bims-mitdis 2023-05-07 papers

bims-mitdis

Biomed News

on Mitochondrial disorders

Issue of 2023–05–07
thirty-six papers selected by
Catalina Vasilescu, Helmholz Munich

Multi-omics identifies large mitoribosomal subunit instability caused by pathogenic MRPL39 variants as a cause of pediatric onset mitochondrial disease.
Evaluation of endpoints for the study and diagnosis of mitochondrial toxicity and disease: A narrative review.
Trends and prospects in mitochondrial genome editing.
Mitochondrial protein import clogging as a mechanism of disease.
A Comprehensive Approach to Sample Preparation for Electron Microscopy and the Assessment of Mitochondrial Morphology in Tissue and Cultured Cells.
Nanopore sequencing identifies a higher frequency and expanded spectrum of mitochondrial DNA deletion mutations in human aging.
Molecular basis of translation termination at noncanonical stop codons in human mitochondria.
Assaying Mitochondrial Function by Multiparametric Flow Cytometry.
Case report: Transient lactate elevation by intravenous insulin infusion therapy for diabetic ketoacidosis in a patient with mitochondrial DNA 3243 A > G mutation: A glycolysis rebooting syndrome?
Assaying Mitochondrial Respiration as an Indicator of Cellular Metabolism and Fitness.
A DddA ortholog-based and transactivator-assisted nuclear and mitochondrial cytosine base editors with expanded target compatibility.
The ER-mitochondria interface, where Ca2+ and cell death meet.
The mitochondrial unfolded protein response regulates hippocampal neural stem cell aging.
A mitochondrial cytopathy presenting with persistent troponin elevation: case report.
Mitochondrial decline in the ageing old world primate retina: Little evidence for difference between the centre and periphery.
Methionine restriction constrains lipoylation and activates mitochondria for nitrogenic synthesis of amino acids.
Deciphering the pathogenesis of retinopathy associated with carnitine palmitoyltransferase I deficiency in zebrafish model.
Oxaloacetate regulates complex II respiration in brown fat: dependence on UCP1 expression.
Activation of the cGAS-STING innate immune response in cells with deficient mitochondrial topoisomerase TOP1MT.
Inter-organelle cross-talk supports acetyl-coenzyme A homeostasis and lipogenesis under metabolic stress.
Development of a fluorescence screening assay for binding partners of the iron-sulfur mitochondrial protein mitoNEET.
Acetylcarnitine shuttling links mitochondrial metabolism to histone acetylation and lipogenesis.
MICU1 occludes the mitochondrial calcium uniporter in divalent-free conditions.
Highly-multiplexed and efficient long-amplicon PacBio and Nanopore sequencing of hundreds of full mitochondrial genomes.
MCU gain-and loss-of-function models define the duality of mitochondrial calcium uptake in heart failure.
Rare-disease researchers pioneer a unique approach to clinical trials.
Mitochondrial Cardiomyopathy: Distinctive Cardiac Phenotype Detected with Cardiovascular MRI.
Cardiogenic shock in a woman with a mitochondrial cardiomyopathy: a case report.
Mitophagy regulation in aging and neurodegenerative disease.
A neuron-specific Isca1 knockout rat developments multiple mitochondrial dysfunction syndromes.
Aberrant splicing prediction across human tissues.
The mitochondrial protein TIMM44 is required for angiogenesis in vitro and in vivo.
Molecular Mechanisms of Neurodegeneration in Parkinson's Disease.
Harnessing mitochondrial transplantation to sustain cardiac function: Another step forward.
Massively parallel base editing to map variant effects in human hematopoiesis.
Machine learning modeling identifies hypertrophic cardiomyopathy subtypes with genetic signature.

Hum Mol Genet. 2023 May 03. pii: ddad069. [Epub ahead of print]

Multi-omics identifies large mitoribosomal subunit instability caused by pathogenic MRPL39 variants as a cause of pediatric onset mitochondrial disease.

Sumudu S C Amarasekera, Daniella H Hock, Nicole J Lake, Sarah E Calvo, Sabine W Grønborg, Emma I Krzesinski, David J Amor, Michael C Fahey, Cas Simons, Flemming Wibrand, Vamsi K Mootha, Monkol Lek, Sebastian Lunke, Zornitza Stark, Elsebet Østergaard, John Christodoulou, David R Thorburn, David A Stroud, Alison G Compton.

MRPL39 encodes one of 52 proteins comprising the large subunit of the mitochondrial ribosome (mitoribosome). In conjunction with 30 proteins in the small subunit, the mitoribosome synthesizes the 13 subunits of the mitochondrial oxidative phosphorylation or OXPHOS system encoded by mitochondrial DNA. We used multi-omics and gene matching to identify three unrelated individuals with biallelic variants in MRPL39 presenting with multisystem diseases with severity ranging from lethal, infantile onset (Leigh syndrome spectrum) to milder with survival into adulthood. Clinical exome sequencing of known disease genes failed to diagnose these patients; however quantitative proteomics identified a specific decrease in the abundance of large but not small mitoribosomal subunits in fibroblasts from the two patients with severe phenotype. Re-analysis of exome sequencing led to the identification of candidate single heterozygous variants in mitoribosomal genes MRPL39 (both patients) and MRPL15. Genome sequencing identified a shared deep intronic MRPL39 variant predicted to generate a cryptic exon, with transcriptomics and targeted studies providing further functional evidence for causation. The patient with milder disease was homozygous for a missense variant identified through trio exome sequencing. Our study highlights the utility of quantitative proteomics in detection of protein signatures and in characterization of gene-disease associations in exome-unsolved patients. We describe Relative Complex Abundance analysis of proteomics data, a sensitive method that can identify defects in OXPHOS disorders to a similar or greater sensitivity to the traditional enzymology. Relative Complex Abundance has potential utility for functional validation or prioritization in many hundreds of inherited rare diseases where protein complex assembly is disrupted.

DOI: https://doi.org/10.1093/hmg/ddad069
Mutagenesis. 2023 May 05. pii: gead010. [Epub ahead of print]

Evaluation of endpoints for the study and diagnosis of mitochondrial toxicity and disease: A narrative review.

Prashamsa Gharti, Jessica F Fletcher, Katherine E Chapman.

Mitochondrial DNA mutation and toxicity have been linked to several inherited and acquired diseases; however, these are challenging to diagnose and characterise due to clinical and genetic heterogeneity. This review investigates current techniques for the analysis of mitochondrial perturbations, and novel, emerging endpoints for routine application within the clinical setting. Particular focus is given to the biochemistry of the mitochondria influencing each endpoint and the relation of these to toxicity. Current approaches such as the use of metabolic markers (e.g., lactate production), and muscle biopsies to measure mitochondrial proteins were found to lack specificity. Newly emerging identified endpoints were: fibroblast growth factor-21, glucose uptake, mitochondrial membrane potential, mitochondrial morphology, mtDNA heteroplasmy, and mutation of mtDNA and nuclear DNA. Owed to the advancement in genetic analysis techniques, it is suggested by this review that genotypic endpoints of mtDNA mutation and heteroplasmy show particular promise as indicators of mitochondrial disease. It is, however, acknowledged that any single endpoint in isolation offers limited information; therefore, it is recommended that analysis of several endpoints simultaneously will offer the greatest benefit in terms of disease diagnosis and study. It is hoped that this review further highlights the need for advancement in understanding mitochondrial disease.

DOI: https://doi.org/10.1093/mutage/gead010
Exp Mol Med. 2023 May 01.

Trends and prospects in mitochondrial genome editing.

Hong Thi Lam Phan, Hyunji Lee, Kyoungmi Kim.

Mitochondria are of fundamental importance in programmed cell death, cellular metabolism, and intracellular calcium concentration modulation, and inheritable mitochondrial disorders via mitochondrial DNA (mtDNA) mutation cause several diseases in various organs and systems. Nevertheless, mtDNA editing, which plays an essential role in the treatment of mitochondrial disorders, still faces several challenges. Recently, programmable editing tools for mtDNA base editing, such as cytosine base editors derived from DddA (DdCBEs), transcription activator-like effector (TALE)-linked deaminase (TALED), and zinc finger deaminase (ZFD), have emerged with considerable potential for correcting pathogenic mtDNA variants. In this review, we depict recent advances in the field, including structural biology and repair mechanisms, and discuss the prospects of using base editing tools on mtDNA to broaden insight into their medical applicability for treating mitochondrial diseases.

DOI: https://doi.org/10.1038/s12276-023-00973-7
Elife. 2023 May 02. pii: e84330. [Epub ahead of print]12

Mitochondrial protein import clogging as a mechanism of disease.

Liam P Coyne, Xiaowen Wang, Jiyao Song, Ebbing de Jong, Karin Schneider, Paul T Massa, Frank A Middleton, Thomas Becker, Xin Jie Chen.

  Mitochondrial biogenesis requires the import of >1,000 mitochondrial preproteins from the cytosol. Most studies on mitochondrial protein import are focused on the core import machinery. Whether and how the biophysical properties of substrate preproteins affect overall import efficiency is underexplored. Here, we show that protein traffic into mitochondria can be disrupted by amino acid substitutions in a single substrate preprotein. Pathogenic missense mutations in ADP/ATP translocase 1 (ANT1), and its yeast homolog Aac2, cause the protein to accumulate along the protein import pathway, thereby obstructing general protein translocation into mitochondria. This impairs mitochondrial respiration, cytosolic proteostasis and cell viability independent of ANT1's nucleotide transport activity. The mutations act synergistically, as double mutant Aac2/ANT1 cause severe clogging primarily at the Translocase of the Outer Membrane (TOM) complex. This confers extreme toxicity in yeast. In mice, expression of a super-clogger ANT1 variant led to neurodegeneration and an age-dependent dominant myopathy that phenocopy ANT1-induced human disease, suggesting clogging as a mechanism of disease. More broadly, this work implies the existence of uncharacterized amino acid requirements for mitochondrial carrier proteins to avoid clogging and subsequent disease.

Keywords:  S. cerevisiae; biochemistry; chemical biology; mouse

DOI:  https://doi.org/10.7554/eLife.84330
Adv Biol (Weinh). 2023 May 04. e2200202

A Comprehensive Approach to Sample Preparation for Electron Microscopy and the Assessment of Mitochondrial Morphology in Tissue and Cultured Cells.

Antentor Hinton, Prasanna Katti, Trace A Christensen, Margaret Mungai, Jianqiang Shao, Liang Zhang, Sergey Trushin, Ahmad Alghanem, Adam Jaspersen, Rachel E Geroux, Kit Neikirk, Michelle Biete, Edgar Garza Lopez, Bryanna Shao, Zer Vue, Larry Vang, Heather K Beasley, Andrea G Marshall, Dominique Stephens, Steven Damo, Jessica Ponce, Christopher K E Bleck, Innes Hicsasmaz, Sandra A Murray, Ranthony A C Edmonds, Andres Dajles, Young Do Koo, Serif Bacevac, Jeffrey L Salisbury, Renata O Pereira, Brian Glancy, Eugenia Trushina, E Dale Abel.

  Mitochondria respond to metabolic demands of the cell and to incremental damage, in part, through dynamic structural changes that include fission (fragmentation), fusion (merging of distinct mitochondria), autophagic degradation (mitophagy), and biogenic interactions with the endoplasmic reticulum (ER). High resolution study of mitochondrial structural and functional relationships requires rapid preservation of specimens to reduce technical artifacts coupled with quantitative assessment of mitochondrial architecture. A practical approach for assessing mitochondrial fine structure using two dimensional and three dimensional high-resolution electron microscopy is presented, and a systematic approach to measure mitochondrial architecture, including volume, length, hyperbranching, cristae morphology, and the number and extent of interaction with the ER is described. These methods are used to assess mitochondrial architecture in cells and tissue with high energy demand, including skeletal muscle cells, mouse brain tissue, and Drosophila muscles. The accuracy of assessment is validated in cells and tissue with deletion of genes involved in mitochondrial dynamics.

Keywords:  automated serial block-face SEM; focused ion beam SEM; mitochondria-endoplasmic reticulum communication; mitochondrial dynamics; mitochondrial morphology; serial-section TEM

DOI:  https://doi.org/10.1002/adbi.202200202
Aging Cell. 2023 May 03. e13842

Nanopore sequencing identifies a higher frequency and expanded spectrum of mitochondrial DNA deletion mutations in human aging.

Amy R Vandiver, Austin N Hoang, Allen Herbst, Cathy C Lee, Judd M Aiken, Debbie McKenzie, Michael A Teitell, Winston Timp, Jonathan Wanagat.

  Mitochondrial DNA (mtDNA) deletion mutations cause many human diseases and are linked to age-induced mitochondrial dysfunction. Mapping the mutation spectrum and quantifying mtDNA deletion mutation frequency is challenging with next-generation sequencing methods. We hypothesized that long-read sequencing of human mtDNA across the lifespan would detect a broader spectrum of mtDNA rearrangements and provide a more accurate measurement of their frequency. We employed nanopore Cas9-targeted sequencing (nCATS) to map and quantitate mtDNA deletion mutations and develop analyses that are fit-for-purpose. We analyzed total DNA from vastus lateralis muscle in 15 males ranging from 20 to 81 years of age and substantia nigra from three 20-year-old and three 79-year-old men. We found that mtDNA deletion mutations detected by nCATS increased exponentially with age and mapped to a wider region of the mitochondrial genome than previously reported. Using simulated data, we observed that large deletions are often reported as chimeric alignments. To address this, we developed two algorithms for deletion identification which yield consistent deletion mapping and identify both previously reported and novel mtDNA deletion breakpoints. The identified mtDNA deletion frequency measured by nCATS correlates strongly with chronological age and predicts the deletion frequency as measured by digital PCR approaches. In substantia nigra, we observed a similar frequency of age-related mtDNA deletions to those observed in muscle samples, but noted a distinct spectrum of deletion breakpoints. NCATS-mtDNA sequencing allows the identification of mtDNA deletions on a single-molecule level, characterizing the strong relationship between mtDNA deletion frequency and chronological aging.

Keywords:  DNA sequencing; aging; human; mitochondrial DNA; skeletal muscle; substantia nigra

DOI:  https://doi.org/10.1111/acel.13842
Science. 2023 May 05. 380(6644): 531-536

Molecular basis of translation termination at noncanonical stop codons in human mitochondria.

Martin Saurer, Marc Leibundgut, Hima Priyanka Nadimpalli, Alain Scaiola, Tanja Schönhut, Richard G Lee, Stefan J Siira, Oliver Rackham, René Dreos, Tea Lenarčič, Eva Kummer, David Gatfield, Aleksandra Filipovska, Nenad Ban.

The genetic code that specifies the identity of amino acids incorporated into proteins during protein synthesis is almost universally conserved. Mitochondrial genomes feature deviations from the standard genetic code, including the reassignment of two arginine codons to stop codons. The protein required for translation termination at these noncanonical stop codons to release the newly synthesized polypeptides is not currently known. In this study, we used gene editing and ribosomal profiling in combination with cryo-electron microscopy to establish that mitochondrial release factor 1 (mtRF1) detects noncanonical stop codons in human mitochondria by a previously unknown mechanism of codon recognition. We discovered that binding of mtRF1 to the decoding center of the ribosome stabilizes a highly unusual conformation in the messenger RNA in which the ribosomal RNA participates in specific recognition of the noncanonical stop codons.

DOI: https://doi.org/10.1126/science.adf9890
Methods Mol Biol. 2023 ;2644 65-80

Assaying Mitochondrial Function by Multiparametric Flow Cytometry.

Hannah C Sheehan, Jonathan L Tilly, Dori C Woods.

  Flow cytometry has been a vital tool in cell biology for decades based on its versatile ability to detect and quantifiably measure both physical and chemical attributes of individual cells within a larger population. More recently, advances in flow cytometry have enabled nanoparticle detection. This is particularly applicable to mitochondria, which, as intracellular organelles have distinct subpopulations that can be evaluated based on differences in functional, physical, and chemical attributes, in a manner analogous to cells. This includes distinctions based on size, mitochondrial membrane potential (ΔΨm), chemical properties, and protein expression on the outer mitochondrial membrane in intact, functional organelles and internally in fixed samples. This method allows for multiparametric analysis of subpopulations of mitochondria, as well as collection for downstream analysis down to the level of a single organelle. The present protocol describes a framework for analysis and sorting mitochondria by flow cytometry, termed fluorescence activated mitochondrial sorting (FAMS), based on the separation of individual mitochondria belonging to subpopulations of interest using fluorescent dyes and antibody labeling.

Keywords:  Analytical tools; Flow cytometry; Mitochondria; Mitochondrial heterogeneity; Organelles

DOI:  https://doi.org/10.1007/978-1-0716-3052-5_5
Front Cardiovasc Med. 2023 ;10 1144925

Case report: Transient lactate elevation by intravenous insulin infusion therapy for diabetic ketoacidosis in a patient with mitochondrial DNA 3243 A > G mutation: A glycolysis rebooting syndrome?

Wataru Ohwada, Hidemichi Kouzu, Tatsuya Sato, Kahomi Sazawa, Azumi Matsui, Nobutaka Nagano, Masayuki Koyama, Noriko Ogasawara, Akifumi Takada, Toshiyuki Yano, Masato Furuhashi.

  Mitochondrial disease, most cases of which are caused by mitochondrial DNA (mtDNA) mutation, is present with multiple phenotypes including diabetes mellitus, sensorineural hearing loss, cardiomyopathy, muscle weakness, renal dysfunction, and encephalopathy, depending on the degree of heteroplasmy. While mitochondria play an important role in intracellular glucose and lactate metabolism in insulin-sensitive tissues such as muscles, appropriate strategies for glycemic control have not yet been established in a patient with mitochondrial disease, which is often complicated by myopathy. Here, we describe the history of a 40-year-old man with mtDNA 3243A > G who had sensorineural hearing loss, cardiomyopathy, muscle wasting, and diabetes mellitus with stage 3 chronic kidney disease. He developed mild diabetic ketoacidosis (DKA) in the process of treatment for poor glycemic control with severe latent hypoglycemia. According to the standard therapy for DKA, he was treated with continuous intravenous insulin infusion therapy, which unexpectedly resulted in an abrupt and transient elevation in blood lactate levels without exacerbation of heart failure and kidney function. Since blood lactate levels are determined by the balance between lactate production and consumption, an abrupt and transient lactate elevation following intravenous insulin injection therapy may reflect not only enhanced glycolysis in insulin-sensitive tissues with mitochondrial dysfunction but also decreased lactate consumption in the sarcopenic skeletal muscle and failing heart. Intravenous insulin infusion therapy in patients with mitochondrial disease may unmask derangements of intracellular glucose metabolism in response to insulin signaling.

Keywords:  cardiomyopathy; case report; diabetic ketoacidosis; glycolysis; lactate metabolism; mitochondrial disease

DOI:  https://doi.org/10.3389/fcvm.2023.1144925
Methods Mol Biol. 2023 ;2644 3-14

Assaying Mitochondrial Respiration as an Indicator of Cellular Metabolism and Fitness.

Natalia Smolina, Aleksandr Khudiakov, Anna Kostareva.

  Mitochondrial respiration is an essential component of cellular metabolism. It is a process of energy conversion through enzymatically mediated reactions, the energy of taken-up substrates transformed to the ATP production. Seahorse equipment allows to measure oxygen consumption in living cells and estimate key parameters of mitochondrial respiration in real-time mode. Four key mitochondrial respiration parameters could be measured: basal respiration, ATP-production coupled respiration, maximal respiration, and proton leak. This approach demands the application of mitochondrial inhibitors-oligomycin to inhibit ATP synthase, FCCP-to uncouple the inner mitochondrial membrane and allow maximum electron flux through the electron transport chain, rotenone, and antimycin A to inhibit complexes I and III, respectively. This chapter describes two protocols of seahorse measurements performed on iPSC-derived cardiomyocytes and TAZ knock-out C2C12 cell line.

Keywords:  Cell viability; Cellular respiration; Knock-out cells; Mitochondrial function; iPSC-derived cardiomyocytes

DOI:  https://doi.org/10.1007/978-1-0716-3052-5_1
Mol Cell. 2023 Apr 30. pii: S1097-2765(23)00283-6. [Epub ahead of print]

A DddA ortholog-based and transactivator-assisted nuclear and mitochondrial cytosine base editors with expanded target compatibility.

Junfan Guo, Wenxia Yu, Min Li, Hongyu Chen, Jie Liu, Xiaowen Xue, Jianxiang Lin, Shisheng Huang, Wenjie Shu, Xingxu Huang, Zhen Liu, Shengqi Wang, Yunbo Qiao.

  Bacterial double-stranded DNA (dsDNA) cytosine deaminase DddAtox-derived cytosine base editor (DdCBE) and its evolved variant, DddA11, guided by transcription-activator-like effector (TALE) proteins, enable mitochondrial DNA (mtDNA) editing at TC or HC (H = A, C, or T) sequence contexts, while it remains relatively unattainable for GC targets. Here, we identified a dsDNA deaminase originated from a Roseburia intestinalis interbacterial toxin (riDddAtox) and generated CRISPR-mediated nuclear DdCBEs (crDdCBEs) and mitochondrial CBEs (mitoCBEs) using split riDddAtox, which catalyzed C-to-T editing at both HC and GC targets in nuclear and mitochondrial genes. Moreover, transactivator (VP64, P65, or Rta) fusion to the tail of DddAtox- or riDddAtox-mediated crDdCBEs and mitoCBEs substantially improved nuclear and mtDNA editing efficiencies by up to 3.5- and 1.7-fold, respectively. We also used riDddAtox-based and Rta-assisted mitoCBE to efficiently stimulate disease-associated mtDNA mutations in cultured cells and in mouse embryos with conversion frequencies of up to 58% at non-TC targets.

Keywords:  DdCBE; Roseburia intestinalis; mitoCBE; mitochondrial DNA; mtDNA; non-TC; nuclear DNA; transactivator

DOI:  https://doi.org/10.1016/j.molcel.2023.04.012
Cell Calcium. 2023 Apr 25. pii: S0143-4160(23)00055-6. [Epub ahead of print]112 102743

The ER-mitochondria interface, where Ca2+ and cell death meet.

Ian de Ridder, Martijn Kerkhofs, Fernanda O Lemos, Jens Loncke, Geert Bultynck, Jan B Parys.

  Endoplasmic reticulum (ER)-mitochondria contact sites are crucial to allow Ca2+ flux between them and a plethora of proteins participate in tethering both organelles together. Inositol 1,4,5-trisphosphate receptors (IP3Rs) play a pivotal role at such contact sites, participating in both ER-mitochondria tethering and as Ca2+-transport system that delivers Ca2+ from the ER towards mitochondria. At the ER-mitochondria contact sites, the IP3Rs function as a multi-protein complex linked to the voltage-dependent anion channel 1 (VDAC1) in the outer mitochondrial membrane, via the chaperone glucose-regulated protein 75 (GRP75). This IP3R-GRP75-VDAC1 complex supports the efficient transfer of Ca2+ from the ER into the mitochondrial intermembrane space, from which the Ca2+ ions can reach the mitochondrial matrix through the mitochondrial calcium uniporter. Under physiological conditions, basal Ca2+ oscillations deliver Ca2+ to the mitochondrial matrix, thereby stimulating mitochondrial oxidative metabolism. However, when mitochondrial Ca2+ overload occurs, the increase in [Ca2+] will induce the opening of the mitochondrial permeability transition pore, thereby provoking cell death. The IP3R-GRP75-VDAC1 complex forms a hub for several other proteins that stabilize the complex and/or regulate the complex's ability to channel Ca2+ into the mitochondria. These proteins and their mechanisms of action are discussed in the present review with special attention for their role in pathological conditions and potential implication for therapeutic strategies.

Keywords:  Apoptosis; ER-mitochondria tether; IP(3) receptor; Metabolism; Mitochondria-associated membranes

DOI:  https://doi.org/10.1016/j.ceca.2023.102743
Cell Metab. 2023 Apr 28. pii: S1550-4131(23)00139-0. [Epub ahead of print]

The mitochondrial unfolded protein response regulates hippocampal neural stem cell aging.

Chih-Ling Wang, Rika Ohkubo, Wei-Chieh Mu, Wei Chen, Jiang Lan Fan, Zehan Song, Ayane Maruichi, Peter H Sudmant, Angela O Pisco, Dena B Dubal, Na Ji, Danica Chen.

  Aging results in a decline in neural stem cells (NSCs), neurogenesis, and cognitive function, and evidence is emerging to demonstrate disrupted adult neurogenesis in the hippocampus of patients with several neurodegenerative disorders. Here, single-cell RNA sequencing of the dentate gyrus of young and old mice shows that the mitochondrial protein folding stress is prominent in activated NSCs/neural progenitors (NPCs) among the neurogenic niche, and it increases with aging accompanying dysregulated cell cycle and mitochondrial activity in activated NSCs/NPCs in the dentate gyrus. Increasing mitochondrial protein folding stress results in compromised NSC maintenance and reduced neurogenesis in the dentate gyrus, neural hyperactivity, and impaired cognitive function. Reducing mitochondrial protein folding stress in the dentate gyrus of old mice improves neurogenesis and cognitive function. These results establish the mitochondrial protein folding stress as a driver of NSC aging and suggest approaches to improve aging-associated cognitive decline.

Keywords:  SIRT1; SIRT2; SIRT3; SIRT6; SIRT7; cognitive aging; mitochondrial unfolded protein response; neural stem cell aging; sirtuin; stem cell aging

DOI:  https://doi.org/10.1016/j.cmet.2023.04.012
Eur Heart J Case Rep. 2023 Apr;7(4): ytad132

A mitochondrial cytopathy presenting with persistent troponin elevation: case report.

Anish Krishnan, Kathy Wu, Laila Girgis, Roger Pamphlett, Susan Tomlinson, Kavitha Muthiah.

   Background: Mitochondrial diseases represent an important potential cause of cardiomyopathy and should be considered in patients presenting with multisystem manifestations. Timely diagnosis of a mitochondrial disorder is needed as it can have reproductive implications for the offspring of the proband.
Case Summary: We describe a case of undifferentiated rising and persistent troponin elevation in a 70-year-old female with only mild heart failure symptoms and signs. An eventual diagnosis of a mitochondrial cytopathy was made after genetic testing, striated muscle, and endomyocardial biopsy. Multidisciplinary involvement was vital in securing the ultimate diagnosis and is a key lesson from this case. On follow up, with institution of heart failure therapy including cardiac resynchronisation device therapy there was improvement in exercise tolerance and symptoms.
Discussion: For discussion is the investigation of undifferentiated cardiomyopathies and consideration of mitochondrial disorders as an important diagnosis to exclude prior to diagnosis as an idiopathic cardiomyopathy.

Keywords:  Cardiovascular magnetic resonance imaging; Case report; Mitochondrial cytopathy; Troponin

DOI:  https://doi.org/10.1093/ehjcr/ytad132
PLoS One. 2023 ;18(5): e0273882

Mitochondrial decline in the ageing old world primate retina: Little evidence for difference between the centre and periphery.

Jaimie Hoh Kam, Harpreet Shinhmar, Michael Barry Powner, Matthew John Hayes, Asmaa Aboelnour, Glen Jeffery.

Mitochondrial decline is a key feature of ageing. The retina has more mitochondria than any other tissue and ages rapidly. To understand human retinal ageing it is critical to examine old world primates that have similar visual systems to humans, and do so across central and peripheral regions, as there is evidence for early central decline. Hence, we examine mitochondrial metrics in young and ageing Macaca fascicularis retinae. In spite of reduced ATP with age, primate mitochondrial complex activity did not decline. But mitochondrial membrane potentials were reduced significantly, and concomitantly, mitochondrial membrane permeability increased. The mitochondrial marker Tom20 declined significantly, consistent with reduced mitochondria number, while VDAC, a voltage dependent anion channel and diffusion pore associated with apoptosis increased significantly. In spite of these clear age-related changes, there was almost no evidence for regional differences between the centre and the periphery in these mitochondrial metrics. Primate cones do not die with age, but many showed marked structural decline with vacuous spaces in proximal inner segments normally occupied by endoplasmic reticulum (ER), that regulate mitochondrial autophagy. In many peripheral cones, ER was displaced by the nucleus that transposed across the outer limiting membrane and could become embedded in mitochondrial populations. These data are consistent with significant changes in retinal mitochondria in old world primate ageing but provide little if any evidence that aged central mitochondria suffer more than those in the periphery.

DOI: https://doi.org/10.1371/journal.pone.0273882
Nat Commun. 2023 May 02. 14(1): 2504

Methionine restriction constrains lipoylation and activates mitochondria for nitrogenic synthesis of amino acids.

Wen Fang, Liu Jiang, Yibing Zhu, Sen Yang, Hong Qiu, Jiou Cheng, Qingxi Liang, Zong-Cai Tu, Cunqi Ye.

Methionine restriction (MR) provides metabolic benefits in many organisms. However, mechanisms underlying the MR-induced effect remain incompletely understood. Here, we show in the budding yeast S. cerevisiae that MR relays a signal of S-adenosylmethionine (SAM) deprivation to adapt bioenergetic mitochondria to nitrogenic anabolism. In particular, decreases in cellular SAM constrain lipoate metabolism and protein lipoylation required for the operation of the tricarboxylic acid (TCA) cycle in the mitochondria, leading to incomplete glucose oxidation with an exit of acetyl-CoA and α-ketoglutarate from the TCA cycle to the syntheses of amino acids, such as arginine and leucine. This mitochondrial response achieves a trade-off between energy metabolism and nitrogenic anabolism, which serves as an effector mechanism promoting cell survival under MR.

DOI: https://doi.org/10.1038/s41467-023-38289-9
Biochem Biophys Res Commun. 2023 Apr 26. pii: S0006-291X(23)00527-2. [Epub ahead of print]664 100-107

Deciphering the pathogenesis of retinopathy associated with carnitine palmitoyltransferase I deficiency in zebrafish model.

Zulvikar Syambani Ulhaq, Yukiko Ogino, William Ka Fai Tse.

  Fatty acid oxidation disorders (FAODs) are a group of rare genetic metabolic disorders caused by mutations in genes responsible for transporting and metabolizing fatty acids in the mitochondria. One crucial enzyme involved in this process is carnitine palmitoyltransferase I (CPT1), which transports long-chain fatty acids to the mitochondrial matrix for beta-oxidation. Defects in beta-oxidation enzymes often lead to pigmentary retinopathy; however, the underlying mechanisms are not entirely understood. To investigate FAOD and its impact on the retina, we employed zebrafish as a model organism. Specifically, we used antisense-mediated knockdown strategies to target the cpt1a gene and examined the resulting retinal phenotypes. We demonstrated that the cpt1a MO-injected fish significantly reduced the length of connecting cilia and severely affected photoreceptor cell development. Moreover, our findings highlight that the loss of functional cpt1a disrupted energy homeostasis in the retina, leading to lipid droplet deposition and promoting ferroptosis, which is likely attributed to the photoreceptor degeneration and visual impairments observed in the cpt1a morphants.

Keywords:  Ciliopathies; Fatty acid oxidation disorders; Ferroptosis; Photoreceptor degeneration

DOI:  https://doi.org/10.1016/j.bbrc.2023.04.096
Am J Physiol Cell Physiol. 2023 May 01.

Oxaloacetate regulates complex II respiration in brown fat: dependence on UCP1 expression.

Ritu Som, Brian D Fink, Liping Yu, William I Sivitz.

  We previously found that skeletal muscle mitochondria incubated at low membrane potential (ΔΨ) or interscapular brown adipose tissue (IBAT) mitochondria, wherein ΔΨ is intrinsically low, accumulate oxaloacetate (OAA) in amounts sufficient to inhibit complex II respiration. We proposed a mechanism wherein low ΔΨ reduces reverse electron transport (RET) to complex I causing a low NADH/NAD+ ratio favoring malate conversion to OAA. To further assess the mechanism and its physiologic relevance we carried out studies of mice with inherently different levels of IBAT mitochondrial inner membrane potential. Isolated complex II (succinate)-energized IBAT mitochondria from obesity resistant 129SVE mice compared to obesity prone C57BL/6J displayed greater UCP1 expression, similar O2 flux despite lower ΔΨ, similar OAA concentrations, and similar NADH/NAD+. When GDP was added to inhibit UCP1, 129SVE IBAT mitochondria, despite their lower ΔΨ, exhibited much lower respiration, 2-fold greater OAA concentrations, much lower RET (as marked by ROS), and much lower NADH and NADH/NAD+ ratios compared to the C57BL/6J IBAT mitochondria. UCP1 knock-out abolished OAA accumulation by succinate-energized mitochondria associated with markedly greater ΔΨ, ROS, and NADH, but equal or greater O2 flux compared to WT mitochondria. GDP addition, compared to no GDP, increased ΔΨ and complex II respiration in wildtype mice associated with much less OAA. Respiration on complex I substrates followed the more classical dynamics of greater respiration at lower ΔΨ. These findings support the above-mentioned mechanism for OAA- and ΔΨ-dependent complex II respiration and support its physiological relevance.

Keywords:  brown adipose tissue; mitochondria; mitochondrial complex II; oxaloacetate; succinate dehydrogenase

DOI:  https://doi.org/10.1152/ajpcell.00565.2022
Hum Mol Genet. 2023 May 02. pii: ddad062. [Epub ahead of print]

Activation of the cGAS-STING innate immune response in cells with deficient mitochondrial topoisomerase TOP1MT.

Iman Al Khatib, Jingti Deng, Yuanjiu Lei, Sylvia Torres-Odio, Gladys R Rojas, Laura E Newman, Brian K Chung, Andrew Symes, Hongliang Zhang, Shar-Yin N Huang, Yves Pommier, Aneal Khan, Gerald S Shadel, A Phillip West, William T Gibson, Timothy E Shutt.

The recognition that cytosolic mtDNA activates cGAS-STING innate immune signaling has unlocked novel disease mechanisms. Here, an uncharacterized variant predicted to affect TOP1MT function, P193L, was discovered in a family with multiple early-onset autoimmune diseases, including Systemic Lupus Erythematosus (SLE). Although there was no previous genetic association between TOP1MT and autoimmune disease, the role of TOP1MT as a regulator of mtDNA led us to investigate whether TOP1MT could mediate the release of mtDNA to the cytosol, where it could then activate the cGAS-STING innate immune pathway known to be activated in SLE and other autoimmune diseases. Through analysis of cells with reduced TOP1MT expression, we show that loss of TOP1MT results in release of mtDNA to the cytosol, which activates the cGAS-STING pathway. We also characterized the P193L variant for its ability to rescue several TOP1MT functions when expressed in TOP1MT knockout cells. We show that the P193L variant is not fully functional, as its re-expression at high levels was unable to rescue mitochondrial respiration deficits, and only showed partial rescue for other functions, including repletion of mtDNA replication following depletion, nucleoid size, steady state mtDNA transcripts levels, and mitochondrial morphology. Additionally, expression of P193L at endogenous levels was unable to rescue mtDNA release-mediated cGAS-STING signaling. Overall, we report a link between TOP1MT and mtDNA release leading to cGAS-STING activation. Moreover, we show that the P193L variant has partial loss of function that may contribute to autoimmune disease susceptibility via cGAS-STING mediated activation of the innate immune system.

DOI: https://doi.org/10.1093/hmg/ddad062
Sci Adv. 2023 May 03. 9(18): eadf0138

Inter-organelle cross-talk supports acetyl-coenzyme A homeostasis and lipogenesis under metabolic stress.

Ramya S Kuna, Avi Kumar, Karl A Wessendorf-Rodriguez, Hector Galvez, Courtney R Green, Grace H McGregor, Thekla Cordes, Reuben J Shaw, Robert U Svensson, Christian M Metallo.

Proliferating cells rely on acetyl-CoA to support membrane biogenesis and acetylation. Several organelle-specific pathways are available for provision of acetyl-CoA as nutrient availability fluctuates, so understanding how cells maintain acetyl-CoA homeostasis under such stresses is critically important. To this end, we applied 13C isotope tracing cell lines deficient in these mitochondrial [ATP-citrate lyase (ACLY)]-, cytosolic [acetyl-CoA synthetase (ACSS2)]-, and peroxisomal [peroxisomal biogenesis factor 5 (PEX5)]-dependent pathways. ACLY knockout in multiple cell lines reduced fatty acid synthesis and increased reliance on extracellular lipids or acetate. Knockout of both ACLY and ACSS2 (DKO) severely stunted but did not entirely block proliferation, suggesting that alternate pathways can support acetyl-CoA homeostasis. Metabolic tracing and PEX5 knockout studies link peroxisomal oxidation of exogenous lipids as a major source of acetyl-CoA for lipogenesis and histone acetylation in cells lacking ACLY, highlighting a role for inter-organelle cross-talk in supporting cell survival in response to nutrient fluctuations.

DOI: https://doi.org/10.1126/sciadv.adf0138
Bioorg Med Chem Lett. 2023 May 01. pii: S0960-894X(23)00188-9. [Epub ahead of print] 129310

Development of a fluorescence screening assay for binding partners of the iron-sulfur mitochondrial protein mitoNEET.

Ebenezer Newton, Sarah A Starcovic, Michael Menze, Mary E Konkle, Timothy E Long, Lori A Hazlehurst, Jason D Huber, Aaron R Robart, Werner J Geldenhuys.

  MitoNEET belongs to the CDGSH Iron-Sulfur Domain (cisd)-gene family of proteins and is a [2Fe-2S] cluster-containing protein found on the outer membrane of mitochondria. The specific functions of mitoNEET/CISD1 remain to be fully elucidated, but the protein is involved in regulating mitochondrial bioenergetics in several metabolic diseases. Unfortunately, drug discovery efforts targeting mitoNEET to improve metabolic disorders are hampered by the lack of ligand-binding assays for this mitochondrial protein. We have developed a protocol amenable for high-throughput screening (HTS) assay, by modifying an ATP fluorescence polarization method to facilitate drug discovery targeting mitoNEET. Based on our observation that adenosine triphosphate (ATP) interacts with mitoNEET, ATP-fluorescein was used during assay development. We established a novel binding assay suitable for both 96- or 384-well plate formats with tolerance for the presence of 2% v/v dimethyl sulfoxide (DMSO). We determined the IC50-values for a set of benzesulfonamide derivatives and found the novel assay reliably ranked the binding-affinities of compounds compared to radioactive binding assay with human recombinant mitoNEET. The developed assay platform is crucial in identifying novel chemical probes for metabolic diseases. It will accelerate drug discovery targeting mitoNEET and potentially other members of the CISD gene family.

Keywords:  CISD1; CISD2; OXPHOS; [2Fe-2S]; cancer; stroke, TBI

DOI:  https://doi.org/10.1016/j.bmcl.2023.129310
Sci Adv. 2023 May 03. 9(18): eadf0115

Acetylcarnitine shuttling links mitochondrial metabolism to histone acetylation and lipogenesis.

Luke T Izzo, Sophie Trefely, Christina Demetriadou, Jack M Drummond, Takuya Mizukami, Nina Kuprasertkul, Aimee T Farria, Phuong T T Nguyen, Nivitha Murali, Lauren Reich, Daniel S Kantner, Joshua Shaffer, Hayley Affronti, Alessandro Carrer, Andrew Andrews, Brian C Capell, Nathaniel W Snyder, Kathryn E Wellen.

The metabolite acetyl-CoA is necessary for both lipid synthesis in the cytosol and histone acetylation in the nucleus. The two canonical precursors to acetyl-CoA in the nuclear-cytoplasmic compartment are citrate and acetate, which are processed to acetyl-CoA by ATP-citrate lyase (ACLY) and acyl-CoA synthetase short-chain 2 (ACSS2), respectively. It is unclear whether other substantial routes to nuclear-cytosolic acetyl-CoA exist. To investigate this, we generated cancer cell lines lacking both ACLY and ACSS2 [double knockout (DKO) cells]. Using stable isotope tracing, we show that both glucose and fatty acids contribute to acetyl-CoA pools and histone acetylation in DKO cells and that acetylcarnitine shuttling can transfer two-carbon units from mitochondria to cytosol. Further, in the absence of ACLY, glucose can feed fatty acid synthesis in a carnitine responsive and carnitine acetyltransferase (CrAT)-dependent manner. The data define acetylcarnitine as an ACLY- and ACSS2-independent precursor to nuclear-cytosolic acetyl-CoA that can support acetylation, fatty acid synthesis, and cell growth.

DOI: https://doi.org/10.1126/sciadv.adf0115
Proc Natl Acad Sci U S A. 2023 May 09. 120(19): e2218999120

MICU1 occludes the mitochondrial calcium uniporter in divalent-free conditions.

Macarena Rodríguez-Prados, Elena Berezhnaya, Maria Teresa Castromonte, Sergio L Menezes-Filho, Melanie Paillard, György Hajnóczky.

  Mitochondrial Ca2+ uptake is mediated by the mitochondrial uniporter complex (mtCU) that includes a tetramer of the pore-forming subunit, MCU, a scaffold protein, EMRE, and the EF-hand regulatory subunit, MICU1 either homodimerized or heterodimerized with MICU2/3. MICU1 has been proposed to regulate Ca2+ uptake via the mtCU by physically occluding the pore and preventing Ca2+ flux at resting cytoplasmic [Ca2+] (free calcium concentration) and to increase Ca2+ flux at high [Ca2+] due to cooperative activation of MICUs EF-hands. However, mtCU and MICU1 functioning when its EF-hands are unoccupied by Ca2+ is poorly studied due to technical limitations. To overcome this barrier, we have studied the mtCU in divalent-free conditions by assessing the Ru265-sensitive Na+ influx using fluorescence-based measurement of mitochondrial matrix [Na+] (free sodium concentration) rise and the ensuing depolarization and swelling. We show an increase in all these measures of Na+ uptake in MICU1KO cells as compared to wild-type (WT) and rescued MICU1KO HEK cells. However, mitochondria in WT cells and MICU1 stable-rescued cells still allowed some Ru265-sensitive Na+ influx that was prevented by MICU1 in excess upon acute overexpression. Thus, MICU1 restricts the cation flux across the mtCU in the absence of Ca2+, but even in cells with high endogenous MICU1 expression such as HEK, some mtCU seem to lack MICU1-dependent gating. We also show rearrangement of the mtCU and altered number of functional channels in MICU1KO and different rescues, and loss of MICU1 during mitoplast preparation, that together might have obscured the pore-blocking function of MICU1 in divalent-free conditions in previous studies.

Keywords:  EMRE; MICU1; Na+; mitochondrial calcium uniporter; mitoplast

DOI:  https://doi.org/10.1073/pnas.2218999120
BMC Genomics. 2023 May 02. 24(1): 229

Highly-multiplexed and efficient long-amplicon PacBio and Nanopore sequencing of hundreds of full mitochondrial genomes.

Benjamin R Karin, Selene Arellano, Laura Wang, Kayla Walzer, Aaron Pomerantz, Juan Manuel Vasquez, Kamalakar Chatla, Peter H Sudmant, Bryan H Bach, Lydia L Smith, Jimmy A McGuire.

   BACKGROUND: Mitochondrial genome sequences have become critical to the study of biodiversity. Genome skimming and other short-read based methods are the most common approaches, but they are not well-suited to scale up to multiplexing hundreds of samples. Here, we report on a new approach to sequence hundreds to thousands of complete mitochondrial genomes in parallel using long-amplicon sequencing. We amplified the mitochondrial genome of 677 specimens in two partially overlapping amplicons and implemented an asymmetric PCR-based indexing approach to multiplex 1,159 long amplicons together on a single PacBio SMRT Sequel II cell. We also tested this method on Oxford Nanopore Technologies (ONT) MinION R9.4 to assess if this method could be applied to other long-read technologies. We implemented several optimizations that make this method significantly more efficient than alternative mitochondrial genome sequencing methods.
RESULTS: With the PacBio sequencing data we recovered at least one of the two fragments for 96% of samples (~ 80-90%) with mean coverage ~ 1,500x. The ONT data recovered less than 50% of input fragments likely due to low throughput and the design of the Barcoded Universal Primers which were optimized for PacBio sequencing. We compared a single mitochondrial gene alignment to half and full mitochondrial genomes and found, as expected, increased tree support with longer alignments, though whole mitochondrial genomes were not significantly better than half mitochondrial genomes.
CONCLUSIONS: This method can effectively capture thousands of long amplicons in a single run and be used to build more robust phylogenies quickly and effectively. We provide several recommendations for future users depending on the evolutionary scale of their system. A natural extension of this method is to collect multi-locus datasets consisting of mitochondrial genomes and several long nuclear loci at once.

Keywords:  DNA barcoding; Long read sequencing; LongAmp; MinION; Plasmid; Third generation sequencing; mtDNA

DOI:  https://doi.org/10.1186/s12864-023-09277-6
bioRxiv. 2023 Apr 18. pii: 2023.04.17.537222. [Epub ahead of print]

MCU gain-and loss-of-function models define the duality of mitochondrial calcium uptake in heart failure.

Joanne F Garbincius, Timothy S Luongo, Jonathan P Lambert, Adam S Mangold, Emma K Murray, Alycia N Hildebrand, Pooja Jadiya, John W Elrod.

Background: Mitochondrial calcium ( m Ca 2+ ) uptake through the mitochondrial calcium uniporter channel (mtCU) stimulates metabolism to meet acute increases in cardiac energy demand. However, excessive m Ca 2+ uptake during stress, as in ischemia-reperfusion, initiates permeability transition and cell death. Despite these often-reported acute physiological and pathological effects, a major unresolved controversy is whether mtCU-dependent m Ca 2+ uptake and long-term elevation of cardiomyocyte m Ca 2+ contributes to the heart's adaptation during sustained increases in workload.
Objective: We tested the hypothesis that mtCU-dependent m Ca 2+ uptake contributes to cardiac adaptation and ventricular remodeling during sustained catecholaminergic stress.
Methods: Mice with tamoxifen-inducible, cardiomyocyte-specific gain (αMHC-MCM x flox-stop-MCU; MCU-Tg) or loss (αMHC-MCM x Mcu fl/fl ; Mcu -cKO) of mtCU function received 2-wk catecholamine infusion.
Results: Cardiac contractility increased after 2d of isoproterenol in control, but not Mcu -cKO mice. Contractility declined and cardiac hypertrophy increased after 1-2-wk of isoproterenol in MCU-Tg mice. MCU-Tg cardiomyocytes displayed increased sensitivity to Ca 2+ - and isoproterenol-induced necrosis. However, loss of the mitochondrial permeability transition pore (mPTP) regulator cyclophilin D failed to attenuate contractile dysfunction and hypertrophic remodeling, and increased isoproterenol-induced cardiomyocyte death in MCU-Tg mice.
Conclusions: mtCU m Ca 2+ uptake is required for early contractile responses to adrenergic signaling, even those occurring over several days. Under sustained adrenergic load excessive MCU-dependent m Ca 2+ uptake drives cardiomyocyte dropout, perhaps independent of classical mitochondrial permeability transition pore opening, and compromises contractile function. These findings suggest divergent consequences for acute versus sustained m Ca 2+ loading, and support distinct functional roles for the mPTP in settings of acute m Ca 2+ overload versus persistent m Ca 2+ stress.

DOI: https://doi.org/10.1101/2023.04.17.537222
Nat Med. 2023 May 05.

Rare-disease researchers pioneer a unique approach to clinical trials.

Mike May.

DOI: https://doi.org/10.1038/s41591-023-02333-4
Radiol Cardiothorac Imaging. 2023 Apr;5(2): e230014

Mitochondrial Cardiomyopathy: Distinctive Cardiac Phenotype Detected with Cardiovascular MRI.

Stephan A C Schoonvelde, Claudine W B Ruijmbeek, Judith M A Verhagen, Debby M E I Hellebrekers, Marcel J M Kofflard, Michelle Michels, Alexander Hirsch.

  Left ventricular hypertrophy (LVH) has a broad differential diagnosis. Pathogenic variants of mitochondrial DNA are a rare cause of LVH, and cardiac MRI is a powerful technique that may aid in differentiating such rare causes. This case report presents three siblings with a pathogenic variant of the mitochondrially encoded tRNA isoleucine (MT-TI) gene. A distinctive cardiac phenotype was detected with cardiac MRI. Extensive LVH and dilatation and decreased ejection fraction were observed with a pattern of increased T2 signal and extensive late gadolinium enhancement, which was remarkably consistent among all three siblings. Keywords: Cardiomyopathies, MR Imaging, Hypertrophic Cardiomyopathy, Mitochondrial, Inherited Cardiomyopathy, Left Ventricular Hypertrophy, Cardiovascular MRI, Late Gadolinium Enhancement Supplemental material is available for this article. © RSNA, 2023.

Keywords:  Cardiomyopathies; Cardiovascular MRI; Hypertrophic Cardiomyopathy; Inherited Cardiomyopathy; Late Gadolinium Enhancement; Left Ventricular Hypertrophy; MR Imaging; Mitochondrial

DOI:  https://doi.org/10.1148/ryct.230014
Eur Heart J Case Rep. 2023 Apr;7(4): ytad183

Cardiogenic shock in a woman with a mitochondrial cardiomyopathy: a case report.

Andrew Girard, Brittain Heindl, Stephen Clarkson, Silvio Litovsky, Eroboghene Ubogu, Coreen Schwartzlow, Jose Tallaj.

   Background: Mitochondrial cardiomyopathy (MCM) is an alteration in cardiac structure and function caused by gene mutations or deletions affecting components of the mitochondrial respiratory chain. We report a case of MCM presenting as cardiogenic shock, ultimately requiring left ventricular assist device (LVAD) placement.
Case summary: A 35-year-old woman with chronic weakness and non-ischaemic cardiomyopathy, on home dobutamine, was referred to our institution for heart transplantation evaluation. She was admitted to the hospital for suspected cardiogenic shock after laboratory tests revealed a lactate level of 5.4 mmol/L (ref: 0.5-2.2 mmol/L). Her hospital course was complicated by persistently undulating lactate levels (0.2-8.6 mmol/L) that increased with exertion and did not correlate with mixed venous oxygen saturation measurements obtained from a pulmonary artery catheter. Electrodiagnostic testing demonstrated a proximal appendicular and axial myopathy. A left deltoid muscle biopsy was performed that demonstrated evidence of a mitochondrial disease on light and electron microscopy. Muscle genetic testing revealed two large-scale mitochondrial deoxyribonucleic acid sequence deletions, confirming the diagnosis of MCM. She subsequently underwent LVAD placement, which was complicated by significant right ventricular failure requiring early mechanical support. She was ultimately discharged home with chronic inotropic support.
Discussion: Mitochondrial cardiomyopathy in adults is a diagnostic and therapeutic challenge. Prompt diagnosis should be made in patients with unknown causes of heart failure via skeletal muscle histopathology guided by electrodiagnostic studies, and targeted genetic testing in affected tissue. Outcomes in adult MCM patients who receive an LVAD are unknown and warrant further investigation.

Keywords:  Advanced heart failure; Case report; Mechanical circulatory support device; Mitochondrial DNA disease; Mitochondrial cardiomyopathy

DOI:  https://doi.org/10.1093/ehjcr/ytad183
Biophys Rev. 2023 Apr;15(2): 239-255

Mitophagy regulation in aging and neurodegenerative disease.

Trupti A Banarase, Shivkumar S Sammeta, Nitu L Wankhede, Shubhada V Mangrulkar, Sandip R Rahangdale, Manish M Aglawe, Brijesh G Taksande, Aman B Upaganlawar, Milind J Umekar, Mayur B Kale.

  Mitochondria are the primary cellular energy generators, supplying the majority of adenosine triphosphate through oxidative phosphorylation, which is necessary for neuron function and survival. Mitophagy is the metabolic process of eliminating dysfunctional or redundant mitochondria. It is a type of autophagy and it is crucial for maintaining mitochondrial and neuronal health. Impaired mitophagy leads to an accumulation of damaged mitochondria and proteins leading to the dysregulation of mitochondrial quality control processes. Recent research shows the vital role of mitophagy in neurons and the pathogenesis of major neurodegenerative diseases. Mitophagy also plays a major role in the process of aging. This review describes the alterations that are being caused in the mitophagy process at the molecular level in aging and in neurodegenerative diseases, particularly Alzheimer's, Parkinson's, and Huntington's diseases and amyotrophic lateral sclerosis, also looks at how mitophagy can be exploited as a therapeutic target for these diseases.

Keywords:  Aging; Alzheimer’s disease; Amyotrophic lateral sclerosis; Mitochondria; Mitophagy; Neurodegenerative diseases; Parkinson’s disease

DOI:  https://doi.org/10.1007/s12551-023-01057-6
Animal Model Exp Med. 2023 Apr;6(2): 155-167

A neuron-specific Isca1 knockout rat developments multiple mitochondrial dysfunction syndromes.

Hanxuan Sheng, Dan Lu, Xiaolong Qi, Yahao Ling, Jing Li, Xu Zhang, Wei Dong, Wei Chen, Shan Gao, Xiang Gao, Li Zhang, Lianfeng Zhang.

   BACKGROUND: Multiple mitochondrial dysfunction syndromes (MMDS) are rare mitochondrial diseases caused by mutation of mitochondrial iron-sulfur cluster synthesis proteins. This study established a rat model simulating MMDS5 disease in the nervous system to investigate its pathological features and neuronal death.
METHODS: We generated neuron-specific Isca1 knockout rat (Isca1flox/flox -NeuN-Cre) using CRISPR-Cas9 technology. The brain structure changes of CKO rats were studied with MRI, and the behavior abnormalities were analyzed through gait analysis and open field tests, Y maze tests and food maze tests. The pathological changes of neurons were analyzed through H&E staining, Nissl staining, and Golgi staining. Mitochondrial damage was assessed by TEM, western blot and ATP assay, and the morphology of neurons was assessed by WGA immunofluorescence to detect the death of neurons.
RESULTS: This study established the disease model of MMDS5 in the nervous system for the first time, and found that after Isca1 loss, the rats suffered from developmental retardation, epilepsy, memory impairment, massive neuronal death, reduced number of Nissl bodies and dendritic spines, mitochondrial fragmentation, cristae fracture, reduced content of respiratory chain complex protein, and reduced production of ATP. Isca1 knockout caused neuronal oncosis.
CONCLUSIONS: This rat model can be used to study the pathogenesis of MMDS. In addition, compared with human MMDS5, the rat model can survive up to 8 weeks of age, effectively extending the window of clinical treatment research, and can be used for the treatment of neurological symptoms in other mitochondrial diseases.

Keywords:  ISCA1; MMDS5; mitochondrial iron-sulfur cluster; neuron oncosis

DOI:  https://doi.org/10.1002/ame2.12318
Nat Genet. 2023 May 04.

Aberrant splicing prediction across human tissues.

Nils Wagner, Muhammed H Çelik, Florian R Hölzlwimmer, Christian Mertes, Holger Prokisch, Vicente A Yépez, Julien Gagneur.

Aberrant splicing is a major cause of genetic disorders but its direct detection in transcriptomes is limited to clinically accessible tissues such as skin or body fluids. While DNA-based machine learning models can prioritize rare variants for affecting splicing, their performance in predicting tissue-specific aberrant splicing remains unassessed. Here we generated an aberrant splicing benchmark dataset, spanning over 8.8 million rare variants in 49 human tissues from the Genotype-Tissue Expression (GTEx) dataset. At 20% recall, state-of-the-art DNA-based models achieve maximum 12% precision. By mapping and quantifying tissue-specific splice site usage transcriptome-wide and modeling isoform competition, we increased precision by threefold at the same recall. Integrating RNA-sequencing data of clinically accessible tissues into our model, AbSplice, brought precision to 60%. These results, replicated in two independent cohorts, substantially contribute to noncoding loss-of-function variant identification and to genetic diagnostics design and analytics.

DOI: https://doi.org/10.1038/s41588-023-01373-3
Cell Death Dis. 2023 May 05. 14(5): 307

The mitochondrial protein TIMM44 is required for angiogenesis in vitro and in vivo.

Zhou-Rui Ma, Hong-Peng Li, Shi-Zhong Cai, Sheng-Yang Du, Xia Chen, Jin Yao, Xu Cao, Yun-Fang Zhen, Qian Wang.

The mitochondrial integrity and function in endothelial cells are essential for angiogenesis. TIMM44 (translocase of inner mitochondrial membrane 44) is essential for integrity and function of mitochondria. Here we explored the potential function and the possible mechanisms of TIMM44 in angiogenesis. In HUVECs, human retinal microvascular endothelial cells and hCMEC/D3 brain endothelial cells, silence of TIMM44 by targeted shRNA largely inhibited cell proliferation, migration and in vitro capillary tube formation. TIMM44 silencing disrupted mitochondrial functions in endothelial cells, causing mitochondrial protein input arrest, ATP reduction, ROS production, and mitochondrial depolarization, and leading to apoptosis activation. TIMM44 knockout, by Cas9-sgRNA strategy, also disrupted mitochondrial functions and inhibited endothelial cell proliferation, migration and in vitro capillary tube formation. Moreover, treatment with MB-10 ("MitoBloCK-10"), a TIMM44 blocker, similarly induced mitochondrial dysfunction and suppressed angiogenic activity in endothelial cells. Contrarily, ectopic overexpression of TIMM44 increased ATP contents and augmented endothelial cell proliferation, migration and in vitro capillary tube formation. In adult mouse retinas, endothelial knockdown of TIMM44, by intravitreous injection of endothelial specific TIMM44 shRNA adenovirus, inhibited retinal angiogenesis, causing vascular leakage, acellular capillary growth, and retinal ganglion cells degeneration. Significant oxidative stress was detected in TIMM44-silenced retinal tissues. Moreover, intravitreous injection of MB-10 similarly induced oxidative injury and inhibited retinal angiogenesis in vivo. Together, the mitochondrial protein TIMM44 is important for angiogenesis in vitro and in vivo, representing as a novel and promising therapeutic target of diseases with abnormal angiogenesis.

DOI: https://doi.org/10.1038/s41419-023-05826-9
J Mol Biol. 2023 Apr 28. pii: S0022-2836(23)00203-6. [Epub ahead of print] 168131

Molecular Mechanisms of Neurodegeneration in Parkinson's Disease.

Jean-François Trempe, Kalle Gehring.

DOI: https://doi.org/10.1016/j.jmb.2023.168131
Mol Ther. 2023 May 03. pii: S1525-0016(23)00201-0. [Epub ahead of print]31(5): 1201-1203

Harnessing mitochondrial transplantation to sustain cardiac function: Another step forward.

Yuhao Li, Kroekkiat Chinda, Yaozu Xiang, Hao Zhou, Dachun Xu, Sang-Ging Ong, Sang-Bing Ong.

DOI: https://doi.org/10.1016/j.ymthe.2023.04.003
Cell. 2023 Apr 27. pii: S0092-8674(23)00332-X. [Epub ahead of print]

Massively parallel base editing to map variant effects in human hematopoiesis.

Jorge D Martin-Rufino, Nicole Castano, Michael Pang, Emanuelle I Grody, Samantha Joubran, Alexis Caulier, Lara Wahlster, Tongqing Li, Xiaojie Qiu, Anna Maria Riera-Escandell, Gregory A Newby, Aziz Al'Khafaji, Santosh Chaudhary, Susan Black, Chen Weng, Glen Munson, David R Liu, Marcin W Wlodarski, Kacie Sims, Jamie H Oakley, Ross M Fasano, Ramnik J Xavier, Eric S Lander, Daryl E Klein, Vijay G Sankaran.

  Systematic evaluation of the impact of genetic variants is critical for the study and treatment of human physiology and disease. While specific mutations can be introduced by genome engineering, we still lack scalable approaches that are applicable to the important setting of primary cells, such as blood and immune cells. Here, we describe the development of massively parallel base-editing screens in human hematopoietic stem and progenitor cells. Such approaches enable functional screens for variant effects across any hematopoietic differentiation state. Moreover, they allow for rich phenotyping through single-cell RNA sequencing readouts and separately for characterization of editing outcomes through pooled single-cell genotyping. We efficiently design improved leukemia immunotherapy approaches, comprehensively identify non-coding variants modulating fetal hemoglobin expression, define mechanisms regulating hematopoietic differentiation, and probe the pathogenicity of uncharacterized disease-associated variants. These strategies will advance effective and high-throughput variant-to-function mapping in human hematopoiesis to identify the causes of diverse diseases.

Keywords:  base editing; differentiation; functional screens; genome engineering; hematopoiesis; hematopoietic stem cell; primary cells; single-cell genomics

DOI:  https://doi.org/10.1016/j.cell.2023.03.035
Front Med. 2023 May 01.

Machine learning modeling identifies hypertrophic cardiomyopathy subtypes with genetic signature.

Jiaqi Dai, Tao Wang, Ke Xu, Yang Sun, Zongzhe Li, Peng Chen, Hong Wang, Dongyang Wu, Yanghui Chen, Lei Xiao, Hao Liu, Haoran Wei, Rui Li, Liyuan Peng, Ting Yu, Yan Wang, Zhongsheng Sun, Dao Wen Wang.

  Previous studies have revealed that patients with hypertrophic cardiomyopathy (HCM) exhibit differences in symptom severity and prognosis, indicating potential HCM subtypes among these patients. Here, 793 patients with HCM were recruited at an average follow-up of 32.78 ± 27.58 months to identify potential HCM subtypes by performing consensus clustering on the basis of their echocardiography features. Furthermore, we proposed a systematic method for illustrating the relationship between the phenotype and genotype of each HCM subtype by using machine learning modeling and interactome network detection techniques based on whole-exome sequencing data. Another independent cohort that consisted of 414 patients with HCM was recruited to replicate the findings. Consequently, two subtypes characterized by different clinical outcomes were identified in HCM. Patients with subtype 2 presented asymmetric septal hypertrophy associated with a stable course, while those with subtype 1 displayed left ventricular systolic dysfunction and aggressive progression. Machine learning modeling based on personal whole-exome data identified 46 genes with mutation burden that could accurately predict subtype propensities. Furthermore, the patients in another cohort predicted as subtype 1 by the 46-gene model presented increased left ventricular end-diastolic diameter and reduced left ventricular ejection fraction. By employing echocardiography and genetic screening for the 46 genes, HCM can be classified into two subtypes with distinct clinical outcomes.

Keywords:  genetic risk; hypertrophic cardiomyopathy; machine learning methods

DOI:  https://doi.org/10.1007/s11684-023-0982-1