bims-mitdis Biomed News
on Mitochondrial disorders
Issue of 2023–10–22
forty-two papers selected by
Catalina Vasilescu, Helmholz Munich
  1. Control of mitochondrial dynamics by a fusogenic lipid.
  2. mtFociCounter for automated single-cell mitochondrial nucleoid quantification and reproducible foci analysis.
  3. Mitochondrial myopathy without extraocular muscle involvement: a unique clinicopathologic profile.
  4. Two mitochondrial HMG-box proteins, Cim1 and Abf2, antagonistically regulate mtDNA copy number in Saccharomyces cerevisiae.
  5. Metabolic Myopathies: Experience of a Reference Center of Inherited Metabolic Diseases.
  6. New insights into the nutritional genomics of adult-onset riboflavin-responsive diseases.
  7. Mitochondrial OPA1 deficiency is associated to reversible defects in spatial memory related to adult neurogenesis in mice.
  8. SIRT4 is a regulator of human skeletal muscle fatty acid metabolism influencing inner and outer mitochondrial-mediated fusion.
  9. A PINK1 input threshold arises from positive feedback in the PINK1/Parkin mitophagy decision circuit.
  10. The metabolic control theory: Its development and its application to mitochondrial oxidative phosphorylation.
  11. Caseinolytic mitochondrial matrix peptidase X is essential for homologous chromosome synapsis and recombination during meiosis of male mouse germ cells.
  12. A rare, likely pathogenic variant causing Leber's hereditary optic neuropathy in three-generation females of an African-American family.
  13. Evidence from 2100 index cases supports genome sequencing as a first-tier genetic test.
  14. Molecular mechanism of glutaminase activation through filamentation and the role of filaments in mitophagy protection.
  15. Mitophagy in neurodegenerative disease pathogenesis.
  16. Long-term super-resolution inner mitochondrial membrane imaging with a lipid probe.
  17. ATAD3 Proteins: Unique Mitochondrial Proteins Essential for Life in Diverse Eukaryotic Lineages.
  18. In fission yeast, 65 non-essential mitochondrial proteins related to respiration and stress become essential in low-glucose conditions.
  19. Super-resolution imaging of mitochondrial cristae using a more hydrophobic far-red Si-rhodamine probe.
  20. Developing mitochondrial base editors with diverse context compatibility and high fidelity via saturated spacer library.
  21. Aspartyl-tRNA synthetase 2 orchestrates iron-sulfur metabolism in hematopoietic stem cells via fine-tuning alternative RNA splicing.
  22. FDX1 is required for the biogenesis of mitochondrial cytochrome c oxidase in mammalian cells.
  23. Lactate dehydrogenase D is a general dehydrogenase for D-2-hydroxyacids and is associated with D-lactic acidosis.
  24. ACOX1 Gain-of-Function Variant in Two German Pediatric Patients, in One Case Mimicking Autoimmune Inflammatory Disease.
  25. Dissecting the roles of the nuclear and mitochondrial genomes in a mouse model of autoimmune diabetes.
  26. Fibrocystin/Polyductin releases a C-terminal fragment that translocates into mitochondria and suppresses cystogenesis.
  27. An optimized protocol to detect ubiquitination modification of exogenous or endogenous proteins.
  28. Membrane remodeling properties of the Parkinson's disease protein LRRK2.
  29. MAO-SiR is a tool for visualizing the inner workings of mitochondria.
  30. Targeting mitochondrial degradation by chimeric autophagy-tethering compounds.
  31. Development of novel cytoprotective small compounds inhibiting mitochondria-dependent cell death.
  32. Combined Oxidative Phosphorylation Deficiency Type-13 with Perinatal Presentation: A Case Report.
  33. Hydroxyapatite-based nano-drug delivery system for nicotinamide mononucleotide (NMN): significantly enhancing NMN bioavailability and replenishing in vivo nicotinamide adenine dinucleotide (NAD+) levels.
  34. Contact sites between host organelles and pathogens: boon or bane?
  35. Identification and quantification of small exon-containing isoforms in long-read RNA sequencing data.
  36. Disturbance of mitochondrial functions caused by N-acetylglutamate and N-acetylmethionine in brain of adolescent rats: Potential relevance in aminoacylase 1 deficiency.
  37. A guide to studying mitochondria transfer.
  38. Methods section too short? Use online protocols to make complex techniques understandable.
  39. MINRbase: a comprehensive database of nuclear- and mitochondrial-ribosomal-RNA-derived fragments (rRFs).
  40. Towards more accurate pharmacogenomic variant effect predictions.
  41. Complex genotypes in family with metachromatic leukodystrophy: Effect of trans and cis mutations distribution on the phenotype variability.
  42. Clinical, Biochemical and Molecular Features of a Cohort of 8 Patients with Inherited Disorders of Vitamin B12 Metabolism in a Metabolic Reference Center.
  1. Trends Cell Biol. 2023 Oct 17. pii: S0962-8924(23)00210-6. [Epub ahead of print]
    Control of mitochondrial dynamics by a fusogenic lipid.
    Zheng Yang, David C Chan.
      Mitochondrial fusion enables cooperation between the mitochondrial population and is critical for mitochondrial function. Phosphatidic acid (PA) on the mitochondrial surface has a key role in mitochondrial fusion. A recent study by Su et al. shows that the nucleoside diphosphate (NDP) kinase NME3 recognizes PA and mediates its effects on mitochondrial dynamics.
    Keywords:  membrane fusion; mitochondria; organelle; phospholipid
    DOI:  https://doi.org/10.1016/j.tcb.2023.10.006
  2. Nucleic Acids Res. 2023 Oct 18. pii: gkad864. [Epub ahead of print]
    mtFociCounter for automated single-cell mitochondrial nucleoid quantification and reproducible foci analysis.
    Timo Rey, Luis Carlos Tábara, Julien Prudent, Michal Minczuk.
      Mitochondrial DNA (mtDNA) encodes the core subunits for OXPHOS, essential in near-all eukaryotes. Packed into distinct foci (nucleoids) inside mitochondria, the number of mtDNA copies differs between cell-types and is affected in several human diseases. Currently, common protocols estimate per-cell mtDNA-molecule numbers by sequencing or qPCR from bulk samples. However, this does not allow insight into cell-to-cell heterogeneity and can mask phenotypical sub-populations. Here, we present mtFociCounter, a single-cell image analysis tool for reproducible quantification of nucleoids and other foci. mtFociCounter is a light-weight, open-source freeware and overcomes current limitations to reproducible single-cell analysis of mitochondrial foci. We demonstrate its use by analysing 2165 single fibroblasts, and observe a large cell-to-cell heterogeneity in nucleoid numbers. In addition, mtFociCounter quantifies mitochondrial content and our results show good correlation (R = 0.90) between nucleoid number and mitochondrial area, and we find nucleoid density is less variable than nucleoid numbers in wild-type cells. Finally, we demonstrate mtFociCounter readily detects differences in foci-numbers upon sample treatment, and applies to Mitochondrial RNA Granules and superresolution microscopy. mtFociCounter provides a versatile solution to reproducibly quantify cellular foci in single cells and our results highlight the importance of accounting for cell-to-cell variance and mitochondrial context in mitochondrial foci analysis.
    DOI:  https://doi.org/10.1093/nar/gkad864
  3. J Neurol. 2023 Oct 17.
    Mitochondrial myopathy without extraocular muscle involvement: a unique clinicopathologic profile.
    Yan Lin, Jiayin Wang, Hong Ren, Xiaotian Ma, Wei Wang, Ying Zhao, Zhihong Xu, Shuangwu Liu, Wenqing Wang, Xuebi Xu, Bin Wang, Dandan Zhao, Dongdong Wang, Wei Li, Fuchen Liu, Yuying Zhao, Jianqiang Lu, Chuanzhu Yan, Kunqian Ji.
       OBJECTIVE: Mitochondrial myopathy without extraocular muscles involvement (MiMy) represents a distinct form of mitochondrial disorder predominantly affecting proximal/distal or axial muscles, with its phenotypic, genotypic features, and long-term prognosis poorly understood.
    METHODS: A cross-sectional study conducted at a national diagnostic center for mitochondrial disease involved 47 MiMy patients, from a cohort of 643 mitochondrial disease cases followed up at Qilu Hospital from January 1, 2000, to January 1, 2021. We compared the clinical, pathological, and genetic features of MiMy to progressive external ophthalmoplegia (PEO) and mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes (MELAS) patients.
    RESULTS: MiMy patients demonstrated a more pronounced muscle involvement syndrome, with lower 6MWT scores, higher FSS, and lower BMI compared to PEO and MELAS patients. Serum levels of creatinine kinase (CK), lactate, and growth and differentiation factor 15 (GDF15) were substantially elevated in MiMy patients. Nearly a third (31.9%) displayed signs of subclinical peripheral neuropathy, mostly axonal neuropathy. Muscle biopsies revealed that cytochrome c oxidase strong (COX-s) ragged-red fibers (RRFs) were a typical pathological feature in MiMy patients. Genetic analysis predominantly revealed mtDNA point pathogenic variants (59.6%) and less frequently single (12.8%) or multiple (4.2%) mtDNA deletions. During the follow-up, a majority (76.1%) of MiMy patients experienced stabilization or improvement after therapeutic intervention.
    CONCLUSIONS: This study provides a comprehensive profile of MiMy through a large patient cohort, elucidating its unique clinical, genetic, and pathological features. These findings offer significant insights into the diagnostic and therapeutic management of MiMy, ultimately aiming to ameliorate patient outcomes and enhance the quality of life.
    Keywords:  Clinicopathologic features; MELAS; MiMy; Mitochondrial disorders; PEO
    DOI:  https://doi.org/10.1007/s00415-023-12005-5
  4. Nucleic Acids Res. 2023 Oct 18. pii: gkad849. [Epub ahead of print]
    Two mitochondrial HMG-box proteins, Cim1 and Abf2, antagonistically regulate mtDNA copy number in Saccharomyces cerevisiae.
    Simon Schrott, Christof Osman.
      The mitochondrial genome, mtDNA, is present in multiple copies in cells and encodes essential subunits of oxidative phosphorylation complexes. mtDNA levels have to change in response to metabolic demands and copy number alterations are implicated in various diseases. The mitochondrial HMG-box proteins Abf2 in yeast and TFAM in mammals are critical for mtDNA maintenance and packaging and have been linked to mtDNA copy number control. Here, we discover the previously unrecognized mitochondrial HMG-box protein Cim1 (copy number influence on mtDNA) in Saccharomyces cerevisiae, which exhibits metabolic state dependent mtDNA association. Surprisingly, in contrast to Abf2's supportive role in mtDNA maintenance, Cim1 negatively regulates mtDNA copy number. Cells lacking Cim1 display increased mtDNA levels and enhanced mitochondrial function, while Cim1 overexpression results in mtDNA loss. Intriguingly, Cim1 deletion alleviates mtDNA maintenance defects associated with loss of Abf2, while defects caused by Cim1 overexpression are mitigated by simultaneous overexpression of Abf2. Moreover, we find that the conserved LON protease Pim1 is essential to maintain low Cim1 levels, thereby preventing its accumulation and concomitant repressive effects on mtDNA. We propose a model in which the protein ratio of antagonistically acting Cim1 and Abf2 determines mtDNA copy number.
    DOI:  https://doi.org/10.1093/nar/gkad849
  5. Endocr Metab Immune Disord Drug Targets. 2023 Oct 18.
    Metabolic Myopathies: Experience of a Reference Center of Inherited Metabolic Diseases.
    Mafalda Rebelo, Madalena Pires, Laura Azurara, Lara Câmara, Márcia Pereira, Augusto Ribeirinho, Gonçalo Padeira, Patrícia Gaspar Silva, Sandra Jacinto, José Pedro Vieira, Ana Cristina Ferreira.
       INTRODUCTION: Metabolic myopathies (MM) are a heterogeneous group of genetic disorders affecting metabolic pathways involved in energy production during rest, exercise and physiologic stress (fever, fasting, …). Impairments in the pathways of glycolysis/ glycogenolysis, fatty acid transport/oxidation or in the mitochondrial respiratory chain present primarily with exercise intolerance, myalgias, weakness, cramps, or rhabdomyolysis. Depending on aetiology, the diagnosis can be made through neonatal screening, pre-symptomatic or in the set of clinical manifestations for which a high level of suspicion is important.
    METHODS: Retrospective descriptive study of the clinical, biochemical, and molecular features of patients with a confirmed diagnosis of MM followed by the multidisciplinary team of the Reference Center of Inherited Metabolic Diseases of Centro Hospitalar Universitário de Lisboa Central from 2009 to 2022.
    RESULTS: Twenty-three patients with MM were included: 9 (39%) glycogen storage diseases (7 McArdle and 2 Pompe), 7 (30%) fatty acid oxidation disorders (3 CPT2, 3 LCHAD and 1 MAD deficiencies), 6 (26%) mitochondrial disease with significant muscle involvement (2 Pearson, 1 Kearns Sayre, 1 VARS2, 1 SUCLA2 and 1 MT-TL1 deficiencies), and 1 myoadenylate deaminase deficiency. Ages varied from 15 months to 35 years. Eighteen (78%) patients were diagnosed by clinical symptoms, 3 by newborn screening (LCHAD) and 2 were asymptomatic (1 Pompe and 1 McArdle). Frequent symptoms were rhabdomyolysis triggered by illness or exercise 12 (52%), fatigue 11 (48%), exercise intolerance 10 (43%), and myalgia 9 (43%). Eight (35%) patients (LCHAD and mitochondrial) had multisystemic involvement. In 20 (87%) patients, the diagnosis was confirmed by biochemical and/or genetic analysis and 3 (McArdle) by muscle biopsy.
    CONCLUSION: MM are a heterogeneous set of disorders, but a careful history may guide the differential diagnosis among biochemical pathways and other etiologies. Nowadays, molecular testing has become a powerful tool for diagnosis confirmation, surpassing muscular biopsy in most cases. Accurate diagnosis is important to identify who may benefit from specific therapeutic options, such as enzyme replacement therapy, restricted diets, emergency regime and cofactors. All patients benefit from adequate lifestyle modifications, individualized exercise prescription, nutritional intervention, and genetic counselling.
    Keywords:  fatty acid; fatty acid oxidation disorders; glycogenosis; metabolic myopathies; mitochondrial diseases; rhabdomyolysis
    DOI:  https://doi.org/10.2174/0118715303279208231012051937
  6. Nutr Metab (Lond). 2023 Oct 16. 20(1): 42
    New insights into the nutritional genomics of adult-onset riboflavin-responsive diseases.
    Chiara Murgia, Ankush Dehlia, Mark A Guthridge.
      Riboflavin, or vitamin B2, is an essential nutrient that serves as a precursor to flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN). The binding of the FAD and/or FMN cofactors to flavoproteins is critical for regulating their assembly and activity. There are over 90 proteins in the human flavoproteome that regulate a diverse array of biochemical pathways including mitochondrial metabolism, riboflavin transport, ubiquinone and FAD synthesis, antioxidant signalling, one-carbon metabolism, nitric oxide signalling and peroxisome oxidative metabolism. The identification of patients with genetic variants in flavoprotein genes that lead to adult-onset pathologies remains a major diagnostic challenge. However, once identified, many patients with adult-onset inborn errors of metabolism demonstrate remarkable responses to riboflavin therapy. We review the structure:function relationships of mutant flavoproteins and propose new mechanistic insights into adult-onset riboflavin-responsive pathologies and metabolic dysregulations that apply to multiple biochemical pathways. We further address the vexing issue of how the inheritance of genetic variants in flavoprotein genes leads to an adult-onset disease with complex symptomologies and varying severities. We also propose a broad clinical framework that may not only improve the current diagnostic rates, but also facilitate a personalized approach to riboflavin therapy that is low cost, safe and lead to transformative outcomes in many patients.
    Keywords:  Metabolism; Micronutrients; Nutrigenomics; Personalised nutrition; Vitamin
    DOI:  https://doi.org/10.1186/s12986-023-00764-x
  7. eNeuro. 2023 Oct 20. pii: ENEURO.0073-23.2023. [Epub ahead of print]
    Mitochondrial OPA1 deficiency is associated to reversible defects in spatial memory related to adult neurogenesis in mice.
    Trinovita Andraini, Lionel Moulédous, Petnoi Petsophonsakul, Cédrick Florian, Sébastien Gauzin, Marlène Botella-Daloyau, Macarena Arrázola, Kamela Nikolla, Adam Philip, Alice Leydier, Manon Marque, Laetitia Arnauné-Pelloquin, Pascale Belenguer, Claire Rampon, Marie-Christine Miquel.
      Mitochondria are integrative hubs central to cellular adaptive pathways. Such pathways are critical in highly differentiated post-mitotic neurons, the plasticity of which sustains brain function. Consequently, defects in mitochondria and in their dynamics appear instrumental in neurodegenerative diseases and may also participate in cognitive impairments. To directly test this hypothesis, we analyzed cognitive performances in a mouse mitochondria-based disease model, due to haploinsufficiency in the mitochondrial optic-atrophy-type-1 (OPA1) protein involved in mitochondrial dynamics. In males, we evaluated adult hippocampal neurogenesis parameters using immunohistochemistry. We performed a battery of tests to assess basal behavioral characteristics and cognitive performances, and tested putative treatments.While in Dominant Optic Atrophy (DOA) mouse models, the known main symptoms are late onset visual deficits, we discovered early impairments in hippocampus-dependent spatial memory attributable to defects in adult neurogenesis. Moreover, less connected adult-born hippocampal neurons showed a decrease in mitochondrial content. Remarkably, voluntary exercise or pharmacological treatment targeting mitochondrial dynamics restored spatial memory in DOA mice. Altogether, our study identifies a crucial role for OPA1-dependent mitochondrial functions in adult neurogenesis, and thus in hippocampal-dependent cognitive functions. More generally, our findings show that adult neurogenesis is highly sensitive to mild mitochondrial defects, generating impairments in spatial memory that can be detected at an early stage and counterbalanced by physical exercise and pharmacological targeting of mitochondrial dynamics. Thus, amplification of mitochondrial function at an early stage appears beneficial for late-onset neurodegenerative diseases.Significance StatementThe adult hippocampus continues to produce new neurons in mammals. These new neurons are highly sensitive to mitochondrial perturbation. Dominant optic atrophy (DOA) is a rare disease mainly caused by mutations in the gene coding the mitochondrial protein OPA1. Using a mouse model of OPA1 deficiency, we found that hippocampal new neurons have dendritic spine density defects and altered mitochondrial content. We further detected impairments in spatial memory capacities relying on adult-neurogenesis. We report that these memory impairments can be corrected by physical exercise and pharmacological treatment targeting mitochondria in mice. Our results indicate that early detection of spatial memory deficits related to adult neurogenesis may allow a precocious action in pathologies involving mitochondria, such as DOA or neurodegenerative diseases.
    Keywords:  Adult Neurogenesis; Dominant Optic Atrophy; Hippocampus; Mitochondria; Pattern separation; Physical exercise
    DOI:  https://doi.org/10.1523/ENEURO.0073-23.2023
  8. Cell Signal. 2023 Oct 17. pii: S0898-6568(23)00346-7. [Epub ahead of print] 110931
    SIRT4 is a regulator of human skeletal muscle fatty acid metabolism influencing inner and outer mitochondrial-mediated fusion.
    John Noone, Keith D Rochfort, Finbarr O'Sullivan, Donal J O'Gorman.
       OBJECTIVE: The mitochondrial phenotype, governed by the balance of fusion-fission, is a key determinant of energy metabolism. The inner and outer mitochondrial membrane (IMM) fusion proteins optic atrophy 1 (OPA1) and Mitofusin 1 and 2 (Mfn1/2) play an important role in this process. Recent evidence also shows that Sirtuin 4 (SIRT4), located within the mitochondria, is involved in the regulation of fatty acid oxidation. The purpose of this study was to determine if SIRT4 expression regulates inner and outer mitochondrial-mediated fusion and substrate utilization within differentiated human skeletal muscle cells (HSkMC).
    MATERIAL AND METHODS: SIRT4 expression was knocked down using small interfering RNA (siRNA) transfection in differentiated HSkMC. Following knockdown, mitochondrial respiration was determined by high-resolution respirometry (HRR) using the Oroboros Oxygraph O2k. Live cell confocal microscopy, quantified using the Mitochondrial Network Analysis (MiNA) toolset, was used to examine mitochondrial morphological change. This was further examined through the measurement of key metabolic and mitochondrial morphological regulators (mRNA and protein) induced by knockdown.
    RESULTS: SIRT4 knockdown resulted in a significant decrease in LEAK respiration, potentially explained by a decrease in ANT1 protein expression. Knockdown further increased oxidative phosphorylation and protein expression of key regulators of fatty acid metabolism. Quantitative analysis of live confocal imaging of fluorescently labelled mitochondria following SIRT4 knockdown supported the role SIRT4 plays in the regulation of mitochondrial morphology, as emphasized by an increase in mitochondrial network branches and junctions. Measurement of key regulators of mitochondrial dynamics illustrated a significant increase in mitochondrial fusion proteins Mfn1, OPA1 respectively, indicative of an increase in mitochondrial size.
    CONCLUSIONS: This study provides evidence of a direct relationship between the mitochondrial phenotype and substrate oxidation in HSkMC. We identify SIRT4 as a key protagonist of energy metabolism via its regulation of IMM and OMM fusion proteins, OPA1 and Mfn1. SIRT4 knockdown increases mitochondrial capacity to oxidize fatty acids, decreasing LEAK respiration and further increasing mitochondrial elongation via its regulation of mitochondrial fusion.
    Keywords:  Metabolism; Mitochondrial dynamics; Mitochondrial function; OPA1; Sirtuin; Skeletal muscle
    DOI:  https://doi.org/10.1016/j.cellsig.2023.110931
  9. Cell Rep. 2023 Oct 17. pii: S2211-1247(23)01272-X. [Epub ahead of print]42(10): 113260
    A PINK1 input threshold arises from positive feedback in the PINK1/Parkin mitophagy decision circuit.
    Christopher S Waters, Sigurd B Angenent, Steven J Altschuler, Lani F Wu.
      Mechanisms that prevent accidental activation of the PINK1/Parkin mitophagy circuit on healthy mitochondria are poorly understood. On the surface of damaged mitochondria, PINK1 accumulates and acts as the input signal to a positive feedback loop of Parkin recruitment, which in turn promotes mitochondrial degradation via mitophagy. However, PINK1 is also present on healthy mitochondria, where it could errantly recruit Parkin and thereby activate this positive feedback loop. Here, we explore emergent properties of the PINK1/Parkin circuit by quantifying the relationship between mitochondrial PINK1 concentrations and Parkin recruitment dynamics. We find that Parkin is recruited to mitochondria only if PINK1 levels exceed a threshold and then only after a delay that is inversely proportional to PINK1 levels. Furthermore, these two regulatory properties arise from the input-coupled positive feedback topology of the PINK1/Parkin circuit. These results outline an intrinsic mechanism by which the PINK1/Parkin circuit can avoid errant activation on healthy mitochondria.
    Keywords:  CP: Molecular biology; PINK1; Parkin; circuit; delay; mathematical model; mitophagy decision; quantitative microscopy; synthetic biology; systems biology; threshold
    DOI:  https://doi.org/10.1016/j.celrep.2023.113260
  10. Biosystems. 2023 Oct 12. pii: S0303-2647(23)00213-7. [Epub ahead of print] 105038
    The metabolic control theory: Its development and its application to mitochondrial oxidative phosphorylation.
    Jean-Pierre Mazat.
      Metabolic Control Theory (MCT) and Metabolic Control Analysis (MCA) are the two sides, theoretical and experimental, of the measurement of the sensitivity of metabolic networks in the vicinity of a steady state. We will describe the birth and the development of this theory from the first analyses of linear pathways up to a global mathematical theory applicable to any metabolic network. We will describe how the theory, given the global nature of mitochondrial oxidative phosphorylation, solved the problem of what controls mitochondrial ATP synthesis and then how it led to a better understanding of the differential tissue expression of human mitochondrial pathologies and of the heteroplasmy of mitochondrial DNA, leading to the concept of the threshold effect.
    Keywords:  Metabolic control analysis; Metabolic control theory; Mitochondrial diseases; Oxidative phoshorylation
    DOI:  https://doi.org/10.1016/j.biosystems.2023.105038
  11. Asian J Androl. 2023 Oct 17.
    Caseinolytic mitochondrial matrix peptidase X is essential for homologous chromosome synapsis and recombination during meiosis of male mouse germ cells.
    Hai-Wei Feng, Yu Zhao, Yan-Ling Gao, Dong-Teng Liu, Li-Jun Huo.
       ABSTRACT: Meiosis is the process of producing haploid gametes through a series of complex chromosomal events and the coordinated action of various proteins. The mitochondrial protease complex (ClpXP), which consists of caseinolytic mitochondrial matrix peptidase X (ClpX) and caseinolytic protease P (ClpP) and mediates the degradation of misfolded, damaged, and oxidized proteins, is essential for maintaining mitochondrial homeostasis. ClpXP has been implicated in meiosis regulation, but its precise role is currently unknown. In this study, we engineered an inducible male germ cell-specific knockout caseinolytic mitochondrial matrix peptidase X (ClpxcKO) mouse model to investigate the function of ClpX in meiosis. We found that disrupting Clpx in male mice induced germ cell apoptosis and led to an absence of sperm in the epididymis. Specifically, it caused asynapsis of homologous chromosomes and impaired meiotic recombination, resulting in meiotic arrest in the zygotene-to-pachytene transition phase. The loss of ClpX compromised the double-strand break (DSB) repair machinery by markedly reducing the recruitment of DNA repair protein RAD51 homolog 1 (RAD51) to DSB sites. This dysfunction may be due to an insufficient supply of energy from the aberrant mitochondria in ClpxcKO spermatocytes, as discerned by electron microscopy. Furthermore, ubiquitination signals on chromosomes and the expression of oxidative phosphorylation subunits were both significantly attenuated in ClpxcKO spermatocytes. Taken together, we propose that ClpX is essential for maintaining mitochondrial protein homeostasis and ensuring homologous chromosome pairing, synapsis, and recombination in spermatocytes during meiotic prophase I.
    DOI:  https://doi.org/10.4103/aja202343
  12. Am J Ophthalmol Case Rep. 2023 Dec;32 101936
    A rare, likely pathogenic variant causing Leber's hereditary optic neuropathy in three-generation females of an African-American family.
    Yanjun Chen, Elizabeth R Kellom.
       Purpose: We report a rare, likely pathogenic variant gene causing Leber's hereditary optic neuropathy (LHON) in three-generation female members of an African-American family.
    Observations: The granddaughter and mother presented with a subacute, painless visual loss in both eyes at age 10 and 42 years to legal blindness. The maternal grandmother presented with a gradual onset of moderate visual loss at age 60. The mother and grandmother reported a history of bariatric surgery and subsequent vitamin deficiencies. All three patients shared similar Optical Coherent Tomography (OCT) findings of profound thinning of ganglion cell complex (GCC) and relatively preserved peripapillary retinal nerve fiber layer thickness (pRNFL). Nuclear and mitochondrial DNA sequencing identified a 14596A > T likely pathogenic variant, p.(Ile26Met), in the MT-ND6 gene, with 100% homoplasmy in the granddaughter and mother and 65% heteroplasmy in the grandmother. The mother and grandmother were treated with idebenone in addition to vitamin supplements, with a slight improvement in their vision.
    Conclusions and Importance: Our patients' presentation stresses the importance of including LHON in the differential diagnosis in females presenting with unexplained bilateral, painless, severe visual loss. The OCT finding of profound GCC thinning with relatively preserved pRNFL thickness can be a red flag for LHON. A collaboration with genetic specialists to utilize expanded gene sequencing may greatly enhance our ability to identify rare pathogenic variants.
    Keywords:  14596A>T variant; Females; Leber's hereditary optic neuropathy (LHON); Nutritional deficiency
    DOI:  https://doi.org/10.1016/j.ajoc.2023.101936
  13. Genet Med. 2023 Oct 12. pii: S1098-3600(23)01011-0. [Epub ahead of print] 100995
    Evidence from 2100 index cases supports genome sequencing as a first-tier genetic test.
    Fen Guo, Ruby Liu, Yinghong Pan, Christin Collins, Lora Bean, Zeqiang Ma, Abhinav Mathur, Cristina Da Silva, Babi Nallamilli, Naga Guruju, Xiangwen Chen-Deutsch, Rizwan Yousaf, Ephrem Chin, Jorune Balciuniene, Madhuri Hegde.
       PURPOSE: Genome sequencing (GS) is one of the most comprehensive assays that interrogate single nucleotide variants, copy number variants, mitochondrial variants, repeat expansions, and structural variants in one assay. Despite the clear technical superiority, the full clinical utility of GS has yet to be determined.
    METHODS: We systematically evaluated 2100 clinical GS index cases performed in our laboratory to explore the diagnostic yield of GS as first-tier and as follow-up testing.
    RESULTS: The overall diagnostic yield was 28% (585/2100). The diagnostic yield for GS as the first-tier test was 26% (294/1146). Among cases with prior non-diagnostic genetic tests, GS provided a diagnosis for 27% (247/910) of cases including 56 cases with prior exome sequencing (ES). Though re-analysis of previous ES might have resolved the diagnosis in 29 cases, diagnoses for 27 cases would have been missed due to the technical inferiority of ES. Moreover, GS further disclosed additional genetic etiology in 3 out of 44 cases with existing partial diagnosis.
    CONCLUSION: We present the largest-to-date GS dataset of a clinically heterogeneous cohort from a single clinical laboratory. Our data demonstrate that GS should be considered as the first-tier genetic test that has the potential to shorten the diagnostic odyssey.
    Keywords:  Clinical utility; Diagnostic yield; Exome sequencing250 Industry Dr; First-tier testing; Genome sequencing
    DOI:  https://doi.org/10.1016/j.gim.2023.100995
  14. Nat Struct Mol Biol. 2023 Oct 19.
    Molecular mechanism of glutaminase activation through filamentation and the role of filaments in mitophagy protection.
    Douglas Adamoski, Marilia Meira Dias, Jose Edwin Neciosup Quesñay, Zhengyi Yang, Ievgeniia Zagoriy, Anna M Steyer, Camila Tanimoto Rodrigues, Alliny Cristiny da Silva Bastos, Bianca Novaes da Silva, Renna Karoline Eloi Costa, Flávia Mayumi Odahara de Abreu, Zeyaul Islam, Alexandre Cassago, Marin Gerard van Heel, Sílvio Roberto Consonni, Simone Mattei, Julia Mahamid, Rodrigo Villares Portugal, Andre Luis Berteli Ambrosio, Sandra Martha Gomes Dias.
      Glutaminase (GLS), which deaminates glutamine to form glutamate, is a mitochondrial tetrameric protein complex. Although inorganic phosphate (Pi) is known to promote GLS filamentation and activation, the molecular basis of this mechanism is unknown. Here we aimed to determine the molecular mechanism of Pi-induced mouse GLS filamentation and its impact on mitochondrial physiology. Single-particle cryogenic electron microscopy revealed an allosteric mechanism in which Pi binding at the tetramer interface and the activation loop is coupled to direct nucleophile activation at the active site. The active conformation is prone to enzyme filamentation. Notably, human GLS filaments form inside tubulated mitochondria following glutamine withdrawal, as shown by in situ cryo-electron tomography of cells thinned by cryo-focused ion beam milling. Mitochondria with GLS filaments exhibit increased protection from mitophagy. We reveal roles of filamentous GLS in mitochondrial morphology and recycling.
    DOI:  https://doi.org/10.1038/s41594-023-01118-0
  15. Neural Regen Res. 2024 May;19(5): 998-1005
    Mitophagy in neurodegenerative disease pathogenesis.
    Kan Yang, Yuqing Yan, Anni Yu, Ru Zhang, Yuefang Zhang, Zilong Qiu, Zhengyi Li, Qianlong Zhang, Shihao Wu, Fei Li.
      Mitochondria are critical cellular energy resources and are central to the life of the neuron. Mitophagy selectively clears damaged or dysfunctional mitochondria through autophagic machinery to maintain mitochondrial quality control and homeostasis. Mature neurons are postmitotic and consume substantial energy, thus require highly efficient mitophagy pathways to turn over damaged or dysfunctional mitochondria. Recent evidence indicates that mitophagy is pivotal to the pathogenesis of neurological diseases. However, more work is needed to study mitophagy pathway components as potential therapeutic targets. In this review, we briefly discuss the characteristics of nonselective autophagy and selective autophagy, including ERphagy, aggrephagy, and mitophagy. We then introduce the mechanisms of Parkin-dependent and Parkin-independent mitophagy pathways under physiological conditions. Next, we summarize the diverse repertoire of mitochondrial membrane receptors and phospholipids that mediate mitophagy. Importantly, we review the critical role of mitophagy in the pathogenesis of neurodegenerative diseases including Alzheimer's disease, Parkinson's disease, and amyotrophic lateral sclerosis. Last, we discuss recent studies considering mitophagy as a potential therapeutic target for treating neurodegenerative diseases. Together, our review may provide novel views to better understand the roles of mitophagy in neurodegenerative disease pathogenesis.
    Keywords:  Alzheimer’s disease; PINK1; Parkin; Parkinson’s disease; amyotrophic lateral sclerosis; autophagy; mitochondria; mitophagy; mitophagy receptor
    DOI:  https://doi.org/10.4103/1673-5374.385281
  16. Nat Chem Biol. 2023 Oct 19.
    Long-term super-resolution inner mitochondrial membrane imaging with a lipid probe.
    Shuai Zheng, Neville Dadina, Deepto Mozumdar, Lauren Lesiak, Kayli N Martinez, Evan W Miller, Alanna Schepartz.
      The inner mitochondrial membrane (IMM) generates power to drive cell function, and its dynamics control mitochondrial health and cellular homeostasis. Here, we describe the cell-permeant, lipid-like small molecule MAO-N3 and use it to assemble high-density environmentally sensitive (HIDE) probes that selectively label and image the IMM in live cells and multiple cell states. MAO-N3 pairs with strain-promoted azide-alkyne click chemistry-reactive fluorophores to support HIDE imaging using confocal, structured illumination, single-molecule localization and stimulated emission depletion microscopy, all with significantly improved resistance to photobleaching. These probes generate images with excellent spatial and temporal resolution, require no genetic manipulations, are non-toxic in model cell lines and primary cardiomyocytes (even under conditions that amplify the effects of mitochondrial toxins) and can visualize mitochondrial dynamics for 12.5 h. This probe will enable comprehensive studies of IMM dynamics with high temporal and spatial resolution.
    DOI:  https://doi.org/10.1038/s41589-023-01450-y
  17. Plant Cell Physiol. 2023 Oct 19. pii: pcad122. [Epub ahead of print]
    ATAD3 Proteins: Unique Mitochondrial Proteins Essential for Life in Diverse Eukaryotic Lineages.
    Elizabeth R Waters, Magdalena Bezanilla, Elizabeth Vierling.
      ATAD3 proteins (ATPase family AAA domain-containing protein 3) are unique mitochondrial proteins that arose deep in the eukaryotic lineage but that are surprisingly absent from the Fungi and Amoebozoa. These ~600 amino acid proteins are anchored in the inner mitochondrial membrane and are essential in metazoans and Arabidopsis thaliana. ATAD3s comprise a C-terminal AAA+ matrix domain and an ATAD3_N domain that is located primarily in the inner membrane space but potentially extends into cytosol to interact with the ER. Sequence and structural alignments indicate ATAD3 proteins are most similar to classic chaperone unfoldases in AAA+ family, suggesting that they operate in mitochondrial protein quality control. A. thaliana has four ATAD3 genes in two distinct clades that appear first in the seed plants, and both clades are essential for viability. The four genes are generally coordinately expressed, and transcripts are highest in growing apices and imbibed seeds. Plants with disrupted ATAD3 have reduced growth, aberrant mitochondrial morphology, diffuse nucleoids and reduced oxidative phosphorylation complex I. These and other pleiotropic phenotypes are also observed in ATAD3 mutants in metazoans. Here we discuss the distribution of ATAD3 proteins as they have evolved in the plant kingdom, their unique structure, what we know about their function in plants, and the challenges in determining their essential roles in mitochondria.
    Keywords:   Arabidopsis thaliana ; eukaryotic evolution; membrane contact sites; nucleoids; oxidative phosphorylation; protein quality control
    DOI:  https://doi.org/10.1093/pcp/pcad122
  18. R Soc Open Sci. 2023 Oct;10(10): 230404
    In fission yeast, 65 non-essential mitochondrial proteins related to respiration and stress become essential in low-glucose conditions.
    Ayaka Mori, Lisa Uehara, Yusuke Toyoda, Fumie Masuda, Saeko Soejima, Shigeaki Saitoh, Mitsuhiro Yanagida.
      Mitochondria perform critical functions, including respiration, ATP production, small molecule metabolism, and anti-oxidation, and they are involved in a number of human diseases. While the mitochondrial genome contains a small number of protein-coding genes, the vast majority of mitochondrial proteins are encoded by nuclear genes. In fission yeast Schizosaccharomyces pombe, we screened 457 deletion (del) mutants deficient in nuclear-encoded mitochondrial proteins, searching for those that fail to form colonies in culture medium containing low glucose (0.03-0.1%; low-glucose sensitive, lgs), but that proliferate in regular 2-3% glucose medium. Sixty-five (14%) of the 457 deletion mutants displayed the lgs phenotype. Thirty-three of them are defective either in dehydrogenases, subunits of respiratory complexes, the citric acid cycle, or in one of the nine steps of the CoQ10 biosynthetic pathway. The remaining 32 lgs mutants do not seem to be directly related to respiration. Fifteen are implicated in translation, and six encode transporters. The remaining 11 function in anti-oxidation, amino acid synthesis, repair of DNA damage, microtubule cytoskeleton, intracellular mitochondrial distribution or unknown functions. These 32 diverse lgs genes collectively maintain mitochondrial functions under low (1/20-1/60× normal) glucose concentrations. Interestingly, 30 of them have homologues associated with human diseases.
    Keywords:  anti-oxidant; coenzyme Q synthesis; human diseases; low-glucose sensitive; mitochondrial mutants; translation
    DOI:  https://doi.org/10.1098/rsos.230404
  19. Chem Commun (Camb). 2023 Oct 16.
    Super-resolution imaging of mitochondrial cristae using a more hydrophobic far-red Si-rhodamine probe.
    Jing Sun, Xichuan Ge, Boya Jin, Shiyi Li, Yiwei Hou, Suyi Zhong, Zikang Yang, Peng Xi, Meiqi Li, Baoxiang Gao.
      Mitochondrial probe SiRPFA was synthesized by attaching a long perfluoroalkyl chain on Si-rhodamine cationic dye. High lipophilicity endowed SiRPFA with mitochondrial membrane potential independent properties. Under stimulated emission depletion microscopy, SiRPFA clearly revealed changes in mitochondrial cristae morphology during autophagy induced by starvation or apoptosis.
    DOI:  https://doi.org/10.1039/d3cc04696f
  20. Nat Commun. 2023 Oct 19. 14(1): 6625
    Developing mitochondrial base editors with diverse context compatibility and high fidelity via saturated spacer library.
    Haifeng Sun, Zhaojun Wang, Limini Shen, Yeling Feng, Lu Han, Xuezhen Qian, Runde Meng, Kangming Ji, Dong Liang, Fei Zhou, Xin Lou, Jun Zhang, Bin Shen.
      DddA-derived cytosine base editors (DdCBEs) greatly facilitated the basic and therapeutic research of mitochondrial DNA mutation diseases. Here we devise a saturated spacer library and successfully identify seven DddA homologs by performing high-throughput sequencing based screen. DddAs of Streptomyces sp. BK438 and Lachnospiraceae bacterium sunii NSJ-8 display high deaminase activity with a strong GC context preference, and DddA of Ruminococcus sp. AF17-6 is highly compatible to AC context. We also find that different split sites result in wide divergence on off-target activity and context preference of DdCBEs derived from these DddA homologs. Additionally, we demonstrate the orthogonality between DddA and DddIA, and successfully minimize the nuclear off-target editing by co-expressing corresponding nuclear-localized DddIA. The current study presents a comprehensive and unbiased strategy for screening and characterizing dsDNA cytidine deaminases, and expands the toolbox for mtDNA editing, providing additional insights for optimizing dsDNA base editors.
    DOI:  https://doi.org/10.1038/s41467-023-42359-3
  21. Cell Rep. 2023 Oct 13. pii: S2211-1247(23)01276-7. [Epub ahead of print]42(10): 113264
    Aspartyl-tRNA synthetase 2 orchestrates iron-sulfur metabolism in hematopoietic stem cells via fine-tuning alternative RNA splicing.
    Xuan Gu, Kailing Li, Meng Zhang, Yandan Chen, Jingchao Zhou, Chunxu Yao, Yong Zang, Jiefeng He, Jun Wan, Bin Guo.
      Aspartyl-tRNA synthetase 2 (Dars2) is involved in the regulation of mitochondrial protein synthesis and tissue-specific mitochondrial unfolded protein response (UPRmt). The role of Dars2 in the self-renewal and differentiation of hematopoietic stem cells (HSCs) is unknown. Here, we show that knockout (KO) of Dars2 significantly impairs the maintenance of hematopoietic stem and progenitor cells (HSPCs) without involving its tRNA synthetase activity. Dars2 KO results in significantly reduced expression of Srsf2/3/6 and impairs multiple events of mRNA alternative splicing (AS). Dars2 directly localizes to Srsf3-labeled spliceosomes in HSPCs and regulates the stability of Srsf3. Dars2-deficient HSPCs exhibit aberrant AS of mTOR and Slc22a17. Dars2 KO greatly suppresses the levels of labile ferrous iron and iron-sulfur cluster-containing proteins, which dampens mitochondrial metabolic activity and DNA damage repair pathways in HSPCs. Our study reveals that Dars2 plays a crucial role in the iron-sulfur metabolism and maintenance of HSPCs by modulating RNA splicing.
    Keywords:  CP: Metabolism; CP: Stem cell research
    DOI:  https://doi.org/10.1016/j.celrep.2023.113264
  22. J Mol Biol. 2023 Oct 17. pii: S0022-2836(23)00428-X. [Epub ahead of print] 168317
    FDX1 is required for the biogenesis of mitochondrial cytochrome c oxidase in mammalian cells.
    Mohammad Zulkifli, Adriana U Okonkwo, Vishal M Gohil.
      Ferredoxins (FDXs) are evolutionarily conserved iron-sulfur (Fe-S) proteins that function as electron transfer proteins in diverse metabolic pathways. Mammalian mitochondria contain two ferredoxins, FDX1 and FDX2, which share a high degree of structural similarity but exhibit different functionalities. Previous studies have established the unique role of FDX2 in the biogenesis of Fe-S clusters; however, FDX1 seems to have multiple targets in vivo, some of which are only recently emerging. Using CRISPR-Cas9-based loss-of-function studies in rat cardiomyocyte cell line, we demonstrate an essential requirement of FDX1 in mitochondrial respiration and energy production. We attribute reduced mitochondrial respiration to a specific decrease in the abundance and assembly of cytochrome c oxidase (CcO), a mitochondrial heme-copper oxidase and the terminal enzyme of the mitochondrial respiratory chain. FDX1 knockout cells have reduced levels of copper and heme a/a3, factors that are essential for the maturation of the CcO enzyme complex. Copper supplementation failed to rescue CcO biogenesis, but overexpression of heme a synthase, COX15, partially rescued COX1 abundance in FDX1 knockouts. This finding links FDX1 function to heme a biosynthesis, and places it upstream of COX15 in CcO biogenesis like its ancestral yeast homolog. Taken together, our work has identified FDX1 as a critical CcO biogenesis factor in mammalian cells.
    Keywords:  COX1; Copper; Heme a; Mitochondria; respiration
    DOI:  https://doi.org/10.1016/j.jmb.2023.168317
  23. Nat Commun. 2023 Oct 20. 14(1): 6638
    Lactate dehydrogenase D is a general dehydrogenase for D-2-hydroxyacids and is associated with D-lactic acidosis.
    Shan Jin, Xingchen Chen, Jun Yang, Jianping Ding.
      Mammalian lactate dehydrogenase D (LDHD) catalyzes the oxidation of D-lactate to pyruvate. LDHD mutations identified in patients with D-lactic acidosis lead to deficient LDHD activity. Here, we perform a systematic biochemical study of mouse LDHD (mLDHD) and determine the crystal structures of mLDHD in FAD-bound form and in complexes with FAD, Mn2+ and a series of substrates or products. We demonstrate that mLDHD is an Mn2+-dependent general dehydrogenase which exhibits catalytic activity for D-lactate and other D-2-hydroxyacids containing hydrophobic moieties, but no activity for their L-isomers or D-2-hydroxyacids containing hydrophilic moieties. The substrate-binding site contains a positively charged pocket to bind the common glycolate moiety and a hydrophobic pocket with some elasticity to bind the varied hydrophobic moieties of substrates. The structural and biochemical data together reveal the molecular basis for the substrate specificity and catalytic mechanism of LDHD, and the functional roles of mutations in the pathogenesis of D-lactic acidosis.
    DOI:  https://doi.org/10.1038/s41467-023-42456-3
  24. Neuropediatrics. 2023 Oct 16.
    ACOX1 Gain-of-Function Variant in Two German Pediatric Patients, in One Case Mimicking Autoimmune Inflammatory Disease.
    C Thiels, T Lücke, T Rothoeft, C Lukas, H P Nguyen, J C von Kleist-Retzow, H Prokisch, M Grimmel, T B Haack, S Hoffjan.
      Mitchell syndrome is a very rare genetic disorder due to a specific de novo gain-of-function variant in acyl-CoA oxidase 1 (ACOX1). So far, only five patients with this disease have been described worldwide. We present here two additional unrelated German patients found to carry the same heterozygous ACOX1 N237S variant through exome sequencing (ES). Both patients showed neurodegenerative clinical features starting from ∼4 to 5 years of age including progressive hearing loss, ataxia, ichthyosis, as well as progressive visual impairment leading to amaurosis, and died at the ages of 16 and 8 years, respectively. The first patient was clinically suspected to have anti-myelin oligodendrocyte glycoprotein-antibody-associated myelitis, but the disease course overall deteriorated despite extensive immunomodulatory therapy. The second patient was originally suspected to have a mitochondrial disorder due to intermittent elevated blood lactate. Since Mitchell syndrome has only been identified in 2020, the diagnosis in this second patient was only established through re-evaluation of ES data years after the original analysis. Comparison of all seven reported patients suggests that Mitchell syndrome often (but not always) clinically mimics autoimmune-inflammatory disease. Therefore, in patients with autoimmune central nervous system disease who do not respond adequately to standard therapies, re-evaluation of this diagnosis is needed and genetic analyses such as trio ES should be considered.
    DOI:  https://doi.org/10.1055/s-0043-1776013
  25. Diabetes. 2023 Oct 17. pii: db230430. [Epub ahead of print]
    Dissecting the roles of the nuclear and mitochondrial genomes in a mouse model of autoimmune diabetes.
    Weiwei Zou, Janaki Chezhian, Tenghui Yu, Wensheng Liu, Jimmy Vu, Jesse Slone, Taosheng Huang.
      Mitochondria, the organelles responsible for generating ATP in eukaryotic cells, have been previously implicated as a contributor to diabetes. However, mitochondrial proteins are encoded by both nuclear DNA (nDNA) and mitochondrial DNA (mtDNA). In order to better understand the relative contribution of each of these genomes to diabetes, a chimeric mitochondrial-nuclear exchange (MNX) mouse was created via pronuclear transfer carrying nDNA from a strain susceptible to type 1 diabetes (NOD/ShiLtJ) and mtDNA from non-diabetic C57BL/6J mice. Inheritance of the resulting "heteroplasmic" mtDNA mixture was then tracked across multiple generations, showing that offspring heteroplasmy generally followed that of the mother, with occasional large shifts consistent with an mtDNA "bottleneck" in the germline. In addition, the survival and incidence of diabetes for MNX mice was tracked and compared to unaltered NOD/ShiLtJ control mice. The results indicate improved survival and a delay in diabetes onset in the MNX mice, demonstrating that the mtDNA holds a critical influence on the disease phenotype. Finally, enzyme activity assays showed that the NOD/ShiLtJ mice have a significant hyperactivity of complex I of the ETC relative to MNX mice, suggesting that a particular mtDNA variant (m.9461T>C) may be responsible for the disease causation in the original NOD/ShiLtJ strain.
    DOI:  https://doi.org/10.2337/db23-0430
  26. Nat Commun. 2023 Oct 16. 14(1): 6513
    Fibrocystin/Polyductin releases a C-terminal fragment that translocates into mitochondria and suppresses cystogenesis.
    Rebecca V Walker, Qin Yao, Hangxue Xu, Anthony Maranto, Kristen F Swaney, Sreekumar Ramachandran, Rong Li, Laura Cassina, Brian M Polster, Patricia Outeda, Alessandra Boletta, Terry Watnick, Feng Qian.
      Fibrocystin/Polyductin (FPC), encoded by PKHD1, is associated with autosomal recessive polycystic kidney disease (ARPKD), yet its precise role in cystogenesis remains unclear. Here we show that FPC undergoes complex proteolytic processing in developing kidneys, generating three soluble C-terminal fragments (ICDs). Notably, ICD15, contains a novel mitochondrial targeting sequence at its N-terminus, facilitating its translocation into mitochondria. This enhances mitochondrial respiration in renal epithelial cells, partially restoring impaired mitochondrial function caused by FPC loss. FPC inactivation leads to abnormal ultrastructural morphology of mitochondria in kidney tubules without cyst formation. Moreover, FPC inactivation significantly exacerbates renal cystogenesis and triggers severe pancreatic cystogenesis in a Pkd1 mouse mutant Pkd1V/V in which cleavage of Pkd1-encoded Polycystin-1 at the GPCR Proteolysis Site is blocked. Deleting ICD15 enhances renal cystogenesis without inducing pancreatic cysts in Pkd1V/V mice. These findings reveal a direct link between FPC and a mitochondrial pathway through ICD15 cleavage, crucial for cystogenesis mechanisms.
    DOI:  https://doi.org/10.1038/s41467-023-42196-4
  27. STAR Protoc. 2023 Oct 18. pii: S2666-1667(23)00617-2. [Epub ahead of print]4(4): 102650
    An optimized protocol to detect ubiquitination modification of exogenous or endogenous proteins.
    Xinyu Li, Wei Jiang, Min Fang.
      Ubiquitination modification is an important post-translational modification that regulates the stability and function of proteins. Here, we present a protocol to detect the K27-linked polyubiquitination of exogenous and endogenous mitochondrial antiviral signaling protein. We describe steps for detecting ubiquitination of exogenous protein, transfecting the encoding plasmid of the protein, and immunoprecipitating the target protein with an antibody. We then detail procedures for detecting ubiquitin of the target protein by western blot. This protocol applies to other proteins of interest. For complete details on the use and execution of this protocol, please refer to Jiang et al. (2023).1.
    Keywords:  Cell Separation/Fractionation; Cell-Based Assays; Immunology; Molecular Biology
    DOI:  https://doi.org/10.1016/j.xpro.2023.102650
  28. Proc Natl Acad Sci U S A. 2023 Oct 24. 120(43): e2309698120
    Membrane remodeling properties of the Parkinson's disease protein LRRK2.
    Xinbo Wang, Javier Espadas, Yumei Wu, Shujun Cai, Jinghua Ge, Lin Shao, Aurélien Roux, Pietro De Camilli.
      Mutations in Leucine-rich repeat kinase 2 (LRRK2) are responsible for late-onset autosomal dominant Parkinson's disease. LRRK2 has been implicated in a wide range of physiological processes including membrane repair in the endolysosomal system. Here, using cell-free systems, we report that purified LRRK2 directly binds acidic lipid bilayers with a preference for highly curved bilayers. While this binding is nucleotide independent, LRRK2 can also deform low-curvature liposomes into narrow tubules in a guanylnucleotide-dependent but Adenosine 5'-triphosphate-independent way. Moreover, assembly of LRRK2 into scaffolds at the surface of lipid tubules can constrict them. We suggest that an interplay between the membrane remodeling and signaling properties of LRRK2 may be key to its physiological function. LRRK2, via its kinase activity, may achieve its signaling role at sites where membrane remodeling occurs.
    Keywords:  GTPase; LRRK2; Parkinson; membrane curvature; tubulation
    DOI:  https://doi.org/10.1073/pnas.2309698120
  29. Nat Chem Biol. 2023 Oct 20.
    MAO-SiR is a tool for visualizing the inner workings of mitochondria.
      
    DOI:  https://doi.org/10.1038/s41589-023-01451-x
  30. Chem Sci. 2023 Oct 18. 14(40): 11192-11202
    Targeting mitochondrial degradation by chimeric autophagy-tethering compounds.
    Zhenqi Liu, Geng Qin, Jie Yang, Wenjie Wang, Wenting Zhang, Boxun Lu, Jinsong Ren, Xiaogang Qu.
      The ability to regulate mitophagy in a living system with small molecules remains a great challenge. We hypothesize that adding fragments specific to the key autophagosome protein LC3 to mitochondria will mimic receptor-mediated mitophagy, thus engaging the autophagy-lysosome pathway to induce mitochondrial degradation. Herein, we develop a general biochemical approach to modulate mitophagy, dubbed mito-ATTECs, which employ chimera molecules composed of LC3-binding moieties linked to mitochondria-targeting ligands. Mito-ATTECs trigger mitophagy via targeting mitochondria to autophagosomes through direct interaction between mito-ATTECs and LC3 on mitochondrial membranes. Subsequently, autophagosomes containing mitochondria rapidly fuse with lysosomes to facilitate the degradation of mitochondria. Therefore, mito-ATTECs circumvent the detrimental effects related to disruption of mitochondrial membrane integrity by inducers routinely used to manipulate mitophagy, and provide a versatile biochemical approach to investigate the physiological roles of mitophagy. Furthermore, we found that sustained mitophagy lead to mitochondrial depletion and autophagic cell death in several malignant cell lines (lethal mitophagy). Among them, apoptosis-resistant malignant melanoma cell lines are particularly sensitive to lethal mitophagy. The therapeutic efficacy of mito-ATTECs has been further evaluated by using subcutaneous and pulmonary metastatic melanoma models. Together, the mitochondrial depletion achieved by mito-ATTECs may demonstrate the general concept of inducing cancer cell lethality through excessive mitochondrial clearance, establishing a promising therapeutic paradigm for apoptosis-resistant tumors.
    DOI:  https://doi.org/10.1039/d3sc03600f
  31. iScience. 2023 Oct 20. 26(10): 107916
    Development of novel cytoprotective small compounds inhibiting mitochondria-dependent cell death.
    Mieko Matsuyama, Joseph T Ortega, Yuri Fedorov, Jonah Scott-McKean, Jeannie Muller-Greven, Matthias Buck, Drew Adams, Beata Jastrzebska, William Greenlee, Shigemi Matsuyama.
      We identified cytoprotective small molecules (CSMs) by a cell-based high-throughput screening of Bax inhibitors. Through a medicinal chemistry program, M109S was developed, which is orally bioactive and penetrates the blood-brain/retina barriers. M109S protected retinal cells in ocular disease mouse models. M109S directly interacted with Bax and inhibited the conformational change and mitochondrial translocation of Bax. M109S inhibited ABT-737-induced apoptosis both in Bax-only and Bak-only mouse embryonic fibroblasts. M109S also inhibited apoptosis induced by staurosporine, etoposide, and obatoclax. M109S decreased maximal mitochondrial oxygen consumption rate and reactive oxygen species production, whereas it increased glycolysis. These effects on cellular metabolism may contribute to the cytoprotective activity of M109S. M109S is a novel small molecule protecting cells from mitochondria-dependent apoptosis both in vitro and in vivo. M109S has the potential to become a research tool for studying cell death mechanisms and to develop therapeutics targeting mitochondria-dependent cell death pathway.
    Keywords:  Cell biology; Cellular physiology; Chemistry
    DOI:  https://doi.org/10.1016/j.isci.2023.107916
  32. Endocr Metab Immune Disord Drug Targets. 2023 Oct 11.
    Combined Oxidative Phosphorylation Deficiency Type-13 with Perinatal Presentation: A Case Report.
    Sílvia Reigada, Constança Santos, Fabiana Ramos, Sílvia Carvalho, Joana Ribeiro, Candida Cancelinha, Luísa Diogo.
       INTRODUCTION: Polynucleotide phosphorylase is involved in RNA processing in mitochondria. Biallelic variants in PNPT1 cause mitochondrial RNA import protein deficiency and heterogeneous clinical manifestations.
    CASE REPORT: The patiest was the first child of remote consanguineous parents, born at 35 weeks by caesarean section due to fetal growth restriction. Apgar index was 9/10/10. Birth weight, length and head circumference were at 3rd, <3rd and 10th percentiles, respectively. In the first hours of life, respiratory distress, hypoglycaemia and seizures ensued. She started invasive mechanic ventilation, phenobarbital and was transferred to ICU. Physical examination showed minor facial dysmorphisms, brief eye-opening, hypotonia and hyporeflexia. Electroencephalogram showed immature pattern and multifocal paroxysmal activity. MRI at D8 of life showed severe reduced brain volume. Normal aminoacid screen was also observed. Expanded newborn screening was negative. Mitochondrial organic aciduria was seen. WES showed a homozygotic likely pathogenic variant in the PNPT1 gene. MRI at 6-months showed brain atrophy, thin corpus callosum, reduced brainstem volume. Bilateral and symmetrical lesions in globi pallidi, compatible with Leigh síndrome were observed. Currently, at 14 months, no neurodevelopment progress, dystonia, visual deficit, sensorineural deafness, hypertrophic cardiomyopathy and microcephaly are observed.
    CONCLUSION: The early and severe Leigh-like presentation of our patient expands the phenotype spectrum of this disease. As far as we know, this is the first reported case of PNPT1 mutation with onset in the perinatal period. Moreover, hypertrophic cardiomyopathy has not yet been described in association with mutation of the PNPT1 gene. WES was the key for early diagnosis in this patient. It should be done in all children with severe clinical presentation of unknown origin.
    Keywords:  PNPT1; Leigh syndrome; oxidative phosphorylation; hypotonia; hypertrophic cardiomyopathy; brain atrophy.
    DOI:  https://doi.org/10.2174/0118715303274239231005105248
  33. J Pharm Pharmacol. 2023 Oct 20. pii: rgad090. [Epub ahead of print]
    Hydroxyapatite-based nano-drug delivery system for nicotinamide mononucleotide (NMN): significantly enhancing NMN bioavailability and replenishing in vivo nicotinamide adenine dinucleotide (NAD+) levels.
    Da Zhang, Lee-Fong Yau, Long-Bo Bai, Tian-Tian Tong, Kai-Yue Cao, Tong-Meng Yan, Ling Zeng, Zhi-Hong Jiang.
       OBJECTIVES: This study addresses the bioavailability challenges associated with oral nicotinamide mononucleotide (NMN) administration by introducing an innovative NMN formulation incorporated with hydroxyapatite (NMN-HAP).
    METHODS: The NMN-HAP was developed using a wet chemical precipitation and physical adsorption method. To assess its superiority over conventional free NMN, we examined NMN, nicotinamide adenine dinucleotide (NAD+), and nicotinamide riboside (NR) levels in mouse plasma and tissues following oral administration of NMN-HAP.
    KEY FINDINGS: NMN-HAP nanoparticles demonstrated a rod-shaped morphology, with an average size of ~50 nm, along with encapsulation efficiency and drug loading capacity exceeding 40%. In vitro, drug release results indicated that NMN-HAP exhibited significantly lower release compared with free NMN. In vivo studies showed that NMN-HAP extended circulation time, improved bioavailability compared with free NMN, and elevated plasma levels of NMN, NAD+, and NR. Moreover, NMN-HAP administration displayed tissue-specific distribution with a substantial accumulation of NMN, NAD+, and NR in the brain and liver.
    CONCLUSION: NMN-HAP represents an ideal formulation for enhancing NMN bioavailability, enabling tissue-specific delivery, and ultimately elevating in vivo NAD+ levels. Considering HAP's biocompatible nature and versatile characteristics, we anticipate that this system has significant potential for various future applications.
    Keywords:  ageing; bioavailability; metabolism; nicotinamide riboside (NR); pharmacokinetic; tissue-specific
    DOI:  https://doi.org/10.1093/jpp/rgad090
  34. mSphere. 2023 Oct 18. e0044823
    Contact sites between host organelles and pathogens: boon or bane?
    Chahat Mehra, Lena Pernas.
      A microbe and its host are in constant communication. An emerging platform for direct communication is the membrane contact sites that form between several pathogens and host organelles. Here, we review our progress on the molecular mechanisms underlying contact sites between host mitochondria and the human parasite Toxoplasma gondii. We discuss open questions regarding their function during infection as well as those formed between the host endoplasmic reticulum and Toxoplasma.
    Keywords:  Toxoplasma gondii; endoplasmic reticulum; membrane; membrane contact sites; mitochondria; pathogens
    DOI:  https://doi.org/10.1128/msphere.00448-23
  35. Nucleic Acids Res. 2023 Oct 16. pii: gkad810. [Epub ahead of print]
    Identification and quantification of small exon-containing isoforms in long-read RNA sequencing data.
    Zhen Liu, Chenchen Zhu, Lars M Steinmetz, Wu Wei.
      Small exons are pervasive in transcriptomes across organisms, and their quantification in RNA isoforms is crucial for understanding gene functions. Although long-read RNA-seq based on Oxford Nanopore Technologies (ONT) offers the advantage of covering transcripts in full length, its lower base accuracy poses challenges for identifying individual exons, particularly microexons (≤ 30 nucleotides). Here, we systematically assess small exons quantification in synthetic and human ONT RNA-seq datasets. We demonstrate that reads containing small exons are often not properly aligned, affecting the quantification of relevant transcripts. Thus, we develop a local-realignment method for misaligned exons (MisER), which remaps reads with misaligned exons to the transcript references. Using synthetic and simulated datasets, we demonstrate the high sensitivity and specificity of MisER for the quantification of transcripts containing small exons. Moreover, MisER enabled us to identify small exons with a higher percent spliced-in index (PSI) in neural, particularly neural-regulated microexons, when comparing 14 neural to 16 non-neural tissues in humans. Our work introduces an improved quantification method for long-read RNA-seq and especially facilitates studies using ONT long-reads to elucidate the regulation of genes involving small exons.
    DOI:  https://doi.org/10.1093/nar/gkad810
  36. Neurochem Int. 2023 Oct 16. pii: S0197-0186(23)00159-6. [Epub ahead of print] 105631
    Disturbance of mitochondrial functions caused by N-acetylglutamate and N-acetylmethionine in brain of adolescent rats: Potential relevance in aminoacylase 1 deficiency.
    Vanessa Trindade Bortoluzzi, Rafael Teixeira Ribeiro, Ângela Beatris Zemniaçak, Sâmela de Azevedo Cunha, Jörn Oliver Sass, Roger Frigério Castilho, Alexandre Umpierrez Amaral, Moacir Wajner.
      Aminoacylase 1 (ACY1) deficiency is a rare genetic disorder that affects the breakdown of short-chain aliphatic N-acetylated amino acids, leading to the accumulation of these amino acid derivatives in the urine of patients. Some of the affected individuals have presented with heterogeneous neurological symptoms such as psychomotor delay, seizures, and intellectual disability. Considering that the pathological mechanisms of brain damage in this disorder remain mostly unknown, here we investigated whether major metabolites accumulating in ACY1 deficiency, namely N-acetylglutamate (NAG) and N-acetylmethionine (NAM), could be toxic to the brain by examining their in vitro effects on important mitochondrial properties. We assessed the effects of NAG and NAM on membrane potential, swelling, reducing equivalents, and Ca2+ retention capacity in purified mitochondrial preparations obtained from the brain of adolescent rats. NAG and NAM decreased mitochondrial membrane potential, reducing equivalents, and calcium retention capacity, and induced swelling in Ca2+-loaded brain mitochondria supported by glutamate plus malate. Notably, these changes were completely prevented by the classical inhibitors of mitochondrial permeability transition (MPT) pore cyclosporin A plus ADP and by ruthenium red, implying the participation of MPT and Ca2+ in these effects. Our findings suggest that NAG- and NAM-induced disruption of mitochondrial functions involving MPT may represent relevant mechanisms of neuropathology in ACY1 deficiency.
    Keywords:  Aminoacylase 1 deficiency; Brain; Mitochondrial functions; Mitochondrial permeability transition; N-acetylglutamate; N-acetylmethionine
    DOI:  https://doi.org/10.1016/j.neuint.2023.105631
  37. Nat Cell Biol. 2023 Oct 18.
    A guide to studying mitochondria transfer.
    Snigdha Tiash, Jonathan Robert Brestoff, Clair Crewe.
      
    DOI:  https://doi.org/10.1038/s41556-023-01246-1
  38. Nature. 2023 Oct 16.
    Methods section too short? Use online protocols to make complex techniques understandable.
    Lars Borm.
      
    Keywords:  Bioinformatics; Brain; Neuroscience; Transcriptomics
    DOI:  https://doi.org/10.1038/d41586-023-03249-2
  39. Nucleic Acids Res. 2023 Oct 16. pii: gkad833. [Epub ahead of print]
    MINRbase: a comprehensive database of nuclear- and mitochondrial-ribosomal-RNA-derived fragments (rRFs).
    Venetia Pliatsika, Tess Cherlin, Phillipe Loher, Panagiotis Vlantis, Parth Nagarkar, Stepan Nersisyan, Isidore Rigoutsos.
      We describe the Mitochondrial and Nuclear rRNA fragment database (MINRbase), a knowledge repository aimed at facilitating the study of ribosomal RNA-derived fragments (rRFs). MINRbase provides interactive access to the profiles of 130 238 expressed rRFs arising from the four human nuclear rRNAs (18S, 5.8S, 28S, 5S), two mitochondrial rRNAs (12S, 16S) or four spacers of 45S pre-rRNA. We compiled these profiles by analyzing 11 632 datasets, including the GEUVADIS and The Cancer Genome Atlas (TCGA) repositories. MINRbase offers a user-friendly interface that lets researchers issue complex queries based on one or more criteria, such as parental rRNA identity, nucleotide sequence, rRF minimum abundance and metadata keywords (e.g. tissue type, disease). A 'summary' page for each rRF provides a granular breakdown of its expression by tissue type, disease, sex, ancestry and other variables; it also allows users to create publication-ready plots at the click of a button. MINRbase has already allowed us to generate support for three novel observations: the internal spacers of 45S are prolific producers of abundant rRFs; many abundant rRFs straddle the known boundaries of rRNAs; rRF production is regimented and depends on 'personal attributes' (sex, ancestry) and 'context' (tissue type, tissue state, disease). MINRbase is available at https://cm.jefferson.edu/MINRbase/.
    DOI:  https://doi.org/10.1093/nar/gkad833
  40. Pharmacogenomics. 2023 Oct 17.
    Towards more accurate pharmacogenomic variant effect predictions.
    Yoomi Park, Volker M Lauschke.
      Tweetable abstract Accurate variant interpretation has become a key bottleneck for the translation of an individual's pharmacogenome into actionable recommendations. We recommend an integrated use of multiplexed assays, structure-based predictions and biobank data to develop more accurate effect predictors.
    Keywords:  Genomic Medicine; Personalized Drug Response; Precision Medicine; Variant of unknown significance
    DOI:  https://doi.org/10.2217/pgs-2023-0187
  41. Int J Dev Neurosci. 2023 Oct 17.
    Complex genotypes in family with metachromatic leukodystrophy: Effect of trans and cis mutations distribution on the phenotype variability.
    Abir Ben Issa, Fatma Kamoun, Wafa Bouchaala, Chahnez Charfi Triki, Faiza Fakhfakh.
      Metachromatic leukodystrophy (MLD) is a severe metabolic disorder caused by the deficient activity of arylsulfatase A due to ARSA gene mutations. According to the age of onset, MLD is classified into three forms: infantile, juvenile, and adult. In our study, we aimed to perform a genetic analysis for two siblings with juvenile MLD for a better characterization of the molecular mechanisms behind the disease. A consanguineous family including two MLD patients (PII.1 and PII.2) was enrolled in our study. The diagnosis was made based on the clinical and neuroimaging investigations. The sequencing of ARSA gene was performed followed by in silico analysis. Besides, the cis/trans distribution of the variants was verified through a PCR-RFLP. The ARSA gene sequencing revealed three known variants, two exonic c.1055A > G and c.1178C > G and an intronic one (c.1524 + 95A > G) in the 3'UTR region. All variants were present at heterozygous state in the two siblings and their mother. The assessment of the cis/trans distribution showed the presence of these variants in cis within the mother, while PII.2 and PII.2 present the c.1055A > G/c.1524 + 95A > G and the c.1178C > G in trans. Additionally, PII.1 harbored a de novo novel missense variant c.1119G > T, whose pathogenicity was supported by our predictive results. Our genetic findings, supported by a clinical examination, confirmed the affection of the mother by the adult MLD. Our results proved the implication of the variable distribution of the found variants in the age of MLD onset. Besides, we described a variable severity between the two siblings due to the de novo pathogenic variant. In conclusion, we identified a complex genotype of ARSA variants within two MLD siblings with a variable severity due to a de novo variant present in one of them. Our results allowed the establishment of an adult MLD diagnosis and highlighted the importance of an assessment of the trans/cis distribution in the cases of complex genotypes.
    Keywords:  MLD; adult form; arylsulfatase A (ARSA); cis/trans allele distribution; juvenile form; late infantile form
    DOI:  https://doi.org/10.1002/jdn.10306
  42. Endocr Metab Immune Disord Drug Targets. 2023 Oct 11.
    Clinical, Biochemical and Molecular Features of a Cohort of 8 Patients with Inherited Disorders of Vitamin B12 Metabolism in a Metabolic Reference Center.
    Gonçalo Padeira, Sandra Jacinto, Augusto Ribeirinho, Ana Cristina Ferreira.
       BACKGROUND: Vitamin B12, or cobalamin (Cbl), undergoes a complex series of absorptive and intracellular processing steps before serving as a cofactor for the enzymes methylmalonyl-CoA mutase and methionine synthase. Disorders of intracellular cobalamin metabolism have variable phenotypes and age of onset related to the location of the defect in the metabolic pathway leading to a combined methylmalonic acidemia and homocystinuria (cblC, cblD, cblF and cblJ), Isolated methylmalonic acidemia (cblA, cblB and cblDv2) and isolated homocystinuria (cblDv1, cblE and cblG).
    OBJECTIVE AND METHODS: We conducted a retrospective study of the clinical biochemical and molecular features of a cohort of patients with disorders of intracellular Cbl metabolism followed in our Reference Centre of Inherited Metabolic Diseases (CR-IMD) for the last 23 years (2000-2023).
    RESULTS: CblC: P1 and P2, pré-newborn screening (NBS), had an early and severe presentation evolving to multiorgan failure and death. P3 was asymptomatic at NBS with an excellent evolution except for nystagmus and retinitis pigmentosa. P4 presented at 19Y with an atypical hemolytic uremic syndrome and is presently on hemodialysis. CblD: P5 had a developmental delay (DD) and hypotonia and presented at 14m with seizures. CblDv2: P6 had DD and failure to thrive (FTT) and presented at 4Y with acute metabolic acidosis. CblDv1: P7 had DD, FTT, and hypotonia and presented at 16m with seizures and anemia. CblG: P8 had DD and FTT and presented at 15m with macrocytic anemia. In all, characteristic biochemical profiles guided the diagnosis, afterward confirmed by genetic analysis (4 MMACHC, 3 MMADHC, 1 MTR). All patients received either betaine, hydroxycobalamin, or both (P3 is on a very high dosage).
    CONCLUSION: Our cohort of patients has similar clinical and biochemical characteristics to the ones described in the literature. Outcomes of patients reinforce the importance of newborn screening and the need for consensus guidelines for optimal doses of parenteral hydroxocobalamin.
    Keywords:  Disorders of intracellular cobalamin metabolism; homocystinuria; methylmalonic acidemia
    DOI:  https://doi.org/10.2174/0118715303272253231004094018
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