also significantly upregulated, suggesting that VPA may induce additional transcription factors. CONCLUSION: Administration of VPA attenuated brain lesion size, reduced brain edema, and induced significant changes in the transcriptome of injured brain within 6 hours. Patterns of differential expression were consistent with the proposed neurogenic and prosurvival effects of VPA treatment.

Deep learning in pharmacogenomics: from gene regulation to patient stratification.

This Perspective provides examples of current and future applications of deep learning in pharmacogenomics, including: identification of novel regulatory variants located in noncoding domains of the genome and their function as applied to pharmacoepigenomics; patient stratification from medical records; and the mechanistic prediction of drug response, targets and their interactions. Deep learning encapsulates a family of machine learning algorithms that has transformed many important subfields of artificial intelligence over the last decade, and has demonstrated breakthrough performance improvements on a wide range of tasks in biomedicine. We anticipate that in the future, deep learning will be widely used to predict personalized drug response and optimize medication selection and dosing, using knowledge extracted from large and complex molecular, epidemiological, clinical and demographic datasets.

3D Shape Modeling for Cell Nuclear Morphological Analysis and Classification.

Quantitative analysis of morphological changes in a cell nucleus is important for the understanding of nuclear architecture and its relationship with pathological conditions such as cancer. However, dimensionality of imaging data, together with a great variability of nuclear shapes, presents challenges for 3D morphological analysis. Thus, there is a compelling need for robust 3D nuclear morphometric techniques to carry out population-wide analysis. We propose a new approach that combines modeling, analysis, and interpretation of morphometric characteristics of cell nuclei and nucleoli in 3D. We used robust surface reconstruction that allows accurate approximation of 3D object boundary. Then, we computed geometric morphological measures characterizing the form of cell nuclei and nucleoli. Using these features, we compared over 450 nuclei with about 1,000 nucleoli of epithelial and mesenchymal prostate cancer cells, as well as 1,000 nuclei with over 2,000 nucleoli from serum-starved and proliferating fibroblast cells. Classification of sets of 9 and 15 cells achieved accuracy of 95.4% and 98%, respectively, for prostate cancer cells, and 95% and 98% for fibroblast cells. To our knowledge, this is the first attempt to combine these methods for 3D nuclear shape modeling and morphometry into a highly parallel pipeline workflow for morphometric analysis of thousands of nuclei and nucleoli in 3D.

Publisher Correction: 3D Shape Modeling for Cell Nuclear Morphological Analysis and Classification.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.

Alterations in the human proteome following administration of valproic acid.

BACKGROUND: High doses of the histone deacetylase inhibitor valproic acid (VPA, 150-400 mg/kg) improve outcomes in animal models of lethal insults. We are conducting a US Food and Drug Administration-approved Phase I, double-blind, placebo-controlled trial to evaluate the safety and tolerability of ascending doses of VPA in human volunteers. We hypothesized that VPA would induce significant changes in the proteome of healthy humans when given at doses lower than those used in prior animal studies. METHODS: Peripheral blood mononuclear cells were obtained from three healthy subjects randomized to receive VPA (120 mg/kg over 1 hour) at baseline and at 4 and 8 hours following infusion. Detailed proteomic analysis was performed using 1D gel electrophoresis, liquid chromatography, and mass spectrometry. Proteins with differential expression were chosen for functional annotation and pathway analysis using Ingenuity Pathway Analysis (Qiagen GmbH, Hilden, Germany) and Panther Gene Ontology. RESULTS: A total of 3,074 unique proteins were identified. The average number of proteins identified per sample was 1,716 ± 459. There were a total of 140 unique differentially expressed proteins (p < 0.05). There was a minor and inconsistent increase in histone and nonhistone protein acetylation. Functional annotation showed significant enrichment of apoptosis (p = 3.5E-43), cell death (p = 9.9E-72), proliferation of cells (p = 1.6E-40), dementia (p = 9.6E-40), amyloidosis (p = 6.3E-38), fatty acid metabolism (p = 4.6E-76), quantity of steroid (p = 4.2E-75), and cell movement (p = 1.9E-64). CONCLUSIONS: Valproic acid induces significant changes to the proteome of healthy humans when given at a dose of 120 mg/kg. It alters the expression of key proteins and pathways, including those related to cell survival, without significant modification of protein acetylation. In the next part of the ongoing Phase I trial, we will study the effects of VPA on trauma patients in hemorrhagic shock. LEVEL OF EVIDENCE: Therapeutic study, level V.

Epigenomic mapping and effect sizes of noncoding variants associated with psychotropic drug response.

AIM: To provide insight into potential regulatory mechanisms of gene expression underlying addiction, analgesia, psychotropic drug response and adverse drug events, genome-wide association studies searching for variants associated with these phenotypes has been undertaken with limited success. We undertook analysis of these results with the aim of applying epigenetic knowledge to aid variant discovery and interpretation. METHODS: We applied conditional imputation to results from 26 genome-wide association studies and three candidate gene-association studies. The analysis workflow included data from chromatin conformation capture, chromatin state annotation, DNase I hypersensitivity, hypomethylation, anatomical localization and biochronicity. We also made use of chromatin state data from the epigenome roadmap, transcription factor-binding data, spatial maps from published Hi-C datasets and 'guilt by association' methods. RESULTS: We identified 31 pharmacoepigenomic SNPs from a total of 2024 variants in linkage disequilibrium with lead SNPs, of which only 6% were coding variants. Interrogation of chromatin state using our workflow and the epigenome roadmap showed agreement on 34 of 35 tissue assignments to regulatory elements including enhancers and promoters. Loop boundary domains were inferred by association with CTCF (CCCTC-binding factor) and cohesin, suggesting proximity to topologically associating domain boundaries and enhancer clusters. Spatial interactions between enhancer-promoter pairs detected both known and previously unknown mechanisms. Addiction and analgesia SNPs were common in relevant populations and exhibited large effect sizes, whereas a SNP located in the promoter of the SLC1A2 gene exhibited a moderate effect size for lithium response in bipolar disorder in patients of European ancestry. SNPs associated with drug-induced organ injury were rare but exhibited the largest effect sizes, consistent with the published literature. CONCLUSION: This work demonstrates that an in silico bioinformatics-based approach using integrative analysis of a diversity of molecular and morphological data types can discover pharmacoepigenomic variants that are suitable candidates for further validation in cell lines, animal models and human clinical trials.

A glutamatergic network mediates lithium response in bipolar disorder as defined by epigenome pathway analysis.

AIM: A regulatory network in the human brain mediating lithium response in bipolar patients was revealed by analysis of functional SNPs from genome-wide association studies (GWAS) and published gene association studies, followed by epigenome mapping. METHODS: An initial set of 23,312 SNPs in linkage disequilibrium with lead SNPs, and sub-threshold GWAS SNPs rescued by pathway analysis, were studied in the same populations. These were assessed using our workflow and annotation by the epigenome roadmap consortium. RESULTS: Twenty-seven percent of 802 SNPs that were associated with lithium response (13 published studies gene association studies and two GWAS) were shared in common with 1281 SNPs from 18 GWAS examining psychiatric disorders and adverse events associated with lithium treatment. Nineteen SNPs were annotated as active regulatory elements such as enhancers and promoters in a tissue-specific manner. They were located within noncoding regions of ten genes: ANK3, ARNTL, CACNA1C, CACNG2, CDKN1A, CREB1, GRIA2, GSK3B, NR1D1 and SLC1A2. Following gene set enrichment and pathway analysis, these genes were found to be significantly associated (p = 10(-27); Fisher exact test) with an AMPA2 glutamate receptor network in human brain. Our workflow results showed concordance with annotation of regulatory elements from the epigenome roadmap. Analysis of cognate mRNA and enhancer RNA exhibited patterns consistent with an integrated pathway in human brain. CONCLUSION: This pharmacoepigenomic regulatory pathway is located in the same brain regions that exhibit tissue volume loss in bipolar disorder. Although in silico analysis requires biological validation, the approach provides value for identification of candidate variants that may be used in pharmacogenomic testing to identify bipolar patients likely to respond to lithium.