bims-mitdis Biomed News
on Mitochondrial disorders
Issue of 2022‒05‒01
seventy papers selected by
Catalina Vasilescu
University of Helsinki

  1. Autophagy. 2022 Apr 26.
      Mitostasis, the process of mitochondrial maintenance by biogenesis and degradative mechanisms, is challenged by the extreme length of axons. PINK1 (PTEN induced putative kinase 1) is a mitochondrial protein that targets damaged mitochondria for mitophagy. In reconciling the short half-life of PINK1 with the need for mitophagy of damaged axonal mitochondria, we found that axonal mitophagy depends on local translation of the Pink1 mRNA. Using live-cell imaging, we detected co-transport of the Pink1 mRNA on mitochondria in neurons, which is crucial for mitophagy in distal parts of the cell. Here we discuss how the coupling of the transcript of a short-lived mitochondrial protein to the movement of its target organelles contributes to our understanding of mitostasis in neurons.
    Keywords:  Axonal biology; RNA transport; local translation; mitochondria; mitophagy
  2. Proc Natl Acad Sci U S A. 2022 May 03. 119(18): e2200549119
      SignificancePrimary mitochondrial diseases (PMDs) are the most prevalent inborn metabolic disorders, affecting an estimated 1 in 4,200 individuals. Endurance exercise is generally known to improve mitochondrial function, but its indication in the heterogeneous group of PMDs is unclear. We determined the relationship between mitochondrial mutations, endurance exercise response, and the underlying molecular pathways in mice with distinct mitochondrial mutations. This revealed that mitochondria are crucial regulators of exercise capacity and exercise response. Endurance exercise proved to be mostly beneficial across the different mitochondrial mutant mice with the exception of a worsened dilated cardiomyopathy in ANT1-deficient mice. Thus, therapeutic exercises, especially in patients with PMDs, should take into account the physical and mitochondrial genetic status of the patient.
    Keywords:  endurance exercise; mitochondrial disease; skeletal muscle adaption
  3. Acta Neuropathol. 2022 Apr 30.
      Frontotemporal lobar degeneration (FTLD) is a common cause of young onset dementia and is characterised by focal neuropathology. The reasons for the regional neuronal vulnerability are not known. Mitochondrial mechanisms have been implicated in the pathogenesis of FTLD, raising the possibility that frontotemporal regional mutations of mitochondrial DNA (mtDNA) are contributory causes. Here we applied dual sequencing of the entire mtDNA at high depth to identify high-fidelity single nucleotide variants (mtSNVs) and mtDNA rearrangements in post mortem brain tissue of people affected by FTLD and age-matched controls. Both mtSNVs and mtDNA rearrangements were elevated in the temporal lobe, with the greatest burden seen in FTLD. mtSNVs found in multiple brain regions also reached a higher heteroplasmy levels in the temporal lobe. The temporal lobe of people with FTLD had a higher burden of ribosomal gene variants predicted to affect intra-mitochondrial protein synthesis, and a higher proportion of missense variants in genes coding for respiratory chain subunits. In conclusion, heteroplasmic mtDNA variants predicted to affect oxidative phosphorylation are enriched in FTLD temporal lobe, and thus may contribute to the regional vulnerability in pathogenesis.
  4. Nat Chem Biol. 2022 May;18(5): 461-469
      Metabolites once considered solely in catabolism or anabolism turn out to have key regulatory functions. Among these, the citric acid cycle intermediate succinate stands out owing to its multiple roles in disparate pathways, its dramatic concentration changes and its selective cell release. Here we propose that succinate has evolved as a signaling modality because its concentration reflects the coenzyme Q (CoQ) pool redox state, a central redox couple confined to the mitochondrial inner membrane. This connection is of general importance because CoQ redox state integrates three bioenergetic parameters: mitochondrial electron supply, oxygen tension and ATP demand. Succinate, by equilibrating with the CoQ pool, enables the status of this central bioenergetic parameter to be communicated from mitochondria to the rest of the cell, into the circulation and to other cells. The logic of this form of regulation explains many emerging roles of succinate in biology, and suggests future research questions.
  5. Cell Rep Methods. 2021 May 24. 1(1): 100002
      Mitochondria sustain the energy demand of the cell. The composition and functional state of the mitochondrial oxidative phosphorylation system are informative indicators of organelle bioenergetic capacity. Here, we describe a highly sensitive and reproducible method for a single-cell quantification of mitochondrial CI- and CIV-containing respiratory supercomplexes (CI∗CIV-SCs) as an alternative means of assessing mitochondrial respiratory chain integrity. We apply a proximity ligation assay (PLA) and stain CI∗CIV-SCs in fixed human and mouse brains, tumorigenic cells, induced pluripotent stem cells (iPSCs) and iPSC-derived neural precursor cells (NPCs), and neurons. Spatial visualization of CI∗CIV-SCs enables the detection of mitochondrial lesions in various experimental models, including complex tissues undergoing degenerative processes. We report that comparative assessments of CI∗CIV-SCs facilitate the quantitative profiling of even subtle mitochondrial variations by overcoming the confounding effects that mixed cell populations have on other measurements. Together, our PLA-based analysis of CI∗CIV-SCs is a sensitive and complementary technique for detecting cell-type-specific mitochondrial perturbations in fixed materials.
    Keywords:  brain; in-situ imaging analysis; mitochondria; mitochondrial diseases; mitochondrial dysfunction; mitochondrial respiratory supercomplexes; proximity ligation assay
  6. STAR Protoc. 2022 Jun 17. 3(2): 101322
      Mitochondrial respiratory chain (MRC) dysfunction is linked to mitochondrial disease as well as other common conditions such as diabetes, neurodegeneration, cancer, and aging. Thus, the evaluation of MRC enzymatic activities is fundamental for diagnostics and research purposes on experimental models. Here, we provide a verified and reliable protocol for mitochondria isolation from various D. melanogaster samples and subsequent measurement of the activity of MRC complexes I-V plus citrate synthase (CS) through UV-VIS spectrophotometry. For complete details on the use and execution of this protocol, please refer to Brischigliaro et al. (2021).
    Keywords:  Cell separation/fractionation; Metabolism; Model Organisms; Protein Biochemistry
  7. Genes Genomics. 2022 Apr 28.
      BACKGROUND: Mitochondrial complex I deficiency (MCID) is the most common biochemical defect identified in childhood with mitochondrial diseases, mainly including Leigh syndrome, encephalopathy, macrocephaly with progressive leukodystrophy, hypertrophic cardiomyopathy and myopathy.OBJECTIVE: To identify genetic cause in a patient with early onset autosomal recessive MCID.
    METHODS: Trio whole-exome sequencing was performed and phenotype-related data analyses were conducted. All candidate mutations were confirmed by Sanger sequencing.
    RESULTS: Here we report a child of Leigh syndrome presented with global developmental delay, progressive muscular hypotonia and myocardial damage. A missense mutation c.118C > T (p.Arg40Trp) and a previously reported mutation c.1157G > A (p.Arg386His) in NDUFV1 have been identified as compound heterozygous in the patient. The mutation p.Arg386His is closely associated with the impairment of 4Fe-4S domain and this mutation has been reported pathogenic. The c.118C > T mutation has not been reported in ClinVar and HGMD database. In silico protein analyses showed that p.Arg40 is highly conserved in a wide range of species, and the amino acid substitution p.Trp40 largely decreases the stability of NDUFV1. In addition, the mutation has not been detected in the Asian populations and it was predicted to be deleterious by numerous prediction tools.
    CONCLUSION: This research expands the mutation spectrum of NDUFV1 and substantially provides an early and accurate diagnosis basis of MCID, which would benefit subsequently effective genetic counseling and prenatal diagnosis for future reproduction of the family.
    Keywords:  Leigh Syndrome; Mitochondrial complex I deficiency; NDUFV1; Whole-exome sequencing
  8. J Biol Chem. 2022 Apr 26. pii: S0021-9258(22)00424-0. [Epub ahead of print] 101984
      A major challenge in eukaryotic cells is the proper distribution of nuclear-encoded proteins to the correct organelles. For a subset of mitochondrial proteins, a signal sequence at the N-terminus (MTS) is recognized by protein complexes to ensure their proper translocation into the organelle. However, the early steps of mitochondrial protein targeting remain undeciphered. The cytosolic chaperone NAC, that in yeast is represented as the two different heterodimers αβ- and αβ'-NAC, has been proposed to be involved during the early steps of mitochondrial protein targeting. We have previously described that the mitochondrial outer membrane protein Sam37 interacts with αβ'-NAC and together promote the import of specific mitochondrial precursor proteins. In this work, we aimed to detect the region in the MTS of mitochondrial precursors relevant for their recognition by αβ'-NAC during their sorting to the mitochondria. We used targeting signals of different mitochondrial proteins (αβ'-NAC-dependent Oxa1 and αβ'-NAC-independent Mdm38) and fused them to green-fluorescent protein (GFP) to study their intracellular localization by biochemical and microscopy methods, and additionally followed their import kinetics in vivo. Our results reveal the presence of a positively charged amino acid cluster in the MTS of select mitochondrial precursors, such as Oxa1 and Fum1, which are crucial for their recognition by αβ'-NAC. Furthermore, we explored the presence of this cluster at the N-terminus of the mitochondrial proteome and propose a set of precursors whose proper localization depends on both αβ'-NAC and Sam37.
  9. Elife. 2022 Apr 25. pii: e75143. [Epub ahead of print]11
      How environmental cues influence peroxisome proliferation, particularly through organelles, remains largely unknown. Yeast peroxisomes metabolize fatty acids (FA), and methylotrophic yeasts also metabolize methanol. NADH and acetyl-CoA, produced by these pathways enter mitochondria for ATP production and for anabolic reactions. During the metabolism of FA and/or methanol, the mitochondrial oxidative phosphorylation (OXPHOS) pathway accepts NADH for ATP production and maintains cellular redox balance. Remarkably, peroxisome proliferation in Pichia pastoris was abolished in NADH shuttling- and OXPHOS mutants affecting complex I or III, or by the mitochondrial uncoupler, 2,4-dinitrophenol (DNP), indicating ATP depletion causes the phenotype. We show that mitochondrial OXPHOS deficiency inhibits expression of several peroxisomal proteins implicated in FA and methanol metabolism, as well as in peroxisome division and proliferation. These genes are regulated by the Snf1 complex (SNF1), a pathway generally activated by a high AMP/ATP ratio. In OXPHOS mutants, Snf1 is activated by phosphorylation, but Gal83, its interacting subunit, fails to translocate to the nucleus. Phenotypic defects in peroxisome proliferation observed in the OXPHOS mutants, and phenocopied by the Dgal83 mutant, were rescued by deletion of three transcriptional repressor genes (MIG1, MIG2 and NRG1) controlled by SNF1 signaling. Our results are interpreted in terms of a mechanism by which peroxisomal and mitochondrial proteins and/or metabolites influence redox and energy metabolism, while also influencing peroxisome biogenesis and proliferation, thereby exemplifying interorganellar communication and interplay involving peroxisomes, mitochondria, cytosol and the nucleus. We discuss the physiological relevance of this work in the context of human OXPHOS deficiencies.
    Keywords:  cell biology
  10. EMBO J. 2022 Apr 25. e111290
      The ability of immune cells to penetrate affected tissues is highly dependent on energy provided by mitochondria, yet their involvement in promoting migration remains unclear. Recent work by Emtenani et al (2022) describes a nuclear Atossa-Porthos axis that adjusts transcription and translation of a small subset of OXPHOS genes to increase mitochondrial bioenergetics and allow macrophage tissue invasion in flies.
  11. MethodsX. 2022 ;9 101685
      Mitochondria are important organelles responsible for energy production. Mitochondrial dysfunction relates to various pathological diseases. The investigation of mitochondrial heath is critical to evaluate the cellular status. Herein, we demonstrated an approach for determining the status of mitochondrial health by observing mitochondrial H2O2 (one type of ROS), membrane potential, and morphology (fragmentation and length) in live primary fibroblast cells. The cells were co-stained with fluorescent dyes (Hoechst 33342 and MITO-ID® Red/MitoPY1/JC-10) and continuously processed by the High Content Imaging System. We employed the Operetta CLSTM to take fluorescent images with its given quickness and high resolution. The CellProfiler image analysis software was further used to identify cell and mitochondrial phenotypes in the thousand fluorescent images.•We could quantitatively analyze fluorescent images with high-throughput and high-speed detection to track the alteration of mitochondrial status.•The MMP assay is sensitive to FCCP even at the concentration of 0.01 µM.•The fibroblast cells treated with stress inducers (H2O2, FCCP, and phenanthroline) revealed a significant change in mitochondrial health parameters, with more ROS accumulation, depolarized MMP, increased fragmentation, and reduced length of mitochondria.
    Keywords:  High content fluorescent imaging; Mitochondrial ROS; Mitochondrial function; Mitochondrial membrane potential; Mitochondrial morphology
  12. Pharmacol Res. 2022 Apr 21. pii: S1043-6618(22)00173-6. [Epub ahead of print] 106228
      A mitochondrial stroke-like event is an evolving subacute neurological syndrome linked to seizure activity and focal metabolic brain derangement in a genetically determined mitochondrial disorder. The acronym "MELAS" (mitochondrial encephalopathy associated with lactic acidosis and stroke-like lesions) identifies subjects with molecular, biochemical and/or histological evidence of mitochondrial disorder who experience stroke-like lesions. MELAS is a rare inherited mitochondrial disease linked to severe multiorgan involvement and stress-induced episodes of metabolic decompensation and lactic acidosis. Unfortunately, there are no etiopathogenetic therapies for stroke-like episodes to date, and the treatment is mainly based on anti-epileptic drugs and supportive therapies. This perspective opinion article discusses the current care standards for MELAS patients and revises current and innovative emerging therapies for mitochondrial stroke-like episodes.
    Keywords:  MELAS; MTTL1; POLG; m.3243A>G; stroke; stroke-like episodes; stroke-like lesion
  13. Neurochem Int. 2022 Apr 25. pii: S0197-0186(22)00069-9. [Epub ahead of print] 105344
      Substantia Nigra Pars-compacta (SNpc), in the basal ganglion region, is a primary source of dopamine release. These dopaminergic neurons require more energy than other neurons, as they are highly arborized and redundant. Neurons meet most of their energy demand (∼90%) from mitochondria. Oxidative phosphorylation (OxPhos) is the primary pathway for energy production. Many genes involved in Parkinson's disease (PD) have been associated with OxPhos, especially complex I. Abrogation in complex I leads to reduced ATP formation in these neurons, succumbing to death by inducing apoptosis. This review discusses the interconnection between complex I-associated PD genes and specific mitochondrial metabolic factors (MMFs) of OxPhos. Interestingly, all the complex I-associated PD genes discussed here have been linked to the Akt signaling pathway; thus, neuron survival is promoted and smooth mitochondrial function is ensured. Any changes in these genes disrupt the Akt pathway, which hampers the opening of the permeability transition pore (PTP) via GSK3β dephosphorylation; promotes destabilization of OxPhos; and triggers the release of pro-apoptotic factors.
    Keywords:  Akt signaling pathway; Mitochondria; Mitochondrial metabolic factors; Neurodegeneration; Oxidative phosphorylation; Parkinson's disease
  14. Cell. 2022 Apr 18. pii: S0092-8674(22)00389-0. [Epub ahead of print]
      Mitochondrial DNA (mtDNA) editing paves the way for disease modeling of mitochondrial genetic disorders in cell lines and animals and also for the treatment of these diseases in the future. Bacterial cytidine deaminase DddA-derived cytosine base editors (DdCBEs) enabling mtDNA editing, however, are largely limited to C-to-T conversions in the 5'-TC context (e.g., TC-to-TT conversions), suitable for generating merely 1/8 of all possible transition (purine-to-purine and pyrimidine-to-pyrimidine) mutations. Here, we present transcription-activator-like effector (TALE)-linked deaminases (TALEDs), composed of custom-designed TALE DNA-binding arrays, a catalytically impaired, full-length DddA variant or split DddA originated from Burkholderia cenocepacia, and an engineered deoxyadenosine deaminase derived from the E. coli TadA protein, which induce targeted A-to-G editing in human mitochondria. Custom-designed TALEDs were highly efficient in human cells, catalyzing A-to-G conversions at a total of 17 target sites in various mitochondrial genes with editing frequencies of up to 49%.
    Keywords:  DNA; DddAtox; TALED; adenine deaminase; base editing; genome editing; mitochondria; organelle
  15. Nat Commun. 2022 Apr 29. 13(1): 2340
      The dynamin-like GTPases Mitofusin 1 and 2 (Mfn1 and Mfn2) are essential for mitochondrial function, which has been principally attributed to their regulation of fission/fusion dynamics. Here, we report that Mfn1 and 2 are critical for glucose-stimulated insulin secretion (GSIS) primarily through control of mitochondrial DNA (mtDNA) content. Whereas Mfn1 and Mfn2 individually were dispensable for glucose homeostasis, combined Mfn1/2 deletion in β-cells reduced mtDNA content, impaired mitochondrial morphology and networking, and decreased respiratory function, ultimately resulting in severe glucose intolerance. Importantly, gene dosage studies unexpectedly revealed that Mfn1/2 control of glucose homeostasis was dependent on maintenance of mtDNA content, rather than mitochondrial structure. Mfn1/2 maintain mtDNA content by regulating the expression of the crucial mitochondrial transcription factor Tfam, as Tfam overexpression ameliorated the reduction in mtDNA content and GSIS in Mfn1/2-deficient β-cells. Thus, the primary physiologic role of Mfn1 and 2 in β-cells is coupled to the preservation of mtDNA content rather than mitochondrial architecture, and Mfn1 and 2 may be promising targets to overcome mitochondrial dysfunction and restore glucose control in diabetes.
  16. Nat Immunol. 2022 Apr 28.
      The NLRP3 inflammasome is linked to sterile and pathogen-dependent inflammation, and its dysregulation underlies many chronic diseases. Mitochondria have been implicated as regulators of the NLRP3 inflammasome through several mechanisms including generation of mitochondrial reactive oxygen species (ROS). Here, we report that mitochondrial electron transport chain (ETC) complex I, II, III and V inhibitors all prevent NLRP3 inflammasome activation. Ectopic expression of Saccharomyces cerevisiae NADH dehydrogenase (NDI1) or Ciona intestinalis alternative oxidase, which can complement the functional loss of mitochondrial complex I or III, respectively, without generation of ROS, rescued NLRP3 inflammasome activation in the absence of endogenous mitochondrial complex I or complex III function. Metabolomics revealed phosphocreatine (PCr), which can sustain ATP levels, as a common metabolite that is diminished by mitochondrial ETC inhibitors. PCr depletion decreased ATP levels and NLRP3 inflammasome activation. Thus, the mitochondrial ETC sustains NLRP3 inflammasome activation through PCr-dependent generation of ATP, but via a ROS-independent mechanism.
  17. Pediatr Transplant. 2022 Apr 24. e14274
      BACKGROUND: MPV17-related mitochondrial DNA maintenance defect (MPV17 deficiency) is a rare, autosomal recessive mitochondrial DNA depletion syndrome with a high mortality rate in infancy and early childhood due to progression to liver failure. Liver transplantation for children with MPV17 deficiency has been considered controversial due to uncertainty about the potential progression of extrahepatic manifestations following liver transplantation.METHODS: We describe our institution's experience for two infants diagnosed with infantile MPV17 deficiency who presented in acute on chronic liver failure, but with normal development and normal neurological status who successfully underwent liver transplantation.
    RESULTS: Both patients underwent successful liver transplantation with normal development and neurological status at 3 years and 16 months post-transplant, respectively.
    CONCLUSIONS: In this rare disease population, we describe two infants with MPV17 deficiency who underwent liver transplantation for acute on chronic liver failure who continue to have normal development, without progression of neurological disease. MPV17 deficiency should not be considered a contraindication to liver transplantation.
    Keywords:  MPV17; MPV17 deficiency; Navajo; acute liver failure; development; hepatocerebral; infantile; liver; mitochondrial; neurohepatopathy; neurological progression; pediatric liver transplant
  18. Cell Rep Methods. 2021 Aug 23. 1(4): 100052
      Engineered synthetic biomolecular devices that integrate elaborate information processing and precisely regulate living cell behavior have potential in various applications. Although devices that directly regulate key biomolecules constituting inherent biological systems exist, no devices have been developed to control intracellular membrane architecture, contributing to the spatiotemporal functions of these biomolecules. This study developed a synthetic biomolecular device, termed inducible counter mitochondrial morphology (iCMM), to manipulate mitochondrial morphology, an emerging informative property for understanding physiopathological cellular behaviors, on a minute timescale by using a chemically inducible dimerization system. Using iCMM, we determined cellular changes by altering mitochondrial morphology in an unprecedented manner. This approach serves as a platform for developing more sophisticated synthetic biomolecular devices to regulate biological systems by extending manipulation targets from conventional biomolecules to mitochondria. Furthermore, iCMM might serve as a tool for uncovering the biological significance of mitochondrial morphology in various physiopathological cellular processes.
    Keywords:  Boolean logic gate; mitochondria; mitochondrial morphology; synthetic biocomputing device; synthetic biology
  19. Front Mol Neurosci. 2022 ;15 841047
      Defective mitochondrial dynamics in axons have been linked to both developmental and late-onset neurological disorders. Axonal trafficking is in large part governed by the microtubule motors kinesin-1 and cytoplasmic dynein 1 (dynein). Dynein is the primary retrograde transport motor in axons, and mutations in dynein and many of its regulators also cause neurological diseases. Depletion of LIS1, famous for linking dynein deregulation to lissencephaly (smooth brain), in adult mice leads to severe neurological phenotypes, demonstrating post-developmental roles. LIS1 stimulates retrograde transport of acidic organelles in cultured adult rat dorsal root ganglion (DRG) axons but findings on its role in mitochondrial trafficking have been inconsistent and have not been reported for adult axons. Here we report that there is an increased number of mitochondria in cross-sections of sciatic nerve axons from adult LIS1+/- mice. This is probably related to reduced dynein activity as axons from adult rat nerves exposed to the dynein inhibitor, ciliobrevin D also had increased numbers of mitochondria. Moreover, LIS1 overexpression (OE) in cultured adult rat DRG axons stimulated retrograde mitochondrial transport while LIS1 knockdown (KD) or expression of a LIS1 dynein-binding mutant (LIS1-K147A) inhibited retrograde transport, as did KD of dynein heavy chain (DHC). These findings are consistent with our report on acidic organelles. However, KD of NDEL1, a LIS1 and dynein binding protein, or expression of a LIS1 NDEL1-binding mutant (LIS1-R212A) also dramatically impacted retrograde mitochondrial transport, which was not the case for acidic organelles. Manipulations that disrupted retrograde mitochondrial transport also increased the average length of axonal mitochondria, suggesting a role for dynein in fusion or fission events. Our data point to cargo specificity in NDEL1 function and raise the possibility that defects in the LIS1/NDEL1 dynein regulatory pathway could contribute to mitochondrial diseases with axonal pathologies.
    Keywords:  DRG; LIS1; NDEL1; axon; dynein; mitochondria; sciatic nerve
  20. Diabetes. 2022 Apr 26. pii: db210834. [Epub ahead of print]
      Mitochondrial dysfunction plays a central role in Type 2 Diabetes (T2D); however, the pathogenic mechanisms in pancreatic β-cells are incompletely elucidated. Succinate dehydrogenase (SDH) is a key mitochondrial enzyme with dual functions in the TCA cycle and electron transport chain (ETC). Using human diabetic samples and a mouse model of β-cell-specific SDH ablation (SDHBβKO), we define SDH deficiency as a driver of mitochondrial dysfunction in β-cell failure and insulinopenic diabetes. β-Cell SDH deficiency impairs glucose-induced respiratory oxidative phosphorylation and mitochondrial membrane potential (ΔΨm) collapse, thereby compromising glucose-stimulated ATP production, insulin secretion and β-cell growth. Mechanistically, metabolomic and transcriptomic studies reveal that the loss of SDH causes excess succinate accumulation, which inappropriately activates mTORC1-regulated metabolic anabolism, including increased SREBP-regulated lipid synthesis. These alterations, which mirror diabetes-associated human β-cell dysfunction, are partially reversed by acute mTOR inhibition with rapamycin. We propose SDH deficiency as a contributing mechanism to the progressive β-cell failure of diabetes and identify mTORC1 inhibition as a potential mitigation strategy.
  21. Protein Sci. 2022 May;31(5): e4312
      Human Suv3 is a unique homodimeric helicase that constitutes the major component of the mitochondrial degradosome to work cooperatively with exoribonuclease PNPase for efficient RNA decay. However, the molecular mechanism of how Suv3 is assembled into a homodimer to unwind RNA remains elusive. Here, we show that dimeric Suv3 preferentially binds to and unwinds DNA-DNA, DNA-RNA, and RNA-RNA duplexes with a long 3' overhang (≥10 nucleotides). The C-terminal tail (CTT)-truncated Suv3 (Suv3ΔC) becomes a monomeric protein that binds to and unwinds duplex substrates with ~six to sevenfold lower activities relative to dimeric Suv3. Only dimeric Suv3, but not monomeric Suv3ΔC, binds RNA independently of ATP or ADP, and is capable of interacting with PNPase, indicating that dimeric Suv3 assembly ensures its continuous association with RNA and PNPase during ATP hydrolysis cycles for efficient RNA degradation. We further determined the crystal structure of the apo-form of Suv3ΔC, and SAXS structures of dimeric Suv3 and PNPase-Suv3 complex, showing that dimeric Suv3 caps on the top of PNPase via interactions with S1 domains, and forms a dumbbell-shaped degradosome complex with PNPase. Overall, this study reveals that Suv3 is assembled into a dimeric helicase by its CTT for efficient and persistent RNA binding and unwinding to facilitate interactions with PNPase, promote RNA degradation, and maintain mitochondrial genome integrity and homeostasis.
    Keywords:  RNA helicase; RNA metabolism; RNA turnover; mitochondrial RNA degradation; mitochondrial homeostasis
  22. J Biol Chem. 2022 Apr 23. pii: S0021-9258(22)00422-7. [Epub ahead of print] 101982
      Friedreich's Ataxia (FRDA) is a degenerative disease caused by a decrease in the mitochondrial protein frataxin, which is involved in iron-sulfur cluster (ISC) synthesis. Diminutions in frataxin result in decreased ISC synthesis, increased mitochondrial iron accumulation, and impaired mitochondrial function. Here we show that conditions that result in increased mitochondrial reactive oxygen species (ROS) in yeast or mammalian cell culture give rise to increased turnover of frataxin, but not of other ISC synthesis proteins. We demonstrate that the mitochondrial Lon protease is involved in frataxin degradation and that iron export through the mitochondrial metal transporter Mmt1 protects yeast frataxin from degradation. We also determined that when FRDA fibroblasts were grown in media containing elevated iron, mitochondrial ROS increased and frataxin decreased compared to WT fibroblasts. Furthermore, we screened a library of FDA-approved compounds and identified 38 compounds that increased yeast frataxin levels, including the azole Bifonazole, antiparasitic Fipronil, anti-tumor compound Dibenzoylmethane (DBM), antihypertensive 4-hydroxychalcone (4'-OHC), and a non-specific anion channel inhibitor 4,4-diisothiocyanostilbene-2,2-sulfonic acid (DIDS). We show that top hits 4'-OHC and DBM increased mRNA levels of transcription factor Nrf2 in FRDA patient-derived fibroblasts, as well as downstream antioxidant targets thioredoxin (TXN), glutathione reductase (GSR), and superoxide dismutase 2 (SOD2). Taken together, these findings reveal that FRDA progression may be in part due to oxidant-mediated decreases in frataxin, and that some approved compounds may be effective in increasing mitochondrial frataxin in FRDA, delaying disease progression.
    Keywords:  ROS; Yfh1; compounds; frataxin; iron; iron-sulfur clusters; mitochondria; screen; yeast
  23. Mol Biol Evol. 2022 Apr 28. pii: msac090. [Epub ahead of print]
      Mitochondria are essential organelles in eukaryotic cells that provide critical support for energetic and metabolic homeostasis. Although the elimination of pathogenic mitochondrial DNA (mtDNA) mutations in somatic cells has been observed, the mechanisms for somatic cells to maintain proper functions despite their mtDNA mutation load are poorly understood. In this study, we analyzed somatic mtDNA mutations in more than 30,000 single human peripheral and bone marrow mononuclear cells. We observed a significant overrepresentation of homoplasmic mtDNA mutations in B, T and NK lymphocytes. Intriguingly, their overall mutational burden was lower than that in hematopoietic progenitors and myeloid cells. This characteristic mtDNA mutational landscape indicates a genetic bottleneck during lymphoid development, as confirmed with single cell datasets from multiple platforms and individuals. We further demonstrated that mtDNA replication lags behind cell proliferation in both pro-B and pre-B progenitor cells, thus likely causing the genetic bottleneck by diluting mtDNA copies per cell. Through computational simulations and approximate Bayesian computation (ABC), we recapitulated this lymphocyte-specific mutational landscape and estimated the minimal mtDNA copies as <30 in T, B, and NK lineages. Our integrative analysis revealed a novel discovery of a lymphoid-specific mtDNA genetic bottleneck, thus illuminating a potential mechanism used by highly metabolically active immune cells to limit their mtDNA mutation load.
  24. Front Biosci (Landmark Ed). 2022 Apr 01. 27(4): 118
      INTRODUCTION: Accumulating evidence suggests that mitochondrial structural and functional defects are present in human placentas affected by pregnancy related disorders, but mitophagy pathways in human trophoblast cells/placental tissues have not been investigated.METHODS: In this study, we investigated three major mitophagy pathways mediated by PRKN, FUNDC1, and BNIP3/BNIP3L in response to AMPK activation by AICAR and knockdown of PRKAA1/2 (AKD) in human trophoblast cell line BeWo and the effect of AKD on mitochondrial membrane potential and ATP production.
    RESULTS: Autophagy flux assay demonstrated that AMPK signaling activation stimulates autophagy, evidenced increased LC3II and SQSTM1 protein abundance in the whole cell lysates and mitochondrial fractions, and mitophagy flux assay demonstrated that the activation of AMPK signaling stimulates mitophagy via PRKN and FUNDC1 mediated but not BNIP3/BNIP3L mediated pathways. The stimulatory regulation of AMPK signaling on mitophagy was confirmed by AKD which reduced the abundance of LC3II, SQSTM1, PRKN, and FUNDC1 proteins, but increased the abundance of BNIP3/BNIP3L proteins. Coincidently, AKD resulted in elevated mitochondrial membrane potential and reduced mitochondrial ATP production, compared to control BeWo cells.
    CONCLUSIONS: In summary, AMPK signaling stimulates mitophagy in human trophoblast cells via PRKN and FUNDC1 mediated mitophagy pathways and AMPK regulated mitophagy contributes to the maintenance of mitochondrial membrane potential and mitochondrial ATP production.
    Keywords:  AMPK; ATP production; BeWo; human; mitochondria; mitophagy; trophoblast
  25. J Neurol. 2022 Apr 29.
      BACKGROUND: There is increasing evidence for the role of inflammation in the pathogenesis of mitochondrial diseases (MDs). However, the mechanisms underlying mutation-induced inflammation in MD remain elusive. Our previous study suggested that mitophagy is impaired in the skeletal muscle of those with MD, likely causing mitochondrial DNA (mtDNA) release and thereby triggering inflammation. We here aimed to decipher the role of the cGAS-STING pathway in inflammatory process in MDs.METHODS: We investigated the levels of circulating cell-free mtDNA (ccf-mtDNA) in the serum of 104 patients with MDs. Immunofluorescence was performed in skeletal muscles in MDs and control. Biochemical analysis of muscle biopsies was conducted with western blot to detect cGAS, STING, TBK1, IRF3 and phosphorylated IRF3 (p-IRF3). RT-qPCR was performed to detect the downstream genes of type I interferon in skeletal muscles. Furthermore, a protein microarray was used to examine the cytokine levels in the serum of patients with MDs.
    RESULTS: We found that ccf-mtDNA levels were significantly increased in those with MDs compared to the controls. Consistently, the immunofluorescent results showed that cytosolic dsDNA levels were increased in the muscle samples of MD patients. Biochemical analysis of muscle biopsies showed that cGAS, IRF3, and TBK1 protein levels were significantly increased in those with MDs, indicating that there was activation of the cGAS-STING pathway. RT-qPCR showed that downstream genes of type I interferon were upregulated in muscle samples of MDs. Protein microarray results showed that a total of six cytokines associated with the cGAS-STING pathway were significantly increased in MD patients (fold change > 1.2, p value < 0.05).
    CONCLUSIONS: These findings suggest that increases in ccf-mtDNA levels is associated with the activation of the cGAS-STING pathway, thereby triggering inflammation in MDs.
    Keywords:  Ccf-mtDNA; Inflammation; Mitochondrial diseases; cGAS-STING pathway
  26. J Neuromuscul Dis. 2022 Apr 22.
      BACKGROUND: Proximal muscle weakness may be the presenting clinical feature of different types of myopathies, including limb girdle muscular dystrophy and primary mitochondrial myopathy. LGMD1B is caused by LMNA mutation. It is characterized by progressive weakness and wasting leading to proximal weakness, cardiomyopathy, and hearth conduction block.OBJECTIVE: In this article, we describe the case of a patient who presented with limb-girdle weakness and a double trouble scenario -mitochondrial DNA single deletion and a new LMNA mutation.
    METHODS: Pathophysiological aspects were investigated with muscle biopsy, Western Blot analysis, NGS nuclear and mtDNA analysis and neuromuscular imaging (muscle and cardiac MRI).
    RESULTS: Although secondary mitochondrial involvement is possible, a "double trouble" syndrome can not be excluded.
    CONCLUSION: Implication deriving from hypothetical coexistence of two different pathological conditions or the possible secondary mitochondrial involvement are discussed.
    Keywords:  LMNA-associated diseases; Muscle disorders; mitochondria; myopathies
  27. Physiol Rev. 2022 Apr 25.
      As a central hub for cellular metabolism and intracellular signalling, the mitochondrion is a pivotal organelle, dysfunction of which has been linked to several human diseases including neurodegenerative disorders, and in particular Parkinson's disease. An inherent challenge that mitochondria face is the continuous exposure to diverse stresses which increase their likelihood of dysregulation. In response, eukaryotic cells have evolved sophisticated quality control mechanisms to monitor, identify, repair and/or eliminate abnormal or misfolded proteins within the mitochondrion and/or the dysfunctional mitochondrion itself. Chaperones identify unstable or otherwise abnormal conformations in mitochondrial proteins and can promote their refolding to recover their correct conformation and stability. However, if repair is not possible, the abnormal protein is selectively degraded to prevent potentially damaging interactions with other proteins or its oligomerization into toxic multimeric complexes. The autophagic-lysosomal system and the ubiquitin-proteasome system mediate the selective and targeted degradation of such abnormal or misfolded protein species. Mitophagy (a specific kind of autophagy) mediates the selective elimination of dysfunctional mitochondria, in order to prevent the deleterious effects the dysfunctional organelles within the cell. Despite our increasing understanding of the molecular responses toward dysfunctional mitochondria, many key aspects remain relatively poorly understood. Herein, we review the emerging mechanisms of mitochondrial quality control including quality control strategies coupled to mitochondrial import mechanisms. In addition, we review the molecular mechanisms regulating mitophagy with an emphasis on the regulation of PINK1/PARKIN-mediated mitophagy in cellular physiology and in the context of Parkinson's disease cell biology.
    Keywords:  PINK1/Parkin; Parkinson's disease; mitochondrial quality control; mitophagy; protein quality control
  28. Mol Genet Genomic Med. 2022 Apr 26. e1955
      BACKGROUND: Mitochondrial encephalomyopathy, lactic acidosis and stroke-like episodes (MELAS) syndrome is one of the most well-known mitochondrial diseases, with most cases attributed to m.3243A>G. MELAS syndrome patients typically present in the first two decades of life with a broad, multi-systemic phenotype that predominantly features neurological manifestations--stroke-like episodes. However, marked phenotypic variability has been observed among paediatric patients, creating a clinical challenge and delaying diagnoses.METHODS: A literature review of paediatric MELAS syndrome patients and a retrospective analysis in a UK tertiary paediatric neurology centre were performed.
    RESULTS: Three children were included in this case series. All patients presented with seizures and had MRI changes not confined to a single vascular territory. Blood heteroplasmy varied considerably, and one patient required a muscle biopsy. Based on a literature review of 114 patients, the mean age of presentation is 8.1 years and seizures are the most prevalent manifestation of stroke-like episodes. Heteroplasmy is higher in a tissue other than blood in most cases.
    CONCLUSION: The threshold for investigating MELAS syndrome in children with suspicious neurological symptoms should be low. If blood m.3243A>G analysis is negative, yet clinical suspicion remains high, invasive testing or further interrogation of the mitochondrial genome should be considered.
    Keywords:  MELAS syndrome; encephalopathy; genetics; lactic acidosis; m.3243A>G; mitochondrial disease; paediatric neurology; stroke-like episodes
  29. Hum Mol Genet. 2022 Apr 25. pii: ddac096. [Epub ahead of print]
      Sensorineural hearing loss often results from damaged or deficient inner ear hair cells. Mitochondrial 12S rRNA 1555A > G mutation has been associated with hearing loss in many families. The m.1555A > G mutation is a primary factor underlying the development of hearing loss and TRMU allele (c.28G > T, p.Ala10Sser) encoding tRNA methyltransferase interact with m.1555A > G mutation to cause hearing loss. However, the tissue specificity of mitochondrial dysfunction remains elusive and there is no highly effective therapy for mitochondrial deafness. We report here the generation of induced pluripotent stem cells (iPSCs) from lymphoblastoid cell lines derived from members of an Arab-Israeli family (asymptomatic individual carrying only m.1555A > G mutation, symptomatic individual bearing both m.1555A > G and c.28G > T mutations, and control subject). The c.28G > T mutation in iPSC lines from a hearing-impaired subject was corrected by CRISPR/Cas9. These iPSCs were differentiated into otic epithelial progenitor (OEP) cells and subsequent inner ear hair cell (HC)-like cells. The iPSCs bearing m.1555A > G mutation exhibited mildly deficient differentiation into OEP and resultant HC-like cells displayed mild defects in morphology and electrophysiological properties. Strikingly, those HC-like cells harboring m.1555A > G and TRMU c.28G > T mutations displayed greater defects in the development, morphology and functions than those in cells bearing only m.1555A > G mutation. Transcriptome analysis of patients-derived HC-like cells revealed altered expressions of genes vital for mechanotransduction of hair cells. Genetic correction of TRMU c.28G > T mutation yielded morphologic and functional recovery of patient derived HC-like cells. These findings provide new insights into pathophysiology of maternally inherited hearing loss and a step toward therapeutic interventions for this disease.
    Keywords:  hearing loss12S rRNA mutationinner ear hair cellsinduced pluripotent stem cellsmitochondrial dysfunction
  30. Pediatr Neurol. 2022 Apr 15. pii: S0887-8994(22)00052-2. [Epub ahead of print]131 30-41
      BACKGROUND: Pontocerebellar hypoplasia type 6 (PCH6) is an early-onset encephalopathy with/without mitochondrial respiratory complex defects caused by recessive mutations in mitochondrial arginyl-tRNA synthetase (RARS2). Highly heterogeneous clinical phenotypes and numerous missense variations of uncertain significance make diagnosis difficult. Pathogenesis of PCH6 remains unclear.METHODS: Facial characteristics of patients were assessed. Genetic tests were performed. Structure prediction was based on the template from AlphaFold Protein Structure Database. Expression of mutant RARS2 was tested in HEK293T cells. Patient-derived induced pluripotent stem cells (iPSCs) were detected for human mitochondrial tRNAArg (hmtRNAArg) steady-state level, mitochondrial respiratory complex (MRC) activity, oxygen consumption rate (OCR), extracellular acidification rate (ECAR), mitochondrial membrane potential (MMP), reactive oxygen species (ROS) abundance, and apoptosis level.
    RESULTS: The three pedigrees were diagnosed as PCH6 caused by compound heterozygous RARS2 variations. Five RARS2 variants were identified: c.3G>C(p.M1?), c.685C>T(p.R229∗), c.1060T>A(p.F354I), c.1210A>G(p.M404V), and c.1369G>A(p.G457R). RARS2 c.3G>C disrupted protein expression. RARS2 c.685C>T created a truncated protein lacking complete catalytic core and anticodon-binding domain. RARS2 c.1060T>A and c.1369G>A were predicted to cause structural abnormality. The hmtRNAArg steady-state abundance in a patient's iPSCs was unaffected. Mitochondrial energy metabolism was normal, including MRC activity, OCR, ECAR, and MMP, while mitochondria-related cellular characteristics, including ROS (P < 0.001) and apoptosis levels (P < 0.001), increased.
    CONCLUSIONS: This study reports five RARS2 variations among which c.3G>C and c.1060T>A are novel. Summarized facial features of PCH6 patients will facilitate diagnosis. Defective mitochondrial energy metabolism may not be key points, but mitochondria-related abnormal cellular physiology, including apoptosis, may be an underlying pathogenesis.
    Keywords:  Apoptosis; Facial feature; Mitochondria; Pontocerebellar hypoplasia type 6; RARS2
  31. Cell Rep Methods. 2021 Aug 23. 1(4): 100044
      Cell membrane deformation is an important feature that occurs during many physiological processes, and its study has been put to good use to investigate cardiomyocyte function. Several methods have been developed to extract information on cardiomyocyte contractility. However, no existing computational framework has provided, in a single platform, a straightforward approach to acquire, process, and quantify this type of cellular dynamics. For this reason, we develop CONTRACTIONWAVE, high-performance software written in Python programming language that allows the user to process large data image files and obtain contractility parameters by analyzing optical flow from images obtained with videomicroscopy. The software was validated by using neonatal, adult-, and human-induced pluripotent stem-cell-derived cardiomyocytes, treated or not with drugs known to affect contractility. Results presented indicate that CONTRACTIONWAVE is an excellent tool for examining changes to cardiac cellular contractility in animal models of disease and for pharmacological and toxicology screening during drug discovery.
    Keywords:  Python software; cardiomyocyte; contractility parameters; contraction average speed; iPSC-CMs; image processing; optical flow; shortening area
  32. Hum Mutat. 2022 Apr 23.
      Most of the pathogenic variants in mitochondrial DNA (mtDNA) exist in a heteroplasmic state (coexistence of mutant and wild-type mtDNA). Understanding how mtDNA is transmitted is crucial for predicting mitochondrial disease risk. Previous studies were based mainly on two-generation pedigree data, which are limited by the randomness in a single transmission. In this study, we analyzed the transmission of heteroplasmies in 16 four-generation families. First, we found that 57.8% of the variants in the great grandmother were transmitted to the fourth generation. The direction and magnitude of the frequency change during transmission appeared to be random. Moreover, no consistent correlation was identified between the frequency changes among the continuous transmissions, suggesting that most variants were functionally neutral or mildly deleterious and thus not subject to strong natural selection. Additionally, we found that the frequency of one nonsynonymous variant (m.15773G>A) showed a consistent increase in one family, suggesting that this variant may confer a fitness advantage to the mitochondrion/cell. We also estimated the effective bottleneck size during transmission to be 21-71. In summary, our study demonstrates the advantages of multigeneration data for studying the transmission of mtDNA for shedding new light on the dynamics of the mutation frequency in successive generations. This article is protected by copyright. All rights reserved.
    Keywords:  heteroplasmy; inheritance; mtDNA; multigeneration pedigrees; transmission
  33. Zhejiang Da Xue Xue Bao Yi Xue Ban. 2022 Mar 25. 50(7): 1-7
      Very long-chain acyl-CoA dehydrogenase (VLCAD) deficiency is a metabolic disease of long chain fatty acid oxidation. The clinical manifestations are heterogeneous, mainly with heart, liver, skeletal muscle and brain damage, and the onset of which can be from newborn to adult. Cardiomyopathy type is more serious with high mortality. The liver failure type and myopathy type would be potentially lethal, but generally the prognosis is relatively good. Recurrent hypoglycemia, energy metabolism disorder, liver dysfunction, cardiomyopathy and serious arrhythmia are the main causes of death. Most patients can be identified through neonatal screening, and the prognosis is usually good in patients with early diagnosis and treatment. The purpose of this consensus is to standardize the diagnosis, treatment and management of VLCAD deficiency, so as to improve the prognosis of patients and reduce death and disability.
    Keywords:  Autosomal recessive disease; Expert consensus; Fatty acid β-oxidation; Neonatal screening; Very long-chain acyl-CoA dehydrogenase deficiency
  34. Elife. 2022 Apr 26. pii: e74308. [Epub ahead of print]11
      The influence of genetic variation on the aging process, including the incidence and severity of age-related diseases, is complex. Here we define the evolutionarily conserved mitochondrial enzyme ALH-6/ALDH4A1 as a predictive biomarker for age-related changes in muscle health by combining C. elegans genetics and a gene-wide association scanning (GeneWAS) from older human participants of the US Health and Retirement Study (HRS). In a screen for mutations that activate oxidative stress responses, specifically in the muscle of C. elegans, we identified 96 independent genetic mutants harboring loss-of-function alleles of alh-6, exclusively. Each of these genetic mutations mapped to the ALH-6 polypeptide and led to the age-dependent loss of muscle health. Intriguingly, genetic variants in ALDH4A1 show associations with age-related muscle-related function in humans. Taken together, our work uncovers mitochondrial alh-6/ALDH4A1 as a critical component to impact normal muscle aging across species and a predictive biomarker for muscle health over the lifespan.
    Keywords:  C. elegans; evolutionary biology; genetics; genomics; human
  35. Cold Spring Harb Mol Case Stud. 2022 Apr;pii: a006196. [Epub ahead of print]8(3):
      The increase in sequencing capacity, reduction in costs, and national and international coordinated efforts have led to the widespread introduction of next-generation sequencing (NGS) technologies in patient care. More generally, human genetics and genomic medicine are gaining importance for more and more patients. Some communities are already discussing the prospect of sequencing each individual's genome at time of birth. Together with digital health records, this shall enable individualized treatments and preventive measures, so-called precision medicine. A central step in this process is the identification of disease causal mutations or variant combinations that make us more susceptible for diseases. Although various technological advances have improved the identification of genetic alterations, the interpretation and ranking of the identified variants remains a major challenge. Based on our knowledge of molecular processes or previously identified disease variants, we can identify potentially functional genetic variants and, using different lines of evidence, we are sometimes able to demonstrate their pathogenicity directly. However, the vast majority of variants are classified as variants of uncertain clinical significance (VUSs) with not enough experimental evidence to determine their pathogenicity. In these cases, computational methods may be used to improve the prioritization and an increasing toolbox of experimental methods is emerging that can be used to assay the molecular effects of VUSs. Here, we discuss how computational and experimental methods can be used to create catalogs of variant effects for a variety of molecular and cellular phenotypes. We discuss the prospects of integrating large-scale functional data with machine learning and clinical knowledge for the development of accurate pathogenicity predictions for clinical applications.
  36. Lab Invest. 2022 Apr 29.
      Abnormal Drp1 activation and subsequent excessive mitochondrial fission play a critical role in ischemia-reperfusion injury (I/RI). Although fibroblast growth factor 21 (FGF21) protects organs against I/RI and regulates metabolism, which indicates that FGF21 is involved in mitochondria homeostasis, the detailed mechanism remains unclear. Herein, we investigated whether FGF21 had an effect on Drp1 activation during skeletal muscle I/RI. Drp1 phosphorylation and its translocation to mitochondria, as regulated by FGF21, was examined in mouse and C2C12 cell I/RI models. Mice overexpressing FGF21 displayed alleviation of serum index, histological lesions and apoptosis levels. Moreover, FGF21 markedly decreased cyclin-dependent kinase 1 (CDK1) and Drp1 phosphorylation at Ser616, accompanied by reduced accumulation in mitochondria. In parallel in vitro studies, cells with FGF21 knockdown displayed enhanced Drp1 activation, and the reverse effect was found when FGF21 was added. More importantly, FGF21 attenuated mitochondrial fission with linear mitochondria rather than fragmented mitochondria. Furthermore, a CDK1 inhibitor reduced Drp1 activation and mitochondrial fission due to FGF21 knockdown. This study shows that FGF21 inhibits Drp1 activation to protect mitochondria from fission, thereby rescuing cells from I/RI-induced apoptosis. Our findings may provide a new therapeutic approach to ameliorate skeletal muscle I/RI.
  37. Life Sci. 2022 Apr 22. pii: S0024-3205(22)00271-5. [Epub ahead of print] 120571
      Mitochondrial complex I (CI), the first multiprotein enzyme complex of the oxidative phosphorylation system, plays a crucial role in cellular energy production. CI deficiency is associated with a variety of clinical phenotypes, including Leigh syndrome. At the cellular level, an increased NAD(P)H concentration is one of the hallmarks in CI-deficiency.AIMS: Here, we aimed to attenuate increased NAD(P)H levels by stimulation of ATP-dependent cassette (ABC)A1 and ABCG1-mediated cellular cholesterol efflux with various PPARα and LXRα agonists.
    MAIN METHODS: Mitochondrial CI-deficient fibroblasts and chemically-induced CI-deficient HeLa cells were used to study the dose-dependent effects of various PPARα and LXRα on cellular NAD(P)H levels and cholesterol efflux.
    KEY FINDINGS: In patient-derived mitochondrial CI-deficient fibroblasts, GW590735, astaxanthin, oleoylethanolamide, and GW3965 significantly reduced the enhanced NAD(P)H levels in CI-deficient fibroblasts. Similar effects were observed in chemically-induced CI-impaired HeLa cells, in which BMS-687453, Wy14643, GW7647, T0901317, DMHCA also demonstrated a beneficial effect. Surprisingly, no effect on ABCA1- and ABCG1-mediated cholesterol efflux in HeLa cells and fibroblasts was found after treatment with these compounds. The reduction in NAD(P)H levels by GW590735 could be partially reversed by inhibition of fatty acid synthase and β-oxidation, which suggests that its beneficial effects are possibly mediated via stimulation of fatty acid metabolism rather than cholesterol efflux.
    SIGNIFICANCE: Collectively, PPARα and LXRα stimulation resulted in attenuated cellular NAD(P)H levels in CI-impaired HeLa cells and patient-derived fibroblasts and could eventually have a therapeutic potential in CI deficiency.
    Keywords:  Liver X receptor α; Mitochondrial complex I deficiency; Nicotinamide adenine dinucleotide (phosphate); Peroxisome proliferator activated receptor α; Redox state
  38. Biomater Sci. 2022 Apr 25.
      Perturbations in mitochondrial membrane stability lead to cytochrome c release and induce caspase-dependent apoptosis. Using synthetic smart chemicals with changeable physicochemical properties to interfere the mitochondrial membrane stability has not yet been reported. Here we show that a thermosensitive anchor-polymer-peptide conjugate (anchor-PPC) destabilizes mitochondrial membranes upon in situ molecule changes from hydrophilic to hydrophobic, which consequently induces apoptosis in a spatiotemporally controlled manner and acts as an antitumor pharmaceutical. The anchor-PPC is composed of a thermosensitive copolymer, a photolabile linker, a hydrophilic HIV Tat-derived peptide both for cell penetration and polymer phase transition temperature (Tt) modulation, and an anchor peptide for intercalating into mitochondrial membranes. The photocontrollable anchor-PPC dehydrates and changes from being hydrophilic to hydrophobic upon photoactivation at body temperature. This cell-penetrable anchor-PPC specifically targets mitochondria and destabilizes mitochondrial membranes upon irradiation, and consequently initiates apoptosis in cells and a complex 3D tumor model. This study provides the first experimental evidence that the synthetic smart chemical can spatiotemporally control the stability of organelle membranes based on its in situ physicochemical property change.
  39. Neurol Int. 2022 Apr 02. 14(2): 337-356
      Ataxia is increasingly being recognized as a cardinal manifestation in primary mitochondrial diseases (PMDs) in both paediatric and adult patients. It can be caused by disruption of cerebellar nuclei or fibres, its connection with the brainstem, or spinal and peripheral lesions leading to proprioceptive loss. Despite mitochondrial ataxias having no specific defining features, they should be included in hereditary ataxias differential diagnosis, given the high prevalence of PMDs. This review focuses on the clinical and neuropathological features and genetic background of PMDs in which ataxia is a prominent manifestation.
    Keywords:  Kearns-Sayre syndrome; MERRF; NARP; POLG1-related ataxia; ataxia; mitochondrial diseases
  40. BMC Neurol. 2022 Apr 27. 22(1): 158
      BACKGROUND: METHODS: The KHENERGYC trial will be a phase II, randomised, double-blinded, placebo-controlled (DBPC), parallel-group study in the paediatric population (birth up to and including 17 years). The study will be recruiting 24 patients suffering from motor symptoms due to genetically confirmed PMD. The trial will be divided into two phases. The first phase of the study will be an adaptive pharmacokinetic (PK) study with four days of treatment, while the second phase will include randomisation of the participants and evaluating the efficacy and safety of sonlicromanol over 6 months.DISCUSSION: Effective novel therapies for treating PMDs in children are an unmet need. This study will assess the pharmacokinetics, efficacy, and safety of sonlicromanol in children with genetically confirmed PMDs, suffering from motor symptoms.
    TRIAL REGISTRATION: NCT04846036 , registered April 15, 2021. European Union Clinical Trial Register (EUDRACT number: 2020-003124-16 ), registered October 20, 2020. CCMO registration: NL75221.091.20, registered on October 7, 2020.
    Keywords:  Children; Clinical study protocol; GMFM; KH176; Mitochondrial diseases; OXPHOS; Redox metabolism; Sonlicromanol
  41. JCI Insight. 2022 Apr 26. pii: e157418. [Epub ahead of print]
      Inherited kidney diseases are the fifth most common cause of the end-stage renal disease. Mitochondrial dysfunction plays a vital role in the progression of inherited kidney diseases, while mt-tRNA variants and their pathogenic contributions to the kidney disease remain largely unclear. In this study, we identified the pathogenic mitochondrial tRNAPhe 616T>C mutation in three families; and documented that m.616T>C showed a high pathogenic threshold, with both heteroplasmy and homoplasmy leading to isolated chronic kidney disease and hyperuricemia without hematuria, proteinuria, or renal cyst formation. Moreover, one proband with homoplamic m.616T>C presented with end-stage renal disease as a child. No symptoms of nervous system evolvement were observed in these families. Lymphoblast cells bearing m. 616T>C exhibited swollen mitochondria, underwent active mitophagy and showed respiratory deficiency, leading to reduced mitochondrial ATP production, diminished membrane potential, and overproduction of mitochondrial reactive oxygen species. Pathogenic m. 616T>C abolished a highly conserved base-pair (A31-U39) in the anticodon stem-loop which altered the structure of mt-tRNAPhe, as confirmed by a decreased melting temperature and slower electrophoretic mobility of the mutant tRNA. Furthermore, the unstable structure of mt-tRNAPhe contributed to a shortage of steady-state mt-tRNAPhe and enhanced aminoacylation efficiency, which resulted in impaired mitochondrial RNA translation and a significant decrease in mt-DNA encoded polypeptides. Collectively, these findings provide new insights into the pathogenesis underlying inherited kidney disease caused by mitochondrial variants.
    Keywords:  Chronic kidney disease; Mitochondria; Nephrology
  42. RNA Biol. 2022 Jan;19(1): 662-677
      In all living cells, the ribosome translates the genetic information carried by messenger RNAs (mRNAs) into proteins. The process of ribosome recycling, a key step during protein synthesis that ensures ribosomal subunits remain available for new rounds of translation, has been largely overlooked. Despite being essential to the survival of the cell, several mechanistic aspects of ribosome recycling remain unclear. In eubacteria and mitochondria, recycling of the ribosome into subunits requires the concerted action of the ribosome recycling factor (RRF) and elongation factor G (EF-G). Recently, the conserved protein HflX was identified in bacteria as an alternative factor that recycles the ribosome under stress growth conditions. The homologue of HflX, the GTP-binding protein 6 (GTPBP6), has a dual role in mitochondrial translation by facilitating ribosome recycling and biogenesis. In this review, mechanisms of ribosome recycling in eubacteria and mitochondria are described based on structural studies of ribosome complexes.
    Keywords:  GTPBP6; HflX; Ribosome; elongation factor G; protein synthesis; ribosome recycling; ribosome recycling factor; tRNA; translation factors
  43. Front Psychiatry. 2022 ;13 864445
      Background: Mitochondrial disorders (MD) are metabolic diseases related to genetic mutations in mitochondrial DNA and nuclear DNA that cause dysfunction of the mitochondrial respiratory chain. Cognitive impairment and psychiatric symptoms are frequently associated with MD in the adult population. The aim of this study is to describe the neuropsychological profile in children and adolescents with MD.Methods: We prospectively recruited a sample of 12 children and adolescents between February 2019 and February 2020 in the Reference Center for Mitochondrial Disorders of Angers (France). Participants and their parents completed an anamnestic form describing socio-demographic data and completed the WISC-V (Wechsler Intelligence Scale for Children, 5th edition) and the Parent and Teacher forms of the BRIEF (Behavior Rating Inventory of Executive Function).
    Results: In our sample, the mean IQ (Intellectual Quotient) score was 87.3 ± 25.3. The score ranged from 52 to 120. Concerning executive functions, a significant global clinical complaint was found for parents (six times more than normal) and to a lesser extent, for teachers (among 3 to 4 times more). Levels of intelligence and executive functioning were globally linked in our cohort but dissociation remains a possibility.
    Conclusion: The results of this study show that MD can be associated to neuropsychological disorders in children and adolescents, especially regarding the intelligence quotient and the executive function. Our study also highlights the need for regular neuropsychological assessments in individuals with MD and developing brains, such as children and adolescents.
    Keywords:  adolescent; child; executive function; intelligence; mitochondrial disorders; neuropsychological profile
  44. Methods Mol Biol. 2022 ;2490 101-140
      Single-cell RNA-sequencing (scRNA-Seq) is a widely used technology to reveal the heterogeneity and dynamics of tissues, organisms, and complex diseases. Here, a workflow is presented for preprocessing of scRNA-seq data to quantify gene abundances in individual cells followed by visualization and annotation of cells.
    Keywords:  Bioinformatics; Gastrulation; Mouse embryogenesis; Workflow; scRNA-Seq
  45. Biol Rev Camb Philos Soc. 2022 Apr 26.
      Protein kinase RNA-like ER kinase (PERK) is an endoplasmic reticulum (ER) stress sensor that responds to the accumulation of misfolded proteins. Once activated, PERK initiates signalling pathways that halt general protein production, increase the efficiency of ER quality control, and maintain redox homeostasis. PERK activation also protects mitochondrial homeostasis during stress. The location of PERK at the contact sites between the ER and the mitochondria creates a PERK-mitochondria axis that allows PERK to detect stress in both organelles, adapt their functions and prevent apoptosis. During ER stress, PERK activation triggers mitochondrial hyperfusion, preventing premature apoptotic fragmentation of the mitochondria. PERK activation also increases the formation of mitochondrial cristae and the assembly of respiratory supercomplexes, enhancing cellular ATP-generating capacity. PERK strengthens mitochondrial quality control during stress by promoting the expression of mitochondrial chaperones and proteases and by increasing mitochondrial biogenesis and mitophagy, resulting in renewal of the mitochondrial network. But how does PERK mediate all these changes in mitochondrial homeostasis? In addition to the classic PERK-eukaryotic translation initiation factor 2α (eIF2α)-activating transcription factor 4 (ATF4) pathway, PERK can activate other protective pathways - PERK-O-linked N-acetyl-glucosamine transferase (OGT), PERK-transcription factor EB (TFEB), and PERK-nuclear factor erythroid 2-related factor 2 (NRF2) - contributing to broader regulation of mitochondrial dynamics, metabolism, and quality control. The pharmacological activation of PERK is protective in models of neurodegenerative and metabolic diseases, such as Huntington's disease, progressive supranuclear palsy and obesity, while the inhibition of PERK was protective in models of Parkinson's and prion diseases and diabetes. In this review, we address the molecular mechanisms by which PERK regulates mitochondrial dynamics, metabolism and quality control, and discuss the therapeutic potential of targeting PERK in neurodegenerative and metabolic diseases.
    Keywords:  PERK; dynamics; endoplasmic reticulum; metabolic diseases; metabolism; mitochondria; neurodegeneration; stress; unfolded protein response
  46. Nat Biomed Eng. 2022 Apr;6(4): 372-388
      The immature physiology of cardiomyocytes derived from human induced pluripotent stem cells (hiPSCs) limits their utility for drug screening and disease modelling. Here we show that suitable combinations of mechanical stimuli and metabolic cues can enhance the maturation of hiPSC-derived cardiomyocytes, and that the maturation-inducing cues have phenotype-dependent effects on the cells' action-potential morphology and calcium handling. By using microfluidic chips that enhanced the alignment and extracellular-matrix production of cardiac microtissues derived from genetically distinct sources of hiPSC-derived cardiomyocytes, we identified fatty-acid-enriched maturation media that improved the cells' mitochondrial structure and calcium handling, and observed divergent cell-source-dependent effects on action-potential duration (APD). Specifically, in the presence of maturation media, tissues with abnormally prolonged APDs exhibited shorter APDs, and tissues with aberrantly short APDs displayed prolonged APDs. Regardless of cell source, tissue maturation reduced variabilities in spontaneous beat rate and in APD, and led to converging cell phenotypes (with APDs within the 300-450 ms range characteristic of human left ventricular cardiomyocytes) that improved the modelling of the effects of pro-arrhythmic drugs on cardiac tissue.
  47. Front Aging Neurosci. 2022 ;14 848919
      Pantothenate kinase-associated neurodegeneration (PKAN) is a rare genetic disorder caused by mutations in the mitochondrial pantothenate kinase 2 (PANK2) gene and displays an inherited autosomal recessive pattern. In this study, we identified eight PANK2 mutations, including three novel mutations (c.1103A > G/p.D368G, c.1696C > G/p.L566V, and c.1470delC/p.R490fs494X), in seven unrelated families with PKAN. All the patients showed an eye-of-the-tiger sign on the MRI, six of seven patients had dystonia, and two of seven patients had Parkinsonism. Biallelic mutations of PANK2 decreased PANK2 protein expression and reduced mitochondrial membrane potential in human embryonic kidney (HEK) 293T cells. The biallelic mutations from patients with early-onset PKAN, a severity phenotype, showed decreased mitochondrial membrane potential more than that from late-onset patients. We systematically reviewed all the reported patients with PKAN with PANK2 mutations. The results indicated that the early-onset patients carried a significantly higher frequency of biallelic loss-of-function (LoF) mutations compared to late-onset patients. In general, patients with LoF mutations showed more severe phenotypes, including earlier onset age and loss of gait. Although there was no significant difference in the frequency of biallelic missense mutations between the early-onset and late-onset patients, we found that patients with missense mutations in the mitochondrial trafficking domain (transit peptide/mitochondrial domain) of PANK2 exhibited the earliest onset age when compared to patients with mutations in the other two domains. Taken together, this study reports three novel mutations and indicates a correlation between the phenotype and mitochondrial dysfunction. This provides new insight for evaluating the clinical severity of patients based on the degree of mitochondrial dysfunction and suggests genetic counseling not just generalized identification of mutated PANK2 in clinics.
    Keywords:  PANK2; mitochondria; mutation; pKAN; severity
  48. STAR Protoc. 2022 Jun 17. 3(2): 101295
      Microglia and astrocytes are implicated in aging and age-related diseases. Here, we present a protocol to isolate and culture these glia cells from the murine brain. The protocol consists of two parts: magnetic sorting of adult microglia and mechanical/magnetic sorting of adult microglia and astrocytes. We then describe the characterization of these glial cells by flow cytometry and immunohistochemistry. Microglia isolated from aged mice maintain age-related phenotype during culture. These purified glia cells can be applied in ex vivo studies.
    Keywords:  Cell Biology; Cell culture; Cell isolation; Flow Cytometry/Mass Cytometry; Immunology; Neuroscience; Single Cell
  49. Cell. 2022 Apr 28. pii: S0092-8674(22)00398-1. [Epub ahead of print]185(9): 1455-1470
      Diet as a whole, encompassing food composition, calorie intake, and the length and frequency of fasting periods, affects the time span in which health and functional capacity are maintained. Here, we analyze aging and nutrition studies in simple organisms, rodents, monkeys, and humans to link longevity to conserved growth and metabolic pathways and outline their role in aging and age-related disease. We focus on feasible nutritional strategies shown to delay aging and/or prevent diseases through epidemiological, model organism, clinical, and centenarian studies and underline the need to avoid malnourishment and frailty. These findings are integrated to define a longevity diet based on a multi-pillar approach adjusted for age and health status to optimize lifespan and healthspan in humans.
  50. Front Physiol. 2022 ;13 772313
      Mitochondrial malfunction is a hallmark of many diseases, including neurodegenerative disorders, cardiovascular and lung diseases, and cancers. We previously found that alveolar progenitor cells, which are more resistant to cigarette smoke-induced injury than the other cells of the lung parenchyma, upregulate the mtDNA-encoded small non-coding RNA mito-ncR-805 after exposure to smoke. The mito-ncR-805 acts as a retrograde signal between the mitochondria and the nucleus. Here, we identified a region of mito-ncR-805 that is conserved in the mammalian mitochondrial genomes and generated shorter versions of mouse and human transcripts (mmu-CR805 and hsa-LDL1, respectively), which differ in a few nucleotides and which we refer to as the "functional bit". Overexpression of mouse and human functional bits in either the mouse or the human lung epithelial cells led to an increase in the activity of the Krebs cycle and oxidative phosphorylation, stabilized the mitochondrial potential, conferred faster cell division, and lowered the levels of proapoptotic pseudokinase, TRIB3. Both oligos, mmu-CR805 and hsa-LDL1 conferred cross-species beneficial effects. Our data indicate a high degree of evolutionary conservation of retrograde signaling via a functional bit of the D-loop transcript, mito-ncR-805, in the mammals. This emphasizes the importance of the pathway and suggests a potential to develop this functional bit into a therapeutic agent that enhances mitochondrial bioenergetics.
    Keywords:  D-loop transcripts; Krebs cycle; OxPhos; mitochondria; mitochondria-to-nucleus retrograde signaling; small ncRNA
  51. Nat Commun. 2022 Apr 26. 13(1): 2253
      Drug-induced cardiotoxicity arises primarily when a compound alters the electrophysiological properties of cardiomyocytes. Features of intracellular action potentials (iAPs) are powerful biomarkers that predict proarrhythmic risks. In the last decade, a number of vertical nanoelectrodes have been demonstrated to achieve parallel and minimally-invasive iAP recordings. However, the large variability in success rate and signal strength have hindered nanoelectrodes from being broadly adopted for proarrhythmia drug assessment. In this work, we develop vertically-aligned nanocrown electrodes that are mechanically robust and achieve > 99% success rates in obtaining intracellular access through electroporation. We validate the accuracy of nanocrown electrode recordings by simultaneous patch clamp recording from the same cell. Finally, we demonstrate that nanocrown electrodes enable prolonged iAP recording for continual monitoring of the same cells upon the sequential addition of four incremental drug doses. Our technology development provides an advancement towards establishing an iAP screening assay for preclinical evaluation of drug-induced arrhythmogenicity.
  52. Immunol Cell Biol. 2022 Apr 29.
      Within the cytoplasm of mammalian cells is a protein called cyclic GMP-AMP synthase (cGAS), which acts to defend against infection and other threats to the host. cGAS operates in this manner through its ability to detect a molecular occurrence that should not exist in healthy cells-the existence of DNA in the cytosol. Upon DNA binding, cGAS synthesizes cyclic GMP-AMP (cGAMP), a cyclic dinucleotide that activates the endoplasmic reticulum-localized protein stimulator of interferon genes (STING). STING-mediated signaling culminates in host defensive responses typified by inflammatory cytokine and interferon expression, and the induction of autophagy. Studies over the last several years have established a consensus in the field of the enzymatic activities of cGAS in vitro, as it relates to DNA-induced production of cGAMP. However, much additional work is needed to understand the regulation of cGAS functions within cells, where multiple sources of DNA can create a problem of self and non-self discrimination. In this review, we provide an overview of how the cGAS-STING pathway mediates innate immune responses during infection and other cellular stresses. We then highlight the recent progress in the understanding of the increasingly diverse ways that this DNA-sensing machinery is regulated inside cells, including how cGAS remains inactive to host-derived DNA under conditions of homeostasis.
    Keywords:  STING; antiviral response; cGAS; innate immune system
  53. Cell Rep Methods. 2022 Feb 28. 2(2): 100155
      Xeno-free culture systems have expanded the clinical and industrial application of human pluripotent stem cells (PSCs). However, reproducibility issues, often arising from variability during passaging steps, remain. Here, we describe an improved method for the subculture of human PSCs. The revised method significantly enhances the viability of human PSCs by lowering DNA damage and apoptosis, resulting in more efficient and reproducible downstream applications such as gene editing and directed differentiation. Furthermore, the method does not alter PSC characteristics after long-term culture and attenuates the growth advantage of abnormal subpopulations. This robust passaging method minimizes experimental error and reduces the rate of PSCs failing quality control of human PSC research and application.
    Keywords:  DNA damage; human pluripotent stem cell; passage; reproducibility; viability; xeno-free culture
  54. Nat Commun. 2022 Apr 25. 13(1): 2232
      ATP synthases are macromolecular machines consisting of an ATP-hydrolysis-driven F1 motor and a proton-translocation-driven FO motor. The F1 and FO motors oppose each other's action on a shared rotor subcomplex and are held stationary relative to each other by a peripheral stalk. Structures of resting mitochondrial ATP synthases revealed a left-handed curvature of the peripheral stalk even though rotation of the rotor, driven by either ATP hydrolysis in F1 or proton translocation through FO, would apply a right-handed bending force to the stalk. We used cryoEM to image yeast mitochondrial ATP synthase under strain during ATP-hydrolysis-driven rotary catalysis, revealing a large deformation of the peripheral stalk. The structures show how the peripheral stalk opposes the bending force and suggests that during ATP synthesis proton translocation causes accumulation of strain in the stalk, which relaxes by driving the relative rotation of the rotor through six sub-steps within F1, leading to catalysis.
  55. Reprod Sci. 2022 Apr 27.
      In humans and most animals, maternal inheritance of mitochondria and mitochondrial DNA (mtDNA) is considered as an universal assumption. Recently, several lines of evidence suggest that different species seem to employ distinct mechanisms to prevent the inheritance of paternal mtDNA. There are few studies in the literature on the molecular basis of sperm mtDNA elimination in mammals and paternal mtDNA transmission in humans. Endonuclease G (ENDOG) is a mitochondrial nuclease encoded by nuclear ENDOG gene. The critical importance of ENDOG gene on paternal mitochondrial elimination (PME) has been previously demonstrated in model organisms such as C. elegans and D. melanogaster. However, its mechanism in human is still unclear. Therefore, we aimed to evaluate whether nuclear ENDOG gene copy number could be a potential marker of paternal mtDNA transmission or not.Male factor infertility patients diagnosed with different infertility subgroups such as azoospermia, oligoteratozoospermia, astheno-teratozoospermia were included in this study: 13 infertile men and 25 healthy men as control group. Quantitative real-time polymerase chain reaction (qPCR) analysis and dual-color Fluorescence in situ hybridization (FISH) method were used to compare the groups. FISH method was applied to verify qPCR results and two signals were observed in nearly all patients. ENDOG gene copy number data were evaluated by comparing them with entire human mtDNA next-generation sequencing (NGS) analysis results obtained through bioinformatics and proteomics tools. Mitochondrial whole genome sequencing (WGS) data allowed determination of novel and reported variations such as single nucleotide polymorphisms (SNPs), multiple nucleotide polymorphism (MNP), insertion/deletion (INDEL). Missense variants causing amino acid substitution were filtered out from patients' mtDNA WGS data.Relative copy number of target ENDOG gene in male infertility patients [0.49 (0.31 - 0.77)] was lower than healthy controls [1.00 (0.66 - 1.51)], and statistical results showed significant differences between the groups (p < 0.01). A total of 38 missense variants were detected in the genes encoding the proteins involved in the respiratory chain complex. Moreover, we detected paternal mtDNA transmissions in the children of these patients who applied to assisted reproductive techniques.In conclusion, this study reveals that ENDOG gene may be an important factor for the PME mechanism in humans. To the best of our knowledge, this is the first study in humans about this topic and assessment of ENDOG gene sequencing and gene expression studies in a larger sample size including patients with male factor infertility would be our future project.
    Keywords:  ENDOG gene; Male infertility; Mitochondrial DNA; Mitochondrial endonuclease G; Paternal mitochondrial elimination
  56. PLoS Genet. 2022 Apr 29. 18(4): e1010191
      Whole genome sequencing is increasingly used to diagnose medical conditions of genetic origin. While both coding and non-coding DNA variants contribute to a wide range of diseases, most patients who receive a WGS-based diagnosis today harbour a protein-coding mutation. Functional interpretation and prioritization of non-coding variants represents a persistent challenge, and disease-causing non-coding variants remain largely unidentified. Depending on the disease, WGS fails to identify a candidate variant in 20-80% of patients, severely limiting the usefulness of sequencing for personalised medicine. Here we present FINSURF, a machine-learning approach to predict the functional impact of non-coding variants in regulatory regions. FINSURF outperforms state-of-the-art methods, owing in particular to optimized control variants selection during training. In addition to ranking candidate variants, FINSURF breaks down the score for each variant into contributions from individual annotations, facilitating the evaluation of their functional relevance. We applied FINSURF to a diverse set of 30 diseases with described causative non-coding mutations, and correctly identified the disease-causative non-coding variant within the ten top hits in 22 cases. FINSURF is implemented as an online server to as well as custom browser tracks, and provides a quick and efficient solution to prioritize candidate non-coding variants in realistic clinical settings.
  57. Trends Genet. 2022 Apr 25. pii: S0168-9525(22)00080-4. [Epub ahead of print]
      Advances in genome sequencing have enabled researchers and clinicians to probe vast numbers of human variants to distinguish pathogenic from benign variants. Model organisms have been crucial in variant assessment and in delineating the molecular mechanisms of some of the diseases caused by these variants. The fruit fly, Drosophila melanogaster, has played a valuable role in this endeavor, taking advantage of its genetic technologies and established biological knowledge. We highlight the utility of the fly in studying the function of genes associated with rare neurological diseases that have led to a better understanding of common disease mechanisms. We emphasize that shared themes emerge among disease mechanisms, including the importance of lipids, in two prominent neurodegenerative diseases: Alzheimer's disease (AD) and Parkinson's disease (PD).
    Keywords:  Alzheimer's disease; Drosophila; Parkinson's disease; lipid; mitochondria; neurodegeneration
  58. Diabetes. 2022 Apr 26. pii: db210800. [Epub ahead of print]
      Mitochondrial glucose metabolism is essential for stimulated insulin release from pancreatic beta cells. Whether mitofusin gene expression, and hence mitochondrial network integrity, is important for glucose or incretin signalling has not previously been explored. Here, we generated mice with beta cell-selective, adult-restricted deletion of the mitofusin genes Mfn1 and Mfn2 (βMfn1/2 dKO). βMfn1/2 dKO mice displayed elevated fed and fasted glycaemia and a >five-fold decrease in plasma insulin. Mitochondrial length, glucose-induced polarisation, ATP synthesis, cytosolic and mitochondrial Ca2+ increases were all reduced in dKO islets. In contrast, oral glucose tolerance was more modestly affected in βMfn1/2 dKO mice and GLP-1 or GIP receptor agonists largely corrected defective GSIS through enhanced EPAC-dependent signalling. Correspondingly, cAMP increases in the cytosol, as measured with an Epac-camps based sensor, were exaggerated in dKO mice. Mitochondrial fusion and fission cycles are thus essential in the beta cell to maintain normal glucose, but not incretin, sensing. These findings broaden our understanding of the roles of mitofusins in beta cells, the potential contributions of altered mitochondrial dynamics to diabetes development and the impact of incretins on this process.
  59. Methods Mol Biol. 2022 ;2504 91-100
      Extracellular vesicles (EVs) secreted by human pluripotent stem cells-derived cardiovascular progenitor cells (hPSC-CVPCs) can improve repair of infarcted hearts in mouse and nonhuman primate myocardial infarction models. To fully achieve their values, it is essential to establish an efficient method for the isolation of EVs from hPSC-CVPCs. Here we describe the protocols for efficient isolation and characterization of EVs from the conditioned medium of hPSC-CVPCs.
    Keywords:  Cardiovascular progenitor cells; Extracellular vesicles; Human embryonic stem cells; Human induced pluripotent stem cells; Human pluripotent stem cells
  60. J Neuroophthalmol. 2022 Apr 27.
      BACKGROUND: Leber hereditary optic neuropathy (LHON) is an inherited mitochondrial disease characterized by severe bilateral vision loss and chronic visual impairment. The objective of this study was to comprehensively explore the impact of LHON on the lives of patients and their relatives at the time of diagnosis and now.METHODS: Qualitative study design with 8 focus group interviews conducted in France, Germany, the United Kingdom, and the United States, involving 17 individuals with m.11778G>A mutation and their relatives. Separate focus groups for patients and their relatives were facilitated by a moderator in French, German, or English. Qualitative analysis of interviews using a pre-defined analytical framework.
    RESULTS: Participants reported feeling devastated by the diagnosis of LHON after a lengthy and worrisome diagnostic journey. Patients were frustrated by the loss of autonomy, which also affected their relatives. Participants described challenges across several domains: physical capabilities, emotional well-being, interpersonal relationships, work and studies, finances, and recreational activities. Access to disability allowances, vision aids, and funded or subsided idebenone varied by country, resulting in unequal financial impact. Patients are hopeful for therapy that would restore autonomy and improve their ability to enjoy a fulfilling life, while alleviating the demands placed on their relatives.
    CONCLUSIONS: The impact of LHON extends beyond vision-related activity limitations. Addressing the psychosocial impact of LHON and helping patients and their relatives adapt and cope with vision loss are vital. As part of this, an accurate and timely diagnosis is important to enable early intervention. Further investigation of specific unmet needs is required.
  61. Stem Cell Reports. 2022 Apr 13. pii: S2213-6711(22)00158-8. [Epub ahead of print]
      Induced pluripotent stem cells (iPSCs) are valuable in disease modeling because of their potential to expand and differentiate into virtually any cell type and recapitulate key aspects of human biology. Functional genomics are genome-wide studies that aim to discover genotype-phenotype relationships, thereby revealing the impact of human genetic diversity on normal and pathophysiology. In this review, we make the case that human iPSCs (hiPSCs) are a powerful tool for functional genomics, since they provide an in vitro platform for the study of population genetics. We describe cutting-edge tools and strategies now available to researchers, including multi-omics technologies, advances in hiPSC culture techniques, and innovations in drug development. Functional genomics approaches based on hiPSCs hold great promise for advancing drug discovery, disease etiology, and the impact of genetic variation on human biology.
    Keywords:  disease modeling; drug screening; functional genomics; genetic variants; hiPSCs
  62. Cell. 2022 Apr 28. pii: S0092-8674(22)00338-5. [Epub ahead of print]185(9): 1618-1618.e1
      Skeletal muscle size is highly plastic and sensitive to a variety of stimuli. Muscle atrophy occurs as the result of changes in multiple signaling pathways that regulate both protein synthesis and degradation. The signaling pathways that are activated or inhibited depend on the specific stimuli that are altered. To view this SnapShot, open of download the PDF.
  63. J Mol Cell Cardiol. 2022 Apr 21. pii: S0022-2828(22)00075-X. [Epub ahead of print]
      Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) have advanced our ability to study the basic function of the heart and model cardiac diseases. Due to the complexities in stem cell culture and differentiation protocols, many researchers source their hiPSC-CMs from collaborators or commercial biobanks. Generally, the field has assumed the health of frozen cardiomyocytes is unchanged if the cells adhere to the substrate and commence beating. However, very few have investigated the effects of cryopreservation on hiPSC-CM's functional and transcriptional health at the cellular and molecular level. Here we review methods and challenges associated with cryopreservation, and examine the effects of cryopreservation on the functionality (contractility and calcium handling) and transcriptome of hiPSC-CMs from six healthy stem cell lines. Utilizing protein patterning methods to template physiological cell aspect ratios (7:1, length:width) in conjunction with polyacrylamide (PA) hydrogels, we measured changes in force generation and calcium handling of single hiPSC-CMs. We observed that cryopreservation altered the functionality and transcriptome of hiPSC-CMs towards larger sizes and contractile force as assessed by increased spread area and volume, single cell traction force microscopy and delayed calcium dynamics. hiPSC-CMs are broadly used for basic science research, regenerative medicine, and testing biological therapeutics. This study informs the design of experiments utilizing hiPSC-CMs to avoid confounding functional changes due to cryopreservation with other treatments.
    Keywords:  Cardiomyocytes; Cryopreservation; Functionality; Human induced pluripotent cells; Viability
  64. Annu Rev Biomed Data Sci. 2022 Apr 28.
      Thousands of common genetic variants in the human population have been associated with disease risk and phenotypic variation by genome-wide association studies (GWAS). However, the majority of GWAS variants fall into noncoding regions of the genome, complicating our understanding of their regulatory functions, and few molecular mechanisms of GWAS variant effects have been clearly elucidated. Here, we set out to review genetic variant effects, focusing on expression quantitative trait loci (eQTLs), including their utility in interpreting GWAS variant mechanisms. We discuss the interrelated challenges and opportunities for eQTL analysis, covering determining causal variants, elucidating molecular mechanisms of action, and understanding context variability. Addressing these questions can enable better functional characterization of disease-associated loci and provide insights into fundamental biological questions of the noncoding genetic regulatory code and its control of gene expression. Expected final online publication date for the Annual Review of Biomedical Data Science, Volume 5 is August 2022. Please see for revised estimates.
  65. Nat Biotechnol. 2022 Apr 25.
      The low photostability of fluorescent proteins is a limiting factor in many applications of fluorescence microscopy. Here we present StayGold, a green fluorescent protein (GFP) derived from the jellyfish Cytaeis uchidae. StayGold is over one order of magnitude more photostable than any currently available fluorescent protein and has a cellular brightness similar to mNeonGreen. We used StayGold to image the dynamics of the endoplasmic reticulum (ER) with high spatiotemporal resolution over several minutes using structured illumination microscopy (SIM) and observed substantially less photobleaching than with a GFP variant optimized for stability in the ER. Using StayGold fusions and SIM, we also imaged the dynamics of mitochondrial fusion and fission and mapped the viral spike proteins in fixed cells infected with severe acute respiratory syndrome coronavirus 2. As StayGold is a dimer, we created a tandem dimer version that allowed us to observe the dynamics of microtubules and the excitatory post-synaptic density in neurons. StayGold will substantially reduce the limitations imposed by photobleaching, especially in live cell or volumetric imaging.
  66. Eur J Neurosci. 2022 Apr 28.
      Mitochondria is an autonomous organelle that plays a crucial role in the metabolic aspects of a cell. Cortical Spreading Depression (CSD) and fluctuations in the cerebral blood flow have for long been mechanisms underlying migraine. It is a neurovascular disorder with a unilateral manifestation of disturbing, throbbing and pulsating head pain. Migraine affects 2.6 and 21.7% of the general population and is the major cause of partial disability in the age group 15-49. Higher mutation rates, imbalance in concentration of physiologically relevant molecules, oxidative stress biomarkers have been the main themes of discussion in determining the role of mitochondrial disability in migraine. The correlation of migraine with other disorders like hemiplegic migraine, MELAS, TTH, CVS, ischemic stroke and hypertension has helped in the assessment of the physiological and morphogenetic basis of migraine. Here, we have reviewed the different nuances of mitochondrial dysfunction and migraine. The different mtDNA polymorphisms that can affect the generation and transmission of nerve impulse has been highlighted and supported with research findings. In addition to this, the genetic basis of migraine pathogenesis as a consequence of mutations in nuclear DNA that can in turn affect the synthesis of defective mitochondrial proteins is discussed along with a brief overview of epigenetic profile. This review gives an overview of the pathophysiology of migraine and explores mitochondrial dysfunction as a potential underlying mechanism. Also, therapeutic supplements for managing migraine have been discussed at different junctures in this paper.
    Keywords:  Mitochondrial dysfunction; impairment; metabolism; migraine; mitochondrial genetics