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Patients with oxidative phosphorylation (OxPhos) defects causing mitochondrial diseases appear particularly vulnerable to infections. Although OxPhos defects modulate cytokine production in vitro and in animal models, little is known about how circulating leukocytes of patients with inherited mitochondrial DNA (mtDNA) defects respond to acute immune challenges. In a small cohort of healthy controls (n=21) and patients (n=12) with either the m.3243A>G mutation or single, large-scale mtDNA deletions, we examined: i) cytokine responses (IL-6, TNF-α, IL-1β) in response to acute lipopolysaccharide (LPS) exposure, and ii) sensitivity to the immunosuppressive effects of glucocorticoid signaling (dexamethasone) on cytokine production. In dose-response experiments to determine the half-maximal effective LPS concentration (EC50), relative to controls, leukocytes from patients with mtDNA deletions showed 74 - 79% lower responses for IL-6 and IL-1β (pIL-6=0.031, pIL-1β=0.009). Moreover, IL-6 response to LPS in presence of GC was also blunted in cells from patients with mtDNA deletions (pIL-6=0.006), but not in leukocytes from patients with the m.3243A>G mutation. Overall, these ex vivo data provide preliminary evidence that some systemic OxPhos defects may compromise immune cytokine responses and glucocorticoid sensitivity. Further work in larger cohorts is needed to define the nature of immune dysregulation in patients with mitochondrial disease, and their potential implications for disease phenotypes.

The transfer of mitochondrial DNA into the nuclear genomes of eukaryotes (Numts) has been linked to lifespan in non-human species1–3and recently demonstrated to occur in rare instances from one human generation to the next4. Here we investigated numtogenesis dynamics in humans in two ways. First, we quantified Numts in 1,187 post-mortem brain and blood samples from different individuals. Compared to circulating immune cells (n=389), post-mitotic brain tissue (n=798) contained more Numts, consistent with their potential somatic accumulation. Within brain samples we observed a 5.5-fold enrichment of somatic Numt insertions in the dorsolateral prefrontal cortex compared to cerebellum samples, suggesting that brain Numts arose spontaneously during development or across the lifespan. Moreover, more brain Numts was linked to earlier mortality. The brains of individuals with no cognitive impairment who died at younger ages carried approximately 2 more Numts per decade of life lost than those who lived longer. Second, we tested the dynamic transfer of Numts using a repeated-measures WGS design in a human fibroblast model that recapitulates several molecular hallmarks of aging5. These longitudinal experiments revealed a gradual accumulation of one Numt every ∼13 days. Numtogenesis was independent of large-scale genomic instability and unlikely driven cell clonality. Targeted pharmacological perturbations including chronic glucocorticoid signaling or impairing mitochondrial oxidative phosphorylation (OxPhos) only modestly increased the rate of numtogenesis, whereas patient-derivedSURF1-mutant cells exhibiting mtDNA instability accumulated Numts 4.7- fold faster than healthy donors. Combined, our data document spontaneous numtogenesis in human cells and demonstrate an association between brain cortical somatic Numts and human lifespan. These findings open the possibility that mito-nuclear horizontal gene transfer among human post-mitotic tissues produce functionally-relevant human Numts over timescales shorter than previously assumed.

Mitochondria contain their own genome that can be released in multiple biofluids such as blood and cerebrospinal fluid, as cell-free mitochondrial DNA (cf-mtDNA). In clinical studies, blood cf-mtDNA predicts mortality and higher cf-mtDNA levels are associated with mental and physical stress. However, the dynamics of cf-mtDNA has not been defined, and whether it can be measured non-invasively like other neuroendocrine markers in saliva has not been examined. Here we report cf-mtDNA in human saliva and establish its natural within-person dynamic behavior across multiple weeks. In a small proof-of-principle cohort of healthy adults, we first develop an approach to rapidly quantify salivary cf-mtDNA without DNA isolation, and demonstrate the existence of salivary cf-mtDNA. We then deploy this approach to perform an intensive repeated-measures analysis of two healthy men studied at 4 daily timepoints over 53-60 consecutive days (n = 212-220 observations each) with parallel measures of steroid hormones, self-reported daily mood, and health-related behaviors. Salivary cf-mtDNA exhibited a robust awakening response reaching up to two orders of magnitude 30-45 min after awakening, varied from day-to-day, and moderately correlated with the cortisol awakening response. In exploratory analyses, no consistent association with self-reported daily mood/health-related behaviors were found, although this requires further examination in future studies. Dynamic variation in cf-mtDNA was inversely related with salivary interleukin 6 (IL-6), inconsistent with a pro-inflammatory effect of salivary cf-mtDNA. The highly dynamic behavior of salivary cf-mtDNA opens the door to non-invasive studies examining the relevance of mtDNA signaling in relation to human health.

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.

Stress triggers anticipatory physiological responses that promote survival, a phenomenon termed allostasis. However, the chronic activation of energy-dependent allostatic responses results in allostatic load, a dysregulated state that predicts functional decline, accelerates aging, and increases mortality in humans. The energetic cost and cellular basis for the damaging effects of allostatic load have not been defined. By longitudinally profiling primary human fibroblasts across their lifespan, we find that chronic glucocorticoid exposure induces a ~60% increase in cellular energy expenditure and a greater reliance on mitochondrial oxidative phosphorylation (OxPhos) rather than glycolysis. We show that this state of stress-induced hypermetabolism is linked to mtDNA instability, affects age-related cytokines secretion, and accelerates cellular aging based on DNA methylation clocks, telomere shortening rate, and reduced lifespan. Pharmacologically normalizing OxPhos activity while further increasing energy expenditure exacerbates the accelerated aging phenotype. Altogether, our findings define bioenergetic and multi-omic recalibrations of stress adaptation, underscoring increased energy expenditure and accelerated cellular aging as interrelated features of cellular allostatic load.

Patients with primary mitochondrial diseases present with fatigue and multi-system disease, are often lean, and die prematurely, but the mechanistic basis for this clinical picture remains unclear. Integrating data from 17 cohorts of patients with mitochondrial diseases (n=690), we find that clinical mitochondrial disorders increase resting energy expenditure, a state termed hypermetabolism. In a longitudinal cellular model of primary patient-derived fibroblasts from multiple donors, we show that genetic and pharmacological disruptions of oxidative phosphorylation (OxPhos) similarly trigger increased energy consumption in a cell-autonomous manner, despite near-normal OxPhos coupling efficiency. Hypermetabolism is associated with mtDNA instability, activation of the integrated stress response, increased extracellular secretion of age-related cytokines and metabokines including GDF15, as well as an accelerated rate of telomere erosion and epigenetic aging, and a reduced Hayflick limit. Together with these dynamic measures, we have generated a longitudinal RNASeq and DNA methylation resource dataset, which reveals conserved, energetically demanding, genome-wide recalibrations in response to OxPhos dysfunction. The increased energetic cost of living, or hypermetabolism, in cells and organisms with OxPhos defects has important biological and clinical implications.

Mitochondria release their genome as cell-free mitochondrial DNA (cf-mtDNA) in multiple biofluids including in the blood, where it predicts mortality and is a marker of mental and physical stress. Here we report cf-mtDNA in human saliva, an accessible biofluid used to study dynamic neuroendocrine changes. To map the natural dynamics of salivary cf-mtDNA over time, we examine cf-mtDNA and steroid hormones in a small cohort of healthy adults, and perform an intensive repeated-measures analysis of two healthy men studied at 4 daily timepoints over 53-60 consecutive days (n=412-420 observations). Salivary cf-mtDNA exhibits a robust awakening response reaching up to two orders of magnitude 30-45 minutes after awakening, varies from day-to-day, and moderately correlates with the cortisol awakening response. Moreover, we find no evidence that salivary cf-mtDNA has pro-inflammatory effects. The dynamic behavior of salivary cf-mtDNA opens the door to non-invasive studies examining the relevance of mtDNA signaling on human health. Significance statement Mitochondria play a key role in the pathophysiology of many human diseases, but researchers currently lack tools to reliably assess mitochondrial function and signaling at scale. Circulating cell-free mitochondrial DNA (cf-mtDNA) in blood is a marker of mental and physical stress that predicts mortality, but it is not easily accessible in large studies. This study reports the existence of cf-mtDNA in human saliva, which exhibits a dynamic behavior, including a robust awakening response and within-person day-to-day variation. We describe a robust measurement approach and document weak associations of cf-mtDNA with other mitochondria-derived steroid hormones, suggesting new avenues to examine the neuroendocrine significance of mitochondrial signaling to human health.

Malignant tumor cells exhibit mitochondrial alterations and are also influenced by biobehavioral processes, but the intersection of biobehavioral factors and mitochondria in malignant tumors remains unexplored. Here we examined multiple biochemical and molecular markers of mitochondrial content and function in benign tissue and in high-grade epithelial ovarian carcinoma (EOC) in parallel with exploratory analyses of biobehavioral factors. First, analysis of a publicly-available database (n = 1435) showed that gene expression of specific mitochondrial proteins in EOC is associated with survival. Quantifying multiple biochemical and molecular markers of mitochondrial content and function in tissue from 51 patients with benign ovarian masses and 128 patients with high-grade EOC revealed that compared to benign tissue, EOCs exhibit 3.3–8.4-fold higher mitochondrial content and respiratory chain enzymatic activities (P

Using a high-throughput mitochondrial phenotyping platform to quantify multiple mitochondrial features among molecularly defined immune cell subtypes, we quantify the natural variation in mitochondrial DNA copy number (mtDNAcn), citrate synthase, and respiratory chain enzymatic activities in human neutrophils, monocytes, B cells, and naïve and memory T lymphocyte subtypes. In mixed peripheral blood mononuclear cells (PBMCs) from the same individuals, we show to what extent mitochondrial measures are confounded by both cell type distributions and contaminating platelets. Cell subtype-specific measures among women and men spanning four decades of life indicate potential age- and sex-related differences, including an age-related elevation in mtDNAcn, which are masked or blunted in mixed PBMCs. Finally, a proof-of-concept, repeated-measures study in a single individual validates cell type differences and also reveals week-to-week changes in mitochondrial activities. Larger studies are required to validate and mechanistically extend these findings. These mitochondrial phenotyping data build upon established immunometabolic differences among leukocyte subpopulations, and provide foundational quantitative knowledge to develop interpretable blood-based assays of mitochondrial health.

Tumor cells exhibit mitochondrial alterations and are also influenced by biobehavioral processes, but the intersection of biobehavioral factors and tumor mitochondria remains unexplored. Here we examined multiple biochemical and molecular markers of mitochondrial content and function in benign and cancerous ovarian tissue in parallel with exploratory analyses of biobehavioral factors. First, analysis of a publicly-available database (n=1,435) showed that gene expression of specific mitochondrial proteins in ovarian tumors is associated with survival. Quantifying multiple biochemical and molecular markers of mitochondrial content and function in 51 benign and 128 high-grade epithelial ovarian tumors revealed that compared to benign tissue, tumors exhibit 3.3-8.4-fold higher mitochondrial content and respiratory chain enzymatic activities (P

Mitochondrial function studies in human leukocytes have mainly focused on peripheral blood mononuclear cells (PBMCs), with the assumption that the immunometabolic properties of different immune cells have a negligible effect on PBMCs. Using a high-throughput mitochondrial phenotyping platform to quantify multiple mitochondrial features among both PBMCs and immunologically-defined immune cell subtypes from the same individuals, we show how mitochondrial activity in PBMCs is confounded by both cell type distributions and contaminating platelets. Applying this cell-specific approach in women and men spanning 4 decades of life, we find that mitochondria exhibit specific age- and sex-related differences, including an age-related elevation in mitochondrial DNA copy number (mtDNAcn), which are masked or blunted in PBMCs. Our purified cell subtypes data in this cohort also define the variation in mtDNAcn, mitochondrial content (citrate synthase), and respiratory chain enzymatic activities among neutrophils, monocytes, B and T lymphocyte subtypes. We then validate these cell type differences and define the natural intra-individual variation in mitochondrial function using an intensive repeated-measures study in a single individual, revealing substantial natural variation over time among cell subtypes and PBMCs. Finally, we introduce multivariate mitochondrial phenotypes – mitotypes – that distinguish lymphoid from myeloid cell types, naive-to-memory lymphocyte states, and moderately differ between women and men, which we propose as potential cell-specific biomarkers for future studies. Together, these findings identify dynamic cell-type specific variation in mitochondrial biology in circulating human leukocytes, providing foundational knowledge to develop interpretable blood-based assays of mitochondrial health.

Hot water epilepsy (HWE) is a rare form of sensory epilepsy where seizures are precipitated by a stimulus of contact with hot water. While earlier studies have suggested causal role of genes for HWE, specific underpinnings are beginning to be explored only recently. We carried out a whole genome-based linkage analysis in a family where most of its members affected by HWE and found evidence of a previously unknown locus at chromosome 9p24.3-p23. Parametric two-point analysis suggested linkage with the greatest LOD score of 3.42 for the marker D9S286 at 9p24.1 at recombination fraction (θ) = 0, 90% penetrance value and 1% phenocopy rate. The highest multipoint LODscore of 3.42 was obtained for same marker at 9p24. The critical genetic interval of about 10 Mb of DNA was defined by the markers D9S917 and D9S168 corresponding to the centromere-distal and centromere-proximal recombination boundaries, respectively. This observation along with our previous findings of hot water genetic loci at 10q21.3-q22.3 (OMIM: 613339) and 4q24-q28 (OMIM: 613340), indicates unanticipated genetic heterogeneity for the disorder in families from a relatively small geographic region in the southern parts of India.

Mitochondria modulate inflammatory processes in various model organisms, but it is unclear how much mitochondria regulate immune responses in human blood leukocytes. Here, we examine the effect of i) experimental perturbations of mitochondrial respiratory chain function, and ii) baseline inter-individual variation in leukocyte mitochondrial energy production capacity on stimulated cytokine release and glucocorticoid (GC) sensitivity. In a first cohort, whole blood from 20 healthy women and men was stimulated with increasing concentrations of the immune agonist lipopolysaccharide (LPS). Four inhibitors of mitochondrial respiratory chain Complexes I, III, IV, and V were used (LPS ​+ ​Mito-Inhibitors) to acutely perturb mitochondrial function, GC sensitivity was quantified using the GC-mimetic dexamethasone (DEX) (LPS ​+ ​DEX), and the resultant cytokine signatures mapped with a 20-cytokine array. Inhibiting mitochondrial respiration caused large inter-individual differences in LPS-stimulated IL-6 reactivity (Cohen’s d ​= ​0.72) and TNF-α (d ​= ​1.55) but only minor alteration in EC50-based LPS sensitivity (d ​= ​0.21). Specifically, inhibiting mitochondrial Complex IV potentiated LPS-induced IL-6 levels by 13%, but inhibited TNF-α induction by 72%, indicating mitochondrial regulation of the IL-6/TNF-α ratio. As expected, DEX treatment suppressed multiple LPS-induced pro-inflammatory cytokines (IFN-γ, IL-6, IL-8, IL-1β, TNF-α) by >85% and increased the anti-inflammatory cytokine IL-10 by 80%. Inhibiting Complex I potentiated DEX suppression of IL-6 by a further 12% (d ​= ​0.73), indicating partial mitochondrial modulation of glucocorticoid sensitivity. Finally, to examine if intrinsic mitochondrial respiratory capacity may explain a portion of immune reactivity differences across individuals, we measured biochemical respiratory chain enzyme activities and mitochondrial DNA copy number in isolated peripheral blood mononuclear cells (PBMCs) from a second cohort of 44 healthy individuals in parallel with LPS-stimulated IL-6 and TNF-α response. Respiratory chain function, particularly Complex IV activity, was positively correlated with LPS-stimulated IL-6 levels (r ​= ​0.45, p ​= ​0.002). Overall, these data provide preliminary evidence that mitochondrial behavior modulates LPS-induced inflammatory cytokine signatures in human blood.

Summary Hearing loss is the most common sensory disorder and is genetically heterogeneous. Apart from nuclear gene mutations, a number of inherited mitochondrial mutations have also been implicated. The m.1555A>G mutation in the mitochondrial MT-RNR1 gene is reported as the most common mutation causing nonsyndromic hearing loss in various ethnic populations. We report here for the first time the clinical, genetic and molecular characterisation of a single large five-generational Tamil-speaking South Indian family with maternally inherited nonsyndromic postlingual hearing loss. Molecular analysis led to identification of m.1555A>G in 28 maternal relatives with variable degree of phenotypic expression. The penetrance of hearing loss among the maternal relatives in this family was 55%. Sequence analysis of the complete mitochondrial genome in 36 members of this pedigree identified 25 known variants and one novel variant co-transmitted along with m.1555A>G mutation. The mtDNA haplotype analysis revealed that the maternal relatives carry the R*T2 haplotype similar to Europeans and South Asians. Sequencing of the coding exon of GJB2 nuclear gene did not show any pathogenic mutations. The results suggest that other nuclear or environmental modifying factors could have played a role in the differential expression of mutation m.1555A>G in postlingual hearing loss in this family.