Circulating cell-free mitochondrial DNA levels and glucocorticoid receptor sensitivity in combat-related PTSD

doi:10.21203/rs.3.rs-2838048/v1

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Circulating cell-free mitochondrial DNA levels and glucocorticoid receptor sensitivity in combat-related PTSD

https://doi.org/10.21203/rs.3.rs-2838048/v1

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published 10 Jan, 2024

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Circulating cell-free mitochondrial DNA (ccf-mtDNA) is a biomarker of cellular injury or cellular stress and is a potential novel biomarker of psychological stress and of various brain, somatic, and psychiatric disorders. No studies have yet analyzed ccf-mtDNA levels in post-traumatic stress disorder (PTSD), despite evidence of mitochondrial dysfunction in this condition. In the current study, we compared plasma ccf-mtDNA levels in combat trauma-exposed male veterans with PTSD (n = 111) with those who did not develop PTSD (n = 121) and also investigated the relationship between glucocorticoid signaling and ccf-mtDNA levels. In unadjusted analyses, ccf-mtDNA levels did not significantly differ between PTSD and non-PTSD groups (t = 1.312, p = 0.191). However, after controlling for the potential confounding variables age, HbA1c, and antidepressant use, the PTSD group had lower ccf-mtDNA levels than did the non-PTSD group (F(1, 221) = 5.509; p = 0.020). We also performed a sensitivity analysis excluding diabetics and antidepressant users and found that the PTSD group still had significantly lower ccf-mtDNA levels (t = 2.577, df = 177, p = 0.011). Across the entire sample, ccf-mtDNA levels were negatively correlated with post-dexamethasone ACTH decline (r=-0.171, p = 0.020) and cortisol decline (r=-0.149, p = 0.034) (viz., greater ACTH and cortisol suppression was associated with lower ccf-mtDNA levels) both with and without controlling for age, antidepressant status and HbA1c. Ccf-mtDNA levels were also significantly positively associated with IC_{50 − DEX}, a measure of lymphocyte glucocorticoid receptor (GR) sensitivity, after controlling for age, antidepressant status, and HbA1c (β = 0.135, p = 0.043), suggesting that increased lymphocyte GR sensitivity is associated with lower ccf-mtDNA levels. Although no overall group differences were found in unadjusted analyses, exclusion of diabetics and antidepressants, which may affect ccf-mtDNA levels, revealed decreased ccf-mtDNA levels in PTSD. In both adjusted and unadjusted analyses, low ccf-mtDNA levels were associated with relatively increased GR sensitivity, often reported in PTSD, suggesting a link between mitochondrial and glucocorticoid signaling abnormalities in PTSD.

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Post-traumatic stress disorder (PTSD) is a debilitating psychiatric disorder that occurs in susceptible individuals as a result of exposure to a severe traumatic event. Presently, it is estimated to affect 1.1% of the global population within any 12-month period [1]. PTSD prevalence is higher among those with combat trauma exposure [2], and prevalence among Operation Iraqi Freedom (OIF) and Operation Enduring Freedom (OEF) veterans has been estimated to be 15.8% [3]. A clearer understanding of biomarkers that accompany PTSD could aid in more effective diagnosis, prognosis, and treatment as well as possibly refining our understanding of PTSD pathophysiology. Circulating cell-free mitochondrial DNA (ccf-mtDNA) is derived and released from cells into the systemic circulation following cellular injury or cellular stress and has received increased attention as a biomarker for a variety of somatic pathologies. Increased blood ccf-mtDNA levels have been associated with conditions including diabetes [4, 5, 6, 7] heart disease [4, 5], and inflammatory diseases [8], as well as with aging [9, 10] and certain cancers [11, 12, 13]. Conversely, decreased ccf-mtDNA levels in cerebrospinal fluid have been observed in some neurodegenerative conditions including some types of Parkinson’s disease and Alzheimer’s disease [14, 15, 16], where they have been suggested to possibly reflect mitochondrial loss in early stages of disease progression [16].

Although ccf-mtDNA has not been specifically investigated in PTSD, some studies have explored the relationship between ccf-mtDNA levels and other psychiatric disorders. Elevated plasma ccf-mtDNA levels have been associated with late-life depression and frailty [17], and with MDD in unmedicated subjects [18]. Notably, antidepressant administration in depressed subjects altered plasma ccf-mtDNA levels differentially in antidepressant treatment responders vs. non-responders [18], being associated with increased ccf-mtDNA levels in non-responders only. Lindqvist et al. [19] found that plasma ccf-mtDNA was significantly increased in recent suicide attempters compared to controls, and ccf-mtDNA levels were positively correlated with post-dexamethasone cortisol levels, suggesting an inverse relationship with glucocorticoid receptor (GR) sensitivity in these individuals. A positive association was also found between acute psychosocial stress and ccf-mtDNA levels in plasma [20] and serum [21]. On the other hand, no significant difference in serum ccf-mtDNA levels was reported in a study of depressed female subjects [22], and two studies found that MDD was associated with lower plasma ccf-mtDNA levels [23, 24], in unmedicated and medicated MDD individuals, respectively. Lower plasma ccf-mtDNA was also reported in subjects with social anxiety disorder [25] and bipolar disorder [23], though two studies assessing serum ccf-mtDNA levels did not find a significant difference between bipolar subjects and controls [26, 27]. A recent meta-analysis of ccf-mtDNA levels and brain disease that included studies of MDD, suicidality, bipolar disorder, and schizophrenia did not find a significant overall difference between psychiatric cases and controls, although certain assorted non-psychiatric neurological conditions were associated with decreased ccf-mtDNA levels [28]. The reasons for the discrepant results thus far are unclear but raise the possibility that individual factors apart from the psychiatric diagnoses per se (e.g., comorbid psychiatric or medical conditions or medications, comorbid metabolic/inflammatory imbalances, or associated hypothalamic-pituitary-adrenal [HPA] axis alterations) may confound certain results [24].

A number of conditions commonly associated with PTSD, including suicidal ideation [29, 30, 31], elevated inflammation [32], and metabolic disorders such as type 2 diabetes [33], have also been associated with elevated ccf-mtDNA, although these have not been specifically studied in individuals with PTSD. On the other hand, increased glucocorticoid receptor sensitivity and increased negative feedback in the HPA axis have been reported in multiple studies of PTSD [34, 35, 2] and based on the positive associations reported between ccf-mtDNA and glucocorticoid levels following physical stress [20] and between post dexamethasone suppression test (DST) cortisol levels and ccf-mtDNA levels in suicide attempters [19], it seems plausible that increased glucocorticoid receptor sensitivity in PTSD subjects could be associated with lower ccf-mtDNA levels.

The main aim of this study was to compare plasma ccf-mtDNA levels between PTSD positive and PTSD negative subjects in a large, well-characterized group of male veterans, all of whom had been exposed to combat trauma. We also created models to control for the potential confounding variables age, hemoglobin A1c (HbA1c), and antidepressant use. Moreover, we analyzed ccf-mtDNA levels in relation to HPA axis activity, glucocorticoid receptor sensitivity, antidepressant medication, and psychometric scores.

Study participants

Male combat trauma-exposed veterans who had served in active duty in Operation Iraqi Freedom (OIF) or Operation Enduring Freedom (OEF) were recruited by New York University (NYU), the Icahn School of Medicine at Mount Sinai (ISMMS), and the James J. Peters Veterans Administration Medical Center (JJPVAMC). Detailed descriptions of the recruitment procedure and exclusion criteria for study participants can be found in prior publications [36, 37, 38]. Briefly, inclusion criteria for the PTSD group were age 20–60 years and current diagnosis of war zone-related PTSD based on the Structured Clinical Interview for DSM-IV-TR [39], which was the DSM version in use at the time of this study, and the Clinician-Administered PTSD Scale (CAPS) [40, 41]. Current PTSD diagnosis was determined by the DSM-IV-TR criteria. Combat trauma-exposed non-PTSD controls were also included if aged between 20 and 60, had previously served in war zones, were free from a lifetime history of PTSD, and had a current CAPS score less than or equal to 20. All subjects (PTSD-positive and PTSD-negative) met DSM-IV diagnostic criterion A of the PTSD diagnosis for combat trauma exposure, and the time since the index trauma was 6 years (± 2.8 years) on average. The exclusion criteria for the study included the following: prominent suicidal or homicidal ideation; a history of alcohol dependence within the past eight months or of substance abuse or dependence other than nicotine within the past year; a lifetime history of any psychiatric disorder with psychotic features, bipolar disorder, or obsessive-compulsive disorder; any neurologic disorder, systemic illness affecting CNS function, a moderate or severe traumatic brain injury (TBI) based on the Ohio State University TBI Identification Method––Short Form [42]; and a history of anemia or recent blood donation in the past 2 months. In addition, individuals who were exposed to ongoing trauma or had been exposed to a traumatic event within the past three months and who were not stable for at least two months on psychiatric medication, anticonvulsants, antihypertensive medication, or sympathomimetic medication were excluded. Subjects with medical conditions that may affect systemic inflammation were also excluded. In total, 232 subjects had available ccf-mtDNA data, and they were grouped into those with (n = 111) and those without (n = 121) PTSD. The study was approved by the Institutional Review Board of the University of California, San Francisco, Medical Center (UCSF), the ISMMS, the JJPVAMC, the NYU Grossman School of Medicine, and the US Army Medical Research and Materiel Command. All subjects provided written informed consent before participation in the study. The study was conducted in accordance with the Declaration of Helsinki (1989).

Evaluation of psychiatric symptoms

PTSD status of all participants was determined by doctoral-level psychologists using the Structured Clinical Interview for DSM-IV Axis I Disorders (First, 1997) and the Clinician Administered PTSD Scale (CAPS)[40]. A review panel including senior experienced clinicians reviewed all diagnoses. Participants also self-reported their PTSD symptoms, depression symptoms, and perceived psychological stress via the PTSD Checklist for DSM-IV (PCL) [43], the Beck Depression Inventory-II (BDI-II) [44], and the Perceived Stress Scale-10 [45], respectively.

Blood collection

Blood was drawn by venipuncture in the morning between 8am to 8:30am after a night of fasting in all subjects. For plasma analyses, blood was collected into tubes containing EDTA. After being inverted 8–10 times, tubes were put on ice for a maximum of 30 minutes and then spun at 1100 x g for 15 minutes at 4°C. Plasma was removed and divided into 500 µL aliquots, which were stored at -80°C for later analysis. Serum samples were collected into serum separation vacutainer tubes (Becton Dickinson, Franklin Lakes, NJ), allowed to clot for 10 min at room temperature, centrifuged for 15 min at 1300 rpm, and then frozen and stored at -80°C.

Ccf-mtDNA purification and quantification

Plasma samples were stored at -80C after collection, thawed, and spun at 10,000 x g for 10 min to remove cellular debris. DNA was isolated from 200µL of plasma using the QIAamp DNA Blood Mini Kit (Cat #51106, Qiagen, Valencia, CA, USA) according to the manufacturer’s instructions. The isolated DNA was eluted in 60µL AE buffer and stored at -80°C before being assayed. The quantitative analysis of cell free-mtDNA was performed using quantitative real-time polymerase chain reaction (qPCR) by amplifying a 161 bp product from the NADH:Ubiquinone Oxidoreductase Core Subunit 2 (MT-ND2) gene. Each 10µL reaction was comprised of 5µL QuantiFast SYBR Green PCR Kit (Qiagen, Valencia, CA, USA), 0.5µL of each primer, and 2.5 µL of extracted DNA. Each reaction was run in triplicate on an LC480 (LightCycler, Roche, Mannheim, Germany) using the following program: Initial denaturation at 95°C for ten minutes, and then 45 Cycles of the following: For melting, 95°C for 10s, annealing at 65°C for 10s, and extension at 72°C for 11s. Finally, a melting curve analysis measured fluorescence continuously from 60°C to 97°. The forward primer sequence was 5’-CACACTCATCACAGCGCTAA-3’, and the reverse primer sequence was5’-GGATTATGGATGCGGTTGCT-3’.

A 7-point, 4-fold serial dilution of a genomic DNA (Roche Life Science, Indianapolis, Indiana, United States) was created as the standard curve to quantify the mitochondrial DNA copy number relative to the genomic DNA using the Roche480’s absolute quantification maximum second derivative method. The highest concentration of the standard curve was 20ng/ul. In order to convert the measured relative copy numbers to absolute copy numbers, the same primers were used to amplify a PCR product from the human genomic DNA (Roche Life Science, Indianapolis, Indiana, United States, cat # 1169111200). The PCR product was purified using the QIAquick PCR Purification Kit (Qiagen, Valencia, CA, USA) protocol. A PicoGreen Assay (Cat ThermoFisher Scientific Inc., Waltham, Massachusetts, USA) was conducted to obtain an accurate concentration of the product. The relative concentration of the PCR product was measured using the human genomic DNA as the reference standard, and the absolute mtDNA copy number concentration of each sample was then converted accordingly. The copy number of the PCR product was calculated first by dividing the mass by its molecular weight, calculated based on its unique sequence (http://www.bioinformatics.org/sms2/dna_mw.html), and multiplied by Avogadro’s constant. The final values are expressed at copy numbers/µL of plasma. The average inter-assay CV from 24 randomly picked samples was 3.8 ± 1.9%.

Clinical Labs

Clinical labs were conducted by a CLIA-certified lab.

Dexamethasone suppression test and neuroendocrine assays

The dexamethasone suppression test (DST) was conducted as previously described (Somvanshi et al., 2020) to measure the negative feedback response of the HPA axis. Dexamethasone (DEX) was administered at a dose of 0.5 mg at 11:00 pm the night before the second blood draw. Plasma cortisol was assayed with a Cortisol ELISA Kit (IBL-America, Minneapolis, MN), with intra- and inter-assay coefficients of variation of 5.3% and 9.8%, respectively. Assay sensitivity was 2.5 ng/mL. Plasma ACTH was assayed using an ACTH ELISA kit (ALPCO Diagnostics, Windham, NH), and the intra- and inter-assay coefficients of variation were 5.0% and 8.7%, respectively. Assay sensitivity was 0.5 pg/mL. Subjects without detectable DEX levels on day 2 were excluded from correlation analyses of post-dexamethasone measures. Declines of cortisol and ACTH from day 1 to day 2 were used as measures of DEX suppression.

Dexamethasone-induced lysozyme suppression (IC_{50 − DEX}) assay

This test was performed as previously described in detail [46, 47] to measure glucocorticoid receptor sensitivity. Briefly, PBMCs were isolated on the day of blood collection via density gradient centrifugation using Ficoll-Paque media (Pharmacia), and the IC_{50 − DEX} assay was performed the same day. The turbidimetric method was used to determine lysozyme inhibition in cells that were incubated with Micrococcus lysodeikticus (Sigma) in DEX concentrations of 0, .5, 1, 2.5, 5, 10, 50, and 100 nmol/L. IC_{50 − DEX} refers to the concentration of DEX at which a 50% reduction in lysozyme activity was observed. Accordingly, higher IC_{50 − DEX} values correspond to lower GR sensitivity. The intra-assay coefficient of variation was 6.9%, and the inter-assay coefficient of variation was 9.8%.

Statistical analysis

Statistical analyses were performed using the Statistical Package for the Social Sciences (SPSS), version 28.0 (SPSS Inc., Chicago, IL, USA). Variables were assessed for normality, and Blom transformation [48] was applied to those with non-normal distribution. Descriptive statistics were calculated for socio-demographic characteristics and biological markers. Continuous variables were summarized with means ± standard deviations (SD) and compared with independent t-tests, and categorical variables were summarized with frequencies and compared with chi-squared tests. To analyze group differences in ccf-mtDNA levels between PTSD-positive and PTSD-negative subjects, two-tailed Student’s t-tests were used.

We selected a priori covariates for our ANCOVA model after conducting a thorough ccf-mtDNA literature review. Age and HbA1c were chosen as covariates because significant associations between both ccf-mtDNA levels and age [9, 10] and ccf-mtDNA levels and diabetes [4, 5, 6, 7] have been reported in more than one study in distinct samples. Antidepressant use was also included as a covariate because 26 of the subjects in the PTSD group were taking antidepressants (compared to 5 in the control group), and preliminary data from a study of MDD subjects [18] suggested higher ccf-mtDNA levels in MDD subjects who did not respond to SSRIs relative to responders. We also conducted a sensitivity analysis excluding diabetics, defined as having HbA1c values greater than 6.5% (PTSD, n = 6; control, n = 3), and antidepressant users (PTSD, n = 30; control, n = 5), and a Student’s t-test was used to compare ccf-mtDNA levels between the remaining PTSD and control subjects.

For analysis of the relationship between ccf-mtDNA levels and glucocorticoid signaling, two-tailed Pearson correlations were performed between ccf-mtDNA levels and continuous variables.

A two-tailed t-test was used to analyze differences in ccf-mtDNA levels between antidepressant users and non-users within the PTSD group, and ANCOVA was used to control for age and HbA1c.

Two-tailed Pearson correlations were used for analyses of the association between ccf-mtDNA levels and age, BMI, HbA1c, and psychometric scores. For all continuous variables analyzed other than HbA1c and age, multiple regression analysis was performed to control for age, HbA1c, and antidepressant use.

Characteristics of subjects and between-group differences

Demographic and clinical characteristics of our sample are shown in Table 1. The PTSD and control groups did not differ significantly in terms of age. There were significant differences in BMI (t=-2.296, p = 0.023), ethnicity (chi square = 12.423, p = 0.029), smoking status (chi square = 17.125, p < 0.001), and years of education (t = 3.296, p < 0.001). Greater antidepressant use (chi square = 24.000, p < 0.001) was found in PTSD positive subjects.

PTSD status and ccf-mtDNA

A between-group t-test did not show a significant difference in ccf-mtDNA levels between the PTSD and control groups (PTSD negative: M = 0.082 (standardized value), SD = 0.934; PTSD positive: M=-0.089 (standardized value), SD = 1.053; t = 1.312, df = 230, p = 0.191). After adjusting for age, HbA1c, and antidepressant use in an ANCOVA model, however, we found significantly lower ccf-mtDNA levels in the PTSD group (F(1, 221) = 5.509; p = 0.020) (Table 2; Fig. 1). Replacing antidepressant use with current MDD status did not result in a significant between-group difference (F(1, 221) = 1.373; p = 0.243), implicating antidepressant medication rather than the diagnosis of MDD. When subjects with antidepressant use and diabetes (defined as HbA1c > 6.5%) were excluded in a sensitivity analysis, a t-test also showed significantly lower ccf-mtDNA levels in the PTSD group (PTSD negative: M = 0.062 (standardized value), SD = 0.947; PTSD positive: M=-0.315 (standardized value), SD = 0.977; t = 2.552, df = 178, p = 0.012).

Ccf-mtDNA and glucocorticoid signaling

Our analyses of DST measures showed that, across the entire group, but not within each individual group, ccf-mtDNA levels were negatively correlated with post-dexamethasone ACTH decline (r=-0.171, p = 0.020) and cortisol decline (r=-0.149, p = 0.034) (Table 2). Specifically, lower ccf-mtDNA levels were associated with larger dexamethasone-associated decreases in ACTH and cortisol, indicating greater GR sensitivity. These correlations remained significant when controlling for age, HbA1c, and antidepressant use (ACTH decline: β =-0.165, p = 0.023; cortisol decline: β=-0.140, p = 0.044) (Table 3; Fig. 1a,b).

IC_{50 − DEX}, a measure of glucocorticoid sensitivity in PBMCs, was significantly lower in PTSD subjects relative to controls (t = 2.506, df = 205.743, p = 0.013; Table 3), indicating greater glucocorticoid receptor sensitivity. Though IC_{50 − DEX} was not significantly associated with ccf-mtDNA levels in unadjusted analyses, after controlling for age, HbA1c, and antidepressant use, IC_{50 − DEX} was significantly positively associated with ccf-mtDNA levels across the entire group (β = 0.135, p = 0.043), but not within each individual group (Table 3; Fig. 1c).

Ccf-mtDNA and antidepressant use in PTSD subjects

Based on our ANCOVA results, we also explored the relationship between antidepressant use and ccf-mtDNA within the PTSD group. Antidepressant users had higher ccf-mtDNA levels, although the difference missed statistical significance (PTSD only: M= -0.199, SD = 1.056; PTSD + Antidepressants: M = 0.211, SD = 1.020; t=-1.832, p = 0.070, Cohen’s d=-0.392). The difference was significant (F(1, 100) = 4.695, p = 0.033), however, when controlling for age and HbA1c. Ccf-mtDNA levels did not differ significantly between PTSD subjects with concurrent MDD and those without concurrent MDD (PTSD only: M=-0.018, SD = 1.137; PTSD + MDD: M=-0.159, SD = 0.969; t = 0.699, p = 0.486, Cohen’s d = 0.133), including when controlling for age, Hba1c, and antidepressant status (F(1, 101) = 0.282, p = 0.596), again implicating antidepressant medication rather than a comorbid MDD diagnosis.

Exploratory correlation analyses

Exploratory analyses were performed to examine potential associations between ccf-mtDNA levels, age, BMI, HbA1c, and psychometric test scores, (see Tables 2 and 3). Ccf-mtDNA levels were significantly positively correlated with age across the entire group (r = 0.187, p = 0.004) and separately within the PTSD negative group (r = 0.181, p = 0.047) and the PTSD positive group (r = 0.204, p = 0.032). Ccf-mtDNA levels were also significantly associated with HbA1c across the entire group (r = 0.245, p < 0.001) and within each group separately (PTSD negative: r = 0.248, p = 0.006; PTSD positive: r = 0.249, p = 0.012). BMI was not significantly associated with ccf-mtDNA levels across the entire group or within either group.

Within the PTSD group, there were no significant associations found between ccf-mtDNA and CAPS lifetime score, PCL score, PTSD duration, BDI-II score, or PSS score, before and after controlling for age, HbA1c, and antidepressant use (Tables 3 and 4). Within the PTSD negative group, CAPS current score was significantly negatively associated with ccf-mtDNA levels (r=-0.221, p = 0.015), including after controlling for age, HbA1c, and antidepressant use (β=-0.203, p = 0.022). Across the entire group, CAPS current score (β=-0.190, p = 0.006), CAPS lifetime score (β=-0.190, p = 0.007), PCL score (β=-0.172, p = 0.013), and BDI-II score (β=-0.168, p = 0.018) were all negatively associated with ccf-mtDNA levels across the entire group after controlling for age, HbA1c and antidepressant use, though this may reflect differences between groups.

To our knowledge, this is the first study to examine the relationship between ccf-mtDNA levels and PTSD. Ccf-mtDNA levels did not differ between PTSD positive subjects and PTSD negative controls in the unadjusted analysis. After controlling for age, HbA1c, and antidepressant use (all variables that may affect ccf-mtDNA levels), however, we found significantly lower ccf-mtDNA in the PTSD positive group. We also found an association between glucocorticoid sensitivity and ccf-mtDNA levels, such that higher GR sensitivity was associated with lower ccf-mtDNA levels. Finally, we replicated previously reported correlations between ccf-mtDNA levels and age [9, 10] and HbA1c [6].

The small number of studies that have examined ccf-mtDNA in psychiatric conditions have been inconclusive [28, 49], and comparing their results is complicated by differences in sample characteristics, exclusion criteria, and ccf-mtDNA purification protocols [49]. Although there have been no prior studies of ccf-mtDNA in PTSD, there have been studies in MDD and in suicidality, which are common PTSD comorbidities and which were associated with elevated plasma ccf-mtDNA in two prior studies by our group, one in unmedicated MDD subjects [18] and another in suicide attempters [19]. In contrast, Fernström et al. [24] reported that current depression and remitted depression were both negatively associated with ccf-mtDNA levels, though over 90% of subjects in that study were on psychotropic medication. Kageyama et al. [23] also reported significantly lower ccf-mtDNA relative to controls in unmedicated MDD subjects. Although the relationship between MDD and ccf-mtDNA remains unclear, because MDD was overrepresented in the PTSD group (n = 56) relative to the control group (n = 4), it seemed plausible that MDD could have affected the association we found between PTSD and ccf-mtDNA. However, ccf-mtDNA levels did not differ significantly between PTSD subjects with and without concurrent MDD, including when controlling for age, HbA1c, and antidepressant use, suggesting that the association we observed between PTSD status and ccf-mtDNA was independent of MDD status. On the other hand, antidepressant users within the PTSD group had significantly higher ccf-mtDNA levels when controlling for age and HbA1c. This finding raising the possibility of a positive association between antidepressant use, itself, and increased ccf-mtDNA levels within the PTSD group may be consistent with prior work showing direct effects of antidepressants on mitochondrial function and integrity [50, 51, 52, 53]. While our data do not offer any clues as to causality, they do suggest that antidepressant use should be considered a relevant variable in mitochondrial studies.

PTSD has been associated with an increased risk of developing Type 2 diabetes [33, 54], and elevated HbA1c was reported to be a risk factor for developing PTSD [55]. Type 2 diabetes is associated with elevated ccf-mtDNA levels [4, 5, 6, 7], and HbA1c has been positively correlated with ccf-mtDNA levels in diabetic patients [6]. There was a higher number of subjects who met criteria for diabetes in the PTSD positive group than the PTSD negative group, though the difference missed significance. Because of the increased prevalence of glucose dysregulation in PTSD, the results from our ANCOVA and sensitivity analyses suggest that future studies of ccf-mtDNA should also take HbA1c or diabetes status into consideration.

The results of our analyses of glucocorticoid sensitivity add support to the growing evidence that glucocorticoid signaling and ccf-mtDNA may be related. Previous studies reported positive correlations between ccf-mtDNA levels and post-dexamethasone cortisol [19] and salivary cortisol following exercise [20]. A hypersensitive negative feedback response in the HPA axis is associated with PTSD, and perhaps contributes to the development of PTSD [2], and PTSD has previously been associated with increased dexamethasone-induced suppression of cortisol [56, 57] and ACTH [58, 59]. Consistent with this, we found heightened cortisol suppression, ACTH suppression, and PBMC GR sensitivity in the PTSD group. Although the precise relationship between glucocorticoids and ccf-mtDNA levels remains unclear, our data are consistent with growing evidence of the importance of glucocorticoid interactions with mitochondria [60, 61, 62, 63]. In particular, based on the negative correlations we found between ccf-mtDNA levels and ACTH and cortisol decline, investigating the associations between ccf-mtDNA and specific molecules involved in GR signaling would be of interest. GRs enter mitochondria and directly interact with mitochondrial DNA [64, 65, 66, 67], and molecules associated with GR entry and activity within mitochondria, such as Bag-1 [68], Bcl-2 [60], FKBP51 [69], HDAC6 [70], and Hsp90 [70]), could be interesting candidates.

Lower ccf-mtDNA levels have been found in the cerebrospinal fluid (CSF) of subjects in the early stages of Parkinson’s and Alzheimer’s disease [14, 15]. In both cases, the mechanisms underlying the lower CSF ccf-mtDNA levels are not currently understood, but it was hypothesized that the reduction in ccf-mtDNA in those cases could accompany a decline in mtDNA resulting from mitochondrial dysfunction that occurs prior to cell death [14, 15, 16]. Alternatively, pathologically decreased ccf-mtDNA levels could be a consequence of the drive to increase cellular mtDNA content by restricting the fraction of mtDNA that is released [71]. PTSD has been associated with an increased risk of neurodegenerative disorders [72], and mitochondrial dysfunction has been implicated in PTSD [73, 74, 75, 32]. Kageyama et al. [76] reported that transgenic mice whose forebrain neurons expressed a mutant form of Plog1, which results in an increase in mtDNA deletions, had a significantly lower C01/D-loop ratio in their plasma ccf-mtDNA relative to controls, suggesting brain-derived mtDNA can enter the plasma. If this is the case in humans, it is plausible that changes in mitochondrial function in the brain could have contributed to the differences observed here, though further research would be required to determine the impact of brain-derived ccf-mtDNA on plasma ccf-mtDNA levels. Importantly, the degree to which peripheral measures of ccf-mtDNA levels reflect central vs. other sources is unknown, and nothing in our data directly implicate central processes.

The present study has several strengths. We recruited a relatively large, well-characterized sample of young and healthy participants, and our exclusion criteria reduced the likelihood that biochemical measurements were influenced by other medical comorbidities. Moreover, because the PTSD and control groups had all experienced combat trauma, we were able to control for the possibility that the differences observed were due to trauma experience itself. On the other hand, however, since the PTSD-negative controls had experienced significant combat trauma, they may have represented an especially resilient group of individuals. The relatively large size of our cohort relative to most other ccf-mtDNA studies to date increased the statistical power of our correlations, making this a significant contribution to the current ccf-mtDNA literature. Our replication of the positive associations between ccf-mtDNA levels and age and HbA1c suggests that these associations may be stable across various populations and should be considered in future studies of ccf-mtDNA levels. Finally, while there have been studies of ccf-mtDNA levels in other psychiatric disorders, this is the first study to investigate ccf-mtDNA levels in PTSD.

Limitations to our study include having only male combat veterans. As a result, it is unclear whether our findings are generalizable to civilians or to females with PTSD. In addition, because studies thus far have had considerable variation in the blood fraction used and DNA purification protocols, our results can only be directly compared with studies using similar protocols. Moreover, ccf-mtDNA levels were only measured once in this study. Because there can be substantial variations in ccf-mtDNA levels within individual subjects over time and depending on psychological state [49], measurements at multiple time points along with a stress assessment at each blood draw would be ideal. Moreover, many subjects were missing smoking data, so smoking was not included in our analyses. The influence of smoking and other possible confounding variables on ccf-mtDNA should be assessed further. Finally, ccf-mtDNA comprises not only non-membrane bound DNA fragments but also mtDNA contained in cell-free intact mitochondria and in extracellular vesicles (EV) such as microvesicles and exosomes, each with potentially distinct mechanisms of release and physiological roles, and different purification protocols result in discrepancies in the type of ccf-mtDNA isolated [77, 49], potentially impacting results. Based on our protocol, our samples should have contained non-membrane bound ccf-mtDNA in addition to that contained in microvesicles and exosomes [49], and we found a clear positive correlation with aging. On the other hand, Lazo et al. [78] analyzed only EV-bound ccf-mtDNA and found a negative association. Future investigations of PTSD’s effect on each type of ccf-mtDNA separately would help clarify the clinical significance of our findings.

In conclusion, using a relatively large, well-phenotyped veteran male sample, we found no overall between-group difference in ccf-mtDNA levels in unadjusted analyses. After controlling for age, HbA1c, and antidepressants, however, those with PTSD showed lower ccf-mtDNA levels than those without PTSD. Thus, while PTSD per se is not associated with altered plasma ccf-mtDNA levels, the subgroup of relatively “pure PTSD,” namely those without diabetes and those not on antidepressants, may show decreased levels. Our results also suggest that increased glucocorticoid sensitivity in PTSD may be associated with lower ccf-mtDNA levels. This observation may tie together the increased GR sensitivity reported in PTSD with our observation of decreased ccf-mtDNA, at least in “pure PTSD.” Finally, our results are consistent with literature suggesting mitochondrial involvement in PTSD [73, 74, 75, 32], at least in its “pure” form as defined here (absent diabetes or antidepressant medication), although the pathophysiological significance of low plasma ccf-mtDNA levels remains uncertain. Although ccf-mtDNA’s usefulness as a diagnostic biomarker of PTSD is doubtful, our results suggest that improving our understanding of ccf-mtDNA in PTSD could aid in elucidating the mechanisms underlying PTSD’S pathophysiology and the relationships between glucocorticoid signaling, antidepressants, and mitochondrial function.

ACKNOWLEDGEMENTS

Disclaimer

Material has been reviewed by the Walter Reed Army Institute of Research. There is no objection to its presentation and/or publication. The opinions or assertions contained herein are the private views of the author, and are not to be construed as official, or as reflecting true views of the Department of the Army or the Department of Defense. The investigators have adhered to the policies for protection of human subjects as prescribed in AR 70-25.

Funding

This work was supported by funding from the U.S. Army Research Office, through award numbers W911NF-13-1-0376, W911NF-17-2- 0086, W911NF-18-2-0056, by the Army Research Laboratory under grant number W911NF-17-1-0069, and from the U.S. Department of Defense under W81XWH-10-1-0021, W81XWH-09-2-0044, and W81XWH-14-1-0043. Daniel Lindqvist is funded by the Swedish Research Council (grant number #2020–01428) and Swedish governmental funding of clinical research (ALF).

CONFLICT OF INTEREST

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

SYSTEMS BIOLOGY PTSD BIOMARKERS CONSORTIUM (SBPBC)

Kerry J. Ressler¹⁰, MD, PhD; Ruoting Yang⁶, PhD; Seid Muhie⁶, PhD; Bernie J. Daigle Jr.¹¹, PhD; Linda M. Bierer⁴, MD; Leroy Hood¹², MD, PhD; Kai Wang¹², PhD; Inyoul Lee¹², PhD; Kelsey R. Dean⁷, PhD; Pramod R. Somvanshi⁷, PhD

¹⁰Department of Psychiatry, Harvard Medical School and McLean Hospital, Belmont, MA, USA ¹¹Departments of Biological Sciences and Computer Science, The University of Memphis, Memphis, TN, USA.

¹²Institute for Systems Biology, Seattle, WA, USA

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Tables 1 to 3 are available in the Supplementary Files section.

The authors have declared there is NO conflict of interest to disclose

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You are reading this latest preprint version

Circulating cell-free mitochondrial DNA levels and glucocorticoid receptor sensitivity in combat-related PTSD

Status:

Journal Publication

Version 1

Abstract

Figures

Introduction

Methods

Study participants

Evaluation of psychiatric symptoms

Blood collection

Ccf-mtDNA purification and quantification

Clinical Labs

Dexamethasone suppression test and neuroendocrine assays

Dexamethasone-induced lysozyme suppression (IC_{50 − DEX}) assay

Statistical analysis

Results

Characteristics of subjects and between-group differences

PTSD status and ccf-mtDNA

Ccf-mtDNA and glucocorticoid signaling

Ccf-mtDNA and antidepressant use in PTSD subjects

Exploratory correlation analyses

Discussion

Declarations

References

Tables

Additional Declarations

Supplementary Files

Status:

Journal Publication

Version 1

Circulating cell-free mitochondrial DNA levels and glucocorticoid receptor sensitivity in combat-related PTSD

Status:

Journal Publication

Version 1

Abstract

Figures

Introduction

Methods

Study participants

Evaluation of psychiatric symptoms

Blood collection

Ccf-mtDNA purification and quantification

Clinical Labs

Dexamethasone suppression test and neuroendocrine assays

Dexamethasone-induced lysozyme suppression (IC50 − DEX) assay

Statistical analysis

Results

Characteristics of subjects and between-group differences

PTSD status and ccf-mtDNA

Ccf-mtDNA and glucocorticoid signaling

Ccf-mtDNA and antidepressant use in PTSD subjects

Exploratory correlation analyses

Discussion

Declarations

References

Tables

Additional Declarations

Supplementary Files

Status:

Journal Publication

Version 1

Dexamethasone-induced lysozyme suppression (IC_{50 − DEX}) assay