bims-mitdis Biomed News
on Mitochondrial disorders
Issue of 2022–08–07
nineteen papers selected by
Catalina Vasilescu, University of Helsinki
  1. Regulation of mitochondrial proteostasis by the proton gradient.
  2. CMT2A-linked mitochondrial hyperfusion-driving mutant MFN2 perturbs ER-mitochondrial associations and Ca2+ homeostasis.
  3. Measuring Single-Cell Mitochondrial DNA Copy Number and Heteroplasmy using Digital Droplet Polymerase Chain Reaction.
  4. Structural insight and characterization of human Twinkle helicase in mitochondrial disease.
  5. The mystery of mitochondrial plasticity: TMBIM5 integrates metabolic state and proteostasis.
  6. Mitochondrial DNA maintenance defects: potential therapeutic strategies.
  7. Multi-mtDNA Variants May Be a Factor Contributing to Mitochondrial Function Variety in the Skin-Derived Fibroblasts of Leber's Hereditary Optic Neuropathy Patients.
  8. Rapamycin rescues mitochondrial dysfunction in cells carrying the m.8344A > G mutation in the mitochondrial tRNALys.
  9. Balancing NAD+ deficits with nicotinamide riboside: therapeutic possibilities and limitations.
  10. Systems biochemistry to "deorphanize" human mitochondrial proteome.
  11. Altered SYNJ2BP-mediated mitochondrial-ER contacts in motor neuron disease.
  12. Metrics of progression and prognosis in untreated adults with thymidine kinase 2 deficiency: An observational study.
  13. Novel DNM1L variants impair mitochondrial dynamics through divergent mechanisms.
  14. Repeat expansions nested within tandem CNVs: A unique structural change in GLS exemplifies the diagnostic challenges of non-coding pathogenic variation.
  15. Noninvasive Ophthalmic Imaging Measures Retinal Degeneration and Vision Deficits in Ndufs4-/- Mouse Model of Mitochondrial Complex I Deficiency.
  16. Nrf2 as a regulator of mitochondrial function: Energy metabolism and beyond.
  17. Transcriptome variation in human tissues revealed by long-read sequencing.
  18. Neuromuscular disorders: finding the missing genetic diagnoses.
  19. Structure-activity relationships of mitochondria-targeted tetrapeptide pharmacological compounds.
  1. EMBO J. 2022 Aug 01. e110476
    Regulation of mitochondrial proteostasis by the proton gradient.
    Maria Patron, Daryna Tarasenko, Hendrik Nolte, Lara Kroczek, Mausumi Ghosh, Yohsuke Ohba, Yvonne Lasarzewski, Zeinab Alsadat Ahmadi, Alfredo Cabrera-Orefice, Akinori Eyiama, Tim Kellermann, Elena I Rugarli, Ulrich Brandt, Michael Meinecke, Thomas Langer.
      Mitochondria adapt to different energetic demands reshaping their proteome. Mitochondrial proteases are emerging as key regulators of these adaptive processes. Here, we use a multiproteomic approach to demonstrate the regulation of the m-AAA protease AFG3L2 by the mitochondrial proton gradient, coupling mitochondrial protein turnover to the energetic status of mitochondria. We identify TMBIM5 (previously also known as GHITM or MICS1) as a Ca2+ /H+ exchanger in the mitochondrial inner membrane, which binds to and inhibits the m-AAA protease. TMBIM5 ensures cell survival and respiration, allowing Ca2+ efflux from mitochondria and limiting mitochondrial hyperpolarization. Persistent hyperpolarization, however, triggers degradation of TMBIM5 and activation of the m-AAA protease. The m-AAA protease broadly remodels the mitochondrial proteome and mediates the proteolytic breakdown of respiratory complex I to confine ROS production and oxidative damage in hyperpolarized mitochondria. TMBIM5 thus integrates mitochondrial Ca2+ signaling and the energetic status of mitochondria with protein turnover rates to reshape the mitochondrial proteome and adjust the cellular metabolism.
    Keywords:  AFG3L2; TMBIM5; mitochondrial calcium; proton gradient; respiratory chain
    DOI:  https://doi.org/10.15252/embj.2021110476
  2. Biol Cell. 2022 Aug 04.
    CMT2A-linked mitochondrial hyperfusion-driving mutant MFN2 perturbs ER-mitochondrial associations and Ca2+ homeostasis.
    Rajdeep Das, Subhrangshu Das, Saikat Chakrabarti, Oishee Chakrabarti.
      Mitofusin2 (MFN2), an important molecular player that regulates mitochondrial fusion, also helps maintain the inter-organellar contact sites, referred as mitochondria associated membranes (MAMs) that exist between the ER and mitochondria. Here we show that a mutant of MFN2, R364W-MFN2, linked with the Charcot Marie Tooth disease, promotes mitochondrial hyperfusion, alters ER mitochondrial associations at the MAM junctions and perturbs inter-organellar calcium homeostasis. Such hyperfused mitochondria are also predisposed towards stress and undergo rapid fission upon induction of mild stress. Thus, here we report that presence of the R364W-MFN2 mutation makes cells susceptible towards stress, thus negatively affecting cellular health. This article is protected by copyright. All rights reserved.
    Keywords:  CMT2A-linked MFN2 mutant; DRP1; ER-mitochondrial associations; mitochondrial hyperfusion
    DOI:  https://doi.org/10.1111/boc.202100098
  3. J Vis Exp. 2022 Jul 12.
    Measuring Single-Cell Mitochondrial DNA Copy Number and Heteroplasmy using Digital Droplet Polymerase Chain Reaction.
    Stephen P Burr, Patrick F Chinnery.
      The mammalian mitochondrial (mt)DNA is a small, circular, double-stranded, intra-mitochondrial DNA molecule, encoding 13 subunits of the electron transport chain. Unlike the diploid nuclear genome, most cells contain many more copies of mtDNA, ranging from less than 100 to over 200,000 copies depending on cell type. MtDNA copy number is increasingly used as a biomarker for a number of age-related degenerative conditions and diseases, and thus, accurate measurement of the mtDNA copy number is becoming a key tool in both research and diagnostic settings. Mutations in the mtDNA, often occurring as single nucleotide polymorphisms (SNPs) or deletions, can either exist in all copies of the mtDNA within the cell (termed homoplasmy) or as a mixture of mutated and WT mtDNA copies (termed heteroplasmy). Heteroplasmic mtDNA mutations are a major cause of clinical mitochondrial pathology, either in rare diseases or in a growing number of common late-onset diseases such as Parkinson's disease. Determining the level of heteroplasmy present in cells is a critical step in the diagnosis of rare mitochondrial diseases and in research aimed at understanding common late-onset disorders where mitochondria may play a role. MtDNA copy number and heteroplasmy have traditionally been measured by quantitative (q)PCR-based assays or deep sequencing. However, the recent introduction of ddPCR technology has provided an alternative method for measuring both parameters. It offers several advantages over existing methods, including the ability to measure absolute mtDNA copy number and sufficient sensitivity to make accurate measurements from single cells even at low copy numbers. Presented here is a detailed protocol describing the measurement of mtDNA copy number in single cells using ddPCR, referred to as droplet generation PCR henceforth, with the option for simultaneous measurement of heteroplasmy in cells with mtDNA deletions. The possibility of expanding this method to measure heteroplasmy in cells with mtDNA SNPs is also discussed.
    DOI:  https://doi.org/10.3791/63870
  4. Proc Natl Acad Sci U S A. 2022 Aug 09. 119(32): e2207459119
    Structural insight and characterization of human Twinkle helicase in mitochondrial disease.
    Amanda A Riccio, Jonathan Bouvette, Lalith Perera, Matthew J Longley, Juno M Krahn, Jason G Williams, Robert Dutcher, Mario J Borgnia, William C Copeland.
      Twinkle is the mammalian helicase vital for replication and integrity of mitochondrial DNA. Over 90 Twinkle helicase disease variants have been linked to progressive external ophthalmoplegia and ataxia neuropathies among other mitochondrial diseases. Despite the biological and clinical importance, Twinkle represents the only remaining component of the human minimal mitochondrial replisome that has yet to be structurally characterized. Here, we present 3-dimensional structures of human Twinkle W315L. Employing cryo-electron microscopy (cryo-EM), we characterize the oligomeric assemblies of human full-length Twinkle W315L, define its multimeric interface, and map clinical variants associated with Twinkle in inherited mitochondrial disease. Cryo-EM, crosslinking-mass spectrometry, and molecular dynamics simulations provide insight into the dynamic movement and molecular consequences of the W315L clinical variant. Collectively, this ensemble of structures outlines a framework for studying Twinkle function in mitochondrial DNA replication and associated disease states.
    Keywords:  Twinkle helicase; cryo-electron microscopy; mitochondrial DNA; mitochondrial DNA replication; progressive external ophthalmoplegia
    DOI:  https://doi.org/10.1073/pnas.2207459119
  5. EMBO J. 2022 Aug 01. e111834
    The mystery of mitochondrial plasticity: TMBIM5 integrates metabolic state and proteostasis.
    Mindong Ren, Michael Schlame.
      Recent work identifies TMBIM5 as inner mitochondrial membrane Ca2+ /H+ exchanger, linking hyperpolarisation regulation to proteome control and energy metabolism.
    DOI:  https://doi.org/10.15252/embj.2022111834
  6. Mol Genet Metab. 2022 Jul 06. pii: S1096-7192(22)00363-8. [Epub ahead of print]137(1-2): 40-48
    Mitochondrial DNA maintenance defects: potential therapeutic strategies.
    Mohammed Almannai, Ayman W El-Hattab, Mahshid S Azamian, May Ali, Fernando Scaglia.
      Mitochondrial DNA (mtDNA) replication depends on the mitochondrial import of hundreds of nuclear encoded proteins that control the mitochondrial genome maintenance and integrity. Defects in these processes result in an expanding group of disorders called mtDNA maintenance defects that are characterized by mtDNA depletion and/or multiple mtDNA deletions with variable phenotypic manifestations. As it applies for mitochondrial disorders in general, current treatment options for mtDNA maintenance defects are limited. Lately, with the development of model organisms, improved understanding of the pathophysiology of these disorders, and a better knowledge of their natural history, the number of preclinical studies and existing and planned clinical trials has been increasing. In this review, we discuss recent preclinical studies and current and future clinical trials concerning potential therapeutic options for the different mtDNA maintenance defects.
    Keywords:  Clinical trials; Mitochondria; Nucleoside bypass therapy; Preclinical studies; mtDNA depletion; mtDNA replication
    DOI:  https://doi.org/10.1016/j.ymgme.2022.07.003
  7. Front Mol Neurosci. 2022 ;15 920221
    Multi-mtDNA Variants May Be a Factor Contributing to Mitochondrial Function Variety in the Skin-Derived Fibroblasts of Leber's Hereditary Optic Neuropathy Patients.
    Shun Yao, Qingru Zhou, Mingzhu Yang, Ya Li, Xiuxiu Jin, Qingge Guo, Lin Yang, Fangyuan Qin, Bo Lei.
      Heterogeneity is a major feature of Leber's hereditary optic neuropathy (LHON) and has a significant impact on the manifestation and diagnosis of the disease. This study explored whether multiple variations in mitochondrial genes were associated with the heterogeneity, mainly phenotypic heterogeneity. Ophthalmic examinations were conducted in two probands with LHON with G11778A and multiple mitochondrial DNA gene (mtDNA) variants. Skin fibroblast cell lines were generated from patients and age- and sex-matched controls. ROS levels, mitochondrial membrane potential, cell energy respiration, and metabolic functions were measured. Flow cytometry and cell viability tests were performed to evaluate the cell apoptosis levels and fate. We found that cells with more mtDNA variants had higher ROS levels, lower mitochondrial membrane potential, and weaker respiratory function. Flow cytometry and cell viability testing showed that multiple mtDNA variants are associated with different levels of cell viability and apoptosis. In conclusion, we found that skin-derived fibroblast cells from G11778A LHON patients could be used as models for LHON research. Multi-mtDNA variants contribute to mitochondrial function variety, which may be associated with heterogeneity in patients with LHON.
    Keywords:  Leber's hereditary optic neuropathy (LHON); cell viability; heterogeneity; mitochondria dysfunction; mtDNA variants
    DOI:  https://doi.org/10.3389/fnmol.2022.920221
  8. Mol Med. 2022 Aug 03. 28(1): 90
    Rapamycin rescues mitochondrial dysfunction in cells carrying the m.8344A > G mutation in the mitochondrial tRNALys.
    Mariantonietta Capristo, Valentina Del Dotto, Concetta Valentina Tropeano, Claudio Fiorini, Leonardo Caporali, Chiara La Morgia, Maria Lucia Valentino, Monica Montopoli, Valerio Carelli, Alessandra Maresca.
       BACKGROUND: Myoclonus, Epilepsy and Ragged-Red-Fibers (MERRF) is a mitochondrial encephalomyopathy due to heteroplasmic mutations in mitochondrial DNA (mtDNA) most frequently affecting the tRNALys gene at position m.8344A > G. Defective tRNALys severely impairs mitochondrial protein synthesis and respiratory chain when a high percentage of mutant heteroplasmy crosses the threshold for full-blown clinical phenotype. Therapy is currently limited to symptomatic management of myoclonic epilepsy, and supportive measures to counteract muscle weakness with co-factors/supplements.
    METHODS: We tested two therapeutic strategies to rescue mitochondrial function in cybrids and fibroblasts carrying different loads of the m.8344A > G mutation. The first strategy was aimed at inducing mitochondrial biogenesis directly, over-expressing the master regulator PGC-1α, or indirectly, through the treatment with nicotinic acid, a NAD+ precursor. The second was aimed at stimulating the removal of damaged mitochondria through prolonged rapamycin treatment.
    RESULTS: The first approach slightly increased mitochondrial protein expression and respiration in the wild type and intermediate-mutation load cells, but was ineffective in high-mutation load cell lines. This suggests that induction of mitochondrial biogenesis may not be sufficient to rescue mitochondrial dysfunction in MERRF cells with high-mutation load. The second approach, when administered chronically (4 weeks), induced a slight increase of mitochondrial respiration in fibroblasts with high-mutation load, and a significant improvement in fibroblasts with intermediate-mutation load, rescuing completely the bioenergetics defect. This effect was mediated by increased mitochondrial biogenesis, possibly related to the rapamycin-induced inhibition of the Mechanistic Target of Rapamycin Complex 1 (mTORC1) and the consequent activation of the Transcription Factor EB (TFEB).
    CONCLUSIONS: Overall, our results point to rapamycin-based therapy as a promising therapeutic option for MERRF.
    Keywords:  MERRF; Mitochondrial DNA; Mitochondrial biogenesis; Mitochondrial dysfunction; Niacin; PGC-1α; Rapamycin; mTORC1
    DOI:  https://doi.org/10.1186/s10020-022-00519-z
  9. Cell Mol Life Sci. 2022 Aug 02. 79(8): 463
    Balancing NAD+ deficits with nicotinamide riboside: therapeutic possibilities and limitations.
    Angelique Cercillieux, Eleonora Ciarlo, Carles Canto.
      Alterations in cellular nicotinamide adenine dinucleotide (NAD+) levels have been observed in multiple lifestyle and age-related medical conditions. This has led to the hypothesis that dietary supplementation with NAD+ precursors, or vitamin B3s, could exert health benefits. Among the different molecules that can act as NAD+ precursors, Nicotinamide Riboside (NR) has gained most attention due to its success in alleviating and treating disease conditions at the pre-clinical level. However, the clinical outcomes for NR supplementation strategies have not yet met the expectations generated in mouse models. In this review we aim to provide a comprehensive view on NAD+ biology, what causes NAD+ deficits and the journey of NR from its discovery to its clinical development. We also discuss what are the current limitations in NR-based therapies and potential ways to overcome them. Overall, this review will not only provide tools to understand NAD+ biology and assess its changes in disease situations, but also to decide which NAD+ precursor could have the best therapeutic potential.
    Keywords:  Metabolic disease; NAD+; Nicotinamide; Nicotinamide riboside; Vitamin B3
    DOI:  https://doi.org/10.1007/s00018-022-04499-5
  10. Mol Cell. 2022 Aug 04. pii: S1097-2765(22)00662-1. [Epub ahead of print]82(15): 2735-2737
    Systems biochemistry to "deorphanize" human mitochondrial proteome.
    Francois Miros, Ran Liu, Hongying Shen.
      Rensvold, Shishkova, et al. (2022) apply an integrated systems biology approach spanning proteomics, lipidomics, and metabolomics to a collection of CRISPR knockout cells targeting 116 distinct human mitochondrial proteins, revealing new mitochondrial biology and guiding orphan disease diagnosis.
    DOI:  https://doi.org/10.1016/j.molcel.2022.07.005
  11. Neurobiol Dis. 2022 Jul 27. pii: S0969-9961(22)00224-8. [Epub ahead of print] 105832
    Altered SYNJ2BP-mediated mitochondrial-ER contacts in motor neuron disease.
    Naemeh Pourshafie, Ester Masati, Amber Lopez, Eric Bunker, Allison Snyder, Nancy A Edwards, Audrey M Winkelsas, Kenneth H Fischbeck, Christopher Grunseich.
      Synaptojanin 2 binding protein (SYNJ2BP) is an outer mitochondrial membrane protein with a cytosolic PDZ domain that functions as a cellular signaling hub. Few studies have evaluated its role in disease. Here we use induced pluripotent stem cell (iPSC)-derived motor neurons and post-mortem tissue from patients with two hereditary motor neuron diseases, spinal and bulbar muscular atrophy (SBMA) and amyotrophic lateral sclerosis type 4 (ALS4), and show that SYNJ2BP expression is increased in diseased motor neurons. Similarly, we show that SYNJ2BP expression increases in iPSC-derived motor neurons undergoing stress. Using proteomic analysis, we found that elevated SYNJ2BP alters the cellular distribution of mitochondria and increases mitochondrial-ER membrane contact sites. Furthermore, decreasing SYNJ2BP levels improves mitochondrial oxidative function in the diseased motor neurons. Together, our observations offer new insight into the molecular pathology of motor neuron disease and the role of SYNJ2BP in mitochondrial dysfunction.
    Keywords:  Motor neuron disease; Polyglutamine disease; Spinal and bulbar muscular atrophy
    DOI:  https://doi.org/10.1016/j.nbd.2022.105832
  12. Neuromuscul Disord. 2022 Jul 16. pii: S0960-8966(22)00601-0. [Epub ahead of print]
    Metrics of progression and prognosis in untreated adults with thymidine kinase 2 deficiency: An observational study.
    Cristina Domínguez-González, Ana Hernández-Voth, Carlos Pablo de Fuenmayor-Fernández de la Hoz, Laura Bermejo Guerrero, Germán Morís, Jorge García-García, Nuria Muelas, Juan Carlos León Hernández, Maria Rabasa, David Lora, Alberto Blázquez, Joaquín Arenas, Miguel Ángel Martin.
      This historical cohort study evaluated clinical characteristics of progression and prognosis in adults with thymidine kinase 2 deficiency (TK2d). Records were available for 17 untreated adults with TK2d (mean age of onset, 32 years), including longitudinal data from 6 patients (mean follow-up duration, 26.5 months). Pearson's correlation assessed associations between standard motor and respiratory assessments, clinical characteristics, and laboratory values. Longitudinal data were assessed by linear regression mixed models. Respiratory involvement progressed at an annual rate of 8.16% decrement in forced vital capacity (FVC). Most patients under noninvasive ventilation (NIV) remained ambulant (12/14, 86%), reduced FVC was not associated with concomitant decline in 6-minute walk test (6MWT), and 6MWT results were not correlated with FVC. Disease severity, assessed by age at NIV onset, correlated most strongly at diagnosis with: creatinine levels (r = 0.8036; P = 0.0009), followed by FVC (r = 0.7265; P = 0.0033), mtDNA levels in muscle (r = 0.7933; P = 0.0188), and age at disease onset (r = 0.7128; P = 0.0042). This population of adults with TK2d demonstrates rapid deterioration of respiratory muscles, which progresses independently of motor impairment. The results support FVC at diagnosis, mtDNA levels in muscle, and age at disease onset as prognostic indicators. Creatinine levels may also be potentially prognostic, as previously reported in other neuromuscular disorders.
    Keywords:  Clinical trials (observational study); Creatinine; Mitochondrial disease; Thymidine kinase 2 deficiency
    DOI:  https://doi.org/10.1016/j.nmd.2022.07.399
  13. Life Sci Alliance. 2022 Dec;pii: e202101284. [Epub ahead of print]5(12):
    Novel DNM1L variants impair mitochondrial dynamics through divergent mechanisms.
    Kelsey A Nolden, John M Egner, Jack J Collier, Oliver M Russell, Charlotte L Alston, Megan C Harwig, Michael E Widlansky, Souphatta Sasorith, Inês A Barbosa, Andrew Gl Douglas, Julia Baptista, Mark Walker, Deirdre E Donnelly, Andrew A Morris, Hui Jeen Tan, Manju A Kurian, Kathleen Gorman, Santosh Mordekar, Charu Deshpande, Rajib Samanta, Robert McFarland, R Blake Hill, Robert W Taylor, Monika Oláhová.
      Imbalances in mitochondrial and peroxisomal dynamics are associated with a spectrum of human neurological disorders. Mitochondrial and peroxisomal fission both involve dynamin-related protein 1 (DRP1) oligomerisation and membrane constriction, although the precise biophysical mechanisms by which distinct DRP1 variants affect the assembly and activity of different DRP1 domains remains largely unexplored. We analysed four unreported de novo heterozygous variants in the dynamin-1-like gene DNM1L, affecting different highly conserved DRP1 domains, leading to developmental delay, seizures, hypotonia, and/or rare cardiac complications in infancy. Single-nucleotide DRP1 stalk domain variants were found to correlate with more severe clinical phenotypes, with in vitro recombinant human DRP1 mutants demonstrating greater impairments in protein oligomerisation, DRP1-peroxisomal recruitment, and both mitochondrial and peroxisomal hyperfusion compared to GTPase or GTPase-effector domain variants. Importantly, we identified a novel mechanism of pathogenesis, where a p.Arg710Gly variant uncouples DRP1 assembly from assembly-stimulated GTP hydrolysis, providing mechanistic insight into how assembly-state information is transmitted to the GTPase domain. Together, these data reveal that discrete, pathological DNM1L variants impair mitochondrial network maintenance by divergent mechanisms.
    DOI:  https://doi.org/10.26508/lsa.202101284
  14. Hum Mol Genet. 2022 Aug 01. pii: ddac173. [Epub ahead of print]
    Repeat expansions nested within tandem CNVs: A unique structural change in GLS exemplifies the diagnostic challenges of non-coding pathogenic variation.
    Sarah Fazal, Matt C Danzi, André B P van Kuilenburg, Selina Reich, Andreas Traschütz, Benjamin Bender, René Leen, Camilo Toro, Karen Usdin, Bruce Hayward, David R Adams, Clara D M van Karnebeek, Carlos R Ferreira, Precilla D'Sousa, Undiagnosed Diseases Network, Mustafa Tekin, Stephan Züchner, Matthis Synofzik.
      Glutaminase deficiency has recently been associated with ataxia and developmental delay due to repeat expansions in the 5'UTR of the glutaminase (GLS) gene. Patients with the described GLS repeat expansion may indeed remain undiagnosed due to the rarity of this variant, the challenge of its detection, and the recency of its discovery. In this study, we combined advanced bioinformatics screening of ~ 3000 genomes and ~ 1500 exomes with optical genome mapping and long-read-sequencing for confirmation studies. We identified two GLS families, previously intensely and unsuccessfully analyzed. One family carries an unusual and complex structural change involving a homozygous repeat expansion nested within a quadruplication event in the 5'UTR of GLS. Glutaminase deficiency and its metabolic consequences were validated by in-depth biochemical analysis. The identified GLS patients showed progressive early-onset ataxia, cognitive deficits, pyramidal tract damage, and optic atrophy, thus demonstrating susceptibility of several specific neuron populations to glutaminase deficiency. This large-scale screening study demonstrates the ability of bioinformatics analysis-validated by latest state-of-the-art technologies (optical genome mapping, long-read-sequencing)-to effectively flag complex repeat expansions using short read datasets and thus facilitate diagnosis of ultra-rare disorders.
    DOI:  https://doi.org/10.1093/hmg/ddac173
  15. Transl Vis Sci Technol. 2022 Aug 01. 11(8): 5
    Noninvasive Ophthalmic Imaging Measures Retinal Degeneration and Vision Deficits in Ndufs4-/- Mouse Model of Mitochondrial Complex I Deficiency.
    Maria I Avrutsky, Jacqueline M Lawson, Jade E Smart, Claire W Chen, Carol M Troy.
       Purpose: To characterize postnatal ocular pathology in a Ndufs4-/- mouse model of complex I deficiency using noninvasive retinal imaging and visual testing.
    Methods: Ndufs4-/- mice and wild-type (WT) littermates were analyzed at 3, 5, and 7 weeks postnatal. Retinal morphology was visualized by optical coherence tomography (OCT). OCT images were analyzed for changes in retinal thickness and reflectivity profiles. Visual function was assessed by electroretinogram (ERG) and optomotor reflex (OMR).
    Results: Ndufs4-/- animals have normal OCT morphology at weaning and develop inner plexiform layer atrophy over weeks 5 to 7. Outer retinal layers show hyporeflectivity of the external limiting membrane (ELM) and photoreceptor ellipsoid zone (EZ). Retinal function is impaired at 3 weeks, with profound deficits in b-wave, a-wave, and oscillatory potential amplitudes. The b-wave and oscillatory potential implicit times are delayed, but the a-wave implicit time is unaffected. Ndufs4-/- animals have normal OMR at 3 weeks and present with increasing acuity and contrast OMR deficits at 5 and 7 weeks. Physiological thinning of inner retinal layers, attenuation of ELM reflectivity, and attenuation of ERG b- and a-wave amplitudes occur in WT C57BL/6 littermates between weeks 3 and 7.
    Conclusions: Noninvasive ocular imaging captures early-onset retinal degeneration in Ndufs4-/- mice and is a tractable approach for investigating retinal pathology subsequent to complex I deficiency.
    Translational Relevance: Ophthalmic imaging captures clinically relevant measures of retinal disease in a fast-progressing mouse model of complex I deficiency consistent with human Leigh syndrome.
    DOI:  https://doi.org/10.1167/tvst.11.8.5
  16. Free Radic Biol Med. 2022 Jul 30. pii: S0891-5849(22)00496-8. [Epub ahead of print]
    Nrf2 as a regulator of mitochondrial function: Energy metabolism and beyond.
    Noemí Esteras, Andrey Y Abramov.
      Mitochondria are unique and essential organelles that mediate many vital cellular processes including energy metabolism and cell death. The transcription factor Nrf2 (NF-E2 p45-related factor 2) has emerged in the last few years as an important modulator of multiple aspects of mitochondrial function. Well-known for controlling cellular redox homeostasis, the cytoprotective effects of Nrf2 extend beyond its ability to regulate a diverse network of antioxidant and detoxification enzymes. Here, we review the role of Nrf2 in the regulation of mitochondrial function and structure. We focus on Nrf2 involvement in promoting mitochondrial quality control and regulation of basic aspects of mitochondrial function, including energy production, reactive oxygen species generation, calcium signalling, and cell death induction. Given the importance of mitochondria in the development of multiple diseases, these findings reinforce the pharmacological activation of Nrf2 as an attractive strategy to counteract mitochondrial dysfunction.
    Keywords:  Calcium; Dynamics; Energy; Fission; Fusion; Mitochondria; Mitochondrial biogenesis; Mitophagy; Nrf2; ROS; mPTP
    DOI:  https://doi.org/10.1016/j.freeradbiomed.2022.07.013
  17. Nature. 2022 Aug 03.
    Transcriptome variation in human tissues revealed by long-read sequencing.
    Dafni A Glinos, Garrett Garborcauskas, Paul Hoffman, Nava Ehsan, Lihua Jiang, Alper Gokden, Xiaoguang Dai, François Aguet, Kathleen L Brown, Kiran Garimella, Tera Bowers, Maura Costello, Kristin Ardlie, Ruiqi Jian, Nathan R Tucker, Patrick T Ellinor, Eoghan D Harrington, Hua Tang, Michael Snyder, Sissel Juul, Pejman Mohammadi, Daniel G MacArthur, Tuuli Lappalainen, Beryl B Cummings.
      Regulation of transcript structure generates transcript diversity and plays an important role in human disease1-7. The advent of long-read sequencing technologies offers the opportunity to study the role of genetic variation in transcript structure8-16. In this Article, we present a large human long-read RNA-seq dataset using the Oxford Nanopore Technologies platform from 88 samples from Genotype-Tissue Expression (GTEx) tissues and cell lines, complementing the GTEx resource. We identified just over 70,000 novel transcripts for annotated genes, and validated the protein expression of 10% of novel transcripts. We developed a new computational package, LORALS, to analyse the genetic effects of rare and common variants on the transcriptome by allele-specific analysis of long reads. We characterized allele-specific expression and transcript structure events, providing new insights into the specific transcript alterations caused by common and rare genetic variants and highlighting the resolution gained from long-read data. We were able to perturb the transcript structure upon knockdown of PTBP1, an RNA binding protein that mediates splicing, thereby finding genetic regulatory effects that are modified by the cellular environment. Finally, we used this dataset to enhance variant interpretation and study rare variants leading to aberrant splicing patterns.
    DOI:  https://doi.org/10.1038/s41586-022-05035-y
  18. Trends Genet. 2022 Jul 28. pii: S0168-9525(22)00178-0. [Epub ahead of print]
    Neuromuscular disorders: finding the missing genetic diagnoses.
    Katherine E Koczwara, Nicole J Lake, Alec M DeSimone, Monkol Lek.
      Neuromuscular disorders (NMDs) are a wide-ranging group of diseases that seriously affect the quality of life of affected individuals. The development of next-generation sequencing revolutionized the diagnosis of NMD, enabling the discovery of hundreds of NMD genes and many more pathogenic variants. However, the diagnostic yield of genetic testing in NMD cohorts remains incomplete, indicating a large number of genetic diagnoses are not identified through current methods. Fortunately, recent advancements in sequencing technologies, analytical tools, and high-throughput functional screening provide an opportunity to circumvent current challenges. Here, we discuss reasons for missing genetic diagnoses in NMD, how emerging technologies and tools can overcome these hurdles, and examine future approaches to improving diagnostic yields in NMD.
    Keywords:  genetic diagnosis; neuromuscular disorders; next-generation sequencing
    DOI:  https://doi.org/10.1016/j.tig.2022.07.001
  19. Elife. 2022 08 01. pii: e75531. [Epub ahead of print]11
    Structure-activity relationships of mitochondria-targeted tetrapeptide pharmacological compounds.
    Wayne Mitchell, Jeffrey D Tamucci, Emery L Ng, Shaoyi Liu, Alexander V Birk, Hazel H Szeto, Eric R May, Andrei T Alexandrescu, Nathan N Alder.
      Mitochondria play a central role in metabolic homeostasis, and dysfunction of this organelle underpins the etiology of many heritable and aging-related diseases. Tetrapeptides with alternating cationic and aromatic residues such as SS-31 (elamipretide) show promise as therapeutic compounds for mitochondrial disorders. In this study, we conducted a quantitative structure-activity analysis of three alternative tetrapeptide analogs, benchmarked against SS-31, that differ with respect to aromatic side chain composition and sequence register. We present the first structural models for this class of compounds, obtained with Nuclear Magnetic Resonance (NMR) and molecular dynamics approaches, showing that all analogs except for SS-31 form compact reverse turn conformations in the membrane-bound state. All peptide analogs bound cardiolipin-containing membranes, yet they had significant differences in equilibrium binding behavior and membrane interactions. Notably, analogs had markedly different effects on membrane surface charge, supporting a mechanism in which modulation of membrane electrostatics is a key feature of their mechanism of action. The peptides had no strict requirement for side chain composition or sequence register to permeate cells and target mitochondria in mammalian cell culture assays. All four peptides were pharmacologically active in serum withdrawal cell stress models yet showed significant differences in their abilities to restore mitochondrial membrane potential, preserve ATP content, and promote cell survival. Within our peptide set, the analog containing tryptophan side chains, SPN10, had the strongest impact on most membrane properties and showed greatest efficacy in cell culture studies. Taken together, these results show that side chain composition and register influence the activity of these mitochondria-targeted peptides, helping provide a framework for the rational design of next-generation therapeutics with enhanced potency.
    Keywords:  NMR structure; S. cerevisiae; biochemistry; cardiolipin; chemical biology; human; medicine; membrane interactions; mitochondria; peptide therapeutics; structure-activity relationship
    DOI:  https://doi.org/10.7554/eLife.75531
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