Transcriptome Alterations in Posttraumatic Stress Disorder

Accepted Manuscript Transcriptome Alterations in Post-traumatic Stress Disorder Matthew J. Girgenti, Ronald S. Duman PII:

S0006-3223(17)32045-0

DOI:

10.1016/j.biopsych.2017.09.023

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BPS 13336

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Biological Psychiatry

Received Date: 21 April 2017 Revised Date:

5 September 2017

Accepted Date: 17 September 2017

Please cite this article as: Girgenti M.J. & Duman R.S., Transcriptome Alterations in Post-traumatic Stress Disorder, Biological Psychiatry (2017), doi: 10.1016/j.biopsych.2017.09.023. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Transcriptome Alterations in Post-traumatic Stress Disorder Matthew J. Girgenti and Ronald S. DumanƢ

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Laboratory of Molecular Psychiatry, Center for Genes and Behavior, Department of Psychiatry, Yale University School of Medicine, New Haven, CT., USA, 06508

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Words in Abstract: 140 Words in Text: 4000 Number of Tables: 2 Number of Figures: 3 Number of Supplemental Figures: zero

Short Title: Transcriptomic Approaches to Characterize PTSD

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Key Words: PTSD; stress; transcriptomics; RNA-sequencing; genomics; prefrontal cortex

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Ƣ Address all correspondences to: Dr. Ronald S. Duman Elizabeth Mears and House Jameson Professor of Psychiatry and Pharmacology Director, Abraham Ribicoff Research Facilities Yale University School of Medicine Phone: (203) 974-7726 E-mail: [email protected]

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ABSTRACT

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Post-traumatic stress disorder (PTSD) is a debilitating psychiatric disorder with a life-time prevalence of nearly 8% in the general population. While the underlying molecular and cellular mechanisms of PTSD remain unknown, recent studies indicate that PTSD is associated with aberrant gene expression in brain as well as peripheral blood cells. The advent of next generation sequencing technologies will allow us to elucidate the gene expression changes occurring in both brain and blood of patients with PTSD. RNA sequencing allows for analysis of the amount of transcript being made as well as alternative splicing, novel transcript identification, micro-RNA, and noncoding RNA quantification. Here we provide an overview of the different types of transcriptomic technologies, the gene expression studies performed in human peripheral blood and animal models of PTSD, and review the human PTSD post-mortem brain gene profiling studies performed to date.

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development remain largely unknown (1). The ability to integrate genetic

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INTRODUCTION The pathophysiology of PTSD and the molecular changes underlying it’s

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information with disease states has been at the forefront of biomedical science

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research with the hope of identifying gene mutations that underlie psychiatric, as

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well as other types of illnesses. However, the possibility that a single mutation, or

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even a small number of mutations are responsible for a complex illness like

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schizophrenia (2), depression (3) or PTSD (4) has largely been disproved.

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An assortment of molecular tools has been developed in the intervening

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years to analyze these genetic changes. Perhaps most notable is the rise of

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technologies to analyze the output of the genetic code: the transcriptional

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profiles. The advent of large-scale massively parallel sequencing technologies

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(deep sequencing) has revolutionized our understanding of the molecular

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underpinnings of disease states. The ability to interrogate a complete read-out of

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the genomic, transcriptomic, and epigenomic landscape of a tissue or even

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individual cells of a particular tissue will give unparalleled insight into the

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molecular causes of many diseases. In addition, these studies hold the promise

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of identifying core biological features and signaling pathways that could be

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influenced by multiple gene mutations that converge to contribute to the neuronal

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abnormalities that underlie PTSD.

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PTSD generally develops from single or repeated exposure to traumatic

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events, and is characterized by intrusive memories, avoidance of stimuli that are

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reminders of the event, recurrent nightmares, and persistent hyperarousal (5).

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Interestingly, the largest GWAS of PTSD individuals revealed significantly higher

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genetic risk overlap with schizophrenia than with MDD or bipolar depression (6).

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Clinical and preclinical studies have demonstrated three principle brain regions

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involved in fear learning and modulation. The amygdala, a brain region involved

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in fear memory (Figure 1) (7-11) is hyperactive in patients with PTSD (12). In

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addition, the hippocampus (13; 14) and medial prefrontal cortex (mPFC) (15-19)

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have been shown to directly modulate fear responsiveness of the amygdala in

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humans as well as in preclinical models. Imaging studies have shown

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dysregulation of both the hippocampus and mPFC in patients with PTSD(20).

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Studies of alterations of gene expression in these regions and the epigenetic

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mechanisms underlying these changes will help to elucidate fundamental

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neurobiological processes of PTSD. It is possible that even though multiple gene

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mutations may contribute to PTSD in different individuals, there may be

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convergent underlying neurobiological gene expression changes that contribute

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to PTSD symptoms. In addition, because of the inherent difficulty of examining

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human brain tissue in patients, studies of gene expression in peripheral blood are

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a necessary proxy, though there are obvious limitations and additional studies of

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post-mortem brain are needed.

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Here we will focus on the approaches for investigating disease-related

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changes in gene expression at the messenger RNA (mRNA) level. We will begin

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by discussing the current technologies available for measuring alterations of

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gene expression. We then summarize the recent publications describing gene

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expression changes in the periphery of patients with PTSD and how they may

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impact our understanding of the disorder.

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differential gene expression studies of preclinical rodent models of PTSD and

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how these results inform and correlate with transcript changes observed in blood.

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Examination of these topics will set the stage for a discussion of studies done in

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post-mortem brains of PTSD subjects and development of a large PTSD brain

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bank to further pursue this work.

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We will then examine current

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Gene Expression Profiling of Neuropsychiatric Disorders The development and maturation of neurons and their connections and

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optimization of circuit formation is critical for fine-tuning all aspects of brain

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function, including cognition, information processing, and complex behaviors.

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One of the most important processes in the development and maintenance of

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these circuits is proper control of gene expression. The significance and impact

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of gene expression on all aspects of brain function has driven the need for

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technical and conceptual breakthroughs that allow for characterization of the

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specific complement of mRNA transcripts.

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Recent advances in our knowledge of the genomic structure of humans

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and other mammals have directed the emergence of sequencing approaches to

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characterize and quantify gene expression products. Next generation sequencing

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(NGS) provides three to four magnitudes more sequencing information than

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previous systems. The key feature of these systems is combining traditional

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DNA sequencing technologies with microfluidics, to allow for optimal spatial

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arrangement of each transcript and efficient scanning of individual base pairs on

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a small flow cell.

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NGS has been applied to many different types of DNA and RNA sequence

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analysis: genome sequencing (DNA-seq)(21), mRNA profiling (RNA-seq) (22;

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23),

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characterizations (26). RNA-seq uses the latest NGS technology to sequence the

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transcriptome and generate gene expression profiles from many types of

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samples. RNA is extracted from the tissue of interest and cDNA libraries are

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generated. Adapter sequences are added to the 3’ and 5’ ends. These linker

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adapters allow the cDNA to attach to the flow cell where sequencing occurs.

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Each cDNA fragment (75-100 bp) is sequenced.

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“reads” are aligned to a known reference genome or can be assembled without

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reference (Figure 1B). This allows for unprecedented analysis of not only what

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is expressed, but how it is expressed (i.e. alternative splicing, exonic SNP

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identification, and novel transcript prediction). RNA-seq has been used to identify

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the transcript changes in schizophrenia (27), major depressive disorder (28; 29),

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bipolar (29), and drug dependence (30). There are currently few studies using

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RNA-seq on human PTSD samples (31; 32). This is due in large part to the lack

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of an organized repository for post-mortem PTSD brain tissue, a limitation

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currently being addressed with the development of a new VA repository

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dedicated to the collection of high quality tissue from PTSD subjects (33).

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interactions

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These resulting sequence

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DNA-protein

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Transcript changes in blood of PTSD patients: dysfunction of immune

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response and glucocorticoid signaling

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Studies to identify biomarkers of PTSD have focused largely on changes

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in gene expression in blood of patients with PTSD (Table 1).

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commonly used cell source for examining gene expression in humans because

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of its easy availability. Although there are obvious limitations for brain disorders,

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Blood is a

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peripheral blood likely reflects some changes occurring in the rest of body and

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can act as proxy for detection of relevant gene transcript alterations and/or may

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reveal biomarkers to aid in diagnosis and treatment response.

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peripheral blood contains cells of the immune system, such as lymphocytes that

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could directly impact brain function (34). Alterations of the hypothalamic-pituitary-

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adrenal (HPA) axis and glucocorticoid function, in particular, have been shown to

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be important in the onset of PTSD. Therefore, it is possible that changes in the

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blood transcriptome may reflect some changes in the brain and offer insight into

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the underlying pathophysiology of PTSD.

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In addition,

A number of studies have examined blood gene expression and

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glucocorticoid activity to identify the molecular underpinnings of PTSD (Table 1

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and Figure 2).

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inflammatory control plays a role in PTSD(35; 36). Several of these studies

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identified down regulation of interluekins-16 and -18 (IL-16 and !L-18) and

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colony-stimulating factor expression (37; 38); three monocyte

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(39), and down regulation of the major histocompatabiity complex II (40). These

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same studies have also identified Fkbp5 and Stat5b gene expression as being

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down regulated in PTSD patients(38; 40; 41).

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known roles in regulating inflammatory and glucocorticoid responses (42; 43).

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Fkbp5 protein in particular is a chaperone of the glucocorticoid receptor (GR)

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(42) that prevents translocation of GR to the nucleus where it regulates many

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genes that contain GR promoter elements(44; 45). Gene association studies

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have identified four SNPs (rs9470080, rs360780, rs3800373, and rs9296158) in

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the Fkbp5 gene that are predictors of adult PTSD onset(38; 46). The significance

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of these studies on blood Fkbp5 gene expression are highlighted by a preliminary

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transcriptomic study of post-mortem brain tissue of PTSD subjects, which also

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demonstrates significantly decreased expression of Fkbp5 expression in the

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subgenual PFC(47).

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specific genes

Fkbp5 and Stat5B have well

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The current literature suggests that disruption of immune-

Glucocorticoid dysfunction is thought to be a contributing factor to the

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development of PTSD.

One study looked at whether components of GC

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signaling could be used as a predictor of PTSD development (48).

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assessed GC receptor (GR) number, and mRNA expression of Fkbp5, and Gilz

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(glucocorticoid-induced leucine zipper) in military personnel prior to deployment.

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They found that high GR number, low Fkbp5 and high Gilz expression predicted

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PTSD development. This same group further investigated whether GC sensitivity

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of leukocytes in soldiers predicted the development of PTSD(49). After their

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return, these soldiers were assessed for level of PTSD, depressive, and fatigue

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symptoms.

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development. One study found that GR sensitivity on leukocytes (regardless of

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GR number, GR subtype, and GR target mRNA levels) and low levels of Fkbp5

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mRNA were predictors of PTSD development(48). The same group reported that

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combat soldiers with high sensitivity of GRs on leukocytes pre-deployment were

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also more likely to develop PTSD(49). Conversely, it has been reported that

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trauma exposed victims have lower GR sensitivity in peripheral blood monocytes

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(PBMCs) and low gene expression levels of GR. A recent study performed RNA-

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seq and microarray analysis on U.S. Marines, both pre- and post-deployment to

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conflict zones (31). This group conducted higher order analysis of gene networks

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rather than focus on individual genes, and identified changes in modules related

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to wound healing and homeostasis. Interestingly, they identified dysregulation of

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an innate immune response module in soldiers pre-deployment who later

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developed PTSD (Figure 3).

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Higher sensitivity of GRs on leukocytes predicted PTSD

Alterations of transcription control mechanisms are also affected in PTSD.

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One study reported decreased levels of blood Dicer transcript that correlated with

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increased activation of the amygdala in response to fearful stimuli (50). Dicer is

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an endonuclease that cleaves double-stranded RNA and is responsible for

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processing pre-microRNA into smaller RNA fragments such as small interfering

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RNAs and microRNAs. It is interesting to note that another study reported that

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the Dicer pathway is also altered in a chronic social defeat stress model of

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depression (51). miRNA dysfunction is an attractive hypothesis as it is possible

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that a broad transcriptional regulator that lies upstream of many other regulated

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genes could underlie PTSD.

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These transcriptional studies of peripheral blood have yielded important

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information, including evidence of impaired glucocorticoid signaling during PTSD,

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transcriptional changes in immune and inflammatory response genes before and

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after the onset of PTSD, and elevated proinflammatory factors associated with

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the pathophysiology of PTSD. These findings could provide biomarkers of illness

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and treatment response and clues to the pathophysiology of PTSD, but it is clear

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that studies of blood cannot fully explain brain pathophysiology.

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There are several established animal models for PTSD that are being

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used to help elucidate the molecular and cellular basis of fear related disorders.

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A powerful feature of animal models is the ability to manipulate all aspects of a

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stressor including time interval, duration, and intensity with no preexisting

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traumatic events and an essentially identical life history leading up to the test (52;

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53).

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conditioning, predator-scent-stress models, and social defeat (54).

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Typical animal models of PTSD include variations of classical fear

Studies of gene expression in animal models of PTSD have focused on

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long-term recovery, with the assumption that sustained alterations could be

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related to the long-lasting changes that underlie PTSD. One group found major

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changes in the amygdala of rats subjected to stress-enhanced fear learning

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(SEFL), in which exposure to stress increases the memory of fearful cues (55).

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Three weeks after foot shock exposure, gene expression changes were

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measured in the lateral and basolateral amygdala. Neuron- and glia-specific

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modules were identified with considerable overlap suggesting a coordination of

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transcription changes that could be mediated by cross talk between these two

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cell types (55).

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One elegant study used predator-scent-stress to identify and correlate

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genome-wide transcript changes in amygdala and hippocampus with gene

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expression changes in peripheral blood (56). They identified transcription factor

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signaling upstream of gene expression changes to describe convergent signaling

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across brain, blood, and sex. Notably, they identified the GR signaling pathway

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as the only convergent pathway. Several other studies have also examined

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correlations between brain and blood transcriptome changes in PTSD and other

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stress models.

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hippocampus after short and long-term recovery to chronic social defeat or

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restraint stress (57). They identified regulation of genes and pathways associated

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with glucocorticoid signaling and inflammation(57). Interestingly, both of these

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studies (56; 57) found regulation of the gene encoding serum/glucocorticoid

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kinase 1 (Sgk1) that was recently reported to be down regulated in postmortem

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dorsolateral PFC of PTSD subjects (58).

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One study mapped gene expression changes in blood and

Another recent report used a social-stress model where mice were

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exposed to an aggressive intruder and were given follow-up reminders (exposure

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without physical contact) of the trauma after rest periods (59). Whole-genome

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arrays were used to map transcription changes across a wide variety of tissues

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from both the aggressor, exposed and control mice and across several time

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points after exposure. They identified alterations of several pathways associated

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with PTSD, including HPA axis function, modulators of GR activity, neuronal

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transmission, and fear memory and extinction.

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Chronic social defeat stress (CSDS) has emerged as a widely used psychosocial stressor in animals.

While generally considered a model of

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depression(60), animals exposed to CSDS exhibit long-term signs of anxiety

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(61) and submissive animals but not dominant animals display elevated plasma

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levels of ACTH and glucocorticoids, suggesting psychological, not just physical

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stress as the cause (62). Two recent papers have used RNA-seq on four brain

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regions in animals that were either resistant or susceptible to CSDS (63). An

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integrative network approach was used to identify transcriptional networks and

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hub genes related to depressive-like symptoms. Transcript changes associated

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with susceptibility of depressive-like phenotypes showed opposite regulation in

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the hippocampus versus the PFC. Another study from the same group identified

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changing transcription patterns for susceptibility and resilience during CSDS and

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overlaid the changes caused by treatment with the conventional antidepressant

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imipramine versus the rapid-acting antidepressant ketamine (64). This uniquely

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large data set boasts numerous comparisons and provides a useful standard for

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future transcriptomic studies of rodent PTSD models. Gene expression profiling in animal models of PTSD can provide

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information on genes and pathways regulated in fear disorders. Because these

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studies are able to directly examine brain tissue many of the regulated pathways

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that were uncovered were different from those identified in peripheral blood.

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Transcripts involved in neuromodulation and neurotransmitter regulation were

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identified in several studies (55; 59; 65). It is interesting to note that the animal

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gene expression studies also identified dysregulation in glucocorticoid and

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inflammatory processes similar to those identified in whole blood (56; 59; 66).

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However, it is important to point out the obvious caveats associated with animal

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studies of complex disorders such as PTSD. Rodents are usually inbred and

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lack genetic diversity, which is likely a major contributing factor to the onset of

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PTSD pathology. Therefore while these results suggest that it is possible to

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validate some molecular changes observed in blood and brain, it is necessary to

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directly examine transcriptional changes occurring in humans with PTSD.

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Transcript profiles of human post-mortem PTSD brain

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Human post-mortem genomic studies of brain tissue promise the best

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source of information to unravel the complex brain molecular underpinnings of

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PTSD. Unfortunately, there have been only a few such studies (see published

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gene expression changes in Table 2, Figure 2). Here we will review the current

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PTSD post-mortem studies and discuss the design and impact of future studies.

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The first PTSD post-mortem study examined a single transcript target in a

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small cohort of subjects, identified up regulation of a multifunction protein p11,

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that interacts with 5-HT receptors in dorsolateral PFC (67). This same PTSD

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cohort was also subjected to the first multi-transcript post-mortem gene

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expression profiling study (58).

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significant up regulation of 42 transcripts and down regulation of 231 transcripts;

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one of the most highly regulated genes was Sgk1, which was decreased by 80

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percent compared to matched control subjects. Further studies in rodent models

Whole genome microarray analysis showed

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showed that viral overexpression of dominant negative Sgk1 (dnSGK1) in an

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auditory fear conditioning model caused higher levels of freezing in the

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contextual memory recall test three days after conditioning. This indicates that

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SGK1 inhibition, and presumably decreased Sgk1 expression, leads to enhanced

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memory of contextual cues associated with fear conditioning.

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overexpression of dnSGK1 also decreased dendritic spine density in medial PFC

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and decreased amplitude and frequency of spontaneous mini excitatory post

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synaptic currents (mEPSCs), suggesting a role for Sgk1 in structural, as well as

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functional changes in PTSD patients.

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Interestingly,

One recent study combined live human imaging and post-mortem gene

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expression studies (47). Photon emission topography (PET) was used to show

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that mGluR5 binding in PFC is increased in patients with PTSD, suggesting

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dysfunction in glutamate cycling and/or transmission. Quantitative PCR analysis

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of post-mortem subgenual PFC did not reveal regulation of mGlur5 transcripts

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but did show significant increases in SH3 and multiple ankyrin repeat domains 1

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(Shank1) transcription. Shank1 protein is responsible for tethering mGluR5 to

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the cell surface and could underlie the increased mGluR5 binding observed in

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PTSD patients. This post-mortem study also identified down regulation of Fkbp5

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in subgenual PFC, consistent with reports of decreased Fkbp5 in blood,

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suggesting a role for glucocorticoid signaling in brain of PTSD subjects.

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A recent report has highlighted the need for integration of gene expression

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analysis with genome wide association studies of PTSD (32). This study tested

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for expression quantitative trait loci (eQTLs) with several positive as well as

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candidate risk alleles. While the study was ultimately unable to find eQTL for

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GWAS positive SNPs, it did identify potential candidate PTSD risk SNPs that

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approached significance. They found that SNP rs363276 in intron 14 of the

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Solute Carrier Family 18 Member A 2 (Slc18a2) is associated with transcript

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levels of this gene as well as those of PDZ Domain Containing 8 (Pdzd8) in the

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dorsolateral PFC and to physiological responses of the amygdala during

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exposure to fearful and angry faces measured by BOLD fMRI..

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preliminary study is promising it should be noted that no PTSD regulated genes

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identified to date contain risk alleles identified in the largest PTSD GWAS study

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conducted recently (6). There is now a major effort supported by the VA and under the direction of

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the National Center for PTSD to develop a brain bank that will provide

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postmortem tissue from PTSD subjects and matched controls for large scale

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deep sequencing, transcriptome studies (33). Future studies should focus on key

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brain regions implicated in PTSD pathophysiology, including the PFC, amygdala,

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hippocampus, and hypothalamus as well as sub-regions of these areas (Figure

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1A).

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increasingly important to develop analysis pipelines that identify the most

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important alterations in gene networks, including key hub genes in these

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networks.

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association studies to transcript levels will likely yield important targets for further

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study. In addition, integration of mRNA and protein data from mass spectrometry

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could provide insights into dysregulated biological processes not detected by

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either technology individually. A further extension will be to integrate DNA

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methylation studies to provide information on epigenetic changes that contribute

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to alterations of gene expression in PTSD (Figure 1C). For example, epigenetic

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alterations could explain how early life trauma increases susceptibility in later life,

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and could even explain transgenerational susceptibility to psychiatric illness

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including fear, anxiety, and PTSD (68).

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As highlighted recently(32), matching previous genome-wide

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RNA-seq studies yield a large amount of data and it will become

Development of Novel Therapeutic Approaches

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Understanding the transcriptome changes of patients with PTSD is a

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critical first step in identifying all of the molecular signaling pathways involved in

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the underlying pathophysiology. In addition, a potential complement to these

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approaches is cellular reprogramming including induced pluripotent stem cells

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(iPSCs) that can be generated from accessible somatic cells from patients

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suffering from psychiatric disorders(69). The exogenous introduction of specific

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transcription factors can generate region and neurotransmitter-specific neural cell

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types that would allow for modeling of psychiatric disorders(70). The ability to

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examine the neurodevelopment of these induced neurons from PTSD patients

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compared to controls could shed light on the molecular changes that underlie

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PTSD. High through-put transcriptomic studies such as RNA-seq can also

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provide an important approach for investigating drug induced expression

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changes, and may help to accelerate the process of identifying novel drug

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targets. In the case of PTSD, it would be particularly interesting to examine

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changes occurring in synaptic or glucocorticoid related transcripts. For example,

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it

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dexamethasone dynamically regulates and normalizes Fkbp5 transcript levels in

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brain tissue of animal models (71) and peripheral blood taken from depressed

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human patients (72). These types of studies can be further refined by targeting

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brain regions that are dysfunctional in PTSD (Figure 1A). For example, based on

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evidence that extinction of traumatic/fearful events requires connectivity of

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infralimbic PFC and amygdala (16), we proposed that the rapid acting

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antidepressant ketamine, which increases synaptic connections in the infralimbic

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PFC, would enhance fear extinction learning. Consistent with this hypothesis, we

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have reported that pretreatment with ketamine enhances fear extinction learning,

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a preclinical model with relevance for understanding the neurobiological

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mechanisms required for extinction of traumatic events(73). This finding is also

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consistent with clinical studies reporting that ketamine rapidly decreases

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symptom severity in PTSD patients (74). This type of information coupled with

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transcriptome and epigenetic analysis will provide critical information for

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development of novel therapeutic interventions.

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Conclusion and Summary Studies to characterize the gene transcription changes that are involved in

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PTSD are still at an early stage, but a hypothesis is starting to emerge from

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currently available results (Figure 3) that is consistent with recent models of

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glucocorticoid signaling and Fkbp5

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patients that are susceptible to or have PTSD. Low blood levels of Fkbp5 and

(1; 42; 75).

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GC levels are low in both

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Gilz (an ant-inflammatory protein regulated by GC signaling) in conjunction with

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altered transcription levels of key innate immune system components predicts

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PTSD susceptibility. This model further shows that after trauma and onset of

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PTSD GC and Fkbp5 levels remain low (levels of Sgk1 are currently being

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examined).

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dissociation of GR and FKBP5, and subsequent translocation of GC-GR complex

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to regulate gene transcription.

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signaling to the nucleus, which then increases Fkbp5 and Sgk1 transcription

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through the GC response element (GRE). In PTSD, low levels of Fkbp5 and

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Sgk1 have been reported in post-mortem PFC. This is expected based on the

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decreased availability of GR after PTSD onset.

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transcription of many genes that contain GREs, as well as other downstream

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signaling pathways. It is possible that these aberrant transcriptional programs

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are related to the observed hypofunction of the PFC, but the mechanistic link

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between low Fkbp5/Sgk1 expression and PFC hypofunction remains unknown.

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Importantly, PFC hypofunction results in hyperfunction of the amygdala and

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contributes to PTSD symptoms, including increased anxiety and fear memory

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and decreased fear extinction.

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Under basal conditions GC binds to it’s receptor(GR), causing

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This includes feedback that increases GR

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This could potentially effect

Gene abundance represents only part of the complexity of the

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transcriptome. RNA splicing and RNA editing, as well as expression levels (76)

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are all critically important for normal brain function and it is likely that dysfunction

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in these processes contribute to the pathophysiology of PTSD. Studies in blood

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have

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dysregulation of GR sensitivity, innate immune, and inflammation pathways.

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Interestingly, many of these findings appear to be confirmed by animal models of

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PTSD.

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demonstrate some agreement with whole blood of PTSD patients and animal

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model studies. Further, we will need to determine how gene expression changes

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observed are influenced by risk variants identified by GWAS. Along this same

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line, it will be important to determine if the changes in transcript levels are a

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cause or a consequence of PTSD . It is likely that we are at the very early stages

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of investigation and more extensive deep sequencing of PTSD brain subregions

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will identify complex gene networks that underlie the pathophysiology of PTSD,

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which could provide novel targets for therapeutic intervention.

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Acknowledgements

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This work is supported by NIMH R01MH93897 (RSD), the State of Connecticut,

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Yale University, and the VA National Center for PTSD.

471 R.S. Duman has consulted and/or received research support from Naurex, Lilly,

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Forest, Johnson & Johnson, Taisho, and Sunovion. M.J. Girgenti reports no

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biomedical financial interests or potential conflicts of interest.

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Figure Legends Figure 1: RNA-seq of human post-mortem brain tissue to identify molecular changes in PTSD neuropathology. (A) Several brain regions have been implicated in PTSD based on imaging studies of PTSD patients, including the vmPFC, amygdala, and hippocampus. To identify the molecular and cellular mechanisms underlying the neurobiology of PTSD, next generation analysis of gene expression must be conducted. (B) We have begun using high through-put RNA-seq to identify the transcriptomic changes in three cortical regions (subgenual, dorsolateral, and orbitofrontal), amygdala and hippocampus. A illustration of the primary steps in the RNA-seq process. Briefly, mRNA is isolated from dissected region-specific brain tissue from PTSD and matched controls. cDNA synthesis and adapter ligation are performed on the RNA and loaded onto a flow cell containing a lawn of matching adapters, allowing the

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individual cDNAs to bind. Cluster growth begins and fluorescently tagged nucleotides are added to the matching base pair of the cDNA. The nucleotides are imaged and the sequence of the colored nucleotides allows for base calling or sequencing. The reads generated are then matched to the current human genome (Hg19) and differential gene expression is measured. (C) Volcano matrix plots for all gene expression changes in PTSD, MDD, and matched controls in human subgenual PFC. Red dots indicate statistically significant genes. RNAseq informs the proteomics assayed using tandem mass spectrometry for protein expression and alternative splicing. Overlay of CpG island methylation on detected transcript changes allows for further identification of the epigenetic alterations that contribute to differential gene expression.

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Figure 2: PTSD associated changes in gene expression in human brain and peripheral blood. Decreased levels of Fkbp5 and Sgk1 gene expression have been identified in subgenual PFC and dorsolateral PFC indicating dysregulation of glucocorticoid signaling in CNS. The mGlur5 anchoring protein Shank1 is increased in subgenual PFC. This correlates with observed increases in mGlur5 protein binding availability, believed to be associated with the avoidance symptoms of PTSD. Studies of peripheral blood have identified gene expression changes associated with susceptibility to developing PTSD (left), as well as transcript changes in patients that were diagnosed with PTSD at the time of analysis (right). In some cases the gene lists were too long to be included in the figure. The differentially expressed genes have been summarized as reported pathways. Collectively, these findings suggest that a predisposition to developing PTSD may be attributed to aberrant expression of innate immune response leading to glucocortidcoid dysfunction during the onset of the disease.

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Figure 3: Proposed Model of Aberrant Transcription in PTSD. Normal levels of GCs are necessary for regulating transcription throughout the body and the CNS. Under basal conditions, GC receptor (GR) is bound to FKBP5 in the cytoplasm and is inactive until sufficient levels of GCs result the bound GC-GR state, leading to dissociation from FKBP5. The activated GC-GR complex then undergoes translocation to the nucleus where GR binds to the GC response element (GRE) in the genome to regulate gene transcription. Low levels of GCs have been reported in patients with PTSD and a preexisting low level prior to trauma is thought to predispose or create a susceptible condition in some patients to develop PTSD. Low levels of GCs could then results in decreased transcriptional regulation of genes with GREs, including Fkbp5 and Sgk1 and their downstream effectors. This may contribute to the observed hypofunction of the vmPFC in PTSD patients, which in turn limits top-down control and hyperfunction of the amygdala. Increased or uncontrolled function of the amygdala is directly related to the symptoms of PTSD, including increased fear learning and decreased fear extinction. There is currently no direct tie between alteration in CNS transcription and the reported changes in blood of the immune and inflammatory response in PTSD, though it is likely that these changes affect one another (indicated by the bidirectional area between blood and PFC).

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ACCEPTED MANUSCRIPT Perturbed Pathway(s) IGF and interleukin

RNA Source Individuals with PTSD

signaling FKBP5, STAT and MHC

Survivors of 9/11

RNA Analysis

Study

Customized

Zieker, et al.,

Microarray

2007 (37)

qRT-PCR

Yehuda, et al., 2009 (40)

Monocyte gene

Trauma victims

signaling

Microarray

Nuclear Factor and

Survivors of 9/11

pathway signaling with

income bracket

FKBP5 SNPs interacts Nuclear factor-ΚB

Female child abuse

signaling increased

victims with PTSD

High expression of GR,

Military personnel

low FKBP5 and high

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GILZ predict PTSD development

Microarray

Sarapas, et al.

Trauma exposed

decreased in PBMCs

military personnel

Changes in Innate

Military personnel

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GR signaling was

Dicer1 expression

Traumatized

reduced in PTSD

individuals. Partial

Mehta, et al.,

2011 (38)

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Individuals in low

PTSD development

2011 (39)

2011 (41)

Interleukin and Stat

expression predicts

Microarray

Neylan et al.,

SC

Stat5b signaling

Immunity gene

Customized

RI PT

signaling

qRT-PCR

qRT-PCR

qRT-PCR

Pace, et al., 2012 (36) van Zuiden, et al., 2012 (48)

Matic, et al., 2013 (77)

Microarray

Breen, et al.,

and RNA-seq

2015 (31)

Microarray

Wingo, et al., 2015 (50)

cohort PTSD with comorbid depression

Table 1. Transcriptomic studies from human peripheral blood

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Perturbed Pathway

RNA Source

RNA Analysis

Study

Prefrontal Cortex Candidate transcript

Zhang, et al., 2008

signaling

(Area 46)

qRT-PCR

(67)

SGK1 signaling

Dorsolateral

Whole Genome

Licznerski, et al.,

PFC

Microarray

Glutamate and

Subgenual PFC

Candidate transcript

Glucocorticoid

(Area 25)

qRT-PCR

Significant eQTL

Healthy

RNA-sequencing

association with

Dorsolateral

PTSD risk SNPs

PFC

RI PT

Glucocorticoid

2015 (58)

Holmes, et al., 2017 (47)

SC

signaling

Bharadwaj, et al.,

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2016 (32)

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Table 2. Transcriptomic studies from post mortem PTSD brain tissue

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Figure 1.! A.!

Dorsolateral PFC!

Hippocampus:! CA1, CA3, dentate ! gyrus!

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Orbitofrontal PFC! Subgenual PFC! Orbitofrontal PFC!

AAAA!

2. cDNA Synthesis/ Adapter Ligation!

AAAA!

RNA-seq!

C.! Mass Spectrometry! Proteomics!

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1. mRNA Isolation!

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Amygdala:! BLA, CeA!

TTTT!

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3. Cluster Growth!

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T! A! C! G! G! C! A! T!

4. Base Calling! AGCT!

Bisulfite Chip! CpG Methylation!

Figure 2.!

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Brain-PTSD! SGK1 !P11! FKBP5 !PZDZ8! SHANK1 !SLC18a2!

SC

Blood-PTSD! FKBP5, IL18, IL16, CSF1, ! STAT5B, MHCII, NFIA, DICER, PF4, SDPR, HIST1H2AC !! Innate Immunity Pathway! Stat Pathway! IGF2! Glutamate Transport!

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Blood- Susceptibility to PTSD! Innate Immunity GR ! !! FKBP5 Pathway! !!   GILZ  

Figure 3.!

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Basal Conditions!

GC! FKBP5!

GILZ!

?!SGK1!

GC! GR!

Innate Immunity!

FKBP5!

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PFC neuron!

GC! FKBP5! ?!SGK1! blood! vessel!

SC

GC!

GR+ FKBP5 !

periphery!

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GC !

endothelial! cells!

PTSD!

PTSD Susceptible!

GR!

GR ! FKBP5! SGK1!

? FKBP5! ? SGK1!

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FKBP5! SGK1!

GRE!

GRE!

GRE!

Transcript Changes! ?!

PFC Hypofunction!

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PFC Top Down Control!

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CNS! ?! Inflammatory! ! Factors!

GC!

?! Amygdala! Neuron!

Amygdala Function!

Amygdala Hyperfunction!

Normal control of ! fear, extinction, and emotion.!

?!

GRE!

PTSD Symptoms! Enhanced Fear Memory! Decreased Fear Extinction!