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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.

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.

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 [SRSF2.sup.P95H/+] 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. [SRSF2.sup.P95H]-induced mitochondrial dysfunction increased PINK1 expression through this mechanism, which is essential for survival of [SRSF2.sup.P95H/+] cells. Inhibition of splicing with a glycogen synthase kinase 3 inhibitor promoted retention of the poison intron, impairing mitophagy and activating apoptosis in [SRSF2.sup.P95H/+] 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 [SRSF2.sup.P95H] mutant MDS and AML., Introduction Myelodysplastic syndrome (MDS) is a clonal hematopoietic disorder characterized by hyperproliferative bone marrow, dysplastic hematopoietic cells, peripheral cytopenias, and high risk of progression to acute myeloid leukemia (AML). Recurrent [...]

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.

BackgroundIdentifying Obstructive Sleep Apnea (OSA) phenotypes among middle-aged and older Hispanics/Latinos can facilitate personalized care, better inform treatment decisions, and could lead to improved clinical outcomes.MethodsWe focused on middle-aged and older adults (ages ≥45-74 years at baseline) with an apnea-hypopnea index (AHI) ≥5 from the HCHS/SOL (2008-2011) (unweighted n = 3,545). We used latent class analyses (LCA) to identify empirical and clinically meaningful OSA phenotypes. Sleep variables included AHI, percent sleep time SpO2

Study objectivesRecent work on US Whites from clinical samples used obstructive sleep apnea (OSA) symptoms to generate phenotypes for individuals with moderate-severe OSA which suggested 3 to 5 symptom classes. However, it is unknown whether similar classes generalize to diverse Hispanics/Latino adults. Therefore, we sought to fill this gap by empirically deriving sleep phenotypes among a large sample of diverse Hispanics/Latinos.MethodsWe used data from The Hispanic Community Health Study/Study of Latinos (HCHS/SOL; 2008-2011), a prospective cohort study designed using a multisite multistage probability sample of adults 18-74 years old. The subpopulation of interest included participants with moderate-severe OSA symptoms (≥15 respiratory event index (REI) events per hour; n = 1,605). We performed latent class analysis for complex survey data using 15 common OSA symptoms (e.g. Epworth Sleepiness Scale) and 4 comorbidities to identify phenotype classes.ResultsAverage age was 52.4 ± 13.9 years and 34.0% were female. Mean REI was 33.8 ± 22.5 events per hour. Fit statistics and clinical significance suggested that a three-class solution provided the best fit to the data. The three phenotypes were: (1) Minimally Symptomatic (47.7%), (2) Excessive sleepiness (37.1%), and (3) Disturbed Sleep (15.2%). Sensitivity models were consistent with the main proposed solution.ConclusionsDerived sleep phenotypes among diverse Hispanic/Latinos were consistent with recent findings from the Sleep Apnea Global Interdisciplinary Consortium, but we found notable differences in class prevalence relative to Whites. Further research is needed to link derived sleep phenotypes to health comorbidities in diverse populations.

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 N1-methylation of guanosine at position 9 (m1G9) 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 m1G9 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 N1-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. Here the authors show that the m1G9 post-transcriptional methylation differentially regulates the stability of the native and the MELAS variants of human mt-Leu(UAA) tRNA, contributing to mitochondrial pathology. [ABSTRACT FROM AUTHOR]

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) infection inhibits mito-chondrial oxidative phosphorylation (OXPHOS) and elevates mitochondrial reactive oxygen species (ROS, mROS) which activates hypoxia-inducible factor-1alpha (HIF-1a), 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. [ABSTRACT FROM AUTHOR]