Acute psychological stress increases serum circulating cell-free mitochondrial DNA

Abstract

Intrinsic biological mechanisms transduce psychological stress into physiological adaptation that requires energy, but the role of mitochondria and mitochondrial DNA (mtDNA) in this process has not been defined in humans. Here, we show that similar to physical injury, exposure to psychological stress increases serum circulating cell-free mtDNA (ccf-mtDNA) levels. Healthy midlife adults exposed on two separate occasions to a brief psychological challenge exhibited a 2-3-fold increase in ccf-mtDNA, with no change in ccf-nuclear DNA levels, establishing the magnitude and specificity for ccf-mtDNA reactivity. In cell-based studies, we show that glucocorticoid signaling - a consequence of psychological stress in humans - is sufficient to induce mtDNA extrusion in a time frame consistent with stress-induced ccf-mtDNA increase. Collectively, these findings provide evidence that acute psychological stress induces ccf-mtDNA and implicate neuroendocrine signaling as a potential trigger for ccf-mtDNA release. Further controlled work is needed to confirm that observed increases in ccf-mtDNA result from stress exposure and to determine the functional significance of this effect.

Keywords: Cell-free DNA; Mitochondria; Mitokine; Neuroendocrine; Psychobiology; Psychosocial stress.

Conflict of interest statement

Conflicts of interests

The authors declare no conflict of interest.

Copyright © 2019 Elsevier Ltd. All rights reserved.

Figures

Fig. 1.. Study design to assess levels of circulating cell-free mitochondrial (ccf-mtDNA) and nuclear DNA (ccf-nDNA) in response to induced psychological stress.

(A) Human cells contain nuclear DNA in the nucleus (purple) and cytoplasmic mitochondria (orange) with multiple copies of their own genome, the mtDNA. (B) Two different mtDNA amplicons, mtDNA1 (mt-ND1) and mtDNA2 (mt-CYTB), and two different nDNA amplicons nDNA1 (B2m) and nDNA2 (Gusb), were amplified from circulating cell-free serum using quantitative PCR. (C) Schematic of the experimental study design with three serial blood draws (pre, post, +30 min). A subsequent validation session 2 which was conducted one month later. (D) Stress-induced elevation of negative (left) and decrease in positive (right) items pre- and post-stress at session 1 and (E) at session 2, confirming the successful experimental manipulation of psychological states. Paired two-tailed student T-tests, n = 49 per session.

Fig. 2.. Psychological stress selectively increases serum circulating cell-free mitochondrial DNA.

(A) Individual (left) and group mean (right) ccf-mtDNA1 (mt-ND1) responses to acute psychological stress at session 1. (B) Validation of the results in (A) in a repeat session 2 one month later. Arrow: a participant with unusually high mtDNA1 baseline values at both sessions. Data are means and SEM. Repeated measure ANOVA on log-transformed data and Least Significant Difference (LSD) pairwise comparisons, n = 31 per session, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Fig. 3.. Psychological stress does not increase serum circulating cell-free nuclear DNA.

(A) Responses to acute psychological stress at session 1 for the nuclear DNA (ccf-nDNA1, B2m) and (B) at the validation session 2. Data are means and SEM. Repeated measure ANOVA on log transformed data and Least Significant Difference (LSD) pairwise comparisons, n = 31 per session, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Fig. 4.. Stress by sex interaction on the levels of circulating cell-free mtDNA.

(A) Adjusted means for ccf-mtDNA1 responses to acute psychological stress. (B) Same as in (A) for ccf-mtDNA2. Repeated measure ANOVA on log transformed data, n = 62 for both sessions combined together.

Fig 5.. Glucocorticoid stimulation causes mtDNA extrusion in human cells.

(A) Schematic of the experimental design. Primary male human fibroblasts were acutely exposed to 100nM dexamethasone (DEX) for 0, 2, 5, 15, 30 and 60 minutes, and mitochondria and mtDNA visualized by immunofluorescence. (B) Representative pictures of human fibroblasts after DEX treatment analyzed for GR subcellular localization. (C) Glucocorticoid receptor (GR) fluorescence was quantified in both the nucleus and the cytoplasm following DEX stimulation, which induced a marked translocation of GR to the nucleus. One Way ANOVA and Least Significant Difference (LSD) pairwise comparisons. (D) A human fibroblast exposed to DEX stimulation for 30 minutes, and dual-labeled by immunofluorescence for a ubiquitous marker of mitochondria (Tom20) and mtDNA. (D’) Higher magnification of boxed area in (D). (E) Cartoon of the mitochondria and nucleoids observed in (D’). Note the two different forms of extruded mtDNA: in contact to the surface of mitochondria (attached), or free floating in the cytoplasm (free floating). (F) Line profile intensity quantification for Tom20 and mtDNA along the yellow line in D’ showing: ➀ A nucleoid inside the mitochondrion; ➁ A nucleoid extruded into the cytoplasm (no mito-Tom20 staining); and ➂ A mitochondrion without mtDNA. (G) Time-course of the number of extruded mtDNA nucleoids after acute DEX stimulation. One Way ANOVA and Least Significant Difference (LSD) pairwise comparisons, n = 82 cells. (H) Time-course of sub-types of mtDNA nucleoids: the number of attached and free floating mtDNA. One Way ANOVA and LSD pairwise comparisons, n = 105 cells. (I) Size of extruded mtDNA (averaged per cell). One Way ANOVA and test for linear trend, n = 105 cells. Data are means ± SEM. Pairwise comparisons presented are relative to DEX 0 minute. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.