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MicroRNAs (miRNAs) have been implicated in human disorders, from cancers to infectious diseases. Targeting miRNAs or their target genes with small molecules offers opportunities to modulate dysregulated cellular processes linked to diseases. Yet, predicting small molecules associated with miRNAs remains challenging due to the small size of small molecule-miRNA datasets. Herein, we develop a generalized deep learning framework, sChemNET, for predicting small molecules affecting miRNA bioactivity based on chemical structure and sequence information. sChemNET overcomes the limitation of sparse chemical information by an objective function that allows the neural network to learn chemical space from a large body of chemical structures yet unknown to affect miRNAs. We experimentally validated small molecules predicted to act on miR-451 or its targets and tested their role in erythrocyte maturation during zebrafish embryogenesis. We also tested small molecules targeting the miR-181 network and other miRNAs using in-vitro and in-vivo experiments. We demonstrate that our machine-learning framework can predict bioactive small molecules targeting miRNAs or their targets in humans and other mammalian organisms., (© 2024. The Author(s).)

DNA methyltransferase and poly(ADP-ribose) polymerase inhibitors (DNMTis, PARPis) induce a stimulator of interferon (IFN) genes (STING)-dependent pathogen mimicry response (PMR) in ovarian (OC) and other cancers. We now show that combining DNMTis and PARPis upregulates expression of a little-studied nucleic-acid sensor, NFX1-type zinc finger-containing 1 protein (ZNFX1). We demonstrate that ZNFX1 is a novel master regulator for PMR induction in mitochondria, serving as a gateway for STING-dependent PMR. In patient OC databases, high ZNFX1 expression levels correlate with advanced stage disease. ZNFX1 expression alone significantly correlates with an increase in overall survival in a phase 3 trial for therapy-resistant OC patients receiving bevacizumab in combination with chemotherapy. In correlative RNA-seq data, inflammasome signaling through ZNFX1 correlates with abnormal vasculogenesis. ZNFX1 controls PMR signaling through the mitochondria and may serve as a biomarker to facilitate offering personalized therapy in OC patients, highlighting the strong translational significance of our findings., Significance Statement: DNA methyltransferase and poly(ADP-ribose) polymerase inhibitors upregulate expression of a novel nucleic-acid sensor, ZNFX1 that serves as a mitochondrial gateway to STING-dependent interferon/inflammasome signaling with tumor suppressor properties in ovarian cancer.

Human mitochondrial tRNAs (mt-tRNAs), critical for mitochondrial biogenesis, are frequently associated with pathogenic mutations. These mt-tRNAs have unusual sequence motifs and require post-transcriptional modifications to stabilize their fragile structures. However, whether a modification that stabilizes a wild-type (WT) mt-tRNA would also stabilize its pathogenic variants is unknown. Here we show that the N 1 -methylation of guanosine at position 9 (m 1 G9) of mt-Leu(UAA), while stabilizing the WT tRNA, has a destabilizing effect on variants associated with MELAS (mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes). This differential effect is further demonstrated, as removal of the m 1 G9 methylation, while damaging to the WT tRNA, is beneficial to the major pathogenic variant, improving the structure and activity of the variant. These results have therapeutic implications, suggesting that the N 1 -methylation of mt-tRNAs at position 9 is a determinant of pathogenicity and that controlling the methylation level is an important modulator of mt-tRNA-associated diseases., (© 2024. The Author(s).)

Although the precise molecular mechanisms responsible for neuronal death and motor dysfunction in late-onset Parkinson's disease (PD) are unknown, evidence suggests that mitochondrial dysfunction and neuroinflammation occur early, leading to a collective increase in reactive oxygen species (ROS) production and oxidative stress. However, the lack of methods for tracking oxidative stress in the living brain has precluded its use as a potential biomarker. The goal of the current study is to address this need through the evaluation of the first superoxide (O 2 •- )-sensitive radioactive tracer, [ 18 F]ROStrace, in a model of late-onset PD. To achieve this goal, MitoPark mice with a dopaminergic (DA) neuron-specific deletion of transcription factor A mitochondrial ( Tfam ) were imaged with [ 18 F]ROStrace from the prodromal phase to the end-stage of PD-like disease. Our data demonstrate [ 18 F]ROStrace was sensitive to increased oxidative stress during the early stages of PD-like pathology in MitoPark mice, which persisted throughout the disease course. Similarly to PD patients, MitoPark males had the most severe parkinsonian symptoms and metabolic impairment. [ 18 F]ROStrace retention was also highest in MitoPark males, suggesting oxidative stress as a potential mechanism underlying the male sex bias of PD. Furthermore, [ 18 F]ROStrace may provide a method to identify patients at risk of Parkinson's before irreparable neurodegeneration occurs and enhance clinical trial design by identifying patients most likely to benefit from antioxidant therapies.

The yellow fever virus 17D (YFV-17D) live attenuated vaccine is considered one of the successful vaccines ever generated associated with high antiviral immunity, yet the signaling mechanisms that drive the response in infected cells are not understood. Here, we provide a molecular understanding of how metabolic stress and innate immune responses are linked to drive type I IFN expression in response to YFV-17D infection. Comparison of YFV-17D replication with its parental virus, YFV-Asibi, and a related dengue virus revealed that IFN expression requires RIG-I-like Receptor signaling through MAVS, as expected. However, YFV-17D uniquely induces mitochondrial respiration and major metabolic perturbations, including hyperactivation of electron transport to fuel ATP synthase. Mitochondrial hyperactivity generates reactive oxygen species (mROS) and peroxynitrite, blocking of which abrogated IFN expression in non-immune cells without reducing YFV-17D replication. Scavenging ROS in YFV-17D-infected human dendritic cells increased cell viability yet globally prevented expression of IFN signaling pathways. Thus, adaptation of YFV-17D for high growth uniquely imparts mitochondrial hyperactivity generating mROS and peroxynitrite as the critical messengers that convert a blunted IFN response into maximal activation of innate immunity essential for vaccine effectiveness., Competing Interests: Declaration of Interests The authors declare no competing interests.

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) infection inhibits mitochondrial oxidative phosphorylation (OXPHOS) and elevates mitochondrial reactive oxygen species (ROS, mROS) which activates hypoxia-inducible factor-1alpha (HIF-1α), shifting metabolism toward glycolysis to drive viral biogenesis but also causing the release of mitochondrial DNA (mtDNA) and activation of innate immunity. To determine whether mitochondrially targeted antioxidants could mitigate these viral effects, we challenged mice expressing human angiotensin-converting enzyme 2 (ACE2) with SARS-CoV-2 and intervened using transgenic and pharmacological mitochondrially targeted catalytic antioxidants. Transgenic expression of mitochondrially targeted catalase (mCAT) or systemic treatment with EUK8 decreased weight loss, clinical severity, and circulating levels of mtDNA; as well as reduced lung levels of HIF-1α, viral proteins, and inflammatory cytokines. RNA-sequencing of infected lungs revealed that mCAT and Eukarion 8 (EUK8) up-regulated OXPHOS gene expression and down-regulated HIF-1α and its target genes as well as innate immune gene expression. These data demonstrate that SARS-CoV-2 pathology can be mitigated by catalytically reducing mROS, potentially providing a unique host-directed pharmacological therapy for COVID-19 which is not subject to viral mutational resistance., Competing Interests: Competing interests statement:D.C.W. is on the scientific advisory boards of Pano Therapeutics, Inc. and Medical Excellent Capital, and published a manuscript with S.E. Schriner in 2022.

Our previous research revealed a key microRNA signature that is associated with spaceflight that can be used as a biomarker and to develop countermeasure treatments to mitigate the damage caused by space radiation. Here, we expand on this work to determine the biological factors rescued by the countermeasure treatment. We performed RNA-sequencing and transcriptomic analysis on 3D microvessel cell cultures exposed to simulated deep space radiation (0.5 Gy of Galactic Cosmic Radiation) with and without the antagonists to three microRNAs: miR-16-5p, miR-125b-5p, and let-7a-5p (i.e., antagomirs). Significant reduction of inflammation and DNA double strand breaks (DSBs) activity and rescue of mitochondria functions are observed after antagomir treatment. Using data from astronaut participants in the NASA Twin Study, Inspiration4, and JAXA missions, we reveal the genes and pathways implicated in the action of these antagomirs are altered in humans. Our findings indicate a countermeasure strategy that can potentially be utilized by astronauts in spaceflight missions to mitigate space radiation damage., (© 2024. The Author(s).)

Impairment of the central nervous system (CNS) poses a significant health risk for astronauts during long-duration space missions. In this study, we employed an innovative approach by integrating single-cell multiomics (transcriptomics and chromatin accessibility) with spatial transcriptomics to elucidate the impact of spaceflight on the mouse brain in female mice. Our comparative analysis between ground control and spaceflight-exposed animals revealed significant alterations in essential brain processes including neurogenesis, synaptogenesis and synaptic transmission, particularly affecting the cortex, hippocampus, striatum and neuroendocrine structures. Additionally, we observed astrocyte activation and signs of immune dysfunction. At the pathway level, some spaceflight-induced changes in the brain exhibit similarities with neurodegenerative disorders, marked by oxidative stress and protein misfolding. Our integrated spatial multiomics approach serves as a stepping stone towards understanding spaceflight-induced CNS impairments at the level of individual brain regions and cell types, and provides a basis for comparison in future spaceflight studies. For broader scientific impact, all datasets from this study are available through an interactive data portal, as well as the National Aeronautics and Space Administration (NASA) Open Science Data Repository (OSDR)., (© 2024. The Author(s).)

Mitochondrial function is important for both energetic and anabolic metabolism. Pathogenic mitochondrial DNA (mtDNA) mutations directly impact these functions, resulting in the detrimental consequences seen in human mitochondrial diseases. The role of pathogenic mtDNA mutations in human cancers is less clear; while pathogenic mtDNA mutations are observed in some cancer types, they are almost absent in others. We report here that the proofreading mutant DNA polymerase gamma ( PolG D256A ) induced a high mtDNA mutation burden in non-small-cell lung cancer (NSCLC), and promoted the accumulation of defective mitochondria, which is responsible for decreased tumor cell proliferation and viability and increased cancer survival. In NSCLC cells, pathogenic mtDNA mutations increased glycolysis and caused dependence on glucose. The glucose dependency sustained mitochondrial energetics but at the cost of a decreased NAD+/NADH ratio that inhibited de novo serine synthesis. Insufficient serine synthesis, in turn, impaired the downstream synthesis of GSH and nucleotides, leading to impaired tumor growth that increased cancer survival. Unlike tumors with intact mitochondrial function, NSCLC with pathogenic mtDNA mutations were sensitive to dietary serine and glycine deprivation. Thus, mitochondrial function in NSCLC is required specifically to sustain sufficient serine synthesis for nucleotide production and redox homeostasis to support tumor growth, explaining why these cancers preserve functional mtDNA., In Brief: High mtDNA mutation burden in non-small-cell lung cancer (NSCLC) leads to the accumulation of respiration-defective mitochondria and dependency on glucose and glycolytic metabolism. Defective respiratory metabolism causes a massive accumulation of cytosolic nicotinamide adenine dinucleotide + hydrogen (NADH), which impedes serine synthesis and, thereby, glutathione (GSH) and nucleotide synthesis, leading to impaired tumor growth and increased survival., Highlights: Proofreading mutations in Polymerase gamma led to a high burden of mitochondrial DNA mutations, promoting the accumulation of mitochondria with respiratory defects in NSCLC.Defective respiration led to reduced proliferation and viability of NSCLC cells increasing survival to cancer.Defective respiration caused glucose dependency to fuel elevated glycolysis.Altered glucose metabolism is associated with high NADH that limits serine synthesis, leading to impaired GSH and nucleotide production.Mitochondrial respiration defects sensitize NSCLC to dietary serine/glycine starvation, further increasing survival.

To determine the effects of SARS-CoV-2 infection on cellular metabolism, we conducted an exhaustive survey of the cellular metabolic pathways modulated by SARS-CoV-2 infection and confirmed their importance for SARS-CoV-2 propagation by cataloging the effects of specific pathway inhibitors. This revealed that SARS-CoV-2 strongly inhibits mitochondrial oxidative phosphorylation (OXPHOS) resulting in increased mitochondrial reactive oxygen species (mROS) production. The elevated mROS stabilizes HIF-1α which redirects carbon molecules from mitochondrial oxidation through glycolysis and the pentose phosphate pathway (PPP) to provide substrates for viral biogenesis. mROS also induces the release of mitochondrial DNA (mtDNA) which activates innate immunity. The restructuring of cellular energy metabolism is mediated in part by SARS-CoV-2 Orf8 and Orf10 whose expression restructures nuclear DNA (nDNA) and mtDNA OXPHOS gene expression. These viral proteins likely alter the epigenome, either by directly altering histone modifications or by modulating mitochondrial metabolite substrates of epigenome modification enzymes, potentially silencing OXPHOS gene expression and contributing to long-COVID., Competing Interests: Declaration of Competing Interest D.C.W. serves on the advisory boards of Plano Therapeutics and Medical Excellence Capital., (Copyright © 2024. Published by Elsevier Ltd.)

Splicing factor mutations are common in myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML), but how they alter cellular functions is unclear. We show that the pathogenic SRSF2P95H/+ mutation disrupts the splicing of mitochondrial mRNAs, impairs mitochondrial complex I function, and robustly increases mitophagy. We also identified a mitochondrial surveillance mechanism by which mitochondrial dysfunction modifies splicing of the mitophagy activator PINK1 to remove a poison intron, increasing the stability and abundance of PINK1 mRNA and protein. SRSF2P95H-induced mitochondrial dysfunction increased PINK1 expression through this mechanism, which is essential for survival of SRSF2P95H/+ cells. Inhibition of splicing with a glycogen synthase kinase 3 inhibitor promoted retention of the poison intron, impairing mitophagy and activating apoptosis in SRSF2P95H/+ cells. These data reveal a homeostatic mechanism for sensing mitochondrial stress through PINK1 splicing and identify increased mitophagy as a disease marker and a therapeutic vulnerability in SRSF2P95H mutant MDS and AML.

NADH autofluorescence imaging is a promising approach for visualizing energy metabolism at the single-cell level. However, it is sensitive to the redox ratio and the total NAD(H) amount, which can change independently from each other, for example with aging. Here, we evaluate the potential of fluorescence lifetime imaging microscopy (FLIM) of NADH to differentiate between these modalities.We perform targeted modifications of the NAD(H) pool size and ratio in cells and mice and assess the impact on NADH FLIM. We show that NADH FLIM is sensitive to NAD(H) pool size, mimicking the effect of redox alterations. However, individual components of the fluorescence lifetime are differently impacted by redox versus pool size changes, allowing us to distinguish both modalities using only FLIM. Our results emphasize NADH FLIM's potential for evaluating cellular metabolism and relative NAD(H) levels with high spatial resolution, providing a crucial tool for our understanding of aging and metabolism., (© 2024. The Author(s).)

Adaptive immunity requires the expansion of high-affinity lymphocytes from a heterogeneous pool. Whereas current models explain this through signal transduction, we hypothesized that antigen affinity tunes discrete metabolic pathways to license clonal lymphocyte dynamics. Here, we identify nicotinamide adenine dinucleotide (NAD) biosynthesis as a biochemical hub for the T cell receptor affinity-dependent metabolome. Through this central anabolic role, we found that NAD biosynthesis governs a quiescence exit checkpoint, thereby pacing proliferation. Normalizing cellular NAD(H) likewise normalizes proliferation across affinities, and enhancing NAD biosynthesis permits the expansion of lower affinity clones. Furthermore, single-cell differences in NAD(H) could predict division potential for both T and B cells, before the first division, unmixing proliferative heterogeneity. We believe that this supports a broader paradigm in which complex signaling networks converge on metabolic pathways to control single-cell behavior.

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.

Photobleaching and phototoxicity can induce detrimental effects on cell viability and compromise the integrity of collected data, particularly in studies utilizing super-resolution microscopes. Given the involvement of multiple factors, it is currently challenging to propose a single set of standards for assessing the potential of phototoxicity. The objective of this paper is to present empirical data on the effects of photobleaching and phototoxicity on mitochondria during super-resolution imaging of mitochondrial structure and function using Airyscan and the fluorescent structure dyes Mitotracker green (MTG), 10-N-nonyl acridine orange (NAO), and voltage dye Tetramethylrhodamine, Ethyl Ester (TMRE). We discern two related phenomena. First, phototoxicity causes a transformation of mitochondria from tubular to spherical shape, accompanied by a reduction in the number of cristae. Second, phototoxicity impacts the mitochondrial membrane potential. Through these parameters, we discovered that upon illumination, NAO is much more phototoxic to mitochondria compared to MTG or TMRE and that these parameters can be used to evaluate the relative phototoxicity of various mitochondrial dye-illumination combinations during mitochondrial imaging., Competing Interests: Declaration of competing interest Dr. Douglas Wallace is the Honorary Editor for Mitochondrial Communications and was not involved in the editorial review or the decision to publish this article. Some of the authors are in the process of founding a company related to microfluidic mitochondrial assays.

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., (© 2023. Springer Nature Limited.)

Background: Mitochondrial dysfunction is involved in several diseases ranging from genetic mitochondrial disorders to chronic metabolic diseases. An emerging approach to potentially treat mitochondrial dysfunction is the transplantation of autologous live mitochondria to promote cell regeneration. We tested the differential filtration-based mitochondrial isolation protocol established by the McCully laboratory for use in cellular models but found whole cell contaminants in the mitochondrial isolate., Methods: Therefore, we explored alternative types of 5-μm filters (filters A and B) for isolation of mitochondria from multiple cell lines including HEK293 cells and induced pluripotent stem cells (iPSCs). MitoTracker™ staining combined with flow cytometry was used to quantify the concentration of viable mitochondria. A proof-of-principle mitochondrial transplant was performed using mitoDsRed2-tagged mitochondria into a H9-derived cerebral organoid., Results: We found that filter B provided the highest quality mitochondria as compared to the 5-μm filter used in the original protocol. Using this method, mitochondria were also successfully isolated from induced pluripotent stem cells. To test for viability, mitoDsRed2-tagged mitochondria were isolated and transplanted into H9-derived cerebral organoids and observed that mitochondria were engulfed as indicated by immunofluorescent co-localization of TOMM20 and MAP2., Conclusions: Thus, use of filter B in a differential filtration approach is ideal for isolating pure and viable mitochondria from cells, allowing us to begin evaluating long-term integration and safety of mitochondrial transplant using cellular sources., (© 2023. BioMed Central Ltd., part of Springer Nature.)

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) viral proteins bind to host mitochondrial proteins, likely inhibiting oxidative phosphorylation (OXPHOS) and stimulating glycolysis. We analyzed mitochondrial gene expression in nasopharyngeal and autopsy tissues from patients with coronavirus disease 2019 (COVID-19). In nasopharyngeal samples with declining viral titers, the virus blocked the transcription of a subset of nuclear DNA (nDNA)-encoded mitochondrial OXPHOS genes, induced the expression of microRNA 2392, activated HIF-1α to induce glycolysis, and activated host immune defenses including the integrated stress response. In autopsy tissues from patients with COVID-19, SARS-CoV-2 was no longer present, and mitochondrial gene transcription had recovered in the lungs. However, nDNA mitochondrial gene expression remained suppressed in autopsy tissue from the heart and, to a lesser extent, kidney, and liver, whereas mitochondrial DNA transcription was induced and host-immune defense pathways were activated. During early SARS-CoV-2 infection of hamsters with peak lung viral load, mitochondrial gene expression in the lung was minimally perturbed but was down-regulated in the cerebellum and up-regulated in the striatum even though no SARS-CoV-2 was detected in the brain. During the mid-phase SARS-CoV-2 infection of mice, mitochondrial gene expression was starting to recover in mouse lungs. These data suggest that when the viral titer first peaks, there is a systemic host response followed by viral suppression of mitochondrial gene transcription and induction of glycolysis leading to the deployment of antiviral immune defenses. Even when the virus was cleared and lung mitochondrial function had recovered, mitochondrial function in the heart, kidney, liver, and lymph nodes remained impaired, potentially leading to severe COVID-19 pathology.

SETD2 is a tumor suppressor that is frequently inactivated in several cancer types. The mechanisms through which SETD2 inactivation promotes cancer are unclear, and whether targetable vulnerabilities exist in these tumors is unknown. Here we identify heightened mTORC1-associated gene expression programs and functionally higher levels of oxidative metabolism and protein synthesis as prominent consequences of Setd2 inactivation in KRAS-driven mouse models of lung adenocarcinoma. Blocking oxidative respiration and mTORC1 signaling abrogates the high rates of tumor cell proliferation and tumor growth specifically in SETD2-deficient tumors. Our data nominate SETD2 deficiency as a functional marker of sensitivity to clinically actionable therapeutics targeting oxidative respiration and mTORC1 signaling., (© 2023. The Author(s).)

Background: Compared to healthy controls, severe COVID19 patients display increased levels of activated NLRP3-inflammasome (NLRP3-I) and interleukin (IL)-1β. SARS-CoV-2 encodes viroporin proteins E and Orf3a(2-E+2-3a) with homologs to SARS-CoV-1, 1-E+1-3a, which elevate NLRP3-I activation; by an unknown mechanism. Thus, we investigated how 2-E+2-3a activates the NLRP3-I to better understand the pathophysiology of severe COVID-19., Methods: We generated a polycistronic expression-vector co-expressing 2-E+2-3a from a single transcript. To elucidate how 2-E+2-3a activates the NLRP3-I, we reconstituted the NLRP3-I in 293T cells and used THP1-derived macrophages to monitor the secretion of mature IL-1β. Mitochondrial physiology was assessed using fluorescent microscopy and plate reader assays, and the release of mitochondrial DNA (mtDNA) was detected from cytosolic-enriched fractions using Real-Time PCR., Results: Expression of 2-E+2-3a in 293T cells increased cytosolic Ca++ and elevated mitochondrial Ca++, taken up through the MCUi11-sensitive mitochondrial calcium uniporter. Increased mitochondrial Ca++ stimulated NADH, mitochondrial reactive oxygen species (mROS) production and the release of mtDNA into the cytosol. Expression of 2-E+2-3a in NLRP3-I reconstituted 293T cells and THP1-derived macrophages displayed increased secretion of IL-1β. Increasing mitochondrial antioxidant defenses via treatment with MnTBAP or genetic expression of mCAT abolished 2-E+2-3a elevation of mROS, cytosolic mtDNA levels, and secretion of NLRP3-activated-IL-1β. The 2-E+2-3a-induced release of mtDNA and the secretion of NLRP3-activated-IL-1β were absent in cells lacking mtDNA and blocked in cells treated with the mitochondrial-permeability-pore(mtPTP)-specific inhibitor NIM811., Conclusion: Our findings revealed that mROS activates the release of mitochondrial DNA via the NIM811-sensitive mitochondrial-permeability-pore(mtPTP), activating the inflammasome. Hence, interventions targeting mROS and the mtPTP may mitigate the severity of COVID-19 cytokine storms., Competing Interests: DW serves of the advisory boards of Plano Therapeutics, Medical Excellent Capital, and has a grant from March Therapeutics. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Guarnieri, Angelin, Murdock, Schaefer, Portluri, Lie, Huang and Wallace.)

Patients with primary mitochondrial oxidative phosphorylation (OxPhos) defects present with fatigue and multi-system disorders, are often lean, and die prematurely, but the mechanistic basis for this clinical picture remains unclear. By integrating data from 17 cohorts of patients with mitochondrial diseases (n = 690) we find evidence that these disorders increase resting energy expenditure, a state termed hypermetabolism. We examine this phenomenon longitudinally in patient-derived fibroblasts from multiple donors. Genetically or pharmacologically disrupting OxPhos approximately doubles cellular energy expenditure. This cell-autonomous state of hypermetabolism occurs despite near-normal OxPhos coupling efficiency, excluding uncoupling as a general mechanism. Instead, hypermetabolism is associated with mitochondrial DNA instability, activation of the integrated stress response (ISR), and increased extracellular secretion of age-related cytokines and metabokines including GDF15. In parallel, OxPhos defects accelerate telomere erosion and epigenetic aging per cell division, consistent with evidence that excess energy expenditure accelerates biological aging. To explore potential mechanisms for these effects, we generate a longitudinal RNASeq and DNA methylation resource dataset, which reveals conserved, energetically demanding, genome-wide recalibrations. Taken together, these findings highlight the need to understand how OxPhos defects influence the energetic cost of living, and the link between hypermetabolism and aging in cells and patients with mitochondrial diseases., (© 2023. The Author(s).)

Mitochondrial dysfunction can be associated with a range of clinical manifestations. Here, we report a family with a complex phenotype including combinations of connective tissue, neurological, and metabolic symptoms that were passed on to all surviving children. Analysis of the maternally inherited mtDNA revealed a novel genotype encompassing the haplogroup J - defining mitochondrial DNA (mtDNA) ND5 m.13708G>A (A458T) variant arising on the mtDNA haplogroup H7A background, an extremely rare combination. Analysis of transmitochondrial cybrids with the 13708A-H7 mtDNA revealed a lower mitochondrial respiration, increased reactive oxygen species production (mROS), and dysregulation of connective tissue gene expression. The mitochondrial dysfunction was exacerbated by histamine, explaining why all eight surviving children inherited the dysfunctional histidine decarboxylase allele (W327X) from the father. Thus, certain combinations of common mtDNA variants can cause mitochondrial dysfunction, mitochondrial dysfunction can affect extracellular matrix gene expression, and histamine-activated mROS production can augment the severity of mitochondrial dysfunction. Most important, we have identified a previously unreported genetic cause of mitochondrial disorder arising from the incompatibility of common, nonpathogenic mtDNA variants.

Objective: Mitochondrial disorders are often characterized by muscle weakness and fatigue. Null mutations in the heart-muscle adenine nucleotide translocator isoform 1 (ANT1) of both humans and mice cause cardiomyopathy and myopathy associated with exercise intolerance and muscle weakness. Here we decipher the molecular underpinnings of ANT1-deficiency-mediated exercise intolerance., Methods: This was achieved by correlating exercise physiology, mitochondrial function and metabolomics of mice deficient in ANT1 and comparing this to control mice., Results: We demonstrate a peripheral limitation of skeletal muscle mitochondrial respiration and a reduced complex I respiration in ANT1-deficient mice. Upon exercise, this results in a lack of NAD + leading to a substrate limitation and stalling of the TCA cycle and mitochondrial respiration, further limiting skeletal muscle mitochondrial respiration. Treatment of ANT1-deficient mice with nicotinamide riboside increased NAD + levels in skeletal muscle and liver, which increased the exercise capacity and the mitochondrial respiration., Conclusion: Increasing NAD +  levels with nicotinamide riboside can alleviate the exercise intolerance associated to ANT1-deficiency, indicating the therapeutic potential of NAD + -stimulating compounds in mitochondrial myopathies., (Copyright © 2022 The Author(s). Published by Elsevier GmbH.. All rights reserved.)

Model organism (MO) research provides a basic understanding of biology and disease due to the evolutionary conservation of the molecular and cellular language of life. MOs have been used to identify and understand the function of orthologous genes, proteins, cells and tissues involved in biological processes, to develop and evaluate techniques and methods, and to perform whole-organism-based chemical screens to test drug efficacy and toxicity. However, a growing richness of datasets and the rising power of computation raise an important question: How do we maximize the value of MOs? In-depth discussions in over 50 virtual presentations organized by the National Institutes of Health across more than 10 weeks yielded important suggestions for improving the rigor, validation, reproducibility and translatability of MO research. The effort clarified challenges and opportunities for developing and integrating tools and resources. Maintenance of critical existing infrastructure and the implementation of suggested improvements will play important roles in maintaining productivity and facilitating the validation of animal models of human biology and disease., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2022. Published by The Company of Biologists Ltd.)

Adenine Nucleotide Translocator isoforms (ANTs) exchange ADP/ATP across the inner mitochondrial membrane, are also voltage-activated proton channels and regulate mitophagy and apoptosis. The ANT1 isoform predominates in heart and muscle while ANT2 is systemic. Here, we report the creation of Ant mutant mouse myoblast cell lines with normal Ant1 and Ant2 genes, deficient in either Ant1 or Ant2, and deficient in both the Ant1 and Ant2 genes. These cell lines are immortal under permissive conditions (IFN-γ + serum at 32 °C) permitting expansion but return to normal myoblasts that can be differentiated into myotubes at 37 °C. With this system we were able to complement our Ant1 mutant studies by demonstrating that ANT2 is important for myoblast to myotube differentiation and myotube mitochondrial respiration. ANT2 is also important in the regulation of mitochondrial biogenesis and antioxidant defenses. ANT2 is also associated with increased oxidative stress response and modulation for Ca ++ sequestration and activation of the mitochondrial permeability transition (mtPTP) pore during cell differentiation., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: Sepsis is the leading cause of death in hospitalized children worldwide. Despite its hypothesized immune-mediated mechanism, targeted immunotherapy for sepsis is not available for clinical use., Objective: To determine the association between longitudinal cytometric, proteomic, bioenergetic, and metabolomic markers of immunometabolic dysregulation and pathogen type in pediatric sepsis., Methods: Serial peripheral blood mononuclear cell (PBMC) samples were obtained from 14 sepsis patients (34 total samples) and 7 control patients for this observational study. Flow cytometry was used to define immunophenotype, including T cell subset frequency and activation state, and assess intracellular cytokine production. Global immune dysfunction was assessed by tumor necrosis factor-α (TNF-α) production capacity and monocyte human leukocyte antigen DR (HLA-DR) expression. Mitochondrial function was assessed by bulk respirometry. Plasma cytokine levels were determined via Luminex assay. Metabolites were measured by liquid chromatography-mass spectrometry. Results were compared by timepoint and pathogen type., Results: Sepsis patients were older (15.9 years vs. 10.4 years, P = 0.02) and had higher illness severity by PRISM-III (12.0 vs. 2.0, P < 0.001) compared to controls; demographics were otherwise similar, though control patients were predominately male. Compared to controls, sepsis patients at timepoint 1 demonstrated lower monocyte HLA-DR expression (75% vs. 92%, P = 0.02), loss of peripheral of non-naïve CD4+ T cells (62.4% vs. 77.6%, P = 0.04), and reduced PBMC mitochondrial spare residual capacity (SRC; 4.0 pmol/s/106 cells vs. 8.4 pmol/s/106 cells, P = 0.01). At sepsis onset, immunoparalysis (defined as TNF-α production capacity < 200 pg/mL) was present in 39% of sepsis patients and not identified among controls. Metabolomic findings in sepsis patients were most pronounced at sepsis onset and included elevated uridine and 2-dehydrogluconate and depleted citrulline. Loss of peripheral non-naïve CD4+ T cells was associated with immune dysfunction and reduced cytokine production despite increased T cell activation. CD4+ T cell differentiation and corresponding pro- and anti-inflammatory cytokines varied by pathogen., Conclusion: Pediatric sepsis patients exhibit a complex, dynamic physiologic state characterized by impaired T cell function and immunometabolic dysregulation which varies by pathogen type., Competing Interests: The authors report no conflicts of interest., (Copyright © 2022 by the Shock Society.)

Butyrate is a short-chain fatty acid that is produced by commensal microbes within the intestinal microbiome through fermentation of dietary fibre. Microbial-derived butyrate has been shown to promote immunologic and metabolic homeostasis, in part through its beneficial effects on mitochondrial function, and thus has been proposed as a possible anti-inflammatory therapy. We tested the hypothesis that butyrate could mitigate the decrease in mitochondrial respiration in immune cells under septic conditions as a preliminary step towards better understanding the potential for butyrate as a novel therapy in sepsis. Mitochondrial respiration and content (measured as citrate synthase activity) were compared within four Epstein-Barr virus-transformed lymphoblast (LB) cell lines exposed to either control media or lipopolysaccharide (LPS) 100 ng/ml. Both co-incubation of LBs with LPS + butyrate and treatment with butyrate after LPS stimulation reversed the decrease in mitochondrial respiration observed in LBs exposed to LPS without butyrate. Neither LPS nor butyrate led to significant changes in citrate synthase activity. The preliminary findings support further investigation of a potential mitochondrial-based mechanism through which butyrate may help to mitigate the immuno-inflammatory response in sepsis., (© 2022 The Authors. Journal of Cellular and Molecular Medicine published by Foundation for Cellular and Molecular Medicine and John Wiley & Sons Ltd.)

We demonstrate a fast and easy-to-use three-dimensional printed microfluidic platform for mitochondria isolation from cell and tissue lysates based on inertial microfluidics. We present and quantify the quality of the isolated mitochondria by measuring the respiration rate under various conditions. We demonstrate that the technology produces vital mitochondria of equal quality to traditional, but more burdensome, differential centrifugation. We anticipate that the availability of improved tools for studies of bioenergetics to the broader biological community will enable these and other links to be explored in more meaningful ways, leading to further understanding of the links between energy, health, and disease.

Rationale: Viral infections are complex processes based on an intricate network of molecular interactions. The infectious agent hijacks components of the cellular machinery for its profit, circumventing the natural defense mechanisms triggered by the infected cell. The successful completion of the replicative viral cycle within a cell depends on the function of viral components versus the cellular defenses. Non-coding RNAs (ncRNAs) are important cellular modulators, either promoting or preventing the progression of viral infections. Among these ncRNAs, the long non-coding RNA (lncRNA) family is especially relevant due to their intrinsic functional properties and ubiquitous biological roles. Specific lncRNAs have been recently characterized as modulators of the cellular response during infection of human host cells by single stranded RNA viruses. However, the role of host lncRNAs in the infection by human RNA coronaviruses such as SARS-CoV-2 remains uncharacterized. Methods: In the present work, we have performed a transcriptomic study of a cohort of patients with different SARS-CoV-2 viral load and analyzed the involvement of lncRNAs in supporting regulatory networks based on their interaction with RNA-binding proteins (RBPs). Results: Our results revealed the existence of a SARS-CoV-2 infection-dependent pattern of transcriptional up-regulation in which specific lncRNAs are an integral component. To determine the role of these lncRNAs, we performed a functional correlation analysis complemented with the study of the validated interactions between lncRNAs and RBPs. This combination of in silico functional association studies and experimental evidence allowed us to identify a lncRNA signature composed of six elements - NRIR, BISPR, MIR155HG, FMR1-IT1, USP30-AS1, and U62317.2 - associated with the regulation of SARS-CoV-2 infection. Conclusions: We propose a competition mechanism between the viral RNA genome and the regulatory lncRNAs in the sequestering of specific RBPs that modulates the interferon response and the regulation of RNA surveillance by nonsense-mediated decay (NMD)., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

Primary mitochondrial diseases (PMDs) are a heterogeneous group of metabolic disorders that can be caused by hundreds of mutations in both mitochondrial DNA (mtDNA) and nuclear DNA (nDNA) genes. Current therapeutic approaches are limited, although one approach has been exercise training. Endurance exercise is known to improve mitochondrial function in heathy subjects and reduce risk for secondary metabolic disorders such as diabetes or neurodegenerative disorders. However, in PMDs the benefit of endurance exercise is unclear, and exercise might be beneficial for some mitochondrial disorders but contraindicated in others. Here we investigate the effect of an endurance exercise regimen in mouse models for PMDs harboring distinct mitochondrial mutations. We show that while an mtDNA ND6 mutation in complex I demonstrated improvement in response to exercise, mice with a CO1 mutation affecting complex IV showed significantly fewer positive effects, and mice with an ND5 complex I mutation did not respond to exercise at all. For mice deficient in the nDNA adenine nucleotide translocase 1 (Ant1), endurance exercise actually worsened the dilated cardiomyopathy. Correlating the gene expression profile of skeletal muscle and heart with the physiologic exercise response identified oxidative phosphorylation, amino acid metabolism, matrisome (extracellular matrix [ECM]) structure, and cell cycle regulation as key pathways in the exercise response. This emphasizes the crucial role of mitochondria in determining the exercise capacity and exercise response. Consequently, the benefit of endurance exercise in PMDs strongly depends on the underlying mutation, although our results suggest a general beneficial effect.

Introduction: Peripheral blood mononuclear cells (PBMCs) are commonly used to compare mitochondrial function in patients with versus without sepsis, but how these measurements in this mixed cell population vary by composition of immune cell subtypes is not known, especially in children. We determined the effect of changing immune cell composition on PBMC mitochondrial respiration and content in children with and without sepsis., Methods: PBMC mitochondrial respiration and citrate synthase (CS) activity, a marker of mitochondrial content, were measured in 167 children with sepsis at three timepoints (day 1-2, 3-5, and 8-14) and once in 19 nonseptic controls. The proportion of lymphocytes and monocytes and T, B, and NK cells was measured using flow cytometry. More specific CD4+ and CD8+ T cell subsets were measured from 13 sepsis patients and 6 controls. Spearman's correlation and simple and mixed effects linear regression were used to determine the association of PBMC mitochondrial measures with proportion of immune cell subtypes., Results: PBMC mitochondrial respiration and CS activity were correlated with proportion of monocytes, lymphocytes, T B, and NK cells in controls, but not in sepsis patients. PBMC mitochondrial respiration was correlated with CD4+ and CD8+ T cell subsets in both groups. After controlling for differences in immune cell composition between groups using linear regression models, PBMC respiration and CS activity remained lower in sepsis patients than controls., Conclusions: Mitochondrial measurements from PBMCs varied with changes in immune cell composition in children with and without sepsis. However, differences in PBMC mitochondrial measurements between sepsis patients and controls were at least partially attributable to the effects of sepsis rather than solely an epiphenomena of variable immune cell composition., Competing Interests: The authors report no conflicts of interest., (Copyright © 2021 The Author(s). Published by Wolters Kluwer Health, Inc. on behalf of the Shock Society.)

Mitochondrial disease diagnosis requires interrogation of both nuclear and mitochondrial (mtDNA) genomes for single-nucleotide variants (SNVs) and copy number alterations, both in the proband and often maternal relatives, together with careful phenotype correlation. We developed a comprehensive mtDNA sequencing test ('MitoGenome') using long-range PCR (LR-PCR) to amplify the full length of the mtDNA genome followed by next generation sequencing (NGS) to accurately detect SNVs and large-scale mtDNA deletions (LSMD), combined with droplet digital PCR (ddPCR) for LSMD heteroplasmy quantification. Overall, MitoGenome tests were performed on 428 samples from 394 patients with suspected or confirmed mitochondrial disease. The positive yield was 11% (43/394), including 34 patients with pathogenic or likely pathogenic SNVs (the most common being m.3243A > G in 8/34 (24%) patients), 8 patients with single LSMD, and 3 patients with multiple LSMD exceeding 10% heteroplasmy levels. Two patients with both LSMD and pathogenic SNV were detected. Overall, this LR-PCR/NGS assay provides a highly accurate and comprehensive diagnostic method for simultaneous mtDNA SNV detection at heteroplasmy levels as low as 1% and LSMD detection at heteroplasmy levels below 10%. Inclusion of maternal samples for variant classification and ddPCR to quantify LSMD heteroplasmy levels further enables accurate pathogenicity assessment and clinical correlation interpretation of mtDNA genome sequence variants and copy number alterations., Competing Interests: Declaration of Competing Interest None., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Neurodegenerative disorders that are triggered by injury typically have variable and unpredictable outcomes due to the complex and multifactorial cascade of events following the injury and during recovery. Hence, several factors beyond the initial injury likely contribute to the disease progression and pathology, and among these are genetic factors. Genetics is a recognized factor in determining the outcome of common neurodegenerative diseases. The role of mitochondrial genetics and function in traditional neurodegenerative diseases, such as Alzheimer's and Parkinson's diseases, is well-established. Much less is known about mitochondrial genetics, however, regarding neurodegenerative diseases that result from injuries such as traumatic brain injury and ischaemic stroke. We discuss the potential role of mitochondrial DNA genetics in the progression and outcome of injury-related neurodegenerative diseases. We present a guide for understanding mitochondrial genetic variation, along with the nuances of quantifying mitochondrial DNA variation. Evidence supporting a role for mitochondrial DNA as a risk factor for neurodegenerative disease is also reviewed and examined. Further research into the impact of mitochondrial DNA on neurodegenerative disease resulting from injury will likely offer key insights into the genetic factors that determine the outcome of these diseases together with potential targets for treatment.

The growing number of next-generation sequencing (NGS) data presents a unique opportunity to study the combined impact of mitochondrial and nuclear-encoded genetic variation in complex disease. Mitochondrial DNA variants and in particular, heteroplasmic variants, are critical for determining human disease severity. While there are approaches for obtaining mitochondrial DNA variants from NGS data, these software do not account for the unique characteristics of mitochondrial genetics and can be inaccurate even for homoplasmic variants. We introduce MitoScape, a novel, big-data, software for extracting mitochondrial DNA sequences from NGS. MitoScape adopts a novel departure from other algorithms by using machine learning to model the unique characteristics of mitochondrial genetics. We also employ a novel approach of using rho-zero (mitochondrial DNA-depleted) data to model nuclear-encoded mitochondrial sequences. We showed that MitoScape produces accurate heteroplasmy estimates using gold-standard mitochondrial DNA data. We provide a comprehensive comparison of the most common tools for obtaining mtDNA variants from NGS and showed that MitoScape had superior performance to compared tools in every statistically category we compared, including false positives and false negatives. By applying MitoScape to common disease examples, we illustrate how MitoScape facilitates important heteroplasmy-disease association discoveries by expanding upon a reported association between hypertrophic cardiomyopathy and mitochondrial haplogroup T in men (adjusted p-value = 0.003). The improved accuracy of mitochondrial DNA variants produced by MitoScape will be instrumental in diagnosing disease in the context of personalized medicine and clinical diagnostics., Competing Interests: The authors have declared that no competing interests exist.

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., Competing Interests: Declaration of interests A.C., P.N., S.V.C., and A.B. have a provisional patent based on the antiviral discovery and design. A.C., P.N., and S.S. are part of the company (Sachi Bioworks Inc.) that has filed a patent on the Nanoligomer technology., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

Importance: Discovery of mechanisms that underlie variable penetrance for neuropsychiatric illness in the context of genetic variants that carry elevated risk can advance novel treatment approaches for these disorders., Objective: To test the hypothesis that mitochondrial compensation is associated with the variable penetrance of schizophrenia in the 22q11.2 deletion syndrome (22q11DS)., Design, Setting, and Participants: This case-control study compared measures of mitochondrial function and the expression of related genes in 14 induced pluripotent stem cell-derived neurons from typically developing control individuals (6 lines) and from adults with 22q11DS (8 lines). The individuals with 22q11DS included 2 groups, those carrying a diagnosis of schizophrenia and those without this diagnosis (4 lines each). Similar measures were made of lymphoblastic cells lines (LCLs) from a separate group of adults with 22q11DS with (10 lines) or without (8 lines) schizophrenia. The study included samples derived from a clinical setting. The induced pluripotent stem cell lines were derived from individuals with 22q11DS with or without a diagnosis of schizophrenia at Stanford University. The LCLs were from adults within the 22q and You Center at the Children's Hospital of Philadelphia. Data were analyzed between July 1, 2019, and January 24, 2021., Main Outcomes and Measures: Total adenosine triphosphate (ATP), oxidative phosphorylation (OXPHOS) complex activity, and messenger RNA expression via reverse transcription-polymerase chain reaction of selected genes encoding for mitochondrial proteins., Results: Study participants included men and women aged 18 to 37 years. Of 32 participants, the mean (SD) age of men was 27 (1.9) years and of women was 29 (1.2) years. Replicating a previous study, neurons from the 22q11DS and schizophrenia (22q+Sz) group had reduced ATP levels (mean [SD], 15.6 [1.5] vs 21.9 [1.4]; P = .02) and reduced OXPHOS activity (ie, complex I; 1.51 [0.1] vs 1.89 [0.1]; P = .01). These deficits were not present in neurons from individuals with 22q11DS without schizophrenia (22q[-]Sz). In this group, the expression of multiple genes encoding OXPHOS subunits was significantly upregulated. For example, compared with control individuals, NDUFV2 expression was increased by 50% in the 22q(-)Sz group (P < .001) but not significantly changed in the 22q+Sz group. Expression of genes driving mitochondrial biogenesis, including PGC1α, showed a similar pattern of upregulation in the 22q(-)Sz group compared with the control and the 22q+Sz groups. Stimulation of mitochondrial biogenesis normalizes the ATP deficit seen in 22q+Sz neurons. Finally, using LCLs from a separate group of adults with 22q11DS, evidence for enhanced mitochondrial biogenesis was again found in the 22q(-)Sz group., Conclusions and Relevance: In this study, an increase in mitochondrial biogenesis and function was associated with the absence of schizophrenia in neurons and LCLs from individuals with 22q11DS, but the deficit in the 22q+Sz group was reversible by agents that enhance mitochondrial biogenesis. Enhancement of mitochondrial biogenesis may provide a targetable opportunity for treatment or prevention of this disorder in individuals with 22q11DS.

Variation in the mitochondrial DNA (mtDNA) sequence is common in certain tumours. Two classes of cancer mtDNA variants can be identified: de novo mutations that act as 'inducers' of carcinogenesis and functional variants that act as 'adaptors', permitting cancer cells to thrive in different environments. These mtDNA variants have three origins: inherited variants, which run in families, somatic mutations arising within each cell or individual, and variants that are also associated with ancient mtDNA lineages (haplogroups) and are thought to permit adaptation to changing tissue or geographic environments. In addition to mtDNA sequence variation, mtDNA copy number and perhaps transfer of mtDNA sequences into the nucleus can contribute to certain cancers. Strong functional relevance of mtDNA variation has been demonstrated in oncocytoma and prostate cancer, while mtDNA variation has been reported in multiple other cancer types. Alterations in nuclear DNA-encoded mitochondrial genes have confirmed the importance of mitochondrial metabolism in cancer, affecting mitochondrial reactive oxygen species production, redox state and mitochondrial intermediates that act as substrates for chromatin-modifying enzymes. Hence, subtle changes in the mitochondrial genotype can have profound effects on the nucleus, as well as carcinogenesis and cancer progression.

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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Niederschweiberer, Schaefer, Singh, Lausser, Bhosale, Hesse, Calzia, Kestler, Rueck, Wallace, von Einem and von Arnim.)

Objectives: The intestinal microbiome can modulate immune function through production of microbial-derived short-chain fatty acids. We explored whether intestinal dysbiosis in children with sepsis leads to changes in microbial-derived short-chain fatty acids in plasma and stool that are associated with immunometabolic dysfunction in peripheral blood mononuclear cells., Design: Prospective observational pilot study., Setting: Single academic PICU., Patients: Forty-three children with sepsis/septic shock and 44 healthy controls., Measurements and Main Results: Stool and plasma samples were serially collected for sepsis patients; stool was collected once for controls. The intestinal microbiome was assessed using 16S ribosomal RNA sequencing and alpha- and beta-diversity were determined. We measured short-chain fatty acids using liquid chromatography, peripheral blood mononuclear cell mitochondrial respiration using high-resolution respirometry, and immune function using ex vivo lipopolysaccharide-stimulated whole blood tumor necrosis factor-α. Sepsis patients exhibited reduced microbial diversity compared with healthy controls, with lower alpha- and beta-diversity. Reduced microbial diversity among sepsis patients (mainly from lower abundance of commensal obligate anaerobes) was associated with increased acetic and propionic acid and decreased butyric, isobutyric, and caproic acid. Decreased levels of plasma butyric acid were further associated with lower peripheral blood mononuclear cell mitochondrial respiration, which in turn, was associated with lower lipopolysaccharide-stimulated tumor necrosis factor-α. However, neither intestinal dysbiosis nor specific patterns of short-chain fatty acids were associated with lipopolysaccharide-stimulated tumor necrosis factor-α., Conclusions: Intestinal dysbiosis was associated with altered short-chain fatty acid metabolites in children with sepsis, but these findings were not linked directly to mitochondrial or immunologic changes. More detailed mechanistic studies are needed to test the role of microbial-derived short-chain fatty acids in the progression of sepsis., Competing Interests: The authors have disclosed that they do not have any potential conflicts of interest., (Copyright © 2021 The Authors. Published by Wolters Kluwer Health, Inc. on behalf of the Society of Critical Care Medicine.)

Autism spectrum disorders (ASDs) are characterized by a deficit in social communication, pathologic repetitive behaviors, restricted interests, and electroencephalogram (EEG) aberrations. While exhaustive analysis of nuclear DNA (nDNA) variation has revealed hundreds of copy number variants (CNVs) and loss-of-function (LOF) mutations, no unifying hypothesis as to the pathophysiology of ASD has yet emerged. Based on biochemical and physiological analyses, it has been hypothesized that ASD may be the result of a systemic mitochondrial deficiency with brain-specific manifestations. This proposal has been supported by recent mitochondrial DNA (mtDNA) analyses identifying both germline and somatic mtDNA variants in ASD. If mitochondrial defects do predispose to ASD, then mice with certain mtDNA mutations should present with autism endophenotypes. To test this prediction, we examined a mouse strain harboring an mtDNA ND6 gene missense mutation (P25L). This mouse manifests impaired social interactions, increased repetitive behaviors and anxiety, EEG alterations, and a decreased seizure threshold, in the absence of reduced hippocampal interneuron numbers. EEG aberrations were most pronounced in the cortex followed by the hippocampus. Aberrations in mitochondrial respiratory function and reactive oxygen species (ROS) levels were also most pronounced in the cortex followed by the hippocampus, but absent in the olfactory bulb. These data demonstrate that mild systemic mitochondrial defects can result in ASD without apparent neuroanatomical defects and that systemic mitochondrial mutations can cause tissue-specific brain defects accompanied by regional neurophysiological alterations., Competing Interests: Competing interest statement: D.C.W. has informal associations with MitoCURIAi and Mitrios. He is a consultant for Pano Therapeutics, Inc. and received funding from SP Accure Lab, Ltd., (Copyright © 2021 the Author(s). Published by PNAS.)

MNRR1 (CHCHD2) is a bi-organellar regulator of mitochondrial function that directly activates cytochrome c oxidase in the mitochondria and functions in the nucleus as a transcriptional activator for hundreds of genes. Since MNRR1 depletion contains features of a mitochondrial disease phenotype, we evaluated the effects of forced expression of MNRR1 on the mitochondrial disease MELAS (mitochondrial encephalomyopathy, lactic acidosis and stroke-like episodes) syndrome. MELAS is a multisystem encephalomyopathy disorder that can result from a heteroplasmic mutation in the mitochondrial DNA (mtDNA; m.3243A > G) at heteroplasmy levels of ∼50 to 90%. Since cybrid cell lines with 73% m.3243A > G heteroplasmy (DW7) display a significant reduction in MNRR1 levels compared to the wild type (0% heteroplasmy) (CL9), we evaluated the effects of MNRR1 levels on mitochondrial functioning. Overexpression of MNRR1 in DW7 cells induces the mitochondrial unfolded protein response (UPR mt ), autophagy, and mitochondrial biogenesis, thereby rescuing the mitochondrial phenotype. It does so primarily as a transcription activator, revealing this function to be a potential therapeutic target. The role of MNRR1 in stimulating UPR mt , which is blunted in MELAS cells, was surprising and further investigation uncovered that under conditions of stress the import of MNRR1 into the mitochondria was blocked, allowing the protein to accumulate in the nucleus to enhance its transcription function. In the mammalian system, ATF5, has been identified as a mediator of UPR mt MNRR1 knockout cells display an ∼40% reduction in the protein levels of ATF5, suggesting that MNRR1 plays an important role upstream of this known mediator of UPR mt ., Competing Interests: Competing interest statement: Company associations for D.C.W. not related to the manuscript include MitoCURia, Mitrios, and Panos.

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., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 The Author(s). Published by Elsevier Inc. All rights reserved.)

Mitochondrial DNA (mtDNA) variant pathogenicity interpretation has special considerations given unique features of the mtDNA genome, including maternal inheritance, variant heteroplasmy, threshold effect, absence of splicing, and contextual effects of haplogroups. Currently, there are insufficient standardized criteria for mtDNA variant assessment, which leads to inconsistencies in clinical variant pathogenicity reporting. An international working group of mtDNA experts was assembled within the Mitochondrial Disease Sequence Data Resource Consortium and obtained Expert Panel status from ClinGen. This group reviewed the 2015 American College of Medical Genetics and Association of Molecular Pathology standards and guidelines that are widely used for clinical interpretation of DNA sequence variants and provided further specifications for additional and specific guidance related to mtDNA variant classification. These Expert Panel consensus specifications allow for consistent consideration of the unique aspects of the mtDNA genome that directly influence variant assessment, including addressing mtDNA genome composition and structure, haplogroups and phylogeny, maternal inheritance, heteroplasmy, and functional analyses unique to mtDNA, as well as specifications for utilization of mtDNA genomic databases and computational algorithms., (© 2020 Wiley Periodicals LLC.)