bims-mitdis Biomed News
on Mitochondrial disorders
Issue of 2023–03–19
forty-five papers selected by
Catalina Vasilescu, Helmholz Munich
  1. Genetic Aberration Analysis of Mitochondrial Respiratory Complex I Implications in the Development of Neurological Disorders and Their Clinical Significance.
  2. Mitochondrial protein transport: Versatility of translocases and mechanisms.
  3. OPA1 disease-causing mutants have domain-specific effects on mitochondrial ultrastructure and fusion.
  4. Role of amino acid metabolism in mitochondrial homeostasis.
  5. Use of dual genomic sequencing to screen mitochondrial diseases in pediatrics: a retrospective analysis.
  6. Inter-tissue communication of mitochondrial stress in aging.
  7. SnapShot: Mitochondrial signaling.
  8. Restoration of mitochondrial function through activation of hypomodified tRNAs with pathogenic mutations associated with mitochondrial diseases.
  9. Highly-purified rapidly expanding clones, RECs, are superior for functional-mitochondrial transfer.
  10. Omics-based approaches for the systematic profiling of mitochondrial biology.
  11. Pptc7 maintains mitochondrial protein content by suppressing receptor-mediated mitophagy.
  12. Mitochondrial-derived vesicles retain membrane potential and contain a functional ATP synthase.
  13. Differential effects of Mendelian GDAP1 clinical variants on mitochondria-lysosome membrane contacts sites.
  14. TRAK adaptors regulate the recruitment and activation of dynein and kinesin in mitochondrial transport.
  15. Mitochondrial pores at the crossroad between cell death and inflammatory signaling.
  16. Activity regulates a cell type-specific mitochondrial phenotype in zebrafish lateral line hair cells.
  17. Incorporating a Polyethyleneglycol Linker to Enhance the Hydrophilicity of Mitochondria-Targeted Triphenylphosphonium Constructs.
  18. Mitochondrial morphology controls fatty acid utilization by changing CPT1 sensitivity to malonyl-CoA.
  19. The use of Nicotinamide and Nicotinamide riboside as an adjunct therapy in the treatment of glaucoma.
  20. Metabolic sensing and control in mitochondria.
  21. NAD+ precursor supplementation prevents mtRNA/RIG-I-dependent inflammation during kidney injury.
  22. Ratiometric NAD+ Sensors Reveal Subcellular NAD+ Modulators.
  23. Role of VPS39, a key tethering protein for endolysosomal trafficking and mitochondria-lysosome crosstalk, in health and disease.
  24. Key Amino Acid Residues of Mitochondrial Transcription Factor A Synergize with Abasic (AP) Site Dynamics To Facilitate AP-Lyase Reactions.
  25. Genetic modifiers modulate phenotypic expression of tafazzin deficiency in a mouse model of Barth syndrome.
  26. Inhibition of ATP synthase reverse activity restores energy homeostasis in mitochondrial pathologies.
  27. A novel approach to measure complex V ATP hydrolysis in frozen cell lysates and tissue homogenates.
  28. Mitophagy in ototoxicity.
  29. Inflammatory and interferon gene expression signatures in patients with mitochondrial disease.
  30. Automated prioritization of sick newborns for whole genome sequencing using clinical natural language processing and machine learning.
  31. Gasdermin-E mediates mitochondrial damage in axons and neurodegeneration.
  32. Lactate regulates cell cycle by remodeling the anaphase promoting complex.
  33. 4E-BP1 counteracts human mesenchymal stem cell senescence via maintaining mitochondrial homeostasis.
  34. SIRT3 improved peroxisomes-mitochondria interplay and prevented cardiac hypertrophy via preserving PEX5 expression.
  35. Genetic association analysis of 77,539 genomes reveals rare disease etiologies.
  36. Identification of a mitochondrial targeting sequence in cathepsin D and its localization in mitochondria.
  37. A protocol for single nucleus RNA-seq from frozen skeletal muscle.
  38. Statistical Methods for Disease Risk Prediction with Genotype Data.
  39. Exploring the role of mitochondria transfer/transplant and their long-non-coding RNAs in regenerative therapies for skin aging.
  40. Spatial epigenome-transcriptome co-profiling of mammalian tissues.
  41. Stepwise use of genomics and transcriptomics technologies increases diagnostic yield in Mendelian disorders.
  42. The multifaceted roles of mitochondria.
  43. nPoRe: n-polymer realigner for improved pileup-based variant calling.
  44. Mutations in DARS2 result in global dysregulation of mRNA metabolism and splicing.
  45. From the 'black box' to 'domino effect' mechanism: what have we learned from the structures of respiratory complex I.
  1. Ageing Res Rev. 2023 Mar 09. pii: S1568-1637(23)00065-X. [Epub ahead of print] 101906
    Genetic Aberration Analysis of Mitochondrial Respiratory Complex I Implications in the Development of Neurological Disorders and Their Clinical Significance.
    Ghulam Mehdi Dar, Ejaj Ahmad, Asgar Ali, Bhawna Mahajan, Ghulam Md Ashraf, Sundeep Singh Saluja.
      Growing neurological diseases pose difficult challenges for modern medicine to diagnose and manage them effectively. Many neurological disorders mainly occur due to genetic alteration in genes encoding mitochondrial proteins. Moreover, mitochondrial genes exhibit a higher rate of mutation due to the generation of Reactive oxygen species (ROS) during oxidative phosphorylation operating in their vicinity. Among the different complexes of Electron transport chain (ETC), NADH: Ubiquinone oxidoreductase (Mitochondrial complex I) is the most important. This multimeric enzyme, composed of 44 subunits, is encoded by both nuclear and mitochondrial genes. It often exhibits mutations resulting in development of various neurological diseases. The most prominent diseases include leigh syndrome (LS), leber hereditary optic neuropathy (LHON), mitochondrial encephalomyopathy with lactic acidosis and stroke-like episodes (MELAS), myoclonic epilepsy associated with ragged-red fibers (MERRF), idiopathic Parkinson's disease (PD) and, Alzheimer's disease (AD). Preliminary data suggest that mitochondrial complex I subunit genes mutated are frequently of nuclear origin; however, most of the mtDNA gene encoding subunits are also primarily involved. In this review, we have discussed the genetic origins of neurological disorders involving mitochondrial complex I and signified recent approaches to unravel the diagnostic and therapeutic potentials and their management.
    Keywords:  Complex I; Electron Transport Chain (ETC); Mitochondrial DNA (mtDNA); Neurological disorders; Oxidative Phosphorylation
    DOI:  https://doi.org/10.1016/j.arr.2023.101906
  2. Mol Cell. 2023 Mar 16. pii: S1097-2765(23)00123-5. [Epub ahead of print]83(6): 890-910
    Mitochondrial protein transport: Versatility of translocases and mechanisms.
    Jakob D Busch, Laura F Fielden, Nikolaus Pfanner, Nils Wiedemann.
      Biogenesis of mitochondria requires the import of approximately 1,000 different precursor proteins into and across the mitochondrial membranes. Mitochondria exhibit a wide variety of mechanisms and machineries for the translocation and sorting of precursor proteins. Five major import pathways that transport proteins to their functional intramitochondrial destination have been elucidated; these pathways range from the classical amino-terminal presequence-directed pathway to pathways using internal or even carboxy-terminal targeting signals in the precursors. Recent studies have provided important insights into the structural organization of membrane-embedded preprotein translocases of mitochondria. A comparison of the different translocases reveals the existence of at least three fundamentally different mechanisms: two-pore-translocase, β-barrel switching, and transport cavities open to the lipid bilayer. In addition, translocases are physically engaged in dynamic interactions with respiratory chain complexes, metabolite transporters, quality control factors, and machineries controlling membrane morphology. Thus, mitochondrial preprotein translocases are integrated into multi-functional networks of mitochondrial and cellular machineries.
    DOI:  https://doi.org/10.1016/j.molcel.2023.02.020
  3. Proc Natl Acad Sci U S A. 2023 Mar 21. 120(12): e2207471120
    OPA1 disease-causing mutants have domain-specific effects on mitochondrial ultrastructure and fusion.
    Benjamín Cartes-Saavedra, Daniel Lagos, Josefa Macuada, Duxan Arancibia, Florence Burté, Marcela K Sjöberg-Herrera, María Estela Andrés, Rita Horvath, Patrick Yu-Wai-Man, György Hajnóczky, Verónica Eisner.
      Inner mitochondrial membrane fusion and cristae shape depend on optic atrophy protein 1, OPA1. Mutations in OPA1 lead to autosomal dominant optic atrophy (ADOA), an important cause of inherited blindness. The Guanosin Triphosphatase (GTPase) and GTPase effector domains (GEDs) of OPA1 are essential for mitochondrial fusion; yet, their specific roles remain elusive. Intriguingly, patients carrying OPA1 GTPase mutations have a higher risk of developing more severe multisystemic symptoms in addition to optic atrophy, suggesting pathogenic contributions for the GTPase and GED domains, respectively. We studied OPA1 GTPase and GED mutations to understand their domain-specific contribution to protein function by analyzing patient-derived cells and gain-of-function paradigms. Mitochondria from OPA1 GTPase (c.870+5G>A and c.889C>T) and GED (c.2713C>T and c.2818+5G>A) mutants display distinct aberrant cristae ultrastructure. While all OPA1 mutants inhibited mitochondrial fusion, some GTPase mutants resulted in elongated mitochondria, suggesting fission inhibition. We show that the GED is dispensable for fusion and OPA1 oligomer formation but necessary for GTPase activity. Finally, splicing defect mutants displayed a posttranslational haploinsufficiency-like phenotype but retained domain-specific dysfunctions. Thus, OPA1 domain-specific mutants result in distinct impairments in mitochondrial dynamics, providing insight into OPA1 function and its contribution to ADOA pathogenesis and severity.
    Keywords:  ADOA; OPA1; cristae; dynamics; mitochondria
    DOI:  https://doi.org/10.1073/pnas.2207471120
  4. Front Cell Dev Biol. 2023 ;11 1127618
    Role of amino acid metabolism in mitochondrial homeostasis.
    Qiaochu Li, Thorsten Hoppe.
      Mitochondria are central hubs for energy production, metabolism and cellular signal transduction in eukaryotic cells. Maintenance of mitochondrial homeostasis is important for cellular function and survival. In particular, cellular metabolic state is in constant communication with mitochondrial homeostasis. One of the most important metabolic processes that provide energy in the cell is amino acid metabolism. Almost all of the 20 amino acids that serve as the building blocks of proteins are produced or degraded in the mitochondria. The synthesis of the amino acids aspartate and arginine depends on the activity of the respiratory chain, which is essential for cell proliferation. The degradation of branched-chain amino acids mainly occurs in the mitochondrial matrix, contributing to energy metabolism, mitochondrial biogenesis, as well as protein quality control in both mitochondria and cytosol. Dietary supplementation or restriction of amino acids in worms, flies and mice modulates lifespan and health, which has been associated with changes in mitochondrial biogenesis, antioxidant response, as well as the activity of tricarboxylic acid cycle and respiratory chain. Consequently, impaired amino acid metabolism has been associated with both primary mitochondrial diseases and diseases with mitochondrial dysfunction such as cancer. Here, we present recent observations on the crosstalk between amino acid metabolism and mitochondrial homeostasis, summarise the underlying molecular mechanisms to date, and discuss their role in cellular functions and organismal physiology.
    Keywords:  TCA cycle; amino acid metabolism; amino acid recycling; lifespan; mitochondrial homeostasis; proteasome; respiratory chain
    DOI:  https://doi.org/10.3389/fcell.2023.1127618
  5. Sci Rep. 2023 Mar 14. 13(1): 4193
    Use of dual genomic sequencing to screen mitochondrial diseases in pediatrics: a retrospective analysis.
    Teng-Hui Wu, Jing Peng, Li Yang, Yan-Hui Chen, Xiu-Lan Lu, Jiao-Tian Huang, Jie-Yu You, Wen-Xian Ou-Yang, Yue-Yu Sun, Yi-Nan Xue, Xiao Mao, Hui-Ming Yan, Rong-Na Ren, Jing Xie, Zhi-Heng Chen, Victor-Wei Zhang, Gui-Zhen Lyu, Fang He.
      Mitochondrial diseases (MDs) were a large group multisystem disorders, attributable in part to the dual genomic control. The advent of massively sequencing has improved diagnostic rates and speed, and was increasingly being used as a first-line diagnostic test. Paediatric patients (aged < 18 years) who underwent dual genomic sequencing were enrolled in this retrospective multicentre study. We evaluated the mitochondrial disease criteria (MDC) and molecular diagnostic yield of dual genomic sequencing. Causative variants were identified in 177 out of 503 (35.2%) patients using dual genomic sequencing. Forty-six patients (9.1%) had mitochondria-related variants, including 25 patients with nuclear DNA (nDNA) variants, 15 with mitochondrial DNA (mtDNA) variants, and six with dual genomic variants (MT-ND6 and POLG; MT-ND5 and RARS2; MT-TL1 and NARS2; MT-CO2 and NDUFS1; MT-CYB and SMARCA2; and CHRNA4 and MT-CO3). Based on the MDC, 15.2% of the patients with mitochondria-related variants were classified as "unlikely to have mitochondrial disorder". Moreover, 4.5% of the patients with non-mitochondria-related variants and 1.43% with negative genetic tests, were classified as "probably having mitochondrial disorder". Dual genomic sequencing in suspected MDs provided a more comprehensive and accurate diagnosis for pediatric patients, especially for patients with dual genomic variants.
    DOI:  https://doi.org/10.1038/s41598-023-31134-5
  6. Yi Chuan. 2023 Mar 20. 45(3): 187-197
    Inter-tissue communication of mitochondrial stress in aging.
    Qian Zhang, Zihao Wang, Ye Tian.
      The protein homeostasis in mitochondria is critical for the normal physiological function of cells. To cope with mitochondrial stress, cells elicit specific stress response named mitochondrial unfolded protein response (UPRmt), to maintain mitochondrial homeostasis and repair mitochondrial function. Although severe damage to mitochondria is detrimental, studies in worms, flies, and mice have shown that mild mitochondrial damage promotes longevity by activating UPRmt. Interestingly, UPRmt can also be induced in a cell non-autonomous manner in cells or tissues which are not directly experiencing mitochondrial stress. The secreted molecules called "mitokine" are responsible for the mitochondrial stress communication between different tissues. This inter-tissue regulation of mitochondrial stress response systematically coordinates the adaptation ability which is closely associated with aging and a variety of diseases such as neurodegeneration and cancer. In this review, we summarize recent advances about inter-tissue mitochondrial stress communications, and introduce the current knowledge about the "mitokine" and its regulation on aging for further studies.
    Keywords:  aging; inter-tissue regulation; mitochondrial unfolded protein response; protein homeostasis
    DOI:  https://doi.org/10.16288/j.yczz.22-416
  7. Mol Cell. 2023 Mar 16. pii: S1097-2765(23)00028-X. [Epub ahead of print]83(6): 1012-1012.e1
    SnapShot: Mitochondrial signaling.
    Taylor A Poor, Navdeep S Chandel.
      Mitochondria have emerged as signaling organelles with roles beyond their well-established function in generating ATP and metabolites for macromolecule synthesis. Healthy mitochondria integrate various physiologic inputs and communicate signals that control cell function or fate as well as adaptation to stress. Dysregulation of these mitochondrial signaling networks are linked to pathology. Here we outline a few modes of signaling between the mitochondrion and the cytoplasm. To view this SnapShot, open or download the PDF.
    DOI:  https://doi.org/10.1016/j.molcel.2023.01.008
  8. Nucleic Acids Res. 2023 Mar 17. pii: gkad139. [Epub ahead of print]
    Restoration of mitochondrial function through activation of hypomodified tRNAs with pathogenic mutations associated with mitochondrial diseases.
    Ena Tomoda, Asuteka Nagao, Yuki Shirai, Kana Asano, Takeo Suzuki, Brendan J Battersby, Tsutomu Suzuki.
      Mutations in mitochondrial (mt-)tRNAs frequently cause mitochondrial dysfunction. Mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes (MELAS), and myoclonus epilepsy associated with ragged red fibers (MERRF) are major clinical subgroups of mitochondrial diseases caused by pathogenic point mutations in tRNA genes encoded in mtDNA. We previously reported a severe reduction in the frequency of 5-taurinomethyluridine (τm5U) and its 2-thiouridine derivative (τm5s2U) in the anticodons of mutant mt-tRNAs isolated from the cells of patients with MELAS and MERRF, respectively. The hypomodified tRNAs fail to decode cognate codons efficiently, resulting in defective translation of respiratory chain proteins in mitochondria. To restore the mitochondrial activity of MELAS patient cells, we overexpressed MTO1, a τm5U-modifying enzyme, in patient-derived myoblasts. We used a newly developed primer extension method and showed that MTO1 overexpression almost completely restored the τm5U modification of the MELAS mutant mt-tRNALeu(UUR). An increase in mitochondrial protein synthesis and oxygen consumption rate suggested that the mitochondrial function of MELAS patient cells can be activated by restoring the τm5U of the mutant tRNA. In addition, we confirmed that MTO1 expression restored the τm5s2U of the mutant mt-tRNALys in MERRF patient cells. These findings pave the way for epitranscriptomic therapies for mitochondrial diseases.
    DOI:  https://doi.org/10.1093/nar/gkad139
  9. Stem Cell Res Ther. 2023 Mar 16. 14(1): 40
    Highly-purified rapidly expanding clones, RECs, are superior for functional-mitochondrial transfer.
    Jiahao Yang, Lu Liu, Yasuaki Oda, Keisuke Wada, Mako Ago, Shinichiro Matsuda, Miho Hattori, Tsukimi Goto, Yuki Kawashima, Yumi Matsuzaki, Takeshi Taketani.
       BACKGROUND: Mitochondrial dysfunction caused by mutations in mitochondrial DNA (mtDNA) or nuclear DNA, which codes for mitochondrial components, are known to be associated with various genetic and congenital disorders. These mitochondrial disorders not only impair energy production but also affect mitochondrial functions and have no effective treatment. Mesenchymal stem cells (MSCs) are known to migrate to damaged sites and carry out mitochondrial transfer. MSCs grown using conventional culture methods exhibit heterogeneous cellular characteristics. In contrast, highly purified MSCs, namely the rapidly expanding clones (RECs) isolated by single-cell sorting, display uniform MSCs functionality. Therefore, we examined the differences between RECs and MSCs to assess the efficacy of mitochondrial transfer.
    METHODS: We established mitochondria-deficient cell lines (ρ0 A549 and ρ0 HeLa cell lines) using ethidium bromide. Mitochondrial transfer from RECs/MSCs to ρ0 cells was confirmed by PCR and flow cytometry analysis. We examined several mitochondrial functions including ATP, reactive oxygen species, mitochondrial membrane potential, and oxygen consumption rate (OCR). The route of mitochondrial transfer was identified using inhibition assays for microtubules/tunneling nanotubes, gap junctions, or microvesicles using transwell assay and molecular inhibitors.
    RESULTS: Co-culture of ρ0 cells with MSCs or RECs led to restoration of the mtDNA content. RECs transferred more mitochondria to ρ0 cells compared to that by MSCs. The recovery of mitochondrial function, including ATP, OCR, mitochondrial membrane potential, and mitochondrial swelling in ρ0 cells co-cultured with RECs was superior than that in cells co-cultured with MSCs. Inhibition assays for each pathway revealed that RECs were sensitive to endocytosis inhibitor, dynasore.
    CONCLUSIONS: RECs might serve as a potential therapeutic strategy for diseases linked to mitochondrial dysfunction by donating healthy mitochondria.
    Keywords:  Mesenchymal stem cells (MSCs); Mitochondrial dysfunction; Mitochondrial transfer; Rapidly expanding clones (RECs)
    DOI:  https://doi.org/10.1186/s13287-023-03274-y
  10. Mol Cell. 2023 Mar 16. pii: S1097-2765(23)00118-1. [Epub ahead of print]83(6): 911-926
    Omics-based approaches for the systematic profiling of mitochondrial biology.
    Jasmin Adriana Schäfer, F X Reymond Sutandy, Christian Münch.
      Mitochondria are essential for cellular functions such as metabolism and apoptosis. They dynamically adapt to the changing environmental demands by adjusting their protein, nucleic acid, metabolite, and lipid contents. In addition, the mitochondrial components are modulated on different levels in response to changes, including abundance, activity, and interaction. A wide range of omics-based approaches has been developed to be able to explore mitochondrial adaptation and how mitochondrial function is compromised in disease contexts. Here, we provide an overview of the omics methods that allow us to systematically investigate the different aspects of mitochondrial biology. In addition, we show examples of how these methods have provided new biological insights. The emerging use of these toolboxes provides a more comprehensive understanding of the processes underlying mitochondrial function.
    DOI:  https://doi.org/10.1016/j.molcel.2023.02.015
  11. bioRxiv. 2023 Mar 01. pii: 2023.02.28.530351. [Epub ahead of print]
    Pptc7 maintains mitochondrial protein content by suppressing receptor-mediated mitophagy.
    Natalie M Niemi, Lia R Serrano, Laura K Muehlbauer, Catie Balnis, Keri-Lyn Kozul, Edrees H Rashan, Evgenia Shishkova, Kathryn L Schueler, Mark P Keller, Alan D Attie, Julia Pagan, Joshua J Coon, David J Pagliarini.
      Pptc7 is a resident mitochondrial phosphatase essential for maintaining proper mitochondrial content and function. Newborn mice lacking Pptc7 exhibit aberrant mitochondrial protein phosphorylation, suffer from a range of metabolic defects, and fail to survive beyond one day after birth. Using an inducible knockout model, we reveal that loss of Pptc7 in adult mice causes marked reduction in mitochondrial mass concomitant with elevation of the mitophagy receptors Bnip3 and Nix. Consistently, Pptc7 -/- mouse embryonic fibroblasts (MEFs) exhibit a major increase in mitophagy that is reversed upon deletion of these receptors. Our phosphoproteomics analyses reveal a common set of elevated phosphosites between perinatal tissues, adult liver, and MEFsâ€" including multiple sites on Bnip3 and Nix. These data suggest that Pptc7 deletion causes mitochondrial dysfunction via dysregulation of several metabolic pathways and that Pptc7 may directly regulate mitophagy receptor function or stability. Overall, our work reveals a significant role for Pptc7 in the mitophagic response and furthers the growing notion that management of mitochondrial protein phosphorylation is essential for ensuring proper organelle content and function.
    DOI:  https://doi.org/10.1101/2023.02.28.530351
  12. EMBO Rep. 2023 Mar 17. e56114
    Mitochondrial-derived vesicles retain membrane potential and contain a functional ATP synthase.
    Reut Hazan Ben-Menachem, Dvora Lintzer, Tamar Ziv, Koyeli Das, Irit Rosenhek-Goldian, Ziv Porat, Hila Ben Ami Pilo, Sharon Karniely, Ann Saada, Neta Regev-Rudzki, Ophry Pines.
      Vesicular transport is a means of communication. While cells can communicate with each other via secretion of extracellular vesicles, less is known regarding organelle-to organelle communication, particularly in the case of mitochondria. Mitochondria are responsible for the production of energy and for essential metabolic pathways in the cell, as well as fundamental processes such as apoptosis and aging. Here, we show that functional mitochondria isolated from Saccharomyces cerevisiae release vesicles, independent of the fission machinery. We isolate these mitochondrial-derived vesicles (MDVs) and find that they are relatively uniform in size, of about 100 nm, and carry selective protein cargo enriched for ATP synthase subunits. Remarkably, we further find that these MDVs harbor a functional ATP synthase complex. We demonstrate that these vesicles have a membrane potential, produce ATP, and seem to fuse with naive mitochondria. Our findings reveal a possible delivery mechanism of ATP-producing vesicles, which can potentially regenerate ATP-deficient mitochondria and may participate in organelle-to-organelle communication.
    Keywords:  ATP synthase; membrane potential; mitochondria; mitochondrial-derived vesicles; protein distribution
    DOI:  https://doi.org/10.15252/embr.202256114
  13. Biol Open. 2023 Mar 13. pii: bio.059707. [Epub ahead of print]
    Differential effects of Mendelian GDAP1 clinical variants on mitochondria-lysosome membrane contacts sites.
    Lara Cantarero, Gisela García-Vargas, Janet Hoenicka, Francesc Palau.
      GDAP1 pathogenic variants cause Charcot-Marie-Tooth (CMT) disease, the most common hereditary motor and sensory neuropathy. CMT-GDAP1 can be axonal or demyelinating, with autosomal dominant or recessive inheritance, leading to phenotypic heterogeneity. Recessive GDAP1 variants cause a severe phenotype, whereas dominant variants are associated with a milder disease course. GDAP1 is an outer mitochondrial membrane protein involved in mitochondrial membrane contact sites (MCSs) with the plasmatic membrane, the endoplasmic reticulum (ER), and lysosomes. In GDAP1-deficient models, the pathophysiology includes morphological defects in mitochondrial network and ER, impaired Ca2+ homeostasis, oxidative stress, and mitochondrial MCSs defects. Nevertheless, the underlying pathophysiology of dominant variants is less understood. Here, we study the effect upon mitochondria-lysosome MCSs of two GDAP1 clinical variants located in the α-loop interaction domain of the protein. p.Thr157Pro dominant variant causes the increase in these MCSs that correlates with a hyper-fissioned mitochondrial network. In contrast, p.Arg161His recessive variant, which is predicted to significantly change the contact surface of GDAP1, causes decreased contacts with more elongated mitochondria. Given that mitochondria-lysosome MCSs regulate Ca2+ transfer from the lysosome to mitochondria, our results support that GDAP1 clinical variants have different consequences for Ca2+ handling and that could be primary insults determining differences in severity between dominant and recessive forms of the disease.
    Keywords:  Charcot-Marie-Tooth (CMT); GDAP1; Lysosome; Membrane Contact Sites (MCSs); Mitochondria
    DOI:  https://doi.org/10.1242/bio.059707
  14. Nat Commun. 2023 Mar 13. 14(1): 1376
    TRAK adaptors regulate the recruitment and activation of dynein and kinesin in mitochondrial transport.
    John T Canty, Andrew Hensley, Merve Aslan, Amanda Jack, Ahmet Yildiz.
      Mitochondrial transport along microtubules is mediated by Miro1 and TRAK adaptors that recruit kinesin-1 and dynein-dynactin. To understand how these opposing motors are regulated during mitochondrial transport, we reconstitute the bidirectional transport of Miro1/TRAK along microtubules in vitro. We show that the coiled-coil domain of TRAK activates dynein-dynactin and enhances the motility of kinesin-1 activated by its cofactor MAP7. We find that TRAK adaptors that recruit both motors move towards kinesin-1's direction, whereas kinesin-1 is excluded from binding TRAK transported by dynein-dynactin, avoiding motor tug-of-war. We also test the predictions of the models that explain how mitochondrial transport stalls in regions with elevated Ca2+. Transport of Miro1/TRAK by kinesin-1 is not affected by Ca2+. Instead, we demonstrate that the microtubule docking protein syntaphilin induces resistive forces that stall kinesin-1 and dynein-driven motility. Our results suggest that mitochondrial transport stalls by Ca2+-mediated recruitment of syntaphilin to the mitochondrial membrane, not by disruption of the transport machinery.
    DOI:  https://doi.org/10.1038/s41467-023-36945-8
  15. Mol Cell. 2023 Mar 16. pii: S1097-2765(23)00124-7. [Epub ahead of print]83(6): 843-856
    Mitochondrial pores at the crossroad between cell death and inflammatory signaling.
    Hector Flores-Romero, Shashank Dadsena, Ana J García-Sáez.
      Mitochondria are cellular organelles with a major role in many cellular processes, including not only energy production, metabolism, and calcium homeostasis but also regulated cell death and innate immunity. Their proteobacterial origin makes them a rich source of potent immune agonists, normally hidden within the mitochondrial membrane barriers. Alteration of mitochondrial permeability through mitochondrial pores thus provides efficient mechanisms not only to communicate mitochondrial stress to the cell but also as a key event in the integration of cellular responses. In this regard, eukaryotic cells have developed diverse signaling networks that sense and respond to the release of mitochondrial components into the cytosol and play a key role in controlling cell death and inflammatory pathways. Modulating pore formation at mitochondria through direct or indirect mechanisms may thus open new opportunities for therapy. In this review, we discuss the current understanding of the structure and molecular mechanisms of mitochondrial pores and how they function at the interface between cell death and inflammatory signaling to regulate cellular outcomes.
    Keywords:  BAK; BAX; VDAC; apoptosis; gasdermin; inflammation; mPTP; membrane pore
    DOI:  https://doi.org/10.1016/j.molcel.2023.02.021
  16. Elife. 2023 Mar 13. pii: e80468. [Epub ahead of print]12
    Activity regulates a cell type-specific mitochondrial phenotype in zebrafish lateral line hair cells.
    Andrea McQuate, Sharmon Knecht, David W Raible.
      Hair cells of the inner ear are particularly sensitive to changes in mitochondria, the subcellular organelles necessary for energy production in all eukaryotic cells. There are over thirty mitochondrial deafness genes, and mitochondria are implicated in hair cell death following noise exposure, aminoglycoside antibiotic exposure, as well as in age-related hearing loss. However, little is known about the basic aspects of hair cell mitochondrial biology. Using hair cells from the zebrafish lateral line as a model and serial block-face scanning electron microscopy, we have quantifiably characterized a unique hair cell mitochondrial phenotype that includes (1) a high mitochondrial volume, and (2) specific mitochondrial architecture: multiple small mitochondria apically, and a reticular mitochondrial network basally. This phenotype develops gradually over the lifetime of the hair cell. Disrupting this mitochondrial phenotype with a mutation in opa1 impacts mitochondrial health and function. While hair cell activity is not required for the high mitochondrial volume, it shapes the mitochondrial architecture, with mechanotransduction necessary for all patterning, and synaptic transmission necessary for development of mitochondrial networks. These results demonstrate the high degree to which hair cells regulate their mitochondria for optimal physiology, and provide new insights into mitochondrial deafness.
    Keywords:  cell biology; neuroscience; zebrafish
    DOI:  https://doi.org/10.7554/eLife.80468
  17. Chembiochem. 2023 Mar 14. e202200774
    Incorporating a Polyethyleneglycol Linker to Enhance the Hydrophilicity of Mitochondria-Targeted Triphenylphosphonium Constructs.
    Shinpei Uno, Alexander Harkiss, Roy Chowdhury, Stuart Caldwell, Tracy Prime, Andrew James, Brendan Gallagher, Julien Prudent, Richard Hartley, Michael Murphy.
      The targeting of bioactive molecules and probes to mitochondria can be achieved by coupling to the lipophilic triphenyl phosphonium (TPP) cation, which accumulates several hundred-fold within mitochondria in response to the mitochondrial membrane potential (Dym). Typically, a simple alkane links the TPP to its "cargo", increasing overall hydrophobicity. As it would be beneficial to enhance the water solubility of mitochondria-targeted compounds we explored the effects of replacing the alkyl linker with a polyethylene glycol (PEG). We found that the use of PEG led to compounds that were readily taken up by isolated mitochondria and by mitochondria inside cells. Within mitochondria the PEG linker greatly decreased adsorption of the TPP constructs to the matrix-facing face of the mitochondrial inner membrane. These findings will allow the distribution of mitochondria-targeted TPP compounds within mitochondria to be fine-tuned.
    Keywords:  biological membrane; lipophilic cation; mitochondria-targeting; polyethylene glycol; triphenylphosphonium
    DOI:  https://doi.org/10.1002/cbic.202200774
  18. EMBO J. 2023 Mar 14. e111901
    Mitochondrial morphology controls fatty acid utilization by changing CPT1 sensitivity to malonyl-CoA.
    Jennifer Ngo, Dong Wook Choi, Illana A Stanley, Linsey Stiles, Anthony J A Molina, Pei-Hsuan Chen, Ana Lako, Isabelle Chiao Han Sung, Rishov Goswami, Min-Young Kim, Nathanael Miller, Siyouneh Baghdasarian, Doyeon Kim-Vasquez, Anthony E Jones, Brett Roach, Vincent Gutierrez, Karel Erion, Ajit S Divakaruni, Marc Liesa, Nika N Danial, Orian S Shirihai.
      Changes in mitochondrial morphology are associated with nutrient utilization, but the precise causalities and the underlying mechanisms remain unknown. Here, using cellular models representing a wide variety of mitochondrial shapes, we show a strong linear correlation between mitochondrial fragmentation and increased fatty acid oxidation (FAO) rates. Forced mitochondrial elongation following MFN2 over-expression or DRP1 depletion diminishes FAO, while forced fragmentation upon knockdown or knockout of MFN2 augments FAO as evident from respirometry and metabolic tracing. Remarkably, the genetic induction of fragmentation phenocopies distinct cell type-specific biological functions of enhanced FAO. These include stimulation of gluconeogenesis in hepatocytes, induction of insulin secretion in islet β-cells exposed to fatty acids, and survival of FAO-dependent lymphoma subtypes. We find that fragmentation increases long-chain but not short-chain FAO, identifying carnitine O-palmitoyltransferase 1 (CPT1) as the downstream effector of mitochondrial morphology in regulation of FAO. Mechanistically, we determined that fragmentation reduces malonyl-CoA inhibition of CPT1, while elongation increases CPT1 sensitivity to malonyl-CoA inhibition. Overall, these findings underscore a physiologic role for fragmentation as a mechanism whereby cellular fuel preference and FAO capacity are determined.
    Keywords:  CPT1; fatty acid oxidation; fission; fusion; mitochondrial dynamics
    DOI:  https://doi.org/10.15252/embj.2022111901
  19. Eur J Ophthalmol. 2023 Mar 14. 11206721231161101
    The use of Nicotinamide and Nicotinamide riboside as an adjunct therapy in the treatment of glaucoma.
    Samuel Goulart Nacácio E Silva, Marcelo Luís Occhiutto, Vital Paulino Costa.
      Glaucoma is an optic neuropathy characterized by death of retinal ganglion cells (RGCs), which leads to progressive visual field loss and may result in blindness. Currently, the only available treatment to avoid or delay progression in glaucoma patients is to decrease intraocular pressure (IOP). However, despite adequate IOP control, approximately 25% of the patients continue to progress. To delay or prevent optic nerve damage in glaucoma, two forms of vitamin B3, nicotinamide (NAM) and nicotinamide riboside (NR) are emerging as viable adjuvant therapies. These compounds are nicotinamide adenine dinucleotide (NAD) precursors. NAD is essential for proper cell functioning and is involved in several metabolic activities, including protection against reactive oxygen species, contribution to the performance of various enzymes, and maintenance of mitochondrial function. Due to its beneficial effects and to the evidence of the reduction of NAD bioavailability with aging, researchers are seeking ways to replenish the cellular NAD pool, by administrating its precursors (NAM and NR), believing that it will reduce the RGC vulnerability to external stressors, such as increased IOP. This article attempts to analyze the current knowledge regarding the use of NAM and NR for the prevention and/or treatment of glaucoma.
    Keywords:  NAD; glaucoma; nicotinamide; nicotinamide riboside
    DOI:  https://doi.org/10.1177/11206721231161101
  20. Mol Cell. 2023 Mar 16. pii: S1097-2765(23)00119-3. [Epub ahead of print]83(6): 877-889
    Metabolic sensing and control in mitochondria.
    Yuyang Liu, Kıvanç Birsoy.
      Mitochondria are membrane-enclosed organelles with endosymbiotic origins, harboring independent genomes and a unique biochemical reaction network. To perform their critical functions, mitochondria must maintain a distinct biochemical environment and coordinate with the cytosolic metabolic networks of the host cell. This coordination requires them to sense and control metabolites and respond to metabolic stresses. Indeed, mitochondria adopt feedback or feedforward control strategies to restrain metabolic toxicity, enable metabolic conservation, ensure stable levels of key metabolites, allow metabolic plasticity, and prevent futile cycles. A diverse panel of metabolic sensors mediates these regulatory circuits whose malfunctioning leads to inborn errors of metabolism with mild to severe clinical manifestations. In this review, we discuss the logic and molecular basis of metabolic sensing and control in mitochondria. The past research outlined recurring patterns in mitochondrial metabolic sensing and control and highlighted key knowledge gaps in this organelle that are potentially addressable with emerging technological breakthroughs.
    DOI:  https://doi.org/10.1016/j.molcel.2023.02.016
  21. Nat Metab. 2023 Mar 13.
    NAD+ precursor supplementation prevents mtRNA/RIG-I-dependent inflammation during kidney injury.
    Tomohito Doke, Sarmistha Mukherjee, Dhanunjay Mukhi, Poonam Dhillon, Amin Abedini, James G Davis, Karthikeyani Chellappa, Beishan Chen, Joseph A Baur, Katalin Susztak.
      Our understanding of how global changes in cellular metabolism contribute to human kidney disease remains incompletely understood. Here we show that nicotinamide adenine dinucleotide (NAD+) deficiency drives mitochondrial dysfunction causing inflammation and kidney disease development. Using unbiased global metabolomics in healthy and diseased human kidneys, we identify NAD+ deficiency as a disease signature. Furthermore using models of cisplatin- or ischaemia-reperfusion induced kidney injury in male mice we observed NAD+ depletion Supplemental nicotinamide riboside or nicotinamide mononucleotide restores NAD+ levels and improved kidney function. We find that cisplatin exposure causes cytosolic leakage of mitochondrial RNA (mtRNA) and activation of the cytosolic pattern recognition receptor retinoic acid-inducible gene I (RIG-I), both of which can be ameliorated by restoring NAD+. Male mice with RIG-I knock-out (KO) are protected from cisplatin-induced kidney disease. In summary, we demonstrate that the cytosolic release of mtRNA and RIG-I activation is an NAD+-sensitive mechanism contributing to kidney disease.
    DOI:  https://doi.org/10.1038/s42255-023-00761-7
  22. ACS Sens. 2023 Mar 17.
    Ratiometric NAD+ Sensors Reveal Subcellular NAD+ Modulators.
    Liuqing Chen, Meiting Chen, Mupeng Luo, Yong Li, Bagen Liao, Min Hu, Qiuliyang Yu.
      Mapping NAD+ dynamics in live cells and human is essential for translating NAD+ interventions into effective therapies. Yet, genetically encoded NAD+ sensors with better specificity and pH resistance are still needed for the cost-effective monitoring of NAD+ in both subcellular compartments and clinical samples. Here, we introduce multicolor, resonance energy transfer-based NAD+ sensors covering nano- to millimolar concentration ranges for clinical NAD+ measurement and subcellular NAD+ visualization. The sensors captured the blood NAD+ increase induced by NMN supplementation and revealed the distinct subcellular effects of NAD+ precursors and modulators. The sensors then enabled high-throughput screenings for mitochondrial and nuclear NAD+ modulators and identified α-GPC, a cognition-related metabolite that induces NAD+ redistribution from mitochondria to the nucleus relative to the total adenine nucleotides, which was further confirmed by NAD+ FRET microscopy.
    Keywords:  BRET; FRET; NAD+; drug screenings; genetically encoded biosensor
    DOI:  https://doi.org/10.1021/acssensors.2c02565
  23. J Cell Biochem. 2023 Mar 16.
    Role of VPS39, a key tethering protein for endolysosomal trafficking and mitochondria-lysosome crosstalk, in health and disease.
    Hanbing Li, Wenwen Gong, Weiyun Sun, Yuanfa Yao, Yubing Han.
      The coordinated interaction between mitochondria and lysosomes, mainly manifested by mitophagy, mitochondria-derived vesicles, and direct physical contact, is essential for maintaining cellular life activities. The VPS39 subunit of the homotypic fusion and protein sorting complex could play a key role in the regulation of organelle dynamics, such as endolysosomal trafficking and mitochondria-vacuole/lysosome crosstalk, thus contributing to a variety of physiological functions. The abnormalities of VPS39 and related subunits have been reported to be involved in the pathological process of some diseases. Here, we analyze the potential mechanisms and the existing problems of VPS39 in regulating organelle dynamics, which, in turn, regulate physiological functions and disease pathogenesis, so as to provide new clues for facilitating the discovery of therapeutic targets for mitochondrial and lysosomal diseases.
    Keywords:  HOPS complex; VPS39; diseases; endolysosomal trafficking; mitochondria-lysosome crosstalk
    DOI:  https://doi.org/10.1002/jcb.30396
  24. ACS Chem Biol. 2023 Mar 17.
    Key Amino Acid Residues of Mitochondrial Transcription Factor A Synergize with Abasic (AP) Site Dynamics To Facilitate AP-Lyase Reactions.
    Wenxin Zhao, Wenyan Xu, Jin Tang, Shivansh Kaushik, Chia-En A Chang, Linlin Zhao.
      Human mitochondrial DNA (mtDNA) encodes 37 essential genes and plays a critical role in mitochondrial and cellular functions. mtDNA is susceptible to damage by endogenous and exogenous chemicals. Damaged mtDNA molecules are counteracted by the redundancy, repair, and degradation of mtDNA. In response to difficult-to-repair or excessive amounts of DNA lesions, mtDNA degradation is a crucial mitochondrial genome maintenance mechanism. Nevertheless, the molecular basis of mtDNA degradation remains incompletely understood. Recently, mitochondrial transcription factor A (TFAM) has emerged as a factor in degrading damaged mtDNA containing abasic (AP) sites. TFAM has AP-lyase activity, which cleaves DNA at AP sites. Human TFAM and its homologs contain a higher abundance of Glu than that of the proteome. To decipher the role of Glu in TFAM-catalyzed AP-DNA cleavage, we constructed TFAM variants and used biochemical assays, kinetic simulations, and molecular dynamics (MD) simulations to probe the functional importance of E187 near a key residue K186. Our previous studies showed that K186 is a primary residue to cleave AP-DNA via Schiff base chemistry. Here, we demonstrate that E187 facilitates β-elimination, key to AP-DNA strand scission. MD simulations showed that extrahelical confirmation of the AP lesion and the flexibility of E187 in TFAM-DNA complexes facilitate AP-lyase reactions. Together, highly abundant Lys and Glu residues in TFAM promote AP-DNA strand scission, supporting the role of TFAM in AP-DNA turnover and implying the breadth of this process across different species.
    DOI:  https://doi.org/10.1021/acschembio.3c00047
  25. Hum Mol Genet. 2023 Mar 14. pii: ddad041. [Epub ahead of print]
    Genetic modifiers modulate phenotypic expression of tafazzin deficiency in a mouse model of Barth syndrome.
    Suya Wang, Erika Yazawa, Erin Keating, Neil Mazumdar, Alexander Hauschild, Qing Ma, Haiyan Wu, Yang Xu, Xu Shi, Douglas Strathdee, Robert E Gerszten, Michael Schlame, William T Pu.
      Barth syndrome is an X-linked disorder caused by loss-of-function mutations in Tafazzin (TAZ), an acyltransferase that catalyzes remodeling of cardiolipin, a signature phospholipid of the inner mitochondrial membrane. Patients develop cardiac and skeletal muscle weakness, growth delay, and neutropenia, although phenotypic expression varies considerably between patients. Taz knockout mice recapitulate many of the hallmark features of the disease. We used mouse genetics to test the hypothesis that genetic modifiers alter the phenotypic manifestations of Taz inactivation. We crossed TazKO/X females in the C57BL6/J inbred strain to males from 8 inbred strains and evaluated the phenotypes of first generation (F1) TazKO/Y progeny, compared to TazWT/Y littermates. We observed that genetic background strongly impacted phenotypic expression. C57BL6/J and CAST/EiJ[F1] TazKO/Y mice developed severe cardiomyopathy, whereas A/J[F1] TazKO/Y mice had normal heart function. C57BL6/J and WSB/EiJ[F1] TazKO/Y mice had severely reduced treadmill endurance, whereas endurance was normal in A/J[F1] and CAST/EiJ[F1] TazKO/Y mice. In all genetic backgrounds, cardiolipin showed similar abnormalities in knockout mice, and transcriptomic and metabolomic investigations identified signatures of mitochondrial uncoupling and activation of the integrated stress response. TazKO/Y cardiac mitochondria were small, clustered, and had reduced cristae density in knockouts in severely affected genetic backgrounds but were relatively preserved in the permissive A/J[F1] strain. Gene expression and mitophagy measurements were consistent with reduced mitophagy in knockout mice in genetic backgrounds intolerant of Taz mutation. Our data demonstrate that genetic modifiers powerfully modulate phenotypic expression of Taz loss-of-function and act downstream of cardiolipin, possibly by altering mitochondrial quality control.
    DOI:  https://doi.org/10.1093/hmg/ddad041
  26. EMBO J. 2023 Mar 13. e111699
    Inhibition of ATP synthase reverse activity restores energy homeostasis in mitochondrial pathologies.
    Rebeca Acin-Perez, Cristiane Benincá, Lucia Fernandez Del Rio, Cynthia Shu, Siyouneh Baghdasarian, Vanessa Zanette, Christoph Gerle, Chimari Jiko, Ramzi Khairallah, Shaharyar Khan, David Rincon Fernandez Pacheco, Byourak Shabane, Karel Erion, Ruchi Masand, Sundeep Dugar, Cristina Ghenoiu, George Schreiner, Linsey Stiles, Marc Liesa, Orian S Shirihai.
      The maintenance of cellular function relies on the close regulation of adenosine triphosphate (ATP) synthesis and hydrolysis. ATP hydrolysis by mitochondrial ATP Synthase (CV) is induced by loss of proton motive force and inhibited by the mitochondrial protein ATPase inhibitor (ATPIF1). The extent of CV hydrolytic activity and its impact on cellular energetics remains unknown due to the lack of selective hydrolysis inhibitors of CV. We find that CV hydrolytic activity takes place in coupled intact mitochondria and is increased by respiratory chain defects. We identified (+)-Epicatechin as a selective inhibitor of ATP hydrolysis that binds CV while preventing the binding of ATPIF1. In cells with Complex-III deficiency, we show that inhibition of CV hydrolytic activity by (+)-Epichatechin is sufficient to restore ATP content without restoring respiratory function. Inhibition of CV-ATP hydrolysis in a mouse model of Duchenne Muscular Dystrophy is sufficient to improve muscle force without any increase in mitochondrial content. We conclude that the impact of compromised mitochondrial respiration can be lessened using hydrolysis-selective inhibitors of CV.
    Keywords:  ATP hydrolysis; ATPase Inhibitor (ATPIF1); Complex V; epicatechin; muscular dystrophy
    DOI:  https://doi.org/10.15252/embj.2022111699
  27. Life Sci Alliance. 2023 Apr;pii: e202201628. [Epub ahead of print]6(4):
    A novel approach to measure complex V ATP hydrolysis in frozen cell lysates and tissue homogenates.
    Lucia Fernandez-Del-Rio, Cristiane Benincá, Frankie Villalobos, Cynthia Shu, Linsey Stiles, Marc Liesa, Ajit S Divakaruni, Rebeca Acin-Perez, Orian S Shirihai.
      Mitochondrial depolarization can initiate reversal activity of ATP synthase, depleting ATP by its hydrolysis. We have recently shown that increased ATP hydrolysis contributes to ATP depletion leading to a maladaptation in mitochondrial disorders, where maximal hydrolytic capacity per CV content is increasing. However, despite its importance, ATP hydrolysis is not a commonly studied parameter because of the limitations of the currently available methods. Methods that measure CV hydrolytic activity indirectly require the isolation of mitochondria and involve the introduction of detergents, preventing their utilization in clinical studies or any high-throughput analyses. Here, we describe a novel approach to assess maximal ATP hydrolytic capacity and maximal respiratory capacity in a single assay in cell lysates, PBMCs, and tissue homogenates that were previously frozen. The methodology described here has the potential to be used in clinical samples to determine adaptive and maladaptive adjustments of CV function in diseases, with the added benefit of being able to use frozen samples in a high-throughput manner and to explore ATP hydrolysis as a drug target for disease treatment.
    DOI:  https://doi.org/10.26508/lsa.202201628
  28. Front Cell Neurosci. 2023 ;17 1140916
    Mitophagy in ototoxicity.
    Hezhou Han, Sainan Hu, Yue Hu, Dongliang Liu, Junbo Zhou, Xiaofang Liu, Xiulan Ma, Yaodong Dong.
      Mitochondrial dysfunction is associated with ototoxicity, which is caused by external factors. Mitophagy plays a key role in maintaining mitochondrial homeostasis and function and is regulated by a series of key mitophagy regulatory proteins and signaling pathways. The results of ototoxicity models indicate the importance of this process in the etiology of ototoxicity. A number of recent investigations of the control of cell fate by mitophagy have enhanced our understanding of the mechanisms by which mitophagy regulates ototoxicity and other hearing-related diseases, providing opportunities for targeting mitochondria to treat ototoxicity.
    Keywords:  PINK1-Parkin; autophagy receptors; mitochondria; mitophagy; ototoxicity
    DOI:  https://doi.org/10.3389/fncel.2023.1140916
  29. Res Sq. 2023 Feb 27. pii: rs.3.rs-2612547. [Epub ahead of print]
    Inflammatory and interferon gene expression signatures in patients with mitochondrial disease.
    Emily Warren, Eliza M Gordon-Lipkin, Foo Cheung, Jinguo Chen, Amrita Mukherjee, Richard Apps, John S Tsang, Jillian Jetmore, Shannon Kruk, Yuanjiu Lei, A Phillip West, Peter J McGuire.
      Background: People with mitochondrial disease (MtD) are susceptible to metabolic decompensation and neurological symptom progression in response to an infection. Increasing evidence suggests that mitochondrial dysfunction may cause chronic inflammation, which may promote hyperresponsiveness to pathogens and neurodegeneration. Methods: We collected whole blood from a cohort of MtD patients and healthy controls and performed RNAseq to examine transcriptomic differences. We performed GSEA analyses to compare our findings against existing studies to identify commonly dysregulated pathways. Results: Gene sets involved in inflammatory signaling, including type I interferons, interleukin-1β and antiviral responses, are enriched in MtD patients compared to controls. Monocyte and dendritic cell gene clusters are also enriched in MtD patients, while T cell and B cell gene sets are negatively enriched. The enrichment of antiviral response corresponds with an independent set of MELAS patients, and two mouse models of mtDNA dysfunction. Conclusions: Through the convergence of our results, we demonstrate translational evidence of systemic peripheral inflammation arising from MtD, predominantly through antiviral response gene sets. This provides key evidence linking mitochondrial dysfunction to inflammation, which may contribute to the pathogenesis of primary MtD and other chronic inflammatory disorders associated with mitochondrial dysfunction.
    DOI:  https://doi.org/10.21203/rs.3.rs-2612547/v1
  30. Genome Med. 2023 Mar 16. 15(1): 18
    Automated prioritization of sick newborns for whole genome sequencing using clinical natural language processing and machine learning.
    Bennet Peterson, Edgar Javier Hernandez, Charlotte Hobbs, Sabrina Malone Jenkins, Barry Moore, Edwin Rosales, Samuel Zoucha, Erica Sanford, Matthew N Bainbridge, Erwin Frise, Albert Oriol, Luca Brunelli, Stephen F Kingsmore, Mark Yandell.
       BACKGROUND: Rapidly and efficiently identifying critically ill infants for whole genome sequencing (WGS) is a costly and challenging task currently performed by scarce, highly trained experts and is a major bottleneck for application of WGS in the NICU. There is a dire need for automated means to prioritize patients for WGS.
    METHODS: Institutional databases of electronic health records (EHRs) are logical starting points for identifying patients with undiagnosed Mendelian diseases. We have developed automated means to prioritize patients for rapid and whole genome sequencing (rWGS and WGS) directly from clinical notes. Our approach combines a clinical natural language processing (CNLP) workflow with a machine learning-based prioritization tool named Mendelian Phenotype Search Engine (MPSE).
    RESULTS: MPSE accurately and robustly identified NICU patients selected for WGS by clinical experts from Rady Children's Hospital in San Diego (AUC 0.86) and the University of Utah (AUC 0.85). In addition to effectively identifying patients for WGS, MPSE scores also strongly prioritize diagnostic cases over non-diagnostic cases, with projected diagnostic yields exceeding 50% throughout the first and second quartiles of score-ranked patients.
    CONCLUSIONS: Our results indicate that an automated pipeline for selecting acutely ill infants in neonatal intensive care units (NICU) for WGS can meet or exceed diagnostic yields obtained through current selection procedures, which require time-consuming manual review of clinical notes and histories by specialized personnel.
    DOI:  https://doi.org/10.1186/s13073-023-01166-7
  31. Neuron. 2023 Mar 03. pii: S0896-6273(23)00123-X. [Epub ahead of print]
    Gasdermin-E mediates mitochondrial damage in axons and neurodegeneration.
    Dylan V Neel, Himanish Basu, Georgia Gunner, Matthew D Bergstresser, Richard M Giadone, Haeji Chung, Rui Miao, Vicky Chou, Eliza Brody, Xin Jiang, Edward Lee, Michelle E Watts, Christine Marques, Aaron Held, Brian Wainger, Clotilde Lagier-Tourenne, Yong-Jie Zhang, Leonard Petrucelli, Tracey L Young-Pearse, Alice S Chen-Plotkin, Lee L Rubin, Judy Lieberman, Isaac M Chiu.
      Mitochondrial dysfunction and axon loss are hallmarks of neurologic diseases. Gasdermin (GSDM) proteins are executioner pore-forming molecules that mediate cell death, yet their roles in the central nervous system (CNS) are not well understood. Here, we find that one GSDM family member, GSDME, is expressed by both mouse and human neurons. GSDME plays a role in mitochondrial damage and axon loss. Mitochondrial neurotoxins induced caspase-dependent GSDME cleavage and rapid localization to mitochondria in axons, where GSDME promoted mitochondrial depolarization, trafficking defects, and neurite retraction. Frontotemporal dementia (FTD)/amyotrophic lateral sclerosis (ALS)-associated proteins TDP-43 and PR-50 induced GSDME-mediated damage to mitochondria and neurite loss. GSDME knockdown protected against neurite loss in ALS patient iPSC-derived motor neurons. Knockout of GSDME in SOD1G93A ALS mice prolonged survival, ameliorated motor dysfunction, rescued motor neuron loss, and reduced neuroinflammation. We identify GSDME as an executioner of neuronal mitochondrial dysfunction that may contribute to neurodegeneration.
    Keywords:  ALS; FTD; axon degeneration; cell death; gasdermins; innate immunity; mitochondria; neurodegeneration; neuroimmunology; pyroptosis
    DOI:  https://doi.org/10.1016/j.neuron.2023.02.019
  32. Nature. 2023 Mar 15.
    Lactate regulates cell cycle by remodeling the anaphase promoting complex.
    Weihai Liu, Yun Wang, Luiz H M Bozi, Patrick Fischer, Mark P Jedrychowski, Haopeng Xiao, Tao Wu, Narek Darabedian, Xiadi He, Evanna L Mills, Nils Burger, Sanghee Shin, Anita Reddy, Hans-Georg Sprenger, Nhien Tran, Sally Winther, Stephen M Hinshaw, Jingnan Shen, Hyuk-Soo Seo, Kijun Song, Andrew Z Xu, Luke Sebastian, Jean Zhao, Sirano Dhe-Paganon, Jianwei Che, Steven P Gygi, Haribabu Arthanari, Edward T Chouchani.
      Lactate is abundant in rapidly dividing cells due to the requirement for elevated glucose catabolism to support proliferation1-6. However, it is not known whether accumulated lactate affects the proliferative state. Here, we deploy a systematic approach to determine lactate-dependent regulation of proteins across the human proteome. From these data, we elucidate a mechanism of cell cycle regulation whereby accumulated lactate remodels the anaphase promoting complex (APC/C). Remodeling of APC/C in this way is caused by direct inhibition of the SUMO protease SENP1 by lactate. We discover that accumulated lactate binds and inhibits SENP1 by forming a complex with zinc in the SENP1 active site. SENP1 inhibition by lactate stabilizes SUMOylation of two residues on APC4, which drives UBE2C binding to APC/C. This direct regulation of APC/C by lactate stimulates timed degradation of cell cycle proteins, and efficient mitotic exit in proliferative human cells. The above mechanism is initiated upon mitotic entry when lactate abundance reaches its apex. In this way, accumulation of lactate communicates the consequences of a nutrient replete growth phase to stimulate timed opening of APC/C, cell division, and proliferation. Conversely, persistent accumulation of lactate drives aberrant APC/C remodeling and can overcome anti-mitotic pharmacology via mitotic slippage. Taken together, we define a biochemical mechanism through which lactate directly regulates protein function to control cell cycle and proliferation.
    DOI:  https://doi.org/10.1038/s41586-023-05939-3
  33. Protein Cell. 2022 Aug 23. pii: pwac037. [Epub ahead of print]
    4E-BP1 counteracts human mesenchymal stem cell senescence via maintaining mitochondrial homeostasis.
    Yifang He, Qianzhao Ji, Zeming Wu, Yusheng Cai, Jian Yin, Yiyuan Zhang, Sheng Zhang, Xiaoqian Liu, Weiqi Zhang, Guang-Hui Liu, Si Wang, Moshi Song, Jing Qu.
      Although the mTOR-4E-BP1 signaling pathway is implicated in aging and aging-related disorders, the role of 4E-BP1 in regulating human stem cell homeostasis remains largely unknown. Here, we report that the expression of 4E-BP1 decreases along with the senescence of human mesenchymal stem cells (hMSCs). Genetic inactivation of 4E-BP1 in hMSCs compromises mitochondrial respiration, increases mitochondrial reactive oxygen species (ROS) production, and accelerates cellular senescence. Mechanistically, the absence of 4E-BP1 destabilizes proteins in mitochondrial respiration complexes, especially several key subunits of complex III including UQCRC2. Ectopic expression of 4E-BP1 attenuates mitochondrial abnormalities and alleviates cellular senescence in 4E-BP1-deficient hMSCs as well as in physiologically aged hMSCs. These f indings together demonstrate that 4E-BP1 functions as a geroprotector to mitigate human stem cell senescence and maintain mitochondrial homeostasis, particularly for the mitochondrial respiration complex III, thus providing a new potential target to counteract human stem cell senescence.
    Keywords:  4E-BP1; aging; mitochondria
    DOI:  https://doi.org/10.1093/procel/pwac037
  34. Redox Biol. 2023 Mar 04. pii: S2213-2317(23)00053-8. [Epub ahead of print]62 102652
    SIRT3 improved peroxisomes-mitochondria interplay and prevented cardiac hypertrophy via preserving PEX5 expression.
    Minghui Wang, Yanqing Ding, Yuehuai Hu, Zeyu Li, Wenwei Luo, Peiqing Liu, Zhuoming Li.
      The present study identified a novel mechanism underlying the protective effect of Sirtuin 3 (SIRT3) against pathological cardiac hypertrophy, beyond its well-accepted role as a deacetylase in mitochondria. SIRT3 modulates the peroxisomes-mitochondria interplay by preserving the expression of peroxisomal biogenesis factor 5 (PEX5), thereby improving mitochondrial function. Downregulation of PEX5 was observed in the hearts of Sirt3-/- mice and angiotensin II-induced cardiac hypertrophic mice, as well as in cardiomyocytes with SIRT3 silencing. PEX5 knockdown abolished the protective effect of SIRT3 against cardiomyocyte hypertrophy, whereas PEX5 overexpression alleviated the hypertrophic response induced by SIRT3 inhibition. PEX5 was involved in the regulation of SIRT3 in mitochondrial homeostasis, including mitochondrial membrane potential, mitochondrial dynamic balance, mitochondrial morphology and ultrastructure, as well as ATP production. In addition, SIRT3 alleviated peroxisomal abnormalities in hypertrophic cardiomyocytes via PEX5, as implied by improvement of peroxisomal biogenesis and ultrastructure, as well as increase of peroxisomal catalase and repression of oxidative stress. Finally, the role of PEX5 as a key regulator of the peroxisomes-mitochondria interplay was confirmed, since peroxisomal defects caused by PEX5 deficiency led to mitochondrial impairment. Taken together, these observations indicate that SIRT3 could maintain mitochondrial homeostasis by preserving the peroxisomes-mitochondria interplay via PEX5. Our findings provide a new understanding of the role of SIRT3 in mitochondrial regulation via interorganelle communication in cardiomyocytes.
    Keywords:  Cardiomyocyte hypertrophy; Mitochondria-peroxisomes crosstalk; PEX5; SIRT3
    DOI:  https://doi.org/10.1016/j.redox.2023.102652
  35. Nat Med. 2023 Mar 16.
    Genetic association analysis of 77,539 genomes reveals rare disease etiologies.
    Genomics England Research Consortium
      The genetic etiologies of more than half of rare diseases remain unknown. Standardized genome sequencing and phenotyping of large patient cohorts provide an opportunity for discovering the unknown etiologies, but this depends on efficient and powerful analytical methods. We built a compact database, the 'Rareservoir', containing the rare variant genotypes and phenotypes of 77,539 participants sequenced by the 100,000 Genomes Project. We then used the Bayesian genetic association method BeviMed to infer associations between genes and each of 269 rare disease classes assigned by clinicians to the participants. We identified 241 known and 19 previously unidentified associations. We validated associations with ERG, PMEPA1 and GPR156 by searching for pedigrees in other cohorts and using bioinformatic and experimental approaches. We provide evidence that (1) loss-of-function variants in the Erythroblast Transformation Specific (ETS)-family transcription factor encoding gene ERG lead to primary lymphoedema, (2) truncating variants in the last exon of transforming growth factor-β regulator PMEPA1 result in Loeys-Dietz syndrome and (3) loss-of-function variants in GPR156 give rise to recessive congenital hearing impairment. The Rareservoir provides a lightweight, flexible and portable system for synthesizing the genetic and phenotypic data required to study rare disease cohorts with tens of thousands of participants.
    DOI:  https://doi.org/10.1038/s41591-023-02211-z
  36. Biochem Biophys Res Commun. 2023 Mar 08. pii: S0006-291X(23)00292-9. [Epub ahead of print]655 25-34
    Identification of a mitochondrial targeting sequence in cathepsin D and its localization in mitochondria.
    Naoki Ikari, Hirofumi Arakawa.
      Cathepsin D (CTSD) is a major lysosomal protease harboring an N-terminal signal peptide (amino acids 1-20) to enable vesicular transport from endoplasmic reticulum to lysosomes. Here, we report the possibility of a mitochondrial targeting sequence and mitochondrial localization of CTSD in cells. Live-cell imaging analysis with C-terminal enhanced green fluorescent protein-tagged CTSD (EGFP-CTSD) indicated that CTSD localizes to mitochondria. CTSD amino acids 21-35 are responsible for its mitochondrial localization, which exhibit typical features of mitochondrial targeting sequences, and are evolutionarily conserved. A proteinase K protection assay and sucrose gradient analysis showed that a small population of endogenous CTSD molecules exists in mitochondria. These results suggest that CTSD is a dual-targeted protein that may localize in both lysosomes and mitochondria.
    Keywords:  Cathepsin D; Dual targeting protein; Live-cell imaging; Mitochondrial localization; Mitochondrial targeting sequence
    DOI:  https://doi.org/10.1016/j.bbrc.2023.03.016
  37. Life Sci Alliance. 2023 May;pii: e202201806. [Epub ahead of print]6(5):
    A protocol for single nucleus RNA-seq from frozen skeletal muscle.
    Tyler Gb Soule, Carly S Pontifex, Nicole Rosin, Matthew M Joel, Sukyoung Lee, Minh Dang Nguyen, Sameer Chhibber, Gerald Pfeffer.
      Single-cell technologies are a method of choice to obtain vast amounts of cell-specific transcriptional information under physiological and diseased states. Myogenic cells are resistant to single-cell RNA sequencing because of their large, multinucleated nature. Here, we report a novel, reliable, and cost-effective method to analyze frozen human skeletal muscle by single-nucleus RNA sequencing. This method yields all expected cell types for human skeletal muscle and works on tissue frozen for long periods of time and with significant pathological changes. Our method is ideal for studying banked samples with the intention of studying human muscle disease.
    DOI:  https://doi.org/10.26508/lsa.202201806
  38. Methods Mol Biol. 2023 ;2629 331-347
    Statistical Methods for Disease Risk Prediction with Genotype Data.
    Xiaoxuan Xia, Yexian Zhang, Yingying Wei, Maggie Haitian Wang.
      Single-nucleotide polymorphism (SNP) is the basic unit to understand the heritability of complex traits. One attractive application of the susceptible SNPs is to construct prediction models for assessing disease risk. Here, we introduce prediction methods for human traits using SNPs data, including the polygenic risk score (PRS), linear mixed models (LMMs), penalized regressions, and methods for controlling population stratification.
    Keywords:  Complex disease prediction; Linear mixed model; Penalized regression; Polygenic risk score; Population stratification; Single-nucleotide polymorphism
    DOI:  https://doi.org/10.1007/978-1-0716-2986-4_15
  39. Mitochondrion. 2023 Mar 13. pii: S1567-7249(23)00029-6. [Epub ahead of print]
    Exploring the role of mitochondria transfer/transplant and their long-non-coding RNAs in regenerative therapies for skin aging.
    Sebastian Peñaherrera, Cristina Ruiz, Verónica Castañeda, Kathryn Livingston, Diego Barba, Verónica A Burzio, Andrés Caicedo, Keshav K Singh.
      Advancing age and environmental stressors lead to mitochondrial dysfunction in the skin, inducing premature aging, impaired regeneration, and greater risk of cancer. Cells rely on the communication between the mitochondria and the nucleus by tight regulation of long non-coding RNAs (lncRNAs) to avoid premature aging and maintain healthy skin. LncRNAs act as key regulators of cell proliferation, differentiation, survival, and maintenance of skin structure. However, research on how the lncRNAs are dysregulated during aging and due to stressors is needed to develop therapies to regenerate skin's function and structure. In this article, we discuss how age and environmental stressors may alter lncRNA homeodynamics, compromising cell survival and skin health, and how these factors may become inducers of skin aging. We describe skin cell types and how they depend on mitochondrial function and lncRNAs. We also provide a list of mitochondria localized and nuclear lncRNAs that can serve to better understand skin aging. Using bioinformatic prediction tools, we predict possible functions of lncRNAs based on their subcellular localization. We also search for experimentally determined protein interactions and the biological processes involved. Finally, we provide therapeutic strategies based on gene editing and mitochondria transfer/transplant (AMT/T) to restore lncRNA regulation and skin health. This article offers a unique perspective in understanding and defining the therapeutic potential of mitochondria localized lncRNAs (mt-lncRNAs) produced and AMT/T to treat skin aging and related diseases.
    Keywords:  AMT/T; Skin; aging; artificial mitochondrial transfer / transplant; gene editing; lncRNAs; mitochondria
    DOI:  https://doi.org/10.1016/j.mito.2023.02.012
  40. Nature. 2023 Mar 15.
    Spatial epigenome-transcriptome co-profiling of mammalian tissues.
    Di Zhang, Yanxiang Deng, Petra Kukanja, Eneritz Agirre, Marek Bartosovic, Mingze Dong, Cong Ma, Sai Ma, Graham Su, Shuozhen Bao, Yang Liu, Yang Xiao, Gorazd B Rosoklija, Andrew J Dwork, J John Mann, Kam W Leong, Maura Boldrini, Liya Wang, Maximilian Haeussler, Benjamin J Raphael, Yuval Kluger, Gonçalo Castelo-Branco, Rong Fan.
      Emerging spatial technologies, including spatial transcriptomics and spatial epigenomics, are becoming powerful tools for profiling of cellular states in the tissue context1-5. However, current methods capture only one layer of omics information at a time, precluding the possibility of examining the mechanistic relationship across the central dogma of molecular biology. Here, we present two technologies for spatially resolved, genome-wide, joint profiling of the epigenome and transcriptome by cosequencing chromatin accessibility and gene expression, or histone modifications (H3K27me3, H3K27ac or H3K4me3) and gene expression on the same tissue section at near-single-cell resolution. These were applied to embryonic and juvenile mouse brain, as well as adult human brain, to map how epigenetic mechanisms control transcriptional phenotype and cell dynamics in tissue. Although highly concordant tissue features were identified by either spatial epigenome or spatial transcriptome we also observed distinct patterns, suggesting their differential roles in defining cell states. Linking epigenome to transcriptome pixel by pixel allows the uncovering of new insights in spatial epigenetic priming, differentiation and gene regulation within the tissue architecture. These technologies are of great interest in life science and biomedical research.
    DOI:  https://doi.org/10.1038/s41586-023-05795-1
  41. Front Cell Dev Biol. 2023 ;11 1021920
    Stepwise use of genomics and transcriptomics technologies increases diagnostic yield in Mendelian disorders.
    Estelle Colin, Yannis Duffourd, Martin Chevarin, Emilie Tisserant, Simon Verdez, Julien Paccaud, Ange-Line Bruel, Frédéric Tran Mau-Them, Anne-Sophie Denommé-Pichon, Julien Thevenon, Hana Safraou, Thomas Besnard, Alice Goldenberg, Benjamin Cogné, Bertrand Isidor, Julian Delanne, Arthur Sorlin, Sébastien Moutton, Mélanie Fradin, Christèle Dubourg, Magali Gorce, Dominique Bonneau, Salima El Chehadeh, François-Guillaume Debray, Martine Doco-Fenzy, Kevin Uguen, Nicolas Chatron, Bernard Aral, Nathalie Marle, Paul Kuentz, Anne Boland, Robert Olaso, Jean-François Deleuze, Damien Sanlaville, Patrick Callier, Christophe Philippe, Christel Thauvin-Robinet, Laurence Faivre, Antonio Vitobello.
      Purpose: Multi-omics offer worthwhile and increasingly accessible technologies to diagnostic laboratories seeking potential second-tier strategies to help patients with unresolved rare diseases, especially patients clinically diagnosed with a rare OMIM (Online Mendelian Inheritance in Man) disease. However, no consensus exists regarding the optimal diagnostic care pathway to adopt after negative results with standard approaches. Methods: In 15 unsolved individuals clinically diagnosed with recognizable OMIM diseases but with negative or inconclusive first-line genetic results, we explored the utility of a multi-step approach using several novel omics technologies to establish a molecular diagnosis. Inclusion criteria included a clinical autosomal recessive disease diagnosis and single heterozygous pathogenic variant in the gene of interest identified by first-line analysis (60%-9/15) or a clinical diagnosis of an X-linked recessive or autosomal dominant disease with no causative variant identified (40%-6/15). We performed a multi-step analysis involving short-read genome sequencing (srGS) and complementary approaches such as mRNA sequencing (mRNA-seq), long-read genome sequencing (lrG), or optical genome mapping (oGM) selected according to the outcome of the GS analysis. Results: SrGS alone or in combination with additional genomic and/or transcriptomic technologies allowed us to resolve 87% of individuals by identifying single nucleotide variants/indels missed by first-line targeted tests, identifying variants affecting transcription, or structural variants sometimes requiring lrGS or oGM for their characterization. Conclusion: Hypothesis-driven implementation of combined omics technologies is particularly effective in identifying molecular etiologies. In this study, we detail our experience of the implementation of genomics and transcriptomics technologies in a pilot cohort of previously investigated patients with a typical clinical diagnosis without molecular etiology.
    Keywords:  RNA-seq; clinical diagnoses; genome sequencing; long-read sequencing; optical genome mapping
    DOI:  https://doi.org/10.3389/fcell.2023.1021920
  42. Mol Cell. 2023 Mar 16. pii: S1097-2765(23)00157-0. [Epub ahead of print]83(6): 819-823
    The multifaceted roles of mitochondria.
    Thomas J Pucadyil, Jerry Edward Chipuk, Ying Liu, Luke O'Neill, Quan Chen.
      Much more than the "powerhouse" of the cell, mitochondria have emerged as critical hubs involved in metabolism, cell death, inflammation, signaling, and stress responses. To open our mitochondria focus issue, we asked several scientists to share the unanswered questions, emerging themes, and topics of investigation that excite them.
    DOI:  https://doi.org/10.1016/j.molcel.2023.02.030
  43. BMC Bioinformatics. 2023 Mar 16. 24(1): 98
    nPoRe: n-polymer realigner for improved pileup-based variant calling.
    Tim Dunn, David Blaauw, Reetuparna Das, Satish Narayanasamy.
      Despite recent improvements in nanopore basecalling accuracy, germline variant calling of small insertions and deletions (INDELs) remains poor. Although precision and recall for single nucleotide polymorphisms (SNPs) now exceeds 99.5%, INDEL recall remains below 80% for standard R9.4.1 flow cells. We show that read phasing and realignment can recover a significant portion of false negative INDELs. In particular, we extend Needleman-Wunsch affine gap alignment by introducing new gap penalties for more accurately aligning repeated n-polymer sequences such as homopolymers ([Formula: see text]) and tandem repeats ([Formula: see text]). At the same precision, haplotype phasing improves INDEL recall from 63.76 to [Formula: see text] and nPoRe realignment improves it further to [Formula: see text].
    Keywords:  Alignment; Copy number; Germline variant calling; Homopolymer; N-polymer; Nanopore sequencing; Short tandem repeat; Variable gap penalty
    DOI:  https://doi.org/10.1186/s12859-023-05193-4
  44. Res Sq. 2023 Feb 27. pii: rs.3.rs-2603446. [Epub ahead of print]
    Mutations in DARS2 result in global dysregulation of mRNA metabolism and splicing.
    Shiqi Guang, Brett O'Brien, Amena Smith Fine, Mingyao Ying, Ali Fatemi, Christina Nemeth.
      Leukoencephalopathy with brain stem and spinal cord involvement and lactate elevation (LBSL) is a rare neurological disorder caused by the mutations in the DARS2 gene, which encodes the mitochondrial aspartyl-tRNA synthetase. The objective of this study was to understand the impact of DARS2 mutations on cell processes through evaluation of LBSL patient stem cell derived cerebral organoids and neurons. We generated human cerebral organoids (hCOs) from induced pluripotent stem cells (iPSCs) of seven LBSL patients and three healthy controls using an unguided protocol. Single cells from 70-day-old hCOs underwent SMART-seq2 sequencing and multiple bioinformatic analysis tools were applied to high-resolution gene and transcript expression analyses. To confirm hCO findings, iPSC-derived neurons (iNs) were generated by overexpressing Neurogenin 2 using lentiviral vector to study neuronal growth, splicing of DARS2 exon 3 and DARS2 protein expression. Global gene expression analysis demonstrated dysregulation of a number of genes involved in mRNA metabolism and splicing processes within LBSL hCOs. Importantly, there were distinct and divergent gene expression profiles based on the nature of the DARS2 mutation. At the transcript level, pervasive differential transcript usage and differential spliced exon events that are involved in protein translation and metabolism were identified in LBSL hCOs. Single-cell analysis of DARS2 (exon 3) showed that some LBSL cells exclusively express transcripts lacking exon 3, indicating that not all LBSL cells can benefit from the "leaky" nature common to splice site mutations. Live cell imaging revealed neuronal growth defects of LBSL iNs, which was consistent with the finding of downregulated expression of genes related to neuronal differentiation in LBSL hCOs. DARS2 protein was downregulated in iNs compared to iPSCs, caused by increased exclusion of exon 3. At the gene- and transcript-level, we uncovered that dysregulated RNA splicing, protein translation and metabolism may underlie at least some of the pathophysiological mechanisms in LBSL. The scope and complexity of our data imply that DARS2 is potentially involved in transcription regulation beyond its canonical role of aminoacylation. Nevertheless, our work highlights transcript-level dysregulation as a critical, and relatively unexplored, mechanism linking genetic data with neurodegenerative disorders.
    DOI:  https://doi.org/10.21203/rs.3.rs-2603446/v1
  45. Biochem J. 2023 Mar 15. 480(5): 319-333
    From the 'black box' to 'domino effect' mechanism: what have we learned from the structures of respiratory complex I.
    Leonid A Sazanov.
      My group and myself have studied respiratory complex I for almost 30 years, starting in 1994 when it was known as a L-shaped giant 'black box' of bioenergetics. First breakthrough was the X-ray structure of the peripheral arm, followed by structures of the membrane arm and finally the entire complex from Thermus thermophilus. The developments in cryo-EM technology allowed us to solve the first complete structure of the twice larger, ∼1 MDa mammalian enzyme in 2016. However, the mechanism coupling, over large distances, the transfer of two electrons to pumping of four protons across the membrane remained an enigma. Recently we have solved high-resolution structures of mammalian and bacterial complex I under a range of redox conditions, including catalytic turnover. This allowed us to propose a robust and universal mechanism for complex I and related protein families. Redox reactions initially drive conformational changes around the quinone cavity and a long-distance transfer of substrate protons. These set up a stage for a series of electrostatically driven proton transfers along the membrane arm ('domino effect'), eventually resulting in proton expulsion from the distal antiporter-like subunit. The mechanism radically differs from previous suggestions, however, it naturally explains all the unusual structural features of complex I. In this review I discuss the state of knowledge on complex I, including the current most controversial issues.
    Keywords:  complex I; electron transfer; membrane protein structure; proton pumping; respiratory chain
    DOI:  https://doi.org/10.1042/BCJ20210285
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