bims-mitdis Biomed News
on Mitochondrial disorders
Issue of 2023‒03‒26
fifty-one papers selected by
Catalina Vasilescu
Helmholz Munich

  1. Cardiol Young. 2023 Mar 23. 1-3
      In contrast to hypertrophic cardiomyopathy caused by maternal diabetes, neonatal mitochondrial cardiomyopathy is rare and has a poor prognosis. We report an infant born to a mother with maternal diabetes with persistent ventricular hypertrophy, who was diagnosed with mitochondrial disease associated with m.3243A>G mutation in a mitochondrial tRNA leucine 1 gene. The hypertrophic cardiomyopathy was his initial and only clinical presentation.
    Keywords:  Hypertrophic cardiomyopathy; MT-TL1 mutation; infant of a diabetic mother; mitochondrial cardiomyopathy
  2. EMBO Rep. 2023 Mar 20. e55760
      Mitochondria play central roles in cellular energy production and metabolism. Most proteins required to carry out these functions are synthesized in the cytosol and imported into mitochondria. A growing number of metabolic disorders arising from mitochondrial dysfunction can be traced to errors in mitochondrial protein import. The mechanisms underlying the import of precursor proteins are commonly studied using radioactively labeled precursor proteins imported into purified mitochondria. Here, we establish a fluorescence-based import assay to analyze protein import into mitochondria. We show that fluorescently labeled precursors enable import analysis with similar sensitivity to those using radioactive precursors, yet they provide the advantage of quantifying import with picomole resolution. We adapted the import assay to a 96-well plate format allowing for fast analysis in a screening-compatible format. Moreover, we show that fluorescently labeled precursors can be used to monitor the assembly of the F1 F0 ATP synthase in purified mitochondria. Thus, we provide a sensitive fluorescence-based import assay that enables quantitative and fast import analysis.
    Keywords:  fluorescent precursor; in vitro import; mitochondria; presequence pathway; protein import
  3. bioRxiv. 2023 Mar 09. pii: 2023.03.09.531795. [Epub ahead of print]
      Mitochondrial diseases are a group of disorders defined by defects in oxidative phosphorylation caused by nuclear- or mitochondrial-encoded gene mutations. A main cellular phenotype of mitochondrial disease mutations are redox imbalances and inflammatory signaling underlying pathogenic signatures of these patients. Depending on the type of mitochondrial mutation, certain mechanisms can efficiently rescue cell death vulnerability. One method is the inhibition of mitochondrial translation elongation using tetracyclines, potent suppressors of cell death in mitochondrial disease mutant cells. However, the mechanisms whereby tetracyclines promote cell survival are unknown. Here, we show that in mitochondrial mutant disease cells, tetracycline-mediated inhibition of mitoribosome elongation promotes survival through suppression of the ER stress IRE1α protein. Tetracyclines increased levels of the splitting factor MALSU1 (Mitochondrial Assembly of Ribosomal Large Subunit 1) at the mitochondria with recruitment to the mitochondrial ribosome (mitoribosome) large subunit. MALSU1, but not other quality control factors, was required for tetracycline-induced cell survival in mitochondrial disease mutant cells during glucose starvation. In these cells, nutrient stress induced cell death through IRE1α activation associated with a strong protein loading in the ER lumen. Notably, tetracyclines rescued cell death through suppression of IRE1α oligomerization and activity. Consistent with MALSU1 requirement, MALSU1 deficient mitochondrial mutant cells were sensitive to glucose-deprivation and exhibited increased ER stress and activation of IRE1α that was not reversed by tetracyclines. These studies show that inhibition of mitoribosome elongation signals to the ER to promote survival, establishing a new interorganelle communication between the mitoribosome and ER with implications in basic mechanisms of cell survival and treatment of mitochondrial diseases.Significance Statement: Mitochondrial diseases are a rare and heterogenous class of diseases that result from mutations in mitochondrial genes. Currently, there are no curative therapies due to a lack of mechanistic insights into pathological transformation and signaling. Our lab has discovered that the class of mitochondrial ribosome targeting antibiotics, tetracyclines, promote survival and fitness in models of mitochondrial disease, establishing a new paradigm of cell survival under nutrient stress conditions. In the current study, we present mechanistic insights into tetracyclines ability to rescue mitochondrial disease cells, detailing an interorganelle communication between mitochondrial protein translation and the unfolded protein response during endoplasmic reticulum stress.
  4. Biochem Soc Trans. 2023 Mar 24. pii: BST20220317. [Epub ahead of print]
      Mitochondrial calcium (Ca2+) signaling has long been known to regulate diverse cellular functions, ranging from ATP production via oxidative phosphorylation, to cytoplasmic Ca2+ signaling to apoptosis. Central to mitochondrial Ca2+ signaling is the mitochondrial Ca2+ uniporter complex (MCUC) which enables Ca2+ flux from the cytosol into the mitochondrial matrix. Several pivotal discoveries over the past 15 years have clarified the identity of the proteins comprising MCUC. Here, we provide an overview of the literature on mitochondrial Ca2+ biology and highlight recent findings on the high-resolution structure, dynamic regulation, and new functions of MCUC, with an emphasis on publications from the last five years. We discuss the importance of these findings for human health and the therapeutic potential of targeting mitochondrial Ca2+ signaling.
    Keywords:  calcium signaling; mitochondria; mitochondrial calcium uniporter; mitochondrial signaling
  5. PLoS Comput Biol. 2023 Mar 23. 19(3): e1010953
      Mitochondria are highly dynamic organelles, containing vital populations of mitochondrial DNA (mtDNA) distributed throughout the cell. Mitochondria form diverse physical structures in different cells, from cell-wide reticulated networks to fragmented individual organelles. These physical structures are known to influence the genetic makeup of mtDNA populations between cell divisions, but their influence on the inheritance of mtDNA at divisions remains less understood. Here, we use statistical and computational models of mtDNA content inside and outside the reticulated network to quantify how mitochondrial network structure can control the variances of inherited mtDNA copy number and mutant load. We assess the use of moment-based approximations to describe heteroplasmy variance and identify several cases where such an approach has shortcomings. We show that biased inclusion of one mtDNA type in the network can substantially increase heteroplasmy variance (acting as a genetic bottleneck), and controlled distribution of network mass and mtDNA through the cell can conversely reduce heteroplasmy variance below a binomial inheritance picture. Network structure also allows the generation of heteroplasmy variance while controlling copy number inheritance to sub-binomial levels, reconciling several observations from the experimental literature. Overall, different network structures and mtDNA arrangements within them can control the variances of key variables to suit a palette of different inheritance priorities.
  6. J Hum Genet. 2023 Mar 23.
      RRM2B encodes the p53-inducible small subunit (p53R2) of ribonucleotide reductase, a key protein for mitochondrial DNA (mtDNA) synthesis. Pathogenic variants in this gene result in familial mitochondrial disease in adults and children, secondary to a maintenance disorder of mtDNA. This study describes two patients, mother and son, with early-onset chronic progressive external ophthalmoplegia (PEO). Skeletal muscle biopsy from the latter was examined: cytochrome c oxidase (COX)-negative fibres were shown, and molecular studies revealed multiple mtDNA deletions. A next-generation sequencing gene panel for nuclear-encoded mitochondrial maintenance genes identified two unreported heterozygous missense variants (c.514 G > A and c.682 G > A) in the clinically affected son. The clinically affected mother harboured the first variant in homozygous state, and the clinically unaffected father harboured the remaining variant in heterozygous state. In silico analyses predicted both variants as deleterious. Cell culture studies revealed that patients' skin fibroblasts, but not fibroblasts from healthy controls, responded to nucleoside supplementation with enhanced mtDNA repopulation, thus suggesting an in vitro functional difference in patients' cells. Our results support the pathogenicity of two novel RRM2B variants found in two patients with autosomal recessive PEO with multiple mtDNA deletions inherited with a pseudodominant pattern.
  7. iScience. 2023 Mar 17. 26(3): 106270
      Charcot-Marie-Tooth disease type 2A (CMT2A), the most common inherited peripheral axonal neuropathy, is associated with more than 100 dominant mutations, including R94Q as the most abundant mutation in the Mitofusin2 (MFN2) gene. CMT2A is characterized by progressive motor and sensory loss, color-vision defects, and progressive loss of visual acuity. We used a well-established transgenic mouse model of CMT2A with R94Q mutation on MFN2 gene (MFN2 R94Q ) to investigate the functional and morphological changes in retina. We documented extensive vision loss due to photoreceptor degeneration, retinal ganglion cell and their axonal loss, retinal secondary neuronal and synaptic alternation, and Müller cell gliosis in the retina of MFN2 R94Q mice. Imbalanced MFN1/MFN2 ratio and dysregulated mitochondrial fusion/fission result in retinal degeneration via P62/LC3B-mediated mitophagy/autophagy in MFN2 R94Q mice. Finally, transgenic MFN1 augmentation (MFN2 R94Q :MFN1) rescued vision and retinal morphology to wild-type level via restoring homeostasis in mitochondrial MFN1/MFN2 ratio, fusion/fission cycle, and PINK1-dependent, Parkin-independent mitophagy.
    Keywords:  Biological sciences; Molecular neuroscience; Neuroscience; Sensory neuroscience
  8. Elife. 2023 Mar 23. pii: e84415. [Epub ahead of print]12
      Respiratory complex I is a proton-pumping oxidoreductase key to bioenergetic metabolism. Biochemical studies have found a divide in the behavior of complex I in metazoans that aligns with the evolutionary split between Protostomia and Deuterostomia. Complex I from Deuterostomia including mammals can adopt a biochemically defined off-pathway 'deactive' state, whereas complex I from Protostomia cannot. The presence of off-pathway states complicates the interpretation of structural results and has led to considerable mechanistic debate. Here, we report the structure of mitochondrial complex I from the thoracic muscles of the model protostome Drosophila melanogaster. We show that although D. melanogaster complex I (Dm-CI) does not have a NEM-sensitive deactive state, it does show slow activation kinetics indicative of an off-pathway resting state. The resting-state structure of Dm-CI from the thoracic muscle reveals multiple conformations. We identify a helix-locked state in which an N-terminal α-helix on the NDUFS4 subunit wedges between the peripheral and membrane arms. Comparison of the Dm-CI structure and conformational states to those observed in bacteria, yeast, and mammals provides insight into the roles of subunits across organisms, explains why the Dm-CI off-pathway resting state is NEM insensitive, and raises questions regarding current mechanistic models of complex I turnover.
    Keywords:  complex I; drosophila melanogaster; electron transport chain; mitochondria; molecular biophysics; respiration; single particle cryoEM; structural biology
  9. FASEB J. 2023 Apr;37(4): e22851
      Sarcopenia is a geriatric syndrome characterized by an age-related decline in skeletal muscle mass and strength. Here, we show that suppression of mitochondrial calcium uniporter (MCU)-mediated Ca2+ influx into mitochondria in the body wall muscles of the nematode Caenorhabditis elegans improved the sarcopenic phenotypes, blunting movement and mitochondrial structural and functional decline with age. We found that normally aged muscle cells exhibited elevated resting mitochondrial Ca2+ levels and increased mitophagy to eliminate damaged mitochondria. Similar to aging muscle, we found that suppressing MCU function in muscular dystrophy improved movement via reducing elevated resting mitochondrial Ca2+ levels. Taken together, our results reveal that elevated resting mitochondrial Ca2+ levels contribute to muscle decline with age and muscular dystrophy. Further, modulation of MCU activity may act as a potential pharmacological target in various conditions involving muscle loss.
    Keywords:  MCU; aging; calcium; dystrophy; mitophagy; sarcopenia
  10. bioRxiv. 2023 Mar 10. pii: 2023.03.09.531996. [Epub ahead of print]
      Background: Cardiomyocyte maturation requires a massive increase in respiratory enzymes and their assembly into long-lived complexes of oxidative phosphorylation (OXPHOS). The molecular mechanisms underlying the maturation of cardiac mitochondria have not been established.Methods: To determine whether the mitochondria-specific lipid cardiolipin is involved in cardiac maturation, we created a cardiomyocyte-restricted knockout (KO) of cardiolipin synthase ( Crls1 ) in mice and studied the postnatal development of the heart. We also measured the turnover rates of proteins and lipids in cardiolipin-deficient flight muscle from Drosophila, a tissue that has mitochondria with high OXPHOS activity like the heart.
    Results: Crls1KO mice survived the prenatal period but failed to accumulate OXPHOS proteins during postnatal maturation and succumbed to heart failure at the age of 2 weeks. Turnover measurements showed that the exceptionally long half-life of OXPHOS proteins is critically dependent on cardiolipin.
    Conclusions: Cardiolipin is essential for the postnatal maturation of cardiomyocytes because it allows mitochondrial cristae to accumulate OXPHOS proteins to a high concentration and to shield them from degradation.
  11. bioRxiv. 2023 Mar 08. pii: 2023.03.06.531392. [Epub ahead of print]
      Mitochondrial genomes co-evolve with the nuclear genome over evolutionary timescales and are shaped by selection in the female germline. Here, we investigate how mismatching between nuclear and mitochondrial ancestry impacts the somatic evolution of the mt-genome in different tissues throughout aging. We used ultra-sensitive Duplex Sequencing to profile ∼2.5 million mt-genomes across five mitochondrial haplotypes and three tissues in young and aged mice, cataloging ∼1.2 million mitochondrial somatic mutations. We identify haplotype-specific mutational patterns and several mutational hotspots, including at the Light Strand Origin of Replication, which consistently exhibits the highest mutation frequency. We show that rodents exhibit a distinct mitochondrial somatic mutational spectrum compared to primates with a surfeit of reactive oxygen species-associated G>T/C>A mutations and that somatic mutations in protein coding genes exhibit strong signatures of positive selection. Lastly, we identify an extensive enrichment in somatic reversion mutations that "re-align" mito-nuclear ancestry within an organism's lifespan. Together, our findings demonstrate that mitochondrial genomes are a dynamically evolving subcellular population shaped by somatic mutation and selection throughout organismal lifetimes.
  12. Biol Chem. 2023 Mar 24.
      The orchestrated activity of the mitochondrial respiratory or electron transport chain (ETC) and ATP synthase convert reduction power (NADH, FADH2) into ATP, the cell's energy currency in a process named oxidative phosphorylation (OXPHOS). Three out of the four ETC complexes are found in supramolecular assemblies: complex I, III, and IV form the respiratory supercomplexes (SC). The plasticity model suggests that SC formation is a form of adaptation to changing conditions such as energy supply, redox state, and stress. Complex I, the NADH-dehydrogenase, is part of the largest supercomplex (CI + CIII2 + CIVn). Here, we demonstrate the role of NDUFB10, a subunit of the membrane arm of complex I, in complex I and supercomplex assembly on the one hand and bioenergetics function on the other. NDUFB10 knockout was correlated with a decrease of SCAF1, a supercomplex assembly factor, and a reduction of respiration and mitochondrial membrane potential. This likely is due to loss of proton pumping since the CI P P -module is downregulated and the P D -module is completely abolished in NDUFB10 knock outs.
    Keywords:  NDUFB10; OXPHOS; complex I; mitochondria; respiratory chain supercomplexes
  13. iScience. 2023 Mar 17. 26(3): 106278
      Replenishing nicotinamide adenine dinucleotide (NAD) via supplementation of nicotinamide riboside (NR) has been shown to confer neuroprotective effects in models of aging and neurodegenerative diseases, including Parkinson's disease (PD). Although generally considered safe, concerns have been raised that NR supplementation could impact methylation dependent reactions, including DNA methylation, because of increased production and methylation dependent breakdown of nicotinamide (NAM). We investigated the effect of NR supplementation on DNA methylation in a double blinded, placebo-controlled trial of 29 human subjects with PD, in blood cells and muscle tissue. Our results show that NR had no impact on DNA methylation homeostasis, including individuals with common pathogenic mutations in the MTHFR gene known to affect one-carbon metabolism. Pathway and methylation variance analyses indicate that there might be minor regulatory responses to NR. We conclude that short-term therapy with high-dose NR for up to 30 days has no deleterious impact on methylation homeostasis.
    Keywords:  Biological sciences; Clinical neuroscience; Molecular neuroscience; Neuroscience
  14. Hum Reprod. 2023 Mar 23. pii: dead052. [Epub ahead of print]
      STUDY QUESTION: Does mitochondrial deficiency affect human embryonic preimplantation development?SUMMARY ANSWER: The presence of a pathogenic mitochondrial variant triggers changes in the gene expression of preimplantation human embryos, compromising their development, cell differentiation, and survival.
    WHAT IS KNOWN ALREADY: Quantitative and qualitative anomalies of mitochondrial DNA (mtDNA) are reportedly associated with impaired human embryonic development, but the underlying mechanisms remain unexplained.
    STUDY DESIGN, SIZE, DURATION: Taking advantage of the preimplantation genetic testing for mitochondrial disorders in at-risk couples, we have compared gene expression of 9 human embryos carrying pathogenic variants in either mtDNA genes or nuclear genes encoding mitochondrial protein to 33 age-matched control embryos.
    PARTICIPANTS/MATERIALS, SETTING, METHODS: Single-embryo transcriptomic analysis was performed on whole human blastocyst embryos donated to research.
    MAIN RESULTS AND THE ROLE OF CHANCE: Specific pathogenic mitochondrial variants downregulate gene expression in preimplantation human embryos [566 genes in oxidative phosphorylation (OXPHOS)-deficient embryos], impacting transcriptional regulators, differentiation factors, and nuclear genes encoding mitochondrial proteins. These changes in gene expression primarily alter OXPHOS and cell survival pathways.
    LIMITATIONS, REASONS FOR CAUTION: The number of OXPHOS-deficient embryos available for the study was limited owing to the rarity of this material. However, the molecular signature shared by all these embryos supports the relevance of the findings.
    WIDER IMPLICATIONS OF THE FINDINGS: While identification of reliable markers of normal embryonic development is urgently needed in ART, our study prompts us to consider under-expression of the targeted genes reported here, as predictive biomarkers of mitochondrial dysfunction during preimplantation development.
    STUDY FUNDING/COMPETING INTEREST(S): This work was supported by the 'Association Française contre les Myopathies (AFM-Téléthon)' and the 'La Fondation Maladies Rares'. No competing interests to declare.
    Keywords:  RNA sequencing; differential gene expression; human embryo development; mitochondrial metabolism; mitochondrial mutation; transcriptome
  15. bioRxiv. 2023 Mar 11. pii: 2023.03.10.532020. [Epub ahead of print]
      Insulin resistance (IR) is a complex metabolic disorder that underlies several human diseases, including type 2 diabetes and cardiovascular disease. Despite extensive research, the precise mechanisms underlying IR development remain poorly understood. Here, we provide new insights into the mechanistic connections between cellular alterations associated with IR, including increased ceramides, deficiency of coenzyme Q (CoQ), mitochondrial dysfunction, and oxidative stress. We demonstrate that elevated levels of ceramide in the mitochondria of skeletal muscle cells results in CoQ depletion and loss of mitochondrial respiratory chain components, leading to mitochondrial dysfunction and IR. Further, decreasing mitochondrial ceramide levels in vitro and in animal models increased CoQ levels and was protective against IR. CoQ supplementation also rescued ceramide-associated IR. Examination of the mitochondrial proteome from human muscle biopsies revealed a strong correlation between the respirasome system and mitochondrial ceramide as key determinants of insulin sensitivity. Our findings highlight the mitochondrial Ceramide-CoQ-respiratory chain nexus as a potential foundation of an IR pathway that may also play a critical role in other conditions associated with ceramide accumulation and mitochondrial dysfunction, such as heart failure, cancer, and aging. These insights may have important clinical implications for the development of novel therapeutic strategies for the treatment of IR and related metabolic disorders.
  16. Cell Genom. 2023 Mar 08. 3(3): 100261
    American Genome Center
      The Foundational Data Initiative for Parkinson Disease (FOUNDIN-PD) is an international collaboration producing fundamental resources for Parkinson disease (PD). FOUNDIN-PD generated a multi-layered molecular dataset in a cohort of induced pluripotent stem cell (iPSC) lines differentiated to dopaminergic (DA) neurons, a major affected cell type in PD. The lines were derived from the Parkinson's Progression Markers Initiative study, which included participants with PD carrying monogenic PD variants, variants with intermediate effects, and variants identified by genome-wide association studies and unaffected individuals. We generated genetic, epigenetic, regulatory, transcriptomic, and longitudinal cellular imaging data from iPSC-derived DA neurons to understand molecular relationships between disease-associated genetic variation and proximate molecular events. These data reveal that iPSC-derived DA neurons provide a valuable cellular context and foundational atlas for modeling PD genetic risk. We have integrated these data into a FOUNDIN-PD data browser as a resource for understanding the molecular pathogenesis of PD.
    Keywords:  Parkinson disease; dopaminergic neurons; genetic risk; induced pluripotent stem cell; omics single-cell RNA sequencing single-cell ATAC sequencing SNCA LRRK2 GBA1
  17. Anal Chem. 2023 Mar 20.
      Mitophagy is a vital cellular process playing vital roles in regulating cellular metabolism and mitochondrial quality control. Mitochondrial viscosity is a key microenvironmental index, closely associated with mitochondrial status. To monitor mitophagy and mitochondrial viscosity, three molecular rotors (Mito-1, Mito-2, and Mito-3) were developed. All probes contain a cationic quinolinium unit and a C12 chain so that they can tightly bind mitochondria and are not affected by the mitochondrial membrane potential. Optical studies showed that all probes are sensitive to viscosity changes with an off-on fluorescence response, and Mito-3 shows the best fluorescence enhancement. Bioimaging studies showed that all these probes can not only tightly locate and visualize mitochondria with near-infrared fluorescence but also effectively monitor the mitochondrial viscosity changes in cells. Moreover, Mito-3 was successfully applied to visualize the mitophagy process induced by starvation, and mitochondrial viscosity was found to show an increase during mitophagy. We expect Mito-3 to become a useful imaging tool for studying mitochondrial viscosity and mitophagy.
  18. Nature. 2023 Mar 22.
      Mitochondrial energy conversion requires an intricate architecture of the inner mitochondrial membrane1. Here we show that a supercomplex containing all four respiratory chain components contributes to membrane curvature induction in ciliates. We report cryo-electron microscopy and cryo-tomography structures of the supercomplex that comprises 150 different proteins and 311 bound lipids, forming a stable 5.8-MDa assembly. Owing to subunit acquisition and extension, complex I associates with a complex IV dimer, generating a wedge-shaped gap that serves as a binding site for complex II. Together with a tilted complex III dimer association, it results in a curved membrane region. Using molecular dynamics simulations, we demonstrate that the divergent supercomplex actively contributes to the membrane curvature induction and tubulation of cristae. Our findings highlight how the evolution of protein subunits of respiratory complexes has led to the I-II-III2-IV2 supercomplex that contributes to the shaping of the bioenergetic membrane, thereby enabling its functional specialization.
  19. Commun Biol. 2023 Mar 22. 6(1): 307
      In mammalian mitochondria, translation of the AUA codon is supported by 5-formylcytidine (f5C) modification in the mitochondrial methionine tRNA anticodon. The 5-formylation is initiated by NSUN3 methylase. Human NSUN3 mutations are associated with mitochondrial diseases. Here we show that Nsun3 is essential for embryonic development in mice with whole-body Nsun3 knockout embryos dying between E10.5 and E12.5. To determine the functions of NSUN3 in adult tissue, we generated heart-specific Nsun3 knockout (Nsun3HKO) mice. Nsun3HKO heart mitochondria were enlarged and contained fragmented cristae. Nsun3HKO resulted in enhanced heart contraction and age-associated mild heart enlargement. In the Nsun3HKO hearts, mitochondrial mRNAs that encode respiratory complex subunits were not down regulated, but the enzymatic activities of the respiratory complexes decreased, especially in older mice. Our study emphasizes that mitochondrial tRNA anticodon modification is essential for mammalian embryonic development and shows that tissue-specific loss of a single mitochondrial tRNA modification can induce tissue aberration that worsens in later adulthood.
  20. J Cell Sci. 2023 Mar 21. pii: jcs.260049. [Epub ahead of print]
      Glucose sensing in pancreatic beta-cells depends on oxidative phosphorylation and mitochondria-derived signals that promote insulin secretion. Using mass spectrometry-based phosphoproteomics to search for down-stream effectors of glucose dependent signal transduction in INS-1E insulinoma cells, we identified the outer mitochondrial membrane protein SLC25A46. Under resting glucose concentrations, SLC25A46 was phosphorylated on a pair of threonine residues (T44/T45) and was dephosphorylated in response to glucose-induced calcium signals. Overexpression of SLC25A46 in INS-1E cells caused complete mitochondrial fragmentation, resulting in a mild mitochondrial defect associated with lowered glucose-induced insulin secretion. In contrast, inactivation of the SLC25A46 gene resulted in dramatic mitochondrial hyperfusion but without affecting respiratory activity or insulin secretion. Consequently, SLC25A46 is not essential for metabolism-secretion coupling under normal nutrient conditions. Importantly, insulin secreting cells lacking SLC25A46 had an exacerbated sensitivity to lipotoxic conditions undergoing massive apoptosis when exposed to palmitate. Therefore, in addition to its role in mitochondrial dynamics, SLC25A46 plays a role in preventing mitochondria-induced apoptosis in INS-E cells exposed to nutrient stress. By protecting mitochondria, SLC25A46 may help to maintain beta-cell mass essential for blood glucose control.
    Keywords:  Beta-cell; Mitochondria; Mitochondrial dynamics
  21. Genes Nutr. 2023 Mar 24. 18(1): 5
      BACKGROUND: Serine hydroxymethyltransferase 2 (SHMT2) catalyzes the reversible conversion of tetrahydrofolate (THF) and serine-producing THF-conjugated one-carbon units and glycine in the mitochondria. Biallelic SHMT2 variants were identified in humans and suggested to alter the protein's active site, potentially disrupting enzymatic function. SHMT2 expression has also been shown to decrease with aging in human fibroblasts. Immortalized cell models of total SHMT2 loss or folate deficiency exhibit decreased oxidative capacity and impaired mitochondrial complex I assembly and protein levels, suggesting folate-mediated one-carbon metabolism (FOCM) and the oxidative phosphorylation system are functionally coordinated. This study examined the role of SHMT2 and folate availability in regulating mitochondrial function, energy metabolism, and cellular proliferative capacity in both heterozygous and homozygous cell models of reduced SHMT2 expression. In this study, primary mouse embryonic fibroblasts (MEF) were isolated from a C57Bl/6J dam crossed with a heterozygous Shmt2+/- male to generate Shmt2+/+ (wild-type) or Shmt2+/- (HET) MEF cells. In addition, haploid chronic myeloid leukemia cells (HAP1, wild-type) or HAP1 cells lacking SHMT2 expression (ΔSHMT2) were cultured for 4 doublings in either low-folate or folate-sufficient culture media. Cells were examined for proliferation, total folate levels, mtDNA content, protein levels of pyruvate kinase and PGC1α, pyruvate kinase enzyme activity, mitochondrial membrane potential, and mitochondrial function.RESULTS: Homozygous loss of SHMT2 in HAP1 cells impaired cellular folate accumulation and altered mitochondrial DNA content, formate production, membrane potential, and basal respiration. Formate rescued proliferation in HAP1, but not ΔSHMT2, cells cultured in low-folate medium. Pyruvate kinase activity and protein levels were impaired in ΔSHMT2 cells and in MEF cells exposed to low-folate medium. Mitochondrial biogenesis protein levels were elevated in Shmt2+/- MEF cells, while mitochondrial mass was increased in both homozygous and heterozygous models of SHMT2 loss.
    CONCLUSIONS: The results from this study indicate disrupted mitochondrial FOCM impairs mitochondrial folate accumulation and respiration, mitochondrial formate production, glycolytic activity, and cellular proliferation. These changes persist even after a potentially compensatory increase in mitochondrial biogenesis as a result of decreased SHMT2 levels.
    Keywords:  Energy metabolism; Folate; One-carbon metabolism; Oxygen consumption rate; Pyruvate kinase; SHMT2
  22. Life Sci Alliance. 2023 Jun;pii: e202201805. [Epub ahead of print]6(6):
      Cellular functionality relies on a well-balanced, but highly dynamic proteome. Dysfunction of mitochondrial protein import leads to the cytosolic accumulation of mitochondrial precursor proteins which compromise cellular proteostasis and trigger a mitoprotein-induced stress response. To dissect the effects of mitochondrial dysfunction on the cellular proteome as a whole, we developed pre-post thermal proteome profiling. This multiplexed time-resolved proteome-wide thermal stability profiling approach with isobaric peptide tags in combination with a pulsed SILAC labelling elucidated dynamic proteostasis changes in several dimensions: In addition to adaptations in protein abundance, we observed rapid modulations of the thermal stability of individual cellular proteins. Different functional groups of proteins showed characteristic response patterns and reacted with group-specific kinetics, allowing the identification of functional modules that are relevant for mitoprotein-induced stress. Thus, our new pre-post thermal proteome profiling approach uncovered a complex response network that orchestrates proteome homeostasis in eukaryotic cells by time-controlled adaptations of the abundance and the conformation of proteins.
  23. bioRxiv. 2023 Mar 10. pii: 2023.03.08.531724. [Epub ahead of print]
      Neurotransmission is an energetically expensive process that underlies cognition. During intense electrical activity or dietary restrictions, glucose levels in the brain plummet, forcing neurons to utilize alternative fuels. However, the molecular mechanisms of neuronal metabolic plasticity remain poorly understood. Here, we demonstrate that glucose-deprived neurons activate the CREB and PGC1α transcriptional program that induces the expression of the mitochondrial deacetylase Sirtuin 3 (Sirt3) both in vitro and in vivo . We show that Sirt3 localizes to axonal mitochondria and stimulates mitochondrial oxidative capacity in hippocampal nerve terminals. Sirt3 plays an essential role in sustaining synaptic transmission in the absence of glucose by powering the retrieval of synaptic vesicles after release. These results demonstrate that the transcriptional induction of Sirt3 ensures the metabolic plasticity of synaptic transmission.Highlights: Glucose deprivation drives transcriptional reprogramming of neuronal metabolism via CREB and PGC1α. Glucose or food deprivation trigger the neuronal expression of mitochondrial deacetylase sirtuin 3 (Sirt3) both in vitro and in vivo . Sirt3 stimulates oxidative ATP synthesis in nerve terminals.Sirt3 sustains the synaptic vesicle cycle in the absence of glucose.
  24. Front Physiol. 2023 ;14 1114595
      Methods of isolating mitochondria commonly utilise mechanical force and shear stress to homogenize tissue followed by purification by multiple rounds of ultracentrifugation. Existing protocols can be time-consuming with some physically impairing integrity of the sensitive mitochondrial double membrane. Here, we describe a method for the recovery of intact, respiring mitochondria from murine skeletal muscle tissue and cell lines using nitrogen cavitation. This protocol results in high-yield, pure and respiring mitochondria without the need for purification gradients or ultracentrifugation. The protocol takes under an hour and requires limited specialised equipment. Our methodology is successful in extracting mitochondria of both cell extracts and skeletal muscle tissue. This represents an improved yield in comparison to many of the existing methods. Western blotting and electron microscopy demonstrate the enrichment of mitochondria with their ultrastructure well-preserved and an absence of contamination from cytoplasmic or nuclear fractions. Using respirometry analysis we show that mitochondria extracted from murine skeletal muscle cell lines (C2C12) and tibialis anterior tissue have an appropriate respiratory control ratio. These measures are indicative of healthy coupled mitochondria. Our method successfully demonstrates the rapid isolation of functional mitochondria and will benefit researchers studying mitochondrial bioenergetics as well as providing greater throughput and application for time-sensitive assays.
    Keywords:  cavitation; methodolody; mitochondria; protocol; skeletal muscle
  25. Front Neurosci. 2023 ;17 1145358
    Keywords:  NADH (NAD+); glucose; glycolysis; lactate; lactate dehydrogenase; mitochondria; oxydative phosphorylation (oxphos); trycarboxylic acid (TCA) cycle
  26. Cell Rep. 2023 Mar 20. pii: S2211-1247(23)00190-0. [Epub ahead of print]42(3): 112179
      The cGAS-STING pathway is central to the interferon response against DNA viruses. However, recent studies are increasingly demonstrating its role in the restriction of some RNA viruses. Here, we show that the cGAS-STING pathway also contributes to the interferon response against noroviruses, currently the commonest causes of infectious gastroenteritis worldwide. We show a significant reduction in interferon-β induction and a corresponding increase in viral replication in norovirus-infected cells after deletion of STING, cGAS, or IFI16. Further, we find that immunostimulatory host genome-derived DNA and mitochondrial DNA accumulate in the cytosol of norovirus-infected cells. Lastly, overexpression of the viral NS4 protein is sufficient to drive the accumulation of cytosolic DNA. Together, our data find a role for cGAS, IFI16, and STING in the restriction of noroviruses and show the utility of host genomic DNA as a damage-associated molecular pattern in cells infected with an RNA virus.
    Keywords:  CP: Immunology; CP: Molecular biology; DNA leakage; IFI16; NS4; STING; VF1; cGAS; cytosolic DNA; genomic DNA; interferon response; mitochondrial DNA; norovirus; p204
  27. J Clin Invest. 2023 Mar 23. pii: e162957. [Epub ahead of print]
      Sphingolipids function as membrane constituents and signaling molecules, with crucial roles in human diseases, from neurodevelopmental to cancer, best exemplified in the inborn errors of sphingolipid metabolism in lysosomes. The dihydroceramide desaturase DEGS1 acts in the last step of a sector of the sphingolipid pathway, de novo ceramide biosynthesis. Defects in DEGS1 cause the recently described hypomyelinating leukodystrophy-18 (HLD18, OMIM #618404). Here, we reveal that DEGS1 is a mitochondria-associated endoplasmic reticulum membrane (MAM)-resident enzyme, refining previous reports locating DEGS1 at the endoplasmic reticulum only. Using patient fibroblasts, multi-omics and enzymatic assays, we show that DEGS1 deficiency disrupts the main core functions of the MAM: i) mitochondrial dynamics, with a hyperfused mitochondrial network associated with decreased activation of dynamin-related protein 1; ii) cholesterol metabolism, with impaired sterol O-acyltransferase activity and decreased cholesteryl esters; iii) phospholipid metabolism, with increased phosphatidic acid and phosphatidylserine and decreased phosphatidylethanolamine; iv) biogenesis of lipid droplets, with increased size and numbers. Moreover, we detected increased mitochondrial superoxide species production in fibroblasts and mitochondrial respiration impairment in patient muscle biopsy tissues. Our findings shed light on the pathophysiology of HLD18 and broaden our understanding of the role of sphingolipid metabolism in MAMs function.
    Keywords:  Bioenergetics; Demyelinating disorders; Lipid rafts; Metabolism; Neuroscience
  28. Case Rep Ophthalmol. 2023 Jan-Dec;14(1):14(1): 99-103
      Mutations to the ND5 gene are uncommonly associated with Leber's hereditary optic neuropathy (LHON). Herein, we describe a 57-year-old man with the m. 13528A>G, p. (Thr398Ala) mutation at the ND5 gene who presented with progressive bilateral vision loss over the course of 3 months. He had a significant history of smoking and alcohol consumption. Visual field testing demonstrated bilateral central scotomas. At 2-year follow-up, his visual acuity improved relative to baseline and temporal optic disc pallor was observed in both eyes. There are scarce reports of this mutation in the literature, and this case report further expands the clinical presentation of the m. 13528A>G mutation at the ND5 gene in patients with LHON phenotype.
    Keywords:  Leber’s hereditary optic neuropathy; Mitochondrial DNA; ND5
  29. J Vis Exp. 2023 Mar 03.
      The quantitative analysis of subcellular organelles such as mitochondria in cell fluorescence microscopy images is a demanding task because of the inherent challenges in the segmentation of these small and morphologically diverse structures. In this article, we demonstrate the use of a machine learning-aided segmentation and analysis pipeline for the quantification of mitochondrial morphology in fluorescence microscopy images of fixed cells. The deep learning-based segmentation tool is trained on simulated images and eliminates the requirement for ground truth annotations for supervised deep learning. We demonstrate the utility of this tool on fluorescence microscopy images of fixed cardiomyoblasts with a stable expression of fluorescent mitochondria markers and employ specific cell culture conditions to induce changes in the mitochondrial morphology.
  30. Nat Immunol. 2023 Mar 20.
      Upon detecting pathogens or cell stress, several NOD-like receptors (NLRs) form inflammasome complexes with the adapter ASC and caspase-1, inducing gasdermin D (GSDMD)-dependent cell death and maturation and release of IL-1β and IL-18. The triggers and activation mechanisms of several inflammasome-forming sensors are not well understood. Here we show that mitochondrial damage activates the NLRP10 inflammasome, leading to ASC speck formation and caspase-1-dependent cytokine release. While the AIM2 inflammasome can also sense mitochondrial demise by detecting mitochondrial DNA (mtDNA) in the cytosol, NLRP10 monitors mitochondrial integrity in an mtDNA-independent manner, suggesting the recognition of distinct molecular entities displayed by the damaged organelles. NLRP10 is highly expressed in differentiated human keratinocytes, in which it can also assemble an inflammasome. Our study shows that this inflammasome surveils mitochondrial integrity. These findings might also lead to a better understanding of mitochondria-linked inflammatory diseases.
  31. J Nutr Biochem. 2023 Mar 22. pii: S0955-2863(23)00055-4. [Epub ahead of print] 109322
      Vitamin D receptor (VDR) is an essential transcription factor (TF) synthesized in different cell types. We hypothesized that VDR might also act as a mitochondrial TF. We conducted the experiments in primary cortical neurons, PC12, HEK293T, SH-SY5Y cell lines, human peripheral blood mononuclear cells (PBMC) and human brain. We showed that vitamin D/VDR affects the expression of mitochondrial DNA (mtDNA) encoded oxidative phosphorylation (OXPHOS) subunits. We observed the co-localization of VDR with mitochondria and the mtDNA with confocal microscopy. mtDNA-chromatin-immunoprecipitation and electrophoretic mobility shift assays indicated that VDR was able to bind to the mtDNA D-loop site in several locations, with a consensus sequence 'MMHKCA'. We also reported the possible interaction between VDR and mitochondrial transcription factor A (TFAM) and their binding sites located in close proximity in mtDNA. Consequently, our results showed for the first time that VDR was able to bind and regulate mtDNA transcription and interact with TFAM even in the human brain. These results not only revealed a novel function of VDR, but also showed that VDR is indispensable for energy demanded cells.
    Keywords:  Vitamin D; mitochondria; mitochondrial DNA; mitochondrial transcription factor; transcription factor; vitamin D receptor
  32. Redox Biol. 2023 Mar 14. pii: S2213-2317(23)00073-3. [Epub ahead of print]62 102672
      The transcription factor Nrf2 and its repressor Keap1 mediate cell stress adaptation by inducing expression of genes regulating cellular detoxification, antioxidant defence and energy metabolism. Energy production and antioxidant defence employ NADH and NADPH respectively as essential metabolic cofactors; both are generated in distinct pathways of glucose metabolism, and both pathways are enhanced by Nrf2 activation. Here, we examined the role of Nrf2 on glucose distribution and the interrelation between NADH production in energy metabolism and NADPH homeostasis using glio-neuronal cultures isolated from wild-type, Nrf2-knockout and Keap1-knockdown mice. Employing advanced microscopy imaging of single live cells, including multiphoton fluorescence lifetime imaging microscopy (FLIM) to discriminate between NADH and NADPH, we found that Nrf2 activation increases glucose uptake into neurons and astrocytes. Glucose consumption is prioritized in brain cells for mitochondrial NADH and energy production, with a smaller contribution to NADPH synthesis in the pentose phosphate pathway for redox reactions. As Nrf2 is suppressed during neuronal development, this strategy leaves neurons reliant on astrocytic Nrf2 to maintain redox balance and energy homeostasis.
    Keywords:  Astrocytes; Brain; Glucose metabolism; Mitochondria; NADH; NADPH; Neurons; Nrf2
  33. Nat Biotechnol. 2023 Mar 23.
      An average shotgun proteomics experiment detects approximately 10,000 human proteins from a single sample. However, individual proteins are typically identified by peptide sequences representing a small fraction of their total amino acids. Hence, an average shotgun experiment fails to distinguish different protein variants and isoforms. Deeper proteome sequencing is therefore required for the global discovery of protein isoforms. Using six different human cell lines, six proteases, deep fractionation and three tandem mass spectrometry fragmentation methods, we identify a million unique peptides from 17,717 protein groups, with a median sequence coverage of approximately 80%. Direct comparison with RNA expression data provides evidence for the translation of most nonsynonymous variants. We have also hypothesized that undetected variants likely arise from mutation-induced protein instability. We further observe comparable detection rates for exon-exon junction peptides representing constitutive and alternative splicing events. Our dataset represents a resource for proteoform discovery and provides direct evidence that most frame-preserving alternatively spliced isoforms are translated.
  34. BMC Ophthalmol. 2023 Mar 24. 23(1): 118
      BACKGROUND: Mitochondrial optic neuropathy is characterized by painless, progressive, symmetrical central vision loss, and dyschromatopsia owing to mitochondrial dysfunction. This report documents a rare case of mitochondrial optic neuropathy due to the SIRT3 gene mutation.CASE PRESENTATION: This report describes a case of a 17-year-old boy who presented with symptoms of bilateral painless, progressive vision decline over several years. Fundus examination revealed temporal pallor of the optic nerve head in both the eyes and an OCT showed considerable thinning of the retinal nerve fiber and ganglion cell layers. Pathogenicity was confirmed by decreased mitochondrial function measured by bioenergetic health index and oxygen consumption rate in this patient. Subsequent NGS revealed a missense mutation of the SIRT3 gene (c.1137G > C, p.Trp379Cys) in the patient.
    CONCLUSIONS: This case describes the clinical manifestation of mitochondrial optic neuropathy due to the SIRT3 gene mutation.
    Keywords:  Case report; Mitochondrial dysfunction; Mitochondrial optic neuropathy; SIRT3 gene mutation
  35. bioRxiv. 2023 Mar 12. pii: 2023.03.11.532186. [Epub ahead of print]
      The integrated stress response (ISR) is a network of eIF2 α kinases, comprising PERK, GCN2, HRI, and PKR, that induce translational and transcriptional signaling in response to diverse insults. The PERK ISR kinase regulates mitochondria in response to endoplasmic reticulum (ER) stress. Deficiencies in PERK signaling lead to mitochondrial dysfunction and contribute to the pathogenesis of numerous diseases. We define the potential for pharmacologic activators of other ISR kinases to rescue ISR signaling and promote mitochondrial adaptation in cells lacking PERK. We show that the HRI activator BtdCPU and the GCN2 activator halofuginone activate ISR signaling and restore ER stress sensitivity in Perk- deficient cells. However, these compounds differentially impact mitochondria. BtdCPU induces mitochondrial depolarization, leading to mitochondrial fragmentation and ISR activation through the OMA1-DELE1-HRI signaling axis. In contrast, halofuginone promotes mitochondrial elongation and altered mitochondrial respiration, mimicking the regulation induced by PERK. This shows halofuginone can compensate for deficiencies in PERK activity and promote adaptive mitochondrial remodeling, highlighting the potential for pharmacologic ISR activation to mitigate mitochondrial dysfunction and motivating the pursuit of highly-selective ISR activators.
  36. Mol Genet Metab Rep. 2023 Jun;35 100963
      NADH dehydrogenase 5 (ND5) is one of 44 subunits composed of Complex I in mitochondrial respiratory chain. Therefore, a mitochondrially encoded ND5 (MT-ND5) gene mutation causes mitochondrial oxidative phosphorylation (OXPHOS) disorder, resulting in the development of mitochondrial diseases. Focal segmental glomerulosclerosis (FSGS) which had podocytes filled with abnormal mitochondria is induced by mitochondrial diseases. An MT-ND5 mutation also causes FSGS. We herein report a Japanese woman who was found to have proteinuria and renal dysfunction in an annual health check-up at 29 years old. Because her proteinuria and renal dysfunction were persistent, she had a kidney biopsy at 33 years of age. The renal histology showed FSGS with podocytes filled with abnormal mitochondria. The podocytes also had foot process effacement and cytoplasmic vacuolization. In addition, the renal pathological findings showed granular swollen epithelial cells (GSECs) in tubular cells, age-inappropriately disarranged and irregularly sized vascular smooth muscle cells (AiDIVs), and red-coloured podocytes (ReCPos) by acidic dye. A genetic analysis using peripheral mononuclear blood cells and urine sediment cells detected the m.13513 G > A variant in the MT-ND5 gene. Therefore, this patient was diagnosed with FSGS due to an MT-ND5 gene mutation. Although this is not the first case report to show that an MT-ND5 gene mutation causes FSGS, this is the first to demonstrate podocyte injuries accompanied with accumulation of abnormal mitochondria in the cytoplasm.
    Keywords:  ATP, adenosine triphosphate; AiDIVs, age-inappropriately disarranged and irregularly sized vascular smooth muscle cells; COX IV, cytochrome c oxidase subunit 4; Case report; Cr, creatinine; FSGS, focal segmental glomerulosclerosis; Focal segmental glomerulosclerosis; GSECs, granular swollen epithelial cells; MELAS, mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes; MRC, mitochondrial respiratory chain; MT-ND5, mitochondrially encoded ND5; Mitochondrial nephropathy; NADH dehydrogenase 5; ND5, NADH dehydrogenase 5; OXPHOS:, oxidative phosphorylation; Podocyte; ReCPos, red-coloured podocytes; eGFR, estimated glomerular filtration rate; mtDNA, mitochondrial DNA; nDNA, nuclear DNA; sCr, serum creatinine
  37. Nat Commun. 2023 Mar 22. 14(1): 1595
      The regulation of the informational flow from the mitochondria to the nucleus (mitonuclear communication) is not fully characterized in the heart. We have determined that mitochondrial ribosomal protein S5 (MRPS5/uS5m) can regulate cardiac function and key pathways to coordinate this process during cardiac stress. We demonstrate that loss of Mrps5 in the developing heart leads to cardiac defects and embryonic lethality while postnatal loss induces cardiac hypertrophy and heart failure. The structure and function of mitochondria is disrupted in Mrps5 mutant cardiomyocytes, impairing mitochondrial protein translation and OXPHOS. We identify Klf15 as a Mrps5 downstream target and demonstrate that exogenous Klf15 is able to rescue the overt defects and re-balance the cardiac metabolome. We further show that Mrps5 represses Klf15 expression through c-myc, together with the metabolite L-phenylalanine. This critical role for Mrps5 in cardiac metabolism and mitonuclear communication highlights its potential as a target for heart failure therapies.
  38. Exp Mol Med. 2023 Mar 24.
      In addition to constituting the genetic material of an organism, DNA is a tracer for the recognition of foreign pathogens and a trigger of the innate immune system. cGAS functions as a sensor of double-stranded DNA fragments and initiates an immune response via the adaptor protein STING. The cGAS-STING pathway not only defends cells against various DNA-containing pathogens but also modulates many pathological processes caused by the immune response to the ectopic localization of self-DNA, such as cytosolic mitochondrial DNA (mtDNA) and extranuclear chromatin. In addition, macrophages can cause inflammation by forming a class of protein complexes called inflammasomes, and the activation of the NLRP3 inflammasome requires the release of oxidized mtDNA. In innate immunity related to inflammasomes, mtDNA release is mediated by macropores that are formed on the outer membrane of mitochondria via VDAC oligomerization. These macropores are specifically formed in response to mitochondrial stress and tissue damage, and the inhibition of VDAC oligomerization mitigates this inflammatory response. The rapidly expanding area of research on the mechanisms by which mtDNA is released and triggers inflammation has revealed new treatment strategies not only for inflammation but also, surprisingly, for neurodegenerative diseases such as amyotrophic lateral sclerosis.
  39. Angiogenesis. 2023 Mar 21.
      Hyperglycemia in early postnatal life of preterm infants with incompletely vascularized retinas is associated with increased risk of potentially blinding neovascular retinopathy of prematurity (ROP). Neovascular ROP (Phase II ROP) is a compensatory but ultimately pathological response to the suppression of physiological postnatal retinal vascular development (Phase I ROP). Hyperglycemia in neonatal mice which suppresses physiological retinal vascular growth is associated with decreased expression of systemic and retinal fibroblast growth factor 21 (FGF21). FGF21 administration promoted and FGF21 deficiency suppressed the physiological retinal vessel growth. FGF21 increased serum adiponectin (APN) levels and loss of APN abolished FGF21 promotion of physiological retinal vascular development. Blocking mitochondrial fatty acid oxidation also abolished FGF21 protection against delayed physiological retinal vessel growth. Clinically, preterm infants developing severe neovascular ROP (versus non-severe ROP) had a lower total lipid intake with more parenteral and less enteral during the first 4 weeks of life. Our data suggest that increasing FGF21 levels in the presence of adequate enteral lipids may help prevent Phase I retinopathy (and therefore prevent neovascular disease).
    Keywords:  Adiponectin; FGF21; Fatty acid oxidation; Hyperglycemia; Retinal vessel; Retinopathy of prematurity
  40. Brain. 2023 Mar 24. pii: awad099. [Epub ahead of print]
      COQ8A-Ataxia is a rare form of neurodegenerative disorder due to mutations in the COQ8A gene. The encoded mitochondrial protein is involved in the regulation of Coenzyme Q10 biosynthesis. Previous studies on the constitutive Coq8a-/-mice indicated specific alterations of cerebellar Purkinje neurons involving altered electrophysiological function and dark cell degeneration. In the present manuscript, we extend our understanding of the contribution of Purkinje neuron dysfunction to the pathology. By generating a Purkinje specific conditional COQ8A knockout, we demonstrate that loss of COQ8A in Purkinje neurons is the main cause of cerebellar ataxia. Furthermore, through in vivo and in vitro approaches, we show that COQ8A-depleted Purkinje neurons have abnormal dendritic arborizations, altered mitochondria function and intracellular calcium dysregulation. Furthermore, we demonstrate that oxidative phosphorylation, in particular Complex IV, is primarily altered at pre-symptomatic stages of the disease. Finally, the morphology of primary Purkinje neurons as well as the mitochondrial dysfunction and calcium dysregulation could be rescued by CoQ10 treatment, suggesting that CoQ10 could be a beneficial treatment for COQ8A-Ataxia.
    Keywords:  Purkinje neurons; ataxia; calcium; coenzyme Q10; mitochondria
  41. Neurochem Res. 2023 Mar 21.
      Parkinson's disease (PD) is a common age-related neurodegenerative disorder whose pathogenesis is not completely understood. Mitochondrial dysfunction and increased oxidative stress have been considered as major causes and central events responsible for the progressive degeneration of dopaminergic (DA) neurons in PD. Therefore, investigating mitochondrial disorders plays a role in understanding the pathogenesis of PD and can be an important therapeutic target for this disease. This study discusses the effect of environmental, genetic and biological factors on mitochondrial dysfunction and also focuses on the mitochondrial molecular mechanisms underlying neurodegeneration, and its possible therapeutic targets in PD, including reactive oxygen species generation, calcium overload, inflammasome activation, apoptosis, mitophagy, mitochondrial biogenesis, and mitochondrial dynamics. Other potential therapeutic strategies such as mitochondrial transfer/transplantation, targeting microRNAs, using stem cells, photobiomodulation, diet, and exercise were also discussed in this review, which may provide valuable insights into clinical aspects. A better understanding of the roles of mitochondria in the pathophysiology of PD may provide a rationale for designing novel therapeutic interventions in our fight against PD.
    Keywords:  Mitochondrial biogenesis; Mitochondrial dynamics; Mitochondrial dysfunction; Mitochondrial transplantation; Parkinson’s disease
  42. Nat Metab. 2023 Mar 23.
      Astrocytes provide key neuronal support, and their phenotypic transformation is implicated in neurodegenerative diseases. Metabolically, astrocytes possess low mitochondrial oxidative phosphorylation (OxPhos) activity, but its pathophysiological role in neurodegeneration remains unclear. Here, we show that the brain critically depends on astrocytic OxPhos to degrade fatty acids (FAs) and maintain lipid homeostasis. Aberrant astrocytic OxPhos induces lipid droplet (LD) accumulation followed by neurodegeneration that recapitulates key features of Alzheimer's disease (AD), including synaptic loss, neuroinflammation, demyelination and cognitive impairment. Mechanistically, when FA load overwhelms astrocytic OxPhos capacity, elevated acetyl-CoA levels induce astrocyte reactivity by enhancing STAT3 acetylation and activation. Intercellularly, lipid-laden reactive astrocytes stimulate neuronal FA oxidation and oxidative stress, activate microglia through IL-3 signalling, and inhibit the biosynthesis of FAs and phospholipids required for myelin replenishment. Along with LD accumulation and impaired FA degradation manifested in an AD mouse model, we reveal a lipid-centric, AD-resembling mechanism by which astrocytic mitochondrial dysfunction progressively induces neuroinflammation and neurodegeneration.
  43. Cell Rep Methods. 2023 Feb 27. 3(2): 100394
      Intracellular long-chain acyl-coenzyme As (LC-acyl-CoAs) are thought to be under tight spatial and temporal controls, yet the ability to image LC-acyl-CoAs in live cells is lacking. Here, we developed a fluorescence resonance energy transfer (FRET) sensor for LC-acyl-CoAs based on the allosterically regulated interaction between α/β hydrolase domain-containing 5 (ABHD5) and Perilipin 5. The genetically encoded sensor rapidly detects intracellular LC-acyl-CoAs generated from exogenous and endogenous fatty acids (FAs), as well as synthetic ABHD5 ligands. Stimulation of lipolysis in brown adipocytes elevated intracellular LC-acyl-CoAs in a cyclic fashion, which was eliminated by inhibiting PNPLA2 (ATGL), the major triglyceride lipase. Interestingly, inhibition of LC-acyl-CoA transport into mitochondria elevated intracellular LC-acyl-CoAs and dampened their cycling. Together, these observations reveal an intimate feedback control between LC-acyl-CoA generation from lipolysis and utilization in mitochondria. We anticipate that this sensor will be an important tool to dissect intracellular LC-acyl-CoA dynamics as well to discover novel synthetic ABHD5 ligands.
    Keywords:  CGI-58; Perilipin; adipose triglyceride lipase; fatty acids; genetically encoded FRET sensor; lipid droplet; long-chain acyl-CoAs; mitochondria; real-time monitoring; α/β hydrolase domain-containing 5
  44. Hear Res. 2023 Mar 13. pii: S0378-5955(23)00052-7. [Epub ahead of print]431 108740
      To process sensory stimuli, intense energy demands are placed on hair cells and primary afferents. Hair cells must both mechanotransduce and maintain pools of synaptic vesicles for neurotransmission. Furthermore, both hair cells and afferent neurons must continually maintain a polarized membrane to propagate sensory information. These processes are energy demanding and therefore both cell types are critically reliant on mitochondrial health and function for their activity and maintenance. Based on these demands, it is not surprising that deficits in mitochondrial health can negatively impact the auditory and vestibular systems. In this review, we reflect on how mitochondrial function and dysfunction are implicated in hair cell-mediated sensory system biology. Specifically, we focus on live imaging approaches that have been applied to study mitochondria using the zebrafish lateral-line system. We highlight the fluorescent dyes and genetically encoded biosensors that have been used to study mitochondria in lateral-line hair cells and afferent neurons. We then describe the impact this in vivo work has had on the field of mitochondrial biology as well as the relationship between mitochondria and sensory system development, function, and survival. Finally, we delineate the areas in need of further exploration. This includes in vivo analyses of mitochondrial dynamics and biogenesis, which will round out our understanding of mitochondrial biology in this sensitive sensory system.
    Keywords:  Afferent neurons; Hair cells; Hearing and balance; In vivo imaging; Lateral line; Mitochondria; Zebrafish
  45. Genet Med. 2023 Mar 16. pii: S1098-3600(23)00843-2. [Epub ahead of print] 100830
      PURPOSE: The analysis of exome and genome sequencing data for the diagnosis of rare diseases is challenging and time-consuming. In this study, we evaluated a machine learning model for automating variant prioritization for diagnosing rare genetic diseases in the Baylor Genetics clinical laboratory.METHODS: The automated analysis model was developed using a supervised learning approach based on thousands of manually curated variants. The model was evaluated on two cohorts. The model accuracy was determined using a retrospective cohort comprised of 180 randomly selected exome cases (57 singletons, 123 trios), all of which were previously diagnosed and solved by manual interpretation. Diagnostic yield with the modified workflow was estimated using a prospective "production" cohort of 334 consecutive clinical cases.
    RESULTS: The model accurately pinpointed all manually reported variants as candidates. The reported variants were ranked in top-ten candidate variants in 98.4% (121/123) of trio cases, in 93.0% (53/57) of single proband cases, and 96.7% (174/180) of all cases. The accuracy of the model was reduced in some cases due to incomplete variant calling (e.g., copy number variants) or incomplete phenotypic description.
    CONCLUSION: The automated model for case analysis assists clinical genetic laboratories in prioritizing candidate variants effectively. The use of such technology may facilitate the interpretation of genomic data for a large number of patients in the era of precision medicine.
    Keywords:  clinical genomics
  46. Life Sci Alliance. 2023 Jun;pii: e202201419. [Epub ahead of print]6(6):
      Autosomal recessive mutations in the Parkin gene cause Parkinson's disease. Parkin encodes an ubiquitin E3 ligase that functions together with the kinase PINK1 in a mitochondrial quality control pathway. Parkin exists in an inactive conformation mediated by autoinhibitory domain interfaces. Thus, Parkin has become a target for the development of therapeutics that activate its ligase activity. Yet, the extent to which different regions of Parkin can be targeted for activation remained unknown. Here, we have used a rational structure-based approach to design new activating mutations in both human and rat Parkin across interdomain interfaces. Out of 31 mutations tested, we identified 11 activating mutations that all cluster near the RING0:RING2 or REP:RING1 interfaces. The activity of these mutants correlates with reduced thermal stability. Furthermore, three mutations V393D, A401D, and W403A rescue a Parkin S65A mutant, defective in mitophagy, in cell-based studies. Overall our data extend previous analysis of Parkin activation mutants and suggests that small molecules that would mimic RING0:RING2 or REP:RING1 destabilisation offer therapeutic potential for Parkinson's disease patients harbouring select Parkin mutations.
  47. Neurochem Res. 2023 Mar 25.
      Mitochondrial dysfunction and oxidative stress are critical to neurodegeneration in Parkinson's disease (PD). Mitochondrial dysfunction in PD entails inhibition of the mitochondrial complex I (CI) in the dopaminergic neurons of substantia nigra. The events contributing to CI inhibition and downstream pathways are not completely elucidated. We conducted proteomic analysis in a dopaminergic neuronal cell line exposed individually to neurotoxic CI inhibitors: rotenone (Rot), paraquat (Pq) and 1-methyl-4-phenylpyridinium (MPP+). Mass spectrometry (MS) revealed the involvement of biological processes including cell death pathways, structural changes and metabolic processes among others, most of which were common across all models. The proteomic changes induced by Pq were significantly higher than those induced by Rot and MPP+. Altered metabolic processes included downregulated mitochondrial proteins such as CI subunits. MS of CI isolated from the models revealed oxidative post-translational modifications with Tryptophan (Trp) oxidation as the predominant modification. Further, 62 peptides in 22 subunits of CI revealed Trp oxidation with 16 subunits common across toxins. NDUFV1 subunit had the greatest number of oxidized Trp and Rot model displayed the highest number of Trp oxidation events compared to the other models. Molecular dynamics simulation (MDS) of NDUFV1 revealed that oxidized Trp 433 altered the local conformation thereby changing the distance between the Fe-S clusters, Fe-S 301(N1a) to Fe-S 502 (N3) and Fe-S 802 (N4) to Fe-S 801 (N5), potentially affecting the efficiency of electron transfer. The events triggered by the neurotoxins represent CI damage, mitochondrial dysfunction and neurodegeneration in PD.
    Keywords:  MPP+ (1-methyl-4-phenylpyridinium); Mass spectrometry; Molecular dynamics; Paraquat; Post-translational Modifications; Rotenone; Tryptophan
  48. Nat Methods. 2023 Mar 23.
      Structural variants (SVs) are a major driver of genetic diversity and disease in the human genome and their discovery is imperative to advances in precision medicine. Existing SV callers rely on hand-engineered features and heuristics to model SVs, which cannot scale to the vast diversity of SVs nor fully harness the information available in sequencing datasets. Here we propose an extensible deep-learning framework, Cue, to call and genotype SVs that can learn complex SV abstractions directly from the data. At a high level, Cue converts alignments to images that encode SV-informative signals and uses a stacked hourglass convolutional neural network to predict the type, genotype and genomic locus of the SVs captured in each image. We show that Cue outperforms the state of the art in the detection of several classes of SVs on synthetic and real short-read data and that it can be easily extended to other sequencing platforms, while achieving competitive performance.
  49. Clin Exp Reprod Med. 2023 Mar;50(1): 1-11
      In reproduction, mitochondria produce bioenergy, help to synthesize biomolecules, and support the ovaries, oogenesis, and preimplantation embryos, thereby facilitating healthy live births. However, the regulatory mechanism of mitochondria in oocytes and embryos during oogenesis and embryo development has not been clearly elucidated. The functional activity of mitochondria is crucial for determining the quality of oocytes and embryos; therefore, the underlying mechanism must be better understood. In this review, we summarize the specific role of mitochondria in reproduction in oocytes and embryos. We also briefly discuss the recovery of mitochondrial function in gametes and zygotes. First, we introduce the general characteristics of mitochondria in cells, including their roles in adenosine triphosphate and reactive oxygen species production, calcium homeostasis, and programmed cell death. Second, we present the unique characteristics of mitochondria in female reproduction, covering the bottleneck theory, mitochondrial shape, and mitochondrial metabolic pathways during oogenesis and preimplantation embryo development. Mitochondrial dysfunction is associated with ovarian aging, a diminished ovarian reserve, a poor ovarian response, and several reproduction problems in gametes and zygotes, such as aneuploidy and genetic disorders. Finally, we briefly describe which factors are involved in mitochondrial dysfunction and how mitochondrial function can be recovered in reproduction. We hope to provide a new viewpoint regarding factors that can overcome mitochondrial dysfunction in the field of reproductive medicine.
    Keywords:  Dysfunction; Mechanism; Mitochondria; Recovery; Reproduction
  50. Nat Genet. 2023 Mar 23.
      Telomere length in humans is associated with lifespan and severe diseases, yet the genetic determinants of telomere length remain incompletely defined. Here we performed genome-wide CRISPR-Cas9 functional telomere length screening and identified thymidine (dT) nucleotide metabolism as a limiting factor in human telomere maintenance. Targeted genetic disruption using CRISPR-Cas9 revealed multiple telomere length control points across the thymidine nucleotide metabolism pathway: decreasing dT nucleotide salvage via deletion of the gene encoding nuclear thymidine kinase (TK1) or de novo production by knockout of the thymidylate synthase gene (TYMS) decreased telomere length, whereas inactivation of the deoxynucleoside triphosphohydrolase-encoding gene SAMHD1 lengthened telomeres. Remarkably, supplementation with dT alone drove robust telomere elongation by telomerase in cells, and thymidine triphosphate stimulated telomerase activity in a substrate-independent manner in vitro. In induced pluripotent stem cells derived from patients with genetic telomere biology disorders, dT supplementation or inhibition of SAMHD1 promoted telomere restoration. Our results demonstrate a critical role of thymidine metabolism in controlling human telomerase and telomere length, which may be therapeutically actionable in patients with fatal degenerative diseases.
  51. Cell Calcium. 2023 Mar 21. pii: S0143-4160(23)00031-3. [Epub ahead of print]111 102719
      The mitochondrial Permeability Transition Pore (PTP) can be defined as a Ca2+ activated mega-channel involved in mitochondrial damage and cell death, making its inhibition a hallmark for therapeutic purposes in many PTP-related paradigms. Although long-lasting PTP openings have been widely studied, the physiological implications of transient openings (also called "flickering" behavior) are still poorly understood. The flickering activity was suggested to play a role in the regulation of Ca2+ and ROS homeostasis, and yet this hypothesis did not reach general consensus. This state of affairs might arise from the lack of unquestionable experimental evidence, due to limitations of the available techniques for capturing transient PTP activity and to a still partial understanding of its molecular identity. In this review we will focus on possible implications of the PTP in physiology, in particular its role as a Ca2+ release pathway, discussing the consequences of its forced inhibition. We will also consider the recent hypothesis of the existence of more permeability pathways and their potential involvement in mitochondrial physiology.
    Keywords:  Calcium; Channels; Cyclophilin D; Mitochondria; Permeability transition; cyclosporin A