bims-mitdis Biomed News
on Mitochondrial disorders
Issue of 2023‒02‒05
sixty-six papers selected by
Catalina Vasilescu
Helmholz Munich


  1. Endocr Rev. 2023 Feb 02. pii: bnad004. [Epub ahead of print]
      Mitochondria both sense biochemical and energetic input in addition to communicating signals regarding the energetic state of the cell. Increasingly, these signaling organelles are key for regulating different cell functions. This review summarizes recent advances in mitochondrial communication in striated muscle, with specific focus on the processes by which mitochondria communicate with each other, other organelles and across distant organ systems. Inter-mitochondrial communication in striated muscle is mediated via conduction of the mitochondrial membrane potential to adjacent mitochondria, physical interactions, mitochondrial fusion or fission and via nannotunnels, allowing for the exchange of proteins, mitochondrial DNA, nucleotides, and peptides. Within striated muscle cells, mitochondria-organelle communication can modulate overall cell function. The various mechanisms in which mitochondria communicate mitochondrial fitness to the rest of the body suggest that extracellular mitochondrial signaling is key during health and disease. Whereas mitochondrial-derived vesicles might excrete mitochondrial-derived endocrine compounds, stimulation of mitochondrial stress can lead to the release of fibroblast growth factor 21 (FGF21) and growth differentiation factor 15 (GDF15) into the circulation to modulate whole-body physiology. Circulating mitochondrial DNA are well-known alarmins that trigger the immune system and may help to explain low-grade inflammation in various chronic diseases. Impaired mitochondrial function and communication are central in common heart and skeletal muscle pathologies, including cardiomyopathies, insulin resistance, and sarcopenia. Lastly, important new advances in research in mitochondrial endocrinology, communication, medical horizons and translational aspects are discussed.
    Keywords:  FGF21; GDF15; mitochondria-organelle interactions; mitochondrial cristae; mitochondrial dynamics; myokines; respiratory supercomplexes
    DOI:  https://doi.org/10.1210/endrev/bnad004
  2. Bioessays. 2023 Jan 29. e2200160
      Mitochondria hold diverse and pivotal roles in fundamental processes that govern cell survival, differentiation, and death, in addition to organismal growth, maintenance, and aging. The mitochondrial protein import system is a major contributor to mitochondrial biogenesis and lies at the crossroads between mitochondrial and cellular homeostasis. Recent findings highlight the mitochondrial protein import system as a signaling hub, receiving inputs from other cellular compartments and adjusting its function accordingly. Impairment of protein import, in a physiological, or disease context, elicits adaptive responses inside and outside mitochondria. In this review, we discuss recent developments, relevant to the mechanisms of mitochondrial protein import regulation, with a particular focus on quality control, proteostatic and metabolic cellular responses, triggered upon impairment of mitochondrial protein import.
    Keywords:  metabolism; mitochondrial protein import; mitochondrial unfolded protein response; mitophagy; proteostasis
    DOI:  https://doi.org/10.1002/bies.202200160
  3. Autophagy. 2023 Feb 01. 1-3
      Age-related human pathologies present with a multitude of molecular and metabolic phenotypes, which individually or synergistically contribute to tissue degeneration. However, current lack of understanding of the interdependence of these molecular pathologies limits the potential range of existing therapeutic intervention strategies. In our study, we set out to understand the chain of molecular events, which underlie the loss of cellular viability in macroautophagy/autophagy deficiency associated with aging and age-related disease. We discover a novel axis linking autophagy, a cellular waste disposal pathway, and a metabolite, nicotinamide adenine dinucleotide (NAD). The axis connects multiple organelles, molecules and stress response pathways mediating cellular demise when autophagy becomes dysfunctional. By elucidating the steps on the path from efficient mitochondrial recycling to NAD maintenance and ultimately cell viability, we highlight targets potentially receptive to therapeutic interventions in a range of genetic and age-related diseases associated with autophagy dysfunction.Abbreviations: IMM: inner mitochondrial membrane; NAD: nicotinamide dinucleotide; OXPHOS: oxidative phosphorylation; PARP: poly(ADP-ribose) polymerase; ROS: reactive oxygen species.
    Keywords:  Aging; DNA damage; NAD; PARP; ROS; autophagy; mitochondria; mitophagy; sirtuins
    DOI:  https://doi.org/10.1080/15548627.2023.2165753
  4. PNAS Nexus. 2022 Sep;1(4): pgac192
      Mitochondria are cellular organelles of crucial relevance for the survival of metazoan organisms. Damage to the mitochondrial DNA can give rise to a variety of mitochondrial diseases and is thought also to be involved in the aging process. The fate of mtDNA mutants is controlled by their synthesis as well as degradation and mathematical models can help to better understand this complex interplay. We present here a model that combines a replicative advantage for mtDNA mutants with selective degradation enabled by mitochondrial fission and fusion processes. The model not only shows that the cell has efficient means to deal with (many) types of mutants but, surprisingly, also predicts that under certain conditions a stable co-existence of mutant and wild-type mtDNAs is possible. We discuss how this new finding might explain how mitochondria can be at the heart of processes with such different phenotypes as mitochondrial diseases and aging.
    Keywords:  aging; mathematical model; mitochondrial disease
    DOI:  https://doi.org/10.1093/pnasnexus/pgac192
  5. Gene. 2023 Jan 27. pii: S0378-1119(23)00070-7. [Epub ahead of print] 147229
      BACKGROUND: The variant m.3571_3572insC/MT-ND1 thus far only reported in oncocytic tumors of different tissues. However, the role of m.3571_3572insC in inherited mitochondrial diseases has yet to be elucidated.METHODS: A patient diagnosed with MELAS syndrome was recruited, and detailed medical records were collected and reviewed. The muscle was biopsied for mitochondrial respiratory chain enzyme activity. Series of fibroblast clones bearing different m.3571_3572insC variant loads were generated from patient-derived fibroblasts and subjected to functional assays.
    RESULTS: Complex I deficiency was confirmed in the patient's muscle via mitochondrial respiratory chain enzyme activity assay. The m.3571_3572insC was filtered for the candidate variant of the patient according to the guidelines for mitochondrial mRNA variants interpretation. Three cell clones with different m.3571_3572insC variant loads were generated to evaluate mitochondrial function. Blue native PAGE analysis revealed that m.3571_3572insC caused a deficiency in the mitochondrial complex I. Oxygen consumption rate, ATP production, and lactate assays found an impairment of cellular bioenergetic capacity due to m.3571_3572insC. Mitochondrial membrane potential was decreased, and mitochondrial reactive oxygen species production was increased with the variant of m.3571_3572insC. According to the competitive cell growth assay, the mutant cells had impaired cell growth capacity compared to wild type.
    CONCLUSIONS: A novel variant m.3571_3572insC was identified in a patient diagnosed with MELAS syndrome, and the variant impaired mitochondrial respiration by decreasing the activity of complex I. In conclusion, the genetic spectrum of mitochondrial diseases was expanded by including m.3571_3572insC/MT-ND1.
    Keywords:  Heteroplasmic; MELAS syndrome; MT-ND1; de novo; m.3571_3572insC
    DOI:  https://doi.org/10.1016/j.gene.2023.147229
  6. bioRxiv. 2023 Jan 11. pii: 2023.01.11.523512. [Epub ahead of print]
      Neuronal activity is an energy-intensive process that is largely sustained by instantaneous fuel utilization and ATP synthesis. However, how neurons couple ATP synthesis rate to fuel availability is largely unknown. Here, we demonstrate that the metabolic sensor enzyme O-GlcNAc transferase regulates neuronal activity-driven mitochondrial bioenergetics. We show that neuronal activity upregulates O-GlcNAcylation mainly in mitochondria. Mitochondrial O-GlcNAcylation is promoted by activity-driven fuel consumption, which allows neurons to compensate for high energy expenditure based on fuel availability. To determine the proteins that are responsible for these adjustments, we mapped the mitochondrial O-GlcNAcome of neurons. Finally, we determine that neurons fail to meet activity-driven metabolic demand when O-GlcNAcylation dynamics are prevented. Our findings suggest that O-GlcNAcylation provides a fuel-dependent feedforward control mechanism in neurons to optimize mitochondrial performance based on neuronal activity. This mechanism thereby couples neuronal metabolism to mitochondrial bioenergetics and plays a key role in sustaining energy homeostasis.
    DOI:  https://doi.org/10.1101/2023.01.11.523512
  7. J Adv Res. 2023 Jan 31. pii: S2090-1232(23)00034-6. [Epub ahead of print]
      INTRODUCTION: Mitochondria are maternally inherited cell organelles with their own genome, and perform various functions in eukaryotic cells such as energy production and cellular homeostasis. Due to their inheritance and manifold biological roles in health and disease, mitochondrial genetics serves a dual purpose of tracing the history as well as disease susceptibility of human populations across the globe. This work requires a comprehensive catalogue of commonly observed genetic variations in the mitochondrial DNAs for all regions throughout the world. So far, however, certain regions, such as North and East Africa have been understudied.OBJECTIVES: To address this shortcoming, we have created the most comprehensive quality-controlled North and East African mitochondrial data set to date and use it for characterizing mitochondrial genetic variation in this region.
    METHODS: We compiled 11 published cohorts with novel data for mitochondrial genomes from 159 Sudanese individuals. We combined these 641 mitochondrial sequences with sequences from the 1000 Genomes (n=2,504) and the Human Genome Diversity Project (n=828) and used the tool haplocheck for extensive quality control and detection of in-sample contamination, as well as Nanopore long read sequencing for haplogroup validation of 18 samples.
    RESULTS: Using a subset of high-coverage mitochondrial sequences, we predict 15 potentially novel haplogroups in North and East African subjects and observe likely phylogenetic deviations from the established PhyloTree reference for haplogroups L0a1 and L2a1.
    CONCLUSION: Our findings demonstrate common hitherto unexplored variants in mitochondrial genomes of North and East Africa that lead to novel phylogenetic relationships between haplogroups present in these regions. These observations call for further in-depth population genetic studies in that region to enable the prospective use of mitochondrial genetic variation for precision medicine.
    Keywords:  energetics; haplogroups; long-read sequencing; mtDNA; next-generation sequencing; phylogenetics
    DOI:  https://doi.org/10.1016/j.jare.2023.01.021
  8. bioRxiv. 2023 Jan 04. pii: 2023.01.04.522744. [Epub ahead of print]
      Circulating cell-free mitochondrial DNA (cf-mtDNA) is an emerging biomarker of psychobiological stress and disease which predicts mortality and is associated with various disease states. To evaluate the contribution of cf-mtDNA to health and disease states, standardized high-throughput procedures are needed to quantify cf-mtDNA in relevant biofluids. Here, we describe MitoQuicLy: Mito chondrial DNA Qu antification in c ell-free samples by Ly sis. We demonstrate high agreement between MitoQuicLy and the commonly used column-based method, although MitoQuicLy is faster, cheaper, and requires a smaller input sample volume. Using 10 µL of input volume with MitoQuicLy, we quantify cf-mtDNA levels from three commonly used plasma tube types, two serum tube types, and saliva. We detect, as expected, significant inter-individual differences in cf-mtDNA across different biofluids. However, cf-mtDNA levels between concurrently collected plasma, serum, and saliva from the same individual differ on average by up to two orders of magnitude and are poorly correlated with one another, pointing to different cf-mtDNA biology or regulation between commonly used biofluids in clinical and research settings. Moreover, in a small sample of healthy women and men (n=34), we show that blood and saliva cf-mtDNAs correlate with clinical biomarkers differently depending on the sample used. The biological divergences revealed between biofluids, together with the lysis-based, cost-effective, and scalable MitoQuicLy protocol for biofluid cf-mtDNA quantification, provide a foundation to examine the biological origin and significance of cf-mtDNA to human health.
    DOI:  https://doi.org/10.1101/2023.01.04.522744
  9. Front Neurol. 2022 ;13 1063733
      Objectives: New-onset refractory status epilepticus (NORSE) is associated with high morbidity and mortality. Despite extensive work-up, the underlying etiology remains unknown in 50% of affected individuals. Mitochondrial disorders represent rare causes of NORSE. Biallelic variants in FASTKD2 were reported as a cause of infantile encephalomyopathy with refractory epilepsy.Case description: In the study, we report a previously healthy 14-year-old with a new, homozygous FASTKD2 variant presenting with NORSE. Following a seizure-free period of 7 years, he experienced another super-refractory SE and subsequently developed drug-resistant focal epilepsy, mild myopathy, optic atrophy, and discrete psychomotor slowing. Structural MRI at the time of NORSE showed right temporo-parieto-occipital FLAIR hyperintensity and diffusion restriction, with extensive right hemispheric atrophy at the age of 22 years. Whole-exome sequencing revealed a novel homozygous loss of function variant [c.(1072C>T);(1072C>T)] [p.(Arg358Ter);(Arg358Ter)] in FASTKD2 (NM_001136193), resulting in a premature termination codon in the protein-coding region and loss of function of FASTKD2. Oxidative phosphorylation (OXPHOS) in muscle and skin fibroblasts was unremarkable.
    Conclusion: This is the first case of a normally developed adolescent with a new homozygous loss of function variant in FASTKD2, manifesting with NORSE. The phenotypical spectrum of FASTKD2-related mitochondrial disease is heterogeneous, ranging from recurrent status epilepticus and refractory focal epilepsy in an adolescent with normal cognitive development to severe forms of infantile mitochondrial encephalopathy. Although mitochondrial diseases are rare causes of NORSE, clinical features such as young age at onset and multi-system involvement should trigger genetic testing. Early diagnosis is essential for counseling and treatment considerations.
    Keywords:  FASTKD2 mutation; drug-resistant epilepsy; genetic epilepsies; mitochondrial disease; new-onset refractory status epilepticus (NORSE)
    DOI:  https://doi.org/10.3389/fneur.2022.1063733
  10. Blood. 2023 Feb 03. pii: blood.2022017584. [Epub ahead of print]
      Metabolic programs contribute to hematopoietic stem and progenitor cell (HSPC) fate, but it is not known whether the metabolic regulation of protein synthesis controls HSPC differentiation. Here, we show that SLC7A1/CAT1-dependent arginine uptake and its catabolism to the polyamine spermidine control human erythroid specification of HSPCs via activation of the eukaryotic translation initiation factor 5A (eIF5A). eIF5A activity is dependent on its hypusination, a post-translational modification resulting from the conjugation of the aminobutyl moiety of spermidine to lysine. Notably, attenuation of hypusine synthesis in erythroid progenitors--by inhibition of deoxyhypusine synthase--abrogates erythropoiesis but not myeloid cell differentiation. Proteomic profiling reveals mitochondrial translation to be a critical target of hypusinated eIF5A and accordingly, progenitors with decreased hypusine activity exhibit diminished oxidative phosphorylation. This impacted pathway is critical for eIF5A-regulated erythropoiesis as interventions augmenting mitochondrial function partially rescue human erythropoiesis under conditions of attenuated hypusination. Levels of mitochondrial ribosomal proteins were especially sensitive to the loss of hypusine and we find that the ineffective erythropoiesis linked to haploinsufficiency of RPS14 in del(5q) myelodysplastic syndrome is associated with a diminished pool of hypusinated eIF5A. Moreover, patients with RPL11-haploinsufficient Diamond-Blackfan anemia as well as CD34+ progenitors with downregulated RPL11 exhibit a markedly decreased hypusination in erythroid progenitors, concomitant with a loss of mitochondrial metabolism. Thus, eIF5A-dependent protein synthesis regulates human erythropoiesis and our data reveal a novel role for RPs in controlling eIF5A hypusination in HSPC, synchronizing mitochondrial metabolism with erythroid differentiation.
    DOI:  https://doi.org/10.1182/blood.2022017584
  11. Elife. 2023 Feb 01. pii: e82283. [Epub ahead of print]12
      Mitochondrial dysfunction has been reported in obesity and insulin resistance, but primary genetic mitochondrial dysfunction is generally not associated with these, arguing against a straightforward causal relationship. A rare exception, recently identified in humans, is a syndrome of lower body adipose loss, leptin-deficient severe upper body adipose overgrowth, and insulin resistance caused by the p.Arg707Trp mutation in MFN2, encoding mitofusin 2. How the resulting selective form of mitochondrial dysfunction leads to tissue- and adipose depot-specific growth abnormalities and systemic biochemical perturbation is unknown. To address this, Mfn2R707W/R707W knock-in mice were generated and phenotyped on chow and high fat diets. Electron microscopy revealed adipose-specific mitochondrial morphological abnormalities. Oxidative phosphorylation measured in isolated mitochondria was unperturbed, but the cellular integrated stress response was activated in adipose tissue. Fat mass and distribution, body weight, and systemic glucose and lipid metabolism were unchanged, however serum leptin and adiponectin concentrations, and their secretion from adipose explants were reduced. Pharmacological induction of the integrated stress response in wild-type adipocytes also reduced secretion of leptin and adiponectin, suggesting an explanation for the in vivo findings. These data suggest that the p.Arg707Trp MFN2 mutation selectively perturbs mitochondrial morphology and activates the integrated stress response in adipose tissue. In mice, this does not disrupt most adipocyte functions or systemic metabolism, whereas in humans it is associated with pathological adipose remodelling and metabolic disease. In both species, disproportionate effects on leptin secretion may relate to cell autonomous induction of the integrated stress response.
    Keywords:  cell biology; mouse
    DOI:  https://doi.org/10.7554/eLife.82283
  12. Eur Heart J. 2023 Feb 03. pii: ehad028. [Epub ahead of print]
      AIMS: Genetic hypertrophic cardiomyopathy (HCM) is caused by mutations in sarcomere protein-encoding genes (i.e. genotype-positive HCM). In an increasing number of patients, HCM occurs in the absence of a mutation (i.e. genotype-negative HCM). Mitochondrial dysfunction is thought to be a key driver of pathological remodelling in HCM. Reports of mitochondrial respiratory function and specific disease-modifying treatment options in patients with HCM are scarce.METHODS AND RESULTS: Respirometry was performed on septal myectomy tissue from patients with HCM (n = 59) to evaluate oxidative phosphorylation and fatty acid oxidation. Mitochondrial dysfunction was most notably reflected by impaired NADH-linked respiration. In genotype-negative patients, but not genotype-positive patients, NADH-linked respiration was markedly depressed in patients with an indexed septal thickness ≥10 compared with <10. Mitochondrial dysfunction was not explained by reduced abundance or fragmentation of mitochondria, as evaluated by transmission electron microscopy. Rather, improper organization of mitochondria relative to myofibrils (expressed as a percentage of disorganized mitochondria) was strongly associated with mitochondrial dysfunction. Pre-incubation with the cardiolipin-stabilizing drug elamipretide and raising mitochondrial NAD+ levels both boosted NADH-linked respiration.
    CONCLUSION: Mitochondrial dysfunction is explained by cardiomyocyte architecture disruption and is linked to septal hypertrophy in genotype-negative HCM. Despite severe myocardial remodelling mitochondria were responsive to treatments aimed at restoring respiratory function, eliciting the mitochondria as a drug target to prevent and ameliorate cardiac disease in HCM. Mitochondria-targeting therapy may particularly benefit genotype-negative patients with HCM, given the tight link between mitochondrial impairment and septal thickening in this subpopulation.
    Keywords:  Cardiomyocyte architecture; Hypertrophic cardiomyopathy; Metabolism; Mitochondrial dysfunction; Mitochondrial therapy
    DOI:  https://doi.org/10.1093/eurheartj/ehad028
  13. Retin Cases Brief Rep. 2022 Dec 22.
      PURPOSE: Kearns-Sayre syndrome (KSS) is a mitochondrial DNA (mtDNA) deletion syndrome that is characterized by the triad of onset commonly before age twenty, pigmentary retinopathy, and chronic progressive external ophthalmoplegia. Here we present a case of KSS masquerading as myasthenia gravis (MG).METHODS: Case report.
    RESULTS: A 15-year-old male with a presumed diagnosis of MG presented with blurry vision, ophthalmoplegia, and ptosis. He was found to have a mitochondrial pigmentary retinopathy and was eventually diagnosed with KSS after mtDNA sequencing revealed a novel large-scale deletion of 7.9kb of mtDNA from nucleotides 6578 to 14,460.
    CONCLUSIONS: We report a case of KSS found to have a novel large-scale mtDNA deletion. The presence of a mitochondrial pigmentary retinopathy found on dilated examination led to reconsideration of the previous diagnosis of MG and ultimately led to the correct diagnosis of KSS.
    DOI:  https://doi.org/10.1097/ICB.0000000000001397
  14. EMBO J. 2023 Feb 02. e112094
      DNA-PKcs is a key regulator of DNA double-strand break repair. Apart from its canonical role in the DNA damage response, DNA-PKcs is involved in the cellular response to oxidative stress (OS), but its exact role remains unclear. Here, we report that DNA-PKcs-deficient human cells display depolarized mitochondria membrane potential (MMP) and reoriented metabolism, supporting a role for DNA-PKcs in oxidative phosphorylation (OXPHOS). DNA-PKcs directly interacts with mitochondria proteins ANT2 and VDAC2, and formation of the DNA-PKcs/ANT2/VDAC2 (DAV) complex supports optimal exchange of ADP and ATP across mitochondrial membranes to energize the cell via OXPHOS and to maintain MMP. Moreover, we demonstrate that the DAV complex temporarily dissociates in response to oxidative stress to attenuate ADP-ATP exchange, a rate-limiting step for OXPHOS. Finally, we found that dissociation of the DAV complex is mediated by phosphorylation of DNA-PKcs at its Thr2609 cluster by ATM kinase. Based on these findings, we propose that the coordination between the DAV complex and ATM serves as a novel oxidative stress checkpoint to decrease ROS production from mitochondrial OXPHOS and to hasten cellular recovery from OS.
    Keywords:  ANT2; ATM; DNA-PKcs; VDAC2; mitochondrial oxidative stress checkpoint
    DOI:  https://doi.org/10.15252/embj.2022112094
  15. Cell Rep. 2023 Jan 31. pii: S2211-1247(23)00078-5. [Epub ahead of print]42(2): 112067
      The generation of neurons from quiescent radial-glia-like neural stem cells (RGLs) in adult brain goes hand in hand with the modulation of cellular metabolism. However, it is still unclear how the exact metabolic program governs the balance between quiescent and activated RGLs. Here, we find that loss of mitochondrial D-2-hydroxyglutarate dehydrogenase (D2HGDH) leads to aberrant accumulation of D-2-hydroxyglutarate (D-2-HG) and impaired RGL activation. Mechanistically, accumulated D-2-HG bonds directly to ATP-citrate lyase and competitively inhibits its enzymatic activity, thereby reducing acetyl-CoA production and diminishing histone acetylation. However, administration of acetate restores the acetyl-CoA levels via acetyl-CoA synthetase-mediated catabolism and rescues the deficiencies in histone acetylation and RGL activation caused by loss of D2HGDH. Therefore, our findings define the role of cross talk between mitochondria and the nucleus via a mitochondrial metabolite, D-2-HG, the aberrant accumulation of which hinders the regulation of histone acetylation in RGL activation and attenuates continuous neurogenesis in adult mammalian brain.
    Keywords:  ATP-citrate lyase; CP: Neuroscience; CP: Stem cell research; D-2-hydroxyglutarate; D-2-hydroxyglutarate dehydrogenase; adult neurogenesis; histone acetylation; neural stem cells
    DOI:  https://doi.org/10.1016/j.celrep.2023.112067
  16. Front Endocrinol (Lausanne). 2022 ;13 1059120
      Background: There is growing interest in the role of DNA methylation in regulating the transcription of mitochondrial genes, particularly in brain disorders characterized by mitochondrial dysfunction. Here, we present a novel approach to interrogate the mitochondrial DNA methylome at single base resolution using targeted bisulfite sequencing. We applied this method to investigate mitochondrial DNA methylation patterns in post-mortem superior temporal gyrus and cerebellum brain tissue from seven human donors.Results: We show that mitochondrial DNA methylation patterns are relatively low but conserved, with peaks in DNA methylation at several sites, such as within the D-LOOP and the genes MT-ND2, MT-ATP6, MT-ND4, MT-ND5 and MT-ND6, predominantly in a non-CpG context. The elevated DNA methylation we observe in the D-LOOP we validate using pyrosequencing. We identify loci that show differential DNA methylation patterns associated with age, sex and brain region. Finally, we replicate previously reported differentially methylated regions between brain regions from a methylated DNA immunoprecipitation sequencing study.
    Conclusions: We have annotated patterns of DNA methylation at single base resolution across the mitochondrial genome in human brain samples. Looking to the future this approach could be utilized to investigate the role of mitochondrial epigenetic mechanisms in disorders that display mitochondrial dysfunction.
    Keywords:  5-Methylcytosine (5mC); Brain; DNA Methylation; Mitochondria; epigenetics; mtDNA
    DOI:  https://doi.org/10.3389/fendo.2022.1059120
  17. PNAS Nexus. 2022 Nov;1(5): pgac276
      Respiratory complex I [NADH:ubiquinone (UQ) oxidoreductase] captures the free energy released from NADH oxidation and UQ reduction to pump four protons across an energy-transducing membrane and power ATP synthesis. Mechanisms for long-range energy coupling in complex I have been proposed from structural data but not yet evaluated by robust biophysical and biochemical analyses. Here, we use the powerful bacterial model system Paracoccus denitrificans to investigate 14 mutations of key residues in the membrane-domain Nqo13/ND4 subunit, defining the rates and reversibility of catalysis and the number of protons pumped per NADH oxidized. We reveal new insights into the roles of highly conserved charged residues in lateral energy transduction, confirm the purely structural role of the Nqo12/ND5 transverse helix, and evaluate a proposed hydrated channel for proton uptake. Importantly, even when catalysis is compromised the enzyme remains strictly coupled (four protons are pumped per NADH oxidized), providing no evidence for escape cycles that circumvent blocked proton-pumping steps.
    Keywords:  NADH:ubiquinone oxidoreductase; biological energy transduction; electron transport chain; proton pumping; respiratory chain
    DOI:  https://doi.org/10.1093/pnasnexus/pgac276
  18. bioRxiv. 2023 Jan 22. pii: 2023.01.22.525042. [Epub ahead of print]
      Proteinaceous cysteines function as essential sensors of cellular redox state. Consequently, defining the cysteine redoxome is a key challenge for functional proteomic studies. While proteome-wide inventories of cysteine oxidation state are readily achieved using established, widely adopted proteomic methods such as OxiCat, Biotin Switch, and SP3-Rox, they typically assay bulk proteomes and therefore fail to capture protein localization-dependent oxidative modifications. To obviate requirements for laborious biochemical fractionation, here, we develop and apply an unprecedented two step cysteine capture method to establish the Local Cysteine Capture (Cys-LoC), and Local Cysteine Oxidation (Cys-LOx) methods, which together yield compartment-specific cysteine capture and quantitation of cysteine oxidation state. Benchmarking of the Cys-LoC method across a panel of subcellular compartments revealed more than 3,500 cysteines not previously captured by whole cell proteomic analysis, together with unexpected non-organelle specific TurboID-catalyzed proximity labeling. This mislabeling was minimized through simultaneous depletion of both endogenous biotin and newly translated TurboID fusion protein. Application of the Cys-LOx method to LPS stimulated murine immortalized bone marrow-derived macrophages (iBMDM), revealed previously unidentified mitochondria-specific inflammation-induced cysteine oxidative modifications including those associated with oxidative phosphorylation. These findings shed light on post-translational mechanisms regulating mitochondrial function during the cellular innate immune response.
    DOI:  https://doi.org/10.1101/2023.01.22.525042
  19. medRxiv. 2023 Jan 19. pii: 2023.01.19.23284696. [Epub ahead of print]
      Human mitochondria contain a high copy number, maternally transmitted genome (mtDNA) that encodes 13 proteins required for oxidative phosphorylation. Heteroplasmy arises when multiple mtDNA variants co-exist in an individual and can exhibit complex dynamics in disease and in aging. As all proteins involved in mtDNA replication and maintenance are nuclear-encoded, heteroplasmy levels can, in principle, be under nuclear genetic control, however this has never been shown in humans. Here, we develop algorithms to quantify mtDNA copy number (mtCN) and heteroplasmy levels using blood-derived whole genome sequences from 274,832 individuals of diverse ancestry and perform GWAS to identify nuclear loci controlling these traits. After careful correction for blood cell composition, we observe that mtCN declines linearly with age and is associated with 92 independent nuclear genetic loci. We find that nearly every individual carries heteroplasmic variants that obey two key patterns: (1) heteroplasmic single nucleotide variants are somatic mutations that accumulate sharply after age 70, while (2) heteroplasmic indels are maternally transmitted as mtDNA mixtures with resulting levels influenced by 42 independent nuclear loci involved in mtDNA replication, maintenance, and novel pathways. These nuclear loci do not appear to act by mtDNA mutagenesis, but rather, likely act by conferring a replicative advantage to specific mtDNA molecules. As an illustrative example, the most common heteroplasmy we identify is a length variant carried by >50% of humans at position m.302 within a G-quadruplex known to serve as a replication switch. We find that this heteroplasmic variant exerts cis -acting genetic control over mtDNA abundance and is itself under trans -acting genetic control of nuclear loci encoding protein components of this regulatory switch. Our study showcases how nuclear haplotype can privilege the replication of specific mtDNA molecules to shape mtCN and heteroplasmy dynamics in the human population.
    DOI:  https://doi.org/10.1101/2023.01.19.23284696
  20. bioRxiv. 2023 Jan 22. pii: 2023.01.22.525071. [Epub ahead of print]
      Cytotrophoblasts fuse to form and renew syncytiotrophoblasts necessary to maintain placental health throughout gestation. During cytotrophoblast to syncytiotrophoblast differentiation, cells undergo regulated metabolic and transcriptional reprogramming. Mitochondria play a critical role in differentiation events in cellular systems, thus we hypothesized that mitochondrial metabolism played a central role in trophoblast differentiation. In this work, we employed static and stable isotope tracing untargeted metabolomics methods along with gene expression and histone acetylation studies in an established cell culture model of trophoblast differentiation. Trophoblast differentiation was associated with increased abundance of the TCA cycle intermediates citrate and α-ketoglutarate. Citrate was preferentially exported from mitochondria in the undifferentiated state but was retained to a larger extent within mitochondria upon differentiation. Correspondingly, differentiation was associated with decreased expression of the mitochondrial citrate transporter (CIC). CRISPR/Cas9 disruption of the mitochondrial citrate carrier showed that CIC is required for biochemical differentiation of trophoblasts. Loss of CIC resulted in broad alterations in gene expression and histone acetylation. These gene expression changes were partially rescued through acetate supplementation. Taken together, these results highlight a central role for mitochondrial citrate metabolism in orchestrating histone acetylation and gene expression during trophoblast differentiation.
    DOI:  https://doi.org/10.1101/2023.01.22.525071
  21. Front Cardiovasc Med. 2022 ;9 1095882
      Background: Mitochondrial intermediate peptidase, encoded by the MIPEP gene, is involved in the processing of precursor mitochondrial proteins related to oxidative phosphorylation. Only a few studies have shown that mutations in MIPEP can cause combined oxidative phosphorylation deficiency-31 (COXPD31), an autosomal recessive multisystem disorder associated with mitochondrial dysfunction. We report herein a rare case of an 8-month-old boy in China with hypertrophic cardiomyopathy (HCM), severe lactic acidosis, and hypotonia caused by novel MIPEP compound heterozygous variants.Methods: Trio-whole-exome sequencing and copy number variation sequencing were performed to identify mutated genetic loci. Sanger sequencing and quantitative real-time PCR were used to validate the candidate single nucleotide variants and copy number variants, respectively.
    Results: The proband was an 8-month-old boy with HCM, severe lactic acidosis, and hypotonia who died 2 months after his first admission. Two novel compound heterozygous variants, c.1081T > A (p. Tyr361Asn) and a whole deletion (Ex1-19 del), were found in the MIPEP gene, which were inherited from his healthy parents respectively. Additionally, his mitochondria DNA copy number was significantly reduced.
    Conclusion: We are the first to report a patient with rare MIPEP variants in China. Our findings expand the mutation spectrum of MIPEP, and provide insights into the genotype-phenotype relationship in COXPD31.
    Keywords:  MIPEP; combined oxidative phosphorylation deficiency-31; hypertrophic cardiomyopathy; mitochondrial disease; oxidative phosphorylation
    DOI:  https://doi.org/10.3389/fcvm.2022.1095882
  22. iScience. 2023 Feb 17. 26(2): 105925
      Aberrant proteostasis is thought to be implicated in Parkinson's disease (PD), but patient-derived evidence is scant. We hypothesized that impaired proteostasis is reflected as altered transcriptome-proteome correlation in the PD brain. We integrated transcriptomic and proteomic data from prefrontal cortex of PD patients and young and aged controls to assess RNA-protein correlations across samples. The aged brain showed a genome-wide decrease in mRNA-protein correlation. Genes encoding synaptic vesicle proteins showed negative correlations, likely reflecting spatial separation of mRNA and protein into soma and synapses. PD showed a broader transcriptome-proteome decoupling, consistent with a proteome-wide decline in proteostasis. Genes showing negative correlation in PD were enriched for proteasome subunits, indicating accentuated spatial separation of transcript and protein in PD neurons. In addition, PD showed positive correlations for mitochondrial respiratory chain genes, suggesting a tighter regulation in the face of mitochondrial dysfunction. Our results support the hypothesis that aberrant proteasomal function is implicated in PD pathogenesis.
    Keywords:  Proteomics; Transcriptomics
    DOI:  https://doi.org/10.1016/j.isci.2023.105925
  23. Eur Heart J Case Rep. 2023 Jan;7(1): ytad028
      Background: Mitochondrial encephalomyopathy with lactic acidosis and stroke-like episodes (MELAS) syndrome is a multi-organ disorder resulting from mitochondrial DNA (mtDNA) mutations. We report a case of suspected MELAS syndrome that progressed to left ventricular dysfunction 24 years after an initial diagnosis of atrioventricular block (AVB).Case summary: A 51-year-old woman was referred to heart failure clinic because of dyspnoea on exertion and progressive cardiomegaly. She had a dual-chamber pacemaker implanted for 24 years because of a high-degree AVB. She was treated for diabetes mellitus for 23 years and used hearing aids for 12 years because of sensorineural hearing loss. Transthoracic echocardiography revealed reduced left ventricular ejection fraction (26%), with increased thickness and unusual texture of the myocardium. The absence of abnormal findings on serum and urine protein electrophoresis suggested that light-chain amyloidosis was unlikely. In addition, 99mTc-3,3-diphosphono-1,2-propanodicarboxylic acid scintigraphy revealed no definite uptake in the myocardium. Endomyocardial biopsy revealed a hypertrophy of myocytes in haematoxylin-eosin staining, and electron microscopy revealed a disarrangement of mitochondrial cristae, which were suggestive of mitochondrial cardiomyopathy. A mtDNA test detected the m.3243A > G mutation in the MT-TL1 gene. According to these findings, MELAS syndrome was the most probable diagnosis despite the absence of common symptoms such as stroke-like episodes or lactic acidosis.
    Discussion: The patient had progressed to heart failure with reduced ejection fraction 24 years after the first cardiac manifestation. An identification of the mutation in the MT-TL1 gene, indicative of MELAS syndrome, enabled the diagnosis of MELAS syndrome without typical manifestations.
    Keywords:  Atrioventricular block; Cardiomyopathy; Case report; Heart failure with reduced ejection fraction; MELAS syndrome; Mitochondrial disease
    DOI:  https://doi.org/10.1093/ehjcr/ytad028
  24. Heliyon. 2023 Jan;9(1): e12987
      As centre of energy production and key regulators of metabolic and cellular signaling pathways, the integrity of mitochondria is essential for mesenchymal stem cell function in tissue regeneration. Alterations in the size, shape and structural organization of mitochondria are correlated with the physiological state of the cell and its environment and could be used as diagnostic biomarkers. Therefore, high-throughput experimental and computational techniques are crucial to ensure adequate correlations between mitochondrial function and disease phenotypes. The emerge of microfluidic technologies can address the shortcomings of traditional methods to determine mitochondrial dimensions for diagnostic and therapeutic use. This review discusses optical detection methods compatible with microfluidics to measure mitochondrial dynamics and their potential for clinical stem cell research targeting mitochondrial dysfunction.
    Keywords:  Automated mitochondrial analysis; MSC-Based therapies; Mesenchymal stem cells; Microfluidic systems; Mitochondrial dynamics
    DOI:  https://doi.org/10.1016/j.heliyon.2023.e12987
  25. Mitochondrion. 2023 Jan 27. pii: S1567-7249(23)00006-5. [Epub ahead of print]
      Leber's Hereditary Optic Neuropathy (LHON) has been mainly (90-95%) associated to one of three variants: m.3460G>A, m.11778G>A, m.14484T>C. Herein, a screening method was developed for its detection, supporting clinical/therapeutics decision. It relies on real-time PCR with High-Resolution Melting (HRM) analysis. Variant classification is made using HRM Software and quality controls. Ninety-four samples were analyzed. All samples were correctly assigned: 58 wild-type, 35 positive for m.11778G>A, 6 positive for m.14484T>C, 2 positive for m.3460G>A. Results presented sensitivity=1, specificity=1, Positive Predictive Value=1 and Negative Predictive Value=1. A new Real-Time PCR/HRM screening method cost-efficient, simple, robust and quick, detecting LHON's top-3 is described.
    Keywords:  High-resolution melting; LHON; idebenone; mtDNA variant; real-time PCR; theranostics
    DOI:  https://doi.org/10.1016/j.mito.2023.01.006
  26. bioRxiv. 2023 Jan 13. pii: 2023.01.13.523698. [Epub ahead of print]
    MoTrPAC Study Group
      Mitochondria are adaptable organelles with diverse cellular functions critical to whole-body metabolic homeostasis. While chronic endurance exercise training is known to alter mitochondrial activity, these adaptations have not yet been systematically characterized. Here, the Molecular Transducers of Physical Activity Consortium (MoTrPAC) mapped the longitudinal, multi-omic changes in mitochondrial analytes across 19 tissues in male and female rats endurance trained for 1, 2, 4 or 8 weeks. Training elicited substantial changes in the adrenal gland, brown adipose, colon, heart and skeletal muscle, while we detected mild responses in the brain, lung, small intestine and testes. The colon response was characterized by non-linear dynamics that resulted in upregulation of mitochondrial function that was more prominent in females. Brown adipose and adrenal tissues were characterized by substantial downregulation of mitochondrial pathways. Training induced a previously unrecognized robust upregulation of mitochondrial protein abundance and acetylation in the liver, and a concomitant shift in lipid metabolism. The striated muscles demonstrated a highly coordinated response to increase oxidative capacity, with the majority of changes occurring in protein abundance and post-translational modifications. We identified exercise upregulated networks that are downregulated in human type 2 diabetes and liver cirrhosis. In both cases HSD17B10, a central dehydrogenase in multiple metabolic pathways and mitochondrial tRNA maturation, was the main hub. In summary, we provide a multi-omic, cross-tissue atlas of the mitochondrial response to training and identify candidates for prevention of disease-associated mitochondrial dysfunction.
    DOI:  https://doi.org/10.1101/2023.01.13.523698
  27. Am J Physiol Regul Integr Comp Physiol. 2023 Jan 30.
      The peroxisome proliferator-activated receptor gamma coactivator-1 (PGC-1) family of transcriptional coactivators are regulators of mitochondrial oxidative capacity and content in skeletal muscle. Many of these conclusions are based primarily on gain-of-function studies using muscle-specific overexpression of PGC1s. We have previously reported that genetic deletion of both PGC-1α and PGC-1β in adult skeletal muscle resulted in a significant reduction in oxidative capacity with no effect on mitochondrial content. However, the contribution of PGC-1-related coactivator (PRC), the third PGC-1 family member, in regulating skeletal muscle mitochondria is unknown. Therefore, we generated an inducible skeletal muscle-specific PRC knockout mouse (iMS-PRC-KO) to assess the contribution of PRC in skeletal muscle mitochondrial function. We measured mRNA expression of electron transport chain (ETC) subunits as well as markers of mitochondrial content in the iMS-PRC-KO animals and observed an increase in ETC gene expression and mitochondrial content. Furthermore, the increase in ETC gene expression and mitochondrial content was associated with increased expression of PGC-1α and PGC-1β. We therefore generated an adult inducible PGC-1 knockout mouse in which all PGC-1 family members are deleted (iMS-PGC-1TKO). The iMS-PGC-1TKO animals exhibited a reduction in ETC mRNA expression and mitochondrial content. These data suggest that in the absence of PRC alone, compensation occurs by increasing PGC-1α and PGC-1β to maintain mitochondrial content. Moreover, removal of all three PGC-1s in skeletal muscle result in a reduction in both ETC mRNA expression and mitochondrial content. Taken together, these results suggests that PRC plays a role in maintaining baseline mitochondrial content in skeletal muscle.
    Keywords:  PRC; mitochondria; mitochondrial biogenesis; skeletal muscle
    DOI:  https://doi.org/10.1152/ajpregu.00241.2022
  28. Neurobiol Dis. 2023 Feb 01. pii: S0969-9961(23)00045-1. [Epub ahead of print] 106031
      Organelle contact sites are multifunctional platforms for maintaining cellular homeostasis. Alternations of the mitochondria-associated membranes (MAM), one of the organelle contact sites where the endoplasmic reticulum (ER) is tethered to the mitochondria, have been involved in the pathogenesis of neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS). However, the detailed mechanisms through which MAM integrity is disrupted in ALS have not been fully elucidated. Here, we examined whether AAA ATPase domain-containing protein 3A (ATAD3A), a mitochondrial membrane AAA ATPase accumulating at the MAM, is involved in ALS. We found that sigma-1 receptor (σ1R), an ER-resident MAM protein causative for inherited juvenile ALS, required ATAD3A to maintain the MAM. In addition, σ1R retained ATAD3A as a monomer, which is associated with an inhibition of mitochondrial fragmentation. ATAD3A dimerization and mitochondrial fragmentation were significantly induced in σ1R-deficient or SOD1-linked ALS mouse spinal cords. Overall, these observations indicate that MAM induction by σ1R depends on ATAD3A and that σ1R maintains ATAD3A as a monomer to inhibit mitochondrial fragmentation. Our findings suggest that targeting σ1R-ATAD3A axis would be promising for a novel therapeutic strategy to treat mitochondrial dysfunction in neurological disorders, including ALS.
    Keywords:  ATPase domain-containing; Lateral sclerosis; Mitochondria-associated membrane/sigma-1; Protein 3A/amyotrophic; Receptor/AAA
    DOI:  https://doi.org/10.1016/j.nbd.2023.106031
  29. Biomed Mater. 2023 Jan 31.
      Nanotopography can promote osseointegration, but how BMMSCs respond to this physical stimulus is unclear. Here, we found that early exposure of BMMSCs to nanotopography (6-hour) caused mitochondrial fission rather than fusion, which was necessary for osseointegration. We analyzed the changes in mitochondrial morphology and function of BMMSCs located on the surfaces of NT100(100-nanometer nanotubes) and ST (Smooth) by super-resolution microscopy and other techniques. Then, we found that both ST and NT100 caused a significant increase in mitochondrial fission early on, but NT100 caused mitochondrial fission much earlier than those on ST. In addition, the mitochondrial functional statuses were good at the 6-hour time point, this is at odds with the conventional wisdom that fusion is good. This fission phenomenon adequately protected mitochondrial membrane potential and respiration and reduced ROS. Interestingly, the mitochondrial membrane potential and oxygen consumption rate of BMMSCs were reduced when mitochondrial fission was inhibited by Mdivi-1 in the early stage. In addition, the effect on osseointegration was significantly worse, and this effect did not improve with time. Taken together, the findings indicate that early mitochondrial fission plays an important role in nanotopography-mediated promotion of osseointegration, which is of great significance to the surface structure design of biomaterials.
    Keywords:  BMMSCs; Mitochondrial fission; Osseointegration; TiO2 nanotubes
    DOI:  https://doi.org/10.1088/1748-605X/acb7bc
  30. bioRxiv. 2023 Jan 19. pii: 2023.01.19.524708. [Epub ahead of print]
      A complex interplay between mRNA translation and cellular respiration has been recently unveiled, but its regulation in humans is poorly characterized in either health or disease. Cancer cells radically reshape both biosynthetic and bioenergetic pathways to sustain their aberrant growth rates. In this regard, we have shown that the molecular chaperone TRAP1 not only regulates the activity of respiratory complexes, behaving alternatively as an oncogene or a tumor suppressor, but also plays a concomitant moonlighting function in mRNA translation regulation. Herein we identify the molecular mechanisms involved, demonstrating that TRAP1: i) binds both mitochondrial and cytosolic ribosomes as well as translation elongation factors, ii) slows down translation elongation rate, and iii) favors localized translation in the proximity of mitochondria. We also provide evidence that TRAP1 is coexpressed in human tissues with the mitochondrial translational machinery, which is responsible for the synthesis of respiratory complex proteins. Altogether, our results show an unprecedented level of complexity in the regulation of cancer cell metabolism, strongly suggesting the existence of a tight feedback loop between protein synthesis and energy metabolism, based on the demonstration that a single molecular chaperone plays a role in both mitochondrial and cytosolic translation, as well as in mitochondrial respiration.
    DOI:  https://doi.org/10.1101/2023.01.19.524708
  31. Exp Gerontol. 2023 Jan 25. pii: S0531-5565(23)00030-X. [Epub ahead of print]173 112109
      Skeletal muscle is a tissue integral to general health. Due to its high abundance and oxidative capacity, its metabolism is intimately linked to whole-body physiology. In the elderly population, mobility correlates positively with life expectancy and survival. Furthermore, regular physical activity is one of the most effective health-promoting interventions that delay the onset of aging-associated chronic diseases. Data from preclinical studies show that aging of various tissues is accompanied by a decrease in the concentration of nicotinamide adenine dinucleotide (NAD), which plays a central role in energy homeostasis. Thus, a hypothesis has emerged that normalization of its content would ameliorate the age-related decline in tissue function and therefore improve health of the elderly. This idea, along with the documented safety and high tolerability of NAD precursor supplementation, makes NAD metabolism a prospective target for anti-aging interventions. Interestingly, muscle NAD biosynthesis pathways are stimulated by exercise training, which suggests that training-induced adaptations rely on tissue NAD levels. However, while the relationship between muscle fitness and regular physical activity is well-characterized, the proposed synergy between muscle NAD replenishment and exercise training has not been established. Here, we review the published data on the role of NAD metabolism in exercise in the context of young and aged skeletal muscle and discuss the current challenges relevant to the field.
    Keywords:  Aging; Exercise training; NAD precursor supplementation; Skeletal muscle
    DOI:  https://doi.org/10.1016/j.exger.2023.112109
  32. bioRxiv. 2023 Jan 15. pii: 2023.01.12.523790. [Epub ahead of print]
    North American Brain Expression Consortium (NABEC)
      Long-read sequencing technologies substantially overcome the limitations of short-reads but to date have not been considered as feasible replacement at scale due to a combination of being too expensive, not scalable enough, or too error-prone. Here, we develop an efficient and scalable wet lab and computational protocol for Oxford Nanopore Technologies (ONT) long-read sequencing that seeks to provide a genuine alternative to short-reads for large-scale genomics projects. We applied our protocol to cell lines and brain tissue samples as part of a pilot project for the NIH Center for Alzheimer’s and Related Dementias (CARD). Using a single PromethION flow cell, we can detect SNPs with F1-score better than Illumina short-read sequencing. Small indel calling remains to be difficult inside homopolymers and tandem repeats, but is comparable to Illumina calls elsewhere. Further, we can discover structural variants with F1-score comparable to state-of-the-art methods involving Pacific Biosciences HiFi sequencing and trio information (but at a lower cost and greater throughput). Using ONT-based phasing, we can then combine and phase small and structural variants at megabase scales. Our protocol also produces highly accurate, haplotype-specific methylation calls. Overall, this makes large-scale long-read sequencing projects feasible; the protocol is currently being used to sequence thousands of brain-based genomes as a part of the NIH CARD initiative. We provide the protocol and software as open-source integrated pipelines for generating phased variant calls and assemblies.
    DOI:  https://doi.org/10.1101/2023.01.12.523790
  33. Nat Metab. 2023 Feb 02.
      The mechanistic target of rapamycin complex 1 (mTORC1) controls cell growth in response to amino acid and glucose levels. However, how mTORC1 senses glucose availability to regulate various downstream signalling pathways remains largely elusive. Here we report that AMP-activated protein kinase (AMPK)-mediated phosphorylation of WDR24, a core component of the GATOR2 complex, has a role in the glucose-sensing capability of mTORC1. Mechanistically, glucose deprivation activates AMPK, which directly phosphorylates WDR24 on S155, subsequently disrupting the integrity of the GATOR2 complex to suppress mTORC1 activation. Phosphomimetic Wdr24S155D knock-in mice exhibit early embryonic lethality and reduced mTORC1 activity. On the other hand, compared to wild-type littermates, phospho-deficient Wdr24S155A knock-in mice are more resistant to fasting and display elevated mTORC1 activity. Our findings reveal that AMPK-mediated phosphorylation of WDR24 modulates glucose-induced mTORC1 activation, thereby providing a rationale for targeting AMPK-WDR24 signalling to fine-tune mTORC1 activation as a potential therapeutic means to combat human diseases with aberrant activation of mTORC1 signalling including cancer.
    DOI:  https://doi.org/10.1038/s42255-022-00732-4
  34. Res Sq. 2023 Jan 16. pii: rs.3.rs-2469234. [Epub ahead of print]
      Nonalcoholic fatty liver disease (NAFLD) encompasses a spectrum of pathologies that includes steatosis, steatohepatitis (NASH) and fibrosis and is strongly associated with insulin resistance and type 2 diabetes. Changes in mitochondrial function are implicated in the pathogenesis of NAFLD, particularly in the transition from steatosis to NASH. Mitophagy is a mitochondrial quality control mechanism that allows for the selective removal of damaged mitochondria from the cell via the autophagy pathway. While past work demonstrated a negative association between liver fat content and rates of mitophagy, when changes in mitophagy occur during the pathogenesis of NAFLD and whether such changes contribute to the primary endpoints associated with the disease are currently poorly defined. We therefore undertook the studies described here to establish when alterations in mitophagy occur during the pathogenesis of NAFLD, as well as to determine the effects of genetic inhibition of mitophagy via conditional deletion of a key mitophagy regulator, PARKIN, on the development of steatosis, insulin resistance, inflammation and fibrosis. We find that loss of mitophagy occurs early in the pathogenesis of NAFLD and that loss of PARKIN hastens the onset but not severity of key NAFLD disease features. These observations suggest that loss of mitochondrial quality control in response to nutritional stress may contribute to mitochondrial dysfunction and the pathogenesis of NAFLD.
    DOI:  https://doi.org/10.21203/rs.3.rs-2469234/v1
  35. Sci Adv. 2023 Feb 03. 9(5): eade8701
      Macrophage metabolic plasticity enables repurposing of electron transport from energy generation to inflammation and host defense. Altered respiratory complex II function has been implicated in cancer, diabetes, and inflammation, but regulatory mechanisms are incompletely understood. Here, we show that macrophage inflammatory activation triggers Complex II disassembly and succinate dehydrogenase subunit B loss through sequestration and selective mitophagy. Mitochondrial fission supported lipopolysaccharide-stimulated succinate dehydrogenase subunit B degradation but not sequestration. We hypothesized that this Complex II regulatory mechanism might be coordinated by the mitochondrial phospholipid cardiolipin. Cardiolipin synthase knockdown prevented lipopolysaccharide-induced metabolic remodeling and Complex II disassembly, sequestration, and degradation. Cardiolipin-depleted macrophages were defective in lipopolysaccharide-induced pro-inflammatory cytokine production, a phenotype partially rescued by Complex II inhibition. Thus, cardiolipin acts as a critical organizer of inflammatory metabolic remodeling.
    DOI:  https://doi.org/10.1126/sciadv.ade8701
  36. Exp Cell Res. 2023 Jan 26. pii: S0014-4827(23)00038-1. [Epub ahead of print]424(1): 113491
      Astrocytes are highly energy-consuming glial cells critical for metabolic support to neurons. A growing body of evidence suggests that mitochondrial dysfunction in astrocytes is involved in age-related neurodegenerative disorders and that fish oil, rich in docosahexaenoic (DHA) and eicosapentaenoic (EPA) fatty acids, may alleviate cognition impairment in Parkinson's and Alzheimer's diseases. The present study examines the effect of DHA and EPA on mitochondrial membrane potential (MMP), apoptosis activation and ATP levels in astrocytes cultured in medium containing glucose or galactose, which limits oxidative phosphorylation (OXPHOS). MMP, expressed as the ratio of red to green JC-10 and MitoTracker fluorescence, increased in EPA-incubated cells in a dose dependent manner and was higher than in DHA-incubated astrocytes, also after uncoupling of OXPHOS by carbonyl cyanide 3-chlorophenylhydrazone (CCCP). In cells cultured in glucose and galactose medium mitochondrial hyperpolarization had no impact on intracellular ATP level. Furthermore, both EPA and DHA elevated mitochondrial cardiolipin content, however only EPA did so in a dose-dependent manner and reduced apoptosis which was analyzed by flow cytometry.
    Keywords:  ATP; Astrocytes; Cardiolipin; Docosahexaenoic acid; Eicosapentaenoic acid; Mitochondrial membrane potential
    DOI:  https://doi.org/10.1016/j.yexcr.2023.113491
  37. JCI Insight. 2023 Feb 02. pii: e165604. [Epub ahead of print]
      The energetic costs of bone formation require osteoblasts to coordinate their activities with tissues, like adipose, that can supply energy-dense macronutrients. In the case of intermittent parathyroid hormone treatment (PTH), a strategy used to reduce fracture risk, bone formation is proceeded by a change in systemic lipid homeostasis. To investigate the requirement for fatty acid oxidation by osteoblasts during PTH-induced bone formation, we subjected mice with osteoblast-specific deficiency of mitochondrial long-chain β-oxidation as well as mice with adipocyte-specific deficiency for the PTH receptor or adipose triglyceride lipase to an anabolic treatment regime. PTH increased the release of fatty acids from adipocytes and B-oxidation by osteoblasts, while the genetic mouse models were resistant to the hormone's anabolic effect. Collectively, these data suggest that PTH's anabolic actions requires coordinated signaling between bone and adipose, wherein a lipolytic response liberates fatty acids that are oxidized by osteoblasts to fuel bone formation.
    Keywords:  Adipose tissue; Bone Biology; Fatty acid oxidation; Osteoporosis
    DOI:  https://doi.org/10.1172/jci.insight.165604
  38. Nat Commun. 2023 Jan 31. 14(1): 512
      The human gut microbiota produces dozens of small molecules that circulate in blood, accumulate to comparable levels as pharmaceutical drugs, and influence host physiology. Despite the importance of these metabolites to human health and disease, the origin of most microbially-produced molecules and their fate in the host remains largely unknown. Here, we uncover a host-microbe co-metabolic pathway for generation of hippuric acid, one of the most abundant organic acids in mammalian urine. Combining stable isotope tracing with bacterial and host genetics, we demonstrate reduction of phenylalanine to phenylpropionic acid by gut bacteria; the host re-oxidizes phenylpropionic acid involving medium-chain acyl-CoA dehydrogenase (MCAD). Generation of germ-free male and female MCAD-/- mice enabled gnotobiotic colonization combined with untargeted metabolomics to identify additional microbial metabolites processed by MCAD in host circulation. Our findings uncover a host-microbe pathway for the abundant, non-toxic phenylalanine metabolite hippurate and identify β-oxidation via MCAD as a novel mechanism by which mammals metabolize microbiota-derived metabolites.
    DOI:  https://doi.org/10.1038/s41467-023-36138-3
  39. Nat Commun. 2023 Feb 02. 14(1): 560
      Amyloid-like aggregates of the microtubule-associated protein Tau are associated with several neurodegenerative disorders including Alzheimer's disease. The existence of cellular machinery for the removal of such aggregates has remained unclear, as specialized disaggregase chaperones are thought to be absent in mammalian cells. Here we show in cell culture and in neurons that the hexameric ATPase valosin-containing protein (VCP) is recruited to ubiquitylated Tau fibrils, resulting in their efficient disaggregation. Aggregate clearance depends on the functional cooperation of VCP with heat shock 70 kDa protein (Hsp70) and the ubiquitin-proteasome machinery. While inhibition of VCP activity stabilizes large Tau aggregates, disaggregation by VCP generates seeding-active Tau species as byproduct. These findings identify VCP as a core component of the machinery for the removal of neurodegenerative disease aggregates and suggest that its activity can be associated with enhanced aggregate spreading in tauopathies.
    DOI:  https://doi.org/10.1038/s41467-023-36058-2
  40. Redox Rep. 2023 Dec;28(1): 2168635
      BACKGROUND: Methionine sulfoxide reductases are found in all aerobic organisms. They function in antioxidant defense, cellular regulation by reversible oxidation of methionine in proteins, and in protein structure. However, very few in vivo binding partners or substrates of the reductases have been identified.METHODS: We implemented a proximity labeling method, TurboID, to covalently link mitochondrial methionine sulfoxide reductase A (MSRA) to its binding partners in HEK293 cells. Proteomic analyses were performed to identify putative binding partners.
    RESULTS: We show that human Ndufaf2, also called mimitin, is a binding partner of MSRA as well as all 3 MSRBs. We found that both methionine residues in Ndufaf2 were susceptible to oxidation by hydrogen peroxide and that the methionine sulfoxide reductases can reduce these methionine sulfoxide residues back to methionine.
    CONCLUSION: Methionine sulfoxide reductases can reduce methionine sulfoxide back to methionine in Ndufaf2. In addition to a repair function, it also creates a mechanism that could regulate cellular processes by modulation of methionine oxidation in Ndufaf2.
    Keywords:  Methionine sulfoxide reductase; Ndufaf2; TurboID proximity labeling; mass spectrometry; methionine oxidation; mimitin; mitochondrial complex I; oxidative stress
    DOI:  https://doi.org/10.1080/13510002.2023.2168635
  41. Nat Commun. 2023 Feb 02. 14(1): 562
      Flavin containing monooxygenases (FMOs) are promiscuous enzymes known for metabolizing a wide range of exogenous compounds. In C. elegans, fmo-2 expression increases lifespan and healthspan downstream of multiple longevity-promoting pathways through an unknown mechanism. Here, we report that, beyond its classification as a xenobiotic enzyme, fmo-2 expression leads to rewiring of endogenous metabolism principally through changes in one carbon metabolism (OCM). These changes are likely relevant, as we find that genetically modifying OCM enzyme expression leads to alterations in longevity that interact with fmo-2 expression. Using computer modeling, we identify decreased methylation as the major OCM flux modified by FMO-2 that is sufficient to recapitulate its longevity benefits. We further find that tryptophan is decreased in multiple mammalian FMO overexpression models and is a validated substrate for FMO-2. Our resulting model connects a single enzyme to two previously unconnected key metabolic pathways and provides a framework for the metabolic interconnectivity of longevity-promoting pathways such as dietary restriction. FMOs are well-conserved enzymes that are also induced by lifespan-extending interventions in mice, supporting a conserved and important role in promoting health and longevity through metabolic remodeling.
    DOI:  https://doi.org/10.1038/s41467-023-36181-0
  42. Mov Disord. 2023 Jan 30.
      BACKGROUND: Alterations in mitochondrial dysfunction have been implicated in the pathogenesis of Parkinson's disease (PD). Mitochondrial energy production is linked to glucose metabolism, and diabetes is associated with PD. However, studies investigating glucose metabolism in vivo in genetically stratified PD patients and controls have yet to be performed.OBJECTIVES: The objectives of this study were to explore glucose production, gluconeogenesis, and the contribution of gluconeogenesis to glucose production in idiopathic and PRKN PD compared with healthy controls with state-of-the-art biochemical methods.
    METHODS: We applied a dried-blood sampling/gas chromatography/mass spectrometry approach to monitor fluxes in the Cori cycle in vivo.
    RESULTS: The contribution of gluconeogenesis to total glucose production is increased in idiopathic PD patients (n = 33), but not in biallelic PRKN mutation carriers (n = 5) compared with healthy controls (n = 13).
    CONCLUSIONS: We provide first-time in vivo evidence for alterations in glucose metabolism in idiopathic PD, in keeping with the epidemiological evidence for an association between PD and diabetes. © 2023 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.
    Keywords:  PRKN; Parkinson's disease; diabetes; glucose metabolism; mitochondrial dysfunction
    DOI:  https://doi.org/10.1002/mds.29333
  43. bioRxiv. 2023 Jan 14. pii: 2023.01.13.523849. [Epub ahead of print]
      Current methods for profiling DNA methylation require costly reagents, sequencing, or labor time. We introduce FML-seq, a sequencing library protocol that greatly reduces all these costs. Relative to other techniques tested on the same human cell lines, FML-seq produces similar measurements of absolute and differential cytosine methylation at a fraction of the price. FML-seq enables inexpensive, high-throughput experimental designs for large-scale epigenetics research projects.
    DOI:  https://doi.org/10.1101/2023.01.13.523849
  44. bioRxiv. 2023 Jan 18. pii: 2023.01.16.524176. [Epub ahead of print]
      Cristae membrane state plays a central role in regulating mitochondrial function and cellular metabolism. The protein Optic atrophy 1 (Opa1) is an important crista remodeler that exists as two forms in the mitochondrion, a membrane-anchored long form (l-Opa1) and a processed short form (s-Opa1). The mechanisms for how Opa1 influences cristae shape have remained unclear due to the lack of native 3D views of cristae morphology. We perform in situ cryo-electron tomography of cryo-focused ion beam milled mouse embryonic fibroblasts with well-defined Opa1 states to understand how each form of Opa1 influences cristae architecture. In our tomograms, we observe elongated mitochondria with a notable stacking phenotype, as well as an absence of tubular cristae, when only l-Opa1 is present. In contrast, when mitochondria contain mainly s-Opa1, we observe irregular cristae packing, an increase in globular cristae, and decreased matrix condensation. Notably, we find the absence of l-Opa1 results in mitochondria with wider cristae junctions. BH3 profiling reveals that absence of l-Opa1 reduces cytochrome c release in response to pro-apoptotic stimuli and protects cells from apoptosis induced by anti-cancer agents. We discuss the implications Opa1-dependent cristae morphologies in cell death initiation.Highlights: In situ ultrastructural characterization of mitochondrial cristae with different forms of Opa1. Mitochondria with predominantly l-Opa1 show cristae stacking, longer cristae compared to WT, but also a reduction of globular cristae and no tubular cristae.Mitochondria with mostly s-Opa1 showed irregular cristae packing with wider cristae junctions and more narrow cristae than WT.BH3 profiling show Opa1-knock-out cells have reduced apoptotic priming and reduced sensitivity to apoptosis-inducing agents, and the presence l-Opa1 restores a WT protective apoptotic response.
    DOI:  https://doi.org/10.1101/2023.01.16.524176
  45. bioRxiv. 2023 Jan 12. pii: 2023.01.12.523808. [Epub ahead of print]
      Network inference is used to model transcriptional, signaling, and metabolic interactions among genes, proteins, and metabolites that identify biological pathways influencing disease pathogenesis. Advances in machine learning (ML)-based inference models exhibit the predictive capabilities of capturing latent patterns in genomic data. Such models are emerging as an alternative to the statistical models identifying causative factors driving complex diseases. We present CoVar, an inference framework that builds upon the properties of existing inference models, to find the central genes driving perturbed gene expression across biological states. We leverage ML-based network inference to find networks that capture the strength of regulatory interactions. Our model first pinpoints a subset of genes, termed variational, whose expression variabilities typify the differences in network connectivity between the control and perturbed data. Variational genes, by being differentially expressed themselves or possessing differentially expressed neighbor genes, capture gene expression variability. CoVar then creates subnetworks comprising variational genes and their strongly connected neighbor genes and identifies core genes central to these subnetworks that influence the bulk of the variational activity. Through the analysis of yeast expression data perturbed by the deletion of the mitochondrial genome, we show that CoVar identifies key genes not found through independent differential expression analysis.
    DOI:  https://doi.org/10.1101/2023.01.12.523808
  46. Nat Metab. 2023 Jan;5(1): 80-95
      Methylmalonic aciduria (MMA) is an inborn error of metabolism with multiple monogenic causes and a poorly understood pathogenesis, leading to the absence of effective causal treatments. Here we employ multi-layered omics profiling combined with biochemical and clinical features of individuals with MMA to reveal a molecular diagnosis for 177 out of 210 (84%) cases, the majority (148) of whom display pathogenic variants in methylmalonyl-CoA mutase (MMUT). Stratification of these data layers by disease severity shows dysregulation of the tricarboxylic acid cycle and its replenishment (anaplerosis) by glutamine. The relevance of these disturbances is evidenced by multi-organ metabolomics of a hemizygous Mmut mouse model as well as through identification of physical interactions between MMUT and glutamine anaplerotic enzymes. Using stable-isotope tracing, we find that treatment with dimethyl-oxoglutarate restores deficient tricarboxylic acid cycling. Our work highlights glutamine anaplerosis as a potential therapeutic intervention point in MMA.
    DOI:  https://doi.org/10.1038/s42255-022-00720-8
  47. Methods Mol Biol. 2023 ;2624 7-18
      Arrays provide a cost-effective platform for the analysis of human DNA methylation. ShinyÉPICo is an interactive, web-based, and graphical tool that allows the user to analyze Illumina DNA methylation arrays (450 k and EPIC), from the user's own computer or from a server. This tool covers the analysis entirely, from the raw data input to the final list of differentially methylated positions or regions. Here, we describe the steps of the analysis, the different parameters available, and useful information to understand and select the best options in each step.
    Keywords:  DNA methylation; Differentially Methylated Positions; Differentially Methylated Regions; Epigenetics; Shiny; Web Interface
    DOI:  https://doi.org/10.1007/978-1-0716-2962-8_2
  48. bioRxiv. 2023 Jan 15. pii: 2023.01.11.523651. [Epub ahead of print]
      Loss of function mutations in PHGDH, PSAT1 , and PSPH cause a set of rare, autosomal recessive diseases known as Neu-Laxova syndrome (NLS) or serine-deficiency disorders. The diseases present with a broad range of phenotypes, including lethality, severe neurological manifestations, seizures, and intellectual disability. However, because L-serine supplementation, especially if started early in pregnancy, can ameliorate and in some cases even prevent symptoms, knowledge of pathogenic variants is highly actionable. Recently, our laboratory established a yeast-based assay for human PSAT1 function. We have now applied it at scale to assay the functional impact of 1,914 SNV-accessible amino acid substitutions. Our results agree well with clinical interpretations and protein structure-function relationships, supporting the use of our data as functional evidence under the ACMG interpretation guidelines. In addition to assaying the functional impact of individual variants in yeast haploid cells, we can assay pairwise combinations of PSAT1 alleles that recapitulate human genotypes, including compound heterozygotes, in yeast diploids. Results from this diploid assay successfully distinguish patient genotypes from those of healthy carriers and agree well with disease severity. Finally, we present a linear model that can accurately predict biallelic function in diploids using information from the individual allele measurements in haploids. Taken together, our work provides an example of how large-scale functional assays in model systems can be powerfully applied to the study of a rare disease.
    DOI:  https://doi.org/10.1101/2023.01.11.523651
  49. Cell Calcium. 2023 Jan 25. pii: S0143-4160(23)00012-X. [Epub ahead of print]110 102700
      The close contacts between endoplasmic reticulum and mitochondria (ERMCs) play a key role in metabolic regulation, Ca2+ homeostasis, reactive oxygen species production, and many other cell functions. Nevertheless, it is not fully clear how these contacts dynamically rearrange to support cell functions. In a recent Nature Communications article [1], Katona et al. elegantly showed that motile IP3Rs can be captured at ERMCs to promptly mediate Ca2+ transfer and stimulate mitochondrial oxidative metabolism.
    Keywords:  Contact sites; Endoplasmic reticulum; IP(3) receptor; Mitochondria
    DOI:  https://doi.org/10.1016/j.ceca.2023.102700
  50. Acta Physiol (Oxf). 2023 Feb 01. e13943
      AIM: Myotonic dystrophy type 1 (DM1) is the second most common muscular dystrophy after Duchenne and is the most prevalent muscular dystrophy in adults. DM1 patients that participate in aerobic exercise training experience several physiological benefits concomitant with improved muscle mitochondrial function without alterations in typical DM1-specific disease mechanisms, which suggests that correcting organelle health is key to ameliorate the DM1 pathology. However, our understanding of the molecular mechanisms of mitochondrial turnover and dynamics in DM1 skeletal muscle is lacking.METHODS: Skeletal muscle tissue were sampled from healthy and DM1 mice under sedentary conditions and at several recovery time-points following an exhaustive treadmill run.
    RESULTS: We demonstrate that DM1 patients exhibit an imbalance in the transcriptional apparatus for mitochondrial turnover and dynamics in skeletal muscle. Additionally, DM1 mice displayed elevated expression of autophagy and mitophagy regulators. A single dose of exercise successfully enhanced canonical exercise molecular pathways and skeletal muscle mitochondrial biogenesis despite failing to alter the cellular pathology in DM1 mice. However, treadmill running stimulated coordinated organelle fusion and fission signaling, as well as improved alternative splicing of Optic atrophy 1. Exercise also evoked autophagy and mitophagy pathways in DM1 skeletal muscle resulting in the normalized expression of autophagy- and lysosome-related machinery responsible for the clearance of dysfunctional organelles.
    CONCLUSION: Collectively, our data indicate that mitochondrial dynamics and turnover processes in DM1 skeletal muscle are initiated with a single dose of exercise, which may underlie the adaptive benefits previously documented in DM1 mice and patients.
    Keywords:  AMP-activated protein kinase; autophagy; biogenesis; dynamics; mitophagy
    DOI:  https://doi.org/10.1111/apha.13943
  51. Neurobiol Dis. 2023 Jan 27. pii: S0969-9961(23)00036-0. [Epub ahead of print]178 106022
      Mitochondrial defects are one of the common underlying causes of neuronal vulnerability in neurodegenerative diseases, such as amyotrophic lateral sclerosis (ALS), and TDP-43 pathology is the most commonly observed proteinopathy. Disrupted inner mitochondrial membrane (IMM) reported in the upper motor neurons (UMNs) of ALS patients with TDP-43 pathology is recapitulated in the UMNs of well-characterized hTDP-43 mouse model of ALS. The construct validity, such as shared and common cellular pathology in mice and human, offers a unique opportunity to test treatment strategies that may translate to patients. SBT-272 is a well-tolerated brain-penetrant small molecule that stabilizes cardiolipin, a phospholipid found in IMM, thereby restoring mitochondrial structure and respiratory function. We investigated whether SBT-272 can improve IMM structure and health in UMNs diseased with TDP-43 pathology in our well-characterized UMN reporter line for ALS. We found that SBT-272 significantly improved mitochondrial structural integrity and restored mitochondrial motility and function. This led to improved health of diseased UMNs in vitro. In comparison to edaravone and AMX0035, SBT-272 appeared more effective in restoring health of diseased UMNs. Chronic treatment of SBT-272 for sixty days starting at an early symptomatic stage of the disease in vivo led to a significant reduction in astrogliosis, microgliosis, and TDP-43 pathology in the ALS motor cortex. Our results underscore the therapeutic potential of SBT-272, especially within the context of TDP-43 pathology and mitochondrial dysfunction.
    Keywords:  ATP; Cardiolipin; Electron transport chain; Motor neuron disease; Proteinopathy; SBT-272; TDP-43
    DOI:  https://doi.org/10.1016/j.nbd.2023.106022
  52. bioRxiv. 2023 Jan 12. pii: 2023.01.11.523633. [Epub ahead of print]
      In metazoans, Polo Kinase (Plk1) controls several mitotic events including nuclear envelope breakdown, centrosome maturation and kinetochore assembly. Here we show that mitotic events regulated by Polo Like Kinase (PLK-1) in early C. elegans embryos depend on the mitochondrial-localized protein SPD-3. spd-3 mutant one-cell embryos contain abnormally positioned mitotic chromosomes and prematurely and asymmetrically disassemble the nuclear lamina. Nuclear envelope breakdown (NEBD) in C. elegans requires direct dephosphorylation of lamin by PLK-1. In spd-3 mutants PLK-1 levels are ~6X higher in comparison to control embryos and PLK-1::GFP was highly accumulated at centrosomes, the nuclear envelope, nucleoplasm, and chromosomes prior to NEBD. Partial depletion of plk-1 in spd-3 mutant embryos rescued mitotic chromosome and spindle positioning defects indicating that these phenotypes result from higher PLK-1 levels and thus activity. Our data suggests that the mitochondrial SPD-3 protein controls NEBD and chromosome positioning by regulating the endogenous levels of PLK-1 during early embryogenesis in C. elegans . This finding suggests a novel link between mitochondria and mitotic events by controlling the amount of a key mitotic regulator, PLK-1 and thus may have further implications in the context of cancers or age-related diseases and infertility as it provides a novel link between mitochondria and mitosis.
    DOI:  https://doi.org/10.1101/2023.01.11.523633
  53. Sci Data. 2023 Feb 02. 10(1): 67
      Developing personalized diagnostic strategies and targeted treatments requires a deep understanding of disease biology and the ability to dissect the relationship between molecular and genetic factors and their phenotypic consequences. However, such knowledge is fragmented across publications, non-standardized repositories, and evolving ontologies describing various scales of biological organization between genotypes and clinical phenotypes. Here, we present PrimeKG, a multimodal knowledge graph for precision medicine analyses. PrimeKG integrates 20 high-quality resources to describe 17,080 diseases with 4,050,249 relationships representing ten major biological scales, including disease-associated protein perturbations, biological processes and pathways, anatomical and phenotypic scales, and the entire range of approved drugs with their therapeutic action, considerably expanding previous efforts in disease-rooted knowledge graphs. PrimeKG contains an abundance of 'indications', 'contradictions', and 'off-label use' drug-disease edges that lack in other knowledge graphs and can support AI analyses of how drugs affect disease-associated networks. We supplement PrimeKG's graph structure with language descriptions of clinical guidelines to enable multimodal analyses and provide instructions for continual updates of PrimeKG as new data become available.
    DOI:  https://doi.org/10.1038/s41597-023-01960-3
  54. Brain. 2023 Jan 30. pii: awac445. [Epub ahead of print]
      Humans accumulate with age the dark-brown pigment neuromelanin inside specific neuronal groups. Neurons with the highest neuromelanin levels are particularly susceptible to degeneration in Parkinson's disease, especially dopaminergic neurons of the substantia nigra, the loss of which leads to characteristic motor Parkinson's disease symptoms. In contrast to humans, neuromelanin does not appear spontaneously in most animals, including rodents, and Parkinson's disease is an exclusively human condition. Using humanized neuromelanin-producing rodents, we recently found that neuromelanin can trigger Parkinson's disease pathology when accumulated above a specific pathogenic threshold. Here, by taking advantage of this newly developed animal model, we assessed whether the intracellular build-up of neuromelanin that occurs with age can be slowed down in vivo to prevent or attenuate Parkinson's disease. Because neuromelanin derives from the oxidation of free cytosolic dopamine, we enhanced dopamine vesicular encapsulation in the substantia nigra of neuromelanin-producing rats by viral vector-mediated overexpression of vesicular monoamine transporter 2 (VMAT2). This strategy reduced the formation of potentially toxic oxidized dopamine species that can convert into neuromelanin and maintained intracellular neuromelanin levels below their pathogenic threshold. Decreased neuromelanin production was associated with an attenuation of Lewy body-like inclusion formation and a long-term preservation of dopamine homeostasis, nigrostriatal neuronal integrity and motor function in these animals. Our results demonstrate the feasibility and therapeutic potential of modulating age-dependent intracellular neuromelanin production in vivo, thereby opening an unexplored path for the treatment of Parkinson's disease and, in a broader sense, brain ageing.
    Keywords:  Parkinson’s disease; VMAT2; dopamine; neuromelanin; substantia nigra
    DOI:  https://doi.org/10.1093/brain/awac445
  55. Bioinform Adv. 2023 ;3(1): vbad002
      Motivation: Previous studies have shown that the heritability of multiple brain-related traits and disorders is highly enriched in transcriptional enhancer regions. However, these regions often contain many individual variants, while only a subset of them are likely to causally contribute to a trait. Statistical fine-mapping techniques can identify putative causal variants, but their resolution is often limited, especially in regions with multiple variants in high linkage disequilibrium. In these cases, alternative computational methods to estimate the impact of individual variants can aid in variant prioritization.Results: Here, we develop a deep learning pipeline to predict cell-type-specific enhancer activity directly from genomic sequences and quantify the impact of individual genetic variants in these regions. We show that the variants highlighted by our deep learning models are targeted by purifying selection in the human population, likely indicating a functional role. We integrate our deep learning predictions with statistical fine-mapping results for 8 brain-related traits, identifying 63 distinct candidate causal variants predicted to contribute to these traits by modulating enhancer activity, representing 6% of all genome-wide association study signals analyzed. Overall, our study provides a valuable computational method that can prioritize individual variants based on their estimated regulatory impact, but also highlights the limitations of existing methods for variant prioritization and fine-mapping.
    Availability and implementation: The data underlying this article, nucleotide-level importance scores, and code for running the deep learning pipeline are available at https://github.com/Pandaman-Ryan/AgentBind-brain.
    Contact: mgymrek@ucsd.edu.
    Supplementary information: Supplementary data are available at Bioinformatics Advances online.
    DOI:  https://doi.org/10.1093/bioadv/vbad002
  56. Immun Ageing. 2023 Jan 30. 20(1): 6
      BACKGROUND: Frailty, a clinical syndrome commencing at midlife, is a risk for morbidity and mortality. Little is known about the factors that contribute to the chronic inflammatory state associated with frailty. Extracellular vesicles (EVs) are small, membrane-bound vesicles that are released into the circulation and are mediators of intercellular communication. We examined whether mitochondrial DNA (mtDNA) and inflammatory proteins in EVs may act as damage-associated molecular pattern (DAMP) molecules in frailty.RESULTS: To address whether EVs and their associated mtDNA and inflammatory protein cargo are altered with frailty, EVs were isolated from non-frail (n = 90) and frail (n = 87) middle-aged (45-55 years) participants from the Healthy Aging in Neighborhoods of Diversity across the Life Span (HANDLS) study. EV concentration was highest in frail White participants. EV mtDNA levels were significantly higher in frail individuals compared to non-frail individuals. The presence of six inflammatory proteins in EVs (FGF-21, HGF, IL-12B, PD-L1, PRDX3, and STAMBP) were significantly associated with frailty. EV inflammatory proteins were significantly altered by frailty status, race, sex, and poverty status. Notably, frail White participants had higher levels of EV-associated CD5, CD8A, CD244, CXCL1, CXCL6, CXCL11, LAP-TGF-beta-1 and MCP-4 compared to frail and non-frail African American participants. Frail White participants living below poverty had higher levels of EV-associated uPA. EV-associated CCL28 levels were highest in non-frail women and CXCL1 were highest in non-frail men. Men living below poverty had higher levels of CD5, CD8A, CXCL1, LAP-TGF-beta-1, and uPA. CXCL6 levels were significantly higher in individuals living above poverty. There was a significant correlation between EV mtDNA levels and the presence of inflammatory proteins.
    CONCLUSIONS: These data suggest that mtDNA within EVs may act as a DAMP molecule in frailty. Its association with chemokines and other inflammatory EV cargo proteins, may contribute to the frailty phenotype. In addition, the social determinant of health, poverty, influences the inflammatory cargo of EVs in midlife.
    Keywords:  Aging; DAMP; EV; Exosome; Frail; Health disparities; Mitochondria, mtDNA, inflammation; Proteins; Social determinants of health
    DOI:  https://doi.org/10.1186/s12979-023-00330-2
  57. Neurotherapeutics. 2023 Jan 30.
      Spinal and bulbar muscular atrophy (SBMA) is characterized by motor neuron (MN) degeneration that leads to slowly progressive muscle weakness. It is considered a neuromuscular disease since muscle has a primary role in disease onset and progression. SBMA is caused by a CAG triplet repeat expansion in the androgen receptor (AR) gene. The translated poly-glutamine (polyQ) tract confers a toxic gain of function to the mutant AR altering its folding, causing its aggregation into intracellular inclusions, and impairing the autophagic flux. In an in vitro SBMA neuronal model, we previously showed that the antiandrogen bicalutamide and trehalose, a natural disaccharide stimulating autophagy, block ARpolyQ activation, reduce its nuclear translocation and toxicity and facilitate the autophagic degradation of cytoplasmic AR aggregates. Here, in a knock-in SBMA mouse model (KI AR113Q), we show that bicalutamide and trehalose ameliorated SBMA pathology. Bicalutamide reversed the formation of the AR insoluble forms in KI AR113Q muscle, preventing autophagic flux blockage. We demonstrated that apoptosis is activated in KI AR113Q muscle, and that both compounds prevented its activation. We detected a decrease of mtDNA and an increase of OXPHOS enzymes, already at early symptomatic stages; these alterations were reverted by trehalose. Overall, bicalutamide and/or trehalose led to a partial recovery of muscle morphology and function, and improved SBMA mouse motor behavior, inducing an extension of their survival. Thus, bicalutamide and trehalose, by counteracting ARpolyQ toxicity in skeletal muscle, are valuable candidates for future clinical trials in SBMA patients.
    Keywords:  Androgen receptor; Autophagy; Motor neuron; SBMA; Skeletal muscle
    DOI:  https://doi.org/10.1007/s13311-023-01343-x
  58. bioRxiv. 2023 Jan 20. pii: 2023.01.19.524841. [Epub ahead of print]
      Pneumonia is a worldwide threat, making discovery of novel means to combat lower respiratory tract infections an urgent need. We have previously shown that manipulating the lungs' intrinsic host defenses by therapeutic delivery of a unique dyad of pathogen-associated molecular patterns protects mice against pneumonia in a reactive oxygen species (ROS)-dependent manner. Here we show that antimicrobial ROS are induced from lung epithelial cells by interactions of CpG oligodeoxynucleotides (ODNs) with mitochondrial voltage-dependent anion channel 1 (VDAC1) without dependence on Toll-like receptor 9 (TLR9). The ODN-VDAC1 interaction alters cellular ATP/ADP/AMP localization, increases delivery of electrons to the electron transport chain (ETC), enhances mitochondrial membrane potential (Δ Ψm ), and differentially modulates ETC complex activities. These combined effects promote leak of electrons from ETC complex III, resulting in superoxide formation. The ODN-induced mitochondrial ROS yield protective antibacterial effects. Together, these studies identify a therapeutic metabolic manipulation strategy that has the potential to broadly protect patients against pneumonia during periods of peak vulnerability without reliance on currently available antibiotics.Author Summary: Pneumonia is a major cause of death worldwide. Increasing antibiotic resistance and expanding immunocompromised populations continue to enhance the clinical urgency to find new strategies to prevent and treat pneumonia. We have identified a novel inhaled therapeutic that stimulates lung epithelial defenses to protect mice against pneumonia in a manner that depends on production of reactive oxygen species (ROS). Here, we report that the induction of protective ROS from lung epithelial mitochondria occurs following the interaction of one component of the treatment, an oligodeoxynucleotide, with the mitochondrial voltage-dependent anion channel 1. This interaction alters energy transfer between the mitochondria and the cytosol, resulting in metabolic reprogramming that drives more electrons into the electron transport chain, then causes electrons to leak from the electron transport chain to form protective ROS. While antioxidant therapies are endorsed in many other disease states, we present here an example of therapeutic induction of ROS that is associated with broad protection against pneumonia without reliance on administration of antibiotics.
    DOI:  https://doi.org/10.1101/2023.01.19.524841
  59. Methods Mol Biol. 2023 ;2624 87-114
      Mapping DNA modifications at the base resolution is now possible at the genome level thanks to advances in sequencing technologies. Long-read sequencing data can be used to identify modified base patterns. However, the downstream analysis of Pacific Biosciences (PacBio) or Oxford Nanopore Technologies (ONT) data requires the integration of genomic annotation and comprehensive filtering to prevent the accumulation of artifact signals. We present in this chapter, a linear workflow to fully analyze modified base patterns using the DNA Modification Annotation (DNAModAnnot) package. This workflow includes a thorough filtering based on sequencing quality and false discovery rate estimation and provides tools for a global analysis of DNA modifications. Here, we provide an application example of this workflow with PacBio data and guide the user by explaining expected outputs via a fully integrated Rmarkdown script. This protocol is presented with tips showing how to adapt the provided code for annotating epigenomes of any organism according to the user needs.
    Keywords:  DNA Methylation; DNA modifications; DNAModAnnot; Epigenome Annotation; Epigenomics; Nanopore technology; PacBio Sequencing
    DOI:  https://doi.org/10.1007/978-1-0716-2962-8_7
  60. J Med Case Rep. 2023 Feb 04. 17(1): 34
      INTRODUCTION: Leber hereditary optic neuropathy is a genetic disease of mitochondrial inheritance characterized by bilateral irreversible vision loss, predominantly affecting males. We report the first genetically authenticated Sri Lankan case of Leber hereditary optic neuropathy, illustrating its characteristic features of male predominance and variable penetrance.CASE PRESENTATION: A 15-year-old previously healthy Sri Lankan boy presented with painless progressive vision loss in his right eye, followed by vision loss in his left eye within 3 months. There was no history of drug or toxin exposure, or a family history of vision loss. His parents were nonconsanguineous. On examination, he could only perceive light. Funduscopy revealed bilateral optic atrophy. Routine hematological and biochemical blood tests, including inflammatory markers, were normal. Cranial magnetic resonance imaging was unremarkable. Optical coherence tomography, and the clinical presentation, suggested a diagnosis of Leber hereditary optic neuropathy, which was confirmed by detection of m.14484T > C pathogenic variant in the MT-ND6 gene through targeted genetic analysis for the three common pathogenic variants in mitochondrial deoxyribonucleic acid. He was homoplasmic for the variant, and his asymptomatic mother and two female siblings were also found to be harboring the variant with homoplasmy.
    CONCLUSIONS: This case report is intended to increase awareness of Leber hereditary optic neuropathy, and highlights the need to consider this rare diagnosis in the appropriate clinical context. It also illustrates the phenomena of incomplete penetrance and male predominance, and suggests the possibility of an X-linked gene governing Leber hereditary optic neuropathy disease expression, which warrants further investigation.
    Keywords:  Blindness; Male predominance; Mitochondrial; Variable penetrance
    DOI:  https://doi.org/10.1186/s13256-023-03763-x
  61. Nat Commun. 2023 Feb 02. 14(1): 555
      Targeted proteomics enables hypothesis-driven research by measuring the cellular expression of protein cohorts related by function, disease, or class after perturbation. Here, we present a pathway-centric approach and an assay builder resource for targeting entire pathways of up to 200 proteins selected from >10,000 expressed proteins to directly measure their abundances, exploiting sample multiplexing to increase throughput by 16-fold. The strategy, termed GoDig, requires only a single-shot LC-MS analysis, ~1 µg combined peptide material, a list of up to 200 proteins, and real-time analytics to trigger simultaneous quantification of up to 16 samples for hundreds of analytes. We apply GoDig to quantify the impact of genetic variation on protein expression in mice fed a high-fat diet. We create several GoDig assays to quantify the expression of multiple protein families (kinases, lipid metabolism- and lipid droplet-associated proteins) across 480 fully-genotyped Diversity Outbred mice, revealing protein quantitative trait loci and establishing potential linkages between specific proteins and lipid homeostasis.
    DOI:  https://doi.org/10.1038/s41467-023-36269-7
  62. J Parkinsons Dis. 2023 Jan 24.
      Regulation of gene expression by epigenetic modifications means lasting and heritable changes in the function of genes without alterations in the DNA sequence. Of all epigenetic mechanisms identified thus far, DNA methylation has been of particular interest in both aging and age-related disease research over the last decade given the consistency of site-specific DNA methylation changes during aging that can predict future health and lifespan. An increasing line of evidence has implied the dynamic nature of DNA (de)methylation events that occur throughout the lifespan has a role in the pathophysiology of aging and age-associated neurodegenerative conditions, including Parkinson's disease (PD). In this regard, PD methylome shows, to some extent, similar genome-wide changes observed in the methylome of healthy individuals of matching age. In this review, we start by providing a brief overview of studies outlining global patterns of DNA methylation, then its mechanisms and regulation, within the context of aging and PD. Considering diverging lines of evidence from different experimental and animal models of neurodegeneration and how they combine to shape our current understanding of tissue-specific changes in DNA methylome in health and disease, we report a high-level comparison of the genomic methylation landscapes of brain, with an emphasis on dopaminergic neurons in PD and in natural aging. We believe this will be particularly useful for systematically dissecting overlapping genome-wide alterations in DNA methylation during PD and healthy aging, and for improving our knowledge of PD-specific changes in methylation patterns independent of aging process.
    Keywords:  DNA methylation; Parkinson’s disease; aging; brain; epigenome; substantia nigra
    DOI:  https://doi.org/10.3233/JPD-223517
  63. Methods Mol Biol. 2023 ;2624 115-126
      DNA methylation is studied extensively for its relations with several biological processes such as transcriptional regulation. While methylation levels are usually estimated per cytosine or genomic region, additional information on methylation heterogeneity can be obtained when considering stretches of successive cytosines on the same reads; however, the majority of methylomes suffer from low coverage of genomic regions with sequencing depths enough for accurate estimation of methylation heterogeneity using existing methods. Here we describe a probabilistic-based imputation method that makes use of methylation information from neighboring sites to recover partially observed methylation patterns. Our method and software are proven to be faster and more accurate among all evaluated. Ultimately, our method allows for a more streamlined monitoring of epigenetic changes within cellular populations and their putative role in disease.
    Keywords:  Bisulfite sequencing; Methylation heterogeneity; Methylation patterns; Probabilistic-based imputation
    DOI:  https://doi.org/10.1007/978-1-0716-2962-8_8
  64. Food Chem Toxicol. 2023 Jan 25. pii: S0278-6915(23)00038-8. [Epub ahead of print]173 113636
      Mitochondrial dysfunction has been implicated in Parkinson's disease. Mic60 is a critical component of mitochondrial crista remodeling and participates in maintaining mitochondrial structure and function. This study investigated whether the carnosic acid (CA) of rosemary protects the mitochondria of SH-SY5Y cells against the neurotoxicity of 6-hydroxydopamine (6-OHDA) by regulating Mic60. Our results showed that CA pretreatment reversed the reduction in the Mic60 and citrate synthase proteins, as well as the protein induction of PKA caused by 6-OHDA. Moreover, Mic60 and PINK1 siRNAs blocked the ability of CA to lessen the release of mitochondrial cytochrome c by 6-OHDA. As shown by immunoprecipitation assay, in 6-OHDA-treated cells, the interaction of Mic60 with its phosphorylated threonine residue was decreased, but the interaction with its phosphorylated serine residue was increased. PINK1 siRNA and forskolin, a PKA activator, reversed these interactions. Moreover, forskolin pretreatment prevented CA from rescuing the interaction of PINK1 and Mic60 and the reduction in cytochrome c release and mitophagy impairment in 6-OHDA-treated cells. In conclusion, CA prevents 6-OHDA-induced cytochrome c release by regulating Mic60 phosphorylation by PINK1 through a downregulation of PKA. The regulation of Mic60 by CA can be considered as a protective mechanism for the prevention of Parkinson's disease.
    Keywords:  Carnosic acid; Mic60; PINK1-mediated mitophagy; PKA; Parkinson's disease
    DOI:  https://doi.org/10.1016/j.fct.2023.113636
  65. Int J Biochem Cell Biol. 2023 Jan 27. pii: S1357-2725(23)00014-6. [Epub ahead of print] 106375
      The activation and proliferation of hepatic stellate cells (HSCs) are critical processes for the treatment of liver fibrosis. It is necessary to identify effective drugs for the treatment of liver fibrosis and elucidate their mechanisms of action. Metformin can inhibit HSCs; however, no systematic studies demonstrating the effects of metformin on mitochondria in HSCs have been reported. This study demonstrated that metformin induces mitochondrial fission by phosphorylating AMPK/DRP1 (S616) in HSCs to decrease the expression of α-SMA and collagen. Additionally, metformin repressed the total ATP production rate, especially the production rate of ATP produced through mitochondrial oxidative phosphorylation, by inhibiting the enzymatic activity of complex I. Further analysis revealed that metformin strongly constrained the transcription of mitochondrial genes (ND1-ND6 and ND4L) that encode the core subunits of respiratory chain I. Upregulation of the mRNA expression of HK2 and GLUT1 slightly enhanced glycolysis. Additionally, metformin increased mitochondrial DNA (mtDNA) copy number to suppress the proliferation and activation of HSCs, indicating that mtDNA copy number can alter the fate of HSCs. In conclusion, metformin can induce mitochondrial fragmentation and low-level energy metabolism in HSCs, thereby suppressing HSCs activation and proliferation to reverse liver fibrosis.
    Keywords:  Hepatic stellate cells (HSCs); Liver fibrosis; Metformin; Mitochondrial dynamics; oxidative phosphorylation
    DOI:  https://doi.org/10.1016/j.biocel.2023.106375