bims-mitdis Biomed News
on Mitochondrial disorders
Issue of 2024–03–17
fifty-two papers selected by
Catalina Vasilescu, Helmholz Munich
  1. Digenic Leigh syndrome on the background of the m.11778G>A Leber hereditary optic neuropathy variant.
  2. Enhancing healthy aging with small molecules: A mitochondrial perspective.
  3. m6A RNA methylation regulates mitochondrial function.
  4. Modular Assembly of Mitochondrial β-Barrel Proteins.
  5. Mitochondria-Targeted Oligomeric α-Synuclein Induces TOM40 Degradation and Mitochondrial Dysfunction in Parkinson's Disease and Parkinsonism-Dementia of Guam.
  6. Structural basis of how MGME1 processes DNA 5' ends to maintain mitochondrial genome integrity.
  7. The genetic landscape of mitochondrial diseases in the next-generation sequencing era: a Portuguese cohort study.
  8. Mitochondria in the Central Nervous System in Health and Disease: The Puzzle of the Therapeutic Potential of Mitochondrial Transplantation.
  9. Revisiting the Mitochondrial Function and Communication in Neurodegenerative Diseases.
  10. The Ubiquitin Ligase RBX2/SAG Regulates Mitochondrial Ubiquitination and Mitophagy.
  11. Role of Mitochondrial DNA in Yeast Replicative Aging.
  12. DNM1L variant presenting as adolescent-onset sensory neuronopathy, spasticity, dystonia, and ataxia.
  13. Generation of mitochondrial replacement monkeys by female pronucleus transfer.
  14. Intracranial calcifications simulating Aicardi-Goutières syndrome in PARS2-related mitochondrial disease.
  15. Mitochondria at the Nanoscale: Physics Meets Biology-What Does It Mean for Medicine?
  16. Tracking the Activity and Position of Mitochondrial β-Barrel Proteins.
  17. The Integrated Stress Response in metabolic adaptation.
  18. Genetic heterogeneity and respiratory chain enzyme analysis in pediatric Indian patients with mitochondrial disorder: Report of novel variants in POLG1 gene and their functional implication using molecular dynamic simulation.
  19. FUS unveiled in mitochondrial DNA repair and targeted ligase-1 expression rescues repair-defects in FUS-linked motor neuron disease.
  20. Activity-dependent mitochondrial ROS signaling regulates recruitment of glutamate receptors to synapses.
  21. Next generation sequencing reveals novel compound heterozygous deletions in NDUFAF2 in a child with mitochondrial complex I deficiency, nuclear type 10.
  22. Federated learning for multi-omics: A performance evaluation in Parkinson's disease.
  23. Mitochondrial dysfunction in type 2 diabetes: A neglected path to skeletal muscle atrophy.
  24. Mitochondrial Dysfunction in Heart Failure: From Pathophysiological Mechanisms to Therapeutic Opportunities.
  25. Genetic ablation of Immt induces a lethal disruption of the MICOS complex.
  26. The loss of OPA1 accelerates intervertebral disc degeneration and osteoarthritis in aged mice.
  27. Study of an FBXO7 patient mutation reveals Fbxo7 and PI31 co-regulate proteasomes and mitochondria.
  28. Genome-wide CRISPR/Cas9 screen shows that loss of GET4 increases mitochondria-endoplasmic reticulum contact sites and is neuroprotective.
  29. How AI is being used to accelerate clinical trials.
  30. Utilizing Extracellular Vesicles for Eliminating 'Unwanted Molecules': Harnessing Nature's Structures in Modern Therapeutic Strategies.
  31. GRAF1 Acts as a Downstream Mediator of Parkin to Regulate Mitophagy in Cardiomyocytes.
  32. Whole genome sequencing increases the diagnostic rate in Charcot-Marie-Tooth disease.
  33. ROS-induced translational regulation-through spatiotemporal differences in codon recognition-is a key driver of brown adipogenesis.
  34. MitoLuc: A Luminescence-Based Assay to Study Real-Time Protein Import into Mitochondria.
  35. Mitophagy defect mediates the aging-associated hallmarks in Hutchinson-Gilford progeria syndrome.
  36. Biallelic NAA60 variants with impaired n-terminal acetylation capacity cause autosomal recessive primary familial brain calcifications.
  37. Fatty acid oxidation drives mitochondrial hydrogen peroxide production by α-ketoglutarate dehydrogenase.
  38. Examining Protein Translocation by β-Barrel Membrane Proteins Using Reconstituted Proteoliposomes.
  39. Long-chain acyl-CoA synthetase 4-mediated mitochondrial fatty acid metabolism and dendritic cell antigen presentation.
  40. Genomes in clinical care.
  41. Neuronal ageing is promoted by the decay of the microtubule cytoskeleton.
  42. Comprehensive proteomics of CSF, plasma, and urine identify DDC and other biomarkers of early Parkinson's disease.
  43. Location Matters: Mitochondrial Arginase 2 as a Treatment for Metabolic Disease?
  44. A novel homozygous mutation, c.662_672del, in DGUOK gene causing mitochondrial DNA depletion syndrome of type hepatocerebral - A case report.
  45. Specific cellular microenvironments for spatiotemporal regulation of StAR and steroid synthesis.
  46. A comparison of methods for detecting DNA methylation from long-read sequencing of human genomes.
  47. High-Throughput Single-Cell, Single-Mitochondrial DNA Assay Using Hydrogel Droplet Microfluidics.
  48. Systematic characterization of protein structural features of alternative splicing isoforms using AlphaFold 2.
  49. Nicotinamide Mononucleotide (NMN) Works in Type 2 Diabetes through Unexpected Effects in Adipose Tissue, Not by Mitochondrial Biogenesis.
  50. Phosphoglycerate mutase 5 aggravates alcoholic liver disease through disrupting VDAC-1-dependent mitochondrial integrity.
  51. Towards a biological diagnosis of PD.
  52. Polarized localization of kinesin-1 and RIC-7 drives axonal mitochondria anterograde transport.
  1. Brain. 2024 Mar 13. pii: awae057. [Epub ahead of print]
    Digenic Leigh syndrome on the background of the m.11778G>A Leber hereditary optic neuropathy variant.
    Beryll Blickhäuser, Sarah L Stenton, Christiane M Neuhofer, Elisa Floride, Victoria Nesbitt, Carl Fratter, Johannes Koch, Birgit Kauffmann, Claudia Catarino, Lea Dewi Schlieben, Robert Kopajtich, Valerio Carelli, Alfredo A Sadun, Robert McFarland, Fang Fang, Chiara La Morgia, Stéphanie Paquay, Marie Cécile Nassogne, Daniele Ghezzi, Costanza Lamperti, Saskia Wortmann, Jo Poulton, Thomas Klopstock, Holger Prokisch.
      Leigh syndrome spectrum (LSS) is a primary mitochondrial disorder defined neuropathologically by a subacute necrotizing encephalomyelopathy and characterised by bilateral basal ganglia and/or brainstem lesions. LSS is associated with variants in several mitochondrial DNA (mtDNA) genes and more than 100 nuclear genes, most often related to mitochondrial complex I (CI) dysfunction. Rarely, LSS has been reported in association with primary Leber hereditary optic neuropathy (LHON) variants of the mtDNA, coding for CI subunits (m.3460G>A in MT-ND1, m.11778G>A in MT-ND4, and m.14484T>C in MT-ND6). The underlying mechanism by which these variants manifest as LSS, a severe neurodegenerative disease, as opposed to the LHON phenotype of isolated optic neuropathy, remains an open question. Here, we analyse the exome sequencing of six probands with LSS carrying primary LHON variants, and report digenic co-occurrence of the m.11778G>A variant with damaging heterozygous variants in nuclear disease genes encoding CI subunits as a plausible explanation. Our findings suggest a digenic mechanism of disease for m.11778G>A-associated LSS, consistent with recent reports of digenic disease in individuals manifesting with LSS due to biallelic variants in the recessive LHON-associated disease gene DNAJC30 in combination with heterozygous variants in CI subunits.
    Keywords:  Leber hereditary optic neuropathy (LHON); Leigh syndrome spectrum (LSS); digenic inheritance; mitochondrial complex I (CI)
    DOI:  https://doi.org/10.1093/brain/awae057
  2. Med Res Rev. 2024 Mar 14.
    Enhancing healthy aging with small molecules: A mitochondrial perspective.
    Xiujiao Qin, Hongyuan Li, Huiying Zhao, Le Fang, Xiaohui Wang.
      The pursuit of enhanced health during aging has prompted the exploration of various strategies focused on reducing the decline associated with the aging process. A key area of this exploration is the management of mitochondrial dysfunction, a notable characteristic of aging. This review sheds light on the crucial role that small molecules play in augmenting healthy aging, particularly through influencing mitochondrial functions. Mitochondrial oxidative damage, a significant aspect of aging, can potentially be lessened through interventions such as coenzyme Q10, alpha-lipoic acid, and a variety of antioxidants. Additionally, this review discusses approaches for enhancing mitochondrial proteostasis, emphasizing the importance of mitochondrial unfolded protein response inducers like doxycycline, and agents that affect mitophagy, such as urolithin A, spermidine, trehalose, and taurine, which are vital for sustaining protein quality control. Of equal importance are methods for modulating mitochondrial energy production, which involve nicotinamide adenine dinucleotide boosters, adenosine 5'-monophosphate-activated protein kinase activators, and compounds like metformin and mitochondria-targeted tamoxifen that enhance metabolic function. Furthermore, the review delves into emerging strategies that encourage mitochondrial biogenesis. Together, these interventions present a promising avenue for addressing age-related mitochondrial degradation, thereby setting the stage for the development of innovative treatment approaches to meet this extensive challenge.
    Keywords:  aging; mitochondrial; mitophagy; redox homeostasis; unfolded protein response
    DOI:  https://doi.org/10.1002/med.22034
  3. Hum Mol Genet. 2024 Mar 14. pii: ddae029. [Epub ahead of print]
    m6A RNA methylation regulates mitochondrial function.
    Michael Kahl, Zhaofa Xu, Saravanan Arumugam, Brittany Edens, Mariafausta Fischietti, Allen C Zhu, Leonidas C Platanias, Chuan He, Xiaoxi Zhuang, Yongchao C Ma.
      RNA methylation of N6-methyladenosine (m6A) is emerging as a fundamental regulator of every aspect of RNA biology. RNA methylation directly impacts protein production to achieve quick modulation of dynamic biological processes. However, whether RNA methylation regulates mitochondrial function is not known, especially in neuronal cells which require a high energy supply and quick reactive responses. Here we show that m6A RNA methylation regulates mitochondrial function through promoting nuclear-encoded mitochondrial complex subunit RNA translation. Conditional genetic knockout of m6A RNA methyltransferase Mettl14 (Methyltransferase like 14) by Nestin-Cre together with metabolomic analysis reveals that Mettl14 knockout-induced m6A depletion significantly downregulates metabolites related to energy metabolism. Furthermore, transcriptome-wide RNA methylation profiling of wild type and Mettl14 knockout mouse brains by m6A-Seq shows enrichment of methylation on mitochondria-related RNA. Importantly, loss of m6A leads to a significant reduction in mitochondrial respiratory capacity and membrane potential. These functional defects are paralleled by the reduced expression of mitochondrial electron transport chain complexes, as well as decreased mitochondrial super-complex assembly and activity. Mechanistically, m6A depletion decreases the translational efficiency of methylated RNA encoding mitochondrial complex subunits through reducing their association with polysomes, while not affecting RNA stability. Together, these findings reveal a novel role for RNA methylation in regulating mitochondrial function. Given that mitochondrial dysfunction and RNA methylation have been increasingly implicate in neurodegenerative disorders, our findings not only provide insights into fundamental mechanisms regulating mitochondrial function, but also open up new avenues for understanding the pathogenesis of neurological diseases.
    Keywords:  m6A RNA methylation; metabolomics; mitochondria; neurological disease; transcriptomics
    DOI:  https://doi.org/10.1093/hmg/ddae029
  4. Methods Mol Biol. 2024 ;2778 201-220
    Modular Assembly of Mitochondrial β-Barrel Proteins.
    Rituparna Bhowmik, Fabian den Brave, Thomas Becker.
      Mitochondrial β-barrel proteins fulfill crucial roles in the biogenesis and function of the cell organelle. They mediate the import and membrane insertion of proteins and transport of small metabolites and ions. All β-barrel proteins are made as precursors on cytosolic ribosomes and are imported into mitochondria. The β-barrel proteins fold and assemble with partner proteins in the outer membrane. The in vitro import of radiolabelled proteins into isolated mitochondria is a powerful tool to investigate the import of β-barrel proteins, the folding of the β-barrel proteins, and their assembly into protein complexes. Altogether, the in vitro import assay is a versatile and crucial assay to analyze the mechanisms of the biogenesis of mitochondrial β-barrel proteins.
    Keywords:  Blue native electrophoresis; Mitochondria; Protein import assay; SAM complex; TOM complex; β-barrel proteins
    DOI:  https://doi.org/10.1007/978-1-0716-3734-0_13
  5. Res Sq. 2024 Feb 21. pii: rs.3.rs-3970470. [Epub ahead of print]
    Mitochondria-Targeted Oligomeric α-Synuclein Induces TOM40 Degradation and Mitochondrial Dysfunction in Parkinson's Disease and Parkinsonism-Dementia of Guam.
    Muralidhar Hegde, Velmarini Vasquez, Manohar Kodavati, Joy Mitra, Indira Vendula, Dale Hamilton, Ralph Garruto, K S Rao.
      Mitochondrial dysfunction is a central aspect of Parkinson's disease (PD) pathology, yet the underlying mechanisms are not fully understood. This study investigates the link between α-Synuclein (α-Syn) pathology and the loss of translocase of the outer mitochondrial membrane 40 (TOM40), unraveling its implications for mitochondrial dysfunctions in neurons. We discovered that TOM40 protein depletion occurs in the brains of patients with Guam Parkinsonism Dementia (Guam PD) and cultured neurons expressing α-Syn proteinopathy, notably, without corresponding changes in TOM40 mRNA levels. Cultured neurons expressing α-Syn mutants, with or without a mitochondria-targeting signal (MTS) underscore the role of α-Syn's mitochondrial localization in inducing TOM40 degradation. Parkinson's Disease related etiological factors, such as 6-hydroxy dopamine or ROS/metal ions stress, which promote α-Syn oligomerization, exacerbate TOM40 depletion in PD patient-derived cells with SNCA gene triplication. Although α-Syn interacts with both TOM40 and TOM20 in the outer mitochondrial membrane, degradation is selective for TOM40, which occurs via the ubiquitin-proteasome system (UPS) pathway. Our comprehensive analyses using Seahorse technology, mitochondrial DNA sequencing, and damage assessments, demonstrate that mutant α-Syn-induced TOM40 loss results in mitochondrial dysfunction, characterized by reduced membrane potential, accumulation of mtDNA damage, deletion/insertion mutations, and altered oxygen consumption rates. Notably, ectopic supplementation of TOM40 or reducing pathological forms of α-Syn using ADP-ribosylation inhibitors ameliorate these mitochondrial defects, suggesting potential therapeutic avenues. In conclusion, our findings provide crucial mechanistic insights into how α-Syn accumulation leads to TOM40 degradation and mitochondrial dysfunction, offering insights for targeted interventions to alleviate mitochondrial defects in PD.
    DOI:  https://doi.org/10.21203/rs.3.rs-3970470/v1
  6. Nucleic Acids Res. 2024 Mar 12. pii: gkae186. [Epub ahead of print]
    Structural basis of how MGME1 processes DNA 5' ends to maintain mitochondrial genome integrity.
    Eric Y C Mao, Han-Yi Yen, Chyuan-Chuan Wu.
      Mitochondrial genome maintenance exonuclease 1 (MGME1) helps to ensure mitochondrial DNA (mtDNA) integrity by serving as an ancillary 5'-exonuclease for DNA polymerase γ. Curiously, MGME1 exhibits unique bidirectionality in vitro, being capable of degrading DNA from either the 5' or 3' end. The structural basis of this bidirectionally and, particularly, how it processes DNA from the 5' end to assist in mtDNA maintenance remain unclear. Here, we present a crystal structure of human MGME1 in complex with a 5'-overhang DNA, revealing that MGME1 functions as a rigid DNA clamp equipped with a single-strand (ss)-selective arch, allowing it to slide on single-stranded DNA in either the 5'-to-3' or 3'-to-5' direction. Using a nuclease activity assay, we have dissected the structural basis of MGME1-derived DNA cleavage patterns in which the arch serves as a ruler to determine the cleavage site. We also reveal that MGME1 displays partial DNA-unwinding ability that helps it to better resolve 5'-DNA flaps, providing insights into MGME1-mediated 5'-end processing of nascent mtDNA. Our study builds on previously solved MGME1-DNA complex structures, finally providing the comprehensive functional mechanism of this bidirectional, ss-specific exonuclease.
    DOI:  https://doi.org/10.1093/nar/gkae186
  7. Front Cell Dev Biol. 2024 ;12 1331351
    The genetic landscape of mitochondrial diseases in the next-generation sequencing era: a Portuguese cohort study.
    C Nogueira, C Pereira, L Silva, Mateus Laranjeira, A Lopes, R Neiva, E Rodrigues, T Campos, E Martins, A Bandeira, M Coelho, M Magalhães, J Damásio, A Gaspar, P Janeiro, A Levy Gomes, A C Ferreira, S Jacinto, J P Vieira, L Diogo, H Santos, C Mendonça, L Vilarinho.
      Introduction: Rare disorders that are genetically and clinically heterogeneous, such as mitochondrial diseases (MDs), have a challenging diagnosis. Nuclear genes codify most proteins involved in mitochondrial biogenesis, despite all mitochondria having their own DNA. The development of next-generation sequencing (NGS) technologies has revolutionized the understanding of many genes involved in the pathogenesis of MDs. In this new genetic era, using the NGS approach, we aimed to identify the genetic etiology for a suspected MD in a cohort of 450 Portuguese patients. Methods: We examined 450 patients using a combined NGS strategy, starting with the analysis of a targeted mitochondrial panel of 213 nuclear genes, and then proceeding to analyze the whole mitochondrial DNA. Results and Discussion: In this study, we identified disease-related variants in 134 (30%) analyzed patients, 88 with nuclear DNA (nDNA) and 46 with mitochondrial DNA (mtDNA) variants, most of them being pediatric patients (66%), of which 77% were identified in nDNA and 23% in mtDNA. The molecular analysis of this cohort revealed 72 already described pathogenic and 20 novel, probably pathogenic, variants, as well as 62 variants of unknown significance. For this cohort of patients with suspected MDs, the use of a customized gene panel provided a molecular diagnosis in a timely and cost-effective manner. Patients who cannot be diagnosed after this initial approach will be further selected for whole-exome sequencing. Conclusion: As a national laboratory for the study and research of MDs, we demonstrated the power of NGS to achieve a molecular etiology, expanding the mutational spectrum and proposing accurate genetic counseling in this group of heterogeneous diseases without therapeutic options.
    Keywords:  mitochondrial DNA; mitochondrial diseases; next-generation sequencing; nuclear DNA; nuclear genes; oxidative phosphorylation; respiratory chain
    DOI:  https://doi.org/10.3389/fcell.2024.1331351
  8. Cells. 2024 Feb 27. pii: 410. [Epub ahead of print]13(5):
    Mitochondria in the Central Nervous System in Health and Disease: The Puzzle of the Therapeutic Potential of Mitochondrial Transplantation.
    Kuldeep Tripathi, Dorit Ben-Shachar.
      Mitochondria, the energy suppliers of the cells, play a central role in a variety of cellular processes essential for survival or leading to cell death. Consequently, mitochondrial dysfunction is implicated in numerous general and CNS disorders. The clinical manifestations of mitochondrial dysfunction include metabolic disorders, dysfunction of the immune system, tumorigenesis, and neuronal and behavioral abnormalities. In this review, we focus on the mitochondrial role in the CNS, which has unique characteristics and is therefore highly dependent on the mitochondria. First, we review the role of mitochondria in neuronal development, synaptogenesis, plasticity, and behavior as well as their adaptation to the intricate connections between the different cell types in the brain. Then, we review the sparse knowledge of the mechanisms of exogenous mitochondrial uptake and describe attempts to determine their half-life and transplantation long-term effects on neuronal sprouting, cellular proteome, and behavior. We further discuss the potential of mitochondrial transplantation to serve as a tool to study the causal link between mitochondria and neuronal activity and behavior. Next, we describe mitochondrial transplantation's therapeutic potential in various CNS disorders. Finally, we discuss the basic and reverse-translation challenges of this approach that currently hinder the clinical use of mitochondrial transplantation.
    Keywords:  CNS; mitochondria; mitochondria and behavior; mitochondrial transplantation; neural development and function; neuropsychiatric disorders
    DOI:  https://doi.org/10.3390/cells13050410
  9. Curr Pharm Des. 2024 Mar 12.
    Revisiting the Mitochondrial Function and Communication in Neurodegenerative Diseases.
    Nitu L Wankhede, Mayur B Kale, Mohit D Umare, Sanket Lokhande, Aman B Upaganlawar, Pranay Wal, Brijesh G Taksande, Milind J Umekar, Prasanna Shama Khandige, Bhupendra Singh, Vandana Sadananda, Seema Ramniwas, Tapan Behl.
      Neurodegenerative disorders are distinguished by the progressive loss of anatomically or physiologically relevant neural systems. Atypical mitochondrial morphology and metabolic malfunction are found in many neurodegenerative disorders. Alteration in mitochondrial function can occur as a result of aberrant mitochondrial DNA, altered nuclear enzymes that interact with mitochondria actively or passively, or due to unexplained reasons. Mitochondria are intimately linked to the Endoplasmic reticulum (ER), and ER-mitochondrial communication governs several of the physiological functions and procedures that are disrupted in neurodegenerative disorders. Numerous researchers have associated these disorders with ER-mitochondrial interaction disturbance. In addition, aberrant mitochondrial DNA mutation and increased ROS production resulting in ionic imbalance and leading to functional and structural alterations in the brain as well as cellular damage may have an essential role in disease progression via mitochondrial malfunction. In this review, we explored the evidence highlighting the role of mitochondrial alterations in neurodegenerative pathways in most serious ailments, including Alzheimer's disease (AD), Parkinson's disease (PD), and Huntington's disease (HD).
    Keywords:  Alzheimer’s disease; Huntington’s disease; Neurodegenerative disorders; Parkinson’s disease; endoplasmic reticulum.; mitochondrial communication
    DOI:  https://doi.org/10.2174/0113816128286655240304070740
  10. bioRxiv. 2024 Feb 28. pii: 2024.02.24.581168. [Epub ahead of print]
    The Ubiquitin Ligase RBX2/SAG Regulates Mitochondrial Ubiquitination and Mitophagy.
    Wenjuan Wang, Ermin Li, Jianqiu Zou, Qu Chen, Juan Ayala, Yuan Wen, Md Sadikul Islam, Neal L Weintraub, David J Fulton, Qiangrong Liang, Jiliang Zhou, Jinbao Liu, Jie Li, Yi Sun, Huabo Su.
      Clearance of damaged mitochondria via mitophagy is crucial for cellular homeostasis. While the role of ubiquitin (Ub) ligase PARKIN in mitophagy has been extensively studied, increasing evidence suggests the existence of PARKIN-independent mitophagy in highly metabolically active organs such as the heart. Here, we identify a crucial role for Cullin-RING Ub ligase 5 (CRL5) in basal mitochondrial turnover in cardiomyocytes. CRL5 is a multi-subunit Ub ligase comprised by the catalytic RING box protein RBX2 (also known as SAG), scaffold protein Cullin 5 (CUL5), and a substrate-recognizing receptor. Analysis of the mitochondrial outer membrane-interacting proteome uncovered a robust association of CRLs with mitochondria. Subcellular fractionation, immunostaining, and immunogold electron microscopy established that RBX2 and Cul5, two core components of CRL5, localizes to mitochondria. Depletion of RBX2 inhibited mitochondrial ubiquitination and turnover, impaired mitochondrial membrane potential and respiration, and increased cell death in cardiomyocytes. In vivo , deletion of the Rbx2 gene in adult mouse hearts suppressed mitophagic activity, provoked accumulation of damaged mitochondria in the myocardium, and disrupted myocardial metabolism, leading to rapid development of dilated cardiomyopathy and heart failure. Similarly, ablation of RBX2 in the developing heart resulted in dilated cardiomyopathy and heart failure. Notably, the action of RBX2 in mitochondria is not dependent on PARKIN, and PARKIN gene deletion had no impact on the onset and progression of cardiomyopathy in RBX2-deficient hearts. Furthermore, RBX2 controls the stability of PINK1 in mitochondria. Proteomics and biochemical analyses further revealed a global impact of RBX2 deficiency on the mitochondrial proteome and identified several mitochondrial proteins as its putative substrates. These findings identify RBX2-CRL5 as a mitochondrial Ub ligase that controls mitophagy under physiological conditions in a PARKIN-independent, PINK1-dependent manner, thereby regulating cardiac homeostasis.
    Non-standard abbreviations and acronyms: RBX2, RING-Box Protein 2; SAG, Sensitive to Apoptosis Gene; Ub, Ubiquitin; pS65-Ub, phosphorylated Ub at serine 65; MAVS, mitochondrial antiviral-signaling protein; AAV, adeno-associated virus; AV, adenovirus; siRNA, Small interfering RNA; GFP, green fluorescent protein; CUL, cullin; RING, Really Interesting New Gene; CRLs, cullin-RING ligases; CSN, COP9 signalosome; APEX2, ascorbate peroxidase 2; mito, mitochondrial; cyto, cytosolic; MOM, mitochondrial outer membrane; CCCP, Carbonyl Cyanide Chlorophenylhydrazone; OMP25, Outer membrane protein 25; PK, proteinase K; HA, hemagglutinin; TMRM, Tetramethylrhodamine methyl ester perchlorate; αMHC,α-myosin heavy chain; CKO, cardiomyocyte-specific knockout; TAM, tamoxifen; TMT, tandem mass tag; KD, knockdown; CTL, control; MCM, MerCreMer; iCKO, inducible cardiomyocyte-specific knockout; BFA, bafilomycin A1; PCA, principle component analysis; MS, Mass spectrometry; DEPs, differentially expressed proteins; FC, fold change; FDR, False Discovery Rate; KEGG, Kyoto encyclopedia of genes and genomes; ER, endoplasmic reticulum; DKO, double knockout; CM, cardiomyocyte; cTnT, cardiac troponin T; NRVCs, neonatal rat ventricular cardiomyocytes; NRVMs, neonatal mouse ventricular cardiomyocytes; NMVFs, neonatal mouse ventricular fibroblasts; HF, heart failure; KO, knockout; MF, Molecular Functions; CC, Cellular Components; BP, Biological Process; TUNEL, Terminal deoxynucleotidyl transferase dUTP nick end labeling; SCF, Skp1-Cullin 1-F-box.
    DOI:  https://doi.org/10.1101/2024.02.24.581168
  11. Biochemistry (Mosc). 2023 Dec;88(12): 1997-2006
    Role of Mitochondrial DNA in Yeast Replicative Aging.
    Aglaia V Azbarova, Dmitry A Knorre.
      Despite the diverse manifestations of aging across different species, some common aging features and underlying mechanisms are shared. In particular, mitochondria appear to be among the most vulnerable systems in both metazoa and fungi. In this review, we discuss how mitochondrial dysfunction is related to replicative aging in the simplest eukaryotic model, the baker's yeast Saccharomyces cerevisiae. We discuss a chain of events that starts from asymmetric distribution of mitochondria between mother and daughter cells. With age, yeast mother cells start to experience a decrease in mitochondrial transmembrane potential and, consequently, a decrease in mitochondrial protein import efficiency. This induces mitochondrial protein precursors in the cytoplasm, the loss of mitochondrial DNA (mtDNA), and at the later stages - cell death. Interestingly, yeast strains without mtDNA can have either increased or decreased lifespan compared to the parental strains with mtDNA. The direction of the effect depends on their ability to activate compensatory mechanisms preventing or mitigating negative consequences of mitochondrial dysfunction. The central role of mitochondria in yeast aging and death indicates that it is one of the most complex and, therefore, deregulation-prone systems in eukaryotic cells.
    Keywords:  aging; development; mitochondrial DNA; mitochondrial dysfunction; yeast
    DOI:  https://doi.org/10.1134/S0006297923120040
  12. J Pediatr Neurol. 2023 Dec;21(6): 475-478
    DNM1L variant presenting as adolescent-onset sensory neuronopathy, spasticity, dystonia, and ataxia.
    Alexander S Wang, Gabrielle Lemire, Grace E VanNoy, Christina Austin-Tse, Anne O'Donnell-Luria, Camilla Kilbane.
      DMN1L encodes for dynamin-like protein 1 (DLP1) which plays a key role in perixosomal and mitochondrial fission. Individuals with heterozygous variants in DNM1L present with a wide range of neurologic symptoms, including encephalopathy, epilepsy, and motor deficits. Here we report on a woman presenting with adolescence onset of sensory neuronopathy, spasticity, dystonia, and ataxia. Trio genome sequencing identified a heterozygous variant in DNM1L (NM_012062.3 c.121G>A/p.Val41Met) which was thought to be pathogenic. This case describes the latest known symptomatic onset of DMN1L-related disease described in literature. We highlight our approach to a challenging diagnostic workup and interpretation of a specific variant that has not been previously reported. Furthermore, the case highlights the diagnostic importance of utilizing genomic sequencing and research studies for patients with rare disease.
    Keywords:  DLP1; DNM1L; ataxia; dystonia; mitochondrial; neuropathy; sensory neuronopathy; spasticity
    DOI:  https://doi.org/10.1055/s-0043-1771352
  13. Zool Res. 2024 Mar 18. pii: 2095-8137(2024)02-0292-07. [Epub ahead of print]45(2): 292-298
    Generation of mitochondrial replacement monkeys by female pronucleus transfer.
    Chun-Yang Li, Xing-Chen Liu, Yu-Zhuo Li, Yan Wang, Yan-Hong Nie, Yu-Ting Xu, Xiao-Tong Zhang, Yong Lu, Qiang Sun.
      Mutations in mitochondrial DNA (mtDNA) are maternally inherited and have the potential to cause severe disorders. Mitochondrial replacement therapies, including spindle, polar body, and pronuclear transfers, are promising strategies for preventing the hereditary transmission of mtDNA diseases. While pronuclear transfer has been used to generate mitochondrial replacement mouse models and human embryos, its application in non-human primates has not been previously reported. In this study, we successfully generated four healthy cynomolgus monkeys ( Macaca fascicularis) via female pronuclear transfer. These individuals all survived for more than two years and exhibited minimal mtDNA carryover (3.8%-6.7%), as well as relatively stable mtDNA heteroplasmy dynamics during development. The successful establishment of this non-human primate model highlights the considerable potential of pronuclear transfer in reducing the risk of inherited mtDNA diseases and provides a valuable preclinical research model for advancing mitochondrial replacement therapies in humans.
    Keywords:  Female pronuclear transfer; Mitochondrial replacement; Non-human primates
    DOI:  https://doi.org/10.24272/j.issn.2095-8137.2023.287
  14. Am J Med Genet A. 2024 Mar 12. e63589
    Intracranial calcifications simulating Aicardi-Goutières syndrome in PARS2-related mitochondrial disease.
    Amanda Gerard, Elizabeth Mizerik, Carrie A Mohila, Sarah AlAwami, Jill V Hunter, Debra L Kearney, Seema R Lalani, Fernando Scaglia.
      PARS2 encodes an aminoacyl-tRNA synthetase that catalyzes the ligation of proline to mitochondrial prolyl-tRNA molecules. Diseases associated with PARS2 primarily affect the central nervous system, causing early infantile developmental epileptic encephalopathies (EIDEE; DEE75; MIM #618437) with infantile-onset neurodegeneration. Dilated cardiomyopathy has also been reported in the affected individuals. About 10 individuals to date have been described with pathogenic biallelic variants in PARS2. While many of the reported individuals succumbed to the disease in the first two decades of life, autopsy findings have not yet been reported. Here, we describe neuropathological findings in a deceased male with evidence of intracranial calcifications in the basal ganglia, thalamus, cerebellum, and white matter, similar to Aicardi-Goutières syndrome. This report describes detailed autopsy findings in a child with PARS2-related mitochondrial disease and provides plausible evidence that intracranial calcifications may be a previously unrecognized feature of this disorder.
    Keywords:  Aicardi-Goutières syndrome; PARS2; intracranial calcifications; mitochondrial disease
    DOI:  https://doi.org/10.1002/ajmg.a.63589
  15. Int J Mol Sci. 2024 Feb 29. pii: 2835. [Epub ahead of print]25(5):
    Mitochondria at the Nanoscale: Physics Meets Biology-What Does It Mean for Medicine?
    Lev Mourokh, Jonathan Friedman.
      Mitochondria are commonly perceived as "cellular power plants". Intriguingly, power conversion is not their only function. In the first part of this paper, we review the role of mitochondria in the evolution of eukaryotic organisms and in the regulation of the human body, specifically focusing on cancer and autism in relation to mitochondrial dysfunction. In the second part, we overview our previous works, revealing the physical principles of operation for proton-pumping complexes in the inner mitochondrial membrane. Our proposed simple models reveal the physical mechanisms of energy exchange. They can be further expanded to answer open questions about mitochondrial functions and the medical treatment of diseases associated with mitochondrial disorders.
    Keywords:  autism spectrum disorder; carcinogenesis; equations of motion; mitochondria; proton-pumping complex; respiratory transport chain
    DOI:  https://doi.org/10.3390/ijms25052835
  16. Methods Mol Biol. 2024 ;2778 221-236
    Tracking the Activity and Position of Mitochondrial β-Barrel Proteins.
    Shuo Wang, Stephan Nussberger.
      Total interference reflection fluorescence (TIRF) microscopy of lipid bilayers is an effective technique for studying the lateral movement and ion channel activity of single integral membrane proteins. Here we describe how to integrate the mitochondrial outer membrane preprotein translocase TOM-CC and its β-barrel protein-conducting channel Tom40 into supported lipid bilayers to identify possible relationships between movement and channel activity. We propose that our approach can be readily applied to membrane protein channels where transient tethering to either membrane-proximal or intramembrane structures is accompanied by a change in channel permeation.
    Keywords:  Channels; Mitochondria; Protein translocation; Single molecule; TIRF microscopy; TOM; β-barrel membrane protein
    DOI:  https://doi.org/10.1007/978-1-0716-3734-0_14
  17. J Biol Chem. 2024 Mar 08. pii: S0021-9258(24)01646-6. [Epub ahead of print] 107151
    The Integrated Stress Response in metabolic adaptation.
    Hyung Don Ryoo.
      The Integrated Stress Response (ISR) refers to signaling pathways initiated by stress-activated eIF2‹ kinases. Distinct eIF2‹ kinases respond to different stress signals, including amino acid deprivation and mitochondrial stress. Such stress-induced eIF2‹ phosphorylation attenuates general mRNA translation and, at the same time, stimulates the preferential translation of specific downstream factors to orchestrate an adaptive gene expression program. In recent years, there have been significant new advances in our understanding of ISR during metabolic stress adaptation. Here, I discuss those advances, reviewing among others the ISR activation mechanisms in response to amino acid deprivation and mitochondrial stress. In addition, I review how ISR regulates the amino acid metabolic pathways and how changes in the ISR impact the physiology and pathology of various disease models.
    Keywords:  ATF4; GCN1; GCN2; HRI; Integrated Stress Response; amino acid deprivation; cysteine; eIF2‹; glutathione; mitochondrial stress; serine biosynthesis
    DOI:  https://doi.org/10.1016/j.jbc.2024.107151
  18. Mitochondrion. 2024 Mar 11. pii: S1567-7249(24)00028-X. [Epub ahead of print]76 101870
    Genetic heterogeneity and respiratory chain enzyme analysis in pediatric Indian patients with mitochondrial disorder: Report of novel variants in POLG1 gene and their functional implication using molecular dynamic simulation.
    Debolina Saha, Sonam Kothari, Shilpa Duttaprasanna Kulkarni, Menaka Thambiraja, Ragothaman M Yennamalli, Dhanjit K Das.
      Mitochondrial disorders are a heterogeneous group of disorders caused by mutations in the mitochondrial DNA or in nuclear genes encoding the mitochondrial proteins and subunits. Polymerase Gamma (POLG) is a nuclear gene and mutation in the POLG gene are one of the major causes of inherited mitochondrial disorders. In this study, 15 pediatric patients, with a wide spectrum of clinical phenotypes were screened using blood samples (n = 15) and muscle samples (n = 4). Respiratory chain enzyme analysis in the muscle samples revealed multi-complex deficiencies with Complex I deficiency present in (1/4) patients, Complex II (2/4), Complex III (3/4) and Complex IV (2/4) patients. Multiple large deletions were observed in 4/15 patients using LR-PCR. Whole exome sequencing (WES) revealed a compound heterozygous mutation consisting of a POLG1 novel variant (NP_002684.1:p.Trp261X) and a missense variant (NP_002684.1:p. Leu304Arg) in one patient and another patient harboring a novel homozygous POLG1 variant (NP_002684.1:p. Phe750Val). These variants (NP_002684.1:p. Leu304Arg) and (NP_002684.1:p. Phe750Val) and their interactions with DNA were modelled using molecular docking and molecular dynamics (MD) simulation studies. The protein conformation was analyzed as root mean square deviation (RMSD), root mean square fluctuation (RMSF) which showed local fluctuations in the mutants compared to the wildtype. However, Solvent Accessible Surface Area (SASA) significantly increased for NP_002684.1:p.Leu304Arg and decreased in NP_002684.1:p.Phe750Val mutants. Further, Contact Order analysis indicated that the Aromatic-sulfur interactions were destabilizing in the mutants. Overall, these in-silico analysis has revealed a destabilizing mutations suggesting pathogenic variants in POLG1 gene.
    Keywords:  Mitochondrial disorders; Molecular docking; POLG1; Respiratory chain enzyme analysis; Sanger sequencing
    DOI:  https://doi.org/10.1016/j.mito.2024.101870
  19. Nat Commun. 2024 Mar 09. 15(1): 2156
    FUS unveiled in mitochondrial DNA repair and targeted ligase-1 expression rescues repair-defects in FUS-linked motor neuron disease.
    Manohar Kodavati, Haibo Wang, Wenting Guo, Joy Mitra, Pavana M Hegde, Vincent Provasek, Vikas H Maloji Rao, Indira Vedula, Aijun Zhang, Sankar Mitra, Alan E Tomkinson, Dale J Hamilton, Ludo Van Den Bosch, Muralidhar L Hegde.
      This study establishes the physiological role of Fused in Sarcoma (FUS) in mitochondrial DNA (mtDNA) repair and highlights its implications to the pathogenesis of FUS-associated neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS). Endogenous FUS interacts with and recruits mtDNA Ligase IIIα (mtLig3) to DNA damage sites within mitochondria, a relationship essential for maintaining mtDNA repair and integrity in healthy cells. Using ALS patient-derived FUS mutant cell lines, a transgenic mouse model, and human autopsy samples, we discovered that compromised FUS functionality hinders mtLig3's repair role, resulting in increased mtDNA damage and mutations. These alterations cause various manifestations of mitochondrial dysfunction, particularly under stress conditions relevant to disease pathology. Importantly, rectifying FUS mutations in patient-derived induced pluripotent cells (iPSCs) preserves mtDNA integrity. Similarly, targeted introduction of human DNA Ligase 1 restores repair mechanisms and mitochondrial activity in FUS mutant cells, suggesting a potential therapeutic approach. Our findings unveil FUS's critical role in mitochondrial health and mtDNA repair, offering valuable insights into the mechanisms underlying mitochondrial dysfunction in FUS-associated motor neuron disease.
    DOI:  https://doi.org/10.1038/s41467-024-45978-6
  20. Elife. 2024 Mar 14. pii: e92376. [Epub ahead of print]13
    Activity-dependent mitochondrial ROS signaling regulates recruitment of glutamate receptors to synapses.
    Rachel L Doser, Kaz M Knight, Ennis W Deihl, Frederic J Hoerndli.
      Our understanding of mitochondrial signaling in the nervous system has been limited by the technical challenge of analyzing mitochondrial function in vivo. In the transparent genetic model Caenorhabditis elegans, we were able to manipulate and measure mitochondrial ROS (reactive oxygen species) signaling of individual mitochondria as well as neuronal activity of single neurons in vivo. Using this approach, we provide evidence supporting a novel role for mitochondrial ROS signaling in dendrites of excitatory glutamatergic C. elegans interneurons. Specifically, we show that following neuronal activity, dendritic mitochondria take up calcium (Ca2+) via the mitochondrial Ca2+ uniporter MCU-1 that results in an upregulation of mitochondrial ROS production. We also observed that mitochondria are positioned in close proximity to synaptic clusters of GLR-1, the C. elegans ortholog of the AMPA subtype of glutamate receptors that mediate neuronal excitation. We show that synaptic recruitment of GLR-1 is upregulated when MCU-1 function is pharmacologically or genetically impaired but is downregulated by mitoROS signaling. Thus, signaling from postsynaptic mitochondria may regulate excitatory synapse function to maintain neuronal homeostasis by preventing excitotoxicity and energy depletion.
    Keywords:  C. elegans; cell biology; neuroscience
    DOI:  https://doi.org/10.7554/eLife.92376
  21. Am J Med Genet A. 2024 Mar 13. e63590
    Next generation sequencing reveals novel compound heterozygous deletions in NDUFAF2 in a child with mitochondrial complex I deficiency, nuclear type 10.
    Aren E Marshall, Lauren Brady, Ed Yeh, Alan J Mears, Melanie Lacaria, Pranesh Chakraborty, Mark A Tarnopolsky, Kristin D Kernohan.
      
    DOI:  https://doi.org/10.1002/ajmg.a.63590
  22. Patterns (N Y). 2024 Mar 08. 5(3): 100945
    Federated learning for multi-omics: A performance evaluation in Parkinson's disease.
    Benjamin P Danek, Mary B Makarious, Anant Dadu, Dan Vitale, Paul Suhwan Lee, Andrew B Singleton, Mike A Nalls, Jimeng Sun, Faraz Faghri.
      While machine learning (ML) research has recently grown more in popularity, its application in the omics domain is constrained by access to sufficiently large, high-quality datasets needed to train ML models. Federated learning (FL) represents an opportunity to enable collaborative curation of such datasets among participating institutions. We compare the simulated performance of several models trained using FL against classically trained ML models on the task of multi-omics Parkinson's disease prediction. We find that FL model performance tracks centrally trained ML models, where the most performant FL model achieves an AUC-PR of 0.876 ± 0.009, 0.014 ± 0.003 less than its centrally trained variation. We also determine that the dispersion of samples within a federation plays a meaningful role in model performance. Our study implements several open-source FL frameworks and aims to highlight some of the challenges and opportunities when applying these collaborative methods in multi-omics studies.
    Keywords:  Parkinson’s disease diagnosis; federated learning; machine learning; omics data analysis
    DOI:  https://doi.org/10.1016/j.patter.2024.100945
  23. World J Orthop. 2024 Feb 18. 15(2): 101-104
    Mitochondrial dysfunction in type 2 diabetes: A neglected path to skeletal muscle atrophy.
    Jian-Jun Wu, Hui-Min Xian, Da-Wei Yang, Fan Yang.
      Over the course of several decades, robust research has firmly established the significance of mitochondrial pathology as a central contributor to the onset of skeletal muscle atrophy in individuals with diabetes. However, the specific intricacies governing this process remain elusive. Extensive evidence highlights that individuals with diabetes regularly confront the severe consequences of skeletal muscle degradation. Deciphering the sophisticated mechanisms at the core of this pathology requires a thorough and meticulous exploration into the nuanced factors intricately associated with mitochondrial dysfunction.
    Keywords:  Diabetes; Mfn-2; Mitochondria metabolism; Oxidative stress; Skeletal muscle atrophy
    DOI:  https://doi.org/10.5312/wjo.v15.i2.101
  24. Int J Mol Sci. 2024 Feb 25. pii: 2667. [Epub ahead of print]25(5):
    Mitochondrial Dysfunction in Heart Failure: From Pathophysiological Mechanisms to Therapeutic Opportunities.
    Giovanna Gallo, Speranza Rubattu, Massimo Volpe.
      Mitochondrial dysfunction, a feature of heart failure, leads to a progressive decline in bioenergetic reserve capacity, consisting in a shift of energy production from mitochondrial fatty acid oxidation to glycolytic pathways. This adaptive process of cardiomyocytes does not represent an effective strategy to increase the energy supply and to restore the energy homeostasis in heart failure, thus contributing to a vicious circle and to disease progression. The increased oxidative stress causes cardiomyocyte apoptosis, dysregulation of calcium homeostasis, damage of proteins and lipids, leakage of mitochondrial DNA, and inflammatory responses, finally stimulating different signaling pathways which lead to cardiac remodeling and failure. Furthermore, the parallel neurohormonal dysregulation with angiotensin II, endothelin-1, and sympatho-adrenergic overactivation, which occurs in heart failure, stimulates ventricular cardiomyocyte hypertrophy and aggravates the cellular damage. In this review, we will discuss the pathophysiological mechanisms related to mitochondrial dysfunction, which are mainly dependent on increased oxidative stress and perturbation of the dynamics of membrane potential and are associated with heart failure development and progression. We will also provide an overview of the potential implication of mitochondria as an attractive therapeutic target in the management and recovery process in heart failure.
    Keywords:  cardiac disease; cardiac rehabilitation; cellular recovery; electron transport chain; heart failure; inflammation; mitochondria; oxidative stress
    DOI:  https://doi.org/10.3390/ijms25052667
  25. Life Sci Alliance. 2024 Jun;pii: e202302329. [Epub ahead of print]7(6):
    Genetic ablation of Immt induces a lethal disruption of the MICOS complex.
    Stephanie M Rockfield, Meghan E Turnis, Ricardo Rodriguez-Enriquez, Madhavi Bathina, Seng Kah Ng, Nathan Kurtz, Nathalie Becerra Mora, Stephane Pelletier, Camenzind G Robinson, Peter Vogel, Joseph T Opferman.
      The mitochondrial contact site and cristae organizing system (MICOS) is important for crista junction formation and for maintaining inner mitochondrial membrane architecture. A key component of the MICOS complex is MIC60, which has been well studied in yeast and cell culture models. However, only one recent study has demonstrated the embryonic lethality of losing Immt (the gene encoding MIC60) expression. Tamoxifen-inducible ROSA-CreERT2-mediated deletion of Immt in adult mice disrupted the MICOS complex, increased mitochondria size, altered cristae morphology, and was lethal within 12 d. Pathologically, these mice displayed defective intestinal muscle function (paralytic ileus) culminating in dehydration. We also identified bone marrow (BM) hypocellularity in Immt-deleted mice, although BM transplants from wild-type mice did not improve survival. Altogether, this inducible mouse model demonstrates the importance of MIC60 in vivo, in both hematopoietic and non-hematopoietic tissues, and provides a valuable resource for future mechanistic investigations into the MICOS complex.
    DOI:  https://doi.org/10.26508/lsa.202302329
  26. Res Sq. 2024 Feb 20. pii: rs.3.rs-3950044. [Epub ahead of print]
    The loss of OPA1 accelerates intervertebral disc degeneration and osteoarthritis in aged mice.
    Makarand Risbud, Vedavathi Madhu, Miriam Hernandez-Meadows, Ashley Coleman, Kimheak Sao, Kameron Inguito, Owen Haslam, Paige Boneski, Hiromi Sesaki, John Collins.
      NP cells of the intervertebral disc and articular chondrocytes reside in avascular and hypoxic tissue niches. As a consequence of these environmental constraints the cells are primarily glycolytic in nature and were long thought to have a minimal reliance on mitochondrial function. Recent studies have challenged this long-held view and highlighted the increasingly important role of mitochondria in the physiology of these tissues. However, the foundational understanding of mechanisms governing mitochondrial dynamics and function in these tissues is lacking. We investigated the role of mitochondrial fusion protein OPA1 in maintaining the spine and knee joint health in mice. OPA1 knockdown in NP cells altered mitochondrial size and cristae shape and increased the oxygen consumption rate without affecting ATP synthesis. OPA1 governed the morphology of multiple organelles, including peroxisomes, early endosomes and cis-Golgi and its loss resulted in the dysregulation of NP cell autophagy. Metabolic profiling and 13 C-flux analyses revealed TCA cycle anaplerosis and altered metabolism in OPA1-deficient NP cells. Noteworthy, Opa1 AcanCreERT2 mice with Opa1 deletion in disc and cartilage showed age-dependent disc degeneration, osteoarthritis, and vertebral osteopenia. Our findings underscore that OPA1 regulation of mitochondrial dynamics and multi-organelle interactions is critical in preserving metabolic homeostasis of disc and cartilage.
    DOI:  https://doi.org/10.21203/rs.3.rs-3950044/v1
  27. FEBS J. 2024 Mar 11.
    Study of an FBXO7 patient mutation reveals Fbxo7 and PI31 co-regulate proteasomes and mitochondria.
    Sara Al Rawi, Lorna Simpson, Guðrún Agnarsdóttir, Neil Q McDonald, Veronika Chernuha, Orly Elpeleg, Massimo Zeviani, Roger A Barker, Ronen Spiegel, Heike Laman.
      Mutations in FBXO7 have been discovered to be associated with an atypical parkinsonism. We report here a new homozygous missense mutation in a paediatric patient that causes an L250P substitution in the dimerisation domain of Fbxo7. This alteration selectively ablates the Fbxo7-PI31 interaction and causes a significant reduction in Fbxo7 and PI31 levels in patient cells. Consistent with their association with proteasomes, patient fibroblasts have reduced proteasome activity and proteasome subunits. We also show PI31 interacts with the MiD49/51 fission adaptor proteins, and unexpectedly, PI31 acts to facilitate SCFFbxo7 -mediated ubiquitination of MiD49. The L250P mutation reduces the SCFFbxo7 ligase-mediated ubiquitination of a subset of its known substrates. Although MiD49/51 expression was reduced in patient cells, there was no effect on the mitochondrial network. However, patient cells show reduced levels of mitochondrial function and mitophagy, higher levels of ROS and are less viable under stress. Our study demonstrates that Fbxo7 and PI31 regulate proteasomes and mitochondria and reveals a new function for PI31 in enhancing the SCFFbxo7 E3 ubiquitin ligase activity.
    Keywords:  E3 ubiquitin ligase; Fbxo7/PARK15; Parkinson's disease; mitochondria; proteasome
    DOI:  https://doi.org/10.1111/febs.17114
  28. Cell Death Dis. 2024 Mar 11. 15(3): 203
    Genome-wide CRISPR/Cas9 screen shows that loss of GET4 increases mitochondria-endoplasmic reticulum contact sites and is neuroprotective.
    Emma L Wilson, Yizhou Yu, Nuno S Leal, James A Woodward, Nikolaos Patikas, Jordan L Morris, Sarah F Field, William Plumbly, Vincent Paupe, Suvagata R Chowdhury, Robin Antrobus, Georgina E Lindop, Yusuf M Adia, Samantha H Y Loh, Julien Prudent, L Miguel Martins, Emmanouil Metzakopian.
      Organelles form membrane contact sites between each other, allowing for the transfer of molecules and signals. Mitochondria-endoplasmic reticulum (ER) contact sites (MERCS) are cellular subdomains characterized by close apposition of mitochondria and ER membranes. They have been implicated in many diseases, including neurodegenerative, metabolic, and cardiac diseases. Although MERCS have been extensively studied, much remains to be explored. To uncover novel regulators of MERCS, we conducted a genome-wide, flow cytometry-based screen using an engineered MERCS reporter cell line. We found 410 genes whose downregulation promotes MERCS and 230 genes whose downregulation decreases MERCS. From these, 29 genes were selected from each population for arrayed screening and 25 were validated from the high population and 13 from the low population. GET4 and BAG6 were highlighted as the top 2 genes that upon suppression increased MERCS from both the pooled and arrayed screens, and these were subjected to further investigation. Multiple microscopy analyses confirmed that loss of GET4 or BAG6 increased MERCS. GET4 and BAG6 were also observed to interact with the known MERCS proteins, inositol 1,4,5-trisphosphate receptors (IP3R) and glucose-regulated protein 75 (GRP75). In addition, we found that loss of GET4 increased mitochondrial calcium uptake upon ER-Ca2+ release and mitochondrial respiration. Finally, we show that loss of GET4 rescues motor ability, improves lifespan and prevents neurodegeneration in a Drosophila model of Alzheimer's disease (Aβ42Arc). Together, these results suggest that GET4 is involved in decreasing MERCS and that its loss is neuroprotective.
    DOI:  https://doi.org/10.1038/s41419-024-06568-y
  29. Nature. 2024 Mar;627(8003): S2-S5
    How AI is being used to accelerate clinical trials.
    Matthew Hutson.
      
    Keywords:  Machine learning; Medical research; Research data; Technology; Therapeutics
    DOI:  https://doi.org/10.1038/d41586-024-00753-x
  30. Molecules. 2024 Feb 21. pii: 948. [Epub ahead of print]29(5):
    Utilizing Extracellular Vesicles for Eliminating 'Unwanted Molecules': Harnessing Nature's Structures in Modern Therapeutic Strategies.
    Monika Kisielewska, Katarzyna Rakoczy, Izabela Skowron, Julia Górczyńska, Julia Kacer, Agata Bocheńska, Anna Choromańska.
      Extracellular vesicles (EVs) are small phospholipid bilayer-bond structures released by diverse cell types into the extracellular environment, maintaining homeostasis of the cell by balancing cellular stress. This article provides a comprehensive overview of extracellular vesicles, their heterogeneity, and diversified roles in cellular processes, emphasizing their importance in the elimination of unwanted molecules. They play a role in regulating oxidative stress, particularly by discarding oxidized toxic molecules. Furthermore, endoplasmic reticulum stress induces the release of EVs, contributing to distinct results, including autophagy or ER stress transmission to following cells. ER stress-induced autophagy is a part of unfolded protein response (UPR) and protects cells from ER stress-related apoptosis. Mitochondrial-derived vesicles (MDVs) also play a role in maintaining homeostasis, as they carry damaged mitochondrial components, thereby preventing inflammation. Moreover, EVs partake in regulating aging-related processes, and therefore they can potentially play a crucial role in anti-aging therapies, including the treatment of age-related diseases such as Alzheimer's disease or cardiovascular conditions. Overall, the purpose of this article is to provide a better understanding of EVs as significant mediators in both physiological and pathological processes, and to shed light on their potential for therapeutic interventions targeting EV-mediated pathways in various pathological conditions, with an emphasis on age-related diseases.
    Keywords:  age-related diseases; aging process; autophagy; endoplasmic reticulum stress; extracellular vesicles; mitochondrial-derived vesicles; oxidative stress
    DOI:  https://doi.org/10.3390/molecules29050948
  31. Cells. 2024 Mar 04. pii: 448. [Epub ahead of print]13(5):
    GRAF1 Acts as a Downstream Mediator of Parkin to Regulate Mitophagy in Cardiomyocytes.
    Qiang Zhu, Matthew E Combs, Dawn E Bowles, Ryan T Gross, Michelle Mendiola Pla, Christopher P Mack, Joan M Taylor.
      Cardiomyocytes rely on proper mitochondrial homeostasis to maintain contractility and achieve optimal cardiac performance. Mitochondrial homeostasis is controlled by mitochondrial fission, fusion, and mitochondrial autophagy (mitophagy). Mitophagy plays a particularly important role in promoting the degradation of dysfunctional mitochondria in terminally differentiated cells. However, the precise mechanisms by which this is achieved in cardiomyocytes remain opaque. Our study identifies GRAF1 as an important mediator in PINK1-Parkin pathway-dependent mitophagy. Depletion of GRAF1 (Arhgap26) in cardiomyocytes results in actin remodeling defects, suboptimal mitochondria clustering, and clearance. Mechanistically, GRAF1 promotes Parkin-LC3 complex formation and directs autophagosomes to damaged mitochondria. Herein, we found that these functions are regulated, at least in part, by the direct binding of GRAF1 to phosphoinositides (PI(3)P, PI(4)P, and PI(5)P) on autophagosomes. In addition, PINK1-dependent phosphorylation of Parkin promotes Parkin-GRAF1-LC3 complex formation, and PINK1-dependent phosphorylation of GRAF1 (on S668 and S671) facilitates the clustering and clearance of mitochondria. Herein, we developed new phosphor-specific antibodies to these sites and showed that these post-translational modifications are differentially modified in human hypertrophic cardiomyopathy and dilated cardiomyopathy. Furthermore, our metabolic studies using serum collected from isoproterenol-treated WT and GRAF1CKO mice revealed defects in mitophagy-dependent cardiomyocyte fuel flexibility that have widespread impacts on systemic metabolism. In summary, our study reveals that GRAF1 co-regulates actin and membrane dynamics to promote cardiomyocyte mitophagy and that dysregulation of GRAF1 post-translational modifications may underlie cardiac disease pathogenesis.
    Keywords:  GRAF1; PINK1-Parkin pathway; cardiomyocytes; metabolism; mitophagy
    DOI:  https://doi.org/10.3390/cells13050448
  32. Brain. 2024 Mar 14. pii: awae064. [Epub ahead of print]
    Whole genome sequencing increases the diagnostic rate in Charcot-Marie-Tooth disease.
    Christopher J Record, Menelaos Pipis, Mariola Skorupinska, Julian Blake, Roy Poh, James M Polke, Kelly Eggleton, Tina Nanji, Stephan Zuchner, Andrea Cortese, Henry Houlden, Alexander M Rossor, Matilde Laura, Mary M Reilly.
      Charcot-Marie-Tooth disease (CMT) is one of the most common and genetically heterogeneous inherited neurological diseases, with more than 130 disease-causing genes. Whole genome sequencing (WGS) has improved diagnosis across genetic diseases, but the diagnostic impact in CMT is yet to be fully reported. We present the diagnostic results from a single specialist inherited neuropathy centre, including the impact of WGS diagnostic testing. Patients were assessed at our specialist inherited neuropathy centre from 2009-2023. Genetic testing was performed using single gene testing, next-generation sequencing targeted panels, research whole exome and whole genome sequencing (WGS), and latterly WGS through the UK National Health Service. Variants were assessed using the American College of Medical Genetics and Genomics and Association for Clinical Genomic Science criteria. Excluding patients with hereditary ATTR amyloidosis, 1515 patients with a clinical diagnosis of CMT and related disorders were recruited. 621 patients had CMT1 (41.0%), 294 CMT2 (19.4%), 205 intermediate CMT (CMTi, 13.5%), 139 hereditary motor neuropathy (HMN, 9.2%), 93 hereditary sensory neuropathy (HSN, 6.1%), 38 sensory ataxic neuropathy (2.5%), 72 hereditary neuropathy with liability to pressure palsies (HNPP, 4.8%) and 53 'complex' neuropathy (3.5%). Overall, a genetic diagnosis was reached in 76.9% (1165/1515). A diagnosis was most likely in CMT1 (96.8%, 601/621), followed by CMTi (81.0%, 166/205) and then HSN (69.9%, 65/93). Diagnostic rates remained less than 50% in CMT2, HMN and complex neuropathies. The most common genetic diagnosis was PMP22 duplication (CMT1A; 505/1165, 43.3%), then GJB1 (CMTX1; 151/1165, 13.0%), PMP22 deletion (HNPP; 72/1165, 6.2%) and MFN2 (CMT2A; 46/1165, 3.9%). We recruited 233 cases to the UK 100,000 Genomes Project (100KGP), of which 74 (31.8%) achieved a diagnosis; 28 had been otherwise diagnosed since recruitment leaving a true diagnostic rate of WGS through the 100KGP of 19.7% (46/233). However, almost half of the solved cases (35/74) received a negative report from the study, and the diagnosis was made through our research access to the WGS data. The overall diagnostic uplift of WGS for the entire cohort was 3.5%. Our diagnostic rate is the highest reported from a single centre, and has benefitted from the use of WGS, particularly access to the raw data. However, almost one quarter of all cases remain unsolved, and a new reference genome and novel technologies will be important to narrow the 'diagnostic gap'.
    Keywords:  CMT; HMSN; WES; WGS; genomics; neurogenetics
    DOI:  https://doi.org/10.1093/brain/awae064
  33. bioRxiv. 2024 Feb 26. pii: 2023.12.22.572954. [Epub ahead of print]
    ROS-induced translational regulation-through spatiotemporal differences in codon recognition-is a key driver of brown adipogenesis.
    Jun Yu Ip, Indrik Wijaya, Li Ting Lee, Yuhua Lim, Deryn En-Jie Teoh, Cheryl Siew Choo Chan, Liang Cui, Thomas J Begley, Peter C Dedon, Huili Guo.
      The role of translational regulation in brown adipogenesis is relatively unknown. Localized translation of mRNAs encoding mitochondrial components enables swift mitochondrial responses, but whether this occurs during brown adipogenesis, which involves massive mitochondrial biogenesis, has not been explored. Here, we used ribosome profiling and RNA-Seq, coupled with cellular fractionation, to obtain spatiotemporal insights into translational regulation. During brown adipogenesis, a translation bias towards G/C-ending codons is triggered first in the mitochondrial vicinity by reactive oxygen species (ROS), which later spreads to the rest of the cell. This translation bias is induced through ROS modulating the activity of the tRNA modification enzyme, ELP3. Intriguingly, functionally relevant mRNAs, including those encoding ROS scavengers, benefit from this bias; in so doing, ROS-induced translation bias both fuels differentiation and concurrently minimizes oxidative damage. These ROS-induced changes could enable sustained mitochondrial biogenesis during brown adipogenesis, and explain in part, the molecular basis for ROS hormesis.
    DOI:  https://doi.org/10.1101/2023.12.22.572954
  34. Methods Mol Biol. 2024 ;2778 185-200
    MitoLuc: A Luminescence-Based Assay to Study Real-Time Protein Import into Mitochondria.
    Holly C Ford, Ian Collinson.
      All but a few mitochondrial proteins are translated into the cytosol and imported in via complicated and varied pathways. These processes occur over short time frames and, as such, are difficult to monitor with classical approaches such as Western blotting or autoradiography that require sample collection at discrete time points. The development of an assay based on a split version of the small luciferase-Mitoluc-has allowed us to monitor the import of proteins into mitochondria in high resolution and real time (Pereira et al., J Mol Biol 431:1689-1699, 2019). Luminescence measurements are acquired using a plate reader in the order of seconds. This allows scores of experiments to be conducted in parallel in a single multi-well plate and permits kinetic analysis yielding information about import mechanisms (Ford et al., Elife 11:e75426, 2022).
    Keywords:  Luminescence; MitoLuc; Mitochondria; NanoLuc; Protein import; Split-luciferase
    DOI:  https://doi.org/10.1007/978-1-0716-3734-0_12
  35. Aging Cell. 2024 Mar 14. e14143
    Mitophagy defect mediates the aging-associated hallmarks in Hutchinson-Gilford progeria syndrome.
    Yingying Sun, Le Xu, Yi Li, Shunze Jia, Gang Wang, Xufeng Cen, Yuyan Xu, Zhongkai Cao, Jingjing Wang, Ning Shen, Lidan Hu, Jin Zhang, Jianhua Mao, Hongguang Xia, Zhihong Liu, Xudong Fu.
      Hutchinson-Gilford progeria syndrome (HGPS) is a rare and fatal disease manifested by premature aging and aging-related phenotypes, making it a disease model for aging. The cellular machinery mediating age-associated phenotypes in HGPS remains largely unknown, resulting in limited therapeutic targets for HGPS. In this study, we showed that mitophagy defects impaired mitochondrial function and contributed to cellular markers associated with aging in mesenchymal stem cells derived from HGPS patients (HGPS-MSCs). Mechanistically, we discovered that mitophagy affected the aging-associated phenotypes of HGPS-MSCs by inhibiting the STING-NF-ĸB pathway and the downstream transcription of senescence-associated secretory phenotypes (SASPs). Furthermore, by utilizing UMI-77, an effective mitophagy inducer, we showed that mitophagy induction alleviated aging-associated phenotypes in HGPS and naturally aged mice. Collectively, our results uncovered that mitophagy defects mediated the aging-associated markers in HGPS, highlighted the function of mitochondrial homeostasis in HGPS progression, and suggested mitophagy as an intervention target for HGPS and aging.
    Keywords:  HGPS; UMI-77; aging; mitophagy
    DOI:  https://doi.org/10.1111/acel.14143
  36. Nat Commun. 2024 Mar 13. 15(1): 2269
    Biallelic NAA60 variants with impaired n-terminal acetylation capacity cause autosomal recessive primary familial brain calcifications.
    SYNaPS Study Group
      Primary familial brain calcification (PFBC) is characterized by calcium deposition in the brain, causing progressive movement disorders, psychiatric symptoms, and cognitive decline. PFBC is a heterogeneous disorder currently linked to variants in six different genes, but most patients remain genetically undiagnosed. Here, we identify biallelic NAA60 variants in ten individuals from seven families with autosomal recessive PFBC. The NAA60 variants lead to loss-of-function with lack of protein N-terminal (Nt)-acetylation activity. We show that the phosphate importer SLC20A2 is a substrate of NAA60 in vitro. In cells, loss of NAA60 caused reduced surface levels of SLC20A2 and a reduction in extracellular phosphate uptake. This study establishes NAA60 as a causal gene for PFBC, provides a possible biochemical explanation of its disease-causing mechanisms and underscores NAA60-mediated Nt-acetylation of transmembrane proteins as a fundamental process for healthy neurobiological functioning.
    DOI:  https://doi.org/10.1038/s41467-024-46354-0
  37. J Biol Chem. 2024 Mar 11. pii: S0021-9258(24)01654-5. [Epub ahead of print] 107159
    Fatty acid oxidation drives mitochondrial hydrogen peroxide production by α-ketoglutarate dehydrogenase.
    Cathryn Grayson, Ben Faerman, Olivia Koufos, Ryan J Mailloux.
      In the present study, we examined the mitochondrial hydrogen peroxide (mH2O2) generating capacity of α-ketoglutarate dehydrogenase (KGDH) and compared it to components of the electron transport chain (ETC) using liver mitochondria isolated from male and female C57BL6N mice. We show for the first time there are some sex dimorphisms in the production of mH2O2 by ETC complexes I and III when mitochondria are fueled with different substrates. However, in our investigations into these sex effects, we made the unexpected discovery that: 1. KGDH serves as a major mH2O2 supplier in male and female liver mitochondria and 2. KGDH can form mH2O2 when mitochondria are energized with fatty acids, but only when malate is used to prime the Krebs cycle. Surprisingly, 2-keto-3-methylvaleric acid (KMV), a site-specific inhibitor for KGDH, nearly abolished mH2O2 generation in both male and female liver mitochondria oxidizing palmitoyl-carnitine. KMV inhibited mH2O2 production in liver mitochondria from male and female mice oxidizing myristoyl-, octanoyl-, or butyryl-carnitine. S1QEL 1.1 (S1) and S3QEL 2 (S3), compounds that inhibit reactive oxygen species (ROS) generation by complexes I and III, respectively, without interfering with OxPhos, had a negligible effect on the rate of mH2O2 production when pyruvate or acyl-carnitines were used as fuels. However, inclusion of KMV in reaction mixtures containing S1 and/or S3 almost abolished mH2O2 generation. Together, our findings suggest KGDH is the main mH2O2 generator in liver mitochondria, even when fatty acids are used as fuel.
    DOI:  https://doi.org/10.1016/j.jbc.2024.107159
  38. Methods Mol Biol. 2024 ;2778 83-99
    Examining Protein Translocation by β-Barrel Membrane Proteins Using Reconstituted Proteoliposomes.
    Minh Sang Huynh, Jiaming Caitlyn Xu, Trevor F Moraes.
      β-barrel membrane proteins populate the outer membrane of Gram-negative bacteria, mitochondria, and chloroplasts, playing significant roles in multiple key cellular pathways. Characterizing the functions of these membrane proteins in vivo is often challenging due to the complex protein network in the periplasm of Gram-negative bacteria (or intermembrane space in mitochondria and chloroplasts) and the presence of other outer membrane proteins. In vitro reconstitution into lipid-bilayer-like environments such as nanodiscs or proteoliposomes provides an excellent method for examining the specific function and mechanism of these membrane proteins in an isolated system. Here, we describe the methodologies employed to investigate Slam, a 14-stranded β-barrel membrane protein also known as the type XI secretion system that is responsible for translocating proteins across the outer membrane of many bacterial species.
    Keywords:   Denatured protein translocation; Gram-negative bacteria; In vitro translocation; Spheroplast-released translocation; Surface lipoprotein translocation; Type XI Secretion System; outer membrane proteins; β-barrel membrane protein; Proteoliposome reconstitution
    DOI:  https://doi.org/10.1007/978-1-0716-3734-0_6
  39. Inflamm Res. 2024 Mar 12.
    Long-chain acyl-CoA synthetase 4-mediated mitochondrial fatty acid metabolism and dendritic cell antigen presentation.
    Yan Li, Wenlong Fu, JinYing Xiang, Yinying Ren, Yuehan Li, Mi Zhou, Jinyue Yu, Zhengxiu Luo, Enmei Liu, Zhou Fu, Bo Liu, Fengxia Ding.
       OBJECTIVE: This study aims to investigate the role of Acyl-CoA synthetase 4 (ACSL4) in mediating mitochondrial fatty acid metabolism and dendritic cell (DC) antigen presentation in the immune response associated with asthma.
    METHODS: RNA sequencing was employed to identify key genes associated with mitochondrial function and fatty acid metabolism in DCs. ELISA was employed to assess the levels of fatty acid metabolism in DCs. Mitochondrial morphology was evaluated using laser confocal microscopy, structured illumination microscopy, and transmission electron microscopy. Flow cytometry and immunofluorescence were utilized to detect changes in mitochondrial superoxide generation in DCs, followed by immunofluorescence co-localization analysis of ACSL4 and the mitochondrial marker protein COXIV. Subsequently, pathological changes and immune responses in mouse lung tissue were observed. ELISA was conducted to measure the levels of fatty acid metabolism in lung tissue DCs. qRT-PCR and western blotting were employed to respectively assess the expression levels of mitochondrial-associated genes (ATP5F1A, VDAC1, COXIV, TFAM, iNOS) and proteins (ATP5F1A, VDAC1, COXIV, TOMM20, iNOS) in lung tissue DCs. Flow cytometry was utilized to analyze changes in the expression of surface antigens presented by DCs in lung tissue, specifically the MHCII molecule and the co-stimulatory molecules CD80/86.
    RESULTS: The sequencing results reveal that ACSL4 is a crucial gene regulating mitochondrial function and fatty acid metabolism in DCs. Inhibiting ACSL4 reduces the levels of fatty acid oxidases in DCs, increases arachidonic acid levels, and decreases A-CoA synthesis. Simultaneously, ACSL4 inhibition leads to an increase in mitochondrial superoxide production (MitoSOX) in DCs, causing mitochondrial rupture, vacuolization, and sparse mitochondrial cristae. In mice, ACSL4 inhibition exacerbates pulmonary pathological changes and immune responses, reducing the fatty acid metabolism levels within lung tissue DCs and the expression of mitochondria-associated genes and proteins. This inhibition induces an increase in the expression of MHCII antigen presentation molecules and co-stimulatory molecules CD80/86 in DCs.
    CONCLUSIONS: The research findings indicate that ACSL4-mediated mitochondrial fatty acid metabolism and dendritic cell antigen presentation play a crucial regulatory role in the immune response of asthma. This discovery holds promise for enhancing our understanding of the mechanisms underlying asthma pathogenesis and potentially identifying novel targets for its prevention and treatment.
    Keywords:  Acyl-CoA synthetase 4; Asthma; Dendritic cells; Mitochondria
    DOI:  https://doi.org/10.1007/s00011-024-01868-7
  40. NPJ Genom Med. 2024 Mar 14. 9(1): 20
    Genomes in clinical care.
    Olaf Riess, Marc Sturm, Benita Menden, Alexandra Liebmann, German Demidov, Dennis Witt, Nicolas Casadei, Jakob Admard, Leon Schütz, Stephan Ossowski, Stacie Taylor, Sven Schaffer, Christopher Schroeder, Andreas Dufke, Tobias Haack.
      In the era of precision medicine, genome sequencing (GS) has become more affordable and the importance of genomics and multi-omics in clinical care is increasingly being recognized. However, how to scale and effectively implement GS on an institutional level remains a challenge for many. Here, we present Genome First and Ge-Med, two clinical implementation studies focused on identifying the key pillars and processes that are required to make routine GS and predictive genomics a reality in the clinical setting. We describe our experience and lessons learned for a variety of topics including test logistics, patient care processes, data reporting, and infrastructure. Our model of providing clinical care and comprehensive genomic analysis from a single source may be used by other centers with a similar structure to facilitate the implementation of omics-based personalized health concepts in medicine.
    DOI:  https://doi.org/10.1038/s41525-024-00402-2
  41. PLoS Biol. 2024 Mar 13. 22(3): e3002504
    Neuronal ageing is promoted by the decay of the microtubule cytoskeleton.
    Pilar Okenve-Ramos, Rory Gosling, Monika Chojnowska-Monga, Kriti Gupta, Samuel Shields, Haifa Alhadyian, Ceryce Collie, Emilia Gregory, Natalia Sanchez-Soriano.
      Natural ageing is accompanied by a decline in motor, sensory, and cognitive functions, all impacting quality of life. Ageing is also the predominant risk factor for many neurodegenerative diseases, including Parkinson's disease and Alzheimer's disease. We need to therefore gain a better understanding of the cellular and physiological processes underlying age-related neuronal decay. However, gaining this understanding is a slow process due to the large amount of time required to age mammalian or vertebrate animal models. Here, we introduce a new cellular model within the Drosophila brain, in which we report classical ageing hallmarks previously observed in the primate brain. These hallmarks include axonal swellings, cytoskeletal decay, a reduction in axonal calibre, and morphological changes arising at synaptic terminals. In the fly brain, these changes begin to occur within a few weeks, ideal to study the underlying mechanisms of ageing. We discovered that the decay of the neuronal microtubule (MT) cytoskeleton precedes the onset of other ageing hallmarks. We showed that the MT-binding factors Tau, EB1, and Shot/MACF1, are necessary for MT maintenance in axons and synapses, and that their functional loss during ageing triggers MT bundle decay, followed by a decline in axons and synaptic terminals. Furthermore, genetic manipulations that improve MT networks slowed down the onset of neuronal ageing hallmarks and confer aged specimens the ability to outperform age-matched controls. Our work suggests that MT networks are a key lesion site in ageing neurons and therefore the MT cytoskeleton offers a promising target to improve neuronal decay in advanced age.
    DOI:  https://doi.org/10.1371/journal.pbio.3002504
  42. Acta Neuropathol. 2024 Mar 11. 147(1): 52
    Comprehensive proteomics of CSF, plasma, and urine identify DDC and other biomarkers of early Parkinson's disease.
    Jarod Rutledge, Benoit Lehallier, Pardis Zarifkar, Patricia Moran Losada, Marian Shahid-Besanti, Dan Western, Priyanka Gorijala, Sephira Ryman, Maya Yutsis, Gayle K Deutsch, Elizabeth Mormino, Alexandra Trelle, Anthony D Wagner, Geoffrey A Kerchner, Lu Tian, Carlos Cruchaga, Victor W Henderson, Thomas J Montine, Per Borghammer, Tony Wyss-Coray, Kathleen L Poston.
      Parkinson's disease (PD) starts at the molecular and cellular level long before motor symptoms appear, yet there are no early-stage molecular biomarkers for diagnosis, prognosis prediction, or monitoring therapeutic response. This lack of biomarkers greatly impedes patient care and translational research-L-DOPA remains the standard of care more than 50 years after its introduction. Here, we performed a large-scale, multi-tissue, and multi-platform proteomics study to identify new biomarkers for early diagnosis and disease monitoring in PD. We analyzed 4877 cerebrospinal fluid, blood plasma, and urine samples from participants across seven cohorts using three orthogonal proteomics methods: Olink proximity extension assay, SomaScan aptamer precipitation assay, and liquid chromatography-mass spectrometry proteomics. We discovered that hundreds of proteins were upregulated in the CSF, blood, or urine of PD patients, prodromal PD patients with DAT deficit and REM sleep behavior disorder or anosmia, and non-manifesting genetic carriers of LRRK2 and GBA mutations. We nominate multiple novel hits across our analyses as promising markers of early PD, including DOPA decarboxylase (DDC), also known as L-aromatic acid decarboxylase (AADC), sulfatase-modifying factor 1 (SUMF1), dipeptidyl peptidase 2/7 (DPP7), glutamyl aminopeptidase (ENPEP), WAP four-disulfide core domain 2 (WFDC2), and others. DDC, which catalyzes the final step in dopamine synthesis, particularly stands out as a novel hit with a compelling mechanistic link to PD pathogenesis. DDC is consistently upregulated in the CSF and urine of treatment-naïve PD, prodromal PD, and GBA or LRRK2 carrier participants by all three proteomics methods. We show that CSF DDC levels correlate with clinical symptom severity in treatment-naïve PD patients and can be used to accurately diagnose PD and prodromal PD. This suggests that urine and CSF DDC could be a promising diagnostic and prognostic marker with utility in both clinical care and translational research.
    DOI:  https://doi.org/10.1007/s00401-024-02706-0
  43. Cell Mol Gastroenterol Hepatol. 2024 Mar 09. pii: S2352-345X(24)00041-9. [Epub ahead of print]
    Location Matters: Mitochondrial Arginase 2 as a Treatment for Metabolic Disease?
    Megan Stefkovich, Paul M Titchenell.
      
    DOI:  https://doi.org/10.1016/j.jcmgh.2024.02.013
  44. Clin Res Hepatol Gastroenterol. 2024 Mar 11. pii: S2210-7401(24)00040-8. [Epub ahead of print]48(4): 102319
    A novel homozygous mutation, c.662_672del, in DGUOK gene causing mitochondrial DNA depletion syndrome of type hepatocerebral - A case report.
    Kandula Radhakrishna, Batchu Rajsekhar, K V Raja Ramesh, Neelima Chitturi, Sandhya Kiran Pemmasani, Anuradha Acharya.
      
    DOI:  https://doi.org/10.1016/j.clinre.2024.102319
  45. J Endocrinol. 2024 Mar 01. pii: JOE-23-0391. [Epub ahead of print]
    Specific cellular microenvironments for spatiotemporal regulation of StAR and steroid synthesis.
    Ana Fernanda Castillo, Cecilia Poderoso, Paula Mariana Maloberti, Fabiana Cornejo Maciel, María Mercedes Mori Sequeiros Garcia, Ulises Daniel Orlando, Pablo Mele, Yanina Benzo, Melina Andrea Dattilo, Jesica Prada, Luciano Quevedo, Matías Belluno, Cristina Paz, Ernesto Jorge Podesta.
      For many years, research in the field of steroid synthesis has aimed to understand the regulation of the rate-limiting step of steroid synthesis, i.e., the transport of cholesterol from the outer to the inner mitochondrial membrane, and identify the protein involved in the conversion of cholesterol into pregnenolone. The extraordinary work by B Clark, J Wells, S R King, and D M Stocco eventually identified this protein and named it steroidogenic acute regulatory protein (StAR). The group's finding was also one of the milestones in understanding the mechanism of non-vesicular lipid transport between organelles. A notable feature of StAR is its high degree of phosphorylation. In fact, StAR phosphorylation in the acute phase is required for full steroid biosynthesis. As a contribution to this subject, our work has led to the characterization of StAR as a substrate of kinases and phosphatases and as an integral part of a mitochondria-associated multi-protein complex, essential for StAR function and cholesterol binding and mitochondrial transport to yield maximum steroid production. Results allow us to postulate the existence of a specific cellular microenvironment where StAR protein synthesis and activation, along with steroid synthesis and secretion, are performed in a compartmentalized manner, at the site of hormone receptor stimulation, and involving the compartmentalized formation of the steroid molecule synthesizing complex.
    DOI:  https://doi.org/10.1530/JOE-23-0391
  46. Genome Biol. 2024 Mar 11. 25(1): 69
    A comparison of methods for detecting DNA methylation from long-read sequencing of human genomes.
    Brynja D Sigurpalsdottir, Olafur A Stefansson, Guillaume Holley, Doruk Beyter, Florian Zink, Marteinn Þ Hardarson, Sverrir Þ Sverrisson, Nina Kristinsdottir, Droplaug N Magnusdottir, Olafur Þ Magnusson, Daniel F Gudbjartsson, Bjarni V Halldorsson, Kari Stefansson.
       BACKGROUND: Long-read sequencing can enable the detection of base modifications, such as CpG methylation, in single molecules of DNA. The most commonly used methods for long-read sequencing are nanopore developed by Oxford Nanopore Technologies (ONT) and single molecule real-time (SMRT) sequencing developed by Pacific Bioscience (PacBio). In this study, we systematically compare the performance of CpG methylation detection from long-read sequencing.
    RESULTS: We demonstrate that CpG methylation detection from 7179 nanopore-sequenced DNA samples is highly accurate and consistent with 132 oxidative bisulfite-sequenced (oxBS) samples, isolated from the same blood draws. We introduce quality filters for CpGs that further enhance the accuracy of CpG methylation detection from nanopore-sequenced DNA, while removing at most 30% of CpGs. We evaluate the per-site performance of CpG methylation detection across different genomic features and CpG methylation rates and demonstrate how the latest R10.4 flowcell chemistry and base-calling algorithms improve methylation detection from nanopore sequencing. Additionally, we show how the methylation detection of 50 SMRT-sequenced genomes compares to nanopore sequencing and oxBS.
    CONCLUSIONS: This study provides the first systematic comparison of CpG methylation detection tools for long-read sequencing methods. We compare two commonly used computational methods for the detection of CpG methylation in a large number of nanopore genomes, including samples sequenced using the latest R10.4 nanopore flowcell chemistry and 50 SMRT sequenced samples. We provide insights into the strengths and limitations of each sequencing method as well as recommendations for standardization and evaluation of tools designed for genome-scale modified base detection using long-read sequencing.
    DOI:  https://doi.org/10.1186/s13059-024-03207-9
  47. Angew Chem Int Ed Engl. 2024 Mar 12. e202401544
    High-Throughput Single-Cell, Single-Mitochondrial DNA Assay Using Hydrogel Droplet Microfluidics.
    Juhwan Park, Parnika S Kadam, Yasemin Atiyas, Bonirath Chhay, Andrew Tsourkas, James H Eberwine, David Issadore.
      There is growing interest in understanding the biological implications of single cell heterogeneity and heteroplasmy of mitochondrial DNA (mtDNA), but current methodologies for single-cell mtDNA analysis limit the scale of analysis to small cell populations. Although droplet microfluidics have increased the throughput of single-cell genomic, RNA, and protein analysis, their application to sub-cellular organelle analysis has remained a largely unsolved challenge. Here, we introduce an agarose-based droplet microfluidic approach for single-cell, single-mtDNA analysis, which allows simultaneous processing of hundreds of individual mtDNA molecules within >10,000 individual cells. Our microfluidic chip encapsulates individual cells in agarose beads, designed to have a sufficiently dense hydrogel network to retain mtDNA after lysis and provide a robust scaffold for subsequent multi-step processing and analysis. To mitigate the impact of the high viscosity of agarose required for mtDNA retention on the throughput of microfluidics, we developed a parallelized device, successfully achieving ~95% mtDNA retention from single cells within our microbeads at >700,000 drops/minute. To demonstrate utility, we analyzed specific regions of the single-mtDNA using a multiplexed rolling circle amplification (RCA) assay. We demonstrated compatibility with both microscopy, for digital counting of individual RCA products, and flow cytometry for higher throughput analysis.
    Keywords:  Droplet microfluidics, High-throughput, Rolling circle amplification, Single cell, Single mitochondrial DNA
    DOI:  https://doi.org/10.1002/anie.202401544
  48. bioRxiv. 2024 Feb 19. pii: 2024.01.30.578053. [Epub ahead of print]
    Systematic characterization of protein structural features of alternative splicing isoforms using AlphaFold 2.
    Yuntao Yang, Yuhan Xie, Zhao Li, Chiamaka Diala, Meer Ali, Rongbin Li, Yi Xu, Albon Wu, Pora Kim, Sayed-Rzgar Hosseini, Erfei Bi, Hongyu Zhao, W Jim Zheng.
      Alternative splicing is an important cellular process in eukaryotes, altering pre-mRNA to yield multiple protein isoforms from a single gene. However, our understanding of the impact of alternative splicing events on protein structures is currently constrained by a lack of sufficient protein structural data. To address this limitation, we employed AlphaFold 2, a cutting-edge protein structure prediction tool, to conduct a comprehensive analysis of alternative splicing for approximately 3,000 human genes, providing valuable insights into its impact on the protein structural. Our investigation employed state of the art high-performance computing infrastructure to systematically characterize structural features in alternatively spliced regions and identified changes in protein structure following alternative splicing events. Notably, we found that alternative splicing tends to alter the structure of residues primarily located in coils and beta-sheets. Our research highlighted a significant enrichment of loops and highly exposed residues within human alternatively spliced regions. Specifically, our examination of the Septin-9 protein revealed potential associations between loops and alternative splicing, providing insights into its evolutionary role. Furthermore, our analysis uncovered two missense mutations in the Tau protein that could influence alternative splicing, potentially contributing to the pathogenesis of Alzheimer's disease. In summary, our work, through a thorough statistical analysis of extensive protein structural data, sheds new light on the intricate relationship between alternative splicing, evolution, and human disease.
    DOI:  https://doi.org/10.1101/2024.01.30.578053
  49. Int J Mol Sci. 2024 Feb 23. pii: 2594. [Epub ahead of print]25(5):
    Nicotinamide Mononucleotide (NMN) Works in Type 2 Diabetes through Unexpected Effects in Adipose Tissue, Not by Mitochondrial Biogenesis.
    Roua Gabriela Popescu, Anca Dinischiotu, Teodoru Soare, Ene Vlase, George Cătălin Marinescu.
      Nicotinamide mononucleotide (NMN) has emerged as a promising therapeutic intervention for age-related disorders, including type 2 diabetes. In this study, we confirmed the previously observed effects of NMN treatment on glucose uptake and investigated its underlying mechanisms in various tissues and cell lines. Through the most comprehensive proteomic analysis to date, we discovered a series of novel organ-specific effects responsible for glucose uptake as measured by the IPGTT: adipose tissue growing (suggested by increased protein synthesis and degradation and mTOR proliferation signaling upregulation). Notably, we observed the upregulation of thermogenic UCP1, promoting enhanced glucose conversion to heat in intermuscular adipose tissue while showing a surprising repressive effect on mitochondrial biogenesis in muscle and the brain. Additionally, liver and muscle cells displayed a unique response, characterized by spliceosome downregulation and concurrent upregulation of chaperones, proteasomes, and ribosomes, leading to mildly impaired and energy-inefficient protein synthesis machinery. Furthermore, our findings revealed remarkable metabolic rewiring in the brain. This involved increased production of ketone bodies, downregulation of mitochondrial OXPHOS and TCA cycle components, as well as the induction of well-known fasting-associated effects. Collectively, our data elucidate the multifaceted nature of NMN action, highlighting its organ-specific effects and their role in improving glucose uptake. These findings deepen our understanding of NMN's therapeutic potential and pave the way for novel strategies in managing metabolic disorders.
    Keywords:  DIA SWATH proteomics; NMN proteomics; NMN type 2 diabetes; nicotinamide mononucleotide (NMN) effects; pathway analysis; protein interaction network
    DOI:  https://doi.org/10.3390/ijms25052594
  50. Int J Med Sci. 2024 ;21(4): 755-764
    Phosphoglycerate mutase 5 aggravates alcoholic liver disease through disrupting VDAC-1-dependent mitochondrial integrity.
    Tian Xia, Jiachi Yu, Ye Chen, Xing Chang, Miao Meng.
      Alcoholic liver disease (ALD) poses a substantial global health challenge, with its pathogenesis deeply rooted in mitochondrial dysfunction. Our study explores the pivotal roles of Phosphoglycerate mutase family member 5 (Pgam5) and Voltage-Dependent Anion Channel 1 (VDAC1) in the progression of ALD, providing novel insights into their interplay and impact on mitochondrial integrity. We demonstrate that Pgam5 silencing preserves hepatocyte viability and attenuates ethanol-induced apoptosis, underscoring its detrimental role in exacerbating hepatocyte dysfunction. Pgam5's influence extends to the regulation of VDAC1 oligomerization, a key process in mitochondrial permeability transition pore (mPTP) opening, mitochondrial swelling, and apoptosis initiation. Notably, the inhibition of VDAC1 oligomerization through Pgam5 silencing or pharmacological intervention (VBIT-12) significantly preserves mitochondrial function, evident in the maintenance of mitochondrial membrane potential and reduced reactive oxygen species (ROS) production. In vivo experiments using hepatocyte-specific Pgam5 knockout (Pgam5hKO) and control mice reveal that Pgam5 deficiency mitigates ethanol-induced liver histopathology, inflammation, lipid peroxidation, and metabolic disorder, further supporting its role in ALD progression. Our findings highlight the critical involvement of Pgam5 and VDAC1 in mitochondrial dysfunction in ALD, suggesting potential therapeutic targets. While promising, these findings necessitate further research, including human studies, to validate their clinical applicability and explore broader implications in liver diseases. Overall, our study provides a significant advancement in understanding ALD pathophysiology, paving the way for novel therapeutic strategies targeting mitochondrial pathways in ALD.
    Keywords:  ALD; Pgam5; VDAC1; mitochondria
    DOI:  https://doi.org/10.7150/ijms.93171
  51. Parkinsonism Relat Disord. 2024 Mar 06. pii: S1353-8020(24)00090-7. [Epub ahead of print] 106078
    Towards a biological diagnosis of PD.
    Avika Chopra, Anthony E Lang, Günter Höglinger, Tiago F Outeiro.
      Since the original description by James Parkinson, Parkinson's disease (PD) has intrigued us for over 200 years. PD is a progressive condition that is incurable so far, and affects millions of people worldwide. Over the years, our knowledge has expanded tremendously, and a range of criteria have been put forward and used to try to define PD. However, owing to the complexity of the problem, it is still not consensual how to diagnose and classify a disease that manifests with diverse features, and that responds differently to existing therapies and to those under development. We are now living a time when 'biological' information is becoming abundant, precise, and accessible enabling us to attempt to incorporate different sources of information to classify different forms of PD. These refinements are essential for basic science, as they will enable us to develop improved models for studying PD, and to implement new findings into clinical practice, as this will be the path towards effective personalized medicine.
    Keywords:  Alpha-synuclein; Biomarker; Genetics; Neurodegeneration; Neuropathology; Parkinson's disease
    DOI:  https://doi.org/10.1016/j.parkreldis.2024.106078
  52. J Cell Biol. 2024 May 06. pii: e202305105. [Epub ahead of print]223(5):
    Polarized localization of kinesin-1 and RIC-7 drives axonal mitochondria anterograde transport.
    Youjun Wu, Chen Ding, Behrang Sharif, Alexis Weinreb, Grace Swaim, Hongyan Hao, Shaul Yogev, Shigeki Watanabe, Marc Hammarlund.
      Mitochondria transport is crucial for axonal mitochondria distribution and is mediated by kinesin-1-based anterograde and dynein-based retrograde motor complexes. While Miro and Milton/TRAK were identified as key adaptors between mitochondria and kinesin-1, recent studies suggest the presence of additional mechanisms. In C. elegans, ric-7 is the only single gene described so far, other than kinesin-1, that is absolutely required for axonal mitochondria localization. Using CRISPR engineering in C. elegans, we find that Miro is important but is not essential for anterograde traffic, whereas it is required for retrograde traffic. Both the endogenous RIC-7 and kinesin-1 act at the leading end to transport mitochondria anterogradely. RIC-7 binding to mitochondria requires its N-terminal domain and partially relies on MIRO-1, whereas RIC-7 accumulation at the leading end depends on its disordered region, kinesin-1, and metaxin2. We conclude that transport complexes containing kinesin-1 and RIC-7 polarize at the leading edge of mitochondria and are required for anterograde axonal transport in C. elegans.
    DOI:  https://doi.org/10.1083/jcb.202305105
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