bims-mitdis Biomed News
on Mitochondrial disorders
Issue of 2023‒04‒23
39 papers selected by
Catalina Vasilescu
Helmholz Munich


  1. Elife. 2023 Apr 19. pii: e87194. [Epub ahead of print]12
      A large-scale study of mutations in mitochondrial DNA has revealed a subset that do not accumulate with age.
    Keywords:  aging; duplex sequencing; genetics; genomics; mitochondrial DNA; mouse; somatic mutations
    DOI:  https://doi.org/10.7554/eLife.87194
  2. Dis Model Mech. 2023 Apr 01. pii: dmm049783. [Epub ahead of print]16(4):
      The list of mitochondrial DNA (mtDNA) variants detected in individuals with neurodegenerative diseases is constantly growing. Evaluating their functional consequences and pathogenicity is not easy, especially when they are found in only a limited number of patients together with wild-type mtDNA (heteroplasmy). Owing to its amenability to mitochondrial genetic transformation and incapacity to stably maintain heteroplasmy, and the strong evolutionary conservation of the proteins encoded in mitochondria, Saccharomyces cerevisiae provides a convenient model to investigate the functional consequences of human mtDNA variants. We herein report the construction and energy-transducing properties of yeast models of eight MT-ATP6 gene variants identified in patients with various disorders: m.8843T>C, m.8950G>A, m.9016A>G, m.9025G>A, m.9029A>G, m.9058A>G, m.9139G>A and m.9160T>C. Significant defect in growth dependent on respiration and deficits in ATP production were observed in yeast models of m.8950G>A, m.9025G>A and m.9029A>G, providing evidence of pathogenicity for these variants. Yeast models of the five other variants showed very mild, if any, effect on mitochondrial function, suggesting that the variants do not have, at least alone, the potential to compromise human health.
    Keywords:   MT-ATP6 ; ATP synthase; LHON; MILS; Mitochondrial DNA mutation; Mitochondrial diseases; Yeast; mtDNA
    DOI:  https://doi.org/10.1242/dmm.049783
  3. Mol Syndromol. 2023 Apr;14(2): 171-174
      Introductıon: Succinate dehydrogenase deficiency, also known as mitochondrial complex II deficiency, is a rare inborn error of metabolism, accounting for approximately 2% of mitochondrial disease. Mutations in the four genes SDHA, B, C,and D have been reported resulting in diverse clinical presentations. The vast majority of clinically affected individuals reported in the literature harbor genetic variants within the SDHA gene and present with a Leigh syndrome phenotype, clinically defined as a subacute necrotizing encephalopathy.Case Report: Herein, we report the first case of a 7-year-old child who was diagnosed as having succinate dehydrogenase deficiency. The affected child presented at 1 year of age with encephalopathy and developmental regression following viral illnesses. MRI changes supported a clinical diagnosis of Leigh syndrome and c.1328C>Q and c.872A>C SDHA variants were identified as compound heterozygous. Mitochondrial cocktail treatment including L-carnitine, riboflavin, thiamine, biotin, and ubiquinone was started. Mild clinical improvement was observed after treatment. He is now unable to walk and speak. The second patient, a 21-year-old woman, presented with generalized muscle weakness, easy fatigability, and cardiomyopathy. Investigations revealed increased lactate level of 67.4 mg/dL (4.5-19.8) with repeatedly increased plasma alanine levels 1,272 µmol/L (200-579). We administered carnitine, coenzyme, riboflavin, and thiamine for empirical therapy with the suspicion of mitochondrial disease. Clinical exome sequencing revealed compound heterozygous variants NM_004168.4:c.1945_1946del (p.Leu649GlufsTer4) at exon 15 of the SDHA gene and NM_004168.4:c.1909-12_1909-11del at intron 14 of SDHA gene.
    Discussion and Conclusion: There are several very different presentations including Leigh syndrome, epileptic encephalopathy, and cardiomyopathy. Some cases present following viral illness; this feature is not specific to mitochondrial complex II deficiency and occurs in many other mitochondrial disease presentations. There is no cure for complex II deficiency, though some reported patients showed clinical improvement following riboflavin therapy. Riboflavin is not the only therapeutic intervention that is available to patients with an isolated complex II deficiency and various other compounds have shown promise in the treatment of symptoms, including L-carnitine and ubiquinone. Treatment alternatives such as parabenzoquinone EPI-743 and rapamycin are under study in the treatment of the disease.
    Keywords:  Leigh syndrome phenotype; Metabolic diseases; Mitochondrial disease; Succinate dehydrogenase deficiency; Whole-exome sequencing
    DOI:  https://doi.org/10.1159/000527538
  4. Methods Mol Biol. 2023 ;2662 53-65
      Measuring the mitochondrial respiratory capacity of brown adipocytes ex vivo is an essential approach to understand the cell-autonomous regulators of mitochondrial uncoupling in brown adipose tissue. Here, we describe two protocols to isolate brown preadipocytes from mice, their ex vivo differentiation to mature brown adipocytes and the quantification of their mitochondrial uncoupling capacity by respirometry.
    Keywords:  Brown adipocytes; Mitochondria; Respirometry; Thermogenesis; Uncoupling
    DOI:  https://doi.org/10.1007/978-1-0716-3167-6_5
  5. PNAS Nexus. 2023 Apr;2(4): pgad105
      Adequate thymidylate [deoxythymidine monophosphate (dTMP) or the "T" base in DNA] levels are essential for stability of mitochondrial DNA (mtDNA) and nuclear DNA (nDNA). Folate and vitamin B12 (B12) are essential cofactors in folate-mediated one-carbon metabolism (FOCM), a metabolic network which supports synthesis of nucleotides (including dTMP) and methionine. Perturbations in FOCM impair dTMP synthesis, causing misincorporation of uracil (or a "U" base) into DNA. During B12 deficiency, cellular folate accumulates as 5-methyltetrahdryfolate (5-methyl-THF), limiting nucleotide synthesis. The purpose of this study was to determine how reduced levels of the B12-dpendent enzyme methionine synthase (MTR) and dietary folate interact to affect mtDNA integrity and mitochondrial function in mouse liver. Folate accumulation, uracil levels, mtDNA content, and oxidative phosphorylation capacity were measured in male Mtr+/+ and Mtr+/- mice weaned onto either a folate-sufficient control (C) diet (2 mg/kg folic acid) or a folate-deficient (FD) diet (lacking folic acid) for 7 weeks. Mtr heterozygosity led to increased liver 5-methyl-THF levels. Mtr+/- mice consuming the C diet also exhibited a 40-fold increase in uracil in liver mtDNA. Mtr+/- mice consuming the FD diet exhibited less uracil accumulation in liver mtDNA as compared to Mtr+/+ mice consuming the FD diet. Furthermore, Mtr+/- mice exhibited 25% lower liver mtDNA content and a 20% lower maximal oxygen consumption rates. Impairments in mitochondrial FOCM are known to lead to increased uracil in mtDNA. This study demonstrates that impaired cytosolic dTMP synthesis, induced by decreased Mtr expression, also leads to increased uracil in mtDNA.
    Keywords:  DNA; folate; methionine synthase; uracil; vitamin B12
    DOI:  https://doi.org/10.1093/pnasnexus/pgad105
  6. Cell Rep. 2023 Apr 20. pii: S2211-1247(23)00383-2. [Epub ahead of print]42(5): 112372
      Autophagy is a homeostatic process critical for cellular survival, and its malfunction is implicated in human diseases including neurodegeneration. Loss of autophagy contributes to cytotoxicity and tissue degeneration, but the mechanistic understanding of this phenomenon remains elusive. Here, we generated autophagy-deficient (ATG5-/-) human embryonic stem cells (hESCs), from which we established a human neuronal platform to investigate how loss of autophagy affects neuronal survival. ATG5-/- neurons exhibit basal cytotoxicity accompanied by metabolic defects. Depletion of nicotinamide adenine dinucleotide (NAD) due to hyperactivation of NAD-consuming enzymes is found to trigger cell death via mitochondrial depolarization in ATG5-/- neurons. Boosting intracellular NAD levels improves cell viability by restoring mitochondrial bioenergetics and proteostasis in ATG5-/- neurons. Our findings elucidate a mechanistic link between autophagy deficiency and neuronal cell death that can be targeted for therapeutic interventions in neurodegenerative and lysosomal storage diseases associated with autophagic defect.
    Keywords:  CP: Cell biology; CP: Metabolism; NAD; NADases; NAM; NMN; NR; autophagy; cell death; cell survival, human embryonic stem cell-derived neurons; mitochondria; nicotinamide; nicotinamide adenine dinucleotide; nicotinamide mononucleotide; nicotinamide riboside
    DOI:  https://doi.org/10.1016/j.celrep.2023.112372
  7. bioRxiv. 2023 Apr 03. pii: 2023.04.02.535296. [Epub ahead of print]
      Healthy mitochondria are critical for reproduction. During aging, both reproductive fitness and mitochondrial homeostasis decline. Mitochondrial metabolism and dynamics are key factors in supporting mitochondrial homeostasis. However, how they are coupled to control reproductive health remains unclear. We report that mitochondrial GTP metabolism acts through mitochondrial dynamics factors to regulate reproductive aging. We discovered that germline-only inactivation of GTP- but not ATP-specific succinyl-CoA synthetase (SCS), promotes reproductive longevity in Caenorhabditis elegans. We further revealed an age-associated increase in mitochondrial clustering surrounding oocyte nuclei, which is attenuated by the GTP-specific SCS inactivation. Germline-only induction of mitochondrial fission factors sufficiently promotes mitochondrial dispersion and reproductive longevity. Moreover, we discovered that bacterial inputs affect mitochondrial GTP and dynamics factors to modulate reproductive aging. These results demonstrate the significance of mitochondrial GTP metabolism in regulating oocyte mitochondrial homeostasis and reproductive longevity and reveal mitochondrial fission induction as an effective strategy to improve reproductive health.
    DOI:  https://doi.org/10.1101/2023.04.02.535296
  8. Geroscience. 2023 Apr 22.
      Altered mitochondrial function is tightly linked to lifespan regulation, but underlying mechanisms remain unclear. Here, we report the chronological and replicative lifespan variation across 167 yeast knock-out strains, each lacking a single nuclear-coded mitochondrial gene, including 144 genes with human homologs, many associated with diseases. We dissected the signatures of observed lifespan differences by analyzing profiles of each strain's proteome, lipidome, and metabolome under fermentative and respiratory culture conditions, which correspond to the metabolic states of replicative and chronologically aging cells, respectively. Examination of the relationships among extended longevity phenotypes, protein, and metabolite levels revealed that although many of these nuclear-encoded mitochondrial genes carry out different functions, their inhibition attenuates a common mechanism that controls cytosolic ribosomal protein abundance, actin dynamics, and proteasome function to regulate lifespan. The principles of lifespan control learned through this work may be applicable to the regulation of lifespan in more complex organisms, since many aspects of mitochondrial function are highly conserved among eukaryotes.
    Keywords:  Aging; Metabolism; Mitochondrial proteins; Omics; Yeast
    DOI:  https://doi.org/10.1007/s11357-023-00796-4
  9. Cell Metab. 2023 Apr 11. pii: S1550-4131(23)00094-3. [Epub ahead of print]
      Even-chain acylcarnitine (AC) metabolites, most of which are generated as byproducts of incomplete fatty acid oxidation (FAO), are viewed as biomarkers of mitochondrial lipid stress attributable to one or more metabolic bottlenecks in the β-oxidation pathway. The origins and functional implications of FAO bottlenecks remain poorly understood. Here, we combined a sophisticated mitochondrial phenotyping platform with state-of-the-art molecular profiling tools and multiple two-state mouse models of respiratory function to uncover a mechanism that connects AC accumulation to lipid intolerance, metabolic inflexibility, and respiratory inefficiency in skeletal muscle mitochondria. These studies also identified a short-chain carbon circuit at the C4 node of FAO wherein reverse flux of glucose-derived acetyl CoA through medium-chain ketothiolase enhances lipid tolerance and redox stability in heart mitochondria by regenerating free CoA and NAD+. The findings help to explain why diminished FAO capacity, AC accumulation, and metabolic inflexibility are tightly linked to poor health outcomes.
    Keywords:  acylcarnitines; bioenergetics; exercise; fatty acid oxidation; heart; ketothiolase; metabolic flexibility; mitochondria; pyruvate; skeletal muscle
    DOI:  https://doi.org/10.1016/j.cmet.2023.03.016
  10. Cerebellum. 2023 Apr 22.
      Different pathogenic variants in the DNA polymerase-gamma2 (POLG2) gene cause a rare, clinically heterogeneous mitochondrial disease. We detected a novel POLG2 variant (c.1270 T > C, p.Ser424Pro) in a family with adult-onset cerebellar ataxia and progressive ophthalmoplegia. We demonstrated altered mitochondrial integrity in patients' fibroblast cultures but no changes of the mitochondrial DNA were found when compared to controls. We consider this novel, segregating POLG2 variant as disease-causing in this family. Moreover, we systematically screened the literature for POLG2-linked phenotypes and re-evaluated all mutations published to date for pathogenicity according to current knowledge. Thereby, we identified twelve published, likely disease-causing variants in 19 patients only. The core features included progressive ophthalmoplegia and cerebellar ataxia; parkinsonism, neuropathy, cognitive decline, and seizures were also repeatedly found in adult-onset heterozygous POLG2-related disease. A severe phenotype relates to biallelic pathogenic variants in POLG2, i.e., newborn-onset liver failure, referred to as mitochondrial depletion syndrome. Our work underlines the broad clinical spectrum of POLG2-related disease and highlights the importance of functional characterization of variants of uncertain significance to enable meaningful genetic counseling.
    Keywords:  Adult-onset ataxia; Mitochondrial dysfunction; POLG2
    DOI:  https://doi.org/10.1007/s12311-023-01557-x
  11. bioRxiv. 2023 Apr 03. pii: 2023.04.03.535288. [Epub ahead of print]
      Efficient metabolic engineering and the development of mitochondrial therapeutics often rely upon the specific and strong import of foreign proteins into mitochondria. Fusing a protein to a mitochondria-bound signal peptide is a common method to localize proteins to mitochondria, but this strategy is not universally effective with particular proteins empirically failing to localize. To help overcome this barrier, this work develops a generalizable and open-source framework to design proteins for mitochondrial import and quantify their specific localization. By using a Python-based pipeline to quantitatively assess the colocalization of different proteins previously used for precise genome editing in a high-throughput manner, we reveal signal peptide-protein combinations that localize well in mitochondria and, more broadly, general trends about the overall reliability of commonly used mitochondrial targeting signals.
    DOI:  https://doi.org/10.1101/2023.04.03.535288
  12. Neurol Genet. 2023 Feb;9(1): e200048
    Care4Rare Canada Consortium,
      Background and Objectives: Coenzyme Q10 (CoQ10) is an important electron carrier and antioxidant. The COQ7 enzyme catalyzes the hydroxylation of 5-demethoxyubiquinone-10 (DMQ10), the second-to-last step in the CoQ10 biosynthesis pathway. We report a consanguineous family presenting with a hereditary motor neuropathy associated with a homozygous c.1A > G p.? variant of COQ7 with abnormal CoQ10 biosynthesis.Methods: Affected family members underwent clinical assessments that included nerve conduction testing, histologic analysis, and MRI. Pathogenicity of the COQ7 variant was assessed in cultured fibroblasts and skeletal muscle using a combination of immunoblots, respirometry, and quinone analysis.
    Results: Three affected siblings, ranging from 12 to 24 years of age, presented with a severe length-dependent motor neuropathy with marked symmetric distal weakness and atrophy with normal sensation. Muscle biopsy of the quadriceps revealed chronic denervation pattern. An MRI examination identified moderate to severe fat infiltration in distal muscles. Exome sequencing demonstrated the homozygous COQ7 c.1A > G p.? variant that is expected to bypass the first 38 amino acid residues at the n-terminus, initiating instead with methionine at position 39. This is predicted to cause the loss of the cleavable mitochondrial targeting sequence and 2 additional amino acids, thereby preventing the incorporation and subsequent folding of COQ7 into the inner mitochondrial membrane. Pathogenicity of the COQ7 variant was demonstrated by diminished COQ7 and CoQ10 levels in muscle and fibroblast samples of affected siblings but not in the father, unaffected sibling, or unrelated controls. In addition, fibroblasts from affected siblings had substantial accumulation of DMQ10, and maximal mitochondrial respiration was impaired in both fibroblasts and muscle.
    Discussion: This report describes a new neurologic phenotype of COQ7-related primary CoQ10 deficiency. Novel aspects of the phenotype presented by this family include pure distal motor neuropathy involvement, as well as the lack of upper motor neuron features, cognitive delay, or sensory involvement in comparison with cases of COQ7-related CoQ10 deficiency previously reported in the literature.
    DOI:  https://doi.org/10.1212/NXG.0000000000200048
  13. Mol Metab. 2023 Apr 14. pii: S2212-8778(23)00061-3. [Epub ahead of print] 101727
      OBJECTIVE: Mitochondrial network dynamics may play role in metabolic homeostasis. Whether mitochondrial network dynamics are involved in adaptations to day-night fluctuations in energy supply and demand is unclear. Here we visualized and quantified the mitochondrial network morphology in human skeletal muscle of young healthy lean and older individuals with obesity over the course of 24 h METHODS: Muscle biopsies taken at 5 timepoints over a 24-hour period obtained from young healthy lean and older metabolically impaired obese males were analyzed for mitochondrial network integrity with confocal laser scanning microscopy. Variation of level of fragmentation over the course of the day were aligned with variation of mitochondrial respiration over the day RESULTS: Young healthy lean individuals displayed a day-night rhythmicity in mitochondrial network morphology, which aligned with the day-night rhythmicity of mitochondrial respiratory capacity, with a more fused network coinciding with higher mitochondrial respiratory capacity. In the older individuals with obesity, the mitochondrial network was more fragmented overall compared to young healthy lean individuals and completely lacked 24 h rhythmicity, which was also true for the mitochondrial respiratory capacity CONCLUSIONS: Our data shows a paralleled rhythmicity between mitochondrial network morphology and mitochondrial oxidative capacity, which oscillates over the course of a mimicked real-life day in human skeletal muscle of young, healthy lean individuals. In older individuals with obesity, the lack of a 24-hour rhythmicity in mitochondrial network connectivity was also aligned with a lack in respiratory capacity. This suggests that 24-hour rhythmicity in mitochondrial network connectivity is a determinant of rhythmicity in mitochondrial respiratory capacity. Thus, restoring mitochondrial network integrity may promote mitochondrial respiratory capacity and hence contribute to blunting the metabolic aberrations in individuals with a disturbed 24-hour rhythmicity in metabolism, like older individuals with obesity.
    Keywords:  Day-night rhythm; Mitochondrial function; Mitochondrial network integrity; Skeletal muscle
    DOI:  https://doi.org/10.1016/j.molmet.2023.101727
  14. Clin Neurol Neurosurg. 2023 Apr 07. pii: S0303-8467(23)00128-2. [Epub ahead of print]229 107712
      Mitochondrial neurogastrointestinal encephalopathy (MNGIE) is a well-known mitochondrial depletion syndrome. Since Van Goethem et al. described MNGIE syndrome with pathogenic POLG1 mutations in 2003, POLG1 gene became a target for MNGIE patients. Cases with POLG1 mutations strikingly differ from classic MNGIE patients due to a lack of leukoencephalopathy. Here we present a female patient with very early onset disease and leukoencephalopathy compatible with classic MNGIE disease who turned out to have homozygous POLG1 mutation compatible with MNGIE-like syndrome, mitochondrial depletion syndrome type 4b.
    Keywords:  Gastroparesis; Inherited peripheral neuropathy; Leukoencephalopathy; MNGIE syndrome; Mitochondrial DNA Depletion Syndrome
    DOI:  https://doi.org/10.1016/j.clineuro.2023.107712
  15. Methods Mol Biol. 2023 ;2662 67-75
      High-resolution respirometry is commonly used to quantify mitochondrial respiratory rates. In the respirometry chamber, a change in oxygen concentration is measured by a polarographic electrode to derive the rate of oxygen consumption (JO2). Here, we describe our adapted protocol to bioenergetically phenotype mitochondria from mouse brown adipose tissue (BAT). Given the presence of uncoupling protein 1 (UCP1), mitochondria from BAT provide unique challenges and opportunities in applying high-resolution respirometry to understand energy transduction through oxidative phosphorylation (OXPHOS).
    Keywords:  Bioenergetics; Mitochondria; Oroboros Oxygraph-2 K respirometry; Oxygen consumption rate; Respiration; Uncoupling protein 1
    DOI:  https://doi.org/10.1007/978-1-0716-3167-6_6
  16. J Cell Sci. 2023 Apr 19. pii: jcs.260578. [Epub ahead of print]
      Mitochondria are essential organelles of eukaryotic cells that are characterized by their unique and complex membrane system. They are confined from the cytosol by an envelope consisting of two membranes. Signals, metabolites, proteins and lipids have to be transferred across these membranes via proteinaceous contact sites to keep mitochondria functional. In the present study we identified a novel mitochondrial contact site that is formed by the inner membrane protein Cqd1 and the outer membrane proteins Por1 and Om14. Similar to the mitochondrial porin, Por1, Cqd1 is highly conserved, suggesting that this complex is conserved in form and function from yeast to human. Cqd1 is a member of the UbiB protein kinase-like family (also called aarF domain containing kinases). It was recently shown that Cqd1 in cooperation with Cqd2 controls the cellular distribution of coenzyme Q by a yet unknown mechanism. Our data suggest that Cqd1 is additionally involved in phospholipid homeostasis. Moreover, overexpression of CQD1 and CQD2 causes tethering of mitochondria to the endoplasmic reticulum, which might explain the ability of Cqd2 to rescue ERMES deletion phenotypes.
    Keywords:  Contact sites; Mitochondria; Mitochondrial biogenesis; Mitochondrial morphology; Phospholipids; UbiB protein family
    DOI:  https://doi.org/10.1242/jcs.260578
  17. Mol Syndromol. 2023 Apr;14(2): 101-108
      Introduction: Antenatal presentation of hypertrophic cardiomyopathy (HCM) is rare. We describe familial recurrence of antenatal HCM associated with intrauterine growth restriction and the diagnostic process undertaken.Methods: Two pregnancies with antenatal HCM were followed up. Biological assessment including metabolic analyses, genetic analyses, and respiratory chain study was performed. We describe the clinical course of these two pregnancies, antenatal manifestations as well as specific histopathological findings, and review the literature.
    Results: The assessment revealed a deficiency in complex I of the respiratory chain and two likely pathogenic variations in the ACAD9 gene.
    Discussion and Conclusion: Antenatal HCM is rare and a diagnosis is not always made. In pregnancies presenting with cardiomyopathy and intrauterine growth restriction, ACAD9 deficiency should be considered as one of the potential underlying diagnoses, and ACAD9 molecular testing should be included among other prenatal investigations.
    Keywords:  ACAD9 mutations; Antenatal hypertrophic cardiomyopathy; Congenital disease; Fetal histopathology; Intrauterine growth restriction; Mitochondrial complex I deficiency
    DOI:  https://doi.org/10.1159/000526022
  18. J Biol Chem. 2023 Apr 13. pii: S0021-9258(23)01736-2. [Epub ahead of print] 104708
      Physiologic Ca2+ entry via the Mitochondrial Calcium Uniporter (MCU) participates in energetic adaption to workload but may also contribute to cell death during Ischemia/Reperfusion (I/R) injury. The MCU has been identified as the primary mode of Ca2+ import into mitochondria. Several groups have tested the hypothesis that Ca2+ import via MCU is detrimental during I/R injury using genetically-engineered mouse models, yet the results from these studies are inconclusive. Furthermore, mitochondria exhibit unstable or oscillatory membrane potentials (ΔΨm) when subjected to stress, such as during I/R, but it is unclear if the primary trigger is excess influx of mitochondrial Ca2+ (mCa2+), reactive oxygen species (ROS) accumulation, or other factors. Here, we critically examine whether MCU-mediated mitochondrial Ca2+ uptake during I/R is involved in ΔΨm instability, or sustained mitochondrial depolarization, during reperfusion by acutely knocking out MCU in neonatal mouse ventricular myocyte (NMVM) monolayers subjected to simulated I/R. Unexpectedly, we find that MCU knockout does not significantly alter mCa2+ import during I/R, nor does it affect ΔΨm recovery during reperfusion. In contrast, blocking the mitochondrial sodium-calcium exchanger (mNCE) suppressed the mCa2+ increase during Ischemia but did not affect ΔΨm recovery or the frequency of ΔΨm oscillations during reperfusion, indicating that mitochondrial ΔΨm instability on reperfusion is not triggered by mCa2+. Interestingly, inhibition of mitochondrial electron transport or supplementation with antioxidants stabilized I/R-induced ΔΨm oscillations. The findings are consistent with mCa2+ overload being mediated by reverse-mode mNCE activity and support ROS-induced ROS release as the primary trigger of ΔΨm instability during reperfusion injury.
    Keywords:  image processing; ischemia; mitochondrial membrane potential; oscillation; oxidative phosphorylation; reperfusion; time-series analysis; wavelet
    DOI:  https://doi.org/10.1016/j.jbc.2023.104708
  19. J Neuromuscul Dis. 2023 Apr 12.
      BACKGROUND: Whole-exome sequencing (WES) facilitates the diagnosis of hereditary neuromuscular disorders. To achieve an accurate diagnosis, physicians should interpret the genetic report carefully along with clinical information and examinations. We described our experience with (1) clinical validation in patients with variants found using WES and (2) a diagnostic approach for those with negative findings from WES.METHODS: WES was performed on patients with the clinical impression of hereditary neuromuscular disorders. Information on clinical manifestations, neurological examination, electrodiagnostic studies, histopathology of muscle and nerve, and laboratory tests were collected.
    RESULTS: Forty-one patients (Male/Female: 18/23, age of onset: 34.5±15.9) accepted WES and were categorized into four scenarios: (1) patients with a positive WES result, (2) patients with an inconclusive WES result but supporting clinical data, (3) negative findings from WES, but a final diagnosis after further work-up, and (4) undetermined etiology from WES and in further work-ups. The yield rate of the initial WES was 63.4% (26/41). Among these, seventeen patients had positive WES result, while the other nine patients had inconclusive WES result but supporting clinical data. Notably, in the fifteen patients with equivocal or negative findings from WES, four patients (26.7%) achieved a diagnosis after further workup: tumor-induced osteomalacia, metabolic myopathy with pathogenic variants in mitochondrial DNA, microsatellite expansion disease, and vasculitis-related neuropathy. The etiologies remained undetermined in eleven patients (myopathy: 7, neuropathy: 4) after WES and further workup.
    CONCLUSIONS: It is essential to design genotype-guided molecular studies to correlate the identified variants with their clinical features. For patients who had negative findings from WES, acquired diseases, mitochondrial DNA disorders and microsatellite expansion diseases should be considered.
    Keywords:  Whole-exome sequencing; motor neuron disease; myopathy; neuromuscular disorders; neuropathy
    DOI:  https://doi.org/10.3233/JND-230013
  20. NPJ Parkinsons Dis. 2023 Apr 18. 9(1): 65
      Homozygous or compound heterozygous (biallelic) variants in PRKN are causal for PD with highly penetrant symptom expression, while the much more common heterozygous variants may predispose to PD with highly reduced penetrance, through altered mitochondrial function. In the presence of pathogenic heterozygous variants, it is therefore important to test for mitochondrial alteration in cells derived from variant carriers to establish potential presymptomatic molecular markers. We generated lymphoblasts (LCLs) and human induced pluripotent stem cell (hiPSC)-derived neurons from non-manifesting heterozygous PRKN variant carriers and tested them for mitochondrial functionality. In LCLs, we detected hyperactive mitochondrial respiration, and, although milder compared to a biallelic PRKN-PD patient, hiPSC-derived neurons of non-manifesting heterozygous variant carriers also displayed several phenotypes of altered mitochondrial function. Overall, we identified molecular phenotypes that might be used to monitor heterozygous PRKN variant carriers during the prodromal phase. Such markers might also be useful to identify individuals at greater risk of eventual disease development and for testing potential mitochondrial function-based neuroprotective therapies before neurodegeneration advances.
    DOI:  https://doi.org/10.1038/s41531-023-00499-9
  21. Cell Rep. 2023 Apr 20. pii: S2211-1247(23)00431-X. [Epub ahead of print]42(5): 112420
      Sun et al. demonstrate that defects in autophagy cause nicotinamide adenine dinucleotide (NAD) depletion and neurotoxicity.1 Restoring NAD levels rescues cytotoxicity in autophagy-deficient neurons, providing a potential therapy for neurodegenerative and lysosomal storage diseases associated with autophagy defects.
    DOI:  https://doi.org/10.1016/j.celrep.2023.112420
  22. Nucleic Acids Res. 2023 Apr 18. pii: gkad284. [Epub ahead of print]
      Over the last decade, Haplogrep has become a standard tool for haplogroup classification in the field of human mitochondrial DNA and is widely used by medical, forensic, and evolutionary researchers. Haplogrep scales well for thousands of samples, supports many file formats and provides an intuitive graphical web interface. Nevertheless, the currently available version has limitations when applying it to large biobank-scale data. In this paper, we present a major upgrade to the software by adding (a) haplogroup summary statistics and variant annotations from various publicly available genome databases, (b) an interface to connect new phylogenetic trees, (c) a new state-of-the-art web framework managing large scale data, (d) algorithmic adaptions to improve FASTA classification using BWA-specific alignment rules and (e) a pre-classification quality control step for VCF samples. These improvements will give researchers the opportunity to classify thousands of samples as usual but providing additional ways to investigate the dataset directly in the browser. The web service and its documentation can be accessed freely without any registration at https://haplogrep.i-med.ac.at.
    DOI:  https://doi.org/10.1093/nar/gkad284
  23. Cell. 2023 Apr 17. pii: S0092-8674(23)00300-8. [Epub ahead of print]
      Functional genomic strategies have become fundamental for annotating gene function and regulatory networks. Here, we combined functional genomics with proteomics by quantifying protein abundances in a genome-scale knockout library in Saccharomyces cerevisiae, using data-independent acquisition mass spectrometry. We find that global protein expression is driven by a complex interplay of (1) general biological properties, including translation rate, protein turnover, the formation of protein complexes, growth rate, and genome architecture, followed by (2) functional properties, such as the connectivity of a protein in genetic, metabolic, and physical interaction networks. Moreover, we show that functional proteomics complements current gene annotation strategies through the assessment of proteome profile similarity, protein covariation, and reverse proteome profiling. Thus, our study reveals principles that govern protein expression and provides a genome-spanning resource for functional annotation.
    Keywords:  Saccharomyces cerevisiae; data-independent acquisition; deletion; functional genomics; functional proteomics; gene annotation; high throughput; knockout; quantitative proteomics; systems biology
    DOI:  https://doi.org/10.1016/j.cell.2023.03.026
  24. Brain. 2023 Apr 18. pii: awad131. [Epub ahead of print]
      Leber Hereditary Optic Neuropathy (LHON) is a primary inherited neurodegenerative disorder of the optic nerve. It has been ascribed to variants in the mitochondrial genome, mainly the m.3460G > A, m.11778G > A and m.14484T > C mutations in ND1, ND4 and ND6 respectively. Nonetheless, inconclusive molecular diagnosis is not uncommon. Recently, biallelic mutations in the NDUFS2, DNAJC30, MCAT and NDUFA12 nuclear genes have been identified in unresolved LHON cases, identifying an autosomal recessive LHON (arLHON, OMIM:619382). The clinical presentation of arLHON copies that of typical mtLHON, with an acute phase of sudden and severe vision loss, telangiectatic and tortuous vessels around the optic nerve, and swelling of the retinal nerve fiber layer (RNFL). This is followed by a chronic phase of RNFL loss, but eventually, affected individuals recovered partial or full visual acuity. Idebenone treatment significantly improved vision recovery in DNAJC30-associated patients. As mtLHON, arLHON predominantly affected male as compared to female carriers. The discovery of arLHON cases breaks with the dogma of exclusive maternal inheritance. It defines a new neuro-ophthalmo-genetic paradigm which should be considered in individuals manifesting a LHON phenotype, but inconclusive molecular diagnosis. NDUFS2, DNAJC30, MCAT and NDUFA12 should be investigated in these individuals, knowing that other arLHON genes might exist.
    Keywords:  Leber; autosomal; optic neuropathy; recessive
    DOI:  https://doi.org/10.1093/brain/awad131
  25. J Gerontol A Biol Sci Med Sci. 2023 Apr 17. pii: glad106. [Epub ahead of print]
      Advancing age and many disease states are associated with declines in nicotinamide adenine dinucleotide (NAD +) levels. Preclinical studies suggest that boosting NAD + abundance with precursor compounds, such as nicotinamide riboside or nicotinamide mononucleotide, has profound effects on physiological function in models of aging and disease. Translation of these compounds for oral supplementation in humans has been increasingly studied within the last ten years; however, the clinical evidence that raising NAD + concentrations can improve physiological function is unclear. The goal of this review was to synthesize the published literature on the effects of chronic oral supplementation with NAD + precursors on healthy aging and age-related chronic diseases. We identified nicotinamide riboside, nicotinamide riboside co-administered with pterostilbene, and nicotinamide mononucleotide as the most common candidates in investigations of NAD +-boosting compounds for improving physiological function in humans. Studies have been performed in generally healthy midlife and older adults, adults with cardiometabolic disease risk factors such as overweight and obesity, and numerous patient populations. Supplementation with these compounds is safe, tolerable, and can increase the abundance of NAD + and related metabolites in multiple tissues. Dosing regimens and study durations vary greatly across interventions, and small sample sizes limit data interpretation of physiological outcomes. Limitations are identified and future research directions are suggested to further our understanding of the potential efficacy of NAD +-boosting compounds for improving physiological function and extending human healthspan.
    Keywords:  Clinical Trials; Nicotinamide Riboside; Nutrition; Physiology; Successful Aging
    DOI:  https://doi.org/10.1093/gerona/glad106
  26. medRxiv. 2023 Apr 03. pii: 2023.04.03.23287612. [Epub ahead of print]
      Background: As availability of genomic testing grows, variant interpretation will increasingly be performed by genomic generalists, rather than domain-specific experts. Demand is rising for laboratories to accurately classify variants in inherited cardiac condition (ICC) genes, including as secondary findings.Methods: We analyse evidence for inheritance patterns, allelic requirement, disease mechanism and disease-relevant variant classes for 65 ClinGen-curated ICC gene-disease pairs. We present this information for the first time in a structured dataset, CardiacG2P, and assess application in genomic variant filtering.
    Results: For 36/65 gene-disease pairs, loss-of-function is not an established disease mechanism, and protein truncating variants are not known to be pathogenic. Using CardiacG2P as an initial variant filter allows for efficient variant prioritisation whilst maintaining a high sensitivity for retaining pathogenic variants compared with two other variant filtering approaches.
    Conclusions: Access to evidence-based structured data representing disease mechanism and allelic requirement aids variant filtering and analysis and is pre-requisite for scalable genomic testing.
    DOI:  https://doi.org/10.1101/2023.04.03.23287612
  27. Sci Rep. 2023 Apr 20. 13(1): 6445
      The retina has the greatest metabolic demand in the body particularly in dark adaptation when its sensitivity is enhanced. This requires elevated level of perfusion to sustain mitochondrial activity. However, mitochondrial performance declines with age leading to reduced adaptive ability. We assessed human retina metabolism in vivo using broad band near-infrared spectroscopy (bNIRS), which records colour changes in mitochondria and blood as retinal metabolism shifts in response to changes in environmental luminance. We demonstrate a significant sustained rise in mitochondrial oxidative metabolism in the first 3 min of darkness in subjects under 50 years old. This was not seen in those over 50 years. Choroidal oxygenation declines in < 50 s as mitochondrial metabolism increases, but gradually rises in the > 50 s. Significant group differences in blood oxygenation are apparent in the first 6 min, consistent with mitochondrial demand leading hemodynamic changes. A greater coupling between mitochondrial oxidative metabolism with hemodynamics is revealed in subjects older than 50, possibly due to reduced capacity in the older retina. Rapid in vivo assessment of retinal metabolism with bNIRS provides a route to understanding fundamental physiology and early identification of retinal disease before pathology is established.
    DOI:  https://doi.org/10.1038/s41598-023-32897-7
  28. Nat Commun. 2023 04 19. 14(1): 2230
      Despite the increasing use of genomic sequencing in clinical practice, the interpretation of rare genetic variants remains challenging even in well-studied disease genes, resulting in many patients with Variants of Uncertain Significance (VUSs). Computational Variant Effect Predictors (VEPs) provide valuable evidence in variant assessment, but they are prone to misclassifying benign variants, contributing to false positives. Here, we develop Deciphering Mutations in Actionable Genes (DeMAG), a supervised classifier for missense variants trained using extensive diagnostic data available in 59 actionable disease genes (American College of Medical Genetics and Genomics Secondary Findings v2.0, ACMG SF v2.0). DeMAG improves performance over existing VEPs by reaching balanced specificity (82%) and sensitivity (94%) on clinical data, and includes a novel epistatic feature, the 'partners score', which leverages evolutionary and structural partnerships of residues. The 'partners score' provides a general framework for modeling epistatic interactions, integrating both clinical and functional information. We provide our tool and predictions for all missense variants in 316 clinically actionable disease genes (demag.org) to facilitate the interpretation of variants and improve clinical decision-making.
    DOI:  https://doi.org/10.1038/s41467-023-37661-z
  29. Mol Cell. 2023 Apr 20. pii: S1097-2765(23)00213-7. [Epub ahead of print]83(8): 1340-1349.e7
      The glycerol-3-phosphate shuttle (G3PS) is a major NADH shuttle that regenerates reducing equivalents in the cytosol and produces energy in the mitochondria. Here, we demonstrate that G3PS is uncoupled in kidney cancer cells where the cytosolic reaction is ∼4.5 times faster than the mitochondrial reaction. The high flux through cytosolic glycerol-3-phosphate dehydrogenase (GPD) is required to maintain redox balance and support lipid synthesis. Interestingly, inhibition of G3PS by knocking down mitochondrial GPD (GPD2) has no effect on mitochondrial respiration. Instead, loss of GPD2 upregulates cytosolic GPD on a transcriptional level and promotes cancer cell proliferation by increasing glycerol-3-phosphate supply. The proliferative advantage of GPD2 knockdown tumor can be abolished by pharmacologic inhibition of lipid synthesis. Taken together, our results suggest that G3PS is not required to run as an intact NADH shuttle but is instead truncated to support complex lipid synthesis in kidney cancer.
    Keywords:  GPD; NAD; glycerol; glycerol-3-phosphate dehydrogenase; glycerol-3-phosphate shuttle; kidney cancer; lipids; metabolism; mitochondria
    DOI:  https://doi.org/10.1016/j.molcel.2023.03.023
  30. Genome Biol. 2023 Apr 21. 24(1): 85
      Detecting and mitigating off-target activity is critical to the practical application of CRISPR-mediated genome and epigenome editing. While numerous methods have been developed to map Cas9 binding specificity genome-wide, they are generally time-consuming and/or expensive, and not applicable to catalytically dead CRISPR enzymes. We have developed CasKAS, a rapid, inexpensive, and facile assay for identifying off-target CRISPR enzyme binding and cleavage by chemically mapping the unwound single-stranded DNA structures formed upon binding of a sgRNA-loaded Cas9 protein. We demonstrate this method in both in vitro and in vivo contexts.
    DOI:  https://doi.org/10.1186/s13059-023-02930-z
  31. Nat Rev Genet. 2023 Apr 21.
      Single-cell genomic technologies are revealing the cellular composition, identities and states in tissues at unprecedented resolution. They have now scaled to the point that it is possible to query samples at the population level, across thousands of individuals. Combining single-cell information with genotype data at this scale provides opportunities to link genetic variation to the cellular processes underpinning key aspects of human biology and disease. This strategy has potential implications for disease diagnosis, risk prediction and development of therapeutic solutions. But, effectively integrating large-scale single-cell genomic data, genetic variation and additional phenotypic data will require advances in data generation and analysis methods. As single-cell genetics begins to emerge as a field in its own right, we review its current state and the challenges and opportunities ahead.
    DOI:  https://doi.org/10.1038/s41576-023-00599-5
  32. Cell Mol Gastroenterol Hepatol. 2023 Apr 19. pii: S2352-345X(23)00054-1. [Epub ahead of print]
      BACKGROUND & AIMS: Alterations in mitochondrial morphology and function and increased oxidative stresses in hepatocytes are well-established in non-alcoholic fatty liver disease (NAFLD). Patients can undergo lifestyle changes, especially in earlier NAFLD stages, to reverse disease-induced phenotypes on a gross level. Yet, little is known about whether mitochondrial function and injuries recover upon reversal. We, thus, elucidated this question and interplays between cytoskeletal network and mitochondria in the development and reversal of steatosis.METHODS: We cultured primary human hepatocytes stably for 2 weeks and used free-fatty-acid (FAA) supplementation to induce steatosis over 7 days and reversed steatosis by FAA withdrawal over the next 7 days. We assessed cytoskeletal and mitochondrial morphologies using immunocytochemistry and confocal microscopy. We evaluated mitochondrial respiration and function via the Seahorse analyzer where we fully optimized reagent dosing specifically for human hepatocytes.
    RESULTS: During early steatosis, intracellular lipid droplets displaced microtubules altering mitochondrial distribution, and disrupted the F-actin network leading to loss of bile canaliculi in steatotic hepatocytes. Basal mitochondrial respiration, maximum respiratory capacity, and resistance to H2O2-induced cell death also increased as an adaptative response. Upon reversal of steatosis, F-actin and bile canaliculi were restored in hepatocytes. Nevertheless, we observed an increase in elongated mitochondrial branches accompanied by decreases in α-tubulin expression, mitochondrial proton leak, and susceptibility to H2O2-induced cell death.
    CONCLUSIONS: Despite the restoration of cytoskeletons morphologically upon reversal of steatosis, mitochondria, in hepatocytes, were impaired due to early adaptative respiratory increase. Hepatocytes were thus highly predisposed to H2O2-induced cell death. These results indicate the persistence of potential health risks for recovering NAFLD patients.
    Keywords:  cytoskeleton; mitochondria; non-alcoholic fatty liver disease; steatosis
    DOI:  https://doi.org/10.1016/j.jcmgh.2023.04.003
  33. Ann Transl Med. 2023 Mar 31. 11(6): 264
      Background and Objective: The mitochondrion is a crucial organelle for aerobic respiration and energy metabolism. It undergoes dynamic changes, including changes in its shape, function, and distribution through fission, fusion, and movement. Under normal conditions, mitochondrial dynamics are in homeostasis. However, once the balance is upset, the nervous system, which has high metabolic demands, will most likely be affected. Recent studies have shown that the imbalance of mitochondrial dynamics is involved in the occurrence and development of various neurological diseases. However, whether the regulation of mitochondrial dynamics can be used to treat neurological diseases is still unclear. We aimed to comprehensively analyze mitochondrial dynamics regulation and its potential role in the treatment of neurological diseases.Methods: A comprehensive literature review was carried out to understand the mechanisms and applications of mitochondrial dynamics in neurological diseases based on the literature available in PubMed, Web of Science, and Google Scholar.
    Key Content and Findings: This review discusses the molecular mechanisms related to mitochondrial dynamics and expounds upon the role of mitochondrial dynamics in the occurrence and development of neurodegenerative diseases, epilepsy, cerebrovascular disease, and brain tumors. Several clinically tested drugs with fewer side effects have been shown to improve the mitochondrial dynamics and nervous system function in neurological diseases.
    Conclusions: Disorders of mitochondrial dynamics can cause various neurological diseases. Elucidation of mechanisms and applications involved in mitochondrial dynamics will inform the development of new therapeutic targets and strategies for neurological diseases. Dynamin-related protein 1 (Drp1), as a highly relevant molecular for mitochondrial dynamics, might be a potential target for treating neurological diseases in the future.
    Keywords:  Alzeimer’s disease; Drp1; Parkinson’s disease; mitochondrial dynamics; mitochondrial fusion protein
    DOI:  https://doi.org/10.21037/atm-22-2401
  34. Front Pediatr. 2023 ;11 1103877
      Background: Neonatal intrahepatic cholestasis caused by citrin deficiency (NICCD) is a common clinical phenotype of citrin deficiency in infants. Its phenotype is atypical, so genetic testing is quite necessary for the diagnosis.Case presentation: We report 4 patients with jaundice and low body weight. Furthermore, the biochemical examination of all showed abnormal liver function and metabolic changes. DNA samples of the patients were extracted and subjected to genetic screening. All candidate pathogenic variants were validated by Sanger sequencing, and CNVs were ascertained by qPCR. The genetic screening revealed 6 variants in 4 patients, and all patients carried compound heterozygous variants of SLC25A13. Importantly, 3 variants were newly discovered: a nonsense mutation in exon17 (c.1803C > G), a frameshift mutation in exon 11(c.1141delG) and a deletion of the whole exon11. Thus, four NICCD patients were clearly caused by variants of SLC25A13. Biochemical indicators of all patients gradually returned to normal after dietary adjustment.
    Conclusions: Our study clarified the genetic etiology of the four infants, expanded the variant spectrum of SLC25A13, and provided a basis for genetic counseling of the family. Early diagnosis and intervention should be given to patients with NICCD.
    Keywords:  NICCD; SLC25A13; citrin deficiency; novel variant; prognosis
    DOI:  https://doi.org/10.3389/fped.2023.1103877
  35. Mov Disord. 2023 Apr;38(4): 515-517
      
    Keywords:  MDS diagnostic criteria; Parkinson's disease; autopsy; diagnosis
    DOI:  https://doi.org/10.1002/mds.29319
  36. Proteomics. 2023 Apr 17. e2200301
      Over the past decade, the majority of the mammalian genome considered to be noncoding has been revealed to be able to produce proteins. Many RNA molecules, mis-annotated as noncoding, actually are predicted to code for proteins. Some of those proteins have been identified and verified to play critical roles in multiple biological processes. The lipid droplet (LD) is a unique cellular organelle bound with a phospholipid monolayer membrane, and is closely associated with cellular lipid metabolism and metabolic disorders. However, it is still unclear how a protein targets to LDs. Here we identified a new protein on LDs, LDANP2, which is encoded by noncoding RNA, through a proteomics-based strategy. The key sequence for its localization on LDs, Truncation 3, is predicted to form an amphipathic helix. Surprisingly, the deletion of the first amino acid in Truncation 3 resulted in mitochondrial localization. How the types of amino acids would determine the LD or mitochondrial localizations of the protein was studied. The findings introduce a useful strategy to mine for new proteins and would provide clues to the understanding of how a protein would find its right organelle, with phospholipid monolayer or bilayer membrane. This article is protected by copyright. All rights reserved.
    Keywords:  LD-associated noncoding RNA-encoded protein; amphipathic helix; lipid droplet; mitochondrion; targeting
    DOI:  https://doi.org/10.1002/pmic.202200301
  37. medRxiv. 2023 Apr 03. pii: 2023.03.31.23287997. [Epub ahead of print]
      Detection of aberrantly spliced genes is an important step in RNA-seq-based rare disease diagnostics. We recently developed FRASER, a denoising autoencoder-based method for aberrant splicing detection that outperformed alternative approaches. However, as FRASER's three splice metrics are partially redundant and tend to be sensitive to sequencing depth, we introduce here a more robust intron excision metric, the Intron Jaccard Index, that combines alternative donor, alternative acceptor, and intron retention signal into a single value. Moreover, we optimized model parameters and filter cutoffs using candidate rare splice-disrupting variants as independent evidence. On 16,213 GTEx samples, our improved algorithm called typically 10 times fewer splicing outliers while increasing the proportion of candidate rare splice-disrupting variants by 10 fold and substantially decreasing the effect of sequencing depth on the number of reported outliers. Application on 303 rare disease samples confirmed the reduction fold-change of the number of outlier calls for a slight loss of sensitivity (only 2 out of 22 previously identified pathogenic splicing cases not recovered). Altogether, these methodological improvements contribute to more effective RNA-seq-based rare diagnostics by a drastic reduction of the amount of splicing outlier calls per sample at minimal loss of sensitivity.
    DOI:  https://doi.org/10.1101/2023.03.31.23287997
  38. Nat Commun. 2023 04 17. 14(1): 2194
      Mitochondria are hubs where bioenergetics, redox homeostasis, and anabolic metabolism pathways integrate through a tightly coordinated flux of metabolites. The contributions of mitochondrial metabolism to tumor growth and therapy resistance are evident, but drugs targeting mitochondrial metabolism have repeatedly failed in the clinic. Our study in pancreatic ductal adenocarcinoma (PDAC) finds that cellular and mitochondrial lipid composition influence cancer cell sensitivity to pharmacological inhibition of electron transport chain complex I. Profiling of patient-derived PDAC models revealed that monounsaturated fatty acids (MUFAs) and MUFA-linked ether phospholipids play a critical role in maintaining ROS homeostasis. We show that ether phospholipids support mitochondrial supercomplex assembly and ROS production; accordingly, blocking de novo ether phospholipid biosynthesis sensitized PDAC cells to complex I inhibition by inducing mitochondrial ROS and lipid peroxidation. These data identify ether phospholipids as a regulator of mitochondrial redox control that contributes to the sensitivity of PDAC cells to complex I inhibition.
    DOI:  https://doi.org/10.1038/s41467-023-37924-9