bims-mitdis Biomed News
on Mitochondrial disorders
Issue of 2022‒05‒15
33 papers selected by
Catalina Vasilescu
University of Helsinki

  1. Endocr Rev. 2022 May 12. 43(3): 583-609
      Mitochondrial diseases are a group of common inherited diseases causing disruption of oxidative phosphorylation. Some patients with mitochondrial disease have endocrine manifestations, with diabetes mellitus being predominant but also include hypogonadism, hypoadrenalism, and hypoparathyroidism. There have been major developments in mitochondrial disease over the past decade that have major implications for all patients. The collection of large cohorts of patients has better defined the phenotype of mitochondrial diseases and the majority of patients with endocrine abnormalities have involvement of several other systems. This means that patients with mitochondrial disease and endocrine manifestations need specialist follow-up because some of the other manifestations, such as stroke-like episodes and cardiomyopathy, are potentially life threatening. Also, the development and follow-up of large cohorts of patients means that there are clinical guidelines for the management of patients with mitochondrial disease. There is also considerable research activity to identify novel therapies for the treatment of mitochondrial disease. The revolution in genetics, with the introduction of next-generation sequencing, has made genetic testing more available and establishing a precise genetic diagnosis is important because it will affect the risk for involvement for different organ systems. Establishing a genetic diagnosis is also crucial because important reproductive options have been developed that will prevent the transmission of mitochondrial disease because of mitochondrial DNA variants to the next generation.
    Keywords:  MIDD; clinical management; diabetes mellitus; genomic testing; mitochondrial DNA; reproductive options
  2. FASEB J. 2022 May;36 Suppl 1
      Phosphorylation has long been appreciated to influence mitochondrial metabolism via the regulation of pyruvate dehydrogenase. However, the extent to which phosphorylation broadly influences mitochondrial function remains unclear, despite the presence of multiple protein phosphatases within the organelle. We recently demonstrated that deletion of the mitochondrial matrix phosphatase Pptc7 unexpectedly caused perinatal lethality in mice, suggesting that the regulation of mitochondrial phosphorylation is essential in mammalian development. Pptc7-/- mice exhibit severe metabolic deficiencies, including hypoglycemia and lactic acidosis, and die within one day of birth. Biochemical and proteomic approaches revealed that Pptc7-/- tissues have decreased mitochondrial function concomitant with a post-transcriptional downregulation of mitochondrial proteins. Multiple elevated mitochondrial protein phosphorylation sites in Pptc7-/- tissues suggest novel functional connections between Pptc7-mediated dephosphorylation and these observed metabolic consequences. Interestingly, these modifications occur on components of the import machinery of the mitochondria and within the mitochondrial targeting sequences of select nuclear-encoded precursor proteins. Collectively, our data reveal an unappreciated role for a matrix-localized phosphatase in the post-translational regulation of the mitochondrial proteome and organismal metabolic homeostasis.
  3. FASEB J. 2022 May;36 Suppl 1
      Mitochondria and peroxisomes are both dynamic signaling organelles that constantly undergo fission. While mitochondrial fission and fusion are known to coordinate cellular metabolism, proliferation, and apoptosis, the physiological relevance of peroxisome dynamics and the implications for cell fate are not fully understood. DRP1 (dynamin-related protein 1) is an essential GTPase that executes both mitochondrial and peroxisomal fission. Patients with de novo heterozygous missense mutations in the gene that encodes DRP1, DNM1L, present with encephalopathy due to mitochondrial and peroxisomal elongation (EMPF). EMPF is a devastating neurodevelopmental disease with no effective treatment. To interrogate the molecular mechanisms by which DRP1 mutations cause developmental defects, we are using patient-derived fibroblasts and iPSC-derived models from patients with mutations in different domains of DRP1 who present with clinically disparate conditions. Using super resolution imaging, we find that patient cells, in addition to displaying elongated mitochondrial and peroxisomal morphology, present with aberrant cristae structure. Given the direct link between cristae morphology and oxidative phosphorylation efficiency, we explored the impact of these mutations on cellular energy production. Patient cells display a lower coupling efficiency of the electron transport chain, increased proton leak, and Complex III deficiency. In addition to these metabolic abnormalities, mitochondrial hyperfusion results in hyperpolarized mitochondrial membrane potential. Intriguingly, human fibroblasts are capable of cellular reprogramming into iPSCs and appear to display peroxisome-mediated mitochondrial adaptations that could help sustain these cell fate transitions. Understanding the mechanism by which DRP1 mutations cause cellular dysfunction will give insight into the role of mitochondrial and peroxisome dynamics in neurodevelopment.
  4. FASEB J. 2022 May;36 Suppl 1
      Barth syndrome (BTHS) is a rare, X-linked disorder of mitochondrial phospholipid metabolism caused by variants in the gene TAFAZZIN.TAFAZZIN is a transacylase involved in the remodeling of cardiolipin (CL), a dimeric phospholipid localized to the inner mitochondrial membrane. Lack of TAFAZZIN-based remodeling results in irregular cardiolipin content, characterized by increased unremodeled CL, increased monoloysocardiolipin (the CL remodeling intermediate), and a decrease in remodeled CL, which is enriched in polyunsaturated fatty acyl chains that are tissue-specific in composition. BTHS is clinically characterized by cardiomyopathy, neutropenia, and myopathy, with a high morbidity and mortality. There are no approved disease-specific therapies. To investigate the cellular pathology and to identify new areas of potential therapeutic intervention, we developed two CRISPR-edited cell lines: TAFAZZIN-knockout (KO) HEK293 cells and iPSCs with which to perform broad-based discovery experiments and to study tissue-specific disease effects, respectively. A combined multi-omics approach including proteomics, lipidomics, and metabolomics in TAZ-KO HEK293 cells revealed diverse mitochondrial abnormalities, including defects in complex I of the respiratory chain, abnormal PDK2 expression, and dysregulation of proteins involved in mitochondrial quality control including PARL and PGAM5. Importantly, we discovered that molecules that bind to cardiolipin (SS-31) or inhibit nascent cardiolipin deacylation (bromoenol lactone), partially remediate these mitochondrial defects. We next explored cell-type specific dysfunction in iPSC-derived TAZ-KO and wild-type cardiomyocytes and neurons via lipidomics, RNA-seq, and functional studies. We identified disturbances in cellular lipid content including an expected increase in the monolysocardiolipin content and a reduction that exhibited cell-type specificity. RNAseq identified dysregulation in pathways regulated by PARL and PGAM5 including Wnt signaling, apoptosis, and autophagy in the undifferentiated state, with differentiated cell types highlighting pathways such as glucose metabolism and response to cellular stimuli. Oxygen consumption studies show impaired maximal respiratory capacity in TAZ-KO cardiomyocytes and neurons. Ongoing investigations aim to address cell type specific mitochondrial dysfunction by characterizing PARL abundance, PGAM5 cleavage, and mitochondrial morphology in TAZ-KO iPSC derived cell types. Additionally, we are currently targeting cardiolipin metabolism in differentiated TAZ-deficient cells with the goal of remediating cellular lipids, mitochondrial gene expression, and oxygen consumption abnormalities.
  5. Bioinformatics. 2022 May 13. 38(10): 2967-2969
      SUMMARY: We present MitoVisualize, a new tool for analysis of the human mitochondrial DNA (mtDNA). MitoVisualize enables visualization of: (i) the position and effect of variants in mitochondrial transfer RNA and ribosomal RNA secondary structures alongside curated variant annotations, (ii) data across RNA structures, such as to show all positions with disease-associated variants or with post-transcriptional modifications and (iii) the position of a base, gene or region in the circular mtDNA map, such as to show the location of a large deletion. All visualizations can be easily downloaded as figures for reuse. MitoVisualize can be useful for anyone interested in exploring mtDNA variation, though is designed to facilitate mtDNA variant interpretation in particular.AVAILABILITY AND IMPLEMENTATION: MitoVisualize can be accessed via The source code is available at
    SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.
  6. PLoS Genet. 2022 May 09. 18(5): e1010190
      Mitochondrial DNA (mtDNA) maintenance disorders are caused by mutations in ubiquitously expressed nuclear genes and lead to syndromes with variable disease severity and tissue-specific phenotypes. Loss of function mutations in the gene encoding the mitochondrial genome and maintenance exonuclease 1 (MGME1) result in deletions and depletion of mtDNA leading to adult-onset multisystem mitochondrial disease in humans. To better understand the in vivo function of MGME1 and the associated disease pathophysiology, we characterized a Mgme1 mouse knockout model by extensive phenotyping of ageing knockout animals. We show that loss of MGME1 leads to de novo formation of linear deleted mtDNA fragments that are constantly made and degraded. These findings contradict previous proposal that MGME1 is essential for degradation of linear mtDNA fragments and instead support a model where MGME1 has a critical role in completion of mtDNA replication. We report that Mgme1 knockout mice develop a dramatic phenotype as they age and display progressive weight loss, cataract and retinopathy. Surprisingly, aged animals also develop kidney inflammation, glomerular changes and severe chronic progressive nephropathy, consistent with nephrotic syndrome. These findings link the faulty mtDNA synthesis to severe inflammatory disease and thus show that defective mtDNA replication can trigger an immune response that causes age-associated progressive pathology in the kidney.
  7. FASEB J. 2022 May;36 Suppl 1
      Recognition of mammalian mitochondrial promoters requires the concerted action of mitochondrial RNA polymerase (mtRNAP) and transcription initiation factors TFAM and TFB2M. In this work, we found that transcript slippage results in heterogeneity of the human mitochondrial transcripts in vivo and in vitro. This allowed us to correctly interpret the RNAseq data, identify the bona fide transcription start sites (TSS), and assign mitochondrial promoters for >50% of mammalian species and some other vertebrates. The divergent structure of the mammalian promoters reveals previously unappreciated aspects of mtDNA evolution. The correct assignment of TSS also enabled us to establish the precise register of the DNA in the initiation complex and permitted investigation of the sequence-specific protein-DNA interactions. We determined the molecular basis of promoter recognition by mtRNAP and TFB2M, which cooperatively recognize bases near TSS in a species-specific manner. Our findings reveal a role of mitochondrial transcription machinery in mitonuclear coevolution and speciation.
  8. Kidney Dis (Basel). 2022 Mar;8(2): 148-159
      Aims: This study aimed to investigate associations between renal and extrarenal manifestations of mitochondrial diseases and their natural history as well as predictors of renal disease severity and overall disease outcome. The secondary aim was to generate a protocol of presymptomatic assessment and monitoring of renal function in patients with a defined mitochondrial disease.Methods: A multicenter, retrospective cohort study was performed by the Mitochondrial Clinical and Research Network (MCRN). Patients of any age with renal manifestations associated with a genetically verified mitochondrial disease were included from 8 expert European centers specializing in mitochondrial diseases: Gothenburg, Oulu, Copenhagen, Bergen, Helsinki, Stockholm, Rotterdam, and Barcelona.
    Results: Of the 36 patients included, two-thirds had mitochondrial DNA-associated disease. Renal manifestations were the first sign of mitochondrial disease in 19%, and renal involvement was first identified by laboratory tests in 57% of patients. Acute kidney injury occurred in 19% of patients and was the first sign of renal disease in the majority of these. The most common renal manifestation was chronic kidney disease (75% with stage 2 or greater), followed by tubulopathy (44.4%), the latter seen mostly among patients with single large-scale mitochondrial DNA deletions. Acute kidney injury and tubulopathy correlated with worse survival outcome. The most common findings on renal imaging were increased echogenicity and renal dysplasia/hypoplasia. Renal histology revealed focal segmental glomerulosclerosis, nephrocalcinosis, and nephronophthisis.
    Conclusion: Acute kidney injury is a distinct renal phenotype in patients with mitochondrial disease. Our results highlight the importance to recognize renal disease as a sign of an underlying mitochondrial disease. Acute kidney injury and tubulopathy are 2 distinct indicators of poor survival in patients with mitochondrial diseases.
    Keywords:  Acute kidney injury; Mitochondrial DNA; Mitochondrial disease; Renal manifestations
  9. Hum Mol Genet. 2022 May 14. pii: ddac109. [Epub ahead of print]
      Leber's hereditary optic neuropathy (LHON) is a maternally inherited eye disease due to mitochondrial DNA (mtDNA) mutations. LHON-linked ND6 14 484 T > C (p.M64V) mutation affected structural components of complex I but its pathophysiology is poorly understood. The structural analysis of complex I revealed that the M64 forms a nonpolar interaction Y59 in the ND6, Y59 in the ND6 interacts with E34 of ND4L, and L60 of ND6 interacts with the Y114 of ND1. These suggested that the m.14484 T > C mutation may perturb the structure and function of complex I. Mutant cybrids constructed by transferring mitochondria from lymphoblastoid cell lines of one Chinese LHON family into mtDNA-less (ρo) cells revealed decreases in the levels of ND6, ND1 and ND4L. The m.14484 T > C mutation may affect mitochondrial mRNA homeostasis, supported by reduced levels of SLIRP and SUPV3L1 involved in mRNA degradation and increasing expression of ND6, ND1 and ND4L genes. These alterations yielded decreased activity of complex I, respiratory deficiency, diminished mitochondrial ATP production and reduced membrane potential, and increased production of reactive oxygen species in the mutant cybrids. Furthermore, the m.14484 T > C mutation promoted apoptosis, evidenced by elevating Annexin V-positive cells, release of cytochrome c into cytosol, levels in apoptotic proteins BAX, caspases 3, 7, 9 and decreasing levels in anti-apoptotic protein Bcl-xL in the mutant cybrids. Moreover, the cybrids bearing the m.14484 T > C mutation exhibited the reduced levels of autophagy protein LC3, increased levels of substrate P62 and impaired PINK1/Parkin-dependent mitophagy. Our findings highlighted the critical role of m.14484 T > C mutation in the pathogenesis of LHON.
  10. Cell Death Dis. 2022 May 09. 13(5): 444
      Mitochondria are highly dynamic organelles that participate in ATP generation and involve calcium homeostasis, oxidative stress response, and apoptosis. Dysfunctional or damaged mitochondria could cause serious consequences even lead to cell death. Therefore, maintaining the homeostasis of mitochondria is critical for cellular functions. Mitophagy is a process of selectively degrading damaged mitochondria under mitochondrial toxicity conditions, which plays an essential role in mitochondrial quality control. The abnormal mitophagy that aggravates mitochondrial dysfunction is closely related to the pathogenesis of many diseases. As the myocardium is a highly oxidative metabolic tissue, mitochondria play a central role in maintaining optimal performance of the heart. Dysfunctional mitochondria accumulation is involved in the pathophysiology of cardiovascular diseases, such as myocardial infarction, cardiomyopathy and heart failure. This review discusses the most recent progress on mitophagy and its role in cardiovascular disease.
  11. FASEB J. 2022 May;36 Suppl 1
      Gitelman syndrome is the most frequent hereditary salt-losing tubulopathy characterized by hypokalemic alkalosis and hypomagnesemia. Gitelman syndrome is caused by biallelic pathogenic variants in SLC12A3, encoding the Na+-Cl- cotransporter (NCC) expressed in the distal convoluted tubule. Pathogenic variants in CLCNKB, HNF1B, FXYD2 or KCNJ10 may result in renal phenocopies of Gitelman syndrome, as they can lead to reduced NCC activity. Nevertheless, ±10% of patients with a Gitelman syndrome phenotype remain genetically unsolved. After identification of mitochondrial DNA (mtDNA) variants in three families with Gitelman syndrome-like electrolyte abnormalities, 156 families were investigated for variants in MT-TI and MT-TF, encoding the transfer RNAs for phenylalanine and isoleucine. Mitochondrial respiratory chain function was assessed in patient fibroblasts. In NCC-expressing HEK293 cells, mitochondrial dysfunction was induced to assess the effect on thiazide-sensitive 22Na+ transport. Genetic investigations revealed four mtDNA variants in 13 families: m.591C>T (n=7), m.616T>C (n=1), m.643A>G (n=1) (all in MT-TF) and m.4291T>C (n=4, in MT-TI). Variants were near homoplasmic in affected individuals. Importantly, affected members of six families with an MT-TF variant additionally suffered from progressive chronic kidney disease. Maximal mitochondrial respiratory capacity was reduced in patient fibroblasts, caused by dysfunction of oxidative phosphorylation complex IV. In vitro pharmacological inhibition of complex IV, mimicking the effect of the mtDNA variants, demonstrated an inhibitory effect on NCC phosphorylation and NCC-mediated sodium uptake. Pathogenic mtDNA variants in MT-TF and MT-TI can cause a Gitelman syndrome-like syndrome. Genetic investigation of mtDNA should be considered in patients with unexplained Gitelman syndrome-like tubulopathies.
  12. Theranostics. 2022 ;12(7): 3237-3250
      Background: Impaired mitochondrial function contributes to non-alcoholic steatohepatitis (NASH). Acylglycerol kinase (AGK) is a subunit of the translocase of the mitochondrial inner membrane 22 (TIM22) protein import complex. AGK mutation is the leading cause of Sengers syndrome, characterized by congenital cataracts, hypertrophic cardiomyopathy, skeletal myopathy, lactic acidosis, and liver dysfunction. The potential roles and mechanisms of AGK in NASH are not yet elucidated. Methods: Hepatic-specific AGK-deficient mice and AGK G126E mutation (AGK kinase activity arrest) mice were on a choline-deficient and high-fat diet (CDAHFD) and a methionine choline-deficient diet (MCD). The mitochondrial function and the molecular mechanisms underlying AGK were investigated in the pathogenesis of NASH. Results: The levels of AGK were significantly downregulated in human NASH liver samples. AGK deficiency led to severe liver damage and lipid accumulation in mice. Aged mice lacking hepatocyte AGK spontaneously developed NASH. AGK G126E mutation did not affect the structure and function of hepatocytes. AGK deficiency, but not AGK G126E mice, aggravated CDAHFD- and MCD-induced NASH symptoms. AGK deficiency-induced liver damage could be attributed to hepatic mitochondrial dysfunction. The mechanism revealed that AGK interacts with mitochondrial respiratory chain complex I subunits, NDUFS2 and NDUFA10, and regulates mitochondrial fatty acid metabolism. Moreover, the AGK DGK domain might directly interact with NDUFS2 and NDUFA10 to maintain the hepatic mitochondrial respiratory chain complex I function. Conclusions: The current study revealed the critical roles of AGK in NASH. AGK interacts with mitochondrial respiratory chain complex I to maintain mitochondrial integrity via the kinase-independent pathway.
    Keywords:  NDUFS2; fatty acid metabolism; mitochondrial ROS; mitochondrial respiratory chain
  13. FASEB J. 2022 May;36 Suppl 1
      Mitochondria play an important role in energy production and cellular metabolism. Mitochondria contain their own DNA (mtDNA), which encodes 13 subunits necessary for oxidative phosphorylation. Over 1500 other mitochondrial proteins, including the 77 remaining subunits required for oxidative phosphorylation and the machinery required for transcription and translation of mtDNA, are encoded by the nuclear genome. Thus, the nuclear and mitochondrial genomes must communicate to respond to the energetic needs of the cell. The mechanism of this communication is unclear. The mitochondrial proteome, including the transcriptional machinery, is subject to post-translational modifications (PTMs) such as phosphorylation of serine, threonine, and tyrosine and acylation of lysine. We hypothesize that PTMs of the mitochondrial transcriptional machinery regulate mitochondrial gene expression, akin to mechanisms controlling nuclear gene expression. Transcription of mtDNA requires three nuclear-encoded proteins: mitochondrial transcription factor A (TFAM), transcription factor B2 (TFB2M), and mitochondrial RNA polymerase (POLRMT). An accessory factor, mitochondrial ribosomal protein L12 (MRPL12), is thought to stabilize POLRMT and may promote transcription. Prior experiments in our lab show phosphorylation mimics of TFB2M have significantly reduced binding affinity for mtDNA and exhibit transcription initiation defects in vitro. Using mass spectrometry POLRMT was previously shown to be acetylated at one lysine and phosphorylated at nine amino acids, while MRPL12 contains five acetylated lysines and one phosphorylated threonine. The biochemical function of these modifications is unknown. PTMs were studied by using site-directed mutagenesis to replace the amino acid of interest to mimic acetylation (lysine to glutamine) or phosphorylation (threonine to glutamate) of POLRMT and MRPL12. Mutated proteins were purified and their mtDNA promoter binding affinity was determined by fluorescence anisotropy experiments. Fluorescence anisotropy revealed that POLRMT PTM mimics had little effect on mtDNA binding. However, when WT MRPL12 was co-incubated with WT POLRMT mtDNA binding affinity was enhanced by 30%. Increased binding affinity was lost with MRPL12 PTM mimics. WT and PTM mimics of MRPL12 were also overexpressed in mammalian cell lines. mtDNA transcript levels and mtDNA content were measured using quantitative PCR. mtDNA content was largely unchanged while some transcript-dependent effects were observed in the presence of MRPL12 protein mutants.
  14. Autophagy. 2022 May 09. 1-2
      The unique cellular organization and metabolic demands of neurons pose a challenge in the maintenance of neuronal homeostasis. A critical element in maintaining neuronal health and homeostasis is mitochondrial quality control via replacement and rejuvenation at the axon. Dysregulation of mitochondrial quality control mechanisms such as mitophagy has been implicated in neurodegenerative diseases including Parkinson disease and amyotrophic lateral sclerosis. To sustain mitophagy at the axon, a continuous supply of PINK1 is required; however, how do neurons maintain a steady supply of this protein at the distal axons? In the study highlighted here, Harbauer et al. show that axonal mitophagy is supported by local translation of Pink1 mRNA that is co-transported with mitochondria to the distal ends of the neuron. This neuronal-specific pathway provides a continuous supply of PINK1 to sustain mitophagy.
    Keywords:  Autophagy; mitochondria; neurodegeneration; neuron; stress
  15. J Inherit Metab Dis. 2022 May 11.
      Primary mitochondrial disorders encompass a wide range of clinical presentations and a spectrum of severity. They currently lack effective disease-modifying therapies and have a high mortality and morbidity rate. It is therefore essential to know that competitively-funded research designed by academics meets core needs of people with mitochondrial disorders and their clinicians. The Priority Setting Partnerships are an established collaborative methodology that brings patients, carers and families, charity representatives and clinicians together to try to establish the most pressing and unanswered research priorities for a particular disease. We developed a web-based questionnaire, requesting all patients affected by primary mitochondrial disease, their carers, and clinicians to pose their research questions. This yielded 709 questions from 147 participants. These were grouped into overarching themes including basic biology, causation, health services, clinical management, social impacts, prognosis, prevention, symptoms, treatment, and psychological impact. Following the removal of 'answered questions' the process resulted in a list of 42 discrete, answerable questions. This was further refined by web-based ranking by the community to 24 questions. These were debated at a face-to-face workshop attended by a diverse range of patients, carers, charity representatives and clinicians to create a definitive 'Top Ten of unanswered research questions for primary mitochondrial disorders'. These Top Ten questions related to understanding biological processes, including triggers of disease onset, mechanisms underlying progression and reasons for differential symptoms between individuals with identical genetic mutations; new treatments; biomarker discovery; psychological support; and optimal management of stroke-like episodes and fatigue. This article is protected by copyright. All rights reserved.
    Keywords:  Primary mitochondrial disease; gene therapy; patient involvement; priority setting partnership; treatment
  16. Elife. 2022 May 13. pii: e74552. [Epub ahead of print]11
      Proliferating cells undergo metabolic changes in synchrony with cell cycle progression and cell division. Mitochondria provide fuel, metabolites, and ATP during different phases of the cell cycle, however it is not completely understood how mitochondrial function and the cell cycle are coordinated. CLUH is a post-transcriptional regulator of mRNAs encoding mitochondrial proteins involved in oxidative phosphorylation and several metabolic pathways. Here, we show a role of CLUH in regulating the expression of astrin, which is involved in metaphase to anaphase progression, centrosome integrity, and mTORC1 inhibition. We find that CLUH binds both the SPAG5 mRNA and its product astrin, and controls the synthesis and the stability of the full-length astrin-1 isoform. We show that CLUH interacts with astrin-1 specifically during interphase. Astrin-depleted cells show mTORC1 hyperactivation and enhanced anabolism. On the other hand, cells lacking CLUH show decreased astrin levels and increased mTORC1 signaling, but cannot sustain anaplerotic and anabolic pathways. In absence of CLUH, cells fail to grow during G1, and progress faster through the cell cycle, indicating dysregulated matching of growth, metabolism and cell cycling. Our data reveal a role of CLUH in coupling growth signaling pathways and mitochondrial metabolism with cell cycle progression.
    Keywords:  cell biology; human
  17. Front Neurosci. 2022 ;16 846425
      To identify conserved components of synapse function that are also associated with human diseases, we conducted a genetic screen. We used the Drosophila melanogaster neuromuscular junction (NMJ) as a model. We employed RNA interference (RNAi) on selected targets and assayed synapse function and plasticity by electrophysiology. We focused our screen on genetic factors known to be conserved from human neurological or muscle functions (300 Drosophila lines screened). From our screen, knockdown of a Mitochondrial Complex I (MCI) subunit gene (ND-20L) lowered levels of NMJ neurotransmission. Due to the severity of the phenotype, we studied MCI function further. Knockdown of core MCI subunits concurrently in neurons and muscle led to impaired neurotransmission. We localized this neurotransmission function to the muscle. Pharmacology targeting MCI phenocopied the impaired neurotransmission phenotype. Finally, MCI subunit knockdowns or pharmacological inhibition led to profound cytological defects, including reduced NMJ growth and altered NMJ morphology. Mitochondria are essential for cellular bioenergetics and produce ATP through oxidative phosphorylation. Five multi-protein complexes achieve this task, and MCI is the largest. Impaired Mitochondrial Complex I subunits in humans are associated with disorders such as Parkinson's disease, Leigh syndrome, and cardiomyopathy. Together, our data present an analysis of Complex I in the context of synapse function and plasticity. We speculate that in the context of human MCI dysfunction, similar neuronal and synaptic defects could contribute to pathogenesis.
    Keywords:  Drosophila; Mitochondrial Complex I; NMJ – neuromuscular junction; neurodevelopment; neurotransmission; synapse; synaptic development; synaptic dysfunction
  18. FASEB J. 2022 May;36 Suppl 1
      The mechanistic target of rapamycin complex 1 (mTORC1) senses diverse signals to regulate cell growth and metabolism. The complex is present at the plasma membrane, nucleus, lysosomes, and the outer mitochondrial membrane. Such spatial compartmentation has been suggested to enhance signaling efficiency and specificity. For instance, we recently discovered nuclear mTORC1 activity, which is distinctly regulated from the canonical lysosomal mTORC1 (Zhou et al., 2020). Previous studies have shown that mTOR is present at the outer mitochondrial membrane (OMM), but it is not clear whether mTORC1 is active at this location and what the functional consequences are. To investigate this, we targeted our FRET-based mTORC1 activity reporter, TORCAR (Zhou et al., 2015), to the OMM and probed the subcellular activity of mTORC1. We found that platelet-derived growth factor (PDGF) stimulation increases mTORC1 activity at the OMM in addition to at the lysosome and in the nucleus, whereas insulin specifically stimulates mTORC1 activity at the OMM without affecting the lysosomal and nuclear activities. We further dissected the regulation of mitochondrial mTORC1 activity and applied a novel approach of identifying new mTORC1 substrates. Elucidating the signaling events that lead to subcellular mTORC1 activity at mitochondria and its downstream functions will increase our understanding of the roles that mTORC1 may play in diseases associated with altered metabolism or mitochondrial dysfunction, such as diabetes and cancer.
  19. J Clin Transl Hepatol. 2022 Apr 28. 10(2): 321-328
      Defects in mitochondria are responsible for various genetic and acquired diseases. Mitochondrial transplantation, a method that involves introduction of healthy donor mitochondria into cells with dysfunctional mitochondria, could offer a novel approach to treat such diseases. Some studies have demonstrated the therapeutic benefit of mitochondrial transplantation and targeted delivery in vivo and in vitro within hepatocytes and the liver. This review discusses the issues regarding isolation and delivery of mitochondria to hepatocytes and the liver, and examines the existing literature in order to elucidate the utility and practicality of mitochondrial transplantation in the treatment of liver disease. Studies reviewed demonstrate that mitochondrial uptake could specifically target hepatocytes, address the challenge of non-specific localization of donor mitochondria, and provide evidence of changes in liver function following injection of mitochondria into mouse and rat disease models. While potential benefits and advantages of mitochondrial transplantation are evident, more research is needed to determine the practicality of mitochondrial transplantation for the treatment of genetic and acquired liver diseases.
    Keywords:  Hepatocytes; In vitro techniques; Liver; Mitochondria; Transplantation
  20. FASEB J. 2022 May;36 Suppl 1
      Mitochondrial DNA (mtDNA) encodes thirteen essential proteins of the oxidative phosphorylation system, responsible for the major production of ATP in the cell. Therefore, damages to the mitochondrial genome result in energy deprivation, which may in turn onset human diseases. Notably, due to its proximity to the electron transport chain, mtDNA remains exposed to damage by reactive oxygen species, thus the maintenance of its integrity requires a robust repair system. Until recently, DNA polymerase gamma (Pol γ) has been the only polymerase identified in mitochondria, bearing responsibility for efficient replication as well as post-replication repair of the genome. We have previously suggested that the division of the roles of Pol γ may be controlled by the association of its catalytic subunit, Pol γA, with the accessory subunit Pol γB, such that the holoenzyme is engaged in the processive mtDNA replication, whereas, alone, Pol γA may serve the repair processes. Recently, the major repair polymerase of the nucleus, Pol β, has been discovered to also localize in mitochondria, which raises the question for its competition or cooperation with Pol γ in the mtDNA repair processes. To address this, we have tested in vitro the efficiency of DNA synthesis by the two polymerases, separately and in combination, using various DNA substrates. In agreement with previous reports, we did not observe any indication of a functional interaction between the Pol γ holoenzyme and Pol β. We did, however, observe a cooperative activity of Pol β with the Pol γA subunit. In conclusion, our results suggest that the repair of mtDNA may entail a synergistic activity of the catalytic subunit of Pol γ and Pol β.
  21. FASEB J. 2022 May;36 Suppl 1
      Mitochondria undergo coordinated rounds of fusion and fission that are critical for maintaining the functional integrity of this essential organelle. While a growing number of proteins have been identified as important regulators of mitochondrial dynamics, the direct role of membrane lipid composition during the fusion and fission processes is poorly understood. To address these shortcomings, we devised a protein-engineering platform that allows for the acute remodeling of structural phospholipids within the outer mitochondrial membrane (OMM) of intact cells. Specifically, we modified a bacterial phospholipase C (Bacillus cereus (Bc)PI-PLC) to initiate the rapid hydrolysis of phosphatidylinositol (PI) and locally generate diacylglycerol (DAG); an important intracellular signaling molecule and metabolic precursor that is used in diverse lipid biosynthetic pathways. Spatial restriction of enzyme activity was achieved using a chemically inducible system consisting of a rapamycin-dependent dimerization module (FKBP-BcPI-PLC) along with an OMM targeting sequence tagged with the FKBP-rapamycin binding domain (OMM-FRB). Using these unique molecular tools, we show that recruitment of FKBP-BcPI-PLC to the OMM not only causes the expected local accumulation of DAG, but also initiates the rapid and uniform fragmentation of the mitochondrial network. Mitochondrial fission induced by FKBP-BcPI-PLC is accompanied by profound swelling of the mitochondrial matrix along with vesiculation of the inner mitochondrial membrane (IMM) and a general loss of cristae, which all occur within minutes of tethering FKBP-BcPI-PLC to the OMM. Expression of dominant-negative constructs targeting essential GTPases known to regulate OMM fission suggest that both dynamin-related protein 1 (Drp1) and dynamin 2 (Dnm2) work together to drive efficient BcPI-PLC-induced mitochondrial division. However, results using a validated Drp1 knockout cell line show that the loss of Drp1 alone is sufficient to prevent the mitochondrial fragmentation initiated by FKBP-BcPI-PLC recruitment, indicating that Drp1 likely functions upstream or independent of Dnm2 in this context. Interestingly, unlike the induced OMM fission, removal of Drp1 from cells does not prevent the matrix swelling or OMM constrictions observed in response to acute generation of DAG within the OMM. Ongoing experiments are now focused on characterizing new methods to sequentially metabolize the DAG generated within the OMM as well as investigate how local lipid composition influences the binding and oligomerization of membrane-shaping proteins that may function in concert with Drp1 to regulate mitochondrial remodeling. Overall, these studies establish a direct relationship between lipid metabolism within the OMM and clinically relevant morphological changes that are known to manifest in mitochondrial-associated diseases.
  22. Sci Adv. 2022 May 13. 8(19): eabl8716
      Several subunits in the matrix domain of mitochondrial complex I (CI) have been posited to be redox sensors for CI, but how elevated levels of reactive oxygen species (ROS) impinge on CI assembly is unknown. We report that genetic disruption of the mitochondrial NADPH-generating enzyme, isocitrate dehydrogenase 2 (IDH2), in Drosophila flight muscles results in elevated ROS levels and impairment of assembly of the oxidative phosphorylation system (OXPHOS). Mechanistically, this begins with an inhibition of biosynthesis of the matrix domain of CI and progresses to involve multiple OXPHOS complexes. Despite activation of multiple compensatory mechanisms, including enhanced coenzyme Q biosynthesis and the mitochondrial unfolded protein response, ferroptotic cell death ensues. Disruption of enzymes that eliminate hydrogen peroxide, but not those that eliminate the superoxide radical, recapitulates the phenotype, thereby implicating hydrogen peroxide as the signaling molecule involved. Thus, IDH2 modulates the assembly of the matrix domain of CI and ultimately that of the entire OXPHOS.
  23. FASEB J. 2022 May;36 Suppl 1
      NAD+ is an essential coenzyme found in all living cells. NAD+ concentrations decline during aging, but whether this reflects impaired production or accelerated consumption remains unclear. Here we employed isotope tracing and mass spectrometry to probe NAD+ metabolism across tissues in aged mice. In 25-month-old mice, we observe modest tissue NAD+ depletion (median decrease ~30%) without significant changes in circulating NAD+ precursors. Isotope tracing showed unimpaired synthesis of circulating nicotinamide from tryptophan, and maintained flux of circulating nicotinamide into tissue NAD+ pools. Although absolute NAD+ biosynthetic flux was maintained in most tissues of aged mice, fractional tissue NAD+ labeling from infused labeled nicotinamide was modestly accelerated, consistent with increased activity of NAD+ consuming enzymes. Long-term calorie restriction partially mitigated age-associated NAD+ decline despite decreasing NAD+ synthesis, suggesting that calorie restriction reduces NAD+ consumption. Acute inflammatory stress induced by LPS decreased NAD+ by impairing synthesis in both young and aged mice. Thus, age-related decline in NAD+ is relatively subtle and driven by increased NAD+ consumer activity rather than impaired production.
  24. FASEB J. 2022 May;36 Suppl 1
      Mitochondria are important organelle which regulate adenosine triphosphate (ATP) production, intracellular calcium buffering, cell survival and apoptosis. They are known to deliver the potential therapeutic role in injured cells through transcellular transfer via extracellular vesicles (EVs), gap junctions, and tunneling nanotubes (TNTs). Astrocytes secrete numerous factors that promote neuron survival, synapse formation, and plasticity. Recent studies have demonstrated that astrocytes transfer mitochondria into damaged neurons to enhance cell viability and recovery. In this study, we observed that treatment of isolated mitochondria from rat primary astrocytes enhance cell viability and ameliorate H2 O2 -damaged neurons. Interestingly, the isolated astrocytic mitochondria increased cell number in damaged neurons but not normal neurons, even though the mitochondrial transfer efficiency was no difference between them. Furthermore, this effect showed in astrocytic mitochondrial transplantation to rat middle cerebral artery occlusion (MCAO) models. These findings suggest that mitochondrial transfer therapy can be used to acute ischemic stroke and other diseases treatment.
  25. Cell Rep. 2022 May 10. pii: S2211-1247(22)00554-X. [Epub ahead of print]39(6): 110787
      The mechanisms that generate robust ionic oscillation in circadian pacemaker neurons are under investigation. Here, we demonstrate critical functions of the mitochondrial cation antiporter leucine zipper-EF-hand-containing transmembrane protein 1 (LETM1), which exchanges K+/H+ in Drosophila and Ca2+/H+ in mammals, in circadian pacemaker neurons. Letm1 knockdown in Drosophila pacemaker neurons reduced circadian cytosolic H+ rhythms and prolonged nuclear PERIOD/TIMELESS expression rhythms and locomotor activity rhythms. In rat pacemaker neurons in the hypothalamic suprachiasmatic nucleus (SCN), circadian rhythms in cytosolic Ca2+ and Bmal1 transcription were dampened by Letm1 knockdown. Mitochondrial Ca2+ uptake peaks late during the day were also observed in rat SCN neurons following photolytic elevation of cytosolic Ca2+. Since cation transport by LETM1 is coupled to mitochondrial energy synthesis, we propose that LETM1 integrates metabolic, ionic, and molecular clock rhythms in the central clock system in both invertebrates and vertebrates.
    Keywords:  CP: Metabolism; CP: Neuroscience; caged Ca(2+) compound; circadian H(+) rhythms; clock genes; lateral neurons; mitochondrial calcium imaging; proton imaging
  26. Genet Med. 2022 May 09. pii: S1098-3600(22)00748-1. [Epub ahead of print]
    Genomics England Research Consortium
      PURPOSE: Exome and genome sequencing have drastically accelerated novel disease gene discoveries. However, discovery is still hindered by myriad variants of uncertain significance found in genes of undetermined biological function. This necessitates intensive functional experiments on genes of equal predicted causality, leading to a major bottleneck.METHODS: We apply the loss-of-function observed/expected upper-bound fraction metric of intolerance to gene inactivation to curate a list of predicted haploinsufficient disease genes. Using data from the 100,000 Genomes Project, we adopt a gene-to-patient approach that matches de novo loss-of-function variants in constrained genes to patients with rare disease. Through large-scale aggregation of data, we reduce excess analytical noise currently hindering novel discoveries.
    RESULTS: Results from 13,949 trios revealed 643 rare, de novo predicted loss-of-function events filtered from 1044 loss-of-function observed/expected upper-bound fraction-constrained genes. A total of 168 variants occurred within 126 genes without a known disease-gene relationship. Of these, 27 genes had >1 kindred affected, and for 18 of these genes, multiple kindreds had overlapping phenotypes. Two years after initial analysis, 11 of 18 (61%) of these genes have been independently published as novel disease gene discoveries.
    CONCLUSION: Using large cohorts and adopting gene-based approaches can rapidly and objectively accelerate dominantly inherited novel gene discovery by targeting the most appropriate genes for functional validation.
    Keywords:  Diagnostic uplift; Disease genes; Genome sequencing; Mendelian disease; Novel gene discovery
  27. FASEB J. 2022 May;36 Suppl 1
      Many human diseases are caused by mutations that perturb metabolism at the cellular level and result in tissue dysfunction. Some metabolic perturbations result in disease by interrupting the canonical functions of the metabolic network: producing energy, generating precursors for macromolecular synthesis, maintaining redox balance, disposing of waste, etc. Others interfere with processes beyond the conventional metabolic network, interfering with signaling and gene expression networks. Understanding these pathological states of metabolic perturbation may help us develop rational approaches to normalize metabolism and restore health. We study two types of diseases characterized by metabolic dysfunction: inborn errors of metabolism and cancer. I will discuss ongoing work in these diseases that seeks to characterize abnormal metabolic states directly in human subjects, then uses experimental models to explore disease mechanisms and propose potential therapies. I will emphasize methods in metabolomics and stable isotope tracing that allow us observe metabolic phenotypes in intact systems relevant to physiology and disease, highlighting recent work on tumor metabolism in patients and genetically-defined metabolic anomalies that interfere with mammalian developmental programs.
  28. Cell Rep. 2022 May 10. pii: S2211-1247(22)00580-0. [Epub ahead of print]39(6): 110809
      Hypertrophic cardiomyopathy (HCM) is a genetic heart disease that is characterized by unexplained segmental hypertrophy that is usually most pronounced in the septum. While sarcomeric gene mutations are often the genetic basis for HCM, the mechanistic origin for the heterogeneous remodeling remains largely unknown. A better understanding of the gene networks driving the cardiomyocyte (CM) hypertrophy is required to improve therapeutic strategies. Patients suffering from HCM often receive a septal myectomy surgery to relieve outflow tract obstruction due to hypertrophy. Using single-cell RNA sequencing (scRNA-seq) on septal myectomy samples from patients with HCM, we identify functional links between genes, transcription factors, and cell size relevant for HCM. The data show the utility of using scRNA-seq on the human hypertrophic heart, highlight CM heterogeneity, and provide a wealth of insights into molecular events involved in HCM that can eventually contribute to the development of enhanced therapies.
    Keywords:  CP: Molecular biology; HCM; cardiomyocyte; cell size; flow cytometry; hypertrophic cardiomyopathy; myectomy; scRNA-seq; sequencing; single cell
  29. Nat Commun. 2022 May 13. 13(1): 2673
      The folded mitochondria inner membrane-cristae is the structural foundation for oxidative phosphorylation (OXPHOS) and energy production. By mechanically simulating mitochondria morphogenesis, we speculate that efficient sculpting of the cristae is organelle non-autonomous. It has long been inferred that folding requires buckling in living systems. However, the tethering force for cristae formation and regulation has not been identified. Combining electron tomography, proteomics strategies, super resolution live cell imaging and mathematical modeling, we reveal that the mitochondria localized actin motor-myosin 19 (Myo19) is critical for maintaining cristae structure, by associating with the SAM-MICOS super complex. We discover that depletion of Myo19 or disruption of its motor activity leads to altered mitochondria membrane potential and decreased OXPHOS. We propose that Myo19 may act as a mechanical tether for effective ridging of the mitochondria cristae, thus sustaining the energy homeostasis essential for various cellular functions.
  30. Nature. 2022 May 12.
      DddA-derived cytosine base editors (DdCBEs), which are fusions of the split-DddA halves and transcription activator-like effector (TALE) array proteins, enable targeted C·G-to- T·A conversions in mitochondrial DNA1. However, its genome-wide specificity is poorly understood. Here we show that the mitochondrial base editor induces extensive off-target editing in the nuclear genome. Genome-wide, unbiased analysis of its editome reveals hundreds of off-target sites that are TALE array sequence (TAS)-dependent or -independent. TAS-dependent off-target sites in the nuclear DNA (nDNA) are often specified by only one of the two TALE repeats, challenging the principle that DdCBEs are guided by a paired TALE proteins positioned in close proximity. TAS-independent nDNA off-target sites are frequently shared among DdCBEs with distinct TALE arrays. Notably, they co-localize strongly with CTCF-binding sites and are enriched in TAD boundaries. We also engineered DdCBE to alleviate such off-target effect. Collectively, our results have implications for the use of DdCBEs in basic research and therapeutic applications, and suggest the need to thoroughly define and evaluate the off-target effects of base editing tools.
  31. Nat Commun. 2022 May 11. 13(1): 2592
      Sequencing cases without matched healthy controls hinders prioritization of germline disease-predisposition genes. To circumvent this problem, genotype summary counts from public data sets can serve as controls. However, systematic inflation and false positives can arise if confounding factors are not controlled. We propose a framework, consistent summary counts based rare variant burden test (CoCoRV), to address these challenges. CoCoRV implements consistent variant quality control and filtering, ethnicity-stratified rare variant association test, accurate estimation of inflation factors, powerful FDR control, and detection of rare variant pairs in high linkage disequilibrium. When we applied CoCoRV to pediatric cancer cohorts, the top genes identified were cancer-predisposition genes. We also applied CoCoRV to identify disease-predisposition genes in adult brain tumors and amyotrophic lateral sclerosis. Given that potential confounding factors were well controlled after applying the framework, CoCoRV provides a cost-effective solution to prioritizing disease-risk genes enriched with rare pathogenic variants.
  32. Curr Heart Fail Rep. 2022 May 13.
      PURPOSE OF THE REVIEW: This review summarizes current understanding on the roles of nicotinamide adenine dinucleotide (NAD+) metabolism in the pathogeneses and treatment development of metabolic and cardiac diseases.RECENT FINDINGS: NAD+ was identified as a redox cofactor in metabolism and a co-substrate for a wide range of NAD+-dependent enzymes. NAD+ redox imbalance and depletion are associated with many pathologies where metabolism plays a key role, for example cardiometabolic diseases. This review is to delineate the current knowledge about harnessing NAD+ metabolism as potential therapy for cardiometabolic diseases. The review has summarized how NAD+ redox imbalance and depletion contribute to the pathogeneses of cardiometabolic diseases. Therapeutic evidence involving activation of NAD+ synthesis in pre-clinical and clinical studies was discussed. While activation of NAD+ synthesis shows great promise for therapy, the field of NAD+ metabolism is rapidly evolving. Therefore, it is expected that new mechanisms will be discovered as therapeutic targets for cardiometabolic diseases.
    Keywords:  Cardiometabolic diseases; Heart failure; NAD+ metabolism; Redox balance
  33. Circ Heart Fail. 2022 May 11. CIRCHEARTFAILURE121009521
      BACKGROUND: Defects in energetics are thought to be central to the pathophysiology of hypertrophic cardiomyopathy (HCM); yet, the determinants of ATP availability are not known. The purpose of this study is to ascertain the nature and extent of metabolic reprogramming in human HCM, and its potential impact on contractile function.METHODS: We conducted proteomic and targeted, quantitative metabolomic analyses on heart tissue from patients with HCM and from nonfailing control human hearts.
    RESULTS: In the proteomic analysis, the greatest differences observed in HCM samples compared with controls were increased abundances of extracellular matrix and intermediate filament proteins and decreased abundances of muscle creatine kinase and mitochondrial proteins involved in fatty acid oxidation. These differences in protein abundance were coupled with marked reductions in acyl carnitines, byproducts of fatty acid oxidation, in HCM samples. Conversely, the ketone body 3-hydroxybutyrate, branched chain amino acids, and their breakdown products, were all significantly increased in HCM hearts. ATP content, phosphocreatine, nicotinamide adenine dinucleotide and its phosphate derivatives, NADP and NADPH, and acetyl CoA were also severely reduced in HCM compared with control hearts. Functional assays performed on human skinned myocardial fibers demonstrated that the magnitude of observed reduction in ATP content in the HCM samples would be expected to decrease the rate of cross-bridge detachment. Moreover, left atrial size, an indicator of diastolic compliance, was inversely correlated with ATP content in hearts from patients with HCM.
    CONCLUSIONS: HCM hearts display profound deficits in nucleotide availability with markedly reduced capacity for fatty acid oxidation and increases in ketone bodies and branched chain amino acids. These results have important therapeutic implications for the future design of metabolic modulators to treat HCM.
    Keywords:  cardiomyopathy, hypertrophic; mitochondrial proteins; phosphate; phosphocreatine; proteomics