Although the functional consequences of mitochondrial DNA (mtDNA) genetic backgrounds (haplotypes, haplogroups) have been demonstrated by both disease association studies and cell culture experiments, it is not clear which of the mutations within the haplogroup carry functional implications and which are "evolutionary silent hitchhikers". We set forth to study the functionality of haplogroup-defining mutations within the mtDNA transcription/replication regulatory region by in vitro transcription, hypothesizing that haplogroup-defining mutations occurring within regulatory motifs of mtDNA could affect these processes. We thus screened >2500 complete human mtDNAs representing all major populations worldwide for natural variation in experimentally established protein binding sites and regulatory regions comprising a total of 241 bp in each mtDNA. Our screen revealed 77/241 sites showing point mutations that could be divided into non-fixed (57/77, 74%) and haplogroup/sub-haplogroup-defining changes (i.e., population fixed changes, 20/77, 26%). The variant defining Caucasian haplogroup J (C295T) increased the binding of TFAM (Electro Mobility Shift Assay) and the capacity of in vitro L-strand transcription, especially of a shorter transcript that maps immediately upstream of conserved sequence block 1 (CSB1), a region associated with RNA priming of mtDNA replication. Consistent with this finding, cybrids (i.e., cells sharing the same nuclear genetic background but differing in their mtDNA backgrounds) harboring haplogroup J mtDNA had a >2 fold increase in mtDNA copy number, as compared to cybrids containing haplogroup H, with no apparent differences in steady state levels of mtDNA-encoded transcripts. Hence, a haplogroup J regulatory region mutation affects mtDNA replication or stability, which may partially account for the phenotypic impact of this haplogroup. Our analysis thus demonstrates, for the first time, the functional impact of particular mtDNA haplogroup-defining control region mutations, paving the path towards assessing the functionality of both fixed and un-fixed genetic variants in the mitochondrial genome.

Mitochondria provide most of the energy for brain cells by the process of oxidative phosphorylation. Mitochondrial abnormalities and deficiencies in oxidative phosphorylation have been reported in individuals with schizophrenia (SZ), bipolar disorder (BD), and major depressive disorder (MDD) in transcriptomic, proteomic, and metabolomic studies. Several mutations in mitochondrial DNA (mtDNA) sequence have been reported in SZ and BD patients.

Despite the central role of amyloid deposition in the development of Alzheimer's disease (AD), the pathogenesis of AD still remains elusive at the molecular level. Increasing evidence suggests that compromised mitochondrial function contributes to the aging process and thus may increase the risk of AD. Dysfunctional mitochondria contribute to reactive oxygen species (ROS) which can lead to extensive macromolecule oxidative damage and the progression of amyloid pathology. Oxidative stress and amyloid toxicity leave neurons chemically vulnerable. Because the brain relies on aerobic metabolism, it is apparent that mitochondria are critical for the cerebral function. Mitochondrial DNA sequence changes could shift cell dynamics and facilitate neuronal vulnerability. Therefore we postulated that mitochondrial DNA sequence polymorphisms may increase the risk of AD. We evaluated the role of mitochondrial haplogroups derived from 138 mitochondrial polymorphisms in 358 Caucasian Alzheimer's Disease Neuroimaging Initiative (ADNI) subjects. Our results indicate that the mitochondrial haplogroup UK may confer genetic susceptibility to AD independently of the apolipoprotein E4 (APOE4) allele.

Traumatic brain injury (TBI) has been shown to activate the peripheral innate immune system and systemic inflammatory response, possibly through the central release of damage associated molecular patterns (DAMPs). Our main purpose was to gain an initial understanding of the peripheral mitochondrial response following TBI, and how this response could be utilized to determine cerebral mitochondrial bioenergetics. We hypothesized that TBI would increase peripheral whole blood relative mtDNA copy number, and that these alterations would be associated with cerebral mitochondrial bioenergetics triggered by TBI.

Success rates for genomic analyses of highly heterogeneous disorders can be greatly improved if a large cohort of patient data is assembled to enhance collective capabilities for accurate sequence variant annotation, analysis, and interpretation. Indeed, molecular diagnostics requires the establishment of robust data resources to enable data sharing that informs accurate understanding of genes, variants, and phenotypes. The "Mitochondrial Disease Sequence Data Resource (MSeqDR) Consortium" is a grass-roots effort facilitated by the United Mitochondrial Disease Foundation to identify and prioritize specific genomic data analysis needs of the global mitochondrial disease clinical and research community. A central Web portal (https://mseqdr.org) facilitates the coherent compilation, organization, annotation, and analysis of sequence data from both nuclear and mitochondrial genomes of individuals and families with suspected mitochondrial disease. This Web portal provides users with a flexible and expandable suite of resources to enable variant-, gene-, and exome-level sequence analysis in a secure, Web-based, and user-friendly fashion. Users can also elect to share data with other MSeqDR Consortium members, or even the general public, either by custom annotation tracks or through the use of a convenient distributed annotation system (DAS) mechanism. A range of data visualization and analysis tools are provided to facilitate user interrogation and understanding of genomic, and ultimately phenotypic, data of relevance to mitochondrial biology and disease. Currently available tools for nuclear and mitochondrial gene analyses include an MSeqDR GBrowse instance that hosts optimized mitochondrial disease and mitochondrial DNA (mtDNA) specific annotation tracks, as well as an MSeqDR locus-specific database (LSDB) that curates variant data on more than 1300 genes that have been implicated in mitochondrial disease and/or encode mitochondria-localized proteins. MSeqDR is integrated with a diverse array of mtDNA data analysis tools that are both freestanding and incorporated into an online exome-level dataset curation and analysis resource (GEM.app) that is being optimized to support needs of the MSeqDR community. In addition, MSeqDR supports mitochondrial disease phenotyping and ontology tools, and provides variant pathogenicity assessment features that enable community review, feedback, and integration with the public ClinVar variant annotation resource. A centralized Web-based informed consent process is being developed, with implementation of a Global Unique Identifier (GUID) system to integrate data deposited on a given individual from different sources. Community-based data deposition into MSeqDR has already begun. Future efforts will enhance capabilities to incorporate phenotypic data that enhance genomic data analyses. MSeqDR will fill the existing void in bioinformatics tools and centralized knowledge that are necessary to enable efficient nuclear and mtDNA genomic data interpretation by a range of shareholders across both clinical diagnostic and research settings. Ultimately, MSeqDR is focused on empowering the global mitochondrial disease community to better define and explore mitochondrial diseases.

Maternal inheritance of mtDNA is the rule in most animals, but the reasons for this pattern remain unclear. To investigate the consequence of overriding uniparental inheritance, we generated mice containing an admixture (heteroplasmy) of NZB and 129S6 mtDNAs in the presence of a congenic C57BL/6J nuclear background. Analysis of the segregation of the two mtDNAs across subsequent maternal generations revealed that proportion of NZB mtDNA was preferentially reduced. Ultimately, this segregation process produced NZB-129 heteroplasmic mice and their NZB or 129 mtDNA homoplasmic counterparts. Phenotypic comparison of these three mtDNA lines demonstrated that the NZB-129 heteroplasmic mice, but neither homoplasmic counterpart, had reduced activity, food intake, respiratory exchange ratio; accentuated stress response; and cognitive impairment. Therefore, admixture of two normal but different mouse mtDNAs can be genetically unstable and can produce adverse physiological effects, factors that may explain the advantage of uniparental inheritance of mtDNA.

Mitochondrial deficiencies with unknown causes have been observed in schizophrenia (SZ) and bipolar disorder (BD) in imaging and postmortem studies. Polymorphisms and somatic mutations in mitochondrial DNA (mtDNA) were investigated as potential causes with next generation sequencing of mtDNA (mtDNA-Seq) and genotyping arrays in subjects with SZ, BD, major depressive disorder (MDD), and controls. The common deletion of 4,977 bp in mtDNA was compared between SZ and controls in 11 different vulnerable brain regions and in blood samples, and in dorsolateral prefrontal cortex (DLPFC) of BD, SZ, and controls. In a separate analysis, association of mitochondria SNPs (mtSNPs) with SZ and BD in European ancestry individuals (n = 6,040) was tested using Genetic Association Information Network (GAIN) and Wellcome Trust Case Control Consortium 2 (WTCCC2) datasets. The common deletion levels were highly variable across brain regions, with a 40-fold increase in some regions (nucleus accumbens, caudate nucleus and amygdala), increased with age, and showed little change in blood samples from the same subjects. The common deletion levels were increased in the DLPFC for BD compared to controls, but not in SZ. Full mtDNA genome resequencing of 23 subjects, showed seven novel homoplasmic mutations, five were novel synonymous coding mutations. By logistic regression analysis there were no significant mtSNPs associated with BD or SZ after genome wide correction. However, nominal association of mtSNPs (p < 0.05) to SZ and BD were found in the hypervariable region of mtDNA to T195C and T16519C. The results confirm prior reports that certain brain regions accumulate somatic mutations at higher levels than blood. The study in mtDNA of common polymorphisms, somatic mutations, and rare mutations in larger populations may lead to a better understanding of the pathophysiology of psychiatric disorders.

Genetic inactivation of the nuclear-encoded mitochondrial heart-muscle adenine nucleotide translocator-1 (ANT1), which exports mitochondrial ATP to the cytosol in both humans (ANT1-/-) and mice (Ant1-/-), results in lactic acidosis and mitochondrial cardiomyopathy and myopathy, the latter involving hyper-proliferation of mitochondria, induction of oxidative phosphorylation (OXPHOS) enzymes, increased reactive oxygen species (ROS), and excessive mtDNA damage. To understand these manifestations, we analyzed Ant1-/- mouse skeletal muscle for changes in gene expression using our custom 644 and 1087 gene MITOCHIP microarrays and for changes in the protein levels of key mitochondrial transcription factors. Thirty-four mRNAs were found to be up-regulated and 29 mRNAs were down-regulated. Up-regulated mRNAs included the mitochondrial DNA (mtDNA) polypeptide and rRNA genes, selected nuclear-encoded OXPHOS genes, and stress-response genes including Mcl-1. Down-regulated mRNAs included glycolytic genes, pro-apoptotic genes, and c-Myc. The mitochondrial regulatory proteins Pgc-1alpha, Nrf-1, Tfam, and myogenin were up-regulated and could account for the induction of the OXPHOS and antioxidant enzymes. By contrast, c-Myc levels were reduced and might account for a reduction in apoptotic potential. Therefore, the Ant1-/- mouse skeletal muscle demonstrates that energy metabolism, antioxidant defenses, and apoptosis form an integrated metabolic network.

Mitochondrial and mechanical alterations in cells have both been shown to be hallmarks of human disease. However, little research has endeavoured to establish connections between these two essential features of cells in both functional and dysfunctional situations. In this work, we hypothesized that a specific genetic alteration in mitochondrial function known to cause human disease would trigger changes in cell mechanics. Using a previously characterized set of mitochondrial cybrid cell lines, we examined the relationship between heteroplasmy for the mitochondrial DNA (mtDNA) 3243A>G mutation, the cell cytoskeleton, and resulting cellular mechanical properties. We found that cells with increasing mitochondrial dysfunction markedly differed from one another in gene expression and protein production of various co-regulated cytoskeletal elements. The intracellular positioning and organization of actin also differed across cell lines. To explore the relationship between these changes and cell mechanics, we then measured cellular mechanical properties using atomic force microscopy and found that cell stiffness correlated with gene expression data for known determinants of cell mechanics, γ-actin, α-actinin and filamin A. This work points towards a mechanism linking mitochondrial genetics to single-cell mechanical properties. The transcriptional and structural regulation of cytoskeletal components by mitochondrial function may explain why energetic and mechanical alterations often coexist in clinical conditions.

Fragile X Syndrome (FXS), the most prevalent form of inherited intellectual disability and the foremost monogenetic cause of autism, is caused by loss of expression of the FMR1 gene . Here, we show that dfmr1 modulates the global metabolome in Drosophila. Despite our previous discovery of increased brain insulin signaling, our results indicate that dfmr1 mutants have reduced carbohydrate and lipid stores and are hypersensitive to starvation stress. The observed metabolic deficits cannot be explained by feeding behavior, as we report that dfmr1 mutants are hyperphagic. Rather, our data identify dfmr1 as a regulator of mitochondrial function. We demonstrate that under supersaturating conditions, dfmr1 mutant mitochondria have significantly increased maximum electron transport system (ETS) capacity. Moreover, electron micrographs of indirect flight muscle reveal striking morphological changes in the dfmr1 mutant mitochondria. Taken together, our results illustrate the importance of dfmr1 for proper maintenance of nutrient homeostasis and mitochondrial function.

Spaceflight is known to impose changes on human physiology with unknown molecular etiologies. To reveal these causes, we used a multi-omics, systems biology analytical approach using biomedical profiles from fifty-nine astronauts and data from NASA's GeneLab derived from hundreds of samples flown in space to determine transcriptomic, proteomic, metabolomic, and epigenetic responses to spaceflight. Overall pathway analyses on the multi-omics datasets showed significant enrichment for mitochondrial processes, as well as innate immunity, chronic inflammation, cell cycle, circadian rhythm, and olfactory functions. Importantly, NASA's Twin Study provided a platform to confirm several of our principal findings. Evidence of altered mitochondrial function and DNA damage was also found in the urine and blood metabolic data compiled from the astronaut cohort and NASA Twin Study data, indicating mitochondrial stress as a consistent phenotype of spaceflight.

Immune cell function is influenced by metabolic conditions. Low-glucose, high-lactate environments, such as the placenta, gastrointestinal tract, and the tumor microenvironment, are immunosuppressive, especially for glycolysis-dependent effector T cells. We report that nicotinamide adenine dinucleotide (NAD+), which is reduced to NADH by lactate dehydrogenase in lactate-rich conditions, is a key point of metabolic control in T cells. Reduced NADH is not available for NAD+-dependent enzymatic reactions involving glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and 3-phosphoglycerate dehydrogenase (PGDH). We show that increased lactate leads to a block at GAPDH and PGDH, leading to the depletion of post-GAPDH glycolytic intermediates, as well as the 3-phosphoglycerate derivative serine that is known to be important for T cell proliferation. Supplementing serine rescues the ability of T cells to proliferate in the presence of lactate-induced reductive stress. Directly targeting the redox state may be a useful approach for developing novel immunotherapies in cancer and therapeutic immunosuppression.

Foxp3+ T-regulatory (Treg) cells are capable of suppressing immune responses. Lysine acetylation is a key mechanism of post-translational control of various transcription factors, and when acetylated, Foxp3 is stabilized and transcriptionally active. Therefore, understanding the roles of various histone/protein deacetylases (HDAC) are key to promoting Treg-based immunotherapy. Several of the 11 classical HDAC enzymes are necessary for optimal Treg function while others are dispensable. We investigated the effect of HDAC10 in murine Tregs. HDAC10 deletion had no adverse effect on the health of mice, which retained normal CD4+ and CD8+ T cell function. However, HDAC10-/- Treg exhibited increased suppressive function in vitro and in vivo. C57BL/6 Rag1-/- mice adoptively transferred with HDAC10-/- but not wild Treg, were protected from developing colitis. HDAC10-/- but not wild-type mice receiving fully MHC-mismatched cardiac transplants became tolerant and showed long-term allograft survival (>100 d). We conclude that targeting of HDAC10 may be of therapeutic value for inflammatory disorders including colitis and also for transplantation.

Alzheimer's disease (AD), the most prevalent form of dementia, affects globally more than 30 million people suffering from cognitive deficits and neuropsychiatric symptoms. Substantial evidence for the involvement of mitochondrial dysfunction in the development and/or progression of AD has been shown in addition to the pathological hallmarks amyloid beta (Aβ) and tau. Still, the selective vulnerability and associated selective mitochondrial dysfunction cannot even be resolved to date. We aimed at optically quantifying mitochondrial function on a single-cell level in primary hippocampal neuron models of AD, unraveling differential involvement of cell and mitochondrial populations in amyloid precursor protein (APP)-associated mitochondrial dysfunction. NADH lifetime imaging is a highly sensitive marker-free method with high spatial resolution. However, deciphering cellular bioenergetics of complex cells like primary neurons has still not succeeded yet. To achieve this, we combined highly sensitive NADH lifetime imaging with respiratory inhibitor treatment, allowing characterization of mitochondrial function down to even the subcellular level in primary neurons. Measuring NADH lifetime of the same neuron before and after respiratory treatment reveals the metabolic delta, which can be taken as a surrogate for cellular redox capacity. Correlating NADH lifetime delta with overexpression strength of Aβ-related proteins on the single-cell level, we could verify the important role of intracellular Aβ-mediated mitochondrial toxicity. Subcellularly, we could demonstrate a higher respiration in neuronal somata in general than dendrites, but a similar impairment of somatic and dendritic mitochondria in our AD models. This illustrates the power of NADH lifetime imaging in revealing mitochondrial function on a single and even subcellular level and its potential to shed light into bioenergetic alterations in neuropsychiatric diseases and beyond.

MicroRNAs (miRNAs) are small non-coding RNAs involved in post-transcriptional gene regulation that have a major impact on many diseases and provide an exciting avenue toward antiviral therapeutics. From patient transcriptomic data, we determined that a circulating miRNA, miR-2392, is directly involved with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) machinery during host infection. Specifically, we show that miR-2392 is key in driving downstream suppression of mitochondrial gene expression, increasing inflammation, glycolysis, and hypoxia, as well as promoting many symptoms associated with coronavirus disease 2019 (COVID-19) infection. We demonstrate that miR-2392 is present in the blood and urine of patients positive for COVID-19 but is not present in patients negative for COVID-19. These findings indicate the potential for developing a minimally invasive COVID-19 detection method. Lastly, using in vitro human and in vivo hamster models, we design a miRNA-based antiviral therapeutic that targets miR-2392, significantly reduces SARS-CoV-2 viability in hamsters, and may potentially inhibit a COVID-19 disease state in humans.

We present super-resolution microscopy of isolated functional mitochondria, enabling real-time studies of structure and function (voltages) in response to pharmacological manipulation. Changes in mitochondrial membrane potential as a function of time and position can be imaged in different metabolic states (not possible in whole cells), created by the addition of substrates and inhibitors of the electron transport chain, enabled by the isolation of vital mitochondria. By careful analysis of structure dyes and voltage dyes (lipophilic cations), we demonstrate that most of the fluorescent signal seen from voltage dyes is due to membrane bound dyes, and develop a model for the membrane potential dependence of the fluorescence contrast for the case of super-resolution imaging, and how it relates to membrane potential. This permits direct analysis of mitochondrial structure and function (voltage) of isolated, individual mitochondria as well as submitochondrial structures in the functional, intact state, a major advance in super-resolution studies of living organelles.

Mitochondrial dysfunction has pleiotropic effects and is frequently caused by mitochondrial DNA mutations. However, factors such as significant variability in clinical manifestations make interpreting the pathogenicity of variants in the mitochondrial genome challenging. Here, we present APOGEE 2, a mitochondrially-centered ensemble method designed to improve the accuracy of pathogenicity predictions for interpreting missense mitochondrial variants. Built on the joint consensus recommendations by the American College of Medical Genetics and Genomics/Association for Molecular Pathology, APOGEE 2 features an improved machine learning method and a curated training set for enhanced performance metrics. It offers region-wise assessments of genome fragility and mechanistic analyses of specific amino acids that cause perceptible long-range effects on protein structure. With clinical and research use in mind, APOGEE 2 scores and pathogenicity probabilities are precompiled and available in MitImpact. APOGEE 2's ability to address challenges in interpreting mitochondrial missense variants makes it an essential tool in the field of mitochondrial genetics.

A unique property of mitochondria in mammalian cells is their ability to physically interact and undergo dynamic events of fusion/fission that remodel their morphology and possibly their function. In cultured cells, metabolic perturbations similar to those incurred during exercise influence mitochondrial fusion and fission processes, but it is unknown whether exercise acutely alters mitochondrial morphology and/or membrane interactions in vivo. To study this question, we subjected mice to a 3-h voluntarily exercise intervention following their normal physical activity patterns, and quantified mitochondrial morphology and membrane interactions in the soleus using a quantitative electron microscopy approach. A single exercise bout effectively decreased blood glucose (P < 0.05) and intramyocellular lipid content (P < 0.01), indicating increased muscle metabolic demand. The number of mitochondria spanning Z-lines and proportion of electron-dense contact sites (EDCS) between adjacent mitochondrial membranes were increased immediately after exercise among both subsarcolemmal (+116%, P < 0.05) and intermyofibrillar mitochondria (+191%, P < 0.001), indicating increased physical interactions. Mitochondrial morphology, and abundance of the mitochondrial pro-fusion proteins Mfn2 and OPA1 were unchanged. Collectively, these results support the notion that mitochondrial membrane dynamics are actively remodelled in skeletal muscle, which may be regulated by contractile activity and the metabolic state. Future studies are required to understand the implications of mitochondrial dynamics in skeletal muscle physiology during exercise and inactivity.

Nuclear-encoded mutations causing metabolic and degenerative diseases have highly variable expressivity. Patients sharing the homozygous mutation (c.523delC) in the adenine nucleotide translocator 1 gene (SLC25A4, ANT1) develop cardiomyopathy that varies from slowly progressive to fulminant. This variability correlates with the mitochondrial DNA (mtDNA) lineage. To confirm that mtDNA variants can modulate the expressivity of nuclear DNA (nDNA)-encoded diseases, we combined in mice the nDNA Slc25a4-/- null mutation with a homoplasmic mtDNA ND6P25L or COIV421A variant. The ND6P25L variant significantly increased the severity of cardiomyopathy while the COIV421A variant was phenotypically neutral. The adverse Slc25a4-/- and ND6P25L combination was associated with impaired mitochondrial complex I activity, increased oxidative damage, decreased l-Opa1, altered mitochondrial morphology, sensitization of the mitochondrial permeability transition pore, augmented somatic mtDNA mutation levels, and shortened lifespan. The strikingly different phenotypic effects of these mild mtDNA variants demonstrate that mtDNA can be an important modulator of autosomal disease.

Bacterial and eukaryotic nuclear RNA polymerases (RNAPs) cap RNA with the oxidized and reduced forms of the metabolic effector nicotinamide adenine dinucleotide, NAD+ and NADH, using NAD+ and NADH as non-canonical initiating nucleotides for transcription initiation. Here, we show that mitochondrial RNAPs (mtRNAPs) cap RNA with NAD+ and NADH, and do so more efficiently than nuclear RNAPs. Direct quantitation of NAD+- and NADH-capped RNA demonstrates remarkably high levels of capping in vivo: up to ~60% NAD+ and NADH capping of yeast mitochondrial transcripts, and up to ~15% NAD+ capping of human mitochondrial transcripts. The capping efficiency is determined by promoter sequence at, and upstream of, the transcription start site and, in yeast and human cells, by intracellular NAD+ and NADH levels. Our findings indicate mtRNAPs serve as both sensors and actuators in coupling cellular metabolism to mitochondrial transcriptional outputs, sensing NAD+ and NADH levels and adjusting transcriptional outputs accordingly.