bims-mitdis Biomed News
on Mitochondrial disorders
Issue of 2023‒09‒24
thirty-six papers selected by
Catalina Vasilescu, Helmholz Munich

  1. Proc Natl Acad Sci U S A. 2023 Sep 26. 120(39): e2304884120
      How does a single amino acid mutation occurring in the blinding disease, Leber's hereditary optic neuropathy (LHON), impair electron shuttling in mitochondria? We investigated changes induced by the m.3460 G>A mutation in mitochondrial protein ND1 using the tools of Molecular Dynamics and Free Energy Perturbation simulations, with the goal of determining the mechanism by which this mutation affects mitochondrial function. A recent analysis suggested that the mutation's replacement of alanine A52 with a threonine perturbs the stability of a region where binding of the electron shuttling protein, Coenzyme Q10, occurs. We found two functionally opposing changes involving the role of Coenzyme Q10. The first showed that quantum electron transfer from the terminal Fe/S complex, N2, to the Coenzyme Q10 headgroup, docked in its binding pocket, is enhanced. However, this positive adjustment is overshadowed by our finding that the mobility of Coenzyme Q10 in its oxidized and reduced states, entering and exiting its binding pocket, is disrupted by the mutation in a manner that leads to conditions promoting the generation of reactive oxygen species. An increase in reactive oxygen species caused by the LHON mutation has been proposed to be responsible for this optic neuropathy.
    Keywords:  Coenzyme Q10; blinding genetic disease; mitochondria; molecular dynamics simulation; quantum electron tunneling
  2. J Med Genet. 2023 Sep 21. pii: jmg-2023-109340. [Epub ahead of print]
      BACKGROUND: Leber's hereditary optic neuropathy (LHON) is a mitochondrial disorder characterised by complex I defect leading to sudden degeneration of retinal ganglion cells. Although typically associated with pathogenic variants in mitochondrial DNA, LHON was recently described in patients carrying biallelic variants in nuclear genes DNAJC30, NDUFS2 and MCAT. MCAT is part of mitochondrial fatty acid synthesis (mtFAS), as also MECR, the mitochondrial trans-2-enoyl-CoA reductase. MECR mutations lead to a recessive childhood-onset syndromic disorder with dystonia, optic atrophy and basal ganglia abnormalities.METHODS: We studied through whole exome sequencing two sisters affected by sudden and painless visual loss at young age, with partial recovery and persistent central scotoma. We modelled the candidate variant in yeast and studied mitochondrial dysfunction in yeast and fibroblasts. We tested protein lipoylation and cell response to oxidative stress in yeast.
    RESULTS: Both sisters carried a homozygous pathogenic variant in MECR (p.Arg258Trp). In yeast, the MECR-R258W mutant showed an impaired oxidative growth, 30% reduction in oxygen consumption rate and 80% decrease in protein levels, pointing to structure destabilisation. Fibroblasts confirmed the reduced amount of MECR protein, but failed to reproduce the OXPHOS defect. Respiratory complexes assembly was normal. Finally, the yeast mutant lacked lipoylation of key metabolic enzymes and was more sensitive to H2O2 treatment. Lipoic Acid supplementation partially rescued the growth defect.
    CONCLUSION: We report the first family with homozygous MECR variant causing an LHON-like optic neuropathy, which pairs the recent MCAT findings, reinforcing the impairment of mtFAS as novel pathogenic mechanism in LHON.
    Keywords:  Genetics, Medical; Neuromuscular Diseases; Ophthalmology
  3. IUBMB Life. 2023 Sep 20.
      Mitochondria are essential for normal cellular function and have emerged as key aging determinants. Indeed, defects in mitochondrial function have been linked to cardiovascular, skeletal muscle and neurodegenerative diseases, premature aging, and age-linked diseases. Here, we describe mechanisms for mitochondrial protein and organelle quality control. These surveillance mechanisms mediate repair or degradation of damaged or mistargeted mitochondrial proteins, segregate mitochondria based on their functional state during asymmetric cell division, and modulate cellular fitness, the response to stress, and lifespan control in yeast and other eukaryotes.
    Keywords:  ageing; mitochondria; mitochondrial reactive oxygen species; oxidative stress; reactive oxygen species
  4. J Biomed Sci. 2023 Sep 22. 30(1): 82
      Mitochondria are essential organelles for cellular metabolism and physiology in eukaryotic cells. Human mitochondria have their own genome (mtDNA), which is maternally inherited with 37 genes, encoding 13 polypeptides for oxidative phosphorylation, and 22 tRNAs and 2 rRNAs for translation. mtDNA mutations are associated with a wide spectrum of degenerative and neuromuscular diseases. However, the pathophysiology of mitochondrial diseases, especially for threshold effect and tissue specificity, is not well understood and there is no effective treatment for these disorders. Especially, the lack of appropriate cell and animal disease models has been significant obstacles for deep elucidating the pathophysiology of maternally transmitted diseases and developing the effective therapy approach. The use of human induced pluripotent stem cells (iPSCs) derived from patients to obtain terminally differentiated specific lineages such as inner ear hair cells is a revolutionary approach to deeply understand pathogenic mechanisms and develop the therapeutic interventions of mitochondrial disorders. Here, we review the recent advances in patients-derived iPSCs as ex vivo models for mitochondrial diseases. Those patients-derived iPSCs have been differentiated into specific targeting cells such as retinal ganglion cells and eventually organoid for the disease modeling. These disease models have advanced our understanding of the pathophysiology of maternally inherited diseases and stepped toward therapeutic interventions for these diseases.
    Keywords:  Maternally inherited diseases; Mitochondria; iPSCs; mtDNA mutations
  5. Nat Genet. 2023 Sep 18.
      Uniparental inheritance of mitochondrial DNA (mtDNA) is an evolutionary trait found in nearly all eukaryotes. In many species, including humans, the sperm mitochondria are introduced to the oocyte during fertilization1,2. The mechanisms hypothesized to prevent paternal mtDNA transmission include ubiquitination of the sperm mitochondria and mitophagy3,4. However, the causative mechanisms of paternal mtDNA elimination have not been defined5,6. We found that mitochondria in human spermatozoa are devoid of intact mtDNA and lack mitochondrial transcription factor A (TFAM)-the major nucleoid protein required to protect, maintain and transcribe mtDNA. During spermatogenesis, sperm cells express an isoform of TFAM, which retains the mitochondrial presequence, ordinarily removed upon mitochondrial import. Phosphorylation of this presequence prevents mitochondrial import and directs TFAM to the spermatozoon nucleus. TFAM relocalization from the mitochondria of spermatogonia to the spermatozoa nucleus directly correlates with the elimination of mtDNA, thereby explaining maternal inheritance in this species.
  6. Front Cell Dev Biol. 2023 ;11 1257651
      The mitochondrion is a major hub of cellular metabolism and involved directly or indirectly in almost all biological processes of the cell. In mitochondrial diseases, compromised respiratory electron transfer and oxidative phosphorylation (OXPHOS) lead to compensatory rewiring of metabolism with resemblance to the Warburg-like metabolic state of cancer cells. The transcription factor MYC (or c-MYC) is a major regulator of metabolic rewiring in cancer, stimulating glycolysis, nucleotide biosynthesis, and glutamine utilization, which are known or predicted to be affected also in mitochondrial diseases. Albeit not widely acknowledged thus far, several cell and mouse models of mitochondrial disease show upregulation of MYC and/or its typical transcriptional signatures. Moreover, gene expression and metabolite-level changes associated with mitochondrial integrated stress response (mt-ISR) show remarkable overlap with those of MYC overexpression. In addition to being a metabolic regulator, MYC promotes cellular proliferation and modifies the cell cycle kinetics and, especially at high expression levels, promotes replication stress and genomic instability, and sensitizes cells to apoptosis. Because cell proliferation requires energy and doubling of the cellular biomass, replicating cells should be particularly sensitive to defective OXPHOS. On the other hand, OXPHOS-defective replicating cells are predicted to be especially vulnerable to high levels of MYC as it facilitates evasion of metabolic checkpoints and accelerates cell cycle progression. Indeed, a few recent studies demonstrate cell cycle defects and nuclear DNA damage in OXPHOS deficiency. Here, we give an overview of key mitochondria-dependent metabolic pathways known to be regulated by MYC, review the current literature on MYC expression in mitochondrial diseases, and speculate how its upregulation may be triggered by OXPHOS deficiency and what implications this has for the pathogenesis of these diseases.
    Keywords:  Warburg effect; cellular senescence; electron transport chain; mitochondrial integrated stress response; oxidative phosphorylation; respiratory complex III
  7. Free Radic Biol Med. 2023 Sep 20. pii: S0891-5849(23)00643-3. [Epub ahead of print]
      Aging is a complex biological process characterized by a progressive decline in cellular and tissue function, ultimately leading to organismal aging. Stem cells, with their regenerative potential, play a crucial role in maintaining tissue homeostasis and repair throughout an organism's lifespan. Mitochondria, the powerhouses of the cell, have emerged as key players in the aging process, impacting stem cell function and contributing to age-related tissue dysfunction. Here are discuss the mechanisms through which mitochondria influence stem cell fate decisions, including energy production, metabolic regulation, ROS signalling, and epigenetic modifications. Therefore, this review highlights the role of mitochondria in driving stem cell senescence and the subsequent impact on tissue function, leading to overall organismal aging and age-related diseases. Finally, we explore potential anti-aging therapies targeting mitochondrial health and discuss their implications for promoting healthy aging. This comprehensive review sheds light on the critical interplay between mitochondrial function, stem cell senescence, and organismal aging, offering insights into potential strategies for attenuating age-related decline and promoting healthy longevity.
    Keywords:  Aging; Anti-aging therapies; Mitochondria; Senescence; Stem cells
  8. Sci Adv. 2023 Sep 22. 9(38): eadh8228
      Breakdown of mitochondrial proteostasis activates quality control pathways including the mitochondrial unfolded protein response (UPRmt) and PINK1/Parkin mitophagy. However, beyond the up-regulation of chaperones and proteases, we have a limited understanding of how the UPRmt remodels and restores damaged mitochondrial proteomes. Here, we have developed a functional proteomics framework, termed MitoPQ (Mitochondrial Proteostasis Quantification), to dissect the UPRmt's role in maintaining proteostasis during stress. We find essential roles for the UPRmt in both protecting and repairing proteostasis, with oxidative phosphorylation metabolism being a central target of the UPRmt. Transcriptome analyses together with MitoPQ reveal that UPRmt transcription factors drive independent signaling arms that act in concert to maintain proteostasis. Unidirectional interplay between the UPRmt and PINK1/Parkin mitophagy was found to promote oxidative phosphorylation recovery when the UPRmt failed. Collectively, this study defines the network of proteostasis mediated by the UPRmt and highlights the value of functional proteomics in decoding stressed proteomes.
  9. Hum Mol Genet. 2023 Sep 18. pii: ddad153. [Epub ahead of print]
      Barth syndrome (BTHS) is a debilitating X-linked cardio-skeletal myopathy caused by loss-of-function mutations in TAFAZZIN, a cardiolipin (CL)-remodeling enzyme required for the maintenance of normal levels of CL species in mitochondrial membranes. At present, how perturbations in CL abundance and composition lead to many debilitating clinical presentations in BTHS patients have not been fully elucidated. Inspired by our recent findings that CL is essential for optimal mitochondrial calcium uptake, we measured the levels of other biologically important metal ions in BTHS mitochondria and found that in addition to calcium, magnesium levels are significantly reduced. Consistent with this observation, we report a decreased abundance of the mitochondrial magnesium influx channel MRS2 in multiple models of BTHS including yeast, murine myoblast, and BTHS patient cells and cardiac tissue. Mechanistically, we attribute reduced steady-state levels of MRS2 to its increased turnover in CL-deficient BTHS models. By expressing Mrs2 in well-characterized yeast mutants of the phospholipid biosynthetic pathways, we demonstrate a specific requirement of CL for Mrs2 abundance and assembly. Finally, we provide in vitro evidence for the direct binding of CL with human MRS2. Together, our study has identified a critical requirement of CL for MRS2 stability and suggests perturbation of mitochondrial magnesium homeostasis as a novel contributing factor to BTHS pathology.
    Keywords:  Barth syndrome; MRS2; cardiolipin; magnesium; mitochondria
  10. PLoS Biol. 2023 Sep 18. 21(9): e3002310
      Decline of mitochondrial function is a hallmark of cellular aging. To counteract this process, some cells inherit mitochondria asymmetrically to rejuvenate daughter cells. The molecular mechanisms that control this process are poorly understood. Here, we made use of matrix-targeted D-amino acid oxidase (Su9-DAO) to selectively trigger oxidative damage in yeast mitochondria. We observed that dysfunctional mitochondria become fusion-incompetent and immotile. Lack of bud-directed movements is caused by defective recruitment of the myosin motor, Myo2. Intriguingly, intact mitochondria that are present in the same cell continue to move into the bud, establishing that quality control occurs directly at the level of the organelle in the mother. The selection of healthy organelles for inheritance no longer works in the absence of the mitochondrial Myo2 adapter protein Mmr1. Together, our data suggest a mechanism in which the combination of blocked fusion and loss of motor protein ensures that damaged mitochondria are retained in the mother cell to ensure rejuvenation of the bud.
  11. Mitochondrion. 2023 Sep 20. pii: S1567-7249(23)00083-1. [Epub ahead of print]
      Allotopic expression is the functional transfer of an organellar gene to the nucleus, followed by synthesis of the gene product in the cytosol and import into the appropriate organellar sub compartment. Here, we focus on mitochondrial genes encoding OXPHOS subunits that were naturally transferred to the nucleus, and critically review experimental evidence that claim their allotopic expression. We emphasize aspects that may have been overlooked before, i.e., when modifying a mitochondrial gene for allotopic expression ━besides adapting the codon usage and including sequences encoding mitochondrial targeting signals━ three additional constraints should be considered: i) the average apparent free energy of membrane insertion (μΔGapp) of the transmembrane stretches (TMS) in proteins earmarked for the inner mitochondrial membrane, ii) the final, functional topology attained by each membrane-bound OXPHOS subunit; and iii) the defined mechanism by which the protein translocator TIM23 sorts cytosol-synthesized precursors. The mechanistic constraints imposed by TIM23 dictate the operation of two pathways through which alpha-helices in TMS are sorted, that eventually determine the final topology of membrane proteins. We used the biological hydrophobicity scale to assign an average apparent free energy of membrane insertion (μΔGapp) and a "traffic light" color code to all TMS of OXPHOS membrane proteins, thereby predicting which are more likely to be internalized into mitochondria if allotopically produced. We propose that the design of proteins for allotopic expression must make allowance for μΔGapp maximization of highly hydrophobic TMS in polypeptides whose corresponding genes have not been transferred to the nucleus in some organisms.
    Keywords:  TIM23; allotopic expression; apparent free energy of membrane insertion; membrane embedded proteins; mitochondrial complexes; oxidative phosphorylation; protein import; transmembrane stretches
  12. Science. 2023 Sep 22. 381(6664): 1287-1288
      Mitochondrial metabolite reduces melanoma growth by boosting antigen presentation.
  13. iScience. 2023 Oct 20. 26(10): 107780
      Mitochondrial networks remodel their connectivity, content, and subcellular localization to support optimized energy production in conditions of increased environmental or cellular stress. Microglia rely on mitochondria to respond to these stressors, however our knowledge about mitochondrial networks and their adaptations in microglia in vivo is limited. Here, we generate a mouse model that selectively labels mitochondria in microglia. We identify that mitochondrial networks are more fragmented with increased content and perinuclear localization in vitro vs. in vivo. Mitochondrial networks adapt similarly in microglia closest to the injury site after optic nerve crush. Preventing microglial UCP2 increase after injury by selective knockout induces cellular stress. This results in mitochondrial hyperfusion in male microglia, a phenotype absent in females due to circulating estrogens. Our results establish the foundation for mitochondrial network analysis of microglia in vivo, emphasizing the importance of mitochondrial-based sex effects of microglia in other pathologies.
    Keywords:  Biological sciences; Natural sciences; Neuroscience; Physiology; Sensory neuroscience; Systems neuroscience
  14. Ultrasound Obstet Gynecol. 2023 Sep 18.
      OBJECTIVES: Mitochondrial complex I deficiency, nuclear type 16 is a rare autosomal recessive disorder caused by biallelic pathogenic variants in NDUFAF5 (C20orf7) [OMIM 618238]. This entity belongs to a genetically and clinically heterogenic group of complex I deficiency which accounts for up to 30% of childhood mitochondrial disorders presenting as Leigh syndrome, leukoencephalopathy, fatal infantile lactic acidosis, and other early-onset neurodegenerative disorders. We present very early, unique, and severe prenatal manifestation of this disorder, previously considered to manifest post-natally.METHODS: We describe five fetuses from three non-related families sharing common sonographic abnormalities including brain cysts, callosal malformations, hydrops fetalis, and growth restriction. Genetic evaluation included chromosomal microarray analysis and exome sequencing. Two fetuses from one family were also available for pathology examination, including electronic microscopy.
    RESULTS: Chromosomal microarray revealed no chromosomal abnormalities. Trio exome sequencing demonstrated that three affected fetuses from three unrelated families were compound heterozygotes or homozygotes for likely pathogenic variants in NDUFAF5. No other causative variants were detected. NDUFAF5 variants association with fetal malformations was further confirmed by segregation studies. Histologic evaluation of fetal tissues and electronic microscopy of muscle, liver, proximal tubules of kidney and heart, demonstrated changes resembling those described in postmortem autopsies of patients with mitochondrial depletion disorders as well as previously undescribed findings.
    CONCLUSIONS: Mitochondrial complex I deficiency and specifically biallelic mutations in NDUFAF5 have a role in abnormal fetal development presenting with severe congenital malformations. Complex I mitochondrial disorders should be considered in the differential diagnosis of callosal malformations and brain cysts, especially when associated with extra central nervous system (CNS) abnormalities such as fetal growth restriction or nonimmune hydrops fetalis. This article is protected by copyright. All rights reserved.
    Keywords:  Brain cysts; Corpus Callosum; Hydrops Fetalis, FGR, Exome sequencing, complex I deficiency; NDUFAF5
  15. Nature. 2023 Sep 19.
    Keywords:  Diseases; Genomics; Machine learning
  16. Nat Commun. 2023 09 18. 14(1): 5781
      The use of exogenous mitochondria to replenish damaged mitochondria has been proposed as a strategy for the treatment of pulmonary fibrosis. However, the success of this strategy is partially restricted by the difficulty of supplying sufficient mitochondria to diseased cells. Herein, we report the generation of high-powered mesenchymal stem cells with promoted mitochondrial biogenesis and facilitated mitochondrial transfer to injured lung cells by the sequential treatment of pioglitazone and iron oxide nanoparticles. This highly efficient mitochondrial transfer is shown to not only restore mitochondrial homeostasis but also reactivate inhibited mitophagy, consequently recovering impaired cellular functions. We perform studies in mouse to show that these high-powered mesenchymal stem cells successfully mitigate fibrotic progression in a progressive fibrosis model, which was further verified in a humanized multicellular lung spheroid model. The present findings provide a potential strategy to overcome the current limitations in mitochondrial replenishment therapy, thereby promoting therapeutic applications for fibrotic intervention.
  17. FEBS J. 2023 Sep 18.
      Mitochondrial outer membrane β-barrel proteins are encoded in the nucleus, translated in the cytosol, and then targeted to and imported into the respective organelles. Detailed studies have uncovered the mechanisms involved in the import of these proteins and identified the targeting signals and the cytosolic factors that govern their proper biogenesis. Recently, de novo designed eight stranded β-barrel proteins (Tmb2.3 and Tmb2.17) were shown to fold and assemble into lipid membranes. To better understand the general aspects of the biogenesis of β-barrel proteins, we investigated the fate of these artificial proteins upon their expression in yeast cells. We demonstrate that although these proteins are de novo designed and are not related to bona fide mitochondrial β-barrel proteins, they were targeted to mitochondria and integrated into the organelle outer membrane. We further studied whether this integration requires components of the yeast mitochondrial import machinery like Tom20, Tom70, Tob55/Sam50, and Mas37/Sam37. Whereas it seems that none of the import receptors was required for the biogenesis of the artificial β-barrel proteins, we observed a strong dependency on the TOB/SAM complex. Collectively, our findings demonstrate that the mitochondrial outer membrane is the preferential location in yeast cells for any membrane embedded β-barrel protein.
    Keywords:  TOB/SAM complex; de novo designed proteins; mitochondria; outer membrane; β-barrel proteins
  18. PLoS One. 2023 ;18(9): e0291442
      Increasing evidence demonstrate that the electron transfer chain plays a critical role in controlling the effector functions of macrophages. In this work, we have generated a Ndufs4-/- murine macrophage cell lines. The Ndufs4 gene, which encodes a supernumerary subunit of complex I, is a mutational hotspot in Leigh syndrome patients. Ndufs4-/- macrophages showed decreased complex I activity, altered complex I assembly, and lower levels of maximal respiration and ATP production. These mitochondrial respiration alterations were associated with a shift towards a pro-inflammatory cytokine profile after lipopolysaccharide challenge and improved ability to phagocytose Gram-negative bacteria.
  19. bioRxiv. 2023 Sep 05. pii: 2023.09.01.555986. [Epub ahead of print]
      Mitochondrial DNA (mtDNA) mutations are frequently observed in cancer, but their contribution to tumor progression is controversial. To evaluate the impact of mtDNA variants on tumor growth and metastasis, we created human melanoma cytoplasmic hybrid (cybrid) cell lines transplanted with wildtype mtDNA or pathogenic mtDNA encoding variants that partially or completely inhibit oxidative phosphorylation. Homoplasmic pathogenic mtDNA cybrids reliably established tumors despite dysfunctional oxidative phosphorylation. However, pathogenic mtDNA variants disrupted spontaneous metastasis of subcutaneous tumors and decreased the abundance of circulating melanoma cells in the blood. Pathogenic mtDNA did not induce anoikis or inhibit organ colonization of melanoma cells following intravenous injections. Instead, migration and invasion were reduced, indicating that limited circulation entry functions as a metastatic bottleneck amidst mtDNA dysfunction. Furthermore, analysis of selective pressure exerted on the mitochondrial genomes of heteroplasmic cybrid lines revealed a suppression of pathogenic mtDNA allelic frequency during melanoma growth. Collectively, these findings demonstrate that functional mtDNA is favored during melanoma growth and enables metastatic entry into the blood.
  20. BMC Genomics. 2023 Sep 22. 24(1): 561
      The mitochondria are central in the cellular response to changing environmental conditions resulting from disease states, environmental exposures or normal physiological processes. Although the influences of environmental stressors upon the nuclear epigenome are well characterized, the existence and role of the mitochondrial epigenome remains contentious. Here, by quantifying the mitochondrial epigenomic response of pineal gland cells to circadian stress, we confirm the presence of extensive cytosine methylation within the mitochondrial genome. Furthermore, we identify distinct epigenetically plastic regions (mtDMRs) which vary in cytosinic methylation, primarily in a non CpG context, in response to stress and in a sex-specific manner. Motifs enriched in mtDMRs contain recognition sites for nuclear-derived DNA-binding factors (ATF4, HNF4A) important in the cellular metabolic stress response, which we found to be conserved across diverse vertebrate taxa. Together, these findings suggest a new layer of mito-nuclear interaction in which the nuclear metabolic stress response could alter mitochondrial transcriptional dynamics through the binding of nuclear-derived transcription factors in a methylation-dependent context.
    Keywords:  DNA-binding; Epigenome; Methylation; Mitochondrial genome; Pineal gland
  21. Am J Hum Genet. 2023 Sep 12. pii: S0002-9297(23)00288-4. [Epub ahead of print]
      Defects in hydroxymethylbilane synthase (HMBS) can cause acute intermittent porphyria (AIP), an acute neurological disease. Although sequencing-based diagnosis can be definitive, ∼⅓ of clinical HMBS variants are missense variants, and most clinically reported HMBS missense variants are designated as "variants of uncertain significance" (VUSs). Using saturation mutagenesis, en masse selection, and sequencing, we applied a multiplexed validated assay to both the erythroid-specific and ubiquitous isoforms of HMBS, obtaining confident functional impact scores for >84% of all possible amino acid substitutions. The resulting variant effect maps generally agreed with biochemical expectations and provide further evidence that HMBS can function as a monomer. Additionally, the maps implicated specific residues as having roles in active site dynamics, which was further supported by molecular dynamics simulations. Most importantly, these maps can help discriminate pathogenic from benign HMBS variants, proactively providing evidence even for yet-to-be-observed clinical missense variants.
    Keywords:  AIP; HMBS,; acute hepatic porphryia; acute intermittent porphryia; clinical variant interpretation; deep mutational scanning; heme biosynthesis; hydroxymethylbilane synthase; molecular dynamics; variant effect mapping
  22. Free Radic Biol Med. 2023 Sep 16. pii: S0891-5849(23)00615-9. [Epub ahead of print]
      Synaptic signaling depends on ATP generated by mitochondria. Dysfunctional mitochondria shift the redox balance towards a more oxidative environment. Due to extensive connectivity, the striatum is especially vulnerable to mitochondrial dysfunction. We found that neuronal calcium-binding protein 2 (NECAB2) plays a role in striatal function and mitochondrial homeostasis. NECAB2 is a predominantly endosomal striatal protein which partially colocalizes with mitochondria. This colocalization is enhanced by mild oxidative stress. Global knockout of Necab2 in the mouse results in increased superoxide levels, increased DNA oxidation and reduced levels of the antioxidant glutathione which correlates with an altered mitochondrial shape and function. Striatal mitochondria from Necab2 knockout mice are more abundant and smaller and characterized by a reduced spare capacity suggestive of intrinsic uncoupling respectively mitochondrial dysfunction. In line with this, we also found an altered stress-induced interaction of endosomes with mitochondria in Necab2 knockout striatal cultures. The predominance of dysfunctional mitochondria and the pro-oxidative redox milieu correlates with a loss of striatal synapses and behavioral changes characteristic of striatal dysfunction like reduced motivation and altered sensory gating. Together this suggests an involvement of NECAB2 in an endosomal pathway of mitochondrial stress response important for striatal function.
  23. Neuron. 2023 Sep 13. pii: S0896-6273(23)00629-3. [Epub ahead of print]
      Parkin-mediated mitophagy has been studied extensively, but whether mutations in parkin contribute to Parkinson's disease pathogenesis through alternative mechanisms remains unexplored. Using patient-derived dopaminergic neurons, we found that phosphorylation of parkin by Ca2+/calmodulin-dependent protein kinase 2 (CaMK2) at Ser9 leads to activation of parkin in a neuronal-activity-dependent manner. Activated parkin ubiquitinates synaptojanin-1, facilitating its interaction with endophilin A1 and synaptic vesicle recycling. Neurons from PD patients with mutant parkin displayed defective recycling of synaptic vesicles, leading to accumulation of toxic oxidized dopamine that was attenuated by boosting endophilin A1 expression. Notably, combined heterozygous parkin and homozygous PTEN-induced kinase 1 (PINK1) mutations led to earlier disease onset compared with homozygous mutant PINK1 alone, further underscoring a PINK1-independent role for parkin in contributing to disease. Thus, this study identifies a pathway for selective activation of parkin at human dopaminergic synapses and highlights the importance of this mechanism in the pathogenesis of Parkinson's disease.
    Keywords:  CaMK2-mediated activation of parkin; PINK1-independent; Parkinson’s disease; human dopaminergic neurons; synaptic dysfunction; toxic oxidized dopamine
  24. J Med Genet. 2023 Sep 21. pii: jmg-2023-109470. [Epub ahead of print]
      PURPOSE: Genome sequencing (GS) is expected to reduce the diagnostic gap in rare disease genetics. We aimed to evaluate a scalable framework for genome-based analyses 'beyond the exome' in regular care of patients with inherited retinal degeneration (IRD) or inherited optic neuropathy (ION).METHODS: PCR-free short-read GS was performed on 1000 consecutive probands with IRD/ION in routine diagnostics. Complementary whole-blood RNA-sequencing (RNA-seq) was done in a subset of 74 patients. An open-source bioinformatics analysis pipeline was optimised for structural variant (SV) calling and combined RNA/DNA variation interpretation.
    RESULTS: A definite genetic diagnosis was established in 57.4% of cases. For another 16.7%, variants of uncertain significance were identified in known IRD/ION genes, while the underlying genetic cause remained unresolved in 25.9%. SVs or alterations in non-coding genomic regions made up for 12.7% of the observed variants. The RNA-seq studies supported the classification of two unclear variants.
    CONCLUSION: GS is feasible in clinical practice and reliably identifies causal variants in a substantial proportion of individuals. GS extends the diagnostic yield to rare non-coding variants and enables precise determination of SVs. The added diagnostic value of RNA-seq is limited by low expression levels of the major IRD disease genes in blood.
    Keywords:  RNA-seq; eye diseases; genomics; molecular diagnostic techniques
  25. Intern Med J. 2023 Sep 21.
      BACKGROUND: The complexities of mitochondrial disease make epidemiological studies challenging, yet this information is important in understanding the healthcare burden and addressing service and educational needs. Existing studies are limited to quaternary centres or focus on a single genotype or phenotype and estimate disease prevalence at 12.5 per 100 000. New Zealand's (NZ) size and partially integrated national healthcare system make it amenable to a nationwide prevalence study.AIM: To estimate the prevalence of molecularly confirmed and suspected mitochondrial disease on 31 December 2015 in NZ.
    METHODS: Cases were identified from subspecialists and laboratory databases and through interrogation of the Ministry of Health National Minimum Dataset with a focus on presentations between 2000 and 2015. Patient records were reviewed, and those with a diagnosis of 'mitochondrial disease' who were alive and residing in NZ on the prevalence date were included. These were divided into molecularly confirmed and clinically suspected cases. Official NZ estimated resident population data were used to calculate prevalence.
    RESULTS: Seven hundred twenty-three unique national health index numbers were identified. Five hundred five were excluded. The minimum combined prevalence for mitochondrial disease was 4.7 per 100 000 (95% confidence interval (CI): 4.1-5.4). The minimum prevalence for molecularly confirmed and suspected disease was 2.9 (95% CI 2.4-3.4) and 1.8 (95% CI 1.4-2.2) cases per 100 000 respectively.
    CONCLUSIONS: Within the limitations of this study, comparison to similar prevalence studies performed by specialist referral centres suggests mitochondrial disease is underdiagnosed in NZ. This highlights a need for improved education and referral pathways for mitochondrial disease in NZ.
    Keywords:  New Zealand/epidemiology*; mitochondrial diseases/diagnosis*; mitochondrial diseases/epidemiology*; mutation/genetics*; prevalence
  26. EBioMedicine. 2023 Sep 20. pii: S2352-3964(23)00375-4. [Epub ahead of print]96 104809
      BACKGROUND: The intestine of children with severe malnutrition (SM) shows structural and functional changes that are linked to increased infection and mortality. SM dysregulates the tryptophan-kynurenine pathway, which may impact processes such as SIRT1- and mTORC1-mediated autophagy and mitochondrial homeostasis. Using a mouse and organoid model of SM, we studied the repercussions of these dysregulations on malnutrition enteropathy and the protective capacity of maintaining autophagy activity and mitochondrial health.METHODS: SM was induced through feeding male weanling C57BL/6 mice a low protein diet (LPD) for 14-days. Mice were either treated with the NAD+-precursor, nicotinamide; an mTORC1-inhibitor, rapamycin; a SIRT1-activator, resveratrol; or SIRT1-inhibitor, EX-527. Malnutrition enteropathy was induced in enteric organoids through amino-acid deprivation. Features of and pathways to malnutrition enteropathy were examined, including paracellular permeability, nutrient absorption, and autophagic, mitochondrial, and reactive-oxygen-species (ROS) abnormalities.
    FINDINGS: LPD-feeding and ensuing low-tryptophan availability led to villus atrophy, nutrient malabsorption, and intestinal barrier dysfunction. In LPD-fed mice, nicotinamide-supplementation was linked to SIRT1-mediated activation of mitophagy, which reduced damaged mitochondria, and improved intestinal barrier function. Inhibition of mTORC1 reduced intestinal barrier dysfunction and nutrient malabsorption. Findings were validated and extended using an organoid model, demonstrating that resolution of mitochondrial ROS resolved barrier dysfunction.
    INTERPRETATION: Malnutrition enteropathy arises from a dysregulation of the SIRT1 and mTORC1 pathways, leading to disrupted autophagy, mitochondrial homeostasis, and ROS. Whether nicotinamide-supplementation in children with SM could ameliorate malnutrition enteropathy should be explored in clinical trials.
    FUNDING: This work was supported by the Bill and Melinda Gates Foundation, the Sickkids Research Institute, the Canadian Institutes of Health Research, and the University Medical Center Groningen.
    Keywords:  Autophagy; Enteropathy; Malnutrition; Mitochondria; SIRT1
  27. Nucleic Acids Res. 2023 Sep 22. pii: gkad773. [Epub ahead of print]
      Translational readthrough of UGA stop codons by selenocysteine-specific tRNA (tRNASec) enables the synthesis of selenoproteins. Seryl-tRNA synthetase (SerRS) charges tRNASec with serine, which is modified into selenocysteine and delivered to the ribosome by a designated elongation factor (eEFSec in eukaryotes). Here we found that components of the human selenocysteine incorporation machinery (SerRS, tRNASec, and eEFSec) also increased translational readthrough of non-selenocysteine genes, including VEGFA, to create C-terminally extended isoforms. SerRS recognizes target mRNAs through a stem-loop structure that resembles the variable loop of its cognate tRNAs. This function of SerRS depends on both its enzymatic activity and a vertebrate-specific domain. Through eCLIP-seq, we identified additional SerRS-interacting mRNAs as potential readthrough genes. Moreover, SerRS overexpression was sufficient to reverse premature termination caused by a pathogenic nonsense mutation. Our findings expand the repertoire of selenoprotein biosynthesis machinery and suggest an avenue for therapeutic targeting of nonsense mutations using endogenous factors.
  28. STAR Protoc. 2023 Sep 21. pii: S2666-1667(23)00557-9. [Epub ahead of print]4(4): 102590
      Yeast mitofissin Atg44 is a mitochondrial intermembrane space protein that causes membrane fission required for mitophagy. Here, we present a protocol for observing Atg44-mediated membrane fission. We describe steps for recombinant Atg44 purification, lipid nanotube preparation as model membranes, and Atg44-mediated membrane fission real-time observation. We then detail procedures for tube radius estimation using confocal microscopy. This protocol can also be adapted to the study of membrane fission by other proteins. For complete details on the use and execution of this protocol, please refer to Fukuda et al. (2023).1.
    Keywords:  Biophysics; Microscopy; Protein Biochemistry
  29. Science. 2023 Sep 22. 381(6664): eadg7492
      The vast majority of missense variants observed in the human genome are of unknown clinical significance. We present AlphaMissense, an adaptation of AlphaFold fine-tuned on human and primate variant population frequency databases to predict missense variant pathogenicity. By combining structural context and evolutionary conservation, our model achieves state-of-the-art results across a wide range of genetic and experimental benchmarks, all without explicitly training on such data. The average pathogenicity score of genes is also predictive for their cell essentiality, capable of identifying short essential genes that existing statistical approaches are underpowered to detect. As a resource to the community, we provide a database of predictions for all possible human single amino acid substitutions and classify 89% of missense variants as either likely benign or likely pathogenic.
  30. Science. 2023 Sep 22. 381(6664): 1316-1323
      Although tumor growth requires the mitochondrial electron transport chain (ETC), the relative contribution of complex I (CI) and complex II (CII), the gatekeepers for initiating electron flow, remains unclear. In this work, we report that the loss of CII, but not that of CI, reduces melanoma tumor growth by increasing antigen presentation and T cell-mediated killing. This is driven by succinate-mediated transcriptional and epigenetic activation of major histocompatibility complex-antigen processing and presentation (MHC-APP) genes independent of interferon signaling. Furthermore, knockout of methylation-controlled J protein (MCJ), to promote electron entry preferentially through CI, provides proof of concept of ETC rewiring to achieve antitumor responses without side effects associated with an overall reduction in mitochondrial respiration in noncancer cells. Our results may hold therapeutic potential for tumors that have reduced MHC-APP expression, a common mechanism of cancer immunoevasion.
  31. Life Sci. 2023 Sep 20. pii: S0024-3205(23)00751-8. [Epub ahead of print] 122116
      AIM: We investigated the effect of mitochondria transfer in high fat diet+streptozotocin (HFD + STZ) induced metabolic syndrome (MeS) in rats. The effect of mitochondria transfer in MeS with co-existing hypertension, hyperlipidaemia, diabetes and fatty liver together, has not been reported.MATERIALS AND METHODS: Heathy mitochondria was transferred intravenously and the effect on several physiological parameters and biochemical parameters were examined in HFD + STZ rats. In addition, RNA-sequencing of healthy liver tissues was performed to elucidate the molecular pathways affected by mitochondria transfer in restoring metabolic health.
    KEY FINDINGS: We observed reduction in both systolic and diastolic blood pressure levels, reduced blood glucose levels, and a marked reduction in serum lipid profiles. The levels of alanine transaminase (ALT) and aspartate transaminase (AST) also improved along with evident restoration of liver morphology demonstrated by histopathological analysis. Enhanced mitochondrial biogenetics and reduction in oxidative stress and inflammatory markers was also observed. The pathway enrichment analysis revealed reduction in insulin resistance, inflammatory markers, regulation of mitochondrial bioenergetics, calcium ion homeostasis, fatty-acid β-oxidation, cytokine immune regulators, and enhanced lipid solubilisation. The significant effect of healthy mitochondria transfer in restoration of metabolic functions was observed by the activation of PI3K-AKT, AMPK/mTOR pathways and cytokine immune regulators, suggesting that inflammatory mediators were also significantly affected after mitochondria transfer.
    SIGNIFICANCE: This study, provides insights on molecular processes triggered by mitochondria transfer in fatty liver regeneration and improvement of overall metabolic health.
    Keywords:  Fatty-acid β oxidation; Metabolic reprogramming; Metabolic syndrome; Mitochondria transplantation; Mitochondrial bioenergetics
  32. Signal Transduct Target Ther. 2023 Sep 21. 8(1): 371
      The innate immune response is the main pathophysiological process of ocular surface diseases exposed to multiple environmental stresses. The epithelium is central to the innate immune response, but whether and how innate immunity is initiated by ocular epithelial cells in response to various environmental stresses in ocular surface diseases, such as dry eye, is still unclear. By utilizing two classic experimental dry eye models-a mouse ocular surface treated with benzalkonium chloride (BAC) and a mouse model with surgically removed extraorbital lachrymal glands, as well as dry eye patient samples-along with human corneal epithelial cells (HCE) exposed to hyperosmolarity, we have discovered a novel innate immune pathway in ocular surface epithelial cells. Under stress, mitochondrial DNA (mtDNA) was released into the cytoplasm through the mitochondrial permeability transition pore (mPTP) and further activated the cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) pathway, aggravating downstream inflammatory responses and ocular surface damage. Genetic deletion or pharmacological suppression of STING and inhibition of mtDNA release reduced inflammatory responses, whereas mtDNA transfection supported cytoplasmic mtDNA-induced inflammatory responses by activating the cGAS-STING pathway. Our study clarified the cGAS-STING pathway-dependent sensing of mitochondrial DNA-mediated ocular surface inflammation, which elucidated a new mechanism of ocular surface diseases in response to multiple environmental stresses.
  33. Electron Mark. 2023 ;33(1): 48
      Data has become an indispensable input, throughput, and output for the healthcare industry. In recent years, omics technologies such as genomics and proteomics have generated vast amounts of new data at the cellular level including molecular, structural, and functional levels. Cellular data holds the potential to innovate therapeutics, vaccines, diagnostics, consumer products, or even ancestry services. However, data at the cellular level is generated with rapidly evolving omics technologies. These technologies use scientific knowledge from resource-rich environments. This raises the question of how new ventures can use cellular-level data from omics technologies to create new products and scale their business. We report on a series of interviews and a focus group discussion with entrepreneurs, investors, and data providers. By conceptualizing omics technologies as external enablers, we show how characteristics of cellular-level data negatively affect the combination mechanisms that drive venture creation and growth. We illustrate how data characteristics set boundary conditions for innovation and entrepreneurship and highlight how ventures seek to mitigate their impact.Supplementary Information: The online version contains supplementary material available at 10.1007/s12525-023-00669-w.
    Keywords:  Digital entrepreneurship; Digital innovation; External enabler; Health data; Omics
  34. STAR Protoc. 2023 Sep 19. pii: S2666-1667(23)00472-0. [Epub ahead of print]4(4): 102505
      We present a detailed protocol for sequencing full-length mRNA isoforms using the Oxford nanopore long-read sequencing technology. We describe steps for poly(A) RNA isolation, library preparation, and cDNA size selection. We then detail procedures for sequencing and processing and a computational framework to identify exon couplings and assign mRNA 5' ends and 3' ends to each other. Our approach enables the identification of links between transcription initiation and co-transcriptional RNA processing events. For complete details on the use and execution of this protocol, please refer to Alfonso-Gonzalez et al.1.
    Keywords:  Bioinformatics; Genomics; Molecular Biology; RNAseq; Sequence Analysis; Sequencing
  35. Nature. 2023 Sep;621(7979): 467-470
    Keywords:  Biotechnology; Drug discovery; Machine learning; Research data
  36. Front Mol Neurosci. 2023 ;16 1241222
      Dominant optic atrophy (DOA) is mainly caused by OPA1 mutations and is characterized by the degeneration of retinal ganglion cells (RGCs), whose axons form the optic nerve. The penetrance of DOA is incomplete and the disease is marked by highly variable expressivity, ranging from asymptomatic patients to some who are totally blind or who suffer from multisystemic effects. No clear genotype-phenotype correlation has been established to date. Taken together, these observations point toward the existence of modifying genetic and/or environmental factors that modulate disease severity. Here, we investigated the influence of genetic background on DOA expressivity by switching the previously described DOA mouse model bearing the c.1065 + 5G → A Opa1 mutation from mixed C3H; C57BL/6 J to a pure C57BL/6 J background. We no longer observed retinal and optic nerve abnormalities; the findings indicated no degeneration, but rather a sex-dependent negative effect on RGC connectivity. This highlights the fact that RGC synaptic alteration might precede neuronal death, as has been proposed in other neurodegenerative diseases, providing new clinical considerations for early diagnosis as well as a new therapeutic window for DOA. Furthermore, our results demonstrate the importance of secondary genetic factors in the variability of DOA expressivity and offer a model for screening for aggravating environmental and genetic factors.
    Keywords:  OPA1; disease severity; genetic modifiers; mitochondria; mouse strains; optic atrophy