Stress and circulating cell-free mitochondrial DNA: A systematic review of human studies, physiological considerations, and technical recommendations

Abstract

Cell-free mitochondrial DNA (cf-mtDNA) is a marker of inflammatory disease and a predictor of mortality, but little is known about cf-mtDNA in relation to psychobiology. A systematic review of the literature reveals that blood cf-mtDNA varies in response to common real-world stressors including psychopathology, acute psychological stress, and exercise. Moreover, cf-mtDNA is inducible within minutes and exhibits high intra-individual day-to-day variation, highlighting the dynamic regulation of cf-mtDNA levels. We discuss current knowledge on the mechanisms of cf-mtDNA release, its forms of transport ("cell-free" does not mean "membrane-free"), potential physiological functions, putative cellular and neuroendocrine triggers, and factors that may contribute to cf-mtDNA removal from the circulation. A review of in vitro, pre-clinical, and clinical studies shows conflicting results around the dogma that physiological forms of cf-mtDNA are pro-inflammatory, opening the possibility of other physiological functions, including the cell-to-cell transfer of whole mitochondria. Finally, to enhance the reproducibility and biological interpretation of human cf-mtDNA research, we propose guidelines for blood collection, cf-mtDNA isolation, quantification, and reporting standards, which can promote concerted advances by the community. Defining the mechanistic basis for cf-mtDNA signaling is an opportunity to elucidate the role of mitochondria in brain-body interactions and psychopathology.

Keywords: Mitochondria; Non-inflammatory effects; Psychosocial stress; Standard protocol; cell-free DNA; mtDNA.

Fig. 1.. Potential forms of blood circulating cell-free mtDNA (cf-mtDNA).

(A) Several forms of cf-mtDNA exist in the circulation. During cf-mtDNA isolation, the time and speed (i.e., g force) of the initial and secondary centrifugations determine the specific forms of cf-mtDNA that are isolated and detected. Platelets, which contain abundant mitochondria and mtDNA, can easily contaminate cf-mtDNA preparations and therefore inflate apparent cf-mtDNA levels when centrifugation forces are low. Note that structures in A are not drawn to scale. (B) The different forms of cf-mtDNA may have different physiological consequences. See Section 5 for discussion. (C) Cf-mtDNA release may occur “passively” with different forms of cell death, or “actively” through regulated processes. (D) Quantifying both cf-nDNA and cf-mtDNA determines whether increases in cf-DNA are specific to mtDNA. The selection of two different mtDNA/nDNA amplicons (mtDNA1 and mtDNA2) helps ensure that results are robust to potential inter-individual mtDNA sequence variations. The example mtDNA and nDNA genes/amplicons are from (Trumpff et al., 2019a). (E) The mtDNA/nDNA amplicons can be quantified in duplex quantitative real-time polymerase chain reaction (qPCR) or using other methods. qPCR measures the exponential amplification of target sequences over several cycles of PCR via fluorescence. The crossing of the resulting function in log linear phase of amplification above the detection threshold is called the cycle threshold (Ct) or crossing point, which depends on the number of the target amplicons (mtDNA or nDNA) in the original sample. Ct values are inversely proportional to the abundance of the amplicons in the original sample. By using multiple dilutions of a known amount of standard DNA, a standard curve can be generated of log concentration against the Ct, and the amount of DNA in a sample can be calculated, yielding the number of copies of mtDNA per mL of plasma.

Fig. 2.. Systematic review of human studies evaluating the effects of psychopathology and acute psychological stress on cfmtDNA levels.

(A) Summary of effects sizes (Hedges’g, 95% confidence intervals) for cf-mtDNA and (B) cf-nDNA. Circles are used for plasma and squares for serum. Results from Zhang et al. (Zhang et al., 2010b) are not displayed in this figure. (C) Magnitude of group differences or acute responses in cf-mtDNA and cf-nDNA represented as fold changes for psychological stress. Note that not all studies measured cf-nDNA. Abbreviations: ASD, autism spectrum disorder; BD, bipolar disorder; HC, healthy controls; MDD, major depressive disorders; SZ, schizophrenia.

Fig. 3.. Systematic review of studies evaluating the effects of exercise training and acute physical activity on cf-mtDNA levels.

(A) Summary of effects sizes (Hedges’g, 95% confidence intervals) in response to exercise for cf-mtDNA levels and (B) cf-nDNA levels. (C) Magnitude of group differences or acute responses in cf-mtDNA and cf-nDNA to exercise stress represented as fold changes. Results from Schockett et al. (2016) are not displayed in these figures.

Fig. 4.. Analysis of inter- and intra- individual variation in cf-mtDNA levels.

(A) Results from inter- and intra-individual variability in baseline cf-mtDNA levels quantified by the coefficient of variation (C.V.) of fold change in plasma (Hummel et al., 2019), and (B) in serum (Trumpff et al., 2019a). Each line represents a healthy participant sampled twice (visits 1 and 2). Note that cf-mtDNA can vary widely (i.e., more than double, in some case ± 80%) across both visits, suggesting that cf-mtDNA exhibits state properties. (C) Diagram representing known kinetics of trait and state variables typically assessed in clinical and psychobiological studies. Current evidence suggests that cfmtDNA levels vary within minutes to hours in healthy individuals. (D) Hypothetical intra-individual variation in cf-mtDNA across a normal day, highlighting the absence of information about potential diurnal variation and minimal information about the influence of acute psychosocial stress on cf-mtDNA dynamics. (E) Illustrative example of three occasions of sampling between two subjects exhibiting normal variation, resulting in three qualitatively different conclusions. As for other neuroendocrine factors (e.g., cortisol), mapping intra-individual cf-mtDNA dynamics and repeated-measures study designs may be required to achieve robust estimates of inter-individual differences.

Fig. 5.. Intracellular and extracellular mtDNA release in relation to pro-inflammatory signaling.

(A) Existing data from experiments on the pro-inflammatory effects of cf-mtDNA have used laboratory DNA extraction methods to remove the membrane and proteins and obtain purified mtDNA, or synthetic (PCR-amplified) oligomers. Therefore, isolated mtDNA using conventional techniques is not biologically equivalent to cf-mtDNA in human circulation. Left: depicted are the most prominent forms of cf-mtDNA in human plasma, which are either whole mitochondria or membrane-encapsulated forms of cf-mtDNA. Additional work is necessary to establish their immunogenic potential. Right: purified or synthetic forms of mtDNA used for in vivo and in vitro adjuvant and co-stimulation experiments, which can act as a DAMPs under certain conditions. Beyond their potential pro-inflammatory effects, physiological forms of cf-mtDNA and cell-free mitochondria could play other signaling roles that remains to be elucidated Section 5. (B) Intracellular release of mtDNA in the cytoplasm cause increased expression of interferon-related pro-inflammatory gene expression through known pathways (see (West and Shadel, 2017) and (Riley and Tait, 2020) for in depth reviews). (C) In humans, the association between physiological forms of cf-mtDNA and inflammation is correlational, and causality has not been established. Studies by Zhang et al. (Zhang et al., 2010a); Pinti et al. (Pinti et al., 2014), Nasi et al. (Nasi et al., 2020) and Kim et al. (Kim et al., 2020) show that purified/naked mtDNA can potentiate the pro-inflammatory effects of bona fide immune agonists like lipopolysaccharide (LPS) and N-Formylmethionyl-leucyl-phenylalanine (fMLF), but found that on its own, extracellular cf-mtDNA is not sufficient to induce pro-inflammatory gene expression or cytokine release.

Fig. 6.. Recommended protocol for cf-mtDNA quantification in human blood.

(A) Experimental steps to quantify cf-mtDNA and cf-nDNA. (B) Left: electron micrograph showing platelets isolated from a healthy individual’s plasma. Middle: magnified image of a single human platelet containing several visible mitochondria (mtDNA) but no nucleus (no nDNA). Right: Single platelet mitochondrion showing the characteristic electron-dense outer and inner membranes, and cristae. (C) Diagram illustrating how platelet abundance influences mtDNA and nDNA quantification and increases cf-mtDNA levels detected in human plasma. (D) Quantitative analysis of studies in the systematic review, showing reported copies of cf-mtDNA (copies per ml) relative to the maximal centrifugation conditions used in each study. Relationship between centrifugation time and speed with the amount of mtDNA detected in healthy subjects at baseline (log transformed plot), linear regression results not statistically significant. (E) Suggested minimum reporting guidelines and (F) standardized technical procedures to increase the robustness and reproducibility of cf-mtDNA research in humans. In addition to these steps, which yield total cf-mtDNA levels (multiple forms of transport), additional centrifugation steps can be applied to to the final clean plasma to isolate additional vehicular and potential non-vesicular forms of transport of cf-mtDNA.