bims-mitdis Biomed News
on Mitochondrial disorders
Issue of 2022–02–13
24 papers selected by
Catalina Vasilescu, University of Helsinki
  1. In vivo mitochondrial base editing via adeno-associated viral delivery to mouse post-mitotic tissue.
  2. Deleterious variants in CRLS1 lead to cardiolipin deficiency and cause an autosomal recessive multi-system mitochondrial disease.
  3. The fate of damaged mitochondrial DNA in the cell.
  4. Protocol to characterize mitochondrial supercomplexes from mouse tissues by combining BN-PAGE and MS-based proteomics.
  5. DNAJC30 defect: a frequent cause of recessive Leber hereditary optic neuropathy and Leigh syndrome.
  6. Imaging Mitochondrial Ca2+ Uptake in Astrocytes and Neurons using Genetically Encoded Ca2+ Indicators (GECIs).
  7. Spatial and temporal control of mitochondrial H2 O2 release in intact human cells.
  8. Biallelic Loss-of-Function NDUFA12 Variants Cause a Wide Phenotypic Spectrum from Leigh/Leigh-Like Syndrome to Isolated Optic Atrophy.
  9. Visualization of Dynamic Mitochondrial Calcium Fluxes in Isolated Cardiomyocytes.
  10. Mitochondrial and metabolic dysfunction in ageing and age-related diseases.
  11. DECIPHER: Supporting the interpretation and sharing of rare disease phenotype-linked variant data to advance diagnosis and research.
  12. New insights into mitochondrial-nuclear interactions revealed through analysis of small RNAs.
  13. NDUFA1 p.Gly32Arg variant in early-onset dementia.
  14. Intermitochondrial signaling regulates the uniform distribution of stationary mitochondria in axons.
  15. Widespread Mislocalization of FUS Is Associated With Mitochondrial Abnormalities in Skeletal Muscle in Amyotrophic Lateral Sclerosis With FUS Mutations.
  16. Deriving waveform parameters from calcium transients in human iPSC-derived cardiomyocytes to predict cardiac activity with machine learning.
  17. The coupling mechanism of mammalian mitochondrial complex I.
  18. Machine Learning Models for Accurate Prioritization of Variants of Uncertain Significance.
  19. Cryo-EM structure of hexameric yeast Lon protease (PIM1) highlights the importance of conserved structural elements.
  20. Whole-cell modeling in yeast predicts compartment-specific proteome constraints that drive metabolic strategies.
  21. Curated variation benchmarks for challenging medically relevant autosomal genes.
  22. NCAM1 and GDF15 are biomarkers of Charcot-Marie-Tooth disease in patients and mice.
  23. Isolation and culture of neural stem cells from adult mouse subventricular zone for genetic and pharmacological treatments with proliferation analysis.
  24. Caloric restriction has a new player.
  1. Nat Commun. 2022 Feb 08. 13(1): 750
    In vivo mitochondrial base editing via adeno-associated viral delivery to mouse post-mitotic tissue.
    Pedro Silva-Pinheiro, Pavel A Nash, Lindsey Van Haute, Christian D Mutti, Keira Turner, Michal Minczuk.
      Mitochondria host key metabolic processes vital for cellular energy provision and are central to cell fate decisions. They are subjected to unique genetic control by both nuclear DNA and their own multi-copy genome - mitochondrial DNA (mtDNA). Mutations in mtDNA often lead to clinically heterogeneous, maternally inherited diseases that display different organ-specific presentation at any stage of life. For a long time, genetic manipulation of mammalian mtDNA has posed a major challenge, impeding our ability to understand the basic mitochondrial biology and mechanisms underpinning mitochondrial disease. However, an important new tool for mtDNA mutagenesis has emerged recently, namely double-stranded DNA deaminase (DddA)-derived cytosine base editor (DdCBE). Here, we test this emerging tool for in vivo use, by delivering DdCBEs into mouse heart using adeno-associated virus (AAV) vectors and show that it can install desired mtDNA edits in adult and neonatal mice. This work provides proof-of-concept for use of DdCBEs to mutagenize mtDNA in vivo in post-mitotic tissues and provides crucial insights into potential translation to human somatic gene correction therapies to treat primary mitochondrial disease phenotypes.
    DOI:  https://doi.org/10.1038/s41467-022-28358-w
  2. Hum Mol Genet. 2022 Feb 11. pii: ddac040. [Epub ahead of print]
    Deleterious variants in CRLS1 lead to cardiolipin deficiency and cause an autosomal recessive multi-system mitochondrial disease.
    Care4Rare Canada Consortium
      Mitochondrial diseases are a group of inherited diseases with highly varied and complex clinical presentations. Here, we report four individuals, including two siblings, affected by a progressive mitochondrial encephalopathy with biallelic variants in the cardiolipin biosynthesis gene CRLS1. Three affected individuals had a similar infantile presentation comprising progressive encephalopathy, bull's eye maculopathy, auditory neuropathy, diabetes insipidus, autonomic instability, cardiac defects and early death. The fourth affected individual presented with chronic encephalopathy with neurodevelopmental regression, congenital nystagmus with decreased vision, sensorineural hearing loss, failure to thrive and acquired microcephaly. Using patient-derived fibroblasts, we characterised cardiolipin synthase 1 (CRLS1) dysfunction that impaired mitochondrial morphology and biogenesis, providing functional evidence that the CRLS1 variants cause a mitochondrial phenotype. Lipid profiling in fibroblasts from two patients further confirmed the functional defect demonstrating reduced cardiolipin levels, altered acyl-chain composition and significantly increased levels of phosphatidylglycerol, the substrate of CRLS1. Proteomic profiling of patient cells and mouse Crls1 knockout cell lines identified both endoplasmic reticular and mitochondrial stress responses, and key features that distinguish between varying degrees of cardiolipin insufficiency. These findings support that deleterious variants in CRLS1 cause an autosomal recessive mitochondrial disease, presenting as a severe encephalopathy with multisystemic involvement. Furthermore, we identify key signatures in cardiolipin and proteome profiles across various degrees of cardiolipin loss, facilitating the use of omics technologies to guide a diagnosis for this mitochondrial disease.
    DOI:  https://doi.org/10.1093/hmg/ddac040
  3. Biochim Biophys Acta Mol Cell Res. 2022 Feb 04. pii: S0167-4889(22)00024-6. [Epub ahead of print] 119233
    The fate of damaged mitochondrial DNA in the cell.
    Siyang Liao, Li Chen, Zhiyin Song, He He.
      Mitochondrion is a double membrane organelle that is responsible for cellular respiration and production of most of the ATP in eukaryotic cells. Mitochondrial DNA (mtDNA) is the genetic material carried by mitochondria, which encodes some essential subunits of respiratory complexes independent of nuclear DNA. Normally, mtDNA binds to certain proteins to form a nucleoid that is stable in mitochondria. Nevertheless, a variety of physiological or pathological stresses can cause mtDNA damage, and the accumulation of damaged mtDNA in mitochondria leads to mitochondrial dysfunction, which triggers the occurrence of mitochondrial diseases in vivo. In response to mtDNA damage, cell initiates multiple pathways including mtDNA repair, degradation, clearance and release, to recover mtDNA, and maintain mitochondrial quality and cell homeostasis. In this review, we provide our current understanding of the fate of damaged mtDNA, focus on the pathways and mechanisms of removing damaged mtDNA in the cell.
    Keywords:  Mitochondria DNA (mtDNA); Mitocytosis; Mitophagy; mtDNA release
    DOI:  https://doi.org/10.1016/j.bbamcr.2022.119233
  4. STAR Protoc. 2022 Mar 18. 3(1): 101135
    Protocol to characterize mitochondrial supercomplexes from mouse tissues by combining BN-PAGE and MS-based proteomics.
    Roger Moreno-Justicia, Alba Gonzalez-Franquesa, Ben Stocks, Atul S Deshmukh.
      The assembly of mitochondrial respiratory complexes into supercomplexes has significant implications for mitochondrial function. This protocol details mitochondrial isolation from mouse tissues and the use of blue native gel electrophoresis (BN-PAGE) to separate pre-identified mitochondrial supercomplexes into different gel bands. We then describe the excision of the individual bands, followed by in-gel protein digestion and peptide desalting for mass spectrometry (MS)-based proteomics. This protocol provides a time-efficient measurement of the abundance and distribution of proteins within known supercomplexes. For complete details on the use and execution of this profile, please refer to Gonzalez-Franquesa et al. (2021).
    Keywords:  Mass Spectrometry; Metabolism; Protein Biochemistry; Proteomics
    DOI:  https://doi.org/10.1016/j.xpro.2022.101135
  5. Brain. 2022 Feb 10. pii: awac052. [Epub ahead of print]
    DNAJC30 defect: a frequent cause of recessive Leber hereditary optic neuropathy and Leigh syndrome.
    Sarah L Stenton, Marketa Tesarova, Natalia L Sheremet, Claudia Catarino, Valerio Carelli, Elżbieta Ciara, Kathryn Curry, Martin Engvall, Leah R Fleming, Peter Freisinger, Katarzyna Iwanicka-Pronicka, Elżbieta Jurkiewicz, Thomas Klopstock, Mary K Koenig, Hana Kolářová, Bohdan Kousal, Tatiana Krylova, Chiara La Morgia, Lenka Nosková, Dorota Piekutowska-Abramczuk, Sam N Russo, Viktor Stránecký, Iveta Tóthová, Frank Träisk, Holger Prokisch.
      The recent description of biallelic DNAJC30 variants in Leber hereditary optic neuropathy (LHON) and Leigh syndrome (LS) challenged the longstanding assumption for LHON to be exclusively maternally inherited and broadened the genetic spectrum of LS, the most frequent paediatric mitochondrial disease. Herein, we characterise 28 so far unreported individuals from 26 families carrying a homozygous DNAJC30 p.Tyr51Cys founder variant, 24 manifesting with LHON, two manifesting with LS, and two remaining asymptomatic. This collection of unreported variant carriers confirms sex-dependent incomplete penetrance of the homozygous variant given a significant male predominance of disease and the report of asymptomatic homozygous variant carriers. The autosomal recessive LHON (arLHON) patients demonstrate an earlier age of disease onset and a higher rate of idebenone-treated and spontaneous recovery of vision in comparison to reported figures for maternally inherited disease (mtLHON). Moreover, the report of two additional patients with childhood- or adult-onset LS further evidences the association of DNAJC30 with LS, previously only reported in a single childhood-onset case.
    Keywords:  DNAJC30; LHON; Leigh syndrome; mitochondrial disease
    DOI:  https://doi.org/10.1093/brain/awac052
  6. J Vis Exp. 2022 Jan 22.
    Imaging Mitochondrial Ca2+ Uptake in Astrocytes and Neurons using Genetically Encoded Ca2+ Indicators (GECIs).
    Nannan Zhang, Zhe Zhang, Ilker Ozden, Shinghua Ding.
      Mitochondrial Ca2+ plays a critical role in controlling cytosolic Ca2+ buffering, energy metabolism, and cellular signal transduction. Overloading of mitochondrial Ca2+ contributes to various pathological conditions, including neurodegeneration and apoptotic cell death in neurological diseases. Here we present a cell-type specific and mitochondria targeting molecular approach for mitochondrial Ca2+ imaging in astrocytes and neurons in vitro and in vivo. We constructed DNA plasmids encoding mitochondria-targeting genetically encoded Ca2+ indicators (GECIs) GCaMP5G or GCaMP6s (GCaMP5G/6s) with astrocyte- and neuron-specific promoters gfaABC1D and CaMKII and mitochondria-targeting sequence (mito-). For in vitro mitochondrial Ca2+ imaging, the plasmids were transfected in cultured astrocytes and neurons to express GCaMP5G/6s. For in vivo mitochondrial Ca2+ imaging, adeno-associated viral vectors (AAVs) were prepared and injected into the mouse brains to express GCaMP5G/6s in mitochondria in astrocytes and neurons. Our approach provides a useful means to image mitochondrial Ca2+ dynamics in astrocytes and neurons to study the relationship between cytosolic and mitochondrial Ca2+ signaling, as well as astrocyte-neuron interactions.
    DOI:  https://doi.org/10.3791/62917
  7. EMBO J. 2022 Feb 11. e109169
    Spatial and temporal control of mitochondrial H2 O2 release in intact human cells.
    Michaela Nicole Hoehne, Lianne J H C Jacobs, Kim Jasmin Lapacz, Gaetano Calabrese, Lena Maria Murschall, Teresa Marker, Harshita Kaul, Aleksandra Trifunovic, Bruce Morgan, Mark Fricker, Vsevolod V Belousov, Jan Riemer.
      Hydrogen peroxide (H2 O2 ) has key signaling roles at physiological levels, while causing molecular damage at elevated concentrations. H2 O2 production by mitochondria is implicated in regulating processes inside and outside these organelles. However, it remains unclear whether and how mitochondria in intact cells release H2 O2 . Here, we employed a genetically encoded high-affinity H2 O2 sensor, HyPer7, in mammalian tissue culture cells to investigate different modes of mitochondrial H2 O2 release. We found substantial heterogeneity of HyPer7 dynamics between individual cells. We further observed mitochondria-released H2 O2 directly at the surface of the organelle and in the bulk cytosol, but not in the nucleus or at the plasma membrane, pointing to steep gradients emanating from mitochondria. Gradient formation is controlled by cytosolic peroxiredoxins, which act redundantly and with a substantial reserve capacity. Dynamic adaptation of cytosolic thioredoxin reductase levels during metabolic changes results in improved H2 O2 handling and explains previously observed differences between cell types. Our data suggest that H2 O2 -mediated signaling is initiated only in close proximity to mitochondria and under specific metabolic conditions.
    Keywords:  HyPer7; hydrogen peroxide release; mitochondria; peroxiredoxin
    DOI:  https://doi.org/10.15252/embj.2021109169
  8. Mov Disord Clin Pract. 2022 Feb;9(2): 218-228
    Biallelic Loss-of-Function NDUFA12 Variants Cause a Wide Phenotypic Spectrum from Leigh/Leigh-Like Syndrome to Isolated Optic Atrophy.
    Francesca Magrinelli, Elisa Cali, Vinícius Lopes Braga, Uluç Yis, Hoda Tomoum, Hanan Shamseldin, Julian Raiman, Christoph Kernstock, Flávio Moura Rezende Filho, Orlando Graziani Povoas Barsottini, Robert W Taylor, Elsebet Østergaard, Abdullah Tamim, Karin Schäferhoff, Juliana Maria Ferraz Sallum, Maha S Zaki, Fernando Kok, Kailash P Bhatia, Bernd Wissinger, Kate Sergeant, Tobias B Haack, Rita Horvath, Semra Hiz, Fowzan S Alkuraya, Henry Houlden, José Luiz Pedroso, Reza Maroofian.
       Background: Biallelic loss-of-function NDUFA12 variants have hitherto been linked to mitochondrial complex I deficiency presenting with heterogeneous clinical and radiological features in nine cases only.
    Objectives: To fully characterize, both phenotypically and genotypically, NDUFA12-related mitochondrial disease.
    Methods: We collected data from cases identified by screening genetic databases of several laboratories worldwide and systematically reviewed the literature.
    Results: Nine unreported NDUFA12 cases from six pedigrees were identified, with presentation ranging from movement disorder phenotypes (dystonia and/or spasticity) to isolated optic atrophy. MRI showed basal ganglia abnormalities (n = 6), optic atrophy (n = 2), or was unremarkable (n = 1). All carried homozygous truncating NDUFA12 variants, three of which are novel.
    Conclusions: Our case series expands phenotype-genotype correlations in NDUFA12-associated mitochondrial disease, providing evidence of intra- and inter-familial clinical heterogeneity for the same variant. It confirms NDUFA12 variants should be included in the diagnostic workup of Leigh/Leigh-like syndromes - particularly with dystonia - as well as isolated optic atrophy.
    Keywords:  Leigh syndrome; NDUFA12; dystonia; optic atrophy; phenotypic heterogeneity
    DOI:  https://doi.org/10.1002/mdc3.13398
  9. Front Physiol. 2021 ;12 808798
    Visualization of Dynamic Mitochondrial Calcium Fluxes in Isolated Cardiomyocytes.
    Anna Maria Krstic, Amelia Sally Power, Marie-Louise Ward.
       Background: Cardiomyocyte contraction requires a constant supply of ATP, which varies depending on work rate. Maintaining ATP supply is particularly important during excitation-contraction coupling, where cytosolic Ca2+ fluxes drive repeated cycles of contraction and relaxation. Ca2+ is one of the key regulators of ATP production, and its uptake into the mitochondrial matrix occurs via the mitochondrial calcium uniporter. Fluorescent indicators are commonly used for detecting cytosolic Ca2+ changes. However, visualizing mitochondrial Ca2+ fluxes using similar methods is more difficult, as the fluorophore must be permeable to both the sarcolemma and the inner mitochondrial membrane. Our aim was therefore to optimize a method using the fluorescent Ca2+ indicator Rhod-2 to visualize beat-to-beat mitochondrial calcium fluxes in rat cardiomyocytes.
    Methods: Healthy, adult male Wistar rat hearts were isolated and enzymatically digested to yield rod-shaped, quiescent ventricular cardiomyocytes. The fluorescent Ca2+ indicator Rhod-2 was reduced to di-hydroRhod-2 and confocal microscopy was used to validate mitochondrial compartmentalization. Cardiomyocytes were subjected to various pharmacological interventions, including caffeine and β-adrenergic stimulation. Upon confirmation of mitochondrial Rhod-2 localization, loaded myocytes were then super-fused with 1.5 mM Ca2+ Tyrodes containing 1 μM isoproterenol and 150 μM spermine. Myocytes were externally stimulated at 0.1, 0.5 and 1 Hz and whole cell recordings of both cytosolic ([Ca2+]cyto) and mitochondrial calcium ([Ca2+] mito ) transients were made.
    Results: Myocytes loaded with di-hydroRhod-2 revealed a distinct mitochondrial pattern when visualized by confocal microscopy. Application of 20 mM caffeine revealed no change in fluorescence, confirming no sarcoplasmic reticulum compartmentalization. Myocytes loaded with di-hydroRhod-2 also showed a large increase in fluorescence within the mitochondria in response to β-adrenergic stimulation (P < 0.05). Beat-to-beat mitochondrial Ca2+ transients were smaller in amplitude and had a slower time to peak and maximum rate of rise relative to cytosolic calcium transients at all stimulation frequencies (P < 0.001).
    Conclusion: Myocytes loaded with di-hydroRhod-2 revealed mitochondrial specific compartmentalization. Mitochondrial Ca2+ transients recorded from di-hydroRhod-2 loaded myocytes were distinct in comparison to the large and rapid Rhod-2 cytosolic transients, indicating different kinetics between [Ca2+]cyto and [Ca2+]mito transients. Overall, our results showed that di-hydroRhod-2 loading is a quick and suitable method for measuring beat-to-beat [Ca2+]mito transients in intact myocytes.
    Keywords:  calcium; cardiomyocytes; di-hydroRhod-2AM; excitation-contraction coupling; fluxes; mitochondria
    DOI:  https://doi.org/10.3389/fphys.2021.808798
  10. Nat Rev Endocrinol. 2022 Feb 10.
    Mitochondrial and metabolic dysfunction in ageing and age-related diseases.
    João A Amorim, Giuseppe Coppotelli, Anabela P Rolo, Carlos M Palmeira, Jaime M Ross, David A Sinclair.
      Organismal ageing is accompanied by progressive loss of cellular function and systemic deterioration of multiple tissues, leading to impaired function and increased vulnerability to death. Mitochondria have become recognized not merely as being energy suppliers but also as having an essential role in the development of diseases associated with ageing, such as neurodegenerative and cardiovascular diseases. A growing body of evidence suggests that ageing and age-related diseases are tightly related to an energy supply and demand imbalance, which might be alleviated by a variety of interventions, including physical activity and calorie restriction, as well as naturally occurring molecules targeting conserved longevity pathways. Here, we review key historical advances and progress from the past few years in our understanding of the role of mitochondria in ageing and age-related metabolic diseases. We also highlight emerging scientific innovations using mitochondria-targeted therapeutic approaches.
    DOI:  https://doi.org/10.1038/s41574-021-00626-7
  11. Hum Mutat. 2022 Feb 10.
    DECIPHER: Supporting the interpretation and sharing of rare disease phenotype-linked variant data to advance diagnosis and research.
    Julia Foreman, Simon Brent, Daniel Perrett, Andrew P Bevan, Sarah E Hunt, Fiona Cunningham, Matthew E Hurles, Helen V Firth.
      DECIPHER (https://www.deciphergenomics.org) is a free web platform for sharing anonymised phenotype-linked variant data from rare disease patients. Its dynamic interpretation interfaces contextualise genomic and phenotypic data to enable more informed variant interpretation, incorporating international standards for variant classification. DECIPHER supports almost all types of germline and mosaic variation in the nuclear and mitochondrial genome: sequence variants, short tandem repeats, copy-number variants and large structural variants. Patient phenotypes are deposited using Human Phenotype Ontology (HPO) terms, supplemented by quantitative data, which is aggregated to derive gene-specific phenotypic summaries. It hosts data from >250 projects from ~40 countries, openly sharing >40,000 patient records containing >51,000 variants and >172,000 phenotype terms. The rich phenotype-linked variant data in DECIPHER drives rare disease research and diagnosis by enabling patient matching within DECIPHER and with other resources, and has been cited in >2,600 publications. In this paper, we describe the types of data deposited to DECIPHER, the variant interpretation tools, and patient matching interfaces which make DECIPHER an invaluable rare disease resource. This article is protected by copyright. All rights reserved.
    Keywords:  Genetic Disorders; Genomic Medicine; Genotype Phenotype Correlation; MatchMaker Exchange; Rare Diseases; Variant Interpretation; Whole Exome sequencing; Whole Genome sequencing
    DOI:  https://doi.org/10.1002/humu.24340
  12. Genome Biol Evol. 2022 Feb 10. pii: evac023. [Epub ahead of print]
    New insights into mitochondrial-nuclear interactions revealed through analysis of small RNAs.
    Andrea Pozzi, Damian K Dowling.
      Mitochondrial sequence variants affect phenotypic function, often through interaction with the nuclear genome. These "mitonuclear" interactions have been linked both to evolutionary processes and human health. The study of these interactions has focused on mechanisms regulating communication between mitochondrial and nuclear proteins; the role of mitochondrial (mt) RNAs has received little attention. Here, we show that small mt-RNAs bind to the nuclear protein Argonaute 2, and that nuclear miRNAs bind to mt-mRNAs. We identify one small mt-RNA that binds to Argonaute 2 in human tissues whose expression and sequence remain unchanged across vertebrates. While analyses of CLEAR-CLIP sequencing datasets of human and mouse did not reveal consistent interactions between small mt-RNAs and nuclear mRNAs, we found that MT-ND4 and MT-ATP6 mRNAs are bound by different nuclear miRNAs in humans and mice. Our work homes in on previously unknown interactions between nuclear and small mt-RNAs, which may play key roles in intergenomic communication.
    Keywords:  AGO2; Mitonuclear communication; mitochondria; mtDNA; small RNAs
    DOI:  https://doi.org/10.1093/gbe/evac023
  13. Neurobiol Aging. 2022 Jan 21. pii: S0197-4580(22)00012-4. [Epub ahead of print]
    NDUFA1 p.Gly32Arg variant in early-onset dementia.
    Samuli Huttula, Henri Väyrynen, Seppo Helisalmi, Laura Kytövuori, Laura Luukkainen, Mikko Hiltunen, Anne M Remes, Johanna Krüger.
      Early-onset dementia (EOD) is highly heritable. However, in many EOD cases the genetic etiology remains unknown. Mitochondrial dysfunction is associated with neurodegeneration and the complex I (CI) deficiency is the most common enzyme deficiency in diseases related to oxidative phosphorylation. The X-chromosomal NDUFA1 gene is essential for the activity of CI. Mutations in NDUFA1 are associated with mitochondrial diseases especially with Leigh syndrome. CI deficiency is also associated with neurodegenerative diseases, such as Alzheimer's disease (AD). The aim of this study was to evaluate the role of NDUFA1 variants in EOD patients. Next-generation sequencing panel was used to screen NDUFA1 variants in a cohort of 37 EOD patients with a family history of dementia or an atypical or rapidly progressive course of disease. We identified a hemizygous p.Gly32Arg variant in two brothers with AD. Subsequent screening of the variant in a larger cohort of EOD patients (n = 279) revealed three additional variant carriers (one male and two heterozygote females), suggesting that NDUFA1 variant p.Gly32Arg may play a role in neurodegenerative dementia.
    Keywords:  Alzheimer's disease; Dementia; Mitochondria; NDUFA1; Neurodegeneration; OXPHOS
    DOI:  https://doi.org/10.1016/j.neurobiolaging.2021.09.026
  14. Mol Cell Neurosci. 2022 Feb 04. pii: S1044-7431(22)00010-0. [Epub ahead of print] 103704
    Intermitochondrial signaling regulates the uniform distribution of stationary mitochondria in axons.
    Nozomu Matsumoto, Ikuma Hori, Masashi K Kajita, Tomoya Murase, Wataru Nakamura, Takahiro Tsuji, Seiji Miyake, Masaru Inatani, Yoshiyuki Konishi.
      In the central nervous system (CNS), many neurons develop axonal arbors that are crucial for information processing. Previous studies have demonstrated that premature axons contain motile and stationary mitochondria, and their balance is important for axonal arborization. However, the mechanisms by which neurons determine the positions of stationary mitochondria as well as their turnover remain to be elucidated. We observed that the distribution of stationary mitochondrial spots along the unmyelinated and nonsynaptic axons is not random but rather relatively uniform both in primary cultured neurons and in tissues. Intriguingly, whereas the positions of each mitochondrial spot changed over time, the overall distribution remained uniform. In addition, local inactivation of mitochondria by KillerRed mediated chromophore-assisted light inactivation (CALI) inhibited the translocation of mitochondrial spots in adjacent axonal regions, suggesting that functional mitochondria enhance the motility of other mitochondria in the vicinity. Signals of ATP:ADP sensor, PercevalHR indicated that the ATP:ADP ratio was relatively high around mitochondria, and treating axons with phosphocreatine (PCr), which supplies ATP, reduced the immobile mitochondria induced by the local mitochondrial inactivation. In a mathematical model, we found that the ATP gradient generated by mitochondria, and ATP dependent regulation of mitochondrial motility could establish uniform mitochondrial distribution. These observations suggest that axons in the CNS possess the system that distributes mitochondria uniformly, and intermitochondrial signaling contribute to the regulation. In addition, our results suggest the possibility that ATP might be one of the molecules mediating the signaling.
    Keywords:  ATP; Axonal transport; Cerebellar granule neurons; Mitochondrial distribution; Retinal ganglion cells; Stationary mitochondria
    DOI:  https://doi.org/10.1016/j.mcn.2022.103704
  15. J Neuropathol Exp Neurol. 2022 Feb 09. pii: nlac004. [Epub ahead of print]
    Widespread Mislocalization of FUS Is Associated With Mitochondrial Abnormalities in Skeletal Muscle in Amyotrophic Lateral Sclerosis With FUS Mutations.
    Meng Yu, Xutong Zhao, Wei Wu, Qingqing Wang, Jing Liu, Wei Zhang, Yun Yuan, Daojun Hong, Zhaoxia Wang, Jianwen Deng.
      Mutations in the fused in sarcoma (FUS) gene have been reported to be the most common genetic cause of early-onset amyotrophic lateral sclerosis (ALS); cytoplasmic inclusions containing FUS protein are the predominant pathological feature. Recent studies indicated that mutant FUS impaired neuromuscular junctions and induced muscle intrinsic toxicity in cell and animal models. However, the role of FUS in muscle degeneration remains unclear. In this study, we investigated FUS protein distribution in skeletal muscle fibers in ALS-FUS. Our data show that cytoplasmic mislocalized FUS in the unaggregated form represented a remarkable pathological feature in affected muscle fibers in ALS-FUS. Additional studies found that cytoplasmic FUS colocalized with some mitochondria and was associated with mitochondrial swelling and disorganized cristae. RNA sequencing and quantitative real-time polymerase chain reaction analyses indicated downregulation of the key subunits of mitochondrial oxidative phosphorylation complexes in the affected skeletal muscle in ALS-FUS patients. Further immunoblot analysis showed increased levels of FUS, but decreased levels of Cox I (subunit of complex IV) in ALS-FUS patients compared with age-matched controls. This is the first demonstration of the close association of cytoplasmic mislocalized FUS with mitochondrial dysfunction in skeletal muscle, implicating the presence of a cell-autonomous mechanism in muscle degeneration in ALS.
    Keywords:  Amyotrophic lateral sclerosis (ALS); Cell autonomous; Fused in sarcoma (FUS); Mislocalization; Mitochondrial damage; Oxidative phosphorylation; Skeletal muscle
    DOI:  https://doi.org/10.1093/jnen/nlac004
  16. Stem Cell Reports. 2022 Feb 04. pii: S2213-6711(22)00053-4. [Epub ahead of print]
    Deriving waveform parameters from calcium transients in human iPSC-derived cardiomyocytes to predict cardiac activity with machine learning.
    Hongbin Yang, Will Stebbeds, Jo Francis, Amy Pointon, Olga Obrezanova, Kylie A Beattie, Peter Clements, James S Harvey, Graham F Smith, Andreas Bender.
      Human induced pluripotent stem cell-derived cardiomyocytes have been established to detect dynamic calcium transients by fast kinetic fluorescence assays that provide insights into specific aspects of clinical cardiac activity. However, the precise derivation and use of waveform parameters to predict cardiac activity merit deeper investigation. In this study, we derived, evaluated, and applied 38 waveform parameters in a novel Python framework, including (among others) peak frequency, peak amplitude, peak widths, and a novel parameter, shoulder-tail ratio. We then trained a random forest model to predict cardiac activity based on the 25 parameters selected by correlation analysis. The area under the curve (AUC) obtained for leave-one-compound-out cross-validation was 0.86, thereby replicating the predictions of conventional methods and outperforming fingerprint-based methods by a large margin. This work demonstrates that machine learning is able to automate the assessment of cardiovascular liability from waveform data, reducing any risk of user-to-user variability and bias.
    Keywords:  calcium transients; cardiac activity; cardiomyocytes; cardiotoxicity; cardiovascular liability; hiPSC-CMs; machine learning; random forest; waveform parameters
    DOI:  https://doi.org/10.1016/j.stemcr.2022.01.009
  17. Nat Struct Mol Biol. 2022 Feb 10.
    The coupling mechanism of mammalian mitochondrial complex I.
    Jinke Gu, Tianya Liu, Runyu Guo, Laixing Zhang, Maojun Yang.
      Mammalian respiratory complex I (CI) is a 45-subunit, redox-driven proton pump that generates an electrochemical gradient across the mitochondrial inner membrane to power ATP synthesis in mitochondria. In the present study, we report cryo-electron microscopy structures of CI from Sus scrofa in six treatment conditions at a resolution of 2.4-3.5 Å, in which CI structures of each condition can be classified into two biochemical classes (active or deactive), with a notably higher proportion of active CI particles. These structures illuminate how hydrophobic ubiquinone-10 (Q10) with its long isoprenoid tail is bound and reduced in a narrow Q chamber comprising four different Q10-binding sites. Structural comparisons of active CI structures from our decylubiquinone-NADH and rotenone-NADH datasets reveal that Q10 reduction at site 1 is not coupled to proton pumping in the membrane arm, which might instead be coupled to Q10 oxidation at site 2. Our data overturn the widely accepted previous proposal about the coupling mechanism of CI.
    DOI:  https://doi.org/10.1038/s41594-022-00722-w
  18. Hum Mutat. 2022 Feb 10.
    Machine Learning Models for Accurate Prioritization of Variants of Uncertain Significance.
    Daniel Mahecha, Haydemar Nuñez, Maria C Lattig, Jorge Duitama.
      The growing use of next generation sequencing technologies on genetic diagnosis has produced an exponential increase in the number of Variants of Uncertain Significance (VUS). In this manuscript we compare three machine learning methods to classify VUS as Pathogenic or No pathogenic, implementing a Random Forest (RF), a Support Vector Machine (SVM), and a Multilayer Perceptron (MLP). To train the models, we extracted high quality variants from ClinVar that were previously classified as VUS. For each variant, we retrieved 9 conservation scores, the loss of function tool and allele frequencies. For the RF and SVM models, hyperparameters were tuned using cross validation with a grid search. The three models were tested on a non-overlapping set of variants that had been classified as VUS any time along the last three years but had been reclassified in august 2020. The three models yielded superior accuracy on this set compared to the benchmarked tools. The RF based model yielded the best performance across different variant types and was used to create VusPrize, an open source software tool for prioritization of variants of uncertain significance. We believe that our model can improve the process of genetic diagnosis in research and clinical settings. This article is protected by copyright. All rights reserved.
    Keywords:  Variants of uncertain significance; genetic diagnosis; machine learning; pathogenicity prediction; variant interpretation
    DOI:  https://doi.org/10.1002/humu.24339
  19. J Biol Chem. 2022 Feb 07. pii: S0021-9258(22)00134-X. [Epub ahead of print] 101694
    Cryo-EM structure of hexameric yeast Lon protease (PIM1) highlights the importance of conserved structural elements.
    Jie Yang, Albert S Song, R Luke Wiseman, Gabriel C Lander.
      Lon protease is a conserved ATP-dependent serine protease composed of an AAA+ domain that mechanically unfolds substrates and a serine protease domain that degrades these unfolded substrates. In yeast, dysregulation of Lon protease (PIM1) attenuates lifespan and leads to gross mitochondrial morphological perturbations. Although structures of the bacterial and human Lon protease reveal a hexameric assembly, yeast PIM1 was speculated to form a heptameric assembly, and is uniquely characterized by a ∼50-residue insertion between the ATPase and protease domains. To further understand the yeast-specific properties of PIM1, we determined a high-resolution cryo-EM structure of PIM1 in a substrate-translocating state. Here, we reveal that PIM1 forms a hexamer, conserved with that of bacterial and human Lon proteases, wherein the ATPase domains form a canonical closed spiral that enables pore loop residues to translocate substrates to the protease chamber. In the substrate-translocating state, PIM1 protease domains form a planar protease chamber in an active conformation and are uniquely characterized by a ∼15-residue C-terminal extension. These additional C-terminal residues form an alpha-helix that is located along the base of the protease domain. Finally, we did not observe density for the yeast-specific insertion between the ATPase and protease domains, likely due to high conformational flexibility. Biochemical studies to investigate the insertion using constructs that truncated or replaced the insertion with a glycine-serine linker suggest that the yeast-specific insertion is dispensable for PIM1's enzymatic function. Altogether, our structural and biochemical studies highlight unique components of PIM1 machinery and demonstrate evolutionary conservation of Lon protease function.
    Keywords:  AAA+ protease; ATP-dependent protease; Lon; cryo-electron microscopy; enzyme structure; mitochondria; proteostasis
    DOI:  https://doi.org/10.1016/j.jbc.2022.101694
  20. Nat Commun. 2022 Feb 10. 13(1): 801
    Whole-cell modeling in yeast predicts compartment-specific proteome constraints that drive metabolic strategies.
    Ibrahim E Elsemman, Angelica Rodriguez Prado, Pranas Grigaitis, Manuel Garcia Albornoz, Victoria Harman, Stephen W Holman, Johan van Heerden, Frank J Bruggeman, Mark M M Bisschops, Nikolaus Sonnenschein, Simon Hubbard, Rob Beynon, Pascale Daran-Lapujade, Jens Nielsen, Bas Teusink.
      When conditions change, unicellular organisms rewire their metabolism to sustain cell maintenance and cellular growth. Such rewiring may be understood as resource re-allocation under cellular constraints. Eukaryal cells contain metabolically active organelles such as mitochondria, competing for cytosolic space and resources, and the nature of the relevant cellular constraints remain to be determined for such cells. Here, we present a comprehensive metabolic model of the yeast cell, based on its full metabolic reaction network extended with protein synthesis and degradation reactions. The model predicts metabolic fluxes and corresponding protein expression by constraining compartment-specific protein pools and maximising growth rate. Comparing model predictions with quantitative experimental data suggests that under glucose limitation, a mitochondrial constraint limits growth at the onset of ethanol formation-known as the Crabtree effect. Under sugar excess, however, a constraint on total cytosolic volume dictates overflow metabolism. Our comprehensive model thus identifies condition-dependent and compartment-specific constraints that can explain metabolic strategies and protein expression profiles from growth rate optimisation, providing a framework to understand metabolic adaptation in eukaryal cells.
    DOI:  https://doi.org/10.1038/s41467-022-28467-6
  21. Nat Biotechnol. 2022 Feb 07.
    Curated variation benchmarks for challenging medically relevant autosomal genes.
    Justin Wagner, Nathan D Olson, Lindsay Harris, Jennifer McDaniel, Haoyu Cheng, Arkarachai Fungtammasan, Yih-Chii Hwang, Richa Gupta, Aaron M Wenger, William J Rowell, Ziad M Khan, Jesse Farek, Yiming Zhu, Aishwarya Pisupati, Medhat Mahmoud, Chunlin Xiao, Byunggil Yoo, Sayed Mohammad Ebrahim Sahraeian, Danny E Miller, David Jáspez, José M Lorenzo-Salazar, Adrián Muñoz-Barrera, Luis A Rubio-Rodríguez, Carlos Flores, Giuseppe Narzisi, Uday Shanker Evani, Wayne E Clarke, Joyce Lee, Christopher E Mason, Stephen E Lincoln, Karen H Miga, Mark T W Ebbert, Alaina Shumate, Heng Li, Chen-Shan Chin, Justin M Zook, Fritz J Sedlazeck.
      The repetitive nature and complexity of some medically relevant genes poses a challenge for their accurate analysis in a clinical setting. The Genome in a Bottle Consortium has provided variant benchmark sets, but these exclude nearly 400 medically relevant genes due to their repetitiveness or polymorphic complexity. Here, we characterize 273 of these 395 challenging autosomal genes using a haplotype-resolved whole-genome assembly. This curated benchmark reports over 17,000 single-nucleotide variations, 3,600 insertions and deletions and 200 structural variations each for human genome reference GRCh37 and GRCh38 across HG002. We show that false duplications in either GRCh37 or GRCh38 result in reference-specific, missed variants for short- and long-read technologies in medically relevant genes, including CBS, CRYAA and KCNE1. When masking these false duplications, variant recall can improve from 8% to 100%. Forming benchmarks from a haplotype-resolved whole-genome assembly may become a prototype for future benchmarks covering the whole genome.
    DOI:  https://doi.org/10.1038/s41587-021-01158-1
  22. Brain. 2022 Feb 10. pii: awac055. [Epub ahead of print]
    NCAM1 and GDF15 are biomarkers of Charcot-Marie-Tooth disease in patients and mice.
    Matthew J Jennings, Alexia Kagiava, Leen Vendredy, Emily L Spaulding, Marina Stavrou, Denisa Hathazi, Anika Grüneboom, Vicky De Winter, Burkhard Gess, Ulrike Schara, Oksana Pogoryelova, Hanns Lochmüller, Christoph H Borchers, Andreas Roos, Robert W Burgess, Vincent Timmerman, Kleopas A Kleopa, Rita Horvath.
      Molecular markers, scalable for clinical use are critical for the development of effective treatments, and for design of clinical trials. Here, we identify proteins in sera of patients and mouse models with Charcot-Marie-Tooth disease (CMT) with characteristics that make them suitable as biomarkers in clinical practice and therapeutic trials. We collected serum from mouse models of CMT1A (C61 het), CMT2D (GarsC201R, GarsP278KY), CMT1X (Gjb1-null), CMT2L (Hspb8K141N) and from CMT patients with genotypes including CMT1A (PMP22d), CMT2D (GARS), CMT2N (AARS) and other rare genetic forms of CMT. The severity of neuropathy in the patients was assessed by the CMT Neuropathy Examination Score (CMTES). We performed multitargeted proteomics on both sample sets to identify proteins elevated across multiple mouse models and CMT patients. Selected proteins and additional potential biomarkers, such as growth differentiation factor 15 (GDF15) and cell free mitochondrial DNA were validated by ELISA and quantitative PCR, respectively. We propose that neural cell adhesion molecule 1 (NCAM1) is a candidate biomarker for CMT, as it was elevated in Gjb1-null, Hspb8K141N, GarsC201R and GarsP278KY mice, as well as in patients with both demyelinating (CMT1A) and axonal (CMT2D, CMT2N) forms of CMT. We show that NCAM1 may reflect disease severity, demonstrated by a progressive increase in mouse models with time and a significant positive correlation with CMTES neuropathy severity in patients. The increase in NCAM1 may reflect muscle regeneration triggered by denervation, which could potentially track disease progression or the effect of treatments. We found that member proteins of the complement system were elevated in Gjb1-null and Hspb8K141Nmouse models, as well as in patients with both demyelinating and axonal CMT, indicating possible complement activation at the impaired nerve terminals. However, complement proteins did not correlate with the severity of neuropathy measured on the CMTES scale. Although the complement system does not seem to be a prognostic biomarker, we do show complement elevation to be a common disease feature of CMT, which may be of interest as a therapeutic target.. We also identify serum GDF15 as a highly sensitive diagnostic biomarker, which was elevated in all CMT genotypes as well as in Hspb8K141N, Gjb1-null, GarsC201R and GarsP278KY mouse models. Although we cannot fully explain its origin, it may reflect increased stress response or metabolic disturbances in CMT. Further large and longitudinal patient studies should be performed to establish the value of these proteins as diagnostic and prognostic molecular biomarkers for CMT.
    Keywords:  Charcot-Marie-Tooth disease (CMT); GDF15; NCAM1; biomarker; mouse models; serum
    DOI:  https://doi.org/10.1093/brain/awac055
  23. STAR Protoc. 2022 Mar 18. 3(1): 101153
    Isolation and culture of neural stem cells from adult mouse subventricular zone for genetic and pharmacological treatments with proliferation analysis.
    Daniel Z Radecki, Jayshree Samanta.
      Neural stem cells (NSCs) from the subventricular zone (SVZ) of the mouse brain can be expanded in vitro and grown as neurospheres, which can be stored long-term in liquid nitrogen. Here, we present a protocol for isolation and culture of NSCs from the adult mouse SVZ. We describe how to grow and expand primary NSCs to neurospheres, followed by differentiation and nucleofection/pharmacological treatments. Finally, we describe RNA extraction, EdU labeling of the cells, and immunofluorescent analysis to examine their proliferation. For complete details on the use and execution of this protocol, please refer to Radecki et al. (2020).
    Keywords:  Cell Biology; Cell culture; Cell isolation; Neuroscience; Stem Cells
    DOI:  https://doi.org/10.1016/j.xpro.2022.101153
  24. Science. 2022 Feb 11. 375(6581): 620-621
    Caloric restriction has a new player.
    Timothy W Rhoads, Rozalyn M Anderson.
      Reverse translation of a human caloric restriction trial finds an immunometabolic regulator.
    DOI:  https://doi.org/10.1126/science.abn6576
This page is being maintained by Thomas Krichel. It was last updated on 2024‒11‒22 at 4:23.