ccf-mtDNA Levels Research Articles

Exploring mitochondrial blood-based and genetic markers in older adults with mild cognitive impairment and remitted major depressive disorder

Mild cognitive impairment (MCI) is a prodromal stage in aging to possible progression to Alzheimer’s disease and related dementia (ADRD), where co-occurrence of major depressive disorder (MDD) accelerates the progression. Metabolic and mitochondrial abnormalities in ADRD and other neurodegenerative disorders have been widely suggested, while possible mitochondrial dysfunction has been associated with etiopathology of both MCI and MDD. Hence, investigation of mitochondrial markers in MCI, MDD, and presence of both conditions is warranted. In total, 332 older adult participants were included: 168 with MCI, 108 with MCI plus remitted MDD (rMDD), and 56 with rMDD but without MCI. We measured plasma circulating mitochondrial DNA (ccf-mtDNA), lactate, and extracted nuclear mitochondrial encoded (NMt) single-nucleotide variants (SNVs) (n = 312). Non-parametric statistical tests on ccf-mtDNA and lactate levels were performed on the diagnosis, clinical and cardiometabolic variables. Binary sequence kernel association test (SKAT-O) and burden test were performed on NMt-SNV, adjusted for age, race, gender, type II diabetes, and APOE genotype. Lower level of lactate was observed in MCI (KW χ2 = 14.8, P = 0.0024), more specifically, significant differences of lower plasma lactate between MCI only and rMDD, but not between MCI+rMDD and MCI were found, suggesting potential roles in MCI driving lactate lower levels. While higher levels of ccf-mtDNA were observed in APOE-ε4 carrier (χ2 = 5.04, P = 0.05). This relationship was present only in MCI (P = 0.043) and MCI+rMDD groups (P = 0.023). No significant nuclear-encoded mitochondrial gene associations were observed with MCI or MDD. The results suggest decreased level of plasma lactate in individuals with MCI and MCI+rMDD, with inverse correlation with ccf-mtDNA, in addition to effect of APOE-ε4 in further increasing ccf-mtDNA specifically in participants with cognitive impairment. These findings contribute to a deeper understanding of the mitochondrial markers in MCI and MDD, warranting further research to explore the precise roles of mitochondrial abnormalities in the development and progression of MCI.

Elevated serum circulating cell-free mitochondrial DNA in amyotrophic lateral sclerosis.

The substantial role of inflammation in amyotrophic lateral sclerosis (ALS) is gaining support from recent research. Studies indicate that circulating cell-free mitochondrial DNA (ccf-mtDNA) can activate the immune system and is associated with neurodegenerative diseases. This research was designed to quantify ccf-mtDNA levels in the serum of ALS patients. The medical records of ALS patients were reviewed. Serum ccf-mtDNA levels of patients with ALS (n = 62) and age-matched healthy controls (n = 46) were measured and compared. Additionally, serum interleukin-6 (IL-6) levels were measured using an enzyme-linked immunosorbent assay in 26 ALS patients. Correlations between variables were analyzed. Serum ccf-mtDNA was notably higher in the patients with ALS. When stratified by genotype, the superoxide dismutase 1 (SOD1) mutation group showed the greatest increase in ccf-mtDNA levels relative to other ALS patients. Among all 108 individuals, a cut-off set at 1.1 × 105 mtDNA copies on a receiver-operating characteristic curve identified patients with ALS with 80.7% sensitivity and 50.0% specificity; the area under the curve was 0.69 (p < 0.001). Furthermore, serum ccf-mtDNA levels correlated negatively with the progression rate of ALS (ΔFS; rs = -0.26, p = 0.044), but not the ALSFRS-R score (rs = 0.06, p = 0.625). Importantly, the correlation between ccf-mtDNA and ΔFS was more pronounced in the SOD1 mutation group (rs = -0.62, p = 0.018). Lastly, a significant positive association was observed between serum ccf-mtDNA levels and IL-6 levels in ALS (r s= 0.41, p = 0.038). Our study found increased serum ccf-mtDNA in ALS patients, suggesting a link to inflammatory processes and disease mechanism. Moreover, ccf-mtDNA could be an indicator for ALS progression, especially in those with the SOD1 mutation.

Pilot study of circulating cell-free mitochondrial DNA in relation to brain structure in youth bipolar disorder

BackgroundMitochondrial dysfunction is implicated in the neuropathology of bipolar disorder (BD). Higher circulating cell-free mitochondrial DNA (ccf-mtDNA), generally reflecting poorer mitochondrial health, has been associated with greater symptoms severity in BD. The current study examines the association of serum ccf-mtDNA and brain structure in relation to youth BD. We hypothesized that higher ccf-mtDNA will be associated with measures of lower brain structure, particularly in the BD group.MethodsParticipants included 40 youth (BD, n = 19; Control group [CG], n = 21; aged 13–20 years). Serum ccf-mtDNA levels were assayed. T1-weighted brain images were acquired using 3T-MRI. Region of interest (ROI) analyses examined prefrontal cortex (PFC) and whole brain gray matter, alongside exploratory vertex-wise analyses. Analyses examined ccf-mtDNA main-effects and ccf-mtDNA-by-diagnosis interaction effects controlling for age, sex, and intracranial volume.ResultsThere was no significant difference in ccf-mtDNA levels between BD and CG. In ROI analyses, higher ccf-mtDNA was associated with higher PFC surface area (SA) (β = 0.32 p < 0.001) and PFC volume (β = 0.32 p = 0.002) in the overall sample. In stratified analyses, higher ccf-mtDNA was associated with higher PFC SA within both subgroups (BD: β = 0.39 p = 0.02; CG: β = 0.24 p = 0.045). Higher ccf-mtDNA was associated with higher PFC volume within the BD group (β = 0.39 p = 0.046). In vertex-wise analyses, higher ccf-mtDNA was associated with higher SA and volume in frontal clusters within the overall sample and within the BD group. There were significant ccf-mtDNA-by-diagnosis interactions in three frontal and parietal clusters, whereby higher ccf-mtDNA was associated with higher neurostructural metrics in the BD group but lower neurostructural metrics in CG.ConclusionsContrasting our hypothesis, higher ccf-mtDNA was consistently associated with higher, rather than lower, regional neuralstructural metrics among youth with BD. While this finding may reflect a compensatory mechanism, future repeated-measures prospective studies evaluating the inter-relationship among ccf-mtDNA, mood, and brain structure across developmental epochs and illness stages are warranted.

Screening mitochondria-related biomarkers in skin and plasma of atopic dermatitis patients by bioinformatics analysis and machine learning.

There is a significant imbalance of mitochondrial activity and oxidative stress (OS) status in patients with atopic dermatitis (AD). This study aims to screen skin and peripheral mitochondria-related biomarkers, providing insights into the underlying mechanisms of mitochondrial dysfunction in AD. Public data were obtained from MitoCarta 3.0 and GEO database. We screened mitochondria-related differentially expressed genes (MitoDEGs) using R language and then performed GO and KEGG pathway analysis on MitoDEGs. PPI and machine learning algorithms were also used to select hub MitoDEGs. Meanwhile, the expression of hub MitoDEGs in clinical samples were verified. Using ROC curve analysis, the diagnostic performance of risk model constructed from these hub MitoDEGs was evaluated in the training and validation sets. Further computer-aided algorithm analyses included gene set enrichment analysis (GSEA), immune infiltration and mitochondrial metabolism, centered on these hub MitoDEGs. We also used real-time PCR and Spearman method to evaluate the relationship between plasma circulating cell-free mitochondrial DNA (ccf-mtDNA) levels and disease severity in AD patients. MitoDEGs in AD were significantly enriched in pathways involved in mitochondrial respiration, mitochondrial metabolism, and mitochondrial membrane transport. Four hub genes (BAX, IDH3A, MRPS6, and GPT2) were selected to take part in the creation of a novel mitochondrial-based risk model for AD prediction. The risk score demonstrated excellent diagnostic performance in both the training cohort (AUC = 1.000) and the validation cohort (AUC = 0.810). Four hub MitoDEGs were also clearly associated with the innate immune cells' infiltration and the molecular modifications of mitochondrial hypermetabolism in AD. We further discovered that AD patients had considerably greater plasma ccf-mtDNA levels than controls (U = 92.0, p< 0.001). Besides, there was a significant relationship between the up-regulation of plasma mtDNA and the severity of AD symptoms. The study highlights BAX, IDH3A, MRPS6 and GPT2 as crucial MitoDEGs and demonstrates their efficiency in identifying AD. Moderate to severe AD is associated with increased markers of mitochondrial damage and cellular stress (ccf=mtDNA). Our study provides data support for the variation in mitochondria-related functional characteristics of AD patients.

Mitochondrial Fraction of Circulating Cell-Free DNA as an Indicator of Human Pathology.

Circulating cell-free DNA (ccfDNA) of mitochondrial origin (ccf-mtDNA) consists of a minor fraction of total ccfDNA in blood or in other biological fluids. Aberrant levels of ccf-mtDNA have been observed in many pathologies. Here, we introduce a simple and effective standardized Taqman probe-based dual-qPCR assay for the simultaneous detection and relative quantification of nuclear and mitochondrial fragments of ccfDNA. Three pathologies of major burden, one malignancy (Breast Cancer, BrCa), one inflammatory (Osteoarthritis, OA) and one metabolic (Type 2 Diabetes, T2D), were studied. Higher levels of ccf-mtDNA were detected both in BrCa and T2D in relation to health, but not in OA. In BrCa, hormonal receptor status was associated with ccf-mtDNA levels. Machine learning analysis of ccf-mtDNA datasets was used to build biosignatures of clinical relevance. (A) a three-feature biosignature discriminating between health and BrCa (AUC: 0.887) and a five-feature biosignature for predicting the overall survival of BrCa patients (Concordance Index: 0.756). (B) a five-feature biosignature stratifying among T2D, prediabetes and health (AUC: 0.772); a five-feature biosignature discriminating between T2D and health (AUC: 0.797); and a four-feature biosignature identifying prediabetes from health (AUC: 0.795). (C) a biosignature including total plasma ccfDNA with very high performance in discriminating OA from health (AUC: 0.934). Aberrant ccf-mtDNA levels could have diagnostic/prognostic potential in BrCa and Diabetes, while the developed multiparameter biosignatures can add value to their clinical management.

Increased circulating cell-free mitochondrial DNA in plasma of first-diagnosed drug-naïve bipolar disorder patients: A case-control and 4-week follow-up study

BackgroundThe study of clinical biological indicators in bipolar disorder (BD) is important. In recent years, basic experiments have associated the pathophysiological mechanism of BD is related to mitochondrial dysfunction, but few clinical studies have confirmed this finding. ObjectThe present study aimed to evaluate whether plasma circulating cell-free mitochondrial DNA (ccf-mtDNA) levels, which can represent the degree of mitochondrial damage in vivo, are altered in patients with BD in early onset and during treatment compared with controls. MethodA total of 75 first-diagnosed drug-naive patients with BD and 60 HCs were recruited and followed up for 1 month. The clinical symptoms were assessed using HAMD, HAMA, and YMRS, and ccf-mtDNA levels were measured by qPCR before and after drug treatment in BD. Result(1) The plasma ccf-mtDNA levels in first-diagnosed drug-naive patients with BD increased compared with those in HCs (p = 0.001). (2) Drug treatment for 1 month can decrease the expression of ccf-mtDNA in BD (p < 0.001). (3) No significant correlation was observed between the changes in ccf-mtDNA levels and the improvement of clinical symptoms in BD after drug treatment. ConclusionThe plasma ccf-mtDNA level was increased in BD, and decreased after pharmacological treatment. These outcomes suggested that plasma ccf-mtDNA level is likely to be sensitive to the drug response in BD, and mitochondrial pathway is a potential target for further therapy.

Quantification of Circulating Cell-Free DNA in Idiopathic Parkinson's Disease Patients.

Parkinson's disease (PD) is one of the most common neurodegenerative disorders globally and leads to an excessive loss of dopaminergic neurons in the substantia nigra of the brain. Circulating cell-free DNA (ccf-DNA) are double-stranded DNA fragments of different sizes and origins that are released into the serum and cerebrospinal fluid (CSF) due to cell death (i.e., necrosis and apoptosis) or are actively released by viable cells via exocytosis and NETosis. Using droplet digital polymerase chain reaction (ddPCR), we comprehensively analyzed and distinguished circulating cell-free mitochondrial DNA (ccf mtDNA) and circulating cell-free nuclear DNA (ccfDNA) in the serum and CSF of PD and control patients. The quantitative analysis of serum ccf-DNA in PD patients demonstrated a significant increase in ccf mtDNA and ccfDNA compared to that in healthy control patients and a significantly higher copy of ccf mtDNA when compared to ccfDNA. Next, the serum ccf mtDNA levels significantly increased in male PD patients compared to those in healthy male controls. Furthermore, CSF ccf mtDNA in PD patients increased significantly compared to ccfDNA, and ccf mtDNA decreased in PD patients more than it did in healthy controls. These decreases were not statistically significant but were in agreement with previous data. Interestingly, ccf mtDNA increased in healthy control patients in both serum and CSF as compared to ccfDNA. The small sample size of serum and CSF were the main limitations of this study. To the best of our knowledge, this is the first comprehensive study on serum and CSF of PD patients using ddPCR to indicate the distribution of the copy number of ccf mtDNA as well as ccfDNA. If validated, we suggest that ccf mtDNA has greater potential than ccfDNA to lead the development of novel treatments for PD patients.

Circulating cell-free mitochondrial DNA levels and glucocorticoid sensitivity in a cohort of male veterans with and without combat-related PTSD

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 ccf mt-DNA levels and glucocorticoid sensitivity. In unadjusted analyses, ccf-mtDNA levels did not differ significantly between the PTSD and non-PTSD groups (t = 1.312, p = 0.191, Cohen’s d = 0.172). In a sensitivity analysis excluding participants with diabetes and those using antidepressant medication and controlling for age, the PTSD group had lower ccf-mtDNA levels than did the non-PTSD group (F(1, 179) = 5.971, p = 0.016, partial η2 = 0.033). Across the entire sample, ccf-mtDNA levels were negatively correlated with post-dexamethasone adrenocorticotropic hormone (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 diabetes status. Ccf-mtDNA levels were also significantly positively associated with IC50-DEX (the concentration of dexamethasone at which 50% of lysozyme activity is inhibited), a measure of lymphocyte glucocorticoid sensitivity, after controlling for age, antidepressant status, and diabetes status (β = 0.142, p = 0.038), suggesting that increased lymphocyte glucocorticoid sensitivity is associated with lower ccf-mtDNA levels. Although no overall group differences were found in unadjusted analyses, excluding subjects with diabetes and those taking antidepressants, which may affect ccf-mtDNA levels, as well as controlling for age, revealed decreased ccf-mtDNA levels in PTSD. In both adjusted and unadjusted analyses, low ccf-mtDNA levels were associated with relatively increased glucocorticoid sensitivity, often reported in PTSD, suggesting a link between mitochondrial and glucocorticoid-related abnormalities in PTSD.

Extracellular vesicle mitochondrial DNA levels are associated with race and mitochondrial DNA haplogroup

Circulating cell-free mitochondrial DNA (ccf-mtDNA) acts as a damage-associated molecular pattern molecule and may be cargo within extracellular vesicles (EVs). ccf-mtDNA and select mitochondrial DNA (mtDNA) haplogroups are associated with cardiovascular disease. We hypothesized that ccf-mtDNA and plasma EV mtDNA would be associated with hypertension, sex, self-identified race, and mtDNA haplogroup ancestry. Participants were normotensive (n= 107) and hypertensive (n= 108) African American and White adults from the Healthy Aging in Neighborhoods of Diversity across the Life Span study. ccf-mtDNA levels were higher in African American participants compared with White participants in both plasma and EVs, but ccf-mtDNA levels were not related to hypertension. EV mtDNA levels were highest in African American participants with African mtDNA haplogroup. Circulating inflammatory protein levels were altered with mtDNA haplogroup, race, and EV mtDNA. Our findings highlight that race is a social construct and that ancestry is crucial when examining health and biomarker differences between groups.

Clinicopathological and circulating cell-free DNA profile in myositis associated with anti-mitochondrial antibody.

Anti-mitochondrial antibodies (AMAs) are associated with idiopathic inflammatory myopathies (IIMs). We aimed to summarize the clinicopathological characteristics, assess circulating cell-free mitochondrial DNA (ccf-mtDNA), and circulating cell-free nuclear DNA (ccf-nDNA) in AMA-associated IIMs. Medical records of 37 IIMs patients with AMAs were reviewed. Circulating cell-free mtDNA and ccf-nDNA levels in sera from IIMs patients with AMAs (n = 21), disease controls (n = 66) and healthy controls (HCs) (n = 23) were measured and compared. Twenty-eight immune-mediated necrotizing myopathy (IMNM) patients, 23 dermatomyositis (DM) patients, and 15 anti-synthetase syndrome (ASS) patients were enrolled as disease controls. Correlations between variables were analyzed. Limb weakness was observed in 75.7% and neck weakness in 56.8% of patients. Cardiac involvement occurred in 51.4% of patients. Muscle pathology revealed 81.1% of IMNM, 5.4% polymyositis, and 13.5% nonspecific myositis. Microinfarction was observed in 8.1% of patients. Serum ccf-mtDNA levels in AMA-associated IIMs were significantly higher than those in HCs (p < 0.001), but no significant differences between AMA-associated IIMs and IMNM, DM, or ASS. Serum ccf-nDNA levels in AMA-associated IIMs were significantly higher than those in HCs (p = 0.02), and significantly lower than those in DM (p = 0.02). Serum ccf-nDNA levels correlated negatively with MMT8 total scores (rs = -0.458, p = 0.037) and positively with mRS scores (rs = 0.486, p = 0.025). Serum ccf-nDNA levels were significantly higher in the non-remission group (p < 0.01). AMA-associated IIMs exhibit distinct clinicopathological features. Serum ccf-nDNA may serve as a potential marker for disease severity and prognosis in AMA-associated IIMs.

Mitochondrial health index correlates with plasma circulating cell-free mitochondrial DNA in bipolar disorder.

Although mitochondrial dysfunction is known to play an essential role in the pathophysiology of bipolar disorder (BD), there is a glaring gap in our understanding of how mitochondrial dysfunction can modulate clinical phenotypes. An emerging paradigm suggests mitochondria play an important non-energetic role in adaptation to stress, impacting cellular resilience and acting as a source of systemic allostatic load. Known as mitochondrial allostatic load, this (phenomenon) occurs when mitochondria are unable to recalibrate and maintain cell homeostasis. This study aimed to evaluate the composite mitochondrial health index (MHI) in BD subjects and non-psychiatry controls. We will also explore whether lower MIH will be related to higher cell-free mtDNA (ccf-mtDNA) levels and poor clinical outcomes. In this study, 14 BD-I patients and 16 age- and sex-matched non-psychiatry controls were enrolled. Peripheral blood mononuclear cells (PBMCs) were used to measure the enzymatic activities of citrate synthase and complexes I, II, and IV and mtDNA copy number. Ccf-mtDNA was evaluated by qPCR in plasma. Mitochondrial quality control (MQC) proteins were evaluated by western blotting. After adjusting for confounding variables, such as age, sex, body mass index (BMI), and smoking status, patients with BD presented lower MHI compared to non-psychiatry controls, as well as higher ccf-mtDNA levels that negatively correlated with MHI. Because the MQC network is essential to maintain mitochondrial health, MHI and ccf-mtDNA were also examined in relation to several MQC-related proteins, such as Fis-1, Opa-1, and LC3. Our results showed that MHI correlated negatively with Fis-1 and positively with Opa-1 and LC3. Accordingly, ccf-mtDNA had a positive correlation with Fis-1 and a negative correlation with Opa-1 and LC3. Furthermore, we found a noteworthy inverse correlation between illness severity and MHI, with lower MHI and higher ccf-mtDNA levels in subjects with a longer illness duration, worse functional status, and higher depressive symptoms. Our findings indicate that mitochondrial allostatic load contributes to BD, suggesting mitochondria represent a potential biological intersection point that could contribute to impaired cellular resilience and increased vulnerability to stress and mood episodes. Ultimately, by linking mitochondrial dysfunction to disease progression and poor outcomes, we might be able to build a predictive marker that explains how mitochondrial function and its regulation contribute to BD development and that may eventually serve as a treatment guide for both old and new therapeutic targets.

Associations between circulating cell-free mitochondrial DNA, inflammatory markers, and cognitive and physical outcomes in community dwelling older adults

BackgroundDementia and frailty are common age-related syndromes often linked to chronic inflammation. Identifying the biological factors and pathways that contribute to chronic inflammation is crucial for developing new therapeutic targets. Circulating cell-free mitochondrial DNA (ccf-mtDNA) has been proposed as an immune stimulator and potential predictor of mortality in acute illnesses. Dementia and frailty are both associated with mitochondrial dysfunction, impaired cellular energetics, and cell death. The size and abundance of ccf-mtDNA fragments may indicate the mechanism of cell death: long fragments typically result from necrosis, while short fragments arise from apoptosis. We hypothesize that increased levels of necrosis-associated long ccf-mtDNA fragments and inflammatory markers in serum are linked to declines in cognitive and physical function, as well as increased mortality risk.ResultsOur study of 672 community-dwelling older adults revealed that inflammatory markers (C-Reactive Protein, soluble tumor necrosis factor alpha, tumor necrosis factor alpha receptor 1 [sTNFR1], and interleukin-6 [IL-6]) positively correlated with ccf-mtDNA levels in serum. Although cross-sectional analysis revealed no significant associations between short and long ccf-mtDNA fragments, longitudinal analysis demonstrated a connection between higher long ccf-mtDNA fragments (necrosis-associated) and worsening composite gait scores over time. Additionally, increased mortality risk was observed only in individuals with elevated sTNFR1 levels.ConclusionIn a community dwelling cohort of older adults, there are cross-sectional and longitudinal associations between ccf-mtDNA and sTNFR1 with impaired physical and cognitive function and increased hazard of death. This work suggests a role for long ccf-mtDNA as a blood-based marker predictive of future physical decline.

Plasma circulating cell-free mitochondrial DNA in social anxiety disorder

ObjectiveTo investigate plasma levels of circulating cell-free mitochondrial DNA (ccf-mtDNA) in patients with social anxiety disorder (SAD) and healthy controls (HC). MethodsIn this study, 88 participants (46 patients with SAD and 42 HCs) were enrolled and both ccf-mtDNA and peripheral blood mononuclear cells (PBMC) mtDNA copy number (mtDNA-cn) were measured at up to three times per individual (9–11 weeks apart). SAD patients also received cognitive behavioral therapy (CBT) between the second and third time-point. ResultsSAD patients had significantly lower ccf-mtDNA compared to HCs at all time points, but ccf-mtDNA did not change significantly after CBT, and was not associated with severity of anxiety symptoms. Plasma ccf-mtDNA did not significantly correlate with PBMC mtDNA-cn in patients. ConclusionThis is the first report of lower ccf-mtDNA in patients with an anxiety disorder. Our findings could reflect a more chronic illness course in SAD patients with prolonged periods of psychological stress leading to decreased levels of ccf-mtDNA, but future longitudinal studies are needed to confirm or refute this hypothesis.

New methods for analysing circulating cell‐free mitochondrial DNA

Trauma, ischemia, inflammatory stress and environmental conditions can cause tissue injury and damage. This damage spurs the release of cellular components and debris into the extracellular environment, including damage associated molecular pattern (DAMP) molecules. These DAMPs are sensed by pattern recognition receptors that activate a cellular damage response to the pathological insult. Mitochondrial components have recently been recognised as potent DAMPs.1, 2 The mitochondrial genome shares features with bacterial genomes, due to their shared origin. Therefore, when mitochondrial DNA (mtDNA) is released into the extracellular space it is recognised as a foreign molecule and initiates an innate immune response.1, 2 Not only have DAMP molecules been of interest in studying the relationships between cellular damage and immune responses, but emerging evidence suggests that DAMPs may serve as ‘liquid biopsies’ to indicate tissue damage or disease status. For example, plasma levels of circulating cell-free mtDNA (ccf-mtDNA) can be isolated and quantified easily using quantitative real-time PCR (qPCR). The results of many studies suggest that there are higher levels of ccf-mtDNA after trauma, acute myocardial infarction, sepsis and in some inflammatory conditions.3 A recent systematic review examined published articles on utilising ccf-mtDNA as a predictor of mortality in critically ill patients.4 The authors noted that in their analysis there are inconsistencies in ccf-mtDNA isolation methods and quantification. For example, most often qPCR primers were designed against subunits of the NADH dehydrogenase (MT-ND, MT-ND1, MT-ND2 or MT-ND6).4 However, some studies use one primer set, others use multiple primer sets and some use primer sets recognising different genes in the mitochondrial genome. This may explain the discrepancies in the findings linking ccf-mtDNA to mortality in some studies but not in all. Therefore, there is a need for the development of technologies that improve upon these current methods to quantify ccf-mtDNA. Daly and colleagues recently developed a protocol that provides a new approach to analysing plasma ccf-mtDNA.5 In their approach, they utilised a target bait-capture kit consisting of biotinylated RNA probes complementary to the entire mitochondrial genome. First, the biotinylated probes bind to mitochondrial sequences and then are enriched, pooled, and sequenced on a standard Illumina instrument. Second, a Workflow Description Language-based computational pipeline aligns the reads to both the nuclear and mitochondrial genome and then custom programs call mitochondrial and nuclear coverage, insert fragment size and mtDNA variants. Nuclear genomes contain sequences of mitochondrial origin, called nuclear mitochondrial DNA segments (NUMTs). NUMT pseudogenes can also be enriched using these methods. Therefore, to exclude the possibility that reads are from the nuclear genome, this method excludes sequences that aligned to both nuclear and mitochondrial genomes. This approach may underestimate the mtDNA levels but increases classification accuracy. In fact, this protocol was shown to enrich for mean coverage of the mtDNA genome compared to whole genome sequencing. Therefore, this protocol allows for the simultaneous quantification of mtDNA levels across the entire mitochondrial genome, differentiation of fragment lengths, and the identification of mtDNA variants.5 The authors utilised their protocol to investigate ccf-mtDNA in comparison to clinical phenotypes of injury and severity scores in a cohort of 30 trauma patients.5 There were no significant differences in ccf-mtDNA abundance between patients with non-severe or severe injuries. The ccf-mtDNA fragment length was significantly shorter in patients with major trauma and in patients where acute lung injury occurred after admission compared to non-severe injury patients. There were also mtDNA variants that were discovered that were associated with various clinical scores of injuries and severity in this cohort. Therefore, the authors utilised samples from this trauma cohort to validate that their protocol can be implemented to examined mtDNA levels, fragment length, and variants.5 This technological advance has advantages over the standard qPCR methods, as the entire mitochondrial genome can be analysed in one protocol. However, additional experiments and validation studies should be performed to directly compare the sensitivity and accuracy of this protocol versus the standard qPCR methods. Nevertheless, these methods will give scientists an additional tool to examine ccf-mtDNA in relation to tissue damage or disease status. This work was funded by the Intramural Research Program of the National Institute on Aging, National Institutes of Health. The authors declare no conflict of interest.

Cell-Free Mitochondrial DNA in Acute Brain Injury.

Traumatic brain injury and aneurysmal subarachnoid haemorrhage are a major cause of morbidity and mortality worldwide. Treatment options remain limited and are hampered by our understanding of the cellular and molecular mechanisms, including the inflammatory response observed in the brain. Mitochondrial DNA (mtDNA) has been shown to activate an innate inflammatory response by acting as a damage-associated molecular pattern (DAMP). Here, we show raised circulating cell-free (ccf) mtDNA levels in both cerebrospinal fluid (CSF) and serum within 48 h of brain injury. CSF ccf-mtDNA levels correlated with clinical severity and the interleukin-6 cytokine response. These findings support the use of ccf-mtDNA as a biomarker after acute brain injury linked to the inflammatory disease mechanism.

Increased level of free-circulating MtDNA in maintenance hemodialysis patients: Possible role in systemic inflammation.

BackgroundMitochondrial DNA (MtDNA) exposed to the extracellular space due to cell death and stress has immunostimulatory properties. However, the clinical significance of circulating MtDNA in maintenance hemodialysis (MHD) patients and the precise mechanism of its emergence have yet to be investigated.MethodsThis cross‐sectional study consisted of 52 MHD patients and 32 age‐ and sex‐matched healthy controls. MHD patients were further categorized into high and low circulating cell‐free MtDNA (ccf‐MtDNA) groups based on the median value. Copy number of MtDNA was quantified using TaqMan‐based qPCR. Plasma cytokines were measured using ELISA kits. Reactive oxygen species (ROS) and mitochondrial membrane potential (Δψm) in peripheral blood mononuclear cells (PBMCs) were detected using DCFH‐DA or JC‐1 staining.ResultsThe copy numbers of ccf‐MtDNA in patients with MHD were higher than those in healthy controls, and these alterations were correlated with changes of cytokines TNF‐α and IL‐6. Adjusted model in multivariate analysis showed that the presence of anuria and longer dialysis vintage were independently associated with higher levels of ccf‐MtDNA. Meanwhile, although not statistically significant, an inverse correlative trend between urinary MtDNA and ccf‐MtDNA was observed in patients with residual urine. Afterward, using PBMCs as surrogates for mitochondria‐rich cells, we found that patients in the high ccf‐MtDNA group exhibited a significantly higher ROS production and lower Δψm in cells.ConclusionsOur data suggested that changes in ccf‐MtDNA correlate with the degree of inflammatory status in MHD patients, and that the excessive MtDNA may be caused by mitochondrial dysfunction and reduced urinary MtDNA excretion.

Circulating inflammatory markers, cell-free mitochondrial DNA, cortisol, endocannabinoids, and N-acylethanolamines in female depressed outpatients

Objectives Major depressive disorder (MDD) involves peripheral low-grade pro-inflammatory activity. This multi-biomarker case-control study characterises the proinflammatory status in MDD beyond C-reactive protein (CRP) and Interleukin (IL)-6 levels through investigating concomitant alterations of immunoregulatory biomolecules. Methods In 20 female MDD patients and 24 non-depressed women, circulating levels of CRP, IL-6, cortisol, selected endocannabinoids (ECs; anandamide [AEA], 2-arachidonylglycerol [2-AG]), and N-acylethanolamines (NAEs), as well as circulating cell-free mitochondrial DNA (ccf-mtDNA) were measured. Results We found higher serum CRP and plasma AEA levels in MDD and a positive association of CRP and AEA levels with current depressive symptoms. Blood levels of cortisol, ccf-mtDNA, 2-AG, and NAEs did depend on MDD diagnosis nor correlated with the severity of current depressive symptoms. CRP correlated positively with AEA, and AEA showed positive associations with 2-AG and NAE levels. Conclusions In this study, female MDD outpatients with mild to moderate disorder severity did not substantially differ from non-depressed controls in the resting levels of multiple immunoregulatory markers in peripheral blood. Instead of investigating resting levels, future research on the role of inflammatory activity in MDD should focus on investigating the reactivity of pathways modulating the immune system upon exposure to physical and psychosocial stressors.

Circulating cell-free mtDNA release is associated with the activation of cGAS-STING pathway and inflammation in mitochondrial diseases.

There is increasing evidence for the role of inflammation in the pathogenesis of mitochondrial diseases (MDs). However, the mechanisms underlying mutation-induced inflammation in MD remain elusive. Our previous study suggested that mitophagy is impaired in the skeletal muscle of those with MD, likely causing mitochondrial DNA (mtDNA) release and thereby triggering inflammation. We here aimed to decipher the role of the cGAS-STING pathway in inflammatory process in MDs. We investigated the levels of circulating cell-free mtDNA (ccf-mtDNA) in the serum of 104 patients with MDs. Immunofluorescence was performed in skeletal muscles in MDs and control. Biochemical analysis of muscle biopsies was conducted with western blot to detect cGAS, STING, TBK1, IRF3 and phosphorylated IRF3 (p-IRF3). RT-qPCR was performed to detect the downstream genes of type I interferon in skeletal muscles. Furthermore, a protein microarray was used to examine the cytokine levels in the serum of patients with MDs. We found that ccf-mtDNA levels were significantly increased in those with MDs compared to the controls. Consistently, the immunofluorescent results showed that cytosolic dsDNA levels were increased in the muscle samples of MD patients. Biochemical analysis of muscle biopsies showed that cGAS, IRF3, and TBK1 protein levels were significantly increased in those with MDs, indicating that there was activation of the cGAS-STING pathway. RT-qPCR showed that downstream genes of type I interferon were upregulated in muscle samples of MDs. Protein microarray results showed that a total of six cytokines associated with the cGAS-STING pathway were significantly increased in MD patients (fold change > 1.2, p value < 0.05). These findings suggest that increases in ccf-mtDNA levels is associated with the activation of the cGAS-STING pathway, thereby triggering inflammation in MDs.

P168. Mitochondrial Health Index Correlates With Plasma Circulating Cell-Free Mitochondrial DNA in Bipolar Disorder

Although mitochondria dysfunction is known to play an important role in the pathophysiology of bipolar disorder (BD), there is a glaring gap in our understanding of how mitochondrial dysfunction moderates clinical phenotypes. This study aimed to evaluate the composite mitochondrial health index (MHI) in BD subjects and healthy controls. We will also explore whether lower MIH will be related to higher cell-free mtDNA (ccf-mtDNA) levels and poor clinical outcomes.

Effects of caloric restriction and aerobic exercise on circulating cell-free mitochondrial DNA in patients with moderate to severe chronic kidney disease.

Circulating cell-free mitochondrial DNA (ccf-mtDNA) may induce systemic inflammation, a common condition in chronic kidney disease (CKD), by acting as a damage-associated molecular pattern. We hypothesized that in patients with moderate to severe CKD, aerobic exercise would reduce ccf-mtDNA levels. We performed a post hoc analysis of a multicenter randomized trial (NCT01150851) measuring plasma concentrations of ccf-mtDNA at baseline and 2 and 4 mo after aerobic exercise and caloric restriction. A total of 99 participants had baseline ccf-mtDNA, and 92 participants completed the study. The median age of the participants was 57 yr, 44% were female and 55% were male, 23% had diabetes, and 92% had hypertension. After adjusting for demographics, blood pressure, body mass index, diabetes, and estimated glomerular filtration rate, median ccf-mtDNA concentrations at baseline, 2 mo, and 4 mo were 3.62, 3.08, and 2.78 pM for the usual activity group and 2.01, 2.20, and 2.67 pM for the aerobic exercise group, respectively. A 16.1% greater increase per month in ccf-mtDNA was seen in aerobic exercise versus usual activity (P = 0.024), which was more pronounced with the combination of aerobic exercise and caloric restriction (29.5% greater increase per month). After 4 mo of intervention, ccf-mtDNA increased in the aerobic exercise group by 81.6% (95% confidence interval: 8.2-204.8, P = 0.024) compared with the usual activity group and was more marked in the aerobic exercise and caloric restriction group (181.7% increase, 95% confidence interval: 41.1-462.2, P = 0.003). There was no statistically significant correlation between markers of oxidative stress and inflammation with ccf-mtDNA. Our data indicate that aerobic exercise increased ccf-mtDNA levels in patients with moderate to severe CKD.NEW & NOTEWORTHY The effects of prolonged exercise on circulating cell-free mitochondrial DNA (ccf-mtDNA) have not been explored in patients with chronic kidney disease (CKD). We showed that 4-mo aerobic exercise is associated with an increase in plasma ccf-mtDNA levels in patients with stages 3 or 4 CKD. These changes were not associated with markers of systemic inflammation. Future studies should determine the mechanisms by which healthy lifestyle interventions influence biomarkers of inflammation and oxidative stress in patients with CKD.