bims-mitdis Biomed News
on Mitochondrial disorders
Issue of 2022‒03‒06
fifty-six papers selected by
Catalina Vasilescu
University of Helsinki

  1. Brain. 2022 Mar 04. pii: awab303. [Epub ahead of print]
      Mitochondria are essential organelles found in every eukaryotic cell, required to convert food into usable energy. Therefore, it is not surprising that mutations in either mtDNA or nuclear DNA-encoded genes of mitochondrial proteins cause diseases affecting the oxidative phosphorylation system, which are heterogeneous from a clinical, genetic, biochemical and molecular perspective and can affect patients at any age. Despite all this, it is surprising that our understanding of the mechanisms governing mitochondrial gene expression and its associated pathologies remain superficial and therapeutic interventions largely unexplored. We recently showed that loss of the mitochondrial matrix protease caseinolytic protease proteolytic subunit (CLPP) ameliorates phenotypes in cells characterized by defects in oxidative phosphorylation maintenance. Here, we build upon this finding by showing that CLPP depletion is indeed beneficial in vivo for various types of neuronal populations, including Purkinje cells in the cerebellum and cortical and hippocampal neurons in the forebrain, as it strongly improves distinct phenotypes of mitochondria encephalopathy, driven by the deficiency of the mitochondrial aspartyl tRNA synthase DARS2. In the absence of CLPP, neurodegeneration of DARS2-deficient neurons is delayed as they present milder oxidative phosphorylation dysfunction. This in turn leads to a decreased neuroinflammatory response and significantly improved motor functions in both double-deficient models (Purkinje cell-specific or forebrain neuron-specific Dars2/Clpp double knockout mice). We propose that diminished turnover of respiratory complex I caused by the loss of CLPP is behind the improved phenotype in Dars2/Clpp double knockout animals, even though this intervention might not restore respiratory complex I activity but rather improve mitochondrial cristae morphology or help maintain the NAD+/NADH ratio inside mitochondria. These results also open the possibility of targeting CLPP activity in many other mitochondrial encephalopathies characterized by respiratory complex I instability.
    Keywords:  CLPP protease; DARS2 deficiency; LBSL; mitochondrial diseases
  2. Front Cardiovasc Med. 2021 ;8 808115
      Mitochondria is a ubiquitous, energy-supplying (ATP-based) organelle found in nearly all eukaryotes. It acts as a "power plant" by producing ATP through oxidative phosphorylation, providing energy for the cell. The bioenergetic functions of mitochondria are regulated by nuclear genes (nDNA). Mitochondrial DNA (mtDNA) and respiratory enzymes lose normal structure and function when nuclear genes encoding the related mitochondrial factors are impaired, resulting in deficiency in energy production. Massive generation of reactive oxygen species and calcium overload are common causes of mitochondrial diseases. The mitochondrial depletion syndrome (MDS) is associated with the mutations of mitochondrial genes in the nucleus. It is a heterogeneous group of progressive disorders characterized by the low mtDNA copy number. TK2, FBXL4, TYPM, and AGK are genes known to be related to MDS. More recent studies identified new mutation loci associated with this disease. Herein, we first summarize the structure and function of mitochondria, and then discuss the characteristics of various types of MDS and its association with cardiac diseases.
    Keywords:  ATP; cardiac disease; mitochondrial DNA depletion syndrome; mtDNA; nuclear gene mutation
  3. Circulation. 2022 Mar 03.
      Background: In most eukaryotic cells, the mitochondrial DNA (mtDNA) is uniparentally transmitted and present in multiple copies derived from the clonal expansion of maternally inherited mtDNA. All copies are therefore near-identical, or homoplasmic. The presence of more than one mtDNA variant in the same cytoplasm can arise naturally or result from new medical technologies aimed at preventing mitochondrial genetic diseases and improving fertility. The latter is called divergent non-pathological mtDNAs heteroplasmy (DNPH). We hypothesized that DNPH is maladaptive and usually prevented by the cell. Methods: We engineered and characterized DNPH mice throughout their lifespan using transcriptomic, metabolomic, biochemical, physiological and phenotyping techniques. We focused on in vivo imaging techniques for non-invasive assessment of cardiac and pulmonary energy metabolism. Results: We show that DNPH impairs mitochondrial function, with profound consequences in critical tissues that cannot resolve heteroplasmy, particularly cardiac and skeletal muscle. Progressive metabolic stress in these tissues leads to severe pathology in adulthood, including pulmonary hypertension and heart failure, skeletal muscle wasting, frailty, and premature death. Symptom severity is strongly modulated by the nuclear context. Conclusions: Medical interventions that may generate DNPH should address potential incompatibilities between donor and recipient mtDNA.
  4. Elife. 2022 Mar 02. pii: e75658. [Epub ahead of print]11
      Mitochondrial biogenesis has two major steps: the transcriptional activation of nuclear genome-encoded mitochondrial proteins and the import of nascent mitochondrial proteins that are synthesized in the cytosol. These nascent mitochondrial proteins are aggregation-prone and can cause cytosolic proteostasis stress. The transcription factor-dependent transcriptional regulations and the TOM-TIM complex-dependent import of nascent mitochondrial proteins have been extensively studied. Yet, little is known regarding how these two steps of mitochondrial biogenesis coordinate with each other to avoid the cytosolic accumulation of these aggregation-prone nascent mitochondrial proteins. Here we show that in budding yeast, Tom70, a conserved receptor of the TOM complex, moonlights to regulate the transcriptional activity of mitochondrial proteins. Tom70's transcription regulatory role is conserved in Drosophila. The dual roles of Tom70 in both transcription/biogenesis and import of mitochondrial proteins allow the cells to accomplish mitochondrial biogenesis without compromising cytosolic proteostasis. The age-related reduction of Tom70, caused by reduced biogenesis and increased degradation of Tom70, is associated with the loss of mitochondrial membrane potential, mtDNA, and mitochondrial proteins. While loss of Tom70 accelerates aging and age-related mitochondrial defects, overexpressing TOM70 delays these mitochondrial dysfunctions and extends the replicative lifespan. Our results reveal unexpected roles of Tom70 in mitochondrial biogenesis and aging.
    Keywords:  S. cerevisiae; cell biology
  5. J Neurol. 2022 Mar 02.
      Mitochondrial disorders are a group of clinically and genetically heterogeneous multisystem disorders and peripheral neuropathy is frequently described in the context of mutations in mitochondrial-related nuclear genes. This study aimed to identify the causative mutations in mitochondrial-related nuclear genes in suspected hereditary peripheral neuropathy patients. We enrolled a large Japanese cohort of clinically suspected hereditary peripheral neuropathy patients who were mutation negative in the prescreening of the known Charcot-Marie-Tooth disease-causing genes. We performed whole-exome sequencing on 247 patients with autosomal recessive or sporadic inheritance for further analysis of 167 mitochondrial-related nuclear genes. We detected novel bi-allelic likely pathogenic/pathogenic variants in four patients, from four mitochondrial-related nuclear genes: pyruvate dehydrogenase beta-polypeptide (PDHB), mitochondrial poly(A) polymerase (MTPAP), hydroxyacyl-CoA dehydrogenase/3-ketoacyl-CoA thiolase/enoyl-CoA hydratase, beta subunit (HADHB), and succinate-CoA ligase ADP-forming beta subunit (SUCLA2). All these patients showed sensory and motor axonal polyneuropathy, combined with central nervous system or multisystem involvements. The pathological analysis of skeletal muscles revealed mild neurogenic changes without significant mitochondrial abnormalities. Targeted screening of mitochondria-related nuclear genes should be considered for patients with complex hereditary axonal polyneuropathy, accompanied by central nervous system dysfunctions, or with unexplainable multisystem disorders.
    Keywords:  Mitochondrial disease; Nuclear genes; Peripheral neuropathy; Whole-exome sequencing
  6. Mitochondrion. 2022 Feb 24. pii: S1567-7249(22)00019-8. [Epub ahead of print]64 45-58
      Mitochondrial diseases are a group of genetic disorders characterized by dysfunctional mitochondria. Within eukaryotic cells, mitochondria contain their own ribosomes, which synthesize small amounts of proteins, all of which are essential for the biogenesis of the oxidative phosphorylation system. The ribosome is an evolutionarily conserved macromolecular machine in nature both from a structural and functional point of view, universally responsible for the synthesis of proteins. Among the diseases afflicting humans, those of ribosomal origin - either cytoplasmic ribosomes (80S) or mitochondrial ribosomes (70S) - are relevant. These are inherited or acquired diseases most commonly caused by either ribosomal protein haploinsufficiency or defects in ribosome biogenesis. Here we review the scientific literature about the recent advances on changes in mitochondrial ribosomal structural and assembly proteins that are implicated in primary mitochondrial diseases and neurodegenerative disorders, and their possible connection with metalloid pollution and toxicity, with a focus on MRPL44, NAM9 (MNA6) and GEP3 (MTG3), whose lack or defect was associated with resistance to tellurite. Finally, we illustrate the suitability of yeast Saccharomyces cerevisiae (S. cerevisiae) and the nematode Caenorhabditis elegans (C. elegans) as model organisms for studying mitochondrial ribosome dysfunctions including those involved in human diseases.
    Keywords:  Alzheimer’s disease; Mitochondrial diseases; Mitochondrial ribosomal protein genes; Mitochondrial ribosome; Yeast and C. elegans model organisms
  7. Mol Cell. 2022 Mar 03. pii: S1097-2765(22)00108-3. [Epub ahead of print]82(5): 1066-1077.e7
      The mitochondrial pyruvate dehydrogenase complex (PDC) translocates into the nucleus, facilitating histone acetylation by producing acetyl-CoA. We describe a noncanonical pathway for nuclear PDC (nPDC) import that does not involve nuclear pore complexes (NPCs). Mitochondria cluster around the nucleus in response to proliferative stimuli and tether onto the nuclear envelope (NE) via mitofusin-2 (MFN2)-enriched contact points. A decrease in nuclear MFN2 levels decreases mitochondria tethering and nPDC levels. Mitochondrial PDC crosses the NE and interacts with lamin A, forming a ring below the NE before crossing through the lamin layer into the nucleoplasm, in areas away from NPCs. Effective blockage of NPC trafficking does not decrease nPDC levels. The PDC-lamin interaction is maintained during cell division, when lamin depolymerizes and disassembles before reforming daughter nuclear envelopes, providing another pathway for nPDC entry during mitosis. Our work provides a different angle to understanding mitochondria-to-nucleus communication and nuclear metabolism.
    Keywords:  acetylation; cell cycle; lamin; metabolism; mitochondria; mitofusin; nucleus; protein trafficking; pyruvate dehydrogenase complex; tethering
  8. Sci Transl Med. 2022 Mar 02. 14(634): eabl6992
      SERAC1 deficiency is associated with the mitochondrial 3-methylglutaconic aciduria with deafness, (hepatopathy), encephalopathy, and Leigh-like disease [MEGD(H)EL] syndrome, but the role of SERAC1 in mitochondrial physiology remains unknown. Here, we generated Serac1-/- mice that mimic the major diagnostic clinical and biochemical phenotypes of the MEGD(H)EL syndrome. We found that SERAC1 localizes to the outer mitochondrial membrane and is a protein component of the one-carbon cycle. By interacting with the mitochondrial serine transporter protein SFXN1, SERAC1 facilitated and was required for SFXN1-mediated serine transport from the cytosol to the mitochondria. Loss of SERAC1 impaired the one-carbon cycle and disrupted the balance of the nucleotide pool, which led to primary mitochondrial DNA (mtDNA) depletion in mice, HEK293T cells, and patient-derived immortalized lymphocyte cells due to insufficient supply of nucleotides. Moreover, both in vitro and in vivo supplementation of nucleosides/nucleotides restored mtDNA content and mitochondrial function. Collectively, our findings suggest that MEGD(H)EL syndrome shares both clinical and molecular features with the mtDNA depletion syndrome, and nucleotide supplementation may be an effective therapeutic strategy for MEGD(H)EL syndrome.
  9. J Vis Exp. 2022 Feb 10.
      Most of the cell's energy is obtained through the degradation of glucose, fatty acids, and amino acids by different pathways that converge on the mitochondrial oxidative phosphorylation (OXPHOS) system, which is regulated in response to cellular demands. The lipid molecule Coenzyme Q (CoQ) is essential in this process by transferring electrons to complex III in the electron transport chain (ETC) through constant oxidation/reduction cycles. Mitochondria status and, ultimately, cellular health can be assessed by measuring ETC oxygen consumption using respirometric assays. These studies are typically performed in established or primary cell lines that have been cultured for several days. In both cases, the respiration parameters obtained may have deviated from normal physiological conditions in any given organ or tissue. Additionally, the intrinsic characteristics of cultured single fibers isolated from skeletal muscle impede this type of analysis. This paper presents an updated and detailed protocol for the analysis of respiration in freshly isolated mitochondria from mouse skeletal muscle. We also provide solutions to potential problems that could arise at any step of the process. The method presented here could be applied to compare oxygen consumption rates in diverse transgenic mouse models and study the mitochondrial response to drug treatments or other factors such as aging or sex. This is a feasible method to respond to crucial questions about mitochondrial bioenergetics metabolism and regulation.
  10. Clin Case Rep. 2022 Feb;10(2): e05401
      Isoleucyl-tRNA synthetase 2 (IARS2) encodes mitochondrial isoleucine-tRNA synthetase. Pathogenic variants in the IARS2 gene are associated with mitochondrial disease. We report a female with IARS2 compound heterozygous variants, p.Val499Glyfs*14 and p.Arg784Trp who presented with infantile spasms, Leigh disease and Wolff-Parkinson White (WPW) pattern. This report expands the phenotypic spectrum of IARS2-related disease.
    Keywords:  CAGSSS; IARS2; WPW; West syndrome; mitochondrial disease
  11. Development. 2022 Mar 03. pii: dev.200458. [Epub ahead of print]
      The mitochondrial matrix AAA+ Lon protease (LONP1) degrades misfolded or unassembled proteins, which play a pivotal role in mitochondrial quality control. During heart development, a metabolic shift from anaerobic glycolysis to mitochondrial oxidative phosphorylation takes place, and this process relies highly on functional mitochondria. However, the relationship between mitochondrial quality control machinery and metabolic shifts is elusive. Here, we interfered with mitochondrial quality control by inactivating Lonp1 in embryonic cardiac tissue and found severely impaired heart development, leading to embryonic lethality. Mitochondrial swelling, cristae loss and abnormal protein aggregates were evident in the mitochondria of Lonp1-deficient cardiomyocytes. Accordingly, the p-eIF2α-ATF4 pathway was triggered, and nuclear translocation of ATF4 was observed. We further demonstrated that ATF4 negatively regulates the expression of Tfam while promoting that of Glut1, which was responsible for the disruption of the metabolic shift to oxidative phosphorylation. Meanwhile, elevated levels of reactive oxygen species were observed in Lonp1 mutant cardiomyocytes. This study revealed that LONP1 safeguards metabolic shifts in the developing heart by controlling mitochondrial protein quality and implies that disrupted mitochondrial quality control may cause prenatal cardiomyopathy.
    Keywords:  ATF4; Glycolysis; Heart development; LONP1; Metabolic shift; Mitochondrial quality control; Oxidative phosphorylation
  12. Mol Cell. 2022 Mar 03. pii: S1097-2765(22)00119-8. [Epub ahead of print]82(5): 886-888
      Zervopoulos et al. (2022) propose a non-canonical nuclear import pathway for the functional mitochondrial pyruvate dehydrogenase complex (PDC), facilitated by dynamic MFN2-mediated tethering of mitochondria to the nuclear envelope upon exposure to proliferative stimuli.
  13. Trends Cell Biol. 2022 Feb 24. pii: S0962-8924(22)00034-4. [Epub ahead of print]
      Intracellular long-lived proteins (LLPs) provide structural support for several highly stable protein complexes and assemblies that play essential roles in ensuring cellular homeostasis and function. Recently, mitochondrial long-lived proteins (mt-LLPs) were discovered within inner mitochondria membranes (IMMs) and cristae invagination in tissues with old postmitotic cells. This observation is at odds with the fact that mitochondria are highly dynamic organelles that are continually remodeled through processes of fission, fusion, biogenesis, and multiple quality control pathways. In this opinion article, we propose that a subset of the mitochondrial proteome persists over long time frames and these mt-LLPs provide key structural support for the lifelong maintenance of mitochondrial structure.
    Keywords:  cristae ultrastructure; long-lived proteins; mitochondria; mitochondrial dynamics; protein turnover; stable structures
  14. Cell Metab. 2022 Mar 01. pii: S1550-4131(22)00045-6. [Epub ahead of print]34(3): 396-407.e6
      We conducted a double-blinded phase I clinical trial to establish whether nicotinamide adenine dinucleotide (NAD) replenishment therapy, via oral intake of nicotinamide riboside (NR), is safe, augments cerebral NAD levels, and impacts cerebral metabolism in Parkinson's disease (PD). Thirty newly diagnosed, treatment-naive patients received 1,000 mg NR or placebo for 30 days. NR treatment was well tolerated and led to a significant, but variable, increase in cerebral NAD levels-measured by 31phosphorous magnetic resonance spectroscopy-and related metabolites in the cerebrospinal fluid. NR recipients showing increased brain NAD levels exhibited altered cerebral metabolism, measured by 18fluoro-deoxyglucose positron emission tomography, and this was associated with mild clinical improvement. NR augmented the NAD metabolome and induced transcriptional upregulation of processes related to mitochondrial, lysosomal, and proteasomal function in blood cells and/or skeletal muscle. Furthermore, NR decreased the levels of inflammatory cytokines in serum and cerebrospinal fluid. Our findings nominate NR as a potential neuroprotective therapy for PD, warranting further investigation in larger trials.
    Keywords:  NAD; NR; disease modifying; experimental therapy; mitochondria; neurodegeneration; neuroprotective; nicotinamide adenine dinucleotide; parkinsonism; treatment
  15. Mol Syndromol. 2022 Feb;13(1): 64-68
      Common causes of hypoglycemia include hyperinsulinism, hormonal deficiencies, fatty acid oxidation disorders, and glycogen storage diseases; however, rare causes should also be considered for the condition. Mitochondrial complex III deficiency shows an autosomal recessive or a mitochondrial inheritance pattern. To date, mitochondrial complex III deficiency, nuclear type 3 attributable to a pathogenic variant of the UQCRB gene (MIM 615158) has been identified in only 2 pediatric patients; both presented with hypoglycemia and lactic acidosis. In this paper, we present a patient with mitochondrial complex III deficiency, nuclear type 3, UQCRB variant associated with acute hypoglycemia and lactic acidosis episodes. The male patient was admitted on the first day of life with tachypnea, metabolic acidosis, and hypoglycemia. Up to 10 years of age, he was admitted 7 times with abdominal pain, vomiting, and fever. His blood tests revealed hypoglycemia, metabolic acidosis, and hyperlactatemia. At 10 years of age, a whole-exome sequencing (WES) analysis was performed identifying a homozygous c.309_313delAGAAA (p.Glu104ArgfsTer10) pathogenic variant of the UQCRB gene. Once the common causes of hypoglycemia are excluded, it is essential to perform a WES analysis for other rare causes. Thus, rare disorders such as mitochondrial complex III deficiency can be diagnosed.
    Keywords:  Hypoglycemia; Lactic acidosis; Mitochondrial complex III deficiency; UQCRB pathogenic variant
  16. Clin Genet. 2022 Mar 04.
      Genetic defect in the nuclear encoded subunits of cytochrome c oxidase are very rare. To date, most deleterious variants affect the mitochondrially encoded subunits of complex IV and the nuclear genes encoded for assembly factors. A biallelic pathogenic variant in the mitochondrial complex IV subunit COX5A was previously reported in a couple of sibs with failure to thrive, lactic acidosis and pulmonary hypertension and a lethal phenotype. Here, we describe a second family with a 11-year-old girl presenting with failure to thrive, lactic acidosis, hypoglycemia and short stature. Clinical exome revealed the homozygous missense variant c.266T>G in COX5A, which produces a drop of the corresponding protein and a reduction of the COX activity. Compared to the previous observation, this girl showed an attenuated metabolic derangement without involvement of the cardiovascular system and neurodevelopment. Our observation confirms that COX5A recessive variants may cause mitochondrial disease and expands the associated phenotype to less severe presentations. This article is protected by copyright. All rights reserved.
    Keywords:  COX5A; cytochrome c oxidase; mitochondrial disorders; supercomplexes
  17. Mol Med Rep. 2022 Apr;pii: 147. [Epub ahead of print]25(4):
      Mitochondria are key organelles of cellular energy metabolism; both mitochondrial function and metabolism determine the physiological function of cells and serve an essential role in immune responses. Key damage‑associated molecular patterns (DAMPs), such as mitochondrial DNA and N‑formyl peptides, released following severe trauma‑induced mitochondrial damage may affect the respiratory chain, enhance oxidative stress and activate systemic inflammatory responses via a variety of inflammation‑associated signaling pathways. Severe trauma can lead to sepsis, multiple organ dysfunction syndrome and death. The present review aimed to summarize the pathophysiological mechanisms underlying the effects of human mitochondrial injury‑released DAMPs on triggering systemic inflammatory responses and to determine their potential future clinical applications in preventing and treating sepsis.
    Keywords:  damage‑associated molecular patterns; intestinal barrier dysfunction; mitochondrial DNA; systemic inflammatory response syndrome
  18. Autophagy. 2022 Feb 27. 1-12
      Mutations in the mitochondrial genome (mtDNA) are ubiquitous in humans and can lead to a broad spectrum of disorders. However, due to the presence of multiple mtDNA molecules in the cell, co-existence of mutant and wild-type mtDNAs (termed heteroplasmy) can mask disease phenotype unless a threshold of mutant molecules is reached. Importantly, the mutant mtDNA level can change across lifespan as mtDNA segregates in an allele- and cell-specific fashion, potentially leading to disease. Segregation of mtDNA is mainly evident in hepatic cells, resulting in an age-dependent increase of mtDNA variants, including non-synonymous potentially deleterious mutations. Here we modeled mtDNA segregation using a well-established heteroplasmic mouse line with mtDNA of NZB/BINJ and C57BL/6N origin on a C57BL/6N nuclear background. This mouse line showed a pronounced age-dependent NZB mtDNA accumulation in the liver, thus leading to enhanced respiration capacity per mtDNA molecule. Remarkably, liver-specific atg7 (autophagy related 7) knockout abolished NZB mtDNA accumulat ion, resulting in close-to-neutral mtDNA segregation through development into adulthood. prkn (parkin RBR E3 ubiquitin protein ligase) knockout also partially prevented NZB mtDNA accumulation in the liver, but to a lesser extent. Hence, we propose that age-related liver mtDNA segregation is a consequence of macroautophagic clearance of the less-fit mtDNA. Considering that NZB/BINJ and C57BL/6N mtDNAs have a level of divergence comparable to that between human Eurasian and African mtDNAs, these findings have potential implications for humans, including the safe use of mitochondrial replacement therapy.
    Keywords:  Atg7; NZB; heteroplasmy; mitochondria; mitophagy; parkin
  19. Genome Med. 2022 Feb 28. 14(1): 23
      Rare diseases affect 30 million people in the USA and more than 300-400 million worldwide, often causing chronic illness, disability, and premature death. Traditional diagnostic techniques rely heavily on heuristic approaches, coupling clinical experience from prior rare disease presentations with the medical literature. A large number of rare disease patients remain undiagnosed for years and many even die without an accurate diagnosis. In recent years, gene panels, microarrays, and exome sequencing have helped to identify the molecular cause of such rare and undiagnosed diseases. These technologies have allowed diagnoses for a sizable proportion (25-35%) of undiagnosed patients, often with actionable findings. However, a large proportion of these patients remain undiagnosed. In this review, we focus on technologies that can be adopted if exome sequencing is unrevealing. We discuss the benefits of sequencing the whole genome and the additional benefit that may be offered by long-read technology, pan-genome reference, transcriptomics, metabolomics, proteomics, and methyl profiling. We highlight computational methods to help identify regionally distant patients with similar phenotypes or similar genetic mutations. Finally, we describe approaches to automate and accelerate genomic analysis. The strategies discussed here are intended to serve as a guide for clinicians and researchers in the next steps when encountering patients with non-diagnostic exomes.
    Keywords:  Diagnosis; Exome-negative; Long read; Omics; Rare
  20. Aging Cell. 2022 Mar 02. e13564
      Aged cardiomyocytes develop a mismatch between energy demand and supply, the severity of which determines the onset of heart failure, and become prone to undergo cell death. The FoF1-ATP synthase is the molecular machine that provides >90% of the ATP consumed by healthy cardiomyocytes and is proposed to form the mitochondrial permeability transition pore (mPTP), an energy-dissipating channel involved in cell death. We investigated whether aging alters FoF1-ATP synthase self-assembly, a fundamental biological process involved in mitochondrial cristae morphology and energy efficiency, and the functional consequences this may have. Purified heart mitochondria and cardiomyocytes from aging mice displayed an impaired dimerization of FoF1-ATP synthase (blue native and proximity ligation assay), associated with abnormal mitochondrial cristae tip curvature (TEM). Defective dimerization did not modify the in vitro hydrolase activity of FoF1-ATP synthase but reduced the efficiency of oxidative phosphorylation in intact mitochondria (in which membrane architecture plays a fundamental role) and increased cardiomyocytes' susceptibility to undergo energy collapse by mPTP. High throughput proteomics and fluorescence immunolabeling identified glycation of 5 subunits of FoF1-ATP synthase as the causative mechanism of the altered dimerization. In vitro induction of FoF1-ATP synthase glycation in H9c2 myoblasts recapitulated the age-related defective FoF1-ATP synthase assembly, reduced the relative contribution of oxidative phosphorylation to cell energy metabolism, and increased mPTP susceptibility. These results identify altered dimerization of FoF1-ATP synthase secondary to enzyme glycation as a novel pathophysiological mechanism involved in mitochondrial cristae remodeling, energy deficiency, and increased vulnerability of cardiomyocytes to undergo mitochondrial failure during aging.
    Keywords:  ATP; ROS; aging; dicarbonyl stress; mitochondria
  21. Autophagy. 2022 Feb 27. 1-14
      Defective mitophagy contributes to normal aging and various neurodegenerative and cardiovascular diseases. The newly developed methodologies to visualize and quantify mitophagy allow for additional progress in defining the pathophysiological significance of mitophagy in various model organisms. However, current knowledge regarding mitophagy relevant to human physiology is still limited. Model organisms such as mice might not be optimal models to recapitulate all the key aspects of human disease phenotypes. The development of the human-induced pluripotent stem cells (hiPSCs) may provide an exquisite approach to bridge the gap between animal mitophagy models and human physiology. To explore this premise, we take advantage of the pH-dependent fluorescent mitophagy reporter, mt-Keima, to assess mitophagy in hiPSCs and hiPSC-derived cardiomyocytes (hiPSC-CMs). We demonstrate that mt-Keima expression does not affect mitochondrial function or cardiomyocytes contractility. Comparison of hiPSCs and hiPSC-CMs during different stages of differentiation revealed significant variations in basal mitophagy. In addition, we have employed the mt-Keima hiPSC-CMs to analyze how mitophagy is altered under certain pathological conditions including treating the hiPSC-CMs with doxorubicin, a chemotherapeutic drug well known to cause life-threatening cardiotoxicity, and hypoxia that stimulates ischemia injury. We have further developed a chemical screening to identify compounds that modulate mitophagy in hiPSC-CMs. The ability to assess mitophagy in hiPSC-CMs suggests that the mt-Keima hiPSCs should be a valuable resource in determining the role mitophagy plays in human physiology and hiPSC-based disease models. The mt-Keima hiPSCs could prove a tremendous asset in the search for pharmacological interventions that promote mitophagy as a therapeutic target.
    Keywords:  Cardiomyocytes; cardiomyopathy; induced pluripotent stem cells; mitochondrial; mitophagy; mt-Keima
  22. Biomed Res Int. 2022 ;2022 7436577
      The mitochondrial unfolded protein response (UPRmt) can repair and remove misfolded or unfolded proteins in mitochondria and enhance mitochondrial protein homeostasis. Reactive oxygen species (ROS) produced by regular exercise is a crucial signal for promoting health, and skeletal muscle mitochondria are the primary source of ROS during exercise. To verify whether UPRmt is related to ROS produced by mitochondria in skeletal muscle during regular exercise, we adapted MitoTEMPO, mitochondrially targeted antioxidants, and ROS production by mitochondria. Our results showed that mitochondrial ROS is the key factor for activating UPRmt in different pathways.
  23. Acta Pharmacol Sin. 2022 Mar 01.
      Both mitochondrial dysfunction and neuroinflammation are implicated in neurodegeneration and neurodegenerative diseases. Accumulating evidence shows multiple links between mitochondrial dysfunction and neuroinflammation. Mitochondrial-derived damage-associated molecular patterns (DAMPs) are recognized by immune receptors of microglia and aggravate neuroinflammation. On the other hand, inflammatory factors released by activated glial cells trigger an intracellular cascade, which regulates mitochondrial metabolism and function. The crosstalk between mitochondrial dysfunction and neuroinflammatory activation is a complex and dynamic process. There is strong evidence that mitochondrial dysfunction precedes neuroinflammation during the progression of diseases. Thus, an in-depth understanding of the specific molecular mechanisms associated with mitochondrial dysfunction and the progression of neuroinflammation in neurodegenerative diseases may contribute to the identification of new targets for the treatment of diseases. In this review, we describe in detail the DAMPs that induce or aggravate neuroinflammation in neurodegenerative diseases including mtDNA, mitochondrial unfolded protein response (mtUPR), mitochondrial reactive oxygen species (mtROS), adenosine triphosphate (ATP), transcription factor A mitochondria (TFAM), cardiolipin, cytochrome c, mitochondrial Ca2+ and iron.
    Keywords:  microglia; mitochondrial dysfunction; mitochondrial-derived damage-associated molecular pattern; neurodegenerative diseases; neuroinflammation
  24. Front Genet. 2022 ;13 780764
      There are recent reports of associations of variants in the HPDL gene with a hereditary neurological disease that presents with a wide spectrum of clinical severity, ranging from severe neonatal encephalopathy with no psychomotor development to adolescent-onset uncomplicated spastic paraplegia. Here, we report two probands from unrelated families presenting with severe and intermediate variations of the clinical course. A homozygous variant in the HPDL gene was detected in each proband; however, there was no known parental consanguinity. We also highlight reductions in citrate synthase and mitochondrial complex I activity detected in both probands in different tissues, reflecting the previously proposed mitochondrial nature of disease pathogenesis associated with HPDL mutations. Further, we speculate on the functional consequences of the detected variants, although the function and substrate of the HPDL enzyme are currently unknown.
    Keywords:  ataxia; brain diseases; citrate-synthase; mitochondrial diseases; spastic paraplegia
  25. Inflamm Bowel Dis. 2022 Mar 05. pii: izac024. [Epub ahead of print]
      Inflammatory bowel disease (IBD) is a chronic recurring inflammation of the intestine which can be debilitating for those with intractable disease. However, the etiopathogenesis of inflammatory bowel disorders remains to be solved. The hypothesis that mitochondrial dysfunction is a crucial factor in the disease process is being validated by an increasing number of recent studies. Thus mitochondrial alteration in conjunction with previously identified genetic predisposition, changes in the immune response, altered gut microbiota, and environmental factors (eg, diet, smoking, and lifestyle) are all posited to contribute to IBD. The implicated factors seem to affect mitochondrial function or are influenced by mitochondrial dysfunction, which explains many of the hallmarks of the disease. This review summarizes the results of studies reporting links between mitochondria and IBD that were available on PubMed through March 2021. The aim of this review is to give an overview of the current understanding of the role of mitochondria in the pathogenesis of IBD.
    Keywords:  energy metabolism; inflammatory bowel disease; mitochondria
  26. Free Radic Biol Med. 2022 Feb 23. pii: S0891-5849(22)00075-2. [Epub ahead of print]
      Professor Bruce Ames demonstrated that nutritional recommendations should be adjusted in order to 'tune-up' metabolism and reduce mitochondria decay, a hallmark of aging and many disease processes. A major subset of tunable nutrients are the minerals, which despite being integral to every aspect of metabolism are often deficient in the typical Western diet. Mitochondria are particularly rich in minerals, where they function as essential cofactors for mitochondrial physiology and overall cellular health. Yet substantial knowledge gaps remain in our understanding of the form and function of these minerals needed for metabolic harmony. Some of the minerals have known activities in the mitochondria but with incomplete regulatory detail, whereas other minerals have no established mitochondrial function at all. A comprehensive metallome of the mitochondria is needed to fully understand the patterns and relationships of minerals within metabolic processes and cellular development. This brief overview serves to highlight the current progress towards understanding mineral homeostasis in the mitochondria and to encourage more research activity in key areas. Future work may likely reveal that adjusting the amounts of specific nutritional minerals has longevity benefits for human health.
    Keywords:  Differentiation; Metals; Minerals; Mitochondria; Redox
  27. Stem Cell Rev Rep. 2022 Mar 01.
      Musculoskeletal system disorders are among the most common age-related conditions worldwide. All associated with a degeneration of the supporting tissues under pro-inflammatory micro- and macro-environments, the erosion of cartilage and later of bones, are the main hallmarks of these pathologies. Affected chondrocytes, osteoblasts and synoviocytes, that are all critical actors in the bone and cartilage defects exhibit mitochondrial dysfunction that develops immediately following cartilage and bone injury, and leads to tissue residing specific cell death, cartilage degeneration, bone erosion, and ultimately post-traumatic musculoskeletal degeneration. Herein, we would like to introduce a novel concept for bone and cartilage related defects treatment based on artificial transfer of exogeneous functional mitochondria (AMT). Particularly, we believe that because mitochondrial failure critically contributes to degenerative disorders onset and progression, replacing malfunctioning mitochondria with their healthy and functional counterparts can represent a novel, and effective therapeutic solution for the management of bone and cartilage related degenerative diseases. Artificial mitochondrial transfer (AMT) may reverse the failed metabolic status of musculoskeletal tissues cells and reduce bone and cartilage tissues defects by restoring mitochondrial bioenergetics.
    Keywords:  ATM; Aging; Bone defect; Mitochondrial Dysfunction; OXPHOS; Synovitis
  28. Nat Methods. 2022 Mar 03.
      Detecting single-cell-regulated splicing from droplet-based technologies is challenging. Here, we introduce the splicing Z score (SpliZ), an annotation-free statistical method to detect regulated splicing in single-cell RNA sequencing. We applied the SpliZ to human lung cells, discovering hundreds of genes with cell-type-specific splicing patterns including ones with potential implications for basic and translational biology.
  29. Front Cell Dev Biol. 2021 ;9 820105
      Neurofilament light (NFL) is one of the proteins forming multimeric neuron-specific intermediate filaments, neurofilaments, which fill the axonal cytoplasm, establish caliber growth, and provide structural support. Dominant missense mutations and recessive nonsense mutations in the neurofilament light gene (NEFL) are among the causes of Charcot-Marie-Tooth (CMT) neuropathy, which affects the peripheral nerves with the longest axons. We previously demonstrated that a neuropathy-causing homozygous nonsense mutation in NEFL led to the absence of NFL in patient-specific neurons. To understand the disease-causing mechanisms, we investigate here the functional effects of NFL loss in human motor neurons differentiated from induced pluripotent stem cells (iPSC). We used genome editing to generate NEFL knockouts and compared them to patient-specific nonsense mutants and isogenic controls. iPSC lacking NFL differentiated efficiently into motor neurons with normal axon growth and regrowth after mechanical axotomy and contained neurofilaments. Electrophysiological analysis revealed that motor neurons without NFL fired spontaneous and evoked action potentials with similar characteristics as controls. However, we found that, in the absence of NFL, human motor neurons 1) had reduced axonal caliber, 2) the amplitude of miniature excitatory postsynaptic currents (mEPSC) was decreased, 3) neurofilament heavy (NFH) levels were reduced and no compensatory increases in other filament subunits were observed, and 4) the movement of mitochondria and to a lesser extent lysosomes was increased. Our findings elaborate the functional roles of NFL in human motor neurons. NFL is not only a structural protein forming neurofilaments and filling the axonal cytoplasm, but our study supports the role of NFL in the regulation of synaptic transmission and organelle trafficking. To rescue the NFL deficiency in the patient-specific nonsense mutant motor neurons, we used three drugs, amlexanox, ataluren (PTC-124), and gentamicin to induce translational read-through or inhibit nonsense-mediated decay. However, the drugs failed to increase the amount of NFL protein to detectable levels and were toxic to iPSC-derived motor neurons.
    Keywords:  Charcot-Marie-Tooth (CMT) disease; axon; induced pluripotent stem cells; motor neurodegeneration; motor neuron (MN); neurofilament light (NfL)
  30. Neurochem Res. 2022 Feb 28.
      Rabies is a fatal encephalitis caused by the Rabies lyssavirus (RABV). The presence of minimal neuropathological changes observed in rabies indicates that neuronal dysfunction, rather than neuronal death contributes to the fatal outcome. The role of mitochondrial changes has been suggested as a possible mechanism for neuronal dysfunction in rabies. However, these findings are mostly based on studies that have employed experimental models and laboratory-adapted virus. Studies on brain tissues from naturally infected human and animal hosts are lacking. The current study investigated the role of mitochondrial changes in rabies by morphological, biochemical and proteomic analysis of RABV-infected human and canine brains. Morphological analysis showed minimal inflammation with preserved neuronal and disrupted mitochondrial structure in both human and canine brains. Proteomic analysis revealed involvement of mitochondrial processes (oxidative phosphorylation, cristae formation, homeostasis and transport), synaptic proteins and autophagic pathways, with over-expression of subunits of mitochondrial respiratory complexes. Consistent with these findings, human and canine brains displayed elevated activities of complexes I (p < 0.05), IV (p < 0.05) and V (p < 0.05). However, this did not result in elevated ATP production (p < 0.0001), probably due to lowered mitochondrial membrane potential as noted in RABV-infected cells in culture. These could lead to mitochondrial dysfunction and mitophagy as indicated by expression of FKBP8 (p < 0.05) and PINK1 (p < 0.001)/PARKIN (p > 0.05) and ensuing autophagy, as shown by the status of LCIII (p < 0.05), LAMP1 (p < 0.001) and pertinent ultrastructural markers. We propose that altered mitochondrial bioenergetics and cristae architecture probably induce mitophagy, leading to autophagy and consequent neuronal dysfunction in rabies.
    Keywords:  Mitochondria; Neuronal dysfunction; Proteomics; Rabies
  31. Mol Cell. 2022 Mar 03. pii: S1097-2765(22)00160-5. [Epub ahead of print]82(5): 882-883
      By comparing the structures of Bax and Bak megapores, Cosentino et al. (2022) reveal new insights suggesting the two pro-apoptotic proteins co-assemble into structures that release DNA from mitochondria and thereby trigger inflammation.
  32. Front Neurosci. 2022 ;16 814445
      Friedreich ataxia is a rare neurodegenerative disorder caused by insufficient levels of the essential mitochondrial protein frataxin. It is a severely debilitating disease that significantly impacts the quality of life of affected patients and reduces their life expectancy, however, an adequate cure is not yet available for patients. Frataxin function, although not thoroughly elucidated, is associated with assembly of iron-sulfur cluster and iron metabolism, therefore insufficient frataxin levels lead to reduced activity of many mitochondrial enzymes involved in the electron transport chain, impaired mitochondrial metabolism, reduced ATP production and inefficient anti-oxidant response. As a consequence, neurons progressively die and patients progressively lose their ability to coordinate movement and perform daily activities. Therapeutic strategies aim at restoring sufficient frataxin levels or at correcting some of the downstream consequences of frataxin deficiency. However, the classical pathways of drug discovery are challenging, require a significant amount of resources and time to reach the final approval, and present a high failure rate. Drug repositioning represents a viable alternative to boost the identification of a therapy, particularly for rare diseases where resources are often limited. In this review we will describe recent efforts aimed at the identification of a therapy for Friedreich ataxia through drug repositioning, and discuss the limitation of such strategies.
    Keywords:  drug development; drug repositioning; frataxin; friedreich ataxia; therapeutics
  33. Free Radic Biol Med. 2022 Feb 23. pii: S0891-5849(22)00074-0. [Epub ahead of print]
      Intermittent fasting (IF) has been studied for its effects on lifespan and lifespan as well as the prevention or delay of age-related diseases upon the regulation of metabolic pathways. Mitochondria participate in key metabolic pathways and play important roles in maintaining intracellular signaling networks that modulate various cellular functions. Mitochondrial dysfunction has been described as an early feature of brain aging and neurodegeneration. Although IF has been shown to prevent brain aging and neurodegeneration, the mechanism is still unclear. This review focuses on the mechanisms by which IF improves mitochondrial function, which plays a central role in brain aging and neurodegenerative diseases, such as Alzheimer's disease, Parkinson's disease, and Huntington's disease. The cellular and molecular mechanisms of IF in brain aging and neurodegeneration involve activation of adaptive cellular stress responses and signaling- and transcriptional pathways, thereby enhancing mitochondrial function, by promoting energy metabolism and reducing oxidant production.
    Keywords:  Brain aging; Intermittent fasting; Mitochondrial dysfunction; Neurodegeneration
  34. Mol Biol Rep. 2022 Feb 28.
      BACKGROUND: Several metabolic disorders follow an autosomal recessive inheritance pattern. Epidemiological information on these disorders is usually limited in developing countries. Our objective is to assess carrier frequencies of rare autosomal recessive metabolic diseases in a cohort of Brazilian patients that underwent molecular investigation with exome sequencing and estimate the overall frequency of these diseases using the Hardy-Weinberg equation.METHODS AND RESULTS: We reviewed the molecular findings of 320 symptomatic patients who had carrier status for recessive diseases actively searched. A total of 205 rare variants were reported in 138 different genes associated with metabolic diseases from 156 patients, which represents that almost half (48.8%) of the patients were carriers of at least one heterozygous pathogenic/likely pathogenic (P/LP) variant for rare metabolic disorders. Most of these variants are harbored by genes associated with multisystemic involvement. We estimated the overall frequency for rare recessive metabolic diseases to be 10.96/10,000 people, while the frequency of metabolic diseases potentially identified by newborn screening was estimated to be 2.93/10,000.
    CONCLUSIONS: This study shows the potential research utility of exome sequencing to determine carrier status for rare metabolic diseases, which may be a possible strategy to evaluate the clinical and social burden of these conditions at the population level and guide the optimization of health policies and newborn screening programs.
    Keywords:  Carrier frequency; Inborn errors of metabolism; Metabolic diseases; Rare diseases; Recessive Mendelian diseases; Whole exome sequencing
  35. Neurochem Res. 2022 Feb 26.
      Parkinson's disease (PD), the main risk factor for which is age, is one of the most common neurodegenerative diseases and imposes a substantial burden on affected individuals and the economy. While the aetiology of PD is still largely unclear, substantial evidence indicates that mitochondrial dysfunction, aggregation of α-synuclein (α-syn), oxidative stress, inflammation, and autophagy play major roles in the pathogenesis of PD. Sirtuins are NAD+-dependent protein deacetylases, includeing seven members, i.e., SIRT1-SIRT7. Among these sirtuins, SIRT3, SIRT4 and SIRT5 are located in mitochondria and are called mitochondrial sirtuins. Mitochondrial sirtuins regulate the activity and biological function of mitochondrial proteins through posttranslational modification of substrate proteins. Increasing evidence shows that mitochondrial sirtuins play an important role in degenerative diseases, including PD. Mitochondrial sirtuins exert a beneficial neuroprotective effect in various models of PD. This paper summarizes a large number of studies and discusses the latest research progress on the role of mitochondrial sirtuins in PD, focusing especially on the regulation of the mitochondrial respiratory chain (MRC), oxidative stress, the inflammatory response and autophagy, to provide new insight into the pathogenesis of PD and new targets for the diagnosis and treatment of the disease.
    Keywords:  Autophagy; Inflammation; Mitochondrial respiratory chain; Mitochondrial sirtuins; PD; ROS
  36. Front Immunol. 2022 ;13 832159
      As the major hub of metabolic activity and an organelle sequestering pro-apoptogenic intermediates, mitochondria lie at the crossroads of cellular decisions of death and survival. Intracellular calcium is a key regulator of these outcomes with rapid, uncontrolled uptake into mitochondria, activating pro-apoptotic cascades that trigger cell death. Here, we show that calcium uptake and mitochondrial metabolism in murine T-regulatory cells (Tregs) is tuned by Notch1 activity. Based on analysis of Tregs and the HEK cell line, we present evidence that modulation of cellular calcium dynamics underpins Notch1 regulation of mitochondrial homeostasis and consequently anti-apoptotic activity. Targeted siRNA-mediated ablations reveal dependency on molecules controlling calcium release from the endoplasmic reticulum (ER) and the chaperone, glucose-regulated protein 75 (Grp75), the associated protein Voltage Dependent Anion Channel (VDAC)1 and the Mitochondrial Calcium Uniporter (MCU), which together facilitate ER calcium transfer and uptake into the mitochondria. Endogenous Notch1 is detected in immune-complexes with Grp75 and VDAC1. Deficits in mitochondrial oxidative and survival in Notch1 deficient Tregs, were corrected by the expression of recombinant Notch1 intracellular domain, and in part by recombinant Grp75. Thus, the modulation of calcium dynamics and consequently mitochondrial metabolism underlies Treg survival in conditions of nutrient stress. This work positions a key role for Notch1 activity in these outcomes.
    Keywords:  Grp75; NOTCH1; Tregs; apoptosis; calcium; mammalian cells; mitochondria; oxphos
  37. Semin Cell Dev Biol. 2022 Feb 28. pii: S1084-9521(22)00039-8. [Epub ahead of print]
      Mitochondria play a major role in apoptotic signaling. In addition to its role in eliminating dysfunctional cells, mitochondrial apoptotic signaling is implicated as a key component of myogenic differentiation and skeletal muscle atrophy. For example, the activation of cysteine-aspartic proteases (caspases; CASP's) can aid in the initial remodeling stages of myogenic differentiation by cleaving protein kinases, transcription factors, and cytoskeletal proteins. Precise regulation of these signals is needed to prevent excessive cell disassemble and subsequent cell death. During skeletal muscle atrophy, the activation of CASP's and mitochondrial derived nucleases participate in myonuclear fragmentation, a potential loss of myonuclei, and cleavage of contractile structures within skeletal muscle. The B cell leukemia/lymphoma 2 (BCL2) family of proteins play a significant role in regulating myogenesis and skeletal muscle atrophy by governing the initiating steps of mitochondrial apoptotic signaling. This review discusses the role of mitochondrial apoptotic signaling in skeletal muscle remodeling during myogenic differentiation and skeletal muscle pathological states, including aging, disuse, and muscular dystrophy.
    Keywords:  Aging; Apoptosis; Atrophy; BCL2; CASP9; Disease; Disuse; Mitochondria; Muscular dystrophy; Myogenesis; Skeletal muscle
  38. iScience. 2022 Mar 18. 25(3): 103901
      Cells transmit their genomes vertically to daughter cells during cell divisions. Here, we demonstrate the occurrence and extent of horizontal mitochondrial (mt)DNA acquisition between cells that are not in a parent-offspring relationship. Extensive single-cell sequencing from various tissues and organs of adult chimeric mice composed of cells carrying distinct mtDNA haplotypes showed that a substantial fraction of individual cardiomyocytes, neurons, glia, intestinal, and spleen cells captured donor mtDNA at high levels. In addition, chimeras composed of cells with wild-type and mutant mtDNA exhibited increased trafficking of wild-type mtDNA to mutant cells, suggesting that horizontal mtDNA transfer may be a compensatory mechanism to restore compromised mitochondrial function. These findings establish the groundwork for further investigations to identify mtDNA donor cells and mechanisms of transfer that could be critical to the development of novel gene therapies.
    Keywords:  Cell biology; Developmental biology; Molecular biology
  39. Methods Mol Biol. 2022 ;2419 301-311
      Mitochondrial function and activity are key indicators of overall cell health and mitochondrial dysfunction is closely associated with disruptions in normal cellular function. Altered mitochondrial function and cellular metabolism has been implicated in processes involved in ageing and associated pathologies. In atherosclerosis, compromised mitochondrial respiration can promote plaque instability and other processes that encourage pathogenesis and dysfunction. For example, increasing respiration promotes vascular smooth muscle cell (VSMC) proliferation and attenuates macrophage and VSMC apoptosis. Use of Agilent Seahorse technology to study mitochondrial bioenergetics has largely replaced previous outdated methods which provided limited insight into mitochondrial function and were associated with various issues. This chapter describes the use of Seahorse Agilent technology (Mito Stress Test) to study key parameters of mitochondrial respiration on cultured cells relevant to atherosclerosis.
    Keywords:  Agilent Seahorse technology; Cellular metabolism; Mito Stress Test; Mitochondrial respiration; XFe96 Analyzer
  40. Adv Exp Med Biol. 2022 ;1361 37-54
      Re-sequencing of the human genome by next-generation sequencing (NGS) has been widely applied to discover pathogenic genetic variants and/or causative genes accounting for various types of diseases including cancers. The advances in NGS have allowed the sequencing of the entire genome of patients and identification of disease-associated variants in a reasonable timeframe and cost. The core of the variant identification relies on accurate variant calling and annotation. Numerous algorithms have been developed to elucidate the repertoire of somatic and germline variants. Each algorithm has its own distinct strengths, weaknesses, and limitations due to the difference in the statistical modeling approach adopted and read information utilized. Accurate variant calling remains challenging due to the presence of sequencing artifacts and read misalignments. All of these can lead to the discordance of the variant calling results and even misinterpretation of the discovery. For somatic variant detection, multiple factors including chromosomal abnormalities, tumor heterogeneity, tumor-normal cross contaminations, unbalanced tumor/normal sample coverage, and variants with low allele frequencies add even more layers of complexity to accurate variant identification. Given the discordances and difficulties, ensemble approaches have emerged by harmonizing information from different algorithms to improve variant calling performance. In this chapter, we first introduce the general scheme of variant calling algorithms and potential challenges at distinct stages. We next review the existing workflows of variant calling and annotation, and finally explore the strategies deployed by different callers as well as their strengths and caveats. Overall, NGS-based variant identification with careful consideration allows reliable detection of pathogenic variant and candidate variant selection for precision medicine.
    Keywords:  Contamination; Ensemble variant calling; Germline variant; Low-frequency variants; Machine learning; Next-generation sequencing; Single-cell sequencing; Somatic variant; Third-generation sequencing; Tumor-only variant calling; Variant annotation; Variant calling; Variant prioritization
  41. Elife. 2022 Mar 04. pii: e57393. [Epub ahead of print]11
      Sustained exposure to a young systemic environment rejuvenates aged organisms and promotes cellular function. However, due to the intrinsic complexity of tissues it remains challenging to pinpoint niche-independent effects of circulating factors on specific cell populations. Here we describe a method for the encapsulation of human and mouse skeletal muscle progenitors in diffusible polyethersulfone hollow fiber capsules that can be used to profile systemic aging in vivo independent of heterogeneous short-range tissue interactions. We observed that circulating long-range signaling factors in the old systemic environment lead to an activation of Myc and E2F transcription factors, induce senescence and suppress myogenic differentiation. Importantly, in vitro profiling using young and old serum in 2D culture does not capture all pathways deregulated in encapsulated cells in aged mice. Thus, in vivo transcriptomic profiling using cell encapsulation allows for the characterization of effector pathways of systemic aging with unparalleled accuracy.
    Keywords:  cell biology; mouse
  42. Curr Cardiol Rep. 2022 Mar 04.
      PURPOSE OF REVIEW: Induced pluripotent stem cells (iPSCs) have become widely adopted tools in cardiovascular biology due to their ability to differentiate into patient-specific cell types. Here, we describe the current protocols, important discoveries, and experimental limitations from the iPSC-derived cell types of the human heart: cardiomyocytes, cardiac fibroblasts, vascular smooth muscle cells, endothelial cells, and pericytes. In addition, we also examine the progress of 3D-based cell culture systems.RECENT FINDINGS: There has been rapid advancement in methods to generate cardiac iPSC-derived cell types. These advancements have led to improved cardiovascular disease modeling, elucidation of interactions among different cell types, and the creation of 3D-based cell culture systems able to provide more physiologically relevant insights into cardiovascular diseases. iPSCs have become an instrumental model system in the toolbox of cardiovascular biologists. Ongoing research continues to advance the use of iPSCs in (1) disease modeling, (2) drug screening, and (3) clinical trials in a dish.
    Keywords:  3D cell culture systems; Cardiac regeneration; Cardiovascular disease modeling; Induced pluripotent stem cells; Precision medicine
  43. Rev Neurosci. 2022 Feb 28.
      The goal of this paper is to provide an overview of our current understanding of mitochondrial function as a framework to motivate the hypothesis that mitochondrial behavior is governed by optimization principles that are constrained by the laws of the physical and biological sciences. Then, mathematical optimization tools can generally be useful to model some of these processes under reasonable assumptions and limitations. We are specifically interested in optimizations via variational methods, which are briefly summarized. Within such an optimization framework, we suggest that the numerous mechanical instigators of cell and intracellular functioning can be modeled utilizing some of the principles of mechanics that govern engineered systems, as well as by the frequently observed feedback and feedforward mechanisms that coordinate the multitude of processes within cells. These mechanical aspects would need to be coupled to governing biochemical rules. Of course, biological systems are significantly more complex than engineered systems, and require considerably more experimentation to ascertain and characterize parameters and subsequent behavior. That complexity requires well-defined limitations and assumptions for any derived models. Optimality is being motivated as a framework to help us understand how cellular decisions are made, especially those that transition between physiological behaviors and dysfunctions along pathophysiological pathways. We elaborate on our interpretation of optimality and cellular decision making within the body of this paper, as we revisit these ideas in the numerous different contexts of mitochondrial functions.
    Keywords:  cristae morphology; energetics; mitochondria; optimization; variational mechanics
  44. Nat Rev Mol Cell Biol. 2022 Feb 28.
      Metabolism has been studied mainly in cultured cells or at the level of whole tissues or whole organisms in vivo. Consequently, our understanding of metabolic heterogeneity among cells within tissues is limited, particularly when it comes to rare cells with biologically distinct properties, such as stem cells. Stem cell function, tissue regeneration and cancer suppression are all metabolically regulated, although it is not yet clear whether there are metabolic mechanisms unique to stem cells that regulate their activity and function. Recent work has, however, provided evidence that stem cells do have a metabolic signature that is distinct from that of restricted progenitors and that metabolic changes influence tissue homeostasis and regeneration. Stem cell maintenance throughout life in many tissues depends upon minimizing anabolic pathway activation and cell division. Consequently, stem cell activation by tissue injury is associated with changes in mitochondrial function, lysosome activity and lipid metabolism, potentially at the cost of eroding self-renewal potential. Stem cell metabolism is also regulated by the environment: stem cells metabolically interact with other cells in their niches and are able to sense and adapt to dietary changes. The accelerating understanding of stem cell metabolism is revealing new aspects of tissue homeostasis with the potential to promote tissue regeneration and cancer suppression.
  45. Hum Mol Genet. 2022 Mar 02. pii: ddac053. [Epub ahead of print]
      BACKGROUND: The endoplasmic reticulum (ER)-membrane protein complex (EMC) is a multi-protein transmembrane complex composed of 10 subunits that functions as a membrane-protein chaperone. Variants in EMC1 lead to neurodevelopmental delay and cerebellar degeneration. Multiple families with biallelic variants have been published, yet to date, only a single report of a monoallelic variant has been described, and functional evidence is sparse.METHODS: Exome sequencing was used to investigate the genetic cause underlying severe developmental delay in three unrelated children. EMC1 variants were modeled in Drosophila, using loss-of-function (LoF) and overexpression studies. Glial-specific and neuronal-specific assays were used to determine whether the dysfunction was specific to one cell type.
    RESULTS: Exome sequencing identified de novo variants in EMC1 in three individuals affected by global developmental delay, hypotonia, seizures, visual impairment, and cerebellar atrophy. All variants were located at Pro582 or Pro584. Drosophila studies indicated that imbalance of EMC1-either overexpression or knockdown-results in pupal lethality and suggest that the tested homologous variants are LoF alleles. In addition, glia-specific gene dosage, overexpression or knockdown, of EMC1 led to lethality, whereas neuron-specific alterations were tolerated.
    DISCUSSION: We establish de novo monoallelic EMC1 variants as causative of a neurological disease trait by providing functional evidence in a Drosophila model. The identified variants failed to rescue the lethality of a null allele. Variations in dosage of the wild-type EMC1, specifically in glia, lead to pupal lethality, which we hypothesize results from the altered stoichiometry of the multi-subunit protein complex EMC.
  46. Elife. 2022 Mar 01. pii: e75821. [Epub ahead of print]11
      Neurotransmitters are generated by de novo synthesis and are essential for sustained, high-frequency synaptic transmission. Histamine, a monoamine neurotransmitter, is synthesized through decarboxylation of histidine by Histidine decarboxylase (Hdc). However, little is known about how histidine is presented to Hdc as a precursor. Here, we identified a specific histidine transporter, TADR (Torn And Diminished Rhabdomeres), which is required for visual transmission in Drosophila. Both TADR and Hdc localized to neuronal terminals, and mutations in tadr reduced levels of histamine, thus disrupting visual synaptic transmission and phototaxis behavior. These results demonstrate that a specific amino acid transporter provides precursors for monoamine neurotransmitters, providing the first genetic evidence that a histidine amino acid transporter plays a critical role in synaptic transmission. These results suggest that TADR-dependent local de novo synthesis of histamine is required for synaptic transmission.
    Keywords:  D. melanogaster; cell biology; neuroscience
  47. J Peripher Nerv Syst. 2022 Feb 28.
      Biallelic mutations in sorbitol dehydrogenase (SORD) have been recently identified as a common cause of recessive axonal Charcot-Marie-Tooth neuropathy (CMT2). We aimed to assess a novel long-read sequencing approach to overcome current limitations in SORD neuropathy diagnostics due to the SORD2P pseudogene and the phasing of biallelic mutations in recessive disease. We conducted a screen of our Australian whole exome sequencing (WES) CMT cohort to identify individuals with homozygous or compound heterozygous SORD variants. Individuals detected with SORD mutations then underwent long-read sequencing, clinical assessment, and serum sorbitol analysis. An individual was detected with compound heterozygous truncating mutations in SORD exon 7, NM_003104.5:c.625C>T (p.Arg209Ter) and NM_003104.5:c.757del (p.Ala253GlnfsTer27). Subsequent Oxford Nanopore Tech (ONT) long-read sequencing was used to successfully differentiate SORD from the highly homologous non-functional SORD2P pseudogene and confirmed that the mutations were biallelic through haplotype-resolved analysis. The patient presented with axonal sensorimotor polyneuropathy (CMT2) and ulnar neuropathy without compression at the elbow. Burning neuropathic pain in the forearms and feet was also reported and was exacerbated by alcohol consumption and improved with alcohol cessation. UPLC-tandem mass spectrometry confirmed that the patient had elevated serum sorbitol levels (12.0 mg/L) consistent with levels previously observed in patients with biallelic SORD mutations. This represents a novel clinical presentation and expands the phenotype associated with biallelic SORD mutations causing CMT2. Our study is the first report of long-read sequencing for an individual with CMT and demonstrates the utility of this approach for clinical genomics.
    Keywords:  Charcot-Marie-Tooth neuropathy; SORD; long-read sequencing
  48. Genome Biol. 2022 Mar 03. 23(1): 69
      BACKGROUND: The detection of physiologically relevant protein isoforms encoded by the human genome is critical to biomedicine. Mass spectrometry (MS)-based proteomics is the preeminent method for protein detection, but isoform-resolved proteomic analysis relies on accurate reference databases that match the sample; neither a subset nor a superset database is ideal. Long-read RNA sequencing (e.g., PacBio or Oxford Nanopore) provides full-length transcripts which can be used to predict full-length protein isoforms.RESULTS: We describe here a long-read proteogenomics approach for integrating sample-matched long-read RNA-seq and MS-based proteomics data to enhance isoform characterization. We introduce a classification scheme for protein isoforms, discover novel protein isoforms, and present the first protein inference algorithm for the direct incorporation of long-read transcriptome data to enable detection of protein isoforms previously intractable to MS-based detection. We have released an open-source Nextflow pipeline that integrates long-read sequencing in a proteomic workflow for isoform-resolved analysis.
    CONCLUSIONS: Our work suggests that the incorporation of long-read sequencing and proteomic data can facilitate improved characterization of human protein isoform diversity. Our first-generation pipeline provides a strong foundation for future development of long-read proteogenomics and its adoption for both basic and translational research.
    Keywords:  Alternative splicing; Iso-Seq; Lifebit CloudOS; Long-read RNA-seq; Mass spectrometry-based proteomics; Nextflow; PacBio; Protein inference; Proteogenomics; SQANTI
  49. Mol Genet Metab Rep. 2022 Mar;30 100847
      As a result of a founder effect, a Leigh syndrome variant called Leigh syndrome, French-Canadian type (LSFC, MIM / 220,111) is more frequent in Saguenay-Lac-Saint-Jean (SLSJ), a geographically isolated region on northeastern Quebec, Canada. LSFC is a rare autosomal recessive mitochondrial neurodegenerative disorder due to damage in mitochondrial energy production. LSFC is caused by pathogenic variants in the nuclear gene leucine-rich pentatricopeptide repeat-containing (LRPPRC). Despite progress understanding the molecular mode of action of LRPPRC gene, there is no treatment for this disease. The present study aims to identify the biological pathways altered in the LSFC disorder through microarray-based transcriptomic profile analysis of twelve LSFC cell lines compared to twelve healthy ones, followed by gene ontology (GO) and pathway analyses. A set of 84 significantly differentially expressed genes were obtained (p ≥ 0.05; Fold change (Flc) ≥ 1.5). 45 genes were more expressed (53.57%) in LSFC cell lines compared to controls and 39 (46.43%) had lower expression levels. Gene ontology analysis highlighted altered expression of genes involved in the mitochondrial respiratory chain and energy production, glucose and lipids metabolism, oncogenesis, inflammation and immune response, cell growth and apoptosis, transcription, and signal transduction. Considering the metabolic nature of LSFC disease, genes included in the mitochondrial respiratory chain and energy production cluster stood out as the most important ones to be involved in LSFC mitochondrial disorder. In addition, the protein-protein interaction network indicated a strong interaction between the genes included in this cluster. The mitochondrial gene NDUFA4L2 (NADH dehydrogenase [ubiquinone] 1 alpha subcomplex, 4-like 2), with higher expression in LSFC cells, represents a target for functional studies to explain the role of this gene in LSFC disease. This work provides, for the first time, the LSFC gene expression profile in fibroblasts isolated from affected individuals. This represents a valuable resource to understand the pathogenic basis and consequences of LRPPRC dysfunction.
    Keywords:  ATP, adénosine-5'-triphosphate; COPD, chronic obstructive pulmonary disease; COX, cytochrome c-oxidase; Cytochrome c oxidase; DMEM, Dubelcco’s Modified Essential Medium; ETC, electron transport chain; Flc, fold change; GO, gene ontology; Gene expression; HES1, hairy and enhancer of split 1; HIF-1, hypoxia inducible factor-1; LRPPRC; LRPPRC, leucine-rich pentatricopeptide repeat-containing; LSFC, Leigh syndrome, French-Canadian type; Leigh syndrome; Leigh syndrome French-Canadian type (LSFC); Microarrays; Mitochondrial chain respiration; NAFLD, non-alcoholic fatty liver disease; ND6, NADH dehydrogenase, subunit 6; NDUFA4L2; NDUFA4L2, NADH dehydrogenase [ubiquinone] 1 alpha subcomplex, 4-like 2; OXPHOS, oxidative phosphorylation; PFKFB4, 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 4; PPI, protein‐protein interaction; RMA, robust multi-array analysis; ROS, reactive oxygen species; RPL13A, ribosomal protein L13a; SLIRP, stem-loop interacting protein; SLSJ, Saguenay–Lac-Saint-Jean; SRA, steroid receptor RNA activator; qRT-PCR, Real-time PCR; rare diseases
  50. Nat Commun. 2022 Feb 28. 13(1): 1074
      Calorie restriction abates aging and cardiometabolic disease by activating metabolic signaling pathways, including nicotinamide adenine dinucleotide (NAD+) biosynthesis and salvage. Nicotinamide phosphoribosyltransferase (NAMPT) is rate-limiting in NAD+ salvage, yet hepatocyte NAMPT actions during fasting and metabolic duress remain unclear. We demonstrate that hepatocyte NAMPT is upregulated in fasting mice, and in isolated hepatocytes subjected to nutrient withdrawal. Mice lacking hepatocyte NAMPT exhibit defective FGF21 activation and thermal regulation during fasting, and are sensitized to diet-induced glucose intolerance. Hepatocyte NAMPT overexpression induced FGF21 and adipose browning, improved glucose homeostasis, and attenuated dyslipidemia in obese mice. Hepatocyte SIRT1 deletion reversed hepatocyte NAMPT effects on dark-cycle thermogenesis, and hepatic FGF21 expression, but SIRT1 was dispensable for NAMPT insulin-sensitizing, anti-dyslipidemic, and light-cycle thermogenic effects. Hepatocyte NAMPT thus conveys key aspects of the fasting response, which selectively dissociate through hepatocyte SIRT1. Modulating hepatocyte NAD+ is thus a potential mechanism through which to attenuate fasting-responsive disease.
  51. Hum Mutat. 2022 Feb 27.
      Next-generation sequencing is a prevalent diagnostic tool for undiagnosed diseases and has played a significant role in rare disease gene discovery. While this technology resolves some cases, others are given a list of possibly damaging genetic variants necessitating functional studies. Productive collaborations between scientists, clinicians, and patients can help resolve such medical mysteries, and provide insights into in vivo function of human genes. Furthermore, facilitating interactions between scientists and research funders, including non-profit organizations or commercial entities, can dramatically reduce the time to translate discoveries from bench to bedside. Several systems designed to connect clinicians and researchers with a shared gene of interest have been successful. However, these platforms exclude some stakeholders based on their role or geography. Here we describe ModelMatcher, a global online matchmaking tool designed to facilitate cross-disciplinary collaborations, especially between scientists and other stakeholders of rare and undiagnosed disease research. ModelMatcher is integrated into the Rare Diseases Models and Mechanisms Network and Matchmaker Exchange, allowing users to identify potential collaborators in other registries. This living database decreases the time from when a scientist or clinician is making discoveries regarding their genes of interest, to when they identify collaborators and sponsors to facilitate translational and therapeutic research. This article is protected by copyright. All rights reserved.
    Keywords:  Collaboration; Functional Studies; Matchmaker Exchange (MME); Matchmaking; Model Organisms; Rare Diseases; Rare Diseases Models and Mechanisms (RDMM) Network; Undiagnosed Diseases; Undiagnosed Diseases Network (UDN); Variants of Unknown Significance (VUS)
  52. J Biol Chem. 2022 Mar 01. pii: S0021-9258(22)00221-6. [Epub ahead of print] 101781
      ClpP is a highly conserved serine protease that is a critical enzyme in maintaining protein homeostasis and is an important drug target in pathogenic bacteria and various cancers. In its functional form, ClpP is a chambered protease composed of two stacked heptameric rings that allow protein degradation to occur internally. ATPase chaperones such as ClpX and ClpA are hexameric ATPases that form larger complexes with ClpP and are responsible for the selection and unfolding of protein substrates prior to their degradation by ClpP. Although individual structures of ClpP and ATPase chaperones have offered mechanistic insights into their function and regulation, their structures together as a complex have only been recently determined to high resolution. Here, we discuss the cryoelectron microscopy structures of ClpP-ATPase complexes and describe findings previously inaccessible from individual Clp structures, including how a hexameric ATPase and a tetradecameric ClpP protease work together in a functional complex. We then discuss the consensus mechanism for substrate unfolding and translocation derived from these structures, consider alternative mechanisms, and present their strengths and limitations. Finally, new insights into the allosteric control of ClpP gained from studies using small molecules and gain or loss-of-function mutations are explored. Overall, this review aims to underscore the multi-layered regulation of ClpP that may present novel ideas for structure-based drug design.
    Keywords:  AAA+ chaperones; ATP-dependent protease; Agonist; Allostery; ClpP; Dysregulation of protein activity; Protein degradation; Small molecule activator
  53. Pediatr Cardiol. 2022 Mar 03.
      Barth Syndrome (BTHS) is an X-linked mitochondrial cardioskeletal myopathy caused by defects in TAFAZZIN, a gene responsible for cardiolipin remodeling. Altered mitochondrial levels of cardiolipin lead to cardiomyopathy (CM), muscle weakness, exercise intolerance, and mortality. Cardiac risk factors predicting outcome are unknown. Therefore, we conducted a longitudinal observational study to determine risk factors for outcome in BTHS. Subjects with minimum two evaluations (or one followed by death or transplant) were included. Cardiac size, function, and QTc data were measured by echocardiography and electrocardiography at 7 time points from 2002 to 2018. Analysis included baseline, continuous, and categorical variables. Categorical risk factors included prolonged QTc, abnormal right ventricle fractional area change (RV FAC), left ventricle (LV) or RV non-compaction, and restrictive CM phenotype. The association between variables and cardiac death or transplant (CD/TX) was assessed. Median enrollment age was 7 years (range 0.5-22; n = 44). Transplant-free survival (TFS) was 74.4% at 15 years from first evaluation. The cohort demonstrated longitudinal declines in LV size and stroke volume z-scores (end-diastolic volume, p = 0.0002; stroke volume p < 0.0001), worsening RV FAC (p = 0.0405), and global longitudinal strain (GLS) (p = 0.0001) with stable ejection (EF) and shortening (FS) fraction. CD/TX subjects (n = 9) displayed worsening LV dilation (p = 0.0066), EF (p ≤ 0.0001), FS (p = 0.0028), and RV FAC (p = .0032) versus stability in TFS. Having ≥ 2 categorical risk factors predicted CD/TX (p = 0.0073). Over 15 years, 25% of BTHS subjects progressed to CD/TX. Those with progressive LV enlargement, dysfunction, and multiple cardiac risk factors warrant increased surveillance and intense therapy.
    Keywords:  Barth syndrome; Heart failure; Mitochondria transplantation; Myopathy; Natural history
  54. Front Mol Biosci. 2022 ;9 854321
      The mitochondrial ATP synthase is responsible for the production of cellular ATP, and it does so by harnessing the membrane potential of the mitochondria that is produced by the sequential oxidation of select cellular metabolites. Since the structural features of ATP synthase were first resolved nearly three decades ago, significant progress has been made in understanding its role in health and disease. Mitochondrial dysfunction is common to neurodegeneration, with elevated oxidative stress a hallmark of this dysfunction. The patterns of this oxidative stress, including molecular targets and the form of oxidative modification, can vary widely. In this mini review we discuss the oxidative modifications of ATP synthase that have been observed in Alzheimer's disease, Parkinson's disease, and Huntington's disease. Oxidative modifications of ATP synthase in Alzheimer's disease are well-documented, and there is a growing body of knowledge on the subject in Parkinson's disease. The consideration of ATP synthase as a pharmacological target in a variety of diseases underlines the importance of understanding these modifications, both as a potential target, and also as inhibitors of any pharmacological intervention.
    Keywords:  ATP synthase; mitochondria; neurodegenarative disease; oxidative phoshorylation; oxidative stress
  55. Mol Med. 2022 Mar 04. 28(1): 28
      BACKGROUND: The underlying pathophysiology of Parkinson's disease is complex, involving different molecular pathways, including brain iron deposition and mitochondrial dysfunction. At a molecular level, these disease mechanisms are likely interconnected. Therefore, they offer potential strategies for disease-modifying treatments. We aimed to investigate subcortical brain iron deposition as a potential predictor of the bioenergetic status in patients with idiopathic Parkinson's disease.METHODS: Thirty patients with idiopathic Parkinson's disease underwent multimodal MR imaging (T1, susceptibility-weighted imaging, SWI) and 31phosphorus magnetic resonance spectroscopy imaging. SWI contrast-to-noise ratios served as a measure for brain iron deposition in the putamen, caudate, globus pallidus, and thalamus and were used in a multiple linear regression model to predict in-vivo energy metabolite ratios.
    RESULTS: Subcortical brain iron deposition, particularly in the putamen and globus pallidus, was highly predictive of the region-specific amount of high-energy-containing phosphorus metabolites in our subjects.
    CONCLUSIONS: Our study suggests that brain iron deposition but not the variability of individual volumetric measurements are highly predictive of mitochondrial impairment in vivo. These findings offer the opportunity, e.g., by using chelating therapies, to improve mitochondrial bioenergetics in patients with idiopathic Parkinson's disease.
    Keywords:  Iron; Mitochondria; Parkinson's disease (PD)
  56. Sci Adv. 2022 Mar 04. 8(9): eabm5386
      More than 50 neurological and neuromuscular diseases are caused by short tandem repeat (STR) expansions, with 37 different genes implicated to date. We describe the use of programmable targeted long-read sequencing with Oxford Nanopore's ReadUntil function for parallel genotyping of all known neuropathogenic STRs in a single assay. Our approach enables accurate, haplotype-resolved assembly and DNA methylation profiling of STR sites, from a list of predetermined candidates. This correctly diagnoses all individuals in a small cohort (n = 37) including patients with various neurogenetic diseases (n = 25). Targeted long-read sequencing solves large and complex STR expansions that confound established molecular tests and short-read sequencing and identifies noncanonical STR motif conformations and internal sequence interruptions. We observe a diversity of STR alleles of known and unknown pathogenicity, suggesting that long-read sequencing will redefine the genetic landscape of repeat disorders. Last, we show how the inclusion of pharmacogenomic genes as secondary ReadUntil targets can further inform patient care.