bims-mitdis Biomed News
on Mitochondrial Disorders
Issue of 2021‒04‒25
sixty papers selected by
Catalina Vasilescu
University of Helsinki

  1. Proc Natl Acad Sci U S A. 2021 Apr 27. pii: e2019665118. [Epub ahead of print]118(17):
      Mitochondrial disease is a debilitating condition with a diverse genetic etiology. Here, we report that TMEM126A, a protein that is mutated in patients with autosomal-recessive optic atrophy, participates directly in the assembly of mitochondrial complex I. Using a combination of genome editing, interaction studies, and quantitative proteomics, we find that loss of TMEM126A results in an isolated complex I deficiency and that TMEM126A interacts with a number of complex I subunits and assembly factors. Pulse-labeling interaction studies reveal that TMEM126A associates with the newly synthesized mitochondrial DNA (mtDNA)-encoded ND4 subunit of complex I. Our findings indicate that TMEM126A is involved in the assembly of the ND4 distal membrane module of complex I. In addition, we find that the function of TMEM126A is distinct from its paralogue TMEM126B, which acts in assembly of the ND2-module of complex I.
    Keywords:  complex I; membrane protein; mitochondria; optic atrophy; oxidative phosphorylation
  2. Acta Physiol (Oxf). 2021 Apr 23. e13666
      AIM: Mitophagy is the regulated process that targets damaged or dysfunctional mitochondria for lysosomal-mediated removal. This process is an essential element of mitochondrial quality control, and dysregulation of mitophagy may contribute to a host of diseases, most notably neurodegenerative conditions such as Parkinson's disease. Mitochondria targeted for mitophagic destruction are molecularly marked by the ubiquitination of several outer mitochondrial membrane (OMM) proteins. This ubiquitination is positively regulated, in part, by the mitochondrial-targeted kinase PINK1 and the E3 ubiquitin ligase Parkin. In contrast, the reverse phenomenon, deubiquitination, removes ubiquitin from Parkin substrates embedded in the OMM proteins, antagonizing mitophagy. Recent evidence suggests that the mitochondrial deubiquitinase USP30 negatively regulates Parkin mediated mitophagy, providing opportunities to identify USP30 inhibitors and test for their effects in augmenting mitophagy. Here we will characterize a USP30 inhibitor and demonstrate how the pharmacological inhibition of USP30 can augment stress-induced mitophagic flux.METHODS: We have conducted mitophagy and mitochondrial analyses in cultured cells. We have determined the plasma pharmacokinetics of the USP30 inhibitor in mice and conducted analyses using the mt-Keima mice to measure in vivo mitophagy directly.
    RESULTS: The compound has minimal mitochondrial toxicity in cultured cells and is tolerated well in mice. Interestingly, we demonstrated tissue-specific induction of mitophagy following USP30 pharmacological inhibition. In particular, pharmacological inhibition of USP30 induces a significant increase in cardiac mitophagy without detriment to cardiac function.
    CONCLUSION: Our data support the evidence that USP30 inhibition may serve as a specific strategy to selectively increase mitophagic flux, allowing for the development of novel therapeutic approaches.
    Keywords:  Mitophagy; PINK1; Parkin; USP30; mitochondrial deubiquitination; mt-Keima
  3. Circ Res. 2021 Apr 22.
      Rationale: Heart failure with preserved ejection fraction (HFpEF) is a mortal clinical syndrome without effective therapies. We recently demonstrated in mice that a combination of metabolic and hypertensive stress recapitulates key features of human HFpEF.Objective: Using this novel preclinical HFpEF model, we set out to define and manipulate metabolic dysregulations occurring in HFpEF myocardium. Methods and Results: We observed impairment in mitochondrial fatty acid oxidation associated with hyperacetylation of key enzymes in the pathway. Down-regulation of sirtuin 3 and deficiency of NAD+ secondary to an impaired NAD+ salvage pathway contribute to this mitochondrial protein hyperacetylation. Impaired expression of genes involved in NAD+ biosynthesis was confirmed in cardiac tissue from HFpEF patients. Supplementing HFpEF mice with nicotinamide riboside or a direct activator of NAD+ biosynthesis led to improvement in mitochondrial function and amelioration of the HFpEF phenotype. Conclusions: Collectively, these studies demonstrate that HFpEF is associated with myocardial mitochondrial dysfunction and unveil NAD+ repletion as a promising therapeutic approach in the syndrome.
  4. Front Cell Dev Biol. 2021 ;9 636506
      As neurons are highly energy-demanding cell, increasing evidence suggests that mitochondria play a large role in several age-related neurodegenerative diseases. Synaptic damage and mitochondrial dysfunction have been associated with early events in the pathogenesis of major neurodegenerative diseases, including Parkinson's disease, atypical parkinsonisms, and Huntington disease. Disruption of mitochondrial structure and dynamic is linked to increased levels of reactive oxygen species production, abnormal intracellular calcium levels, and reduced mitochondrial ATP production. However, recent research has uncovered a much more complex involvement of mitochondria in such disorders than has previously been appreciated, and a remarkable number of genes and proteins that contribute to the neurodegeneration cascade interact with mitochondria or affect mitochondrial function. In this review, we aim to summarize and discuss the deep interconnections between mitochondrial dysfunction and basal ganglia disorders, with an emphasis into the molecular triggers to the disease process. Understanding the regulation of mitochondrial pathways may be beneficial in finding pharmacological or non-pharmacological interventions to delay the onset of neurodegenerative diseases.
    Keywords:  Huntington disease; Parkinson’s disease; Pink1/parkin pathway; alpha-synuclein; atypical parkinsonism; mitochondrial dysfunction; neurodegenerative diseases
  5. Biol Cell. 2021 Apr 18.
      Mitochondria are organelles involved in various functions related to cellular metabolism and homeostasis. Though mitochondria contain own genome, their nuclear counterparts encode most of the different mitochondrial proteins. These are synthesized as precursors in the cytosol and have to be delivered into the mitochondria. These organelles hence have elaborate machineries for the import of precursor proteins from cytosol. The protein import machineries present in both mitochondrial membrane and aqueous compartments show great variability in pre-protein recognition, translocation and sorting across or into it. Mitochondrial protein import machineries also interact transiently with other protein complexes of the respiratory chain or those involved in the maintenance of membrane architecture. Hence mitochondrial protein translocation is an indispensable part of the regulatory network that maintains protein biogenesis, bioenergetics, membrane dynamics and quality control of the organelle. Various stress conditions and diseases that are associated with mitochondrial import defects lead to changes in cellular transcriptomic and proteomic profiles. Dysfunction in mitochondrial protein import also causes over-accumulation of precursor proteins and their aggregation in the cytosol. Multiple pathways may be activated for buffering these harmful consequences. Here we present a comprehensive picture of import machinery and its role in cellular quality control in response to defective mitochondrial import. We also discuss the pathological consequences of dysfunctional mitochondrial protein import in neurodegeneration and cancer. This article is protected by copyright. All rights reserved.
    Keywords:  Intracellular compartmentalization; Mitochondria; Protein degradation/proteases
  6. Expert Rev Proteomics. 2021 Apr 19.
      Mitochondrial dysfunction is involved in Alzheimer's disease (AD) pathogenesis. Mitochondria have their own genetic material; however, most of their proteins (∼99%) are synthesized as precursors on cytosolic ribosomes, and then imported into the mitochondria. Therefore, exploring proteome changes in these organelles can yield valuable information and shed light on the molecular mechanisms underlying mitochondrial dysfunction in AD. Here we review AD associated mitochondrial changes including the effects of amyloid beta and tau protein accumulation on the mitochondrial proteome. We also discuss the relationship of ApoE genetic polymorphism with mitochondrial changes, and present a meta-analysis of various differentially expressed proteins in the mitochondria in AD.AREA COVERED: Proteomics studies and their contribution to our understanding of mitochondrial dysfunction in AD pathogenesis.
    EXPERT OPINION: Proteomics has proved to be an efficient tool to uncover various aspects of this complex organelle, which will broaden our understanding of mitochondrial dysfunction in AD. Evidently, mitochondrial dysfunction is an early biochemical event that might play a central role in driving AD pathogenesis.
    Keywords:  Alzheimer’s disease; Mitochondrial dysfunction; Proteomics
  7. J Cell Biol. 2021 May 03. pii: e202010004. [Epub ahead of print]220(5):
      Mitochondria, which are excluded from the secretory pathway, depend on lipid transport proteins for their lipid supply from the ER, where most lipids are synthesized. In yeast, the outer mitochondrial membrane GTPase Gem1 is an accessory factor of ERMES, an ER-mitochondria tethering complex that contains lipid transport domains and that functions, partially redundantly with Vps13, in lipid transfer between the two organelles. In metazoa, where VPS13, but not ERMES, is present, the Gem1 orthologue Miro was linked to mitochondrial dynamics but not to lipid transport. Here we show that Miro, including its peroxisome-enriched splice variant, recruits the lipid transport protein VPS13D, which in turn binds the ER in a VAP-dependent way and thus could provide a lipid conduit between the ER and mitochondria. These findings reveal a so far missing link between function(s) of Gem1/Miro in yeast and higher eukaryotes, where Miro is a Parkin substrate, with potential implications for Parkinson's disease pathogenesis.
  8. Mol Cell. 2021 Apr 09. pii: S1097-2765(21)00263-X. [Epub ahead of print]
      The mitochondrial translation system originates from a bacterial ancestor but has substantially diverged in the course of evolution. Here, we use single-particle cryo-electron microscopy (cryo-EM) as a screening tool to identify mitochondrial translation termination mechanisms and to describe them in molecular detail. We show how mitochondrial release factor 1a releases the nascent chain from the ribosome when it encounters the canonical stop codons UAA and UAG. Furthermore, we define how the peptidyl-tRNA hydrolase ICT1 acts as a rescue factor on mitoribosomes that have stalled on truncated messages to recover them for protein synthesis. Finally, we present structural models detailing the process of mitochondrial ribosome recycling to explain how a dedicated elongation factor, mitochondrial EFG2 (mtEFG2), has specialized for cooperation with the mitochondrial ribosome recycling factor to dissociate the mitoribosomal subunits at the end of the translation process.
    Keywords:  ICT1; cryo-EM; mitochondria; mtEFG2; mtRF1a; mtRRF; recycling; ribosome; termination; translation
  9. Front Genet. 2021 ;12 627050
      Gene editing of the mitochondrial genome using the CRISPR-Cas9 system is highly challenging mainly due to sub-efficient delivery of guide RNA and Cas9 enzyme complexes into the mitochondria. In this study, we were able to perform gene editing in the mitochondrial DNA by appending an NADH-ubiquinone oxidoreductase chain 4 (ND4) targeting guide RNA to an RNA transport-derived stem loop element (RP-loop) and expressing the Cas9 enzyme with a preceding mitochondrial localization sequence. We observe mitochondrial colocalization of RP-loop gRNA and a marked reduction of ND4 expression in the cells carrying a 11205G variant in their ND4 sequence coincidently decreasing the mtDNA levels. This proof-of-concept study suggests that a stem-loop element added sgRNA can be transported to the mitochondria and functionally interact with Cas9 to mediate sequence-specific mtDNA cleavage. Using this novel approach to target the mtDNA, our results provide further evidence that CRISPR-Cas9-mediated gene editing might potentially be used to treat mitochondrial-related diseases.
    Keywords:  PNPase; RP-loop; chimeric guide RNA; heteroplasmic mutations; mitochondria
  10. Hum Mol Genet. 2021 Apr 22. pii: ddab116. [Epub ahead of print]
      UBQLN2 mutations cause amyotrophic lateral sclerosis (ALS) with frontotemporal dementia (FTD), but the pathogenic mechanisms by which they cause disease remain unclear. Proteomic profiling identified 'mitochondrial proteins' as comprising the largest category of protein changes in the spinal cord (SC) of the P497S UBQLN2 mouse model of ALS/FTD. Immunoblots confirmed P497S animals have global changes in proteins predictive of a severe decline in mitochondrial health, including oxidative phosphorylation (OXPHOS), mitochondrial protein import, and network dynamics. Functional studies confirmed mitochondria purified from the SC of P497S animals have age-dependent decline in nearly all steps of OXPHOS. Mitochondria cristae deformities were evident in spinal motor neurons of aged P497S animals. Knockout (KO) of UBQLN2 in HeLa cells resulted in changes in mitochondrial proteins and OXPHOS activity similar to those seen in the SC. KO of UBQLN2 also compromised targeting and processing of the mitochondrial import factor, TIMM44, resulting in accumulation in abnormal foci. The functional OXPHOS deficits and TIMM44 targeting defects were rescued by re-expression of WT UBQLN2 but not by ALS/FTD mutant UBQLN2 proteins. In-vitro binding assays revealed ALS/FTD mutant UBQLN2 proteins bind weaker with TIMM44 than WT UBQLN2 protein, suggesting that the loss of UBQLN2 binding may underlie the import and/or delivery defect of TIMM44 to mitochondria. Our studies indicate a potential key pathogenic disturbance in mitochondrial health caused by UBQLN2 mutations.
  11. Biochim Biophys Acta Mol Basis Dis. 2021 Apr 15. pii: S0925-4439(21)00080-6. [Epub ahead of print] 166147
      The mitochondrial respiratory chain (MRC) complex III (CIII) associates with complexes I and IV (CI and CIV) into supercomplexes. We identified a novel homozygous missense mutation (c.665G>C; p.Gly222Ala) in UQCRC2 coding for structural subunit Core 2 in a patient with severe encephalomyopathy. The structural data suggest that the Gly222Ala exchange might result in an altered spatial arrangement in part of the UQCRC2 subunit, which could impact specific protein-protein interactions. Accordingly, we have found decreased levels of CIII and accumulation of CIII-specific subassemblies comprising MT-CYB, UQCRB, UQCRQ, UQCR10 and CYC1 subunits, but devoid of UQCRC1, UQCRC2, and UQCRFS1 in the patient's fibroblasts. The lack of UQCRC1 subunit-containing subassemblies could result from an impaired interaction with mutant UQCRC2Gly222Ala and subsequent degradation of both subunits by mitochondrial proteases. Indeed, we show an elevated amount of matrix CLPP protease, suggesting the activation of the mitochondrial protein quality control machinery in UQCRC2Gly222Ala fibroblasts. In line with growing evidence, we observed a rate-limiting character of CIII availability for the supercomplex formation, accompanied by a diminished amount of CI. Furthermore, we found impaired electron flux between CI and CIII in skeletal muscle and fibroblasts of the UQCRC2Gly222Ala patient. The ectopic expression of wild-type UQCRC2 in patient cells rescued maximal respiration rate, demonstrating the deleterious effect of the mutation on MRC. Our study expands the phenotypic spectrum of human disease caused by CIII Core protein deficiency, provides insight into the assembly pathway of human CIII, and supports the requirement of assembled CIII for a proper accumulation of CI.
    Keywords:  Core 2); Mitochondrial dysfunction; UQCRC2 (Core2; caseinolytic mitochondrial matrix peptidase proteolytic subunit (CLPP); mitochondrial complex III; mitochondrial protein quality control; respiratory supercomplexes
  12. Dev Cell. 2021 Apr 19. pii: S1534-5807(21)00268-9. [Epub ahead of print]56(8): 1080-1082
      During symmetric cell division, it is important that daughter cells receive not only equal genetic information, but also equal allocations of organelles. Recently, in Nature,Moore et al. (2021) identify three complementary F-actin networks that help ensure proper mixing and distribution of functionally equivalent mitochondria to daughter cells.
  13. Neurology. 2021 Apr 21. pii: 10.1212/WNL.0000000000012033. [Epub ahead of print]
      OBJECTIVE: To explore the possibilities of radioligands against the mitochondrial outer membrane protein TSPO as biomarkers for mitochondrial disease, we performed PET (PET)-MR brain imaging with [11C]PK11195 in 14 patients with genetically confirmed mitochondrial disease and 33 matched controls.METHODS: A case-control study of PET-MR imaging with the TSPO radioligand [11C]PK11195.
    RESULTS: Forty-six percent of symptomatic patients had volumes of abnormal radiotracer binding greater than the 95th percentile in controls. [11C]PK11195 binding was generally greater in grey matter and significantly decreased in white matter. This was most striking in patients with nuclear TYMP or mitochondrial m.3243A>G MT-TL1 mutations, in keeping with differences in mitochondrial density seen post mortem. Some regional binding patterns corresponded to clinical presentation and underlying mutation, even in the absence of structural changes on MRI. This was most obvious for the cerebellum, where patients with ataxia had decreased binding in the cerebellar cortex, but not necessarily volume loss. Overall, there was a positive correlation between aberrant [11C]PK11195 binding and clinical severity.
    CONCLUSION: These findings endorse the use of PET imaging with TSPO radioligands as a non-invasive in vivo biomarker of mitochondrial pathology.
    CLASSIFICATION OF EVIDENCE: This study provides Class III evidence that PET-MR brain imaging with TSPO radioligands identifies mitochondrial pathology.
  14. Stem Cell Reports. 2021 Apr 13. pii: S2213-6711(21)00161-2. [Epub ahead of print]
      Sequestosome-1 (SQSTM1/p62) is involved in cellular processes such as autophagy and metabolic reprogramming. Mutations resulting in the loss of function of SQSTM1 lead to neurodegenerative diseases including frontotemporal dementia. The pathogenic mechanism that contributes to SQSTM1-related neurodegeneration has been linked to its role as an autophagy adaptor, but this is poorly understood, and its precise role in mitochondrial function and clearance remains to be clarified. Here, we assessed the importance of SQSTM1 in human induced pluripotent stem cell (iPSC)-derived cortical neurons through the knockout of SQSTM1. We show that SQSTM1 depletion causes altered mitochondrial gene expression and functionality, as well as autophagy flux, in iPSC-derived neurons. However, SQSTM1 is not essential for mitophagy despite having a significant impact on early PINK1-dependent mitophagy processes including PINK1 recruitment and phosphorylation of ubiquitin on depolarized mitochondria. These findings suggest that SQSTM1 is important for mitochondrial function rather than clearance.
    Keywords:  FTD; SQSTM1; iPSC disease modeling; mitochondria
  15. Clin Drug Investig. 2021 Apr 22.
      Riboflavin is classified as one of the water-soluble B vitamins. It is part of the functional group of flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD) cofactors and is required for numerous flavoprotein-catalysed reactions. Riboflavin has important antioxidant properties, essential for correct cell functioning. It is required for the conversion of oxidised glutathione to the reduced form and for the mitochondrial respiratory chain as complexes I and II contain flavoprotein reductases and electron transferring flavoproteins. Riboflavin deficiency has been demonstrated to impair the oxidative state of the body, especially in relation to lipid peroxidation status, in both animal and human studies. In the nervous system, riboflavin is essential for the synthesis of myelin and its deficiency can determine the disruption of myelin lamellae. The inherited condition of restricted riboflavin absorption and utilisation, reported in about 10-15% of world population, warrants further investigation in relation to its association with the main neurodegenerative diseases. Several successful trials testing riboflavin for migraine prevention were performed, and this drug is currently classified as a Level B medication for migraine according to the American Academy of Neurology evidence-based rating, with evidence supporting its efficacy. Brown-Vialetto-Van Laere syndrome and Fazio-Londe diseases are now renamed as "riboflavin transporter deficiency" because these are autosomal recessive diseases caused by mutations of SLC52A2 and SLC52A3 genes that encode riboflavin transporters. High doses of riboflavin represent the mainstay of the therapy of these diseases and high doses of riboflavin should be rapidly started as soon as the diagnosis is suspected and continued lifelong. Remarkably, some mitochondrial diseases respond to supplementation with riboflavin. These include multiple acyl-CoA-dehydrogenase deficiency (which is caused by ETFDH gene mutations in the majority of the cases, or mutations in the ETFA and ETFB genes in a minority), mutations of ACAD9 gene, mutations of AIFM1 gene, mutations of the NDUFV1 and NDUFV2 genes. Therapeutic riboflavin administration has been tried in other neurological diseases, including stroke, multiple sclerosis, Friedreich's ataxia and Parkinson's disease. Unfortunately, the design of these clinical trials was not uniform, not allowing to accurately assess the real effects of this molecule on the disease course. In this review we analyse the properties of riboflavin and its possible effects on the pathogenesis of different neurological diseases, and we will review the current indications of this vitamin as a therapeutic intervention in neurology.
  16. J Vis Exp. 2021 Apr 04.
      Caenorhabditis elegans is widely recognized for its central utility as a translational animal model to efficiently interrogate mechanisms and therapies of diverse human diseases. Worms are particularly well-suited for high-throughput genetic and drug screens to gain deeper insight into therapeutic targets and therapies by exploiting their fast development cycle, large brood size, short lifespan, microscopic transparency, low maintenance costs, robust suite of genomic tools, mutant repositories, and experimental methodologies to interrogate both in vivo and ex vivo physiology. Worm locomotor activity represents a particularly relevant phenotype that is frequently impaired in mitochondrial disease, which is highly heterogeneous in causes and manifestations but collectively shares an impaired capacity to produce cellular energy. While a suite of different methodologies may be used to interrogate worm behavior, these vary greatly in experimental costs, complexity, and utility for genomic or drug high-throughput screens. Here, the relative throughput, advantages, and limitations of 16 different activity analysis methodologies were compared that quantify nematode locomotion, thrashing, pharyngeal pumping, and/or chemotaxis in single worms or worm populations of C. elegans at different stages, ages, and experimental durations. Detailed protocols were demonstrated for two semi-automated methods to quantify nematode locomotor activity that represent novel applications of available software tools, namely, ZebraLab (a medium-throughput approach) and WormScan (a high-throughput approach). Data from applying these methods demonstrated similar degrees of reduced animal activity occurred at the L4 larval stage, and progressed in day 1 adults, in mitochondrial complex I disease (gas-1(fc21)) mutant worms relative to wild-type (N2 Bristol) C. elegans. This data validates the utility for these novel applications of using the ZebraLab or WormScan software tools to quantify worm locomotor activity efficiently and objectively, with variable capacity to support high-throughput drug screening on worm behavior in preclinical animal models of mitochondrial disease.
  17. Nutr Neurosci. 2021 Apr 05. 1-13
      Objectives: Human nutrition plays an important role in prevention or at least slowing down the progression of age- and diet-related diseases. Thereby, mitochondrial dysfunction represents one common underlying mechanism, which is being investigated in mouse models. However, the influence of the selected diets in preclinical studies on cognition and mitochondrial function has not yet been reported cohesively.Methods: Therefore, we present the results of three different studies that addressed this question. First, we investigated the influence of two standard control chow diets and a special diet low in antioxidants over 6 months in aged NMRI mice. Additionally, a 70% high-fat (HF) chow diet as well as a western-style diet (WSD) rich in lard and fructose were examined in C57/BL6 mice. Cognitive performance, mitochondrial function and bioenergetics in the brain were investigated. Moreover, cerebral expression of genes involved in biogenesis and antioxidant defence (citrate synthase, complex I, complex IV, SOD2, Cat1, GPx-1) were quantified.Results: The results show that a modified, low antioxidant diet increased ATP levels in the brain of aged mice, while cognitive functions remained largely unaffected. A HF diet also showed significant effects on ATP levels and gene expression levels of relevant antioxidant markers, while the WSD had marginal effects on mitochondrial function and bioenergetics in the brain.Discussion: Our results indicate that standard- and special diets have an impact on cognition and mitochondrial function in the brain. Thus, appropriate caution is warranted when selecting a suitable diet for preclinical studies in mice.
    Keywords:  Mitochondria; ageing; antioxidants; cognitive function; energy metabolism; high-fat diet; nutrition; standard diets
  18. Methods Mol Biol. 2021 ;2302 1-20
      Rhomboid proteases are a ubiquitous superfamily of serine intramembrane peptidases that play a role in a wide variety of cellular processes. The mammalian mitochondrial rhomboid protease, Presenilin-Associated Rhomboid Like (PARL), is a critical regulator of mitochondrial homeostasis through the cleavage of its substrates, which have roles in mitochondrial quality control and apoptosis. However, neither structural nor functional information for this important protease is available, because the expression of eukaryotic membrane proteins to sufficient levels in an active form often represents a major bottleneck for in vitro studies. Here we present an optimized protocol for expression and purification of the human PARL protease using the eukaryotic expression host Pichia pastoris. The PARL gene construct was generated in tandem with green fluorescent protein (GFP), which allowed for the selection of high expressing clones and monitoring during the large-scale expression and purification steps. We discuss the production protocol with precise details for each step. The protocol yields 1 mg of pure PARL per liter of yeast culture.
    Keywords:  Detergent; Intramembrane serine proteases; Membrane protein expression; PARL; PINK1; Pichia pastoris; Polytopic membrane protein; Rhomboid
  19. FEBS J. 2021 Apr 18.
      The mammalian sirtuin family consists of seven proteins, three of which (SIRT3, SIRT4, and SIRT5) localise specifically within mitochondria and preserve mitochondrial function and homeostasis. Mitochondrial sirtuins are involved in diverse functions such as deacetylation, ADP-ribosylation, demalonylation and desuccinylation, thus affecting various aspect of cell fate. Intriguingly, mitochondrial sirtuins are able to manage these delicate processes with accuracy mediated by cross-talk between the nucleus and mitochondria. Previous studies have provided ample information about their substrates and targets, whereas less is known about their role in cancer and stem cells. Here, we review and discuss recent advances in our understanding of the structural and functional properties of mitochondrial sirtuins, including their targets in cancer and stem cells. These advances could help to improve the understanding of their interplay with signalling cascades and pathways, leading to new avenues for developing novel drugs for sirtuin-related disease treatments. We also highlight the complex network of mitochondrial sirtuins in cancer and stem cells, which may be important in deciphering the molecular mechanism for their activation and inhibition.
    Keywords:   SIRT3 ; SIRT4 ; SIRT5 ; Cancer; Mitochondria; Sirtuins; Stem cells
  20. Neurotoxicology. 2021 Apr 17. pii: S0161-813X(21)00035-8. [Epub ahead of print]
      Mitochondrial biogenesis relies on different protein import machinery, as mitochondrial proteins are imported from the cytosol. The mitochondrial intermembrane space assembly (MIA) pathway consists of GFER/ALR and CHCHD4/Mia40, responsible for importing proteins and their oxidative folding inside the mitochondria. The MIA pathway plays an essential role in complex IV (COX IV) biogenesis via importing copper chaperone COX17, associated with the respiratory chain. BPA, an environmental toxicant, found in consumable plastics, causes neurotoxicity via impairment in mitochondrial dynamics, neurogenesis, and cognitive functions. We studied the levels of key regulatory proteins of mitochondrial import pathways and mitochondrial biogenesis after BPA exposure in the rat hippocampus. BPA caused a significant reduction in the levels of mitochondrial biogenesis proteins (PGC1α, and TFAM) and mitochondrial import protein (GFER). Immunohistochemical analysis showed reduced co-localization of NeuN with GFER, PGC-1α, and TFAM suggesting impaired mitochondrial biogenesis and protein import. BPA exposure resulted in damaged mitochondria with distorted cristae in neurons and caused a significant reduction in GFER localization inside IMS as depicted by immunogold electron microscopy. The reduced levels of GFER resulted in defective COX17 import. The translocation of cytochrome c into the cytosol and increased cleaved caspase-3 levels triggered apoptosis due to BPA toxicity. Overall, our study implicates GFER as a potential target for impaired mitochondrial protein machinery, biogenesis, and apoptosis against BPA neurotoxicity in the rat hippocampus.
    Keywords:  Bisphenol-A; GFER; Hippocampus; Mitochondria import machinery; Mitochondrial biogenesis; PGC-1α; TFAM
  21. Nat Commun. 2021 Apr 22. 12(1): 2382
      Conventional approaches to identify secreted factors that regulate homeostasis are limited in their abilities to identify the tissues/cells of origin and destination. We established a platform to identify secreted protein trafficking between organs using an engineered biotin ligase (BirA*G3) that biotinylates, promiscuously, proteins in a subcellular compartment of one tissue. Subsequently, biotinylated proteins are affinity-enriched and identified from distal organs using quantitative mass spectrometry. Applying this approach in Drosophila, we identify 51 muscle-secreted proteins from heads and 269 fat body-secreted proteins from legs/muscles, including CG2145 (human ortholog ENDOU) that binds directly to muscles and promotes activity. In addition, in mice, we identify 291 serum proteins secreted from conditional BirA*G3 embryo stem cell-derived teratomas, including low-abundance proteins with hormonal properties. Our findings indicate that the communication network of secreted proteins is vast. This approach has broad potential across different model systems to identify cell-specific secretomes and mediators of interorgan communication in health or disease.
  22. Dev Cell. 2021 Apr 16. pii: S1534-5807(21)00308-7. [Epub ahead of print]
      The ancient, dynamic, and multifaceted functions of the mitochondrial network are essential for organismal homeostasis and contribute to numerous human diseases. As central hubs for metabolism, ion transport, and multiple macromolecular synthesis pathways, mitochondria establish and control extensive signaling networks to ensure cellular survival. In this review, we explore how these same mitochondrial functions also participate in the control of regulated cell death (RCD). We discuss the complementary essential mitochondrial functions as compartments that participate in the production and presentation of key molecules and platforms that actively enable, initiate, and execute RCD.
    Keywords:  cell biology; mitochondrial function; programed cell death; regulated cell death; signal transduction; stress signaling
  23. Sci Adv. 2021 Apr;pii: eabd8407. [Epub ahead of print]7(17):
      Critical early steps in human embryonic development include polarization of the inner cell mass, followed by formation of an expanded lumen that will become the epiblast cavity. Recently described three-dimensional (3D) human pluripotent stem cell-derived cyst (hPSC-cyst) structures can replicate these processes. To gain mechanistic insights into the poorly understood machinery involved in epiblast cavity formation, we interrogated the proteomes of apical and basolateral membrane territories in 3D human hPSC-cysts. APEX2-based proximity bioinylation, followed by quantitative mass spectrometry, revealed a variety of proteins without previous annotation to specific membrane subdomains. Functional experiments validated the requirement for several apically enriched proteins in cyst morphogenesis. In particular, we found a key role for the AP-1 clathrin adaptor complex in expanding the apical membrane domains during lumen establishment. These findings highlight the robust power of this proximity labeling approach for discovering novel regulators of epithelial morphogenesis in 3D stem cell-based models.
  24. Science. 2021 Apr 22. pii: eabd5491. [Epub ahead of print]
      The coenzyme nicotinamide adenine dinucleotide phosphate (NADP+) and its reduced form (NADPH) regulate reductive metabolism in a subcellularly compartmentalized manner. Mitochondrial NADP(H) production depends on the phosphorylation of NAD(H) by NAD kinase 2 (NADK2). Deletion of NADK2 in human cell lines did not alter mitochondrial folate pathway activity, tricarboxylic acid cycle activity, or mitochondrial oxidative stress, but led to impaired cell proliferation in minimal medium. This growth defect was rescued by proline supplementation. NADK2-mediated mitochondrial NADP(H) generation was required for the reduction of glutamate and hence proline biosynthesis. Furthermore, mitochondrial NADP(H) availability determined the production of collagen proteins by cells of mesenchymal lineage. Thus, a primary function of the mitochondrial NADP(H) pool is to support proline biosynthesis for use in cytosolic protein synthesis.
  25. Biomed Pharmacother. 2021 Apr 14. pii: S0753-3322(21)00378-4. [Epub ahead of print]139 111593
      BACKGROUND: Cerebral ischemic events, comprising of excitotoxicity, reactive oxygen production, and inflammation, adversely impact the metabolic-redox circuit in highly active neuronal metabolic profile which maintains energy-dependent brain activities. Therefore, we investigated neuro-regenerative potential of melatonin (Mel), a natural biomaterial secreted by pineal gland.METHODS: We specifically determined whether Mel could influence tunneling nanotubes (TNTs)-mediated transfer of functional mitochondria (Mito) which in turn may alter membrane potential, oxidative stress and apoptotic factors. In vitro studies assessed the effects of Mito on levels of cytochrome C, mitochondrial transfer, reactive oxygen species, membrane potential and mass, which were all further enhanced by Mel pre-treatment, whereas in vivo studies examined brain infarct area (BIA), neurological function, inflammation, brain edema and integrity of neurons and myelin sheath in control, ischemia stroke (IS), IS + Mito and IS + Mel-Mito group rats.
    RESULTS: Results showed that Mel pre-treatment significantly increased mitochondrial transfer and antioxidants, and inhibited apoptosis. Mel-pretreated Mito also significantly reduced BIA with improved neurological function. Apoptotic, oxidative-stress, autophagic, mitochondrial/DNA-damaged biomarkers indices were also improved.
    CONCLUSION: Conclusively, Mel is a potent biomaterial which could potentially impart neurogenesis through repairing impaired metabolic-redox circuit via enhanced TNT-mediated mitochondrial transfer, anti-oxidation, and anti-apoptotic activities in ischemia.
    Keywords:  Acute ischemic stroke; Apoptosis; Melatonin; Mitochondria; Oxidative stress
  26. Br J Pharmacol. 2021 Apr 05.
      Metabolism is dynamically regulated to accompany immune cell function, and altered immunometabolism can result in impaired immune responses. Concomitantly, the pharmacological manipulation of metabolic processes offers an opportunity for therapeutic intervention in inflammatory disorders. The nicotinamide adenine dinucleotide (NAD+ ) is a critical metabolic intermediate that serve as enzyme cofactor in redox reactions, which is also used as a co-substrate by multiple enzymes such as sirtuins, adenosine diphosphate ribose transferases and synthases. Through these activities, NAD+ metabolism regulates a broad spectrum of cellular functions such as energy metabolism, DNA repair, regulation of the epigenetic landscape and inflammation. Thus, the manipulation of NAD+ availability using pharmacological compounds such as NAD+ precursors can have immune-modulatory properties in inflammation. Herein, we discuss how the NAD+ metabolism contributes to the immune response and inflammatory conditions, with a special focus on multiple sclerosis, inflammatory bowel diseases and inflammageing.
  27. Brain. 2021 Apr 23. pii: awab071. [Epub ahead of print]
      Peroxiredoxin 3 (PRDX3) belongs to a superfamily of peroxidases that function as protective antioxidant enzymes. Among the six isoforms (PRDX1-PRDX6), PRDX3 is the only protein exclusively localized to the mitochondria, which are the main source of reactive oxygen species. Excessive levels of reactive oxygen species are harmful to cells, inducing mitochondrial dysfunction, DNA damage, lipid and protein oxidation and ultimately apoptosis. Neuronal cell damage induced by oxidative stress has been associated with numerous neurodegenerative disorders including Alzheimer's and Parkinson's diseases.  Leveraging the large aggregation of genomic ataxia datasets from the PREPARE (Preparing for Therapies in Autosomal Recessive Ataxias) network, we identified recessive mutations in PRDX3 as the genetic cause of cerebellar ataxia in five unrelated families, providing further evidence for oxidative stress in the pathogenesis of neurodegeneration. The clinical presentation of individuals with PRDX3 mutations consists of mild-to-moderate progressive cerebellar ataxia with concomitant hyper- and hypokinetic movement disorders, severe early-onset cerebellar atrophy, and in part olivary and brainstem degeneration. Patient fibroblasts showed a lack of PRDX3 protein, resulting in decreased glutathione peroxidase activity and decreased mitochondrial maximal respiratory capacity. Moreover, PRDX3 knockdown in cerebellar medulloblastoma cells resulted in significantly decreased cell viability, increased H2O2 levels and increased susceptibility to apoptosis triggered by reactive oxygen species. Pan-neuronal and pan-glial in vivo models of Drosophila revealed aberrant locomotor phenotypes and reduced survival times upon exposure to oxidative stress.  Our findings reveal a central role for mitochondria and the implication of oxidative stress in PRDX3 disease pathogenesis and cerebellar vulnerability and suggest targets for future therapeutic approaches.
    Keywords:  PRDX3; ROS; ataxia
  28. JPEN J Parenter Enteral Nutr. 2021 Apr 21.
      BACKGROUND: Ketogenic diet (KD) is a low carbohydrate, high fat diet used  as a non-pharmacological treatment in many neurological and metabolic disorders effectively for a long time. The effectiveness of the KD  is revealed in mitochondrial disorders mainly in the pyruvate dehydrogenase deficiency.CONCLUSION: This is the first case of effectively treated mitochondrial depletion syndrome with parenteral ketogenic diet for lactic acidosis. This article is protected by copyright. All rights reserved.
    Keywords:  enteral ketogenic diet; lactic acidosis; mitochondrial depletion syndrome; parenteral ketogenic diet
  29. iScience. 2021 Apr 23. 24(4): 102324
      Mitochondria are key organelles inside the cell that house a wide range of molecular pathways involved in energy metabolism, ions homeostasis, and cell death. Several databases characterize the different mitochondrial aspects and thus support basic and clinical research. Here we present MitopatHs, a web-based data set that allows navigating among the biochemical signaling pathways (PatHs) of human (H) mitochondria (Mito). MitopatHs is designed to visualize and comprehend virtually all types of pathways in two complementary ways: a logical view, where the sequence of biochemical reactions is presented as logical deductions, and an intuitive graphical visualization, which enables the examination and the analysis of each step of the pathway. MitopatHs is a manually curated, open access and collaborative tool, whose goal is to enable the visualization and comprehension of complicated molecular routes in an easy and fast way.
    Keywords:  Cell Biology; Molecular Network; Software
  30. Arch Biochem Biophys. 2021 Apr 14. pii: S0003-9861(21)00127-2. [Epub ahead of print] 108877
      The molecular mechanisms underlying the degeneration and neuronal death associated with Parkinson's disease (PD) are not clearly understood. Several pathways and models have been explored in an overwhelming number of studies. Overall, from these studies, mitochondrial dysfunction and nitroxidative stress have emerged as major contributors to degeneration of dopaminergic neurons in PD. In addition, an excessive or inappropriate production of nitric oxide (•NO) and an abnormal metabolism of dopamine have been independently implicated in both processes. However, the participation of •NO in reactions with dopamine relevant to neurotoxicity strongly suggests that dopamine or its metabolites may be potential targets for •NO, affecting the physiological chemistry of both, •NO and dopamine. In this short review, we provide a critical and integrative appraisal of the nitric oxide-dopamine pathway we have previously suggested and that might be operative in PD. This pathway emphasizes a connection between abnormal dopamine and •NO metabolism, which may potentially converge in an integrated mechanism with toxic cellular outcomes. In particular, it encompasses the synergistic interaction of •NO with 3,4-dihydroxyphenylacetic acid (DOPAC), a major dopamine metabolite, leading to dopaminergic cell death via mechanisms that involve mitochondrial dysfunction, gluthathione depletion and nitroxidative stress.
    Keywords:  DOPAC; Glutathione; Mitochondrial dysfunction; Nitric oxide; Nitroxidative stress; Parkinson's disease
  31. Autophagy. 2021 Apr 18. 1-4
      Whether macroautophagy/autophagy is physiologically relevant to regulate mitochondrial function for a rapid and dynamic adaptation of yeast cells to respiratory growth was not fully understood until recently. May et al. (2020. Nat Commun) report that bulk autophagy provides serine as a one-carbon (1C) metabolite that controls respiratory growth onset by initiating mitochondrial initiator tRNAMet modification and mitochondrial translation linking autophagy mechanistically to mitochondrial function. We discuss the mechanistic interplay between autophagy, one-carbon-metabolism, and mitochondrial function and the possible implications in neurodegeneration, aging, and carcinogenesis.
    Keywords:  Autophagy; mitochondrial respiration; mitochondrial translation; one-carbon metabolism; respiratory growth
  32. Cell. 2021 Apr 13. pii: S0092-8674(21)00381-0. [Epub ahead of print]
      Long non-coding RNA (lncRNA) genes have well-established and important impacts on molecular and cellular functions. However, among the thousands of lncRNA genes, it is still a major challenge to identify the subset with disease or trait relevance. To systematically characterize these lncRNA genes, we used Genotype Tissue Expression (GTEx) project v8 genetic and multi-tissue transcriptomic data to profile the expression, genetic regulation, cellular contexts, and trait associations of 14,100 lncRNA genes across 49 tissues for 101 distinct complex genetic traits. Using these approaches, we identified 1,432 lncRNA gene-trait associations, 800 of which were not explained by stronger effects of neighboring protein-coding genes. This included associations between lncRNA quantitative trait loci and inflammatory bowel disease, type 1 and type 2 diabetes, and coronary artery disease, as well as rare variant associations to body mass index.
    Keywords:  GTEx; co-expression; colocalization; complex trait; disease; eQTL; expression quantitative trait loci; lncRNA; long non-coding RNA
  33. Cell Rep. 2021 Apr 20. pii: S2211-1247(21)00316-8. [Epub ahead of print]35(3): 109002
      Complex I (CI) is the largest enzyme of the mitochondrial respiratory chain, and its defects are the main cause of mitochondrial disease. To understand the mechanisms regulating the extremely intricate biogenesis of this fundamental bioenergetic machine, we analyze the structural and functional consequences of the ablation of NDUFS3, a non-catalytic core subunit. We show that, in diverse mammalian cell types, a small amount of functional CI can still be detected in the complete absence of NDUFS3. In addition, we determine the dynamics of CI disassembly when the amount of NDUFS3 is gradually decreased. The process of degradation of the complex occurs in a hierarchical and modular fashion in which the ND4 module remains stable and bound to TMEM126A. We, thus, uncover the function of TMEM126A, the product of a disease gene causing recessive optic atrophy as a factor necessary for the correct assembly and function of CI.
    Keywords:  CI; CI modules; NDUFS3; SILAC; TMEM126A; assembly factor; optic atrophy type 7; respiratory complex I
  34. Life Sci. 2021 Apr 14. pii: S0024-3205(21)00496-3. [Epub ahead of print] 119511
      Effective Ca2+ dependent mitochondrial energy supply is imperative for proper cardiac contractile activity, while disruption of Ca2+ homeostasis participates in the pathogenesis of multiple human diseases. This phenomenon is particularly prominent in cardiac ischemia and reperfusion (I/R) and heart failure, both of which require strict clinical intervention. The interface between endoplasmic reticula (ER) and mitochondria, designated the mitochondria-associated membrane (MAM), is now regarded as a crucial mediator of Ca2+ transportation. Thus, interventions targeting this physical and functional coupling between mitochondria and the ER are highly desirable. Increasing evidence supports the notion that restoration, and maintenance, of the physiological contact between these two organelles can improve mitochondrial function, while inhibiting cell death, thereby sufficiently ameliorating I/R injury and heart failure development. A better understanding regarding the underlying mechanism of MAM-mediated transport will pave the way for identification of novel treatment approaches for heart disease. Therefore, in this review, we summarize the crucial functions and potential mechanisms of MAMs in the pathogenesis of I/R and heart failure.
    Keywords:  Calcium transfer; Heart failure; Mitochondria; Mitochondria-associated membranes; Myocardial ischemia-reperfusion injury
  35. J Biol Chem. 2021 Apr 14. pii: S0021-9258(21)00458-0. [Epub ahead of print] 100669
      The mitochondrial calcium uniporter (MCU) and cyclophilin D (CyD) are key players in induction of the permeability transition pore (PTP), which leads to mitochondrial depolarization and swelling, the major sings of Ca2+-induced mitochondrial damage. Mitochondrial depolarization inhibits ATP production, whereas swelling results in the release of mitochondrial pro-apoptotic proteins. The extent to which simultaneous deletion of MCU and CyD inhibits PTP induction and prevents damage of brain mitochondria is not clear. Here, we investigated the effects of MCU and CyD deletion on the propensity for PTP induction using mitochondria isolated from the brains of MCU-KO, CyD-KO, and newly created MCU/CyD-double knockout (DKO) mice. Neither deletion of MCU nor of CyD affected respiration or membrane potential in mitochondria isolated from the brains of these mice. Mitochondria from MCU-KO and MCU/CyD-DKO mice displayed reduced Ca2+ uptake and diminished extent of PTP induction. The Ca2+ uptake by mitochondria from CyD-KO mice was increased compared to mitochondria from wild-type mice. Deletion of CyD prevented mitochondrial swelling and resulted in transient depolarization in response to Ca2+, but it did not prevent Ca2+-induced delayed mitochondrial depolarization. Mitochondria from MCU/CyD-DKO mice did not swell in response to Ca2+, but they did exhibit mild sustained depolarization. Dibucaine, an inhibitor of the Ca2+-activated mitochondrial phospholipase A2, attenuated, and bovine serum albumin completely eliminated the sustained depolarization. This suggests the involvement of phospholipase A2 and free fatty acids. Thus, in addition to induction of the classical PTP, alternative deleterious mechanisms may contribute to mitochondrial damage following exposure to elevated Ca2+.
    Keywords:  calcium; cyclophilin D; membrane potential; mitochondria; mitochondrial calcium uniporter; permeability transition pore; respiration
  36. BMC Bioinformatics. 2021 Apr 20. 22(1): 201
      BACKGROUND: A major challenge in evaluating quantitative ChIP-seq analyses, such as peak calling and differential binding, is a lack of reliable ground truth data. Accurate simulation of ChIP-seq data can mitigate this challenge, but existing frameworks are either too cumbersome to apply genome-wide or unable to model a number of important experimental conditions in ChIP-seq.RESULTS: We present ChIPs, a toolkit for rapidly simulating ChIP-seq data using statistical models of key experimental steps. We demonstrate how ChIPs can be used for a range of applications, including benchmarking analysis tools and evaluating the impact of various experimental parameters. ChIPs is implemented as a standalone command-line program written in C++ and is available from .
    CONCLUSIONS: ChIPs is an efficient ChIP-seq simulation framework that generates realistic datasets over a flexible range of experimental conditions. It can serve as an important component in various ChIP-seq analyses where ground truth data are needed.
    Keywords:  Bioinformatics; ChIP-sequencing; Command-line program; Epigenomics; Simulation tool
  37. Biofactors. 2021 Apr 20.
      Coenzyme Q10 (CoQ10 ) deficiency is a rare disease characterized by a decreased accumulation of CoQ10 in cell membranes. Considering that CoQ10 synthesis and most of its functions are carried out in mitochondria, CoQ10 deficiency cases are usually considered a mitochondrial disease. A relevant feature of CoQ10 deficiency is that it is the only mitochondrial disease with a successful therapy available, the CoQ10 supplementation. Defects in components of the synthesis machinery caused by mutations in COQ genes generate the primary deficiency of CoQ10 . Mutations in genes that are not directly related to the synthesis machinery cause secondary deficiency. Cases of CoQ10 deficiency without genetic origin are also considered a secondary deficiency. Both types of deficiency can lead to similar clinical manifestations, but the knowledge about primary deficiency is deeper than secondary. However, secondary deficiency cases may be underestimated since many of their clinical manifestations are shared with other pathologies. This review shows the current state of secondary CoQ10 deficiency, which could be even more relevant than primary deficiency for clinical activity. The analysis covers the fundamental features of CoQ10 deficiency, which are necessary to understand the biological and clinical differences between primary and secondary CoQ10 deficiencies. Further, a more in-depth analysis of CoQ10 secondary deficiency was undertaken to consider its origins, introduce a new way of classification, and include aging as a form of secondary deficiency.
    Keywords:  CoQ10 deficiency; aging; coenzyme CoQ10; mitochondrial dysfunction; rare diseases
  38. Mol Genet Metab Rep. 2021 Apr 14. 100756
      The novel SARS-CoV-2 has infected over 48 million persons around the world. Children have been spared with regards to symptoms and sequelae of this highly infectious virus and in those with neurologic issues, the virus has not been present in the cerebrospinal fluid. Here, the authors present the first case of metabolic stroke-like episode with SARS-CoV-2 present in the cerebrospinal fluid in a child with a FARS2 deficiency. This report suggests a possible association of SARS-COV-2 infection and metabolic stroke-like episode, even in the absence of a phenotype classically associated with metabolic stroke-like episodes.
    Keywords:  Cerebrospinal fluid; FARS2; Mitochondrial; PCR, (polymerase chain reaction); Pediatric; SARS-CoV-2; Stroke
  39. Cell Rep. 2021 Apr 20. pii: S2211-1247(21)00332-6. [Epub ahead of print]35(3): 109018
      Physical exercise has profound effects on quality of life and susceptibility to chronic disease; however, the regulation of skeletal muscle function at the molecular level after exercise remains unclear. We tested the hypothesis that the benefits of exercise on muscle function are linked partly to microtraumatic events that result in accumulation of circulating heme. Effective metabolism of heme is controlled by Heme Oxygenase-1 (HO-1, Hmox1), and we find that mouse skeletal muscle-specific HO-1 deletion (Tam-Cre-HSA-Hmox1fl/fl) shifts the proportion of muscle fibers from type IIA to type IIB concomitant with a disruption in mitochondrial content and function. In addition to a significant impairment in running performance and response to exercise training, Tam-Cre-HSA-Hmox1fl/fl mice show remarkable muscle atrophy compared to Hmox1fl/fl controls. Collectively, these data define a role for heme and HO-1 as central regulators in the physiologic response of skeletal muscle to exercise.
    Keywords:  DAMP; exercise training; heme; heme oxygenase-1; hemopexin; mitochondrial dysfunction; muscle atrophy; muscle microtrauma; satellite cells
  40. Curr Neuropharmacol. 2021 Apr 20.
      Uncoupling protein 2 (UCP2) is a mitochondrial protein that acts as an anion carrier. It is involved in the regulation of several processes including mitochondrial membrane potential, generation of reactive oxygen species within the inner mitochondrial membrane and calcium homeostasis. UCP2 expression can be regulated at different levels: genetic (gene variants), transcriptional [by peroxisome proliferator-activated receptors (PPARs) and microRNAs], and post-translational. Experimental evidence indicates that activation of UCP2 expression through the AMPK/PPAR-α axis exerts a protective effect toward renal damage and stroke occurrence in an animal model of ischemic stroke (IS) associated with hypertension. UCP2 plays a key role in heart diseases (myocardial infarction and cardiac hypertrophy) and metabolic disorders (obesity and diabetes). In humans, UCP2 genetic variants (-866G/A and Ala55Val) associate with an increased risk of type 2 diabetes mellitus and of IS development. Over the last few years, many agents that modulate UCP2 expression have been identified. Some of them are natural compounds of plant origin such as Brassica oleracea, curcumin, berberine and resveratrol. Other molecules, currently used in clinical practice, include anti-diabetic (gliptin) and chemotherapeutic (doxorubicin and taxol) drugs. This evidence highlights the relevant role of UCP2 for the treatment of a wide range of diseases, which affect the national health systems of the Western countries. We will review current knowledge on the physiological and pathological implications of UCP2 with particular regard to cardiovascular and metabolic disorders and will focus on the available therapeutic approaches affecting UCP2 level for the treatment of human diseases.
    Keywords:  UCP2; cardiovascular disease; diabetes; obesity; stroke; therapeutics
  41. J Neurosci. 2021 Apr 23. pii: JN-RM-2770-20. [Epub ahead of print]
      Parkinson's disease (PD) is characterized by progressive dopamine (DA) neuron loss in the substantia nigra pars compacta (SNc). In contrast, DA neurons in the ventral tegmental area (VTA) are relatively protected from neurodegeneration, but the underlying mechanisms for this resilience remain poorly understood. Recent work suggests that expression of the vesicular glutamate transporter 2 (VGLUT2) selectively impacts midbrain DA neuron vulnerability. We investigated whether altered DA neuron VGLUT2 expression determines neuronal resilience in rats exposed to rotenone, a mitochondrial complex I inhibitor and toxicant model of PD. We discovered that VTA/SNc DA neurons that expressed VGLUT2 are more resilient to rotenone-induced DA neurodegeneration. Surprisingly, the density of neurons with detectable VGLUT2 expression in the VTA and SNc increases in response to rotenone. Furthermore, dopaminergic terminals within the nucleus accumbens, where the majority of VGLUT2-expressing DA neurons project, exhibit greater resilience compared to DA terminals in the caudate/putamen. More broadly, VGLUT2-expressing terminals are protected throughout the striatum from rotenone-induced degeneration. Together, our data demonstrate that a distinct subpopulation of VGLUT2-expressing DA neurons are relatively protected from rotenone neurotoxicity. Rotenone-induced upregulation of the glutamatergic machinery in VTA and SNc neurons and their projections may be part of a broader neuroprotective mechanism. These findings offer a putative new target for neuronal resilience that can be manipulated to prevent toxicant-induced DA neurodegeneration in PD.SIGNIFICANCE STATEMENT:Environmental exposures to pesticides contribute significantly to pathological processes that culminate in Parkinson's disease (PD). The pesticide rotenone has been used to generate a PD model that replicates key features of the illness including dopamine neurodegeneration. To date, longstanding questions remain: are there dopamine neuron subpopulations resilient to rotenone, and if so, what are the molecular determinants of this resilience? Here we show that the subpopulation of midbrain dopaminergic neurons that express the vesicular glutamate transporter 2 (VGLUT2) are more resilient to rotenone-induced neurodegeneration. Rotenone also upregulates VGLUT2 more broadly in the midbrain, suggesting VGLUT2 expression generally confers increased resilience to rotenone. VGLUT2 may therefore be a new target for boosting neuronal resilience to prevent toxicant-induced DA neurodegeneration in PD.
  42. JCI Insight. 2021 Apr 20. pii: 146728. [Epub ahead of print]
      Altered mitochondria activity in osteoblasts and osteoclast has been implicated in the loss of bone mass associated with aging and estrogen deficiency - the two most common causes of osteoporosis. However, the mechanisms that control mitochondrial metabolism in bone cells during health or disease remain unknown. The mitochondrial deacetylase Sirtuin-3 (Sirt3) has been earlier implicated in age-related diseases. Here, we show that deletion of Sirt3 had no effect on the skeleton of young mice but attenuated the age-related loss of bone mass in both sexes. This effect was associated with impaired bone resorption. Osteoclast progenitors from aged Sirt3 null mice were able to differentiate into osteoclasts. Albeit, the differentiated cells exhibited impaired polykaryon formation and resorptive activity as well as decreased oxidative phosphorylation and mitophagy. The Sirt3 inhibitor LC-0296 recapitulated the effects of Sirt3 deletion in osteoclast formation and mitochondrial function, and its administration to aging mice increased bone mass. Deletion of Sirt3 also attenuated the increase in bone resorption and loss of bone mass caused by estrogen deficiency. These findings suggest that Sirt3 inhibition and the resulting impairment of osteoclast mitochondrial function could be a novel therapeutic intervention for the two most important causes of osteoporosis.
    Keywords:  Bone Biology; Mitochondria; Osteoclast/osteoblast biology; Osteoporosis
  43. Elife. 2021 Apr 20. pii: e62952. [Epub ahead of print]10
      Aging is associated with distinct phenotypical, physiological, and functional changes, leading to disease and death. The progression of aging-related traits varies widely among individuals, influenced by their environment, lifestyle, and genetics. In this study, we conducted physiologic and functional tests cross-sectionally throughout the entire lifespan of male C57BL/6N mice. In parallel, metabolomics analyses in serum, brain, liver, heart, and skeletal muscle were also performed to identify signatures associated with frailty and age-dependent functional decline. Our findings indicate that declines in gait speed as a function of age and frailty are associated with a dramatic increase in the energetic cost of physical activity and decreases in working capacity. Aging and functional decline prompt organs to rewire their metabolism and substrate selection and towards redox-related pathways, mainly in liver and heart. Collectively, the data provide a framework to further understand and characterize processes of aging at the individual organism and organ levels.
    Keywords:  computational biology; mouse; systems biology
  44. Genomics. 2021 Apr 17. pii: S0888-7543(21)00153-1. [Epub ahead of print]
      Chromatin immunoprecipitation followed by sequencing (ChIP-seq) is the primary protocol for detecting genome-wide DNA-protein interactions, and therefore a key tool for understanding transcriptional regulation. A number of factors, including low specificity of antibody and cellular heterogeneity of sample, may cause "peak" callers to output noise and experimental artefacts. Statistically combining multiple experimental replicates from the same condition could significantly enhance our ability to distinguish actual transcription factor binding events, even when peak caller accuracy and consistency of detection are compromised. We adapted the rank-product test to statistically evaluate the reproducibility from any number of ChIP-seq experimental replicates. We demonstrate over a number of benchmarks that our adaptation "ChIP-R" (pronounced 'chipper') performs as well as or better than comparable approaches on recovering transcription factor binding sites in ChIP-seq peak data. We also show ChIP-R extends to evaluate ATAC-seq peaks, finding reproducible peak sets even at low sequencing depth. ChIP-R decomposes peaks across replicates into "fragments" which either form part of a peak in a replicate, or not. We show that by re-analysing existing data sets, ChIP-R reconstructs reproducible peaks from fragments with enhanced biological enrichment relative to current strategies.
    Keywords:  ATAC-seq; ChIP-seq; Reproducibility; Transcription factor; Transcriptional regulation
  45. Proc Natl Acad Sci U S A. 2021 Apr 27. pii: e2018538118. [Epub ahead of print]118(17):
      Secondary infections typically worsen outcomes of patients recovering from septic shock. Neutrophil [polymorphonuclear leukocytes (PMNs)] migration to secondarily inoculated sites may play a key role in inhibiting progression from local bacterial inoculation to secondary infection. Mitochondrial N-formyl peptide (mtFP) occupancy of formyl peptide receptor-1 (FPR1) has been shown to suppress PMN chemotaxis. Therefore, we studied the association between circulating mtFPs and the development of secondary infection in patients with septic shock. We collected clinical data and plasma samples from patients with septic shock admitted to the intensive care unit for longer than 72 h. Impacts of circulating nicotinamide adenine dinucleotide dehydrogenase subunit-6 (ND6) upon clinical outcomes were analyzed. Next, the role of ND6 in PMN chemotaxis was investigated using isolated human PMNs. Studying plasma samples from 97 patients with septic shock, we found that circulating ND6 levels at admission were independently and highly associated with the development of secondary infection (odds ratio = 30.317, 95% CI: 2.904 to 316.407, P = 0.004) and increased 90-d mortality (odds ratio = 1.572, 95% CI: 1.002 to 2.465, P = 0.049). In ex vivo experiments, ND6 pretreatment suppressed FPR1-mediated PMN chemotactic responses to bacterial peptides in the presence of multiple cytokines and chemokines, despite increased nondirectional PMN movements. Circulating mtFPs appear to contribute to the development of secondary infection and increased mortality in patients with septic shock who survive their early hyperinflammatory phase. The increased susceptibility to secondary infection is probably partly mediated by the suppression of FPR1-mediated PMN chemotaxis to secondary infected sites.
    Keywords:  formyl peptide; infections; neutrophils; nosocomial; sepsis
  46. Am J Ophthalmol Case Rep. 2021 Jun;22 101073
      Purpose: To describe two patients with bilateral ptosis, ophthalmoplegia, cataracts and corneal endothelial disease requiring corneal transplantation.Observations: Histopathological analysis of muscle biopsy samples from both patients identified features consistent with a mitochondrial cytopathy. A single multigenic mitochondrial deoxyribonucleic acid (DNA) deletion was detected in the first patient. Pathogenic mutations in the POLG gene which codes for mitochondrial DNA polymerase, tasked with replicating the mitochondrial genome were identified in the second patient.
    Conclusion: The collection of clinical features present in both cases described can be explained by a diagnosis of mitochondrial disease.
    Importance: Corneal endothelial disease, in addition to ptosis, ophthalmoplegia, cataract, pigmentary retinopathy and optic atrophy should be recognised as a feature of mitochondrial disease.
    Keywords:  ATP, Adenosine triphosphate; CHED, Congenital hereditary endothelial dystrophy; COX, Cytochrome oxidase; CPEO, Chronic progressive external ophthalmoplegia; Corneal endothelial disease; DNA, Deoxyribonucleic acid; DSAEK, Descemet's stripping automated endothelial keratoplasty; FECD, Fuchs endothelial corneal dystrophy; LF, Levator palpebrae superioris function; MELAS syndrome, Mitochondrial myopathy, encephalopathy, lactic acidosis and stroke; MRD1, Margin reflex 1 distance; MT-ATP6, Mitochondrially encoded adenosine triphosphate synthase membrane subunit 6; MT-TP, Mitochondrially encoded transfer ribonucleic acid proline; Mitochondrial disease; Ophthalmoplegia; Ptosis; RNA, Ribonucleic acid; SDH, Succinic dehydrogenase; TRNA, Transfer ribonucleic acid
  47. Trends Genet. 2021 Apr 20. pii: S0168-9525(21)00079-2. [Epub ahead of print]
      Like protein-coding genes, long noncoding RNA (lncRNA) genes are composed of introns and exons. After their transcription, lncRNAs are subject to constitutive and/or alternative splicing. Here, we describe the current knowledge on lncRNA splice variants and their functional implications in cell biology.
    Keywords:  alternative splicing; long noncoding RNA; splice variant
  48. iScience. 2021 Apr 23. 24(4): 102357
      Microglia play a key role in brain development, normal homeostasis, and neurodegenerative disorders. Single-cell technologies have led to important findings about microglia, with many animal model studies using single-cell RNA sequencing (scRNA-seq), whereas most human specimen studies using archived frozen brains for single-nucleus RNA sequencing (snRNA-seq). However, microglia compose a small proportion of the total brain tissue; snRNAseq depletes expression of microglia activation genes that characterize many diseases. Here we examine the use of purified, cryopreserved microglia for scRNA-seq. Comparison of scRNA-seq on paired fresh and cryopreserved microglia from rhesus monkeys revealed a high level of correlation of gene expression between the two conditions. Disease-related genes were relatively unaffected, but an increase in immediate-early gene expression was present in cryopreserved cells. Regardless, changes in immediate-early gene expression are still detectable. Cryopreservation of microglia is a suitable procedure for prospectively archiving samples.
    Keywords:  Cellular Neuroscience; Molecular Neuroscience; Transcriptomics
  49. Nat Commun. 2021 Apr 23. 12(1): 2437
      CRISPR-Cas9 cytidine and adenosine base editors (CBEs and ABEs) can disrupt genes without introducing double-stranded breaks by inactivating splice sites (BE-splice) or by introducing premature stop (pmSTOP) codons. However, no in-depth comparison of these methods or a modular tool for designing BE-splice sgRNAs exists. To address these needs, we develop SpliceR ( ) to design and rank BE-splice sgRNAs for any Ensembl annotated genome, and compared disruption approaches in T cells using a screen against the TCR-CD3 MHC Class I immune synapse. Among the targeted genes, we find that targeting splice-donors is the most reliable disruption method, followed by targeting splice-acceptors, and introducing pmSTOPs. Further, the CBE BE4 is more effective for disruption than the ABE ABE7.10, however this disparity is eliminated by employing ABE8e. Collectively, we demonstrate a robust method for gene disruption, accompanied by a modular design tool that is of use to basic and translational researchers alike.
  50. Cell Discov. 2021 Apr 19. 7(1): 25
      High-throughput sequencing reveals the complex landscape of small noncoding RNAs (sRNAs). However, it is limited by requiring 5'-monophosphate and 3'-hydroxyl in RNAs for adapter ligation and hindered by methylated nucleosides that interfere with reverse transcription. Here we develop Cap-Clip acid pyrophosphatase (Cap-Clip), T4 polynucleotide kinase (PNK) and AlkB/AlkB(D135S)-facilitated small ncRNA sequencing (CPA-seq) to detect and quantify sRNAs with terminus multiplicities and nucleoside methylations. CPA-seq identified a large number of previously undetected sRNAs. Comparison of sRNAs with or without AlkB/AlkB(D135S) treatment reveals nucleoside methylations on sRNAs. Using CPA-seq, we profiled the sRNA transcriptomes (sRNomes) of nine mouse tissues and reported the extensive tissue-specific differences of sRNAs. We also observed the transition of sRNomes during hepatic reprogramming. Knockdown of mesenchymal stem cell-enriched U1-5' snsRNA promoted hepatic reprogramming. CPA-seq is a powerful tool with high sensitivity and specificity for profiling sRNAs with methylated nucleosides and diverse termini.
  51. Crit Rev Clin Lab Sci. 2021 Apr 21. 1-31
      Vitamin B12 (cobalamin) is an essential cofactor for two metabolic pathways. It is obtained principally from food of animal origin. Cobalamin becomes bioavailable through a series of steps pertaining to its release from dietary protein, intrinsic factor-mediated absorption, haptocorrin or transcobalamin-mediated transport, cellular uptake, and two enzymatic conversions (via methionine synthase and methylmalonyl-CoA-mutase) into cofactor forms: methylcobalamin and adenosylcobalamin. Vitamin B12 deficiency can masquerade as a multitude of illnesses, presenting different perspectives from the point of view of the hematologist, neurologist, gastroenterologist, general physician, or dietician. Increased physician vigilance and heightened patient awareness often account for its early presentation, and testing sometimes occurs during a phase of vitamin B12 insufficiency before the main onset of the disease. The chosen test often depends on its availability rather than on the diagnostic performance and sensitivity to irrelevant factors interfering with vitamin B12 markers. Although serum B12 is still the most commonly used and widely available test, diagnostics by holotranscobalamin, serum methylmalonic acid, and plasma homocysteine measurements have grown in the last several years in routine practice. The lack of a robust absorption test, coupled with compromised sensitivity and specificity of other tests (intrinsic factor and gastric parietal cell antibodies), hinders determination of the cause for depleted B12 status. This can lead to incorrect supplementation regimes and uncertainty regarding later treatment. This review discusses currently available knowledge on vitamin B12, informs the reader about the pitfalls of tests for assessing its deficiency, reviews B12 status in various populations at different disease stages, and provides recommendations for interpretation, treatment, and associated risks. Future directions for diagnostics of B12 status and health interventions are also discussed.
    Keywords:  Vitamin B12; cobalamin holotranscobalamin methylmalonic acid homocysteine
  52. Neurosciences (Riyadh). 2021 Apr;26(2): 128-133
      The m.3243A >G mutation in the tRNA Leu (UUR) gene (MT-TL1) of the mitochondrial DNA is the most widely seen pathogenic mtDNA mutation which has major phenotypic variations. The clinical phenotype involves various organs such as the brain and nerves, skeletal muscles, heart, endocrine system, gastrointestinal tract, and skin. Some phenotypes conform to well established syndromes, while most of the symptoms appear individually or concomitant to other syndromes, making identification difficult. Furthermore, some progress has been made on cardiac manifestations as well as complications during pregnancy and perinatal period. This article provides a systematic review of the non-syndromic phenotypes and latest developments in m.3243A>G mutation.
  53. Nat Commun. 2021 Apr 23. 12(1): 2403
      The ability to design functional sequences and predict effects of variation is central to protein engineering and biotherapeutics. State-of-art computational methods rely on models that leverage evolutionary information but are inadequate for important applications where multiple sequence alignments are not robust. Such applications include the prediction of variant effects of indels, disordered proteins, and the design of proteins such as antibodies due to the highly variable complementarity determining regions. We introduce a deep generative model adapted from natural language processing for prediction and design of diverse functional sequences without the need for alignments. The model performs state-of-art prediction of missense and indel effects and we successfully design and test a diverse 105-nanobody library that shows better expression than a 1000-fold larger synthetic library. Our results demonstrate the power of the alignment-free autoregressive model in generalizing to regions of sequence space traditionally considered beyond the reach of prediction and design.
  54. Brief Bioinform. 2021 Apr 19. pii: bbab123. [Epub ahead of print]
      Although synonymous mutations do not alter the encoded amino acids, they may impact protein function by interfering with the regulation of RNA splicing or altering transcript splicing. New progress on next-generation sequencing technologies has put the exploration of synonymous mutations at the forefront of precision medicine. Several approaches have been proposed for predicting the deleterious synonymous mutations specifically, but their performance is limited by imbalance of the positive and negative samples. In this study, we firstly expanded the number of samples greatly from various data sources and compared six undersampling strategies to solve the problem of the imbalanced datasets. The results suggested that cluster centroid is the most effective scheme. Secondly, we presented a computational model, undersampling scheme based method for deleterious synonymous mutation (usDSM) prediction, using 14-dimensional biology features and random forest classifier to detect the deleterious synonymous mutation. The results on the test datasets indicated that the proposed usDSM model can attain superior performance in comparison with other state-of-the-art machine learning methods. Lastly, we found that the deep learning model did not play a substantial role in deleterious synonymous mutation prediction through a lot of experiments, although it achieves superior results in other fields. In conclusion, we hope our work will contribute to the future development of computational methods for a more accurate prediction of the deleterious effect of human synonymous mutation. The web server of usDSM is freely accessible at
    Keywords:  deep learning; deleterious synonymous mutation; machine learning; undersampling scheme
  55. Drug Discov Today. 2021 Apr 20. pii: S1359-6446(21)00203-8. [Epub ahead of print]
      Pharmacogenomics (PGx) has essential roles in identifying optimal drug responders, optimizing dosage regimens and avoiding adverse events. Population-specific therapeutic interventions that tackle the genetic root causes of clinical outcomes are an important precision medicine strategy. In this perspective, we discuss next-generation sequencing genotyping and its significance for population-specific PGx applications. We emphasize the potential of NGS for preemptive pharmacogenotyping, which is crucial to population-specific clinical studies and patient care. We also provide examples that use publicly available population-based genomics data for population-specific PGx studies. Last, we discuss the remaining challenges and regulatory efforts towards improvements in this field.
    Keywords:  Biomarkers; Next-generation sequencing; Pharmacogenomics; population group
  56. Neurosci Lett. 2021 Apr 15. pii: S0304-3940(21)00277-9. [Epub ahead of print]754 135899
      The volitional movement of skeletal is controlled by the motor neuron at the site of neuromuscular junction (NMJ) where the retrograde signals are also passed back from muscle to the motor neuron. As the normal function of muscle largely depends on mitochondria that determine the fate of a skeletal muscle myofiber, there must exist a fine-controlled functional coupling between NMJ and mitochondria in myofibers. This mini-review discusses recent publications that reveal how spatiotemporal profiles of intracellular free Ca2+ could couple mitochondrial function with the activity of NMJ in skeletal muscle myofibers.
    Keywords:  Ca(2+) signaling; Mitochondria; Neuromuscular junction; Skeletal muscle
  57. Biophys J. 2021 Apr 19. pii: S0006-3495(21)00283-6. [Epub ahead of print]
      Parkinson's disease-associated LRRK2 is a multi-domain Ser/Thr kinase that phosphorylates a subset of Rab GTPases to control their effector functions. Rab GTPases are the prime regulators of membrane trafficking in eukaryotic cells. Rabs exert their biological effects by recruitment of effector proteins to subcellular compartments via their Rab-binding domain (RBD). Effectors are modular and typically contain additional domains that regulate various aspects of vesicle formation, trafficking, fusion and organelle dynamics. The RBD of effectors is typically an α-helical coiled coil that recognizes the GTP conformation of the switch 1 and switch 2 motifs of Rabs. LRRK2 phosphorylates Rab8a at T72 (pT72) of its switch 2 α-helix. This post-translational modification enables recruitment of RILPL2, an effector that regulates ciliogenesis in model cell lines. A newly identified RBD motif of RILPL2, termed the X-cap, has been shown to recognize the phosphate via direct interactions between an arginine residue (R132) and pT72 of Rab8a. Here we show that a second 'distal' arginine (R130) is also essential for phospho-Rab binding by RILPL2. Through structural, biophysical and cellular studies, we find that R130 stabilizes the primary R132:pT72 salt bridge through favorable enthalpic contributions to the binding affinity. These findings may have implications for the mechanism by which LRRK2 activation leads to assembly of phospho-Rab complexes and subsequent control of their membrane trafficking functions in cells.
    Keywords:  Leucine-rich repeat kinase 2; RILP-like protein 2; Rab8a GTPase; X-ray crystallography; effector; effector recruitment; isothermal titration calorimetry; membrane trafficking; thermodynamics
  58. Cell Death Dis. 2021 Apr 20. 12(5): 413
      Retinal degeneration diseases (RDDs) are common and devastating eye diseases characterized by the degeneration of photoreceptors, which are highly associated with oxidative stress. Previous studies reported that mitochondrial dysfunction is associated with various neurodegenerative diseases. However, the role of mitochondrial proteostasis mainly regulated by mitophagy and mitochondrial unfolded protein response (mtUPR) in RDDs is unclear. We hypothesized that the mitochondrial proteostasis is neuroprotective against oxidative injury in RDDs. In this study, the data from our hydrogen peroxide (H2O2)-treated mouse retinal cone cell line (661w) model of RDDs showed that nicotinamide riboside (NR)-activated mitophagy increased the expression of LC3B II and PINK1, and promoted the co-localization of LC3 and mitochondria, as well as PINK1 and Parkin in the H2O2-treated 661w cells. However, the NR-induced mitophagy was remarkably reversed by chloroquine (CQ) and cyclosporine A (CsA), mitophagic inhibitors. In addition, doxycycline (DOX), an inducer of mtUPR, up-regulated the expression of HSP60 and CHOP, the key proteins of mtUPR. Activation of both mitophagy and mtUPR increased the cell viability and reduced the level of apoptosis and oxidative damage in the H2O2-treated 661w cells. Furthermore, both mitophagy and mtUPR played a protective effect on mitochondria by increasing mitochondrial membrane potential and maintaining mitochondrial mass. By contrast, the inhibition of mitophagy by CQ or CsA reversed the beneficial effect of mitophagy in the H2O2-treated 661w cells. Together, our study suggests that the mitophagy and mtUPR pathways may serve as new therapeutic targets to delay the progression of RDDs through enhancing mitochondrial proteostasis.
  59. Cell Rep. 2021 Apr 20. pii: S2211-1247(21)00339-9. [Epub ahead of print]35(3): 109025
      Ablation of Slc22a14 causes male infertility in mice, but the underlying mechanisms remain unknown. Here, we show that SLC22A14 is a riboflavin transporter localized at the inner mitochondrial membrane of the spermatozoa mid-piece and show by genetic, biochemical, multi-omic, and nutritional evidence that riboflavin transport deficiency suppresses the oxidative phosphorylation and reprograms spermatozoa energy metabolism by disrupting flavoenzyme functions. Specifically, we find that fatty acid β-oxidation (FAO) is defective with significantly reduced levels of acyl-carnitines and metabolites from the TCA cycle (the citric acid cycle) but accumulated triglycerides and free fatty acids in Slc22a14 knockout spermatozoa. We demonstrate that Slc22a14-mediated FAO is essential for spermatozoa energy generation and motility. Furthermore, sperm from wild-type mice treated with a riboflavin-deficient diet mimics those in Slc22a14 knockout mice, confirming that an altered riboflavin level causes spermatozoa morphological and bioenergetic defects. Beyond substantially advancing our understanding of spermatozoa energy metabolism, our study provides an attractive target for the development of male contraceptives.
    Keywords:  SLC22A14 transporter; energy metabolism; fatty acid β-oxidation; male infertility; riboflavin