Trama and Health - Master

Global Challenges

Trauma Post Traumatic Stress Disorder and Health

Literature-Based Guided Assessment (LGA)

 

Needed background and learning goals

This LGA is designed for students at the end of a one or two-semester biochemistry class.  Any additional background in molecular biology would be helpful.  This LGA should help students to:

• describe the complexity and molecular signatures of trauma and PTSD
• analyze transcriptome data and their statistical analyses, a topic not often covered in one-semester biochemistry courses
• create and interpret molecular models to address the structure/function relationships of proteins
• analyze simplified signaling networks and their regulation

This LGA is not only a formative/summative assessment for students, but also is a tutorial on some aspects of signaling and analysis of the transcriptome.  Data comes not from just one paper but many.

Introduction

Trauma (and Post Traumatic Stress Disorder (PTSD) arising from it) is a global issue.  Many people who have experienced combat, lived in a war zone, witnessed violent events, experienced environmental catastrophes or severe accidents, or who have experienced physical, emotional, or sexual abuse experience some symptoms of PTSD.  The young, including small children through young adults (the latter whom society, unfortunately, chooses to send to war) with developing brains are especially susceptible to PTSD.  Even one traumatic event may trigger PTSD.  From 15-43% of boys and girls have experienced at least one traumatic event.   In the U.S.,  Latinos, African Americans, and Indigenous peoples have higher rates than whites. 

Of those boys and girls, 3-15% of girls and 1-6% of boys will likely develop PTSD.  Repetitive trauma (such as emotional, sexual, and physical abuse) in children also increases the rates.  Girls have a higher rate of PTSD after experiencing trauma.   PTSD rates as high as 69-85% have been reported for adults who experienced sexual/physical abuse as children. In those who have experienced war, PTSD has historically been called by different names including soldier's heart, shell shock, and war neurosis.  It is not a weakness of character that causes some to get PTSD.  Rather it is a a physical, neurological, emotional, and behavioral outcome in some people who have been exposed to events people should never experience.

The Diagnostic and Statistical Manual of Mental Disorders (DSM-5) of the American Psychiatric Association states that PTSD is defined by four symptoms, including re-experiencing (flashbacks), dissociation, avoidance, hyperarousal and hypervigilance, and negative alterations in mood and cognition.  Other common features include recurring nightmares and sleep disturbance.  Seemingly innocuous clues such as sights and smells can trigger people into flashbacks and other PTSD symptoms. Here are the diagnostic criteria for PTSD from the DSM-5.

Although around 90% of people in the US have had at least one exposure to a traumatizing event, the rate of PTSD in the general population is about 8% but if the trauma results are severe (as in combat veterans and those sexually assaulted), it can rise to 25-35%, or even higher in victims of torture or prolong childhood abuse.  Not everyone exposed to such stresses gets diagnosable PTSD. The severity of the trauma, levels, and history of previous stresses, psychological and social support mechanisms, and other traits affect the likelihood of getting PTSD.  Genetic factors play a role with 30-40% inheritability reported for women who are twice as likely to get PTSD and have more severe and persistent symptoms. At a neurobiological level, the mental health effects must involve circuits in the brain that are involved in the learning and extinction of fear.  Even mice display sex differences in fear conditioning.  Brain areas involved in fear conditioning (prefrontal cortex, amygdala, and the hippocampus) and sex differences in those regions likely play a role in differential susceptibility to PTSD.   

As we will see below, PTSD affects all body systems and not just the brain. Abused children typically have poorer general health in later life.  In the Adverse Childhood Experiences (ACES) study, Felitti et al. documented that over 50% of 9508 respondents to a question given after a standard health care exam self-reported one episode and 25% more than 1 episode of childhood exposure in these categories: psychological, physical, or sexual abuse; violence against mother; or living with household members who were substance abusers, mentally ill or suicidal, or ever imprisoned. Those who had experienced such episodes in 4 or more categories had 4-12 times the risk for drug abuse, alcoholism, depression, and suicidality.  In addition, they smoked at a 2-4 increased rate and had over 50 sexual partners.  The study found a strong relationship between childhood exposure to the abuse and dysfunction listed above and the development of adult diseases (heart, lung, and liver diseases as well as cancer)  which are among the leading causes of death in adulthood.  

Keywords

Here are some references.

• Felitti VJ, Anda RF, Nordenberg D, et al. Relationship of childhood abuse and household dysfunction to many of the leading causes of death in adults. The Adverse Childhood Experiences (ACE) Study. Am J Prev Med. 1998;14(4):245-258. doi:10.1016/s0749-3797(98)00017-8
• https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3181586/).
• https://www.ptsd.va.gov/understand/c...ma%20survivors.
• https://theconversation.com/brain-sc...se-ptsd-115669
• Kritikos, M., Clouston, S.A.P., Huang, C. et al. Cortical complexity in World Trade Center responders with chronic posttraumatic stress disorder. Transl Psychiatry 11, 597 (2021). https://doi.org/10.1038/s41398-021-01719-7.  Creative Commons Attribution 4.0 International License. http://creativecommons.org/licenses/by/4.0/.
• https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3181586/
• https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5918463/
• https://www.nature.com/articles/s41380-022-01498-7
• ACES:  https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6220625/

Figure \(\PageIndex{1}\) shows brain regions implicated in interpersonal violence (IPV), childhood trauma and sexual assault.

Figure \(\PageIndex{1}\): Brain regions implicated in IPV, childhood trauma, and sexual assault. Eder-Moreau Elizabeth et al., Neurobiological Alterations in Females With PTSD: A Systematic Review,  Frontiers in Psychiatry, 13, 2022.   https://www.frontiersin.org/articles...yt.2022.862476.  Creative Commons Attribution License (CC BY).     

The general functions of each region are listed below.

• frontoparietal network - the center of cognitive control
• anterior cingulate cortex - cognitive functions as well as emotional expression, control of attention, and regulation of mood
• amygdala - formation of memories (especially emotion-associated ones) and emotion regulation
• hippocampus - involved in the regulation of learning and memory including its consolidation
• medial prefrontal cortex - integrates information from various brain regions and is involved in cognition, emotion, motivation, socialization 
• Caudate nucleus - controls movement of the voluntary skeleton and is involved also in learning, memory, emotion and motivation  

Brain scans show differences between people with and without a PTSD diagnosis.  Changes in the cerebral cortex can be measured using a parameter called fractal dimension (FD) obtained from MRI brain scans.  It is determined using multiple attributes that relate to shape complexity.   Figure \(\PageIndex{1}\) below shows FD values in the brains of PTSD-negative to PTSD-positive first responders from the 9/11 attacks in New York City. PTSD diagnoses were based on interview and their evaluation using the DSM-IV symptom designation (reexperiencing symptoms, avoidance, hyperarousal, and negative life experiences). 

 

Figure \(\PageIndex{1}\):  Kritikos, M., Clouston, S.A.P., Huang, C. et al. Cortical complexity in World Trade Center responders with chronic posttraumatic stress disorder. Transl Psychiatry 11, 597 (2021). https://doi.org/10.1038/s41398-021-01719-7.  Creative Commons Attribution 4.0 International License. http://creativecommons.org/licenses/by/4.0/.

Gray indicates no significant differences with statistically significant differences colored from dark blue (P = 0.05) to green/yellow (P ≈ 0.005) to dark red (P ≈ 0.0005).

A review of T and P values

If you compare two sets of data, and you wish to determine if the two means are equal, you can determine P and T values.  For a t-test, you define two hypotheses:

• H₀ or the null hypothesis: the population means are equal (μ₁ = μ₂₎ (the two population means are equal)
• H₁ alternative hypothesis: the population means are NOT equal (μ₁ ≠ μ₂)

You then perform a t-test which determines the difference in the means compared to the variation in each sample.  Effectively it is the difference expressed in standard error units.  The higher the T value, the more likely the population means are different and you can reject the null hypothesis.  A T value close to zero supports the null hypothesis that the means are the same.  The figure below shows t-values ranging from 3-7.5 showing that the scans from PTSD victims are different than control (combat veterans without PTSD).

You can then calculate the p-value that corresponds to the calculated t-value.  We mentioned p-values above.  A p-value < 0.05 suggests that the null hypothesis is correct and the same means are the same with a high probability (> 95%).  The number <0.05  suggests that the risk of concluding that the means are different is less than 5%.  The p values represented in the colored regions in the figure above are all <0.5)

Two Sample t-test Calculator 

Another technique, magnetoencephalography (MEG) has also been used to compare those with PTSD and controls. A flow of ions across a neural membrane produces a magnetic field, just as does the flow of electrons through a wire.  If many neurons (50,000 - 100,000) neurons are active, a magnetic field can be measured in that region.  Changes in the summative magnetic field can be measured at the millisecond time scale.  Hence MEG provides direct spatial and temporal measures of brain activity.  This compares to functional MRI (fMRI) which is more indirect and involves measuring oxgyen levels from blood flow in brain regions where neurons are active when people think about their trauma or view traumatic images.  MEG measurement can be measured when subjects are sleeping as well.  

Figure \(\PageIndex{1}\) below shows differences in magnetoencephalography (MEG) in the resting state of controls (combat veterans without PTSD) and combat victims with PTSD.

 Figure \(\PageIndex{1}\): 3D Renditions showing rain regions with significant resting-state neuronal activity in compabte veterans without PTSD (top) and with PTSD (bottom row).   Badura-Brack Amy S et al., Resting-State Neurophysiological Abnormalities in Posttraumatic Stress Disorder: A Magnetoencephalography Study.  Frontiers in Human Neuroscience. 11, 12-17 (2017). https://www.frontiersin.org/articles...hum.2017.00205.  Creative Commons Attribution License (CC BY).     

All group-level maps have been thresholded at (p < 0.001, corrected); a color scale bar showing respective t-values appears on the far right. Brain regions with significant activity in veterans without PTSD included bilateral occipital regions, inferior parietal cortices, postcentral gyri, posterior cingulate, parahippocampal gyri and other medial temporal areas. For veterans with PTSD, bilateral activity was observed in the primary motor and somatosensory cortices (i.e., precentral and postcentral gyri), superior parietal cortices, inferior parietal cortices, occipital regions, dorsolateral prefrontal cortices, inferior temporal sulci, posterior cingulate and bilateral medial temporal structures including parahippocampal gyri, hippocampi and the amygdala.

 Figure \(\PageIndex{1}\) below shows the results of MEG brain scans documenting brain regions that differ in activity (red stronger in PTSD, blue stronger in control) in combat veterans.

Figure \(\PageIndex{1}\): Group differences in resting-state neuronal activity between combat veterans with and without PTSD.  Badura-Brack Amy S et al. Ibid.

In the top row, brain areas exhibiting significantly (p < 0.01, corrected) stronger activity in veterans with PTSD are shown in red, whereas regions with significantly weaker activity in these patients appear in blue. As shown, veterans with PTSD had stronger resting-state neuronal activity in bilateral medial temporal areas (e.g., amygdalae, parahippocampal), sensorimotor, temporal, and prefrontal regions, along with significantly weaker activity in left and right lateral occipital cortices.

These studies clearly show significant changes in neuronal activity in cohorts with PTSD compared to controls.  However, as you will learn below, PTSD leads to widespread systemic changes outside of the nervous system in people with PTSD, with a plethora of biochemical changes and effects.  We could focus this section on a specific documented neuronal change to excitatory glutamate receptors such as the N-methyl-D-aspartate receptor (NMDAR) which is involved in synaptic plasticity critical learning and memory.    We'll reserve that for another problem set and instead focus on the systemic changes seen in victims of PTSD to gain a broader understanding of its impact on people.

Effects of trauma on individuals -  Soldiers with PTSD

Seid Muhie et al. Signatures of post-traumatic stress disorder in war-zone-exposed veteran and active-duty soldiers. Cell Reports - Medicine.  4, May 16, 2023.  DOI:https://doi.org/10.1016/j.xcrm.2023.101045.  Creative Commons Attribution (CC BY 4.0).  

Something as complicated as trauma effects on an organism is bound to produce a large number of changes in key metabolites, transcripts, proteins and enzymes.  Hence before we focus on one enzyme as an example, let's look at changes in whole systems within traumatized people.  Several key studies have focused on active duty and veteran US soldiers with diagnoses of PTSD from their experiences in active combat in Iraq and Afghanistan. There have been large-scale PTSD genome-wide association studies (GWAS).  In addition, the study by Muhie et used more defined cohorts and assessment measures: 

• The Systems Biology Consortium (SBC) - 340 veterans (300 males and 40 females)
• Fort Campbell Cohort (FCC) cohorts - 180 active-duty service members (159 males and 21 females); 26 followed 13 month
• All were exposed in PTSD A defined by the military as follows: The person was exposed to: death, threatened death, actual or threatened serious injury, or actual or threatened sexual violence, in the following way(s): Direct exposure. Witnessing the trauma. Learning that the trauma happened to a close relative or close friend.)
• Clinician-Administered PTSD Scale (CAPS)

They analyzed

• whole blood, plasma, serum, buffy-coats (white blood cells)
• 1,305 proteins were assayed in serum samples
• DNA methylation (focusing on cis-regulatory sites, probes for DNA within 1,500 bp of the promoter regions)

Changes in gene expression (and other types of -omics data) are often measured using microchips to measure genes, transcripts, and proteins in samples. The processed data is usually color-coded along the visible spectra.  This offers a visual impression of whether the expression is increased (red, orange, or pink) or decreased (violet, blue, or green).  This visual data can be misleading if changes are expressed on a linear scale.  Let's say the expression of a control (C) and sample (S) are determined and the ratio of S/C is calculated.  From a mathematical sense, a linear scale has a potential flaw.

• If the expression of S is doubled with respect to C, then x = S/C =2, a +1 increase in the "signal"
• If the expression of S is halved with respect to C, then x = S/C = 0.5, a -1/2 decrease in the"signal".

We could "normalize" the data and results by using the log₁₀(x).  If expression increased 10 fold, x= S/C=10, and log₁₀(10) = +1.  If it decreased 10 fold, then x= S/C=0.1, and log₁₀(0.1) = -1. This gives up and down expression values of equal "visual" weight for the same fold-change in expression.

To better cover changes around the 2-fold increases/decreases (halved) range, log₂(x) is used instead.  This gives a bigger visual change in the calculated expression data, as shown in the table below.  A doubling of expression now gives a log₂(2) = +1, the same number as a 10-fold increase gives if expressed as log₁₀(10) = +1

doubling or 2x increase ↑	10-fold increase ↑	halving  or 0.5 decrease ↓	10 fold decrease ↓
log2(2) = 1	log2(10) = 3.3	log2(0.5) = -1	log2(0.1) = -3.3

From the analysis, Muhie et al. found that several major pathways are altered in traumatized soldiers.  Figure \(\PageIndex{1}\) below shows multi-omics (metabolites, proteins, mRNA, microRNA, and DNA methylation) changes in two major sets of pathways, inflammatory responses/oxidative stress (A, top) and angiogenesis (growth of new blood vessels/epithelial cell dysfunction (B, bottom).

Figure \(\PageIndex{1}\):  Integrated multi-omics showing regulatory and functional relations (horizontally from right to left) across genetic variants, epigenetic marks, microRNAs, mRNAs proteins, and metabolites.  Seid Muhie et al. Signatures of post-traumatic stress disorder in war-zone-exposed veteran and active-duty soldiers. Cell Reports - Medicine.  4, May 16, 2023.  DOI:https://doi.org/10.1016/j.xcrm.2023.101045.  Creative Commons Attribution (CC BY 4.0).

Panel (A and B) show differentially expressed proteins (DEPs) that were persistent across PTSD cohorts and associated with (A) activated inflammatory response or oxidative stress, (B) impaired angiogenesis, epithelial dysfunction, or cardiovascular function were integrated with multi-omics datasets and compared across SBC Training (109/109 PTSD+/−), SBC Testing (43/39 PTSD+/−), FCC Validation (47/44 PTSD+/−), FCC Longitudinal (26/26 PTSD+/−), and FCC Subthreshold (68/44 PTSD subclinical/controls) cohorts. Vertical lanes of the protein heatmap correspond to the fold changes of each protein from each group of cohorts (as shown by the labels). SBC: Systems Biology Consortium (veteran cohort), FCC: Fort Campbell (active-duty) Cohort; for brain regions (postmortem mRNA brain data): dIPFC, dorsolateral pre-frontal cortex (PFC); ACC, anterior cingulate cortex (ACC); dACC, dorsal ACC; sgPFC, subgenual PFC; OFC, orbito-frontal cortex

 

Exercise \(\PageIndex{1}\)

Look at the changes in the protein and mRNA for inflammatory response and oxidative stress.  Are these expression responses generally increased or decreased?

a.  inflammatory response and oxidative stress - proteins

b.  inflammatory response and oxidative stress - mRNA

c.  angiogenesis and epithelial dysfunction -  proteins

d.  angiogenesis and epithelial dysfunction -  mRNA

Answer

a. and b.  increase

c. and d.  decrease

 

Exercise \(\PageIndex{2}\)

What happens to the expression of these genes? What is their function (find it at Uniprot)

a.  CDH5

b.  SOD2

c.  CCL5

Answer

a.  CDH5 expression is decreased. It encodes the gene for Cadherin-5.  Caherins are calcium-dependent cell adhesion proteins and it may play a important role in endothelial cell biology through control of the cohesion and organization of the intercellular junctions. Hence repair of the vasculature might be impaired.

b.  SOD2 expression is decreased.  It encodes mitochondrial superoxide dismutase [Mn] which destroys superoxide anion radicals which are normally produced within the cells and which are toxic to biological system.  Hence its decrease likely promotes inflammation and causes decreased production of ATP.

c.  CCL5 expression is decreased.  It encodes the C-C motif chemokine 5.  It is a Chemoattractant for blood monocytes, memory T-helper cells and eosinophils. It causes the release of histamine from basophils and activates eosinophils. It may activate several chemokine receptors including CCR1, CCR3, CCR4 and CCR5. It is a prognostic marker for survival and an indicator for Immune Checkpoint Therapies in Small Cell Lung Cancer.  Low levels are associated with higher cardiovascular risk.

 

Exercise \(\PageIndex{3}\)

Answer these questions on micro-RNA expression.

a.  In general compare the expression of mi-RNAs in inflammatory response/oxidative stress and in angiogenesis/epithelial dysfunction

b.  Do a web search to find out the role of mi-RNAs 146a (in inflammatory response/oxidative stress).

c.  Do a web search to find out the role of mi-RNA 32 (in angiogenesis and epithelial dysfunction).

Answer

a. expression of mi-RNAs in inflammatory response/oxidative stress generally decreases but increases in angiogenesis/epithelial dysfunction

b.  146a: One of the most important miRNAs to orchestrate immune and inflammatory signaling, often through its recognized target genes, IRAK1 and TRAF6, is microRNA-146a (miR-146a). MiR-146a is one, of a small number of miRNAs, whose expression is strongly induced following the challenge of cells with bacterial endotoxin, and prolonged expression has been linked to immune tolerance, implying that it acts as a fine-tuning mechanism to prevent overstimulation of the inflammatory response. 

c.  mi-RNA 34;  tumor suppressor. Under physiological conditions, miR-34a has an inhibitory effect on all processes related to cell proliferation by targeting many proto-oncogenes and silencing them at the post-transcriptional level

 

The authors state that "Although PTSD has primarily been conceptualized as a brain disease, it is increasingly being recognized as a systemic condition affecting multiple physiological parts and associated with divergent chronic medical conditions.   Thus, PTSD is coming to be seen as a systemic disorder rather than as a purely psychological illness."

 

Serine/threonine-protein kinase Sgk1 Function

Let's focus on just one enzyme, serum/glucocorticoid-regulated kinase 1(SGK1), also called serine/threonine-protein kinase1, that seems to play a significant role in many pathways that are affected by trauma.   Let's try to interpret and understand data from several research papers that discuss the role of SGK1 in several signaling pathways.  As a Ser/Thr protein kinase, SGK phosphorylates many protein substrates, some of which are protein kinases (to activate/inhibit them), enzymes other than kinases, and transcription factors, as shown in Figure \(\PageIndex{1}\) below. 

Figure \(\PageIndex{1}\):  Signaling pathway of SGK1 in oncology.  The prospect of serum and glucocorticoid-inducible kinase 1 (SGK1) in cancer therapy: a rising star.  Ruizhe Zhu, Gang Yang, Zhe Cao, Kexin Shen, Lianfang Zheng, Jianchun Xiao, Lei You, and Taiping Zhang. Therapeutic Advances in Medical Oncology 2020 10.1177/1758835920940946. Creative Commons Non-Commercial CC BY-NC.  

The ones we will discuss below have a red star (★) by them.  SGK1 is activated by phosphorylation by two major kinases, PDK1★ and mTORC2★. We will discuss those later when we study the structure/function of the molecule in depth. 

1.  SGK1 regulates the immune system

The immune system is extremely complicated (for a detailed review see Chapter 5.4).  Several major types of immune cells exist:

• Antigen-presenting cells - cells like macrophages, and dendritic and microglial cells in the brain that engulf foreign pathogens or cellular.  In addition, virally infected and cancer cells have "tumor" antigens presented on their surfaces for recognition by the immune system. 
• B cells - produce antibodies that target foreign extracellular or surface molecules of bacteria (for example).
• T cells - recognize virally-infected or cancer cells by binding to fragments of viral or tumor antigens presented on the cell surface.  Two major types are T Helper cells that express a protein called CD4. These bind to cells like macrophages that express MHCII protein.  Another set of effector T lymphocytes (for example T Cytotoxic, Natural Killer cells) express CD8. These cells express MHCI proteins and kill virus-infected cells and produce antiviral cytokine. Other CD8 cells suppress the immune response.

Signaling among these cells occurs through the release of cytokines from T cells that amplify or dampen immune cell response.

SGK1, downstream of mTORC2 is a regulator of T Helper or T_H cells. There are two major types:

• T_H1:  generally produces proinflammatory cytokines (for example interleukin-2 or IL2, interferon-γ, and tumor necrosis factor β or TNF-β) that are especially involved in immune responses to intracellular pathogens (viruses and bacteria)  and in prolonged autoimmune reactions against self (such as in Type I diabetes and MS).
• T_H2:  generally produces "antiinflammatory" cytokines such as IL 4, 5, and 13 involved in antibody responses to some extracellular parasites and allergic responses to "persistent" antigens (such as in asthma, dermatitis, etc.) and IL 10, which damps down the immune response, in part by turning down expression of T_H1 cytokines. 

Each also regulates the other to fine-tune the immune response. They derive from a common precursor T_Hp or T_H0 cell which differentiates into the two"poles" (T cell polarization), T_H1 and T_H2, each with their own phenotype.

To test the role of SGK1 in Tcell activation, the gene was deleted to produce Sgk1^−/−' progeny. Under different "polarizing" sets of conditions, these cells can differentiate from a precursor T_H0  cell into either T_H1 or T_H2 cells.   In the absence of polarizing cytokines, T cells from differentiated into TH1 cells. 

 

Exercise \(\PageIndex{4}\)

Appropriate factors were added to T_H0 wild-type (WT) or T-Sgk1^-/- cells to differentiate into the cells shown in the table below. The data is approximate based on graphs in the paper.

Cell Type	TH0 WT	TH0 T-Sgk1^-/-	TH1 WT	TH1 T-Sgk1^-/-	TH2 WT	TH2 T-Sgk1^-/-
IL-4 (ng/mL)	ND	ND	0.20	ND	1.8	0.55

Heikamp, E., Patel, C., Collins, S. et al. The AGC kinase SGK1 regulates T_H1 and T_H2 differentiation downstream of the mTORC2 complex. Nat Immunol 15, 457–464 (2014). https://doi.org/10.1038/ni.2867

What conclusions can you draw from these data?

Answer

As expected, wild-type T cells stimulated under T_H2-polarizing conditions (T_H2-WT)  produced LOTS OF IL-4. However, T-Sgk1^−/− T cells produced about 1/3 of the amount of IL-4 under the same conditions. Furthermore, lack of SGK1 resulted in lower production of the T_H2 cytokines IL-5 and IL-13 (data not shown)

 

 

Exercise \(\PageIndex{5}\)

In another set of experiments, the same cell types (stimulated as above) were stained for intracellular IFN-γ in CD4+ T cells in the presence of an inhibitor of protein transport across the membrane.  The results show that all the T cells had substantial IFN-γ expression. 

a.  Why did they include an inhibitor of protein transport in their experiments.

b.  How do these results compare to the expression of IL-4 above

c.  In a mouse model of asthma, an allergic disease, what might you expect if SGK1 was not present?

Answer

a. To keep IFN-γ in cells for staining.

b.  One would expect little IFN-γ expression under T_H2 polarizing conditions from SGK1^- cells which should have favored IL-4, 5 and 13 expression and not IFN-γ expression expected from T_H1 cells. The lack of SGK1 led to less IL-4 production and inappropriate production of IFN-γ in conditions expected to enhance IL4 and inhibit IFN-γ production. 

c.  This should decrease the production of IL4 through a lowered T_H2 response, whose normal role helps a sustained inflammatory response in asthma.  Also there would be the "unexpected "increase in IFN-γ.  The net effect would be resistance to asthma but unexpectedly greater resistance to viral infection and tumor cells from the unexpected release of IFN-γ .  Hence T_H2 cell–mediated diseases decrease in CD4 cells which lack SGK1.  In contrast, cells that lack SGK1 also have a greater immune response to viral infections and tumors through increased IFN-γ production

 

 

Exercise \(\PageIndex{6}\)

How does SGK1 affect T_H2 cells and the eventual production of IL-4?  It appears to do so through Nedd4 and Jun Bas illustrated in the partial signaling pathway below.

a.  Is Nedd4 an enzyme?  A kinase? Both?

b.  Does SGK1 inhibit or activate Nedd?  How specifically?

c.  Search Uniprot to find out what type of enzyme human Nedd4 is.

d.  What is the net effect of SGK1 on Jun B, a transcription factor, and IL-4 concentrations?

e.  Jun B is higher in WT T cells cultured to produce T_H2 than in T_H1 cells.  Compare Jun B levels in SGK1-deficient T cells under both T_H1- and T_H2-polarizing conditions.

 

Answer

a.  Nedd is an enzyme but not a kinase as it is not color-coded orange like SGK1

b.  SGK1 inhibits the enzyme Nedd4 by post-translational modification through phosphorylation.  The blunt arrow from SGK1 indicated inhibition.  It phosphorylated Nedd4 at Ser342 and Ser 448.  

c.Nedd 4 is a E3 ubiquitin-protein ligase NEDD4 so it covalently attached the small protein ubiquitin to JunB which targets it for degradation by the proteasome. 

d.  SGK1 inhibits the inhibitor of Jun B (i.e a net activation) so both the active transcription factor Jun B and the transcribed and translated IL4 will increase. 

e.  Jun B concentrations will decrease as its ubiquitination by Nedd4 is not inhibited by SGK1. Jun B is essential for T_H2 development. JunB will lead to increased expression of IL4.

 

 

Here is an expanded pathway showing signaling effects in wildtype and Sgk1^−/− cells.

 

 

Exercise \(\PageIndex{7}\)

Summarize how the lack of SGK1 causes β-catenin and JunB degradation. 

Answer

In the absence of SGK1, both GSK-3β (a kinase) and Nedd2 (a ubiquitinating enzyme) are active. 

• active (unphosphorylated) GSK-3β phosphorylates β-catenin making it a target for RBX1, which ubiquitinates it, marking it for proteolysis
• active (unphosphorylated) Nedd2 ubiquitinated JunB, which marks it for proteolysis.  

 

From these brief questions, it should be clear that the effects of changing the expression of just one protein (SGK1 in this case) are really complicated.  SGK1 is involved in:

• the control of protein synthesis and proliferation in endothelial cells (and hence the vasculature)

• major depressive disorders

• development and progression of tumors

• promotion of energy production in detached cells

• skeletal muscle homeostasis

• stress-induced cognitive impairment and PTSD

• symptoms in Parkinson's disease animal models

So let's look at one case of SGK1's involvement in brain function.

---

2.  SGK1 affects brain glial cells in neurodegenerative diseases

The protein is widely expressed in the brain with increasing levels for a variety of brain diseases.  Its name derives from the fact that its expression increased in rat tumor cells with the addition of serum and glucocorticoids.  Parkinson's disease (PD) is associated with the death of dopaminergic neurons (DA). Damaged neurons, extracellular protein aggregates, and microbial agents are removed by microglial cells, which are a specialized type of macrophage in the central nervous system.  For instance, they can initiate cell death in damaged cells.  

SGK1 is elevated in the midbrain and increased expression leads to death of DA neurons.  The increased expression appears to occur in the microglial cell.  Hence the inhibition of microglial cell SGK1 would be a useful treatment in PD and other brain neurodegenerative diseases.

The inflammatory activities of microglial arise from the activation of a key transcription factor, NFκB, whose activation leads to the expression of inflammatory cytokines and the inflammasome.  NFκB is perhaps the key regulator of the inflammatory response, which as shown above is elevated in PTSD.  It is a dimer of two proteins P50/P65 (also called NFkB1/RelA), which binds to promoter regions for inflammatory genes in the nucleus.   Cytoplasmic NFkB1 is inactive when bound to an inhibitor, IKB, which keeps it from translocating to the nucleus. Inflammatory signals lead to the phosphorylation of IKB by IkB kinase (or IKK, a dimer of IKKα and IKKβ), leading to the dissociation of IKB and the translocation of the  NFκB into the nucleus and subsequent expression of inflammatory cytokines.

SGK1 increases NFκB signaling and the deleterious inflammatory response in glial cells as illustrated in Figure \(\PageIndex{1}\) below. For simplicity, both NFκB and IKKβ are shown as monomers

 

 

Figure \(\PageIndex{1}\):  Modified from Kwon et al. SGK1 inhibition in glia ameliorates pathologies and symptoms in Parkinson disease animal models.  EMBO Molecular Medicine, 2021.  https://doi.org/10.15252/emmm.202013076.   Published under the terms of the CC BY 4.0 license

 

Exercise \(\PageIndex{8}\)

From the simplified pathway diagram above, how does SGK1 promote inflammation?

Answer

SGK1, a kinase, phosphorylates IKKβ subunit, activating the IKK kinase, which phosphorylates IKβ, promoting the dissociation of NFKB.  NFKB now enters the nucleus where it acts as a transcription factor in the expression of inflammatory genes.

 

Hence inflammation can be decreased if SGK1 can be negatively regulated at the transcription or post-transcriptional levels.  In glial cells, two other proteins, Nurr1 (N) and Foxa2 (F), are involved in the regulation of SGK1 transcription. To test their effects, glial cells were transduced (genes added) with the N and F genes, and in a mock control without them.   The results are shown in the figure below.

Panel A shows that Sgk1 is significantly downregulated (drop in expression, red arrow, red rectangle) in the glial cells transduced with Nurr1 (N) and Foxa2 (F).

Panel B and Panel show fold change (FC) or actual level changes (red count).

Panel D shows RT-PCR mRNA expression levels. 

Exercise \(\PageIndex{9}\)

What are the effects of the addition of the gene for  N, F, and N+F compared to the sham control?

Answer

Both N and F by themselves decrease SGK1 gene expression.  Both N and F together have a greater than additive effect on the downregulation of SGK1 (i.e. N+F have a synergistic effect)

 

Panel E below shows a Western blot of PAGE gels showing protein levels and graphs of the Western blot data showing quantitative values.  P65 is one of the subunits of NFKB.  β-actin gene is a "housing-keeping" gene whose levels are not expected to change.

 

Exercise \(\PageIndex{10}\)

What happens to the concentration of these proteins on the treatment of cells with N+F? The small red letter p indicates that a phospho group is attached as a posttranslational modification.  (p65 is the name of that protein and the p indicates no particular phosphorylation state.) Interpret the results.

SGK1	p-IKKB	p-IKBα	p65 (NFKB) total cell	p65 (NFKB) nucleus	β-actin
?	?	?	?	?	?

Answer

See the table below.

SGK1	p-IKKB	p-IKBα	p65 (NFKB) total cell	p65 (NFKB) nucleus	β-actin
decreases	decreases	decreased	constant	decreased	constant

The N + F-mediated downregulation of NFκB signaling is attained by inhibiting IκB phosphorylation (E) and thus blocking the release of NFκB from the NFκB-IκB inhibitory complex

 

What happens to the actual expression of inflammatory cytokines if inhibitory RNA (that block SGK1 translation by binding to SGK1 mRNA) are added to cells or if an "extra" gene is added to overexpress SGK1?  The results for inhibition are shown in Panels J-M and for overexpression in N below.

Exercise \(\PageIndex{11}\)

Interpret the results

Answer

Compared to the addition of nonspecific inhibitory RNA control, the addition of small RNA inhibitors of SGK1 mRNA translation significantly reduced the expression of inflammatory cytokines. The opposite effect was seen when an extra gene was added for overexpression.

 

 

Exercise \(\PageIndex{12}\)

The figure below summarizes the effect of NURR1, FOXA2 on SGK1-mediated expression of cytokines in glial cells.  Summarize these effects in words.

Answer

N+F inhibits SGK1 expression which arises from decreased phosphorylation of IKKβ, which decreases phosphorylation of IKβ, which decreases NFKB movement into the nucleus,  which then decreases the expression of pro-inflammatory genes.

---

Structure/Function of SGK1

Structure information is derived from:

Zhao B, Lehr R, Smallwood AM, Ho TF, Maley K, Randall T, Head MS, Koretke KK, Schnackenberg CG. Crystal structure of the kinase domain of serum and glucocorticoid-regulated kinase 1 in complex with AMP PNP. Protein Sci. 2007 Dec;16(12):2761-9. doi: 10.1110/ps.073161707. Epub 2007 Oct 26. PMID: 17965184; PMCID: PMC2222817. NO CREATIVE COMMONS PERMISSIONS

Serum and glucocorticoid-regulated kinase 1, SGK 1, a member of the AGC kinase family which includes protein kinases A (PKA), G (PKG), and C (PKC), is a serine/threonine kinase.  It has 3 domains, an N-terminal PK-like domain, a kinase domain, and a C-terminal hydrophobic domain.   Its catalytic domain is around 50% homologous to other members.

The protein is regulated at the transcriptional and also at the post-transcription level, the latter by phosphorylation.  Two are required. One occurs at Ser 422 in the C-terminal hydrophobic domain by mTORC2.  This allows its recruitment to membrane phosphatidylinositol 3, 4, 5-trisphosphate-dependent kinase (PDK1), which phosphorylates SGK 1 in a second location, Thr 256, located in a conformationally flexible activation loop in the catalytic domain, leading to the formation of a competent active site in the enzyme. 

All AGC kinases bind ATP and phosphorylate Ser/Thr side chains on target proteins.  Hence the conformations of the catalytic domain of the active protein kinases are similar.  Their inactive structures diverge more, which allows them to bind to specific target domains or proteins in a process that provides specificity for the phosphorylation.  Such is the case with SGK 1.

Figure \(\PageIndex{6}\)s shows an interactive iCn3D model of the catalytic domain of inactive serum and glucocorticoid-regulated kinase 1 (SGK 1) in complex with AMP-PNP (2R5T).

Figure \(\PageIndex{4}\): Catalytic Domain of Inactive Serum and Glucocorticoid- Regulated Kinase 1 in Complex with AMP-PNP (2R5T).. (Copyright; author via source). Click the image for a popup or use this external link: https://structure.ncbi.nlm.nih.gov/i...695vFs5jzQjH67

As with other AGC kinases, the catalytic domain of SGK1 has an N-terminal (light blue, mostly antiparallel beta strands) lobe and C-terminal (salmon, mostly loops and alpha helices) lobe, with ATP and its substrate analog AMP-PNP binding in the groove between them.  Key amino acids involved in catalysis are shown. The ATP binding site consists of residues 104-136 with key catalytic residues at 218-230.

The first phosphorylation occurs at Ser 422 found in the hydrophobic/aromatic motif FLGFSYA.  This sequence is conserved in AGC kinases, which have the general consensus sequence FXXFS/TF/Y.  This entire motif is not seen in the structure above.  The second phosphorylation occurs at Thr 256 (CPK-colored sticks, labeled).   

Figure \(\PageIndex{6}\) shows another interactive iCn3D model of the inactive SGK 1 and AMP-PNP highlight activation and catalytic features (2R5T).

Figure \(\PageIndex{4}\): Inactive SGK 1 and AMP-PNP highlighting activation and catalytic features (2R5T). (Copyright; author via source).
Click the image for a popup or use this external link: https://structure.ncbi.nlm.nih.gov/icn3d/share.html?MioLpk9FqXoVEbBp8

The activation loop is shown in blue.  Activation of the kinase requires phosphorylation of the activation loop Thr 256 (shown in sticks and labeled) by PDK1.  (Note that it is next to a SO₄^2-, part of ammonium sulfate used in the crystallization process.) Key residues involved in the binding of AMP-PNP in the active site are shown as CPK-colored sticks and labeled.

Catalysis requires binding of substrate, in this case ATP (or in the model above AMP-PNP) and any activating conformational changes that position catalytic residues around the substrate:

• Glu 146 and Lys 127:  E146 (E91 in PKA), is found in the N-terminal lobe αC-Helix of most AGC kinases. It interacts with a key catalytic residue K127 (K72 in PKA) in the catalytic loop. However, the equivalent αC-Helix is not found in the inactive SGK1.  Rather it forms a beta-strand.  This is perhaps the main feature differentiating inactive SGK1 (along with the activation loop) from other AGC kinases.  In AGC kinases, the activation loop is connected to the N-lobe through the αC helix.
• Lys 72 which stabilizes the β phosphate of ATP.
• Lys127, Glu183, Asp222, Lys224, Glu226, and Asn227:  These mostly charged side chains interact with the phosphates in the substrate (analog) 
• Asp 240, Phe 241, and Gly 242 (DFG motif): This motif is in the activation loop and helps in catalysis by positioning a molecule of ATP bound to magnesium or manganese for phosphoryl transfer which helps the SGK1 to switch between DFG-out (inactive) and DFG-in
• Cys193 and Cys258:  These might also affect the activity. 

 

 

Exercise \(\PageIndex{13}\)

Zoom into the iCn3D model and localize E146 and K127.  What did you find?  Offer an explanation.  With what does K127 interact? 

Answer

K127 interacts with ATP and is present but E146 in the "αC-Helix" is missing. This suggests that it is too conformational flexible to detect electron density and localize it in the crystal structure.

 

Exercise \(\PageIndex{14}\)

Zoom into the iCn3D model and answer the following questions. 

a. What is the role of K127 in binding and in catalysis?

b.  What is the role of K224?

c.  E226:

d.  N227

e.  E183

f.  D222

 

Answer

a.  K127 interacts with the β-phosphate of AMP–PNP. and stabilizes the - charge in the substrate and the extra negatived arising in the bound cleavage product, ADP.  As such it would also stabilize the developing - charge in the transition state.   

b. K224 stabilizes the γ-phosphate and also the developing charge in the transition state.

c.  E226 interacts with the oxygen atom between the α- and β-phosphates of AMP–PNP through the Mg²⁺ ion.

d. N227  interacts with the nitrogen between the β- and γ-phosphates

e.  E183 forms a hydrogen bond with one of the hydroxyl groups of the ribose

f.  D222 (equiv to general base D166 in PKA) points toward the γ-phosphate but helps deprotonate the target protein Ser/Thr-OH to enhance nucleophilic attack

 

Cys 258 near T256 and it forms a S-S with S193 to form a covalent homodimer :  

Exercise \(\PageIndex{15}\)

Locate Cys 258 and C193 in the iCn3D above.  Propose a mechanism in which they might alter activity. 

Answer

A disulfide bond between these two Cys on different monomers results in a covalent homodimer.  One of the Cys is near the Thr 256 which is phosphorylated by PDK1 to activate SGK 1.  This might not occur in the covalent dimer.  Also, oxidative modification of Cys 258 might affect the binding site on SGK1 for PDK1 altering its post-translation phosphorylation and the activity of the enzyme.

 

 

 

Exercise \(\PageIndex{16}\)

One clue to conformational flexibility is to color code the model based on the B-factor. 

Use this link and do the following in iCn3D:

• Choose Analysis, Seq. and Annotations, and then the Details tab.
• Under Proteins, click 2R5T_A to highlight the protein
• Choose Color, B-Factor, Original

What do the B-factors tell you about the conformational flexibility of the inactive protein?

 

Answer

Here is a snip of the structure color-coded by B factors.  The higher B factors (red) are found in the N-lobe and the activation loop of the kinase domains suggesting that these are more conformationally flexible, consistent with the changes required for kinase activation.

 

Exercise \(\PageIndex{17}\)

Make an iCn3D model of 2R5T. 

• Select these amino acids by choosing  Analysis, Seq and Annotations, and then the details tab:  Ile104, Val112, Ala125, Val160, Leu176, Asp177, Tyr178, Ile179, Leu229, Thr239  (Click/sweep each amino acids to highlight them)
• Select, save selection, and give it a name
• Style, Sidechains, Stick
• Color, Atom
• Analysis, Label, Per residue and number
• Analysis, lab scale, 3.0 
• Style, background, transparent
• Select, Clear selection (to remove yellow highlights)
• Files, Share Link, Lifetime short link, then copy it and save it in a file documenting your work

What role do these amino acids play in the SGK 1 catalytic domain?

Answer

Here is a link showing the rendered structure based on the instructions above

These mostly nonpolar side chains surround the mostly nonpolar adenosine ring in the substrate analog.

 

Investigators studied the phosphorylation and the activity of the protein in vitro. The structure reported above and used in the phosphorylation studies contained planned mutations.

 

Exercise \(\PageIndex{18}\)

Investigators make these mutations in the protein.  Explain why they did.

a.  S74A, S78A, S397A, S401A. 

b.  S422D mutation.

c.  R192A

 

Answer

a.  These would get rid of "nonspecific" phosphorylation sites and help direct the in vitro phosphorylation to T256 in the activation loop.The resulting phosphoprotein would be more homogenous as well.

b.  The S422D mutation mimics the charge state of phospho-Ser422 which is necessary for in vivo recruitment of the protein to the membrane where it would be phosphorylated and activated by PDK1

c.  R192 is near C193 which is involved in an interchain S-S bond.  R192A mutation decreases the conformational flexibility (entropy) of the side chain and increases the likelihood of S-S bond formation with C258 for the covalent homodimer described above. 

 

The inactive SGK1 structure, with its unique conformation in the αC and activation loop regions, may indicate that αC and the activation loop are important for substrate recognition and binding and thus are two key regulatory elements within the kinase domain. Upon activation, SGK1 will adopt a conformation similar to other active forms of AGC family kinases.

The amino acid sequence of SGK1 provides a plausible explanation for the disordered αC helix. Six successive large hydrophilic residues (KKKEEK, residues 136–141) are located at the beginning of the segment corresponding to αC, while there are only three hydrophilic residues in this region in AKT2 and four in PKA. These large hydrophilic residues are generally quite solvent-exposed and may make the peptide chain more flexible.

 

Exercise \(\PageIndex{19}\)

What happens on the phosphorylation of Thr 256 in the activation loop of SGK1?  A comparison with analogous residue, Thr 197) in Protein Kinase A (PKA)  would be helpful to know.

• In PKA, Thr 197 becomes phosphorylated and interacts with Arg165 and Lys189 in active PKA
• In SGK 1, Thr 256 becomes phosphorylated and likely interacts with Arg 221 and Lys 245 (the equivalent of Arg 165 and Lys 189 in PKA) in active SGK1.

a.  What type of noncovalent interactions occur between pThr197 and Arg 165/Lys 189?

b.  Measure the distance between Thr 256 and Arg 221/Lys 245 in this rendered structure of SGK 1 and describe what must happened to these residues in the likely active state of SGK 1.

In the iCn3D model,

• Choose Analysis, Seq and Annotation, the Detail tab, and then select Arg 221/Lys 245 with your mouse.
• Select, Save Selection, and name it R221K245.
• Style, Side Chains, Sticks
• Color, Atom
• Analysis, Label, Per Residue and Number
• Analysis, Distance, Between 2 sets
• Pick the predefined T256 as 1 set, and R221K245 as the second
• Choose Display to see the distance between them in Angstroms.

 

Answer

a.  ion ... ion or salt bridges between the phosphate (-2 charge) on pT256 and the + charges on Arg and Lys

b.  Here is a screen snip that shows the distance between T257 and R221/K245 in the inactive SGK 1. Compare this to the average length of a salt bridge in protein, 4.0 Å.  On phosphorylation of Thr256, the activation loop must undergo a large conformational change to shift the pT256 close enough to R221/K245 to enable the activation of the kinase.

 

 

 

Bashir A. Akhoon, Neha S. Gandhi, Rakesh Pandey, Computational insights into the active structure of SGK1 and its implication for ligand design, Biochimie,
Volume 165, 2019, Pages 57-66, ISSN 0300-9084, https://doi.org/10.1016/j.biochi.2019.07.007.  NO CREATIVE COMMONS PERMISSIONS

 

Additional Results

1.  Transcriptomic studies of four prefrontal cortex subregions from postmortem tissue of people with PTSD show that PTSD has different molecular traits than major depressive disorders.  32 unique genes were identified.  A few keys are listed below.

• ELFN1 (downregulated):  Synaptic adhesion, allosteric regulator of metabotropic glutamate (excitatory) receptors. It's required for formation of synapses.
• GABA (downregulated):  GAD2 encoding glutamic acid decarboxylase-2 required for GABA synthesis, and SLC32A1 which is a vesicular transporter for GABA and glycine. These suggest a downregulation of GABA (an inhibitory neurotransmitter) signaling 
• UBA7 (downregulated):  ubiquitin-like activating enzyme involved in the expression of inflammatory genes.
• FKBP5 (upregulated):  a glucocorticoid receptor chaperone. Key driver on females

---

Licznerski P, Duric V, Banasr M, Alavian KN, Ota KT, Kang HJ, et al. (2015) Decreased SGK1 Expression and Function Contributes to Behavioral Deficits Induced by Traumatic Stress. PLoS Biol 13(10): e1002282. https://doi.org/10.1371/journal.pbio.1002282.   Creative Commons CC0 public domain

This group looked at SGK1 expression in the postmortem prefrontal cortex of PTSD subjects (who died with PTSD) and in rat models of PTSD when rats were subjected to stimuli that resulted in symptoms of helplessness (exposure to acute foot shock stress without the possibility of escape) and anhedonia (lose of ability to experience pleasure as measured by decreased preference for a sweetened sucrose solution).  

Results of the studies of mRNA levels for SGK1 and two isozymes, SGK 2 and 3 from postmortem samples are shown below.

Panel (A) shows microarray gene expression analysis of postmortem dorsolateral PFC (DLPFC) samples collected from patients with PTSD. The * indicates significant results. The dashed line indicates healthy controls.

Panel (B) shows real-time qPCR was conducted to verify the microarray findings. Data are expressed as a mean fold change ± standard error of the mean (SEM) switch student’s t test values of *p < 0.05, **p < 0.01, ***p < 0.001].

Panel (C) shows expression levels of stress- and glucocorticoid-regulated genes were also examined. Changes are shown as a mean fold change. Asterisk indicates a significant p-value (*p < 0.05, FDR adjusted).  NR3C1, glucocorticoid receptor; NR3C2, mineralocorticoid receptor; GMEB1, glucocorticoid modulatory element binding protein 1; GRLF1, glucocorticoid receptor DNA binding factor 1; CRH, corticotropin-releasing hormone; CRHR1, corticotropin-releasing hormone receptor 1; CRHR2, corticotropin-releasing hormone receptor 2; CRHBP, corticotropin-releasing hormone binding protein; UCN, urocortin; UCN3, stresscopin (urocortin 3); FKBP5, FK506 binding protein 5.

 

Exercise \(\PageIndex{20}\)

Interpret the results.

Answer

Panels A and B clearly show statistically significant decreases in SGK1 compared to the other isozymes. Only 1 of the stress- and glucocorticoid-regulated genes showed a statistical increase with the rest hinting at decreases. 

 

Rat brain SGK1 from either low or high escapers from shock (a model for learned helplessness) was run on PAGE gels.  The results are shown below.

dfdfd

Panel (A): Rats were exposed to inescapable shock (day 1), tested in active avoidance (AA) (day 4), and then sacrificed (day 8) as indicated. Animals were separated into low- and high-escape groups according to their performance in AA test. Data are the mean ± SEM [t(12) = 14.39, Student’s t test, ****p < 0.0001].

Panel (B):  SGK1 and (C) PSD-95 protein levels in dissected PFC are decreased in the low-escape group. Data are mean ± SEM percent change over control group (naive, n = 5; high escape, n = 5; low escape, n = 9).

 

Exercise \(\PageIndex{21}\)

Interpret the results.  Why did they run GAPDH (glyceraldehyde-3-phosphate dehydrogenase) on the gels?  PSD-95 is the postsynaptic density protein-95 and GluR1 is the glutamate receptor gene product.  

Answer

Panels A and B show that brain tissue from rat brains from low escapers had less SGK1.  GADPDH levels serve as a control.  This enzyme is from glycolysis and is not expected to change between groups.  It also shows that equivalent amounts of samples were added to each lane. PSD-95 is significantly decreased and GluR1 trended downward, which might indicate a lowered number of function synapses in the low escape rats.  

 

To determine the role of SGK1 in the behavior of live animals,  investigators made a dominant negative mutated form of SGK1 (S422A) which was referred to as dnSGK1. The gene was added to an adeno-associated virus (AAV) to express the protein in live animals and then observed their behavior.  Some results are shown below. 

 

.

Effects of rAAV-dnSGK1 infusion into medial PFC on behavior were tested in (F) AA (escape failures), (G) sucrose preference, (H) total fluid consumption, and (I) locomotor activity. Data are shown as mean ± SEM (controls n = 11; dnSGK1 n = 9). (t(18) = 2.61 for AA and t(18) = 2.795 for SPT, Student’s t test, *p < 0.05]. (J) AA (escape failures) for rAAV-wtSGK1 injected rats (controls n = 10; wtSGK1 n = 10). (t(18) = 1.933, one-tailed Student’s t test, *p < 0.05.)

 

Exercise \(\PageIndex{22}\)

Answer these questions.

a.  What structure/activity changes in SGK1 would you expect in the mutant?

b.  Interpret the results

Answer

a.  The S422A mutant removes the phosphorylation site for mTORC2, one of two required phosphorylation for activation of the kinase (search for 422 in the text above for more information).  Hence the dominant form of SGK1 expressed would be inactive.

b.  In mice infected with the AAV with dnSGK1 expressed,  there were statistically increased escape failures (F), consistent with learned helplessness, and decreased % of sucrose consumed/total fluid (G), consistent with anhedonia.  (Note the total decrease in fluid consumption (H) was not statistically significant. Panel G shows that if the WT SGK1 gene was placed in the AAV genome, the number of escape failures decreased.  

 

 

 

---

Seah, C., Breen, M.S., Rusielewicz, T. et al. Modeling gene × environment interactions in PTSD using human neurons reveals diagnosis-specific glucocorticoid-induced gene expression. Nat Neurosci 25, 1434–1445 (2022). https://doi.org/10.1038/s41593-022-01161-y.  Creative Commons Attribution 4.0 International License.  http://creativecommons.org/licenses/by/4.0/.
This is a U.S. Government

Now let's return to whole genome/transcriptome studies.  To gain new insight, it would be useful to analyze a more specific set of cells to get clues about the effects of PTSD on their biochemistry.  The first study mentioned above looked at "omics" analyses of whole blood, plasma, serum, and buffy-coats.  A newer approach is to study specific cell types from people with and without PTSD.  We've clearly showed that PTSD is a systems disease, but at its heart are neurological effects, so it makes sense to study neurons (not just post-mortem brain tissue or do whole brain scans).  Instead of isolating neurons from people,  investigators have made glutamineric neurons in culture from stem cells isolated from people so the effects of PTSD can be studied in "petri dishes". 

How can you mimic PTSD conditions in culture?  One way is to add glucocorticoids (like the drug dexamethasone or hydrocortisone) to the cells from donors with PTSD and as controls, those who don't.  We've seen repeatedly in the above examples the effects of changes in SGK1, which as its name implies, is affected by glucocorticoids.  Transcriptomic studies to determine changes in gene expression have been done to compare the following sets of cells and PTSD mimics.

1.  Peripheral blood mononuclear cells (PBMCs) from combat veterans with PTSD and controls without PTSD

This study used dexamethasone (DEX) as the cell "stressor".  To interpret the graphs you need to know some definitions which are likely unfamiliar to biochemistry students who haven't yet studied large scale omic analyses.  

• FDR (False Discovery Rate).  This is the expected # false predictions/ # total predictions, so it's a statistical measure of the likeliness that the results are not what is hypothesized (or the null hypothesis). It's like a p-value except for multiple tests.  It is the rate at which features that are determined to be significant are in fact not.  For example, an FDR of 0.05 means that 5% of all features determined to be significant are NOT (so 95 out of 100 are).  Hence the lower the FDR, the high likely that the features are significant (in this case that they are associated with PTSD).  The table below shows some examples of different FDR values.  Often they are expressed as -log FDR(fx) so the larger this value, the more likely an outcome is significant.

FDR (fx)	FDR (% determined significant that aren't)	-log FDR (fx)
0.01	1%	2
0.05	5%	1.3
0.1	10%	1
0.25	25%	0.60

• DEGS: differentially expressed genes
• LogFC:  differences observed between vehicle and hydrocortisone exposure (for example)

Results from the PBMC studies +/- DEX are shown in the figure below.  

Fig. 1: Transcriptional response to DEX in PBMCs.  

Panel a shows how PBMCs from 20 PTSD cases and 20 combat-exposed controls were treated with DEX for 72 h and RNA-seq was performed. 

Pane b shows the number of differentially expressed genes (DEGs) observed in batch A (this study) and batch B (another published study) are upregulated and downregulated (y axis) across three different concentrations of DEX conditioning (x axis).

Panel c shows  Meta-analysis of expression logFC (differences observed between vehicle and DEX exposure, x axis) was plotted against –log₁₀(FDR) for each gene. Red points indicate significant DEGs in the meta-analysis. (Since the graph in the paper shows logFC and not log₁₀FC, we will assume, as in our previous discussions, that logFC implies log₂FC, which is customary.) Panel c is called a volcano (scatter) plot that shows the statistical significance (-log₁₀FDR or otherwise P value) vs a measure of the magnitude of the change (here logFC)

Exercise \(\PageIndex{23}\)

Which part of Figure C, 50 nM DEX shows the genes are most statistically downregulated?  Upregulated?  Interpret the overall result

Answer

Although all the red dots are significant, the region shown in blue is statistically most significant as they have the highest + (-log₁₀FDR) for the down-regulated genes (remember that logFC is a negative number for down-regulation.  The red circle highlights the most activated gene (logFC highest) with the most + (-log₁₀FDR)

There are 1000s of genes that are upregulated and 1000s downregulated by dexamethasone in Peripheral blood mononuclear cells (PBMCs) from veterans with and without  PTSD.  Experiments described below will differentiate between veterans with and without PTSD.

Changes were high in genes for immune signaling (this make sense since most nucleated cells in the blood are immune cells) and also in glucocorticoid sensitivity. 

2.  Neurons from combat veterans with PTSD and controls that without PTSD

Next investigators did the same experiments using glutaminergic neurons derived from pluripotent stem cells induced with neurogenin 2 (NGN2) to produce NGN2-neurons with glutamate receptors, glutamate transporters, and the neurotransmitter glutamate.

Results from the NGN2 neutrons +/- hydrocortisone (a glucocorticoid) are shown in the figure below.  

 

Fig. 2: Gene expression changes to HCort in hiPSC-derived neurons.  

Panel a, hiPSC-derived NGN2-neurons were treated with HCort for 24 h and RNA-seq performed.

Panel b, NGN2-neurons stained for neuronal markers NESTIN and MAP2, nucleic marker HOECHST and green fluorescent protein to confirm neuronal identity and morphology across all conditions. 

Panel c, Meta-analyzed DEGs in response to increasing concentrations of HCort shows robust changes in NGN2-neurons. A comparative analysis of transcriptome-wide log₂FC in response to different concentrations of HCort in NGN2-neurons shows similar responses, indicating a conserved response across all donors to HCort in NGN2-neurons. 

Panel e, Morphological analysis of neurite outgrowth in day 7 NGN2-neurons showing a dose-dependent decrease in neurite outgrowth with HCort exposure.

Exercise \(\PageIndex{24}\)

What conclusions can be derived from

• Panel B
• Panel C
• Panel E

Answer

• Panel B - The treatment with HCort acts as a control to show that it did not change/damage the morphology and hence the viability and functions of the cell
• Panel C - Gene regulation was dependent on the concentration of HCort, with higher Hcort leading to both greater inhibition and activation of gene expresssion. Both sets of Hcort concentrations had similar fractions of underexpressed and overexpressed genes
• Panel E - The lengths of neurites (axons and dendrites) projecting from the main neural cell body clearly decrease as the Hcort exposure increases. Decreasesd neurite length indicate possible signs of neuron degeneration or impaired function.

Some genes/pathways that were underexpressed include acetylcholine signaling, ubiquitin ligation, specific cell type development and differentiation.  Some that were overexpressed include protein methylation, immune cell function, histone modification, and regulation of gene expression.

3.  Direct comparison of gene expression in neurons from active duty/veterans with +/- PTSD

Results from these studies are shown below.

  

Fig. 4: PTSD(+) specific responses to HCort in NGN2-neurons.  

Panel a shows genes that differ in their response to HCort in PTSD(+) donors compared with PTSD(–) donors, here termed DRGs, were detected in both the 100 nM and 1,000 nM dose, indicating PTSD diagnosis-specific responses to HCort. 

Panel b, Significant NGN2-DRGs correctly classify PTSD(+) from PTSD(–) participants using an "unsupervised (machine learning) approach"  (does not require selection of input parameters a prior.  Just raw data is used).

 

Exercise \(\PageIndex{25}\)

Interpret Panels a and b.

Answer

Panel a:  Once again the PTSD stress "mimic" Hcort neurons show a large number of genes that differ when exposed to HCort, but with unexpectedly fewer changes at very high concentrations.

Panel b: At 100 nM HCort, the bottom left quadrant shows that in control neurons (without PTSD) many more genes were underexpressed while in the top left quadrant, in PTSD neurons many more were overexpressed.  Clear patterns emerged in both groups showing dysregulation in the +PTSD group.  The results were not just a random pattern but concentrated in certain pathways.

 

 

Here is a summary from the paper:

"Both blood and neuronal glucocorticoid responses were significantly enriched for immune response genes; neuronal glucocorticoid responses were also associated with brain development and neurodevelopmental disorder genes. Although a PTSD diagnosis-specific signature was not detectable at baseline in either cell type, glucocorticoid hypersensitivity in PTSD was observed.  These findings are consistent with the glucocorticoid hypersensitivity hypothesis; for example, patients with PTSD have altered blood sensitivity to glucocorticoids⁶ and perturbations in glucocorticoid receptor signaling have been shown for PTSD in PM brain tissue¹⁰. 

Stress impacts the risk to psychiatric disorders throughout the lifespan—across prenatal development^62,63, childhood⁵² and adulthood^52,64,65. Moreover, the significant and positive relationship between our observed associations in NGN2-neurons and previously demonstrated transcriptional PTSD signatures in PM brains suggests that some impacts of glucocorticoid exposure may persist through to adulthood. Notably, glucocorticoid stimulation is not a specific model for PTSD; stress response is comorbid in many psychiatric disorders. HCort-responsive genes therefore likely represent aspects of stress response shared across PTSD and other neuropsychiatric disorders, such as the shared impact on social cognition between PTSD and ASD^66,67. Combined analysis of context-dependent hiPSC models with cross-lifespan datasets, such as PM brains, may uncover long-term glucocorticoid-dependent PTSD signatures with which to refine hallmarks of PTSD susceptibility following combat exposure."

Other related studies:

• SGK1 knockdown in the medial prefrontal cortex reduces resistance to stress-induced memory impairment
• Glucocorticoids Can Induce PTSD-Like Memory Impairments in Mice

 

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https://www.nature.com/articles/s41380-022-01498-7

 

 

 

Lee, B., Pothula, S., Wu, M. et al. Positive modulation of N-methyl-D-aspartate receptors in the mPFC reduces the spontaneous recovery of fear. Mol Psychiatry 27, 2580–2589 (2022). https://doi.org/10.1038/s41380-022-01498-7.  Creative Commons Attribution 4.0 International License.  http://creativecommons.org/licenses/by/4.0/.

 

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Epigenetic effects of trauma

The studies - a note of caution

Epigenetic effects of trauma would have the potential to be passed on to offspring and could lead to multigenerational trauma effects.  It is very difficult to conduct quality studies to show effects in subsequent generations.  Subjects have to be identified and consent to participation in the study.  Few studies have been done on the biochemical effects of childhood trauma on victims and intergenerational effects.   Perhaps the best studies derive from war victims (soldiers, civilian populations, mostly from WWII) and their descendants.  It is also difficult to disentangle possible epigenetic effects from environmental causes such as those that might arise from children brought up by a parent who has or has had PTSD.  Studies have also been done after events such as 9/11 and even the Covid pandemic.   Animal trauma models have also been explored and even those have ethical concerns.

Epigenetics -  short review

Trauma might also be encoded by a myriad of epigenetic changes that may or may not be passed onto offspring. Figure \(\PageIndex{1}\) below shows different types of epigenetic changes in trauma victims.

Figure \(\PageIndex{1}\):  Four different epigenetic regulatory mechanisms.  Wu, YL., Lin, ZJ., Li, CC. et al. Epigenetic regulation in metabolic diseases: mechanisms and advances in clinical study. Sig Transduct Target Ther 8, 98 (2023). https://doi.org/10.1038/s41392-023-01333-7.  Creative Commons Attribution 4.0 International License.  http://creativecommons.org/licenses/by/4.0/.

The figure shows 4 kinds of epigenetic changes.

DNA methylation

This occurs on cytosine bases in CpG motifs (island). Methylation is abundant in telomers, centromers, repetitive sequences, and on the inactivated X chromosomes in females. It can affect genome stability and silencing (of expression).  5meC is the most common but other modifications (hydroyxmethy, formyl, and carboxyl) also occur at the 5' position of cytosine bases.  The methylation is catalyzed by several different DNA methyltransferases (DNMTs) using S-adenosylmethionine (SAM) as the methyl donor.  DNMT1 maintains methylation states while DNMT3A and DNMT3B create new meCs.

Histone modifications

Two copies each of the positively charged histones H2A, H2B, H3, and H4 comprise the nucleosome core around which negatively charged dsDNA wraps. The histones can be post-translationally modified by acetylation, methylation, lactylation, phosphorylation, dopaminylation, and ubiquitination. Acetylation removes the + charge on histones and decreases DNA binding affinity. These modifications also add binding sites for proteins which affect the overall packing of chromatin.  Overall, these modifications can change the accessibility of sites on chromatin required for gene transcription and hence they can regulate gene transcription.

Acetylations are catalyzed by histone acetyltransferases (HATs) and reversed by histone deacetylases (HDACs).  Most modifications occur on the N-terminal sections of H3 and H4. Proteins with bromodomains act as histone acetylation "readers" and are involved in transcriptional regulation and chromatin remodeling. The N-termini of H3 and H4 are also the site of methylation of lysine or arginine residues, which can produce mono-, di-, or trimethylated lysine residues.  Some methylations activate transcription, others inhibit it. The enzymes involved are lysine methyl transferase (KMTs) and lysine demethylases (KDMs), while protein arginine methyltransferases (PRMTs) modify arginines.

Chromatin remodeling

DNA wound around a nucleosome is not accessible to RNA polymerase (about the size of the nucleosome core) for transcription.  Even worse for accessibility is condensed chromation.  Remodeling of the chromatin, which requires ATP hydrolysis, can open it for transcription by increasing accessibility. Several families of remodelers (SWI/SNF, ISWI, chromodomain helicase DNA binding or CHD and inositol requiring 80 (INO80) can remodel chromatin.  

Noncoding RNAs (ncRNAs)

Two types are described below

• endogenous miRNA of about 23 NT length can inhibit gene expression by inhibiting mRNA translation.  RNA polymerase II is used in their synthesis and the inhibitor act to inhibit translation through the RNA-induced silencing complex (RISC) and binding to the 3′-untranslated regions (3′-UTRs) of specific mRNAs. The synthesis and hence expression of specific miRNA can hence be regulated by the epigenetic mechanism described above. More directly, but they can also regulate the key enzymes involved in epigenetic modifications including HDACs and DNMTs. diabetes.
• lncRNA are longer than 200 nucleotides and include long intergenic non-coding RNAs (lincRNAs), antisense RNA, and others.  They can affect chromatin structure and even enzyme activity through specific binding interactions. 

Here are some references that describe the epigenetic effects of trauma.

• A review of epigenetic contributions to post-traumatic stress disorder


• Epigenetic Modifications in Stress Response Genes Associated With Childhood Trauma

• Methyl-CpG binding protein 2 expression is associated with symptom severity in patients with PTSD in a sex-dependent manner

• Epigenetics of childhood trauma: Long-term sequelae and potential for treatment

• GABAergic mechanisms regulated by miR-33 encode state-dependent fear

• Epigenetic mechanisms in fear conditioning: Implications for treating post-traumatic stress disorder

• Intergenerational transmission of trauma effects: putative role of epigenetic mechanisms

• The public reception of putative epigenetic mechanisms in the transgenerational effects of trauma