bims-mitdis Biomed News
on Mitochondrial Disorders
Issue of 2021‒03‒28
fifty-four papers selected by
Catalina Vasilescu
University of Helsinki

  1. Am J Physiol Cell Physiol. 2021 Mar 24.
      The nuclear genome-encoded mitochondrial DNA (mtDNA) transcription factor A (TFAM) is indispensable for mitochondrial energy production in the developing and postnatal heart; a similar role for TFAM is inferred in adult heart. Here, we provide evidence that challenges this long-standing paradigm. Unexpectedly, conditionalTfam ablation in vivo in adult mouse cardiomyocytes resulted in a prolonged period of functional resilience characterized by preserved mtDNA content, mitochondrial function, and cardiac function, despite mitochondrial structural alterations and decreased transcript abundance. Remarkably, TFAM protein levels did not directly dictate mtDNA content in the adult heart, and mitochondrial translation was preserved with acute TFAM inactivation, suggesting maintenance of respiratory chain assembly/function. Long-term Tfam inactivation, however, downregulated the core mtDNA transcription and replication machinery, leading to mitochondrial dysfunction and cardiomyopathy. Collectively, in contrast to the developing heart, these data reveal a striking resilience of the differentiated adult heart to acute insults to mtDNA regulation.
    Keywords:  heart; mitochondria; mtDNA
  2. Mol Brain. 2021 Mar 23. 14(1): 58
      Mitochondrial structural changes are associated with the regulation of mitochondrial function, apoptosis, and neurodegenerative diseases. PRKN is known to be involved with various mechanisms of mitochondrial quality control including mitochondrial structural changes. Parkinson's disease (PD) with PRKN mutations is characterized by the preferential degeneration of dopaminergic neurons in the substantia nigra pars compacta, which has been suggested to result from the accumulation of damaged mitochondria. However, ultrastructural changes of mitochondria specifically in dopaminergic neurons derived from iPSC have rarely been analyzed. The main reason for this would be that the dopaminergic neurons cannot be distinguished directly among a mixture of iPSC-derived differentiated cells under electron microscopy. To selectively label dopaminergic neurons and analyze mitochondrial morphology at the ultrastructural level, we generated control and PRKN-mutated patient tyrosine hydroxylase reporter (TH-GFP) induced pluripotent stem cell (iPSC) lines. Correlative light-electron microscopy analysis and live cell imaging of GFP-expressing dopaminergic neurons indicated that iPSC-derived dopaminergic neurons had smaller and less functional mitochondria than those in non-dopaminergic neurons. Furthermore, the formation of spheroid-shaped mitochondria, which was induced in control dopaminergic neurons by a mitochondrial uncoupler, was inhibited in the PRKN-mutated dopaminergic neurons. These results indicate that our established TH-GFP iPSC lines are useful for characterizing mitochondrial morphology, such as spheroid-shaped mitochondria, in dopaminergic neurons among a mixture of various cell types. Our in vitro model would provide insights into the vulnerability of dopaminergic neurons and the processes leading to the preferential loss of dopaminergic neurons in patients with PRKN mutations.
    Keywords:  Dopaminergic neurons; IPSC; Mitochondria; PRKN; Ultrastructure
  3. Nat Commun. 2021 03 22. 12(1): 1807
      Mitochondria-lysosome contacts are recently identified sites for mediating crosstalk between both organelles, but their role in normal and diseased human neurons remains unknown. In this study, we demonstrate that mitochondria-lysosome contacts can dynamically form in the soma, axons, and dendrites of human neurons, allowing for their bidirectional crosstalk. Parkinson's disease patient derived neurons harboring mutant GBA1 exhibited prolonged mitochondria-lysosome contacts due to defective modulation of the untethering protein TBC1D15, which mediates Rab7 GTP hydrolysis for contact untethering. This dysregulation was due to decreased GBA1 (β-glucocerebrosidase (GCase)) lysosomal enzyme activity in patient derived neurons, and could be rescued by increasing enzyme activity with a GCase modulator. These defects resulted in disrupted mitochondrial distribution and function, and could be further rescued by TBC1D15 in Parkinson's patient derived GBA1-linked neurons. Together, our work demonstrates a potential role of mitochondria-lysosome contacts as an upstream regulator of mitochondrial function and dynamics in midbrain dopaminergic neurons in GBA1-linked Parkinson's disease.
  4. Cell Metab. 2021 Mar 23. pii: S1550-4131(21)00110-8. [Epub ahead of print]
      Mitochondria have an independent genome (mtDNA) and protein synthesis machinery that coordinately activate for mitochondrial generation. Here, we report that the Krebs cycle intermediate fumarate links metabolism to mitobiogenesis through binding to malic enzyme 2 (ME2). Mechanistically, fumarate binds ME2 with two complementary consequences. First, promoting the formation of ME2 dimers, which activate deoxyuridine 5'-triphosphate nucleotidohydrolase (DUT). DUT fosters thymidine generation and an increase of mtDNA. Second, fumarate-induced ME2 dimers abrogate ME2 monomer binding to mitochondrial ribosome protein L45, freeing it for mitoribosome assembly and mtDNA-encoded protein production. Methylation of the ME2-fumarate binding site by protein arginine methyltransferase-1 inhibits fumarate signaling to constrain mitobiogenesis. Notably, acute myeloid leukemia is highly dependent on mitochondrial function and is sensitive to targeting of the fumarate-ME2 axis. Therefore, mitobiogenesis can be manipulated in normal and malignant cells through ME2, an unanticipated governor of mitochondrial biomass production that senses nutrient availability through fumarate.
    Keywords:  acute myeloid leukemia; arginine methylation; deoxyuridine 5′-triphosphate nucleotidohydrolase; fumarate; malic enzyme 2; mitobiogenesis; mitochondrial ribosome; mitochondrial ribosome protein L45; protein arginine methyltransferase 1
  5. BMC Biol. 2021 Mar 24. 19(1): 57
      BACKGROUND: Mitochondrial dysfunction is a common feature of aging, neurodegeneration, and metabolic diseases. Hence, mitotherapeutics may be valuable disease modifiers for a large number of conditions. In this study, we have set up a large-scale screening platform for mitochondrial-based modulators with promising therapeutic potential.RESULTS: Using differentiated human neuroblastoma cells, we screened 1200 FDA-approved compounds and identified 61 molecules that significantly increased cellular ATP without any cytotoxic effect. Following dose response curve-dependent selection, we identified the flavonoid luteolin as a primary hit. Further validation in neuronal models indicated that luteolin increased mitochondrial respiration in primary neurons, despite not affecting mitochondrial mass, structure, or mitochondria-derived reactive oxygen species. However, we found that luteolin increased contacts between mitochondria and endoplasmic reticulum (ER), contributing to increased mitochondrial calcium (Ca2+) and Ca2+-dependent pyruvate dehydrogenase activity. This signaling pathway likely contributed to the observed effect of luteolin on enhanced mitochondrial complexes I and II activities. Importantly, we observed that increased mitochondrial functions were dependent on the activity of ER Ca2+-releasing channels inositol 1,4,5-trisphosphate receptors (IP3Rs) both in neurons and in isolated synaptosomes. Additionally, luteolin treatment improved mitochondrial and locomotory activities in primary neurons and Caenorhabditis elegans expressing an expanded polyglutamine tract of the huntingtin protein.
    CONCLUSION: We provide a new screening platform for drug discovery validated in vitro and ex vivo. In addition, we describe a novel mechanism through which luteolin modulates mitochondrial activity in neuronal models with potential therapeutic validity for treatment of a variety of human diseases.
    Keywords:  High-throughput screen; Luteolin; Mitochondria; Mitochondria-ER contacts; Mitochondrial calcium
  6. Proc Natl Acad Sci U S A. 2021 Mar 30. pii: e2100558118. [Epub ahead of print]118(13):
      Human mitochondrial ATP synthase is a molecular machine with a rotary action bound in the inner organellar membranes. Turning of the rotor, driven by a proton motive force, provides energy to make ATP from ADP and phosphate. Among the 29 component proteins of 18 kinds, ATP6 and ATP8 are mitochondrial gene products, and the rest are nuclear gene products that are imported into the organelle. The ATP synthase is assembled from them via intermediate modules representing the main structural elements of the enzyme. One such module is the c8-ring, which provides the membrane sector of the enzyme's rotor, and its assembly is influenced by another transmembrane (TMEM) protein, TMEM70. We have shown that subunit c interacts with TMEM70 and another hitherto unidentified mitochondrial transmembrane protein, TMEM242. Deletion of TMEM242, similar to deletion of TMEM70, affects but does not completely eliminate the assembly of ATP synthase, and to a lesser degree the assembly of respiratory enzyme complexes I, III, and IV. Deletion of TMEM70 and TMEM242 together prevents assembly of ATP synthase and the impact on complex I is enhanced. Removal of TMEM242, but not of TMEM70, also affects the introduction of subunits ATP6, ATP8, j, and k into the enzyme. TMEM70 and TMEM242 interact with the mitochondrial complex I assembly (the MCIA) complex that supports assembly of the membrane arm of complex I. The interactions of TMEM70 and TMEM242 with MCIA could be part of either the assembly of ATP synthase and complex I or the regulation of their levels.
    Keywords:  ATP synthase; TMEM242; TMEM70; assembly; human mitochondria
  7. Nat Commun. 2021 Mar 26. 12(1): 1929
      Leigh syndrome (LS) is a severe manifestation of mitochondrial disease in children and is currently incurable. The lack of effective models hampers our understanding of the mechanisms underlying the neuronal pathology of LS. Using patient-derived induced pluripotent stem cells and CRISPR/Cas9 engineering, we developed a human model of LS caused by mutations in the complex IV assembly gene SURF1. Single-cell RNA-sequencing and multi-omics analysis revealed compromised neuronal morphogenesis in mutant neural cultures and brain organoids. The defects emerged at the level of neural progenitor cells (NPCs), which retained a glycolytic proliferative state that failed to instruct neuronal morphogenesis. LS NPCs carrying mutations in the complex I gene NDUFS4 recapitulated morphogenesis defects. SURF1 gene augmentation and PGC1A induction via bezafibrate treatment supported the metabolic programming of LS NPCs, leading to restored neuronal morphogenesis. Our findings provide mechanistic insights and suggest potential interventional strategies for a rare mitochondrial disease.
  8. J Lipid Res. 2021 Mar 20. pii: S0022-2275(21)00051-1. [Epub ahead of print] 100069
      Long-chain fatty acid oxidation is frequently impaired in primary and systemic metabolic diseases affecting the heart, thus therapeutically increasing reliance on normally minor energetic substrates, such as ketones and medium chain fatty acids, could benefit cardiac health. However, the molecular fundamentals of this therapy are not fully known. Here, we explored the ability of octanoate, an eight-carbon medium-chain fatty acid known as an unregulated mitochondrial energetic substrate, to ameliorate cardiac hypertrophy in long-chain fatty acid oxidation deficient hearts due to carnitine palmitoyltransferase 2 deletion (Cpt2M-/-). CPT2 converts acylcarnitines to acyl-CoAs in the mitochondrial matrix for oxidative bioenergetic metabolism. In Cpt2M-/- mice, high octanoate-ketogenic diet failed to alleviate myocardial hypertrophy, dysfunction, and acylcarnitine accumulation suggesting that this alternative substrate is not sufficiently compensatory for energy provision. Aligning this outcome, we identified a major metabolic distinction between muscles and liver, wherein heart and skeletal muscle mitochondria were unable to oxidize free octanoate but liver was able to oxidize free octanoate. Liver mitochondria, but not heart or muscle, highly expressed medium-chain acyl-CoA synthetases, potentially enabling octanoate activation for oxidation and circumventing acylcarnitine-shuttling. Conversely, octanoylcarnitine was oxidized by liver, skeletal muscle, and heart, with rates in heart 4-fold greater than liver and, in muscles, was not dependent upon CPT2. Together, these data suggest that dietary octanoate cannot rescue CPT2-deficient cardiac disease. These data also suggest the existence of tissue-specific mechanisms for octanoate oxidative metabolism, with liver being independent of free carnitine availability while cardiac and skeletal muscles depend on carnitine but not on CPT2.
    Keywords:  carnitine palmitoyltransferase; carnitine-shuttle; fatty acid oxidation; medium-chain fatty acids; mitochondria
  9. Brain Res. 2021 Mar 18. pii: S0006-8993(21)00291-2. [Epub ahead of print] 147434
      Parkinson's disease (PD) is a progressive neurodegenerative disease manifesting both motor and non-motor symptoms. The motor features are generally ascribed to the selective loss of dopamine neurons within the substantia nigra pars compacta. While the precise etiology of PD remains elusive, multiple genetic and environmental elements have emerged as contributing factors. The discovery of MPTP-induced parkinsonism directed intense inquiry towards mitochondrial pathways, with a specific focus on mitochondrial complex I. Consisting of more than 40 subunits, complex I is the first enzyme of the electron transport chain that is required for mitochondrial ATP production. In this review, we present a critical analysis of studies assessing the prevalence and specificity of mitochondrial complex I deficiency in PD. In addition, we take the novel view of incorporating the features of genetically-defined bona fide complex I disorders and the prevalence of nigral involvement in such cases. Through this innovative bi-directional view, we consider both complex I changes in a disease of the substantia nigra and nigral changes in diseases of complex I. We assess the strength of association between nigral cell loss and complex I deficits, as well as the oft under-appreciated heterogeneity of complex I deficiency disorders and the variability of the PD data.
    Keywords:  MPTP; NDUFAF2; PINK1; Parkinson’s Disease; mitochondrial complex I
  10. Hum Mol Genet. 2021 Mar 22. pii: ddab078. [Epub ahead of print]
      Mutations in CHCHD10, coding for a mitochondrial intermembrane space protein, are a rare cause of autosomal dominant amyotrophic lateral sclerosis (ALS). Mutation-specific toxic gain of function or haploinsufficiency models have been proposed to explain pathogenicity. To decipher the metabolic dysfunction associated with the haploinsufficient p.R15L variant we integrated transcriptomic, metabolomic and proteomic data sets in patient cells subjected to an energetic stress that forces the cells to rely on oxidative phosphorylation for ATP production. Patient cells had a complex I deficiency that resulted in an increased NADH/NAD+ ratio, diminished TCA cycle activity, a reorganization of one carbon metabolism, and an increased AMP/ATP ratio leading to phosphorylation of AMPK and inhibition of mTORC1. These metabolic changes activated the unfolded protein response (UPR) in the ER through the IRE1/XBP1 pathway, upregulating downstream targets including ATF3, ATF4, CHOP and EGLN3, and two cytokine markers of mitochondrial disease, GDF15 and FGF21. Activation of the mitochondrial UPR was mediated through an upregulation of the transcription factors ATF4 and ATF5, leading to increased expression of mitochondrial proteases and heat shock proteins. There was a striking transcriptional up regulation of at least seven dual specific phosphatases, associated with an almost complete dephosphorylation of JNK isoforms, suggesting a concerted deactivation of MAP kinase pathways. This study demonstrates that loss of CHCHD10 function elicits an energy deficit that activates unique responses to nutrient stress in both the mitochondria and ER, which may contribute to the selective vulnerability of motor neurons.
  11. Front Neurol. 2021 ;12 601307
      Objective: Mitochondrial 13513G>A mutation presenting as isolated Leber's hereditary optic neuropathy (LHON) without any extraocular pathology has not been reported in literature. We herein evaluate the clinical characteristics and heteroplasmy of m.13513G>A mutation manifesting as isolated LHON. Methods: Seven members of a Chinese family were enrolled in this study. All subjects underwent detailed systemic and ophthalmic examinations. Mitochondrial DNA in their blood was assessed by targeted PCR amplifications, next generation sequencing (NGS), and pyrosequencing. One hundred of blood samples from ethnic-matched healthy volunteers were tested by NGS and pyrosequencing as normal controls. Results: Isolated LHON without any other ocular or extraocular pathology was identified in a 16 year old patient in this family. Heteroplasmic m.13513G>A mutation was detected by NGS of the full mtDNA genome in the patient with mutant load of 33.56%, and of 26% 3 months and 3 years after the onset of LHON, respectively. No m.13513G>A mutation was detected in all his relatives by NGS. Pyrosequencing revealed the mutant load of m.13513G>A mutation of the LHON patient, his mother, father and sister were 22.4, 1.9, 0, and 0%, respectively. None of 100 healthy control subjects was detected to harbor m.13513G>A mutation either by NGS or by pyrosequencing of the full mt DNA genome. Conclusions: We first report m.13513G>A mutation with low mutant load presenting as isolated LHON. NGS of the full mitochondrial DNA genome is highly recommended for LHON suspects when targeted PCR amplification for main primary point mutations of LHON was negative.
    Keywords:  Leber's hereditary optic neuropathy; gene mutation; m13513G>A; mitochondrial DNA; optic atrophy
  12. Cell Mol Life Sci. 2021 Mar 22.
      The remodeling of the mitochondrial network is a critical process in maintaining cellular homeostasis and is intimately related to mitochondrial function. The interplay between the formation of new mitochondria (biogenesis) and the removal of damaged mitochondria (mitophagy) provide a means for the repopulation of the mitochondrial network. Additionally, mitochondrial fission and fusion serve as a bridge between biogenesis and mitophagy. In recent years, the importance of these processes has been characterised in multiple tissue- and cell-types, and under various conditions. In skeletal muscle, the robust remodeling of the mitochondrial network is observed, particularly after injury where large portions of the tissue/cell structures are damaged. The significance of mitochondrial remodeling in regulating skeletal muscle regeneration has been widely studied, with alterations in mitochondrial remodeling processes leading to incomplete regeneration and impaired skeletal muscle function. Needless to say, important questions related to mitochondrial remodeling and skeletal muscle regeneration still remain unanswered and require further investigation. Therefore, this review will discuss the known molecular mechanisms of mitochondrial network remodeling, as well as integrate these mechanisms and discuss their relevance in myogenesis and regenerating skeletal muscle.
    Keywords:  Biogenesis; Fission; Fusion; Mitochondria; Mitophagy; Regeneration; Skeletal muscle; Skeletal muscle stem cells
  13. Exp Neurol. 2021 Mar 19. pii: S0014-4886(21)00113-8. [Epub ahead of print] 113707
      The cardinal pathophysiological finding of Parkinson's disease (PD) is a chronic, progressive degeneration of dopamine (DA) neurons in the substantia nigra, which is responsible for the motor and some of the non-motor symptomatology. While the primary causes of nigrostriatal degeneration are hotly debated, considerable evidence supports a central role for impaired mitochondrial function. Postmortem analysis of PD patients reveals impaired respiratory chains and increased mutations of mitochondrial DNA (mtDNA), in addition to increased markers of oxidative stress indicative of mitochondrial impairment. Most animal models of PD, both genetic and toxin-based, target some component of mitochondrial function to reproduce aspects of the human disease. One model that continues to gain attention is the MitoPark mouse, created through a cell type-specific knockout of mitochondrial transcription factor A specifically in midbrain DA neurons. This model effectively recapitulates the slowly developing, adult onset motor decline seen in PD due to mass loss of DA neurons. MitoPark mice therefore represent an effective tool for studying the sequence of events that occurs in the early stages of DA neuron degeneration following mitochondrial impairment, as well as for testing the efficacy of potential disease-modifying therapies in a progressive model of neurodegeneration. A targeted review of key findings from MitoPark mice has not been published since the early years following the initial report of the model in 2007. The current review synthesizes findings from several groups that are exploring MitoPark mice and discusses implications for the future identification of disease-modifying treatments for PD.
    Keywords:  Dopamine neurons; Electrophysiology; MitoPark; Mitochondria; Mitochondrial transcription factor A; Model; Parkinson's disease; Striatum; Substantia nigra
  14. Hum Genet. 2021 Mar 21.
      While newborns and children with hearing loss are routinely offered genetic testing, adults are rarely clinically tested for a genetic etiology. One clinically actionable result from genetic testing in children is the discovery of variants in syndromic hearing loss genes. EYA4 is a known hearing loss gene which is also involved in important pathways in cardiac tissue. The pleiotropic effects of rare EYA4 variants are poorly understood and their prevalence in a large cohort has not been previously reported. We investigated cardio-auditory phenotypes in 11,451 individuals in a large biobank using a rare variant, genome-first approach to EYA4. We filtered 256 EYA4 variants carried by 6737 participants to 26 rare and predicted deleterious variants carried by 42 heterozygotes. We aggregated predicted deleterious EYA4 gene variants into a combined variable (i.e. "gene burden") and performed association studies across phenotypes compared to wildtype controls. We validated findings with replication in three independent cohorts and human tissue expression data. EYA4 gene burden was significantly associated with audiometric-proven HL (p = [Formula: see text], Mobitz Type II AV block (p = [Formula: see text]) and the syndromic presentation of HL and primary cardiomyopathy (p = 0.0194). Analyses on audiogram, echocardiogram, and electrocardiogram data validated these associations. Prior reports have focused on identifying variants in families with severe or syndromic phenotypes. In contrast, we found, using a genotype-first approach, that gene burden in EYA4 is associated with more subtle cardio-auditory phenotypes in an adult medical biobank population, including cardiac conduction disorders which have not been previously reported. We show the value of using a focused approach to uncover human disease related to pleiotropic gene variants and suggest a role for genetic testing in adults presenting with hearing loss.
  15. Front Aging Neurosci. 2021 ;13 650038
      Mitochondria are organelles responsible for bioenergetic metabolism, calcium homeostasis, and signal transmission essential for neurons due to their high energy consumption. Accumulating evidence has demonstrated that mitochondria play a key role in axon degeneration and regeneration under physiological and pathological conditions. Mitochondrial dysfunction occurs at an early stage of axon degeneration and involves oxidative stress, energy deficiency, imbalance of mitochondrial dynamics, defects in mitochondrial transport, and mitophagy dysregulation. The restoration of these defective mitochondria by enhancing mitochondrial transport, clearance of reactive oxidative species (ROS), and improving bioenergetic can greatly contribute to axon regeneration. In this paper, we focus on the biological behavior of axonal mitochondria in aging, injury (e.g., traumatic brain and spinal cord injury), and neurodegenerative diseases (Alzheimer's disease, AD; Parkinson's disease, PD; Amyotrophic lateral sclerosis, ALS) and consider the role of mitochondria in axon regeneration. We also compare the behavior of mitochondria in different diseases and outline novel therapeutic strategies for addressing abnormal mitochondrial biological behavior to promote axonal regeneration in neurological diseases and injuries.
    Keywords:  Alzheimer's disease; Amyotrophic lateral sclerosis; Parkinson's disease; aging; axon regeneration; mitochondria; spinal cord injury; traumatic brain injury
  16. Mol Genet Metab. 2021 Mar 13. pii: S1096-7192(21)00072-X. [Epub ahead of print]
      Mutations in the HADHB gene lead to Mitochondrial Trifunctional Protein (MTP) deficiency. MTP deficiency is a rare autosomal recessive disorder affecting long-chain fatty acid oxidation. Patients affected by MTP deficiency are unable to metabolize long-chain fatty-acids and suffer a variety of symptoms exacerbated during fasting. The three phenotypes associated with complete MTP deficiency are an early-onset cardiomyopathy and early death, an intermediate form with recurrent hypoketotic hypoglycemia and a sensorimotor neuropathy with episodic rhabdomyolysis with small amount of residual enzyme activities. This review aims to discuss the pathophysiological mechanisms and clinical manifestations of each phenotype, which appears different and linked to HADHB expression levels. Notably, the pathophysiology of the sensorimotor neuropathy is relatively unknown and we provide a hypothesis on the qualitative aspect of the role of acylcarnitine buildup in Schwann cells in MTP deficiency patients. We propose that acylcarnitine may exit the Schwann cell and alter membrane properties of nearby axons leading to axonal degeneration based on recent findings in different metabolic disorders.
    Keywords:  HADHB; Mitochondrial trifunctional protein; Myopathy; Peripheral neuropathy; Rhabdomyolysis
  17. Mitochondrion. 2021 Mar 17. pii: S1567-7249(21)00035-0. [Epub ahead of print]
      Complex I is the largest and most intricate of the protein complexes of mitochondrial electron transport chain (ETC). This L-shaped enzyme consists of a peripheral hydrophilic matrix domain and a membrane-bound orthogonal hydrophobic domain. The interfacial region between these two arms is known to be critical for binding of ubiquinone moieties and has also been shown to be the binding site of Complex I inhibitors. Knowledge on specific roles of the ETC interfacial region proteins is scarce due to lack of knockout cell lines and animal models. Here we mutated nuclear encoded NADH dehydrogenase [ubiquinone] iron-sulfur protein 2 (NDUFS2), one of three protein subunits of the interfacial region, in a human embryonic kidney cell line 293 using a CRISPR/Cas9 procedure. Disruption of NDUFS2 significantly decreased cell growth in medium, Complex I specific respiration, glycolytic capacity, ATP pool and cell-membrane integrity, but significantly increased Complex II respiration, ROS generation, apoptosis, and necrosis. Treatment with idebenone, a clinical benzoquinone currently being investigated in other indications, partially restored growth, ATP pool, and oxygen consumption of the mutant. Overall, our results suggest that NDUFS2 is vital for growth and metabolism of mammalian cells, and respiratory defects of NDUFS2 dysfunction can be partially corrected with treatment of an established mitochondrial therapeutic candidate. This is the first report to use CRISPR/Cas9 approach to construct a knockout NDUFS2 cell line and use the constructed mutant to evaluate the efficacy of a known mitochondrial therapeutic to enhance bioenergetic capacity.
    Keywords:  ATP synthesis; CRISPR/Cas9; Complex I; Electron transport chain; ROS; apoptosis; glycolysis; idebenone; necrosis; oxygen consumption; respiration
  18. Autophagy. 2021 Mar 23.
      Mitochondria are the main cellular energy powerhouses and supply most of the energy in the form of ATP to fuel essential neuronal functions through oxidative phosphorylation (OXPHOS). In Alzheimer disease (AD), metabolic and mitochondrial disruptions are an early feature preceding any histopathological and clinical manifestations. Mitochondrial malfunction is also linked to synaptic defects in early AD. Mitophagy serves as a key cellular quality control mechanism involving sequestration of damaged mitochondria within autophagosomes and their subsequent degradation in lysosomes. However, it remains largely unknown whether mitophagy is involved in the regulation of energy metabolism in neurons, and if so, whether metabolic deficiency in AD is attributed to mitophagy dysfunction. Here we reveal that mitophagy is broadly activated in metabolically enhanced neurons upon OXPHOS stimulation, which sustains high energetic activity by increasing mitochondrial turnover and hence facilitating mitochondrial maintenance. Unexpectedly, in AD-related mutant HsAPP Tg mouse brains, early stimulation of OXPHOS activity fails to correct energy deficits but exacerbates synapse loss as a consequence of mitophagy failure. Excitingly, lysosomal enhancement in AD neurons restores impaired metabolic function by promoting elimination of damaged mitochondria, protecting against synaptic damage in AD mouse brains. Taken together, we propose a new mechanism by which mitophagy controls bioenergetic status in neurons, furthering our understanding of the direct impact of mitophagy defects on AD-linked metabolic deficits and shedding light on the development of novel therapeutic strategies to treat AD by the early stimulation of mitochondrial metabolism combined with elevation of lysosomal proteolytic activity.
    Keywords:  Alzheimer; bioenergetics; energy metabolism; lysosomal proteolysis; metabolic deficiency; mitochondrial stress; mitophagosome; neuronal mitophagy; retrograde transport; synapse loss
  19. Hum Mol Genet. 2021 Mar 22. pii: ddab080. [Epub ahead of print]
      Human neurodegenerative proteinopathies are disorders associated with abnormal protein depositions in brain neurons. They include polyglutamine (polyQ) conditions such as Huntington's disease (HD) and α-synucleinopathies such as Parkinson's disease (PD). Overexpression of NMNAT/Nma1, an enzyme in the NAD+ biosynthetic salvage pathway, acts as an efficient suppressor of proteotoxicities in yeast, fly, and mouse models. Screens in yeast models of HD and PD allowed us to identify three additional enzymes of the same pathway that achieve similar protection against proteotoxic stress: Npt1, Pnc1, and Qns1. The mechanism by which these proteins maintain proteostasis has not been identified. Here, we report that their ability to maintain proteostasis in yeast models of HD and PD is independent of their catalytic activity and does not require cellular protein quality control systems such as the proteasome or autophagy. Furthermore, we show that, under proteotoxic stress, the four proteins are recruited as molecular chaperones with holdase and foldase activities. The NAD+ salvage proteins act by preventing misfolding and, together with the Hsp90 chaperone, promoting the refolding of extended polyQ domains and α-synuclein (α-Syn). Our results illustrate the existence of an evolutionarily conserved strategy of repurposing or moonlighting housekeeping enzymes under stress conditions to maintain proteostasis. We conclude that the entire salvage NAD+ biosynthetic pathway links NAD+ metabolism and proteostasis and emerges as a target for therapeutics to combat age-associated neurodegenerative proteotoxicities.
  20. Mol Genet Metab. 2021 Mar 18. pii: S1096-7192(21)00076-7. [Epub ahead of print]
      GBA variations are common risk factors for Parkinson's disease (PD), and are found in 21.7% of Ashkenazi PD patients (AJ-PD), 4.23% of them carry an allele, 370Rec, which is different from the common GBA-N370S allele. Using whole-genome-sequencing of 370Rec carriers, N370S carriers, and non-carriers, we characterize the unique 370Rec haplotype in AJ-PDs, and show that it harbors a missense variant replacing the highly conserved methionine-27 with valine in the transmembrane domain of the mitochondrial SLC25A44.
    Keywords:  GBA; N370S; Parkinson's disease; Risk allele
  21. Cell Rep. 2021 Mar 23. pii: S2211-1247(21)00209-6. [Epub ahead of print]34(12): 108895
      Here, we examine the cellular changes triggered by tumor necrosis factor alpha (TNF-α) and different alpha-synuclein (αSYN) species in astrocytes derived from induced pluripotent stem cells. Human astrocytes treated with TNF-α display a strong reactive pro-inflammatory phenotype with upregulation of pro-inflammatory gene networks, activation of the nuclear factor κB (NF-κB) pathway, and release of pro-inflammatory cytokines, whereas those treated with high-molecular-weight αSYN fibrils acquire a reactive antigen (cross)-presenting phenotype with upregulation of major histocompatibility complex (MHC) genes and increased human leukocyte antigen (HLA) molecules at the cell surface. Surprisingly, the cell surface location of MHC proteins is abrogated by larger F110 fibrillar polymorphs, despite the upregulation of MHC genes. Interestingly, TNF-α and αSYN fibrils compete to drive the astrocyte immune reactive response. The astrocyte immune responses are accompanied by an impaired mitochondrial respiration, which is exacerbated in Parkinson's disease (PD) astrocytes. Our data provide evidence for astrocytic involvement in PD pathogenesis and reveal their complex immune reactive responses to exogenous stressors.
    Keywords:  HLA genes; Parkinson’s disease; alpha-synuclein; astrocytes; iPSC; reactivity
  22. Cell Biol Int. 2021 Mar 25.
      During myoblast differentiation, mitochondria undergo numerous changes that are necessary for the progression of the myogenic program. Notably, we previously showed that alteration in mitochondrial activity were able to control the expression of keys regulator of cell cycle withdrawal and terminal differentiation. Here, we assessed whether inhibition of one of the respiratory complexes was a key factor in the regulation of myogenic differentiation in C2C12 cells, and was associated with alteration in ROS production. C2C12 cells were treated from proliferation to differentiation with specific inhibitors of mitochondrial complexes at concentration that were inhibiting respiration but not altering cell morphology. Proliferation was significantly repressed with inhibition of complexes I, II and III, or mitochondrial protein synthesis (using CHL treatment), while complex IV inhibition did not alter myoblast proliferation compared to control cells. Moreover, inhibition of complex I and II altered cell cycle regulators, with p21 protein expression upregulated since proliferation and p27 protein expression reduced at differentiation. Myotubes formation and myogenin expression were blunted with complex I and II inhibitors while MyoD protein expression was maintained suggesting an alteration in its transcriptional activity. Finally, a decrease in overall ROS production was observed with continuous inhibition of mitochondrial complexes I to IV. In summary, our data provide evidence that complexes I and II may be the primary regulators of C2C12 myogenic differentiation. This occurs through specific regulation of myogenic rather than cell cycle regulators expression, and ROS production at mitochondrial rather than cell level. This article is protected by copyright. All rights reserved.
    Keywords:  ROS; mitochondria; myogenic differentiation; respiratory complexes
  23. Free Radic Biol Med. 2021 Mar 19. pii: S0891-5849(21)00163-5. [Epub ahead of print]
      Coenzyme Q (CoQ) is ubiquitously embedded in lipid bilayers of various cellular organelles. As a redox cycler, CoQ shuttles electrons between mitochondrial complexes and extramitochondrial reductases and oxidases. In this way, CoQ is crucial for maintaining the mitochondrial function, ATP synthesis, and redox homeostasis. Cardiomyocytes have a high metabolic rate and rely heavily on mitochondria to provide energy. CoQ levels, in both plasma and the heart, correlate with heart failure in patients, indicating that CoQ is critical for cardiac function. Moreover, CoQ supplementation in clinics showed promising results for treating heart failure. This review provides a comprehensive view of CoQ metabolism and its interaction with redox enzymes and reactive species. We summarize the clinical trials and applications of CoQ in heart failure and discuss the caveats and future directions to improve CoQ therapeutics.
    Keywords:  CoQ; heart failure; redox
  24. Oxid Med Cell Longev. 2021 ;2021 5545261
      Mitochondrial dysfunction has been suggested to be the key factor in the development and progression of cardiac hypertrophy. The onset of mitochondrial dysfunction and the mechanisms underlying the development of cardiac hypertrophy (CH) are incompletely understood. The present study is based on the use of multiple bioinformatics analyses for the organization and analysis of scRNA-seq and microarray datasets from a transverse aortic constriction (TAC) model to examine the potential role of mitochondrial dysfunction in the pathophysiology of CH. The results showed that NADH:ubiquinone oxidoreductase core subunit S1- (Ndufs1-) dependent mitochondrial dysfunction plays a key role in pressure overload-induced CH. Furthermore, in vivo animal studies using a TAC mouse model of CH showed that Ndufs1 expression was significantly downregulated in hypertrophic heart tissue compared to that in normal controls. In an in vitro model of angiotensin II- (Ang II-) induced cardiomyocyte hypertrophy, Ang II treatment significantly downregulated the expression of Ndufs1 in cardiomyocytes. In vitro mechanistic studies showed that Ndufs1 knockdown induced CH; decreased the mitochondrial DNA content, mitochondrial membrane potential (MMP), and mitochondrial mass; and increased the production of mitochondrial reactive oxygen species (ROS) in cardiomyocytes. On the other hand, Ang II treatment upregulated the expression levels of atrial natriuretic peptide, brain natriuretic peptide, and myosin heavy chain beta; decreased the mitochondrial DNA content, MMP, and mitochondrial mass; and increased mitochondrial ROS production in cardiomyocytes. The Ang II-mediated effects were significantly attenuated by overexpression of Ndufs1 in rat cardiomyocytes. In conclusion, our results demonstrate downregulation of Ndufs1 in hypertrophic heart tissue, and the results of mechanistic studies suggest that Ndufs1 deficiency may cause mitochondrial dysfunction in cardiomyocytes, which may be associated with the development and progression of CH.
  25. Nat Rev Mol Cell Biol. 2021 Mar 23.
      Brown and beige adipocytes are mitochondria-enriched cells capable of dissipating energy in the form of heat. These thermogenic fat cells were originally considered to function solely in heat generation through the action of the mitochondrial protein uncoupling protein 1 (UCP1). In recent years, significant advances have been made in our understanding of the ontogeny, bioenergetics and physiological functions of thermogenic fat. Distinct subtypes of thermogenic adipocytes have been identified with unique developmental origins, which have been increasingly dissected in cellular and molecular detail. Moreover, several UCP1-independent thermogenic mechanisms have been described, expanding the role of these cells in energy homeostasis. Recent studies have also delineated roles for these cells beyond the regulation of thermogenesis, including as dynamic secretory cells and as a metabolic sink. This Review presents our current understanding of thermogenic adipocytes with an emphasis on their development, biological functions and roles in systemic physiology.
  26. Methods Mol Biol. 2021 Mar 24.
      Disease-specific induced pluripotent stem (iPS) cells are useful tools to analyze the pathology of neurodegenerative diseases. In this chapter, we describe a procedure to efficiently induce small-scale differentiation of patient iPS cells into midbrain dopaminergic neurons to detect cell death and mitochondrial clearance by using immunostaining. A combination of our method described here and an image analysis system, such as the IN Cell Analyzer, will enable the quantitative assessment of cell vulnerability and mitochondrial quality control abnormalities in cells derived from patients with Parkinson's disease; this set-up might be used to perform drug screening.
    Keywords:  Disease modeling; Dopaminergic neurons; Drug discovery; Parkinson’s disease; Small scale; iPS cells
  27. Proc Natl Acad Sci U S A. 2021 Mar 30. pii: e2021385118. [Epub ahead of print]118(13):
      Conventional T cell fate and function are determined by coordination between cellular signaling and mitochondrial metabolism. Invariant natural killer T (iNKT) cells are an important subset of "innate-like" T cells that exist in a preactivated effector state, and their dependence on mitochondrial metabolism has not been previously defined genetically or in vivo. Here, we show that mature iNKT cells have reduced mitochondrial respiratory reserve and iNKT cell development was highly sensitive to perturbation of mitochondrial function. Mice with T cell-specific ablation of Rieske iron-sulfur protein (RISP; T-Uqcrfs1 -/- ), an essential subunit of mitochondrial complex III, had a dramatic reduction of iNKT cells in the thymus and periphery, but no significant perturbation on the development of conventional T cells. The impaired development observed in T-Uqcrfs1 -/- mice stems from a cell-autonomous defect in iNKT cells, resulting in a differentiation block at the early stages of iNKT cell development. Residual iNKT cells in T-Uqcrfs1 -/- mice displayed increased apoptosis but retained the ability to proliferate in vivo, suggesting that their bioenergetic and biosynthetic demands were not compromised. However, they exhibited reduced expression of activation markers, decreased T cell receptor (TCR) signaling and impaired responses to TCR and interleukin-15 stimulation. Furthermore, knocking down RISP in mature iNKT cells diminished their cytokine production, correlating with reduced NFATc2 activity. Collectively, our data provide evidence for a critical role of mitochondrial metabolism in iNKT cell development and activation outside of its traditional role in supporting cellular bioenergetic demands.
    Keywords:  CD1; NKT cells; T cell development; knockout mice; mitochondrial metabolism
  28. Cardiovasc Res. 2021 Mar 22. pii: cvab112. [Epub ahead of print]
      AIMS: Autophagy protects against the development of cardiac hypertrophy and failure. While aberrant Ca2+ handling promotes myocardial remodelling and contributes to contractile dysfunction, the role of autophagy in maintaining Ca2+ homeostasis remains elusive. Here, we examined whether Atg5 deficiency-mediated autophagy promotes early changes in subcellular Ca2+ handling in ventricular cardiomyocytes, and whether those alterations associate with compromised cardiac reserve capacity, which commonly precedes the onset of heart failure.METHODS AND RESULTS: RT-qPCR and immunoblotting demonstrated reduced Atg5 gene and protein expression and decreased abundancy of autophagy markers in hypertrophied and failing human hearts. The function of ATG5 was examined using cardiomyocyte-specific Atg5-knockout mice (Atg5-/-). Before manifesting cardiac dysfunction, Atg5-/- mice showed compromised cardiac reserve in response to β-adrenergic stimulation. Consequently, effort intolerance and maximal oxygen consumption were reduced during treadmill-based exercise tolerance testing. Mechanistically, cellular imaging revealed that Atg5 deprivation did not alter spatial and functional organization of intracellular Ca2+ stores or affect Ca2+ cycling in response to slow pacing or upon acute isoprenaline administration. However, high frequency stimulation exposed stunted amplitude of Ca2+ transients, augmented nucleoplasmic Ca2+ load and increased CaMKII activity, especially in the nuclear region of hypertrophied Atg5-/- cardiomyocytes. These changes in Ca2+ cycling were recapitulated in hypertrophied human cardiomyocytes. Finally, ultrastructural analysis revealed accumulation of mitochondria with reduced volume and size distribution, meanwhile functional measurements showed impaired redox balance in Atg5-/- cardiomyocytes, implying energetic unsustainability due to overcompensation of single mitochondria, particularly under increased workload.
    CONCLUSION: Loss of cardiac Atg5-dependent autophagy reduces mitochondrial abundance and causes subtle alterations in subcellular Ca2+ cycling upon increased workload in mice. Autophagy-related impairment of Ca2+ handling is progressively worsened by β-adrenergic signalling in ventricular cardiomyocytes, thereby leading to energetic exhaustion and compromised cardiac reserve.
    Keywords:  autophagy; beta-adrenergic signalling; calcium; cardiomyocytes; mitochondria
  29. BMC Bioinformatics. 2021 Mar 09. 22(1): 115
      BACKGROUND: Today an unprecedented amount of genetic sequence data is stored in publicly available repositories. For decades now, mitochondrial DNA (mtDNA) has been the workhorse of genetic studies, and as a result, there is a large volume of mtDNA data available in these repositories for a wide range of species. Indeed, whilst whole genome sequencing is an exciting prospect for the future, for most non-model organisms' classical markers such as mtDNA remain widely used. By compiling existing data from multiple original studies, it is possible to build powerful new datasets capable of exploring many questions in ecology, evolution and conservation biology. One key question that these data can help inform is what happened in a species' demographic past. However, compiling data in this manner is not trivial, there are many complexities associated with data extraction, data quality and data handling.RESULTS: Here we present the mtDNAcombine package, a collection of tools developed to manage some of the major decisions associated with handling multi-study sequence data with a particular focus on preparing sequence data for Bayesian skyline plot demographic reconstructions.
    CONCLUSIONS: There is now more genetic information available than ever before and large meta-data sets offer great opportunities to explore new and exciting avenues of research. However, compiling multi-study datasets still remains a technically challenging prospect. The mtDNAcombine package provides a pipeline to streamline the process of downloading, curating, and analysing sequence data, guiding the process of compiling data sets from the online database GenBank.
    Keywords:  Bayesian skyline plots; Demographic history; Mitochondrial DNA; Public datasets; R package
  30. Front Cell Dev Biol. 2021 ;9 644667
      Human induced pluripotent stem cell (iPSC)-derived cardiomyocytes (CMs) (hiPSC-CMs) are a promising cell source for disease modeling, myocardial regeneration, and drug assessment. However, hiPSC-CMs have certain immature fetal CM-like properties that are different from the characteristics of adult CMs in several aspects, including cellular structure, mitochondrial function, and metabolism, thus limiting their applications. Adenosine 5'-monophosphate (AMP)-activated protein kinase (AMPK) is an energy-sensing protein kinase involved in the regulation of fatty acid oxidation and mitochondrial biogenesis in cardiomyocytes. This study investigated the effects of AMPK on the maturation of hiPSC-CMs. Activation of AMPK in hiPSC-CMs significantly increased the expression of CM-specific markers and resulted in a more mature myocardial structure compared to that in the control cells. We found that activation of AMPK improved mitochondrial oxidative phosphorylation (OxPhos) and the oxygen consumption rate (OCR). Additionally, our data demonstrated that activation of AMPK increased mitochondrial fusion to promote the maturation of mitochondrial structure and function. Overall, activation of AMPK is an effective approach to promote hiPSC-CMs maturation, which may enhance the utility of hiPSC-CMs in clinical applications.
    Keywords:  AMPK; cardiomyocyte maturation; hiPSC-CMs; hiPSCs; mitochondrial
  31. FASEB J. 2021 Apr;35(4): e21278
      Mitochondria share attributes of vesicular transport with their bacterial ancestors given their ability to form mitochondrial-derived vesicles (MDVs). MDVs are involved in mitochondrial quality control and their formation is enhanced with stress and may, therefore, play a potential role in mitochondrial-cellular communication. However, MDV proteomic cargo has remained mostly undefined. In this study, we strategically used an in vitro MDV budding/reconstitution assay on cardiac mitochondria, followed by graded oxidative stress, to identify and characterize the MDV proteome. Our results confirmed previously identified cardiac MDV markers, while also revealing a complete map of the MDV proteome, paving the way to a better understanding of the role of MDVs. The oxidative stress vulnerability of proteins directed the cargo loading of MDVs, which was enhanced by antimycin A (Ant-A). Among OXPHOS complexes, complexes III and V were found to be Ant-A-sensitive. Proteins from metabolic pathways such as the TCA cycle and fatty acid metabolism, along with Fe-S cluster, antioxidant response proteins, and autophagy were also found to be Ant-A sensitive. Intriguingly, proteins containing hyper-reactive cysteine residues, metabolic redox switches, including professional redox enzymes and those that mediate iron metabolism, were found to be components of MDV cargo with Ant-A sensitivity. Last, we revealed a possible contribution of MDVs to the formation of extracellular vesicles, which may indicate mitochondrial stress. In conclusion, our study provides an MDV proteomics signature that delineates MDV cargo selectivity and hints at the potential for MDVs and their novel protein cargo to serve as vital biomarkers during mitochondrial stress and related pathologies.
    Keywords:  hyper-reactive cysteine residues; mitochondrial iron transport; mitochondrial quality control; mitochondrial stress; mitochondrial-derived vesicle proteome
  32. Clin Case Rep. 2021 Mar;9(3): 1711-1715
      Pyruvate dehydrogenase complex (PDHC) deficiency is a mitochondrial disorder. We report two cases of PDHC deficiency with clinical symptoms and brain imaging findings reminiscent of FOXG1 syndrome, suggesting a phenotypic overlap of these disorders.
    Keywords:  FOXG1 syndrome; MRI; PDHC deficiency; phenotype
  33. Development. 2021 Mar 25. pii: dev.199026. [Epub ahead of print]
      Mammalian heart development relies hugely on cardiomyocyte mitochondrial maturation and metabolism. Embryonic cardiomyocytes make metabolic shift from anaerobic glycolysis to oxidative metabolism by mid-gestation. The VHL-HIF signaling favors anaerobic glycolysis but this process subsides by E14.5. Meanwhile, the oxidative metabolism becomes activated but its regulation is largely elusive. Here, we first pinpointed a critical temporal window for mitochondrial maturation and metabolic shift, and uncovered the pivotal role of the SRCAP chromatin remodeling complex in these processes. Disruption of this complex massively suppressed the transcription of key genes required for the tricarboxylic acid (TCA) cycle, fatty acid β-oxidation and ubiquinone biosynthesis, and destroyed respirasome stability. Furthermore, we found that the SRCAP complex functioned through H2A.Z deposition to activate transcription of metabolic genes. These findings unveiled the important physiological functions of SRCAP complex in regulating mitochondrial maturation and promoting oxidative metabolism during heart development, and shed new light on the transcriptional regulation of ubiquinone biosynthesis.
    Keywords:  H2A.Z; Heart development; Metabolism; Mitochondria; SRCAP chromatin remodeling complex; Znhit1
  34. Elife. 2021 Mar 25. pii: e66079. [Epub ahead of print]10
      Strategies have not been available until recently to uncover interacting protein networks specific to key cell types, their subcellular compartments, and their major regulators during complex in vivo events. Here we apply BioID2 proximity labeling to capture protein networks acting within cardiomyocytes during a key model of innate heart regeneration in zebrafish. Transgenic zebrafish expressing a promiscuous BirA2 localized to the entire myocardial cell or membrane compartment were generated, each identifying distinct proteomes in adult cardiomyocytes that became altered during regeneration. BioID2 profiling for interactors with ErbB2, a co-receptor for the cardiomyocyte mitogen Nrg1, implicated Rho A as a target of ErbB2 signaling in cardiomyocytes. Blockade of Rho A during heart regeneration, or during cardiogenic stimulation by the mitogenic influences Nrg1, Vegfaa or Vitamin D, disrupted muscle creation. Our findings reveal proximity labeling as a useful resource to interrogate cell proteomes and signaling networks during tissue regeneration in zebrafish.
    Keywords:  developmental biology; zebrafish
  35. Nat Metab. 2021 Mar;3(3): 394-409
      Both obesity and sarcopenia are frequently associated in ageing, and together may promote the progression of related conditions such as diabetes and frailty. However, little is known about the pathophysiological mechanisms underpinning this association. Here we show that systemic alanine metabolism is linked to glycaemic control. We find that expression of alanine aminotransferases is increased in the liver in mice with obesity and diabetes, as well as in humans with type 2 diabetes. Hepatocyte-selective silencing of both alanine aminotransferase enzymes in mice with obesity and diabetes retards hyperglycaemia and reverses skeletal muscle atrophy through restoration of skeletal muscle protein synthesis. Mechanistically, liver alanine catabolism driven by chronic glucocorticoid and glucagon signalling promotes hyperglycaemia and skeletal muscle wasting. We further provide evidence for amino acid-induced metabolic cross-talk between the liver and skeletal muscle in ex vivo experiments. Taken together, we reveal a metabolic inter-tissue cross-talk that links skeletal muscle atrophy and hyperglycaemia in type 2 diabetes.
  36. Methods Mol Biol. 2021 ;2252 175-188
      Robust mechanisms exist that serve to dynamically regulate the translation of mRNA into proteins across heterogeneous tissues. These processes ensure timely generation of proteins in quantities that scale with the demands of specific cell types. Importantly, this translational regulation occurs with spatiotemporal precision and is capable of recalibration as conditions change. Aberrant regulation of translation contributes to and exacerbates a wide range of diseases. Although dynamic control of translation is an essential and fundamental process shared by organisms, specific tissues and cell types can be differentially impacted by circumstances that challenge and impair basal translation, highlighting the heterogeneous nature of translational regulation. To understand how translation is differentially regulated during changing environments and across specific cells and tissues, methods capable of profiling translation in specific tissues and cells are crucial. Here, we describe a method for profiling genome-wide translation in specific tissues or cell types in Drosophila melanogaster, in which we combine ribosome affinity purification with ribosome profiling to enable a simplified protocol for robust analysis of translation in specific tissues.
    Keywords:  Drosophila; Genome-wide; Muscle; Next-generation sequencing; Ribosome profiling; Tissue-specific; Translational regulation
  37. Elife. 2021 Mar 24. pii: e59687. [Epub ahead of print]10
      Endoplasmic reticulum (ER) and mitochondria form close physical associations to facilitate calcium transfer, thereby regulating mitochondrial function. Neurons with high metabolic demands, such as sensory hair cells, are especially dependent on precisely regulated ER-mitochondria associations. We previously showed that the secreted metalloprotease Pregnancy associated plasma protein-aa (Pappaa) regulates mitochondrial function in zebrafish lateral line hair cells (Alassaf et al., 2019). Here, we show that pappaa mutant hair cells exhibit excessive and abnormally close ER-mitochondria associations, suggesting increased ER-mitochondria calcium transfer. pappaa mutant hair cells are more vulnerable to pharmacological induction of ER-calcium transfer. Additionally, pappaa mutant hair cells display ER stress and dysfunctional downstream processes of the ER-mitochondria axis including altered mitochondrial morphology and reduced autophagy. We further show that Pappaa influences ER-calcium transfer and autophagy via its ability to stimulate insulin-like growth factor-1 bioavailability. Together our results identify Pappaa as a novel regulator of the ER-mitochondria axis.
    Keywords:  cell biology; neuroscience; zebrafish
  38. Mol Genet Metab. 2021 Mar 11. pii: S1096-7192(21)00062-7. [Epub ahead of print]
      Leigh syndrome is a severe mitochondrial neurodegenerative disease with no effective treatment. In the Ndufs4-/- mouse model of Leigh syndrome, continuously breathing 11% O2 (hypoxia) prevents neurodegeneration and leads to a dramatic extension (~5-fold) in lifespan. We investigated the effect of hypoxia on the brain metabolism of Ndufs4-/- mice by studying blood gas tensions and metabolite levels in simultaneously sampled arterial and cerebral internal jugular venous (IJV) blood. Relatively healthy Ndufs4-/- and wildtype (WT) mice breathing air until postnatal age ~38 d were compared to Ndufs4-/- and WT mice breathing air until ~38 days old followed by 4-weeks of breathing 11% O2. Compared to WT control mice, Ndufs4-/- mice breathing air have reduced brain O2 consumption as evidenced by an elevated partial pressure of O2 in IJV blood (PijvO2) despite a normal PO2 in arterial blood, and higher lactate/pyruvate (L/P) ratios in IJV plasma revealed by metabolic profiling. In Ndufs4-/- mice, hypoxia treatment normalized the cerebral venous PijvO2 and L/P ratios, and decreased levels of nicotinate in IJV plasma. Brain concentrations of nicotinamide adenine dinucleotide (NAD+) were lower in Ndufs4-/- mice breathing air than in WT mice, but preserved at WT levels with hypoxia treatment. Although mild hypoxia (17% O2) has been shown to be an ineffective therapy for Ndufs4-/- mice, we find that when combined with nicotinic acid supplementation it provides a modest improvement in neurodegeneration and lifespan. Therapies targeting both brain hyperoxia and NAD+ deficiency may hold promise for treating Leigh syndrome.
    Keywords:  A-V difference; Arterial-venous difference; Arteriovenous difference; Brain; Hypoxia; Leigh syndrome; Metabolism; Metabolomics; NAD; Ndufs4; Niacin; Nicotinamide adenine dinucleotide; Nicotinic acid; O(2); Oxygen
  39. Brain. 2021 Mar 22. pii: awab135. [Epub ahead of print]
      Sensory neuronopathies are a rare and distinct subgroup of peripheral neuropathies, characterized by degeneration of the dorsal root ganglia neurons. About 50% of sensory neuronopathies are idiopathic and genetic causes remain to be clarified. Through a combination of homozygosity mapping and whole exome sequencing, we linked an autosomal recessive sensory neuronopathy to pathogenic variants in COX20 gene. We identified 8 unrelated families from the eastern China population carrying a founder variant c.41A>G (p. Lys14Arg) within COX20 in either a homozygous or compound heterozygous state. All patients displayed sensory ataxia with non-length-dependent sensory potentials decrease. COX20 encodes a key transmembrane protein implicated in the assembly of mitochondrial complex IV. We showed that COX20 variants lead to reduction of COX20 protein in patient's fibroblasts and transfected cell lines, consistent with a loss-of-function mechanism. Knockdown of COX20 expression in ND7/23 sensory neuron cells resulted in complex IV deficiency and perturbed assembly of complex IV, which subsequently compromised cell spare respiratory capacity and reduced cell proliferation under metabolic stress. Consistent with mitochondrial dysfunction in knockdown cells, reduced complex IV assembly, enzyme activity and oxygen consumption rate were also found in patients' fibroblasts. We speculated that the mechanism of COX20 was similar to other causative genes (e.g. SURF1, COX6A1, COA3 and SCO2) for peripheral neuropathies, all of which were functionally important in the structure and assembly of complex IV. Our study identifies a novel causative gene for the autosomal recessive sensory neuronopathy, whose vital function in complex IV and high expression in the proprioceptive sensory neuron further underlines loss of COX20 contributing to mitochondrial bioenergetic dysfunction as a mechanism in peripheral sensory neuron disease.
    Keywords:  COX20 gene; complex IV deficiency; loss of function; sensory neuronopathy
  40. FASEB J. 2021 Apr;35(4): e21553
      The role of mitofusin 2 (Mfn2) in the regulation of skeletal muscle (SM) mitochondria-sarcoplasmic (SR) juxtaposition, mitochondrial morphology, mitochondrial cristae density (MCD), and SM quality has not been studied in humans. In in vitro studies, whether Mfn2 increases or decreases mitochondria-SR juxtaposition remains controversial. Transmission electron microscopy (TEM) images are commonly used to measure the organelle juxtaposition, but the measurements are performed "by-hand," thus potentially leading to between-rater differences. The purposes of this study were to: (1) examine the repeatability and reproducibility of mitochondrial-SR juxtaposition measurement from TEM images of human SM between three raters with different experience and (2) compare the mitochondrial-SR juxtaposition, mitochondrial morphology, MCD (stereological-method), and SM quality (cross-sectional area [CSA] and the maximum voluntary contraction [MVC]) between subjects with high abundance (Mfn2-HA; n = 6) and low abundance (Mfn2-LA; n = 6) of Mfn2 protein. The mitochondria-SR juxtaposition had moderate repeatability and reproducibility, with the most experienced raters showing the best values. There were no differences between Mfn2-HA and Mfn2-LA groups in mitochondrial size, distance from mitochondria to SR, CSA, or MVC. Nevertheless, the Mfn2-LA group showed lower mitochondria-SR interaction, MCD, and VO2max . In conclusion, mitochondrial-SR juxtaposition measurement depends on the experience of the rater, and Mfn2 protein seems to play a role in the metabolic control of human men SM, by regulating the mitochondria-SR interaction.
    Keywords:  MICOS complex; mitochondria dynamics; organelle communication; repeatability; reproducibility; transmission electron microscopy
  41. BMC Genomics. 2021 Mar 24. 22(1): 213
      BACKGROUND: In addition to their well characterized role in cellular energy production, new evidence has revealed the involvement of mitochondria in diverse signaling pathways that regulate a broad array of cellular functions. The mitochondrial genome (mtDNA) encodes essential components of the oxidative phosphorylation (OXPHOS) pathway whose expression must be coordinated with the components transcribed from the nuclear genome. Mitochondrial dysfunction is associated with disorders including cancer and neurodegenerative diseases, yet the role of the complex interactions between the mitochondrial and nuclear genomes are poorly understood.RESULTS: Using a Drosophila model in which alternative mtDNAs are present on a common nuclear background, we studied the effects of this altered mitonuclear communication on the transcriptomic response to altered nutrient status. Adult flies with the 'native' and 'disrupted' genotypes were re-fed following brief starvation, with or without exposure to rapamycin, the cognate inhibitor of the nutrient-sensing target of rapamycin (TOR). RNAseq showed that alternative mtDNA genotypes affect the temporal transcriptional response to nutrients in a rapamycin-dependent manner. Pathways most greatly affected were OXPHOS, protein metabolism and fatty acid metabolism. A distinct set of testis-specific genes was also differentially regulated in the experiment.
    CONCLUSIONS: Many of the differentially expressed genes between alternative mitonuclear genotypes have no direct interaction with mtDNA gene products, suggesting that the mtDNA genotype contributes to retrograde signaling from mitochondria to the nucleus. The interaction of mitochondrial genotype (mtDNA) with rapamycin treatment identifies new links between mitochondria and the nutrient-sensing mTORC1 (mechanistic target of rapamycin complex 1) signaling pathway.
    Keywords:  Mitochondrial introgression; Mitonuclear genotype; Rapamycin; mTORC1
  42. Nat Commun. 2021 03 09. 12(1): 1515
      Ribosome-profiling has uncovered pervasive translation in non-canonical open reading frames, however the biological significance of this phenomenon remains unclear. Using genetic variation from 71,702 human genomes, we assess patterns of selection in translated upstream open reading frames (uORFs) in 5'UTRs. We show that uORF variants introducing new stop codons, or strengthening existing stop codons, are under strong negative selection comparable to protein-coding missense variants. Using these variants, we map and validate gene-disease associations in two independent biobanks containing exome sequencing from 10,900 and 32,268 individuals, respectively, and elucidate their impact on protein expression in human cells. Our results suggest translation disrupting mechanisms relating uORF variation to reduced protein expression, and demonstrate that translation at uORFs is genetically constrained in 50% of human genes.
  43. J Hepatol. 2021 Mar 18. pii: S0168-8278(21)00174-4. [Epub ahead of print]
      BACKGROUND & AIMS: We have previously reported that the mitochondrial dicarboxylate carrier (mDIC) is predominantly expressed in the white adipose tissue (WAT) and subject to regulation by metabolic cues. The specific physiological functions of mDIC and the reasons for its abundant presence in adipocytes are however poorly understood.METHODS: To systemically investigate the impact of mDIC function in adipocytes in vivo, we generated loss- and gain-of-function mouse models, selectively eliminating or overexpressing mDIC in mature adipocytes, respectively.
    RESULTS: In in vitro differentiated white adipocytes, mDIC is responsible for succinate transport from the mitochondrial matrix to the cytosol, from where succinate can act on the succinate receptor SUCNR1 and inhibit lipolysis by dampening the cAMP- phosphorylated hormone-sensitive lipase (pHSL) pathway. We eliminated mDIC expression in adipocytes in a doxycycline (dox)-inducible manner (mDICiKO) and demonstrated that such a deletion results in enhanced adipocyte lipolysis and promotes high-fat diet (HFD)-induced adipocyte dysfunction, liver lipotoxicity, and systemic insulin resistance. Conversely, in a mouse model with dox-inducible, adipocyte-specific overexpression of mDIC (mDICiOE), we observed suppression of adipocyte lipolysis both in vivo and ex vivo. mDICiOE mice are potently protected from liver lipotoxicity upon HFD feeding. Furthermore, they show resistance to HFD-induced weight gain and adipose tissue expansion with concomitant improvements in glucose tolerance and insulin sensitivity. Beyond our data in rodents, we found that human WAT mDIC mRNA levels are positively correlated with insulin sensitivity and negatively correlated with intrahepatic triglyceride levels, suggesting a critical role of mDIC in regulating overall metabolic homeostasis in humans as well.
    CONCLUSIONS: In summary, we highlight that mDIC plays an essential role in governing adipocyte lipolysis and preventing liver lipotoxicity under a HFD challenge.
    LAY SUMMARY: Dysfunctional fat tissue plays an important role in the development of fatty liver disease and liver injury. Our present study identifies a mitochondrial transporter, mDIC, that tightly controls the release of free fatty acids from adipocytes to the liver through the export of succinate from mitochondria. We believe this mDIC-succinate axis has potential as an area for therapeutic intervention in fatty liver disease.
    Keywords:  NAFLD; NASH; adipocytes; dicarboxylate carrier; insulin resistance; lipolysis; lipotoxicity; mitochondria; succinate
  44. Genome Med. 2021 Mar 09. 13(1): 39
      BACKGROUND: Macular telangiectasia type 2 (MacTel) is a rare, heritable and largely untreatable retinal disorder, often comorbid with diabetes. Genetic risk loci subtend retinal vascular calibre and glycine/serine/threonine metabolism genes. Serine deficiency may contribute to MacTel via neurotoxic deoxysphingolipid production; however, an independent vascular contribution is also suspected. Here, we use statistical genetics to dissect the causal mechanisms underpinning this complex disease.METHODS: We integrated genetic markers for MacTel, vascular and metabolic traits, and applied Mendelian randomisation and conditional and interaction genome-wide association analyses to discover the causal contributors to both disease and spatial retinal imaging sub-phenotypes.
    RESULTS: Genetically induced serine deficiency is the primary causal metabolic driver of disease occurrence and progression, with a lesser, but significant, causal contribution of type 2 diabetes genetic risk. Conversely, glycine, threonine and retinal vascular traits are unlikely to be causal for MacTel. Conditional regression analysis identified three novel disease loci independent of endogenous serine biosynthetic capacity. By aggregating spatial retinal phenotypes into endophenotypes, we demonstrate that SNPs constituting independent risk loci act via related endophenotypes.
    CONCLUSIONS: Follow-up studies after GWAS integrating publicly available data with deep phenotyping are still rare. Here, we describe such analysis, where we integrated retinal imaging data with MacTel and other traits genomics data to identify biochemical mechanisms likely causing this disorder. Our findings will aid in early diagnosis and accurate prognosis of MacTel and improve prospects for effective therapeutic intervention. Our integrative genetics approach also serves as a useful template for post-GWAS analyses in other disorders.
    Keywords:  GWAS; Mendelian randomisation; Metabolomics; Retinal disease; Serine
  45. Am J Hum Genet. 2021 Mar 17. pii: S0002-9297(21)00094-X. [Epub ahead of print]
      The increasing scope of genetic testing allowed by next-generation sequencing (NGS) dramatically increased the number of genetic variants to be interpreted as pathogenic or benign for adequate patient management. Still, the interpretation process often fails to deliver a clear classification, resulting in either variants of unknown significance (VUSs) or variants with conflicting interpretation of pathogenicity (CIP); these represent a major clinical problem because they do not provide useful information for decision-making, causing a large fraction of genetically determined disease to remain undertreated. We developed a machine learning (random forest)-based tool, RENOVO, that classifies variants as pathogenic or benign on the basis of publicly available information and provides a pathogenicity likelihood score (PLS). Using the same feature classes recommended by guidelines, we trained RENOVO on established pathogenic/benign variants in ClinVar (training set accuracy = 99%) and tested its performance on variants whose interpretation has changed over time (test set accuracy = 95%). We further validated the algorithm on additional datasets including unreported variants validated either through expert consensus (ENIGMA) or laboratory-based functional techniques (on BRCA1/2 and SCN5A). On all datasets, RENOVO outperformed existing automated interpretation tools. On the basis of the above validation metrics, we assigned a defined PLS to all existing ClinVar VUSs, proposing a reclassification for 67% with >90% estimated precision. RENOVO provides a validated tool to reduce the fraction of uninterpreted or misinterpreted variants, tackling an area of unmet need in modern clinical genetics.
    Keywords:  ClinVar; VUS; machine learning; reclassification; variant interpretation
  46. Nat Rev Mol Cell Biol. 2021 Mar 26.
      In animals, systemic control of metabolism is conducted by metabolic tissues and relies on the regulated circulation of a plethora of molecules, such as hormones and lipoprotein complexes. MicroRNAs (miRNAs) are a family of post-transcriptional gene repressors that are present throughout the animal kingdom and have been widely associated with the regulation of gene expression in various contexts, including virtually all aspects of systemic control of metabolism. Here we focus on glucose and lipid metabolism and review current knowledge of the role of miRNAs in their systemic regulation. We survey miRNA-mediated regulation of healthy metabolism as well as the contribution of miRNAs to metabolic dysfunction in disease, particularly diabetes, obesity and liver disease. Although most miRNAs act on the tissue they are produced in, it is now well established that miRNAs can also circulate in bodily fluids, including their intercellular transport by extracellular vesicles, and we discuss the role of such extracellular miRNAs in systemic metabolic control and as potential biomarkers of metabolic status and metabolic disease.
  47. Nat Commun. 2021 Mar 26. 12(1): 1905
      Brown and beige adipose tissue are emerging as distinct endocrine organs. These tissues are functionally associated with skeletal muscle, adipose tissue metabolism and systemic energy expenditure, suggesting an interorgan signaling network. Using metabolomics, we identify 3-methyl-2-oxovaleric acid, 5-oxoproline, and β-hydroxyisobutyric acid as small molecule metabokines synthesized in browning adipocytes and secreted via monocarboxylate transporters. 3-methyl-2-oxovaleric acid, 5-oxoproline and β-hydroxyisobutyric acid induce a brown adipocyte-specific phenotype in white adipocytes and mitochondrial oxidative energy metabolism in skeletal myocytes both in vitro and in vivo. 3-methyl-2-oxovaleric acid and 5-oxoproline signal through cAMP-PKA-p38 MAPK and β-hydroxyisobutyric acid via mTOR. In humans, plasma and adipose tissue 3-methyl-2-oxovaleric acid, 5-oxoproline and β-hydroxyisobutyric acid concentrations correlate with markers of adipose browning and inversely associate with body mass index. These metabolites reduce adiposity, increase energy expenditure and improve glucose and insulin homeostasis in mouse models of obesity and diabetes. Our findings identify beige adipose-brown adipose-muscle physiological metabokine crosstalk.
  48. Nat Metab. 2021 Mar;3(3): 428-441
      Obesity reduces adipocyte mitochondrial function, and expanding adipocyte oxidative capacity is an emerging strategy to improve systemic metabolism. Here, we report that serine/threonine-protein kinase 3 (STK3) and STK4 are key physiological suppressors of mitochondrial capacity in brown, beige and white adipose tissues. Levels of STK3 and STK4, kinases in the Hippo signalling pathway, are greater in white than brown adipose tissues, and levels in brown adipose tissue are suppressed by cold exposure and greatly elevated by surgical denervation. Genetic inactivation of Stk3 and Stk4 increases mitochondrial mass and function, stabilizes uncoupling protein 1 in beige adipose tissue and confers resistance to metabolic dysfunction induced by high-fat diet feeding. Mechanistically, STK3 and STK4 increase adipocyte mitophagy in part by regulating the phosphorylation and dimerization status of the mitophagy receptor BNIP3. STK3 and STK4 expression levels are elevated in human obesity, and pharmacological inhibition improves metabolic profiles in a mouse model of obesity, suggesting STK3 and STK4 as potential targets for treating obesity-related diseases.
  49. Sci Adv. 2021 Mar;pii: eabc1834. [Epub ahead of print]7(13):
      Cyclic GMP-AMP synthase (cGAS) functions as an essential DNA sensor, which senses the cytoplasmic double-stranded DNA and activates the antiviral response. However, the posttranslational modification of cGAS remains to be fully understood and whether it has arginine methylation modification remains unknown. Here, we identified protein arginine methyltransferase 5 (PRMT5) as a direct binding partner of cGAS, and it catalyzed the arginine symmetrical dimethylation of cGAS at the Arg124 residue. Further investigation demonstrated that methylation of cGAS by PRMT5 attenuated cGAS-mediated antiviral immune response by blocking the DNA binding ability of cGAS. Oral administration of PRMT5 inhibitors significantly protected mice from HSV-1 infection and prolonged the survival time of these infected mice. Therefore, our findings revealed an essential regulatory effect of PRMT5 on cGAS-mediated antiviral immune response and provided a promising potential antiviral strategy by modulating PRMT5.
  50. Eur J Med Genet. 2021 Mar 18. pii: S1769-7212(21)00061-6. [Epub ahead of print] 104195
      The cytochrome c-oxidase (COX) enzyme, also known as mitochondrial complex IV (MT-C4D), is a transmembrane protein complex found in mitochondria. COX deficiency is one of the most frequent causes of electron transport chain defects in humans. Therefore, high energy demand organs and tissues are affected in patients with mutations in the COX15 gene, with variable phenotypic expressiveness. We describe the case of a male newborn with hypertrophic cardiomyopathy and serum and cerebrospinal fluid hyperlacticaemia, whose exome sequencing revealed two variants in a compound heterozygous state: c.232G>A;p.(Gly78Arg), classified as likely pathogenic, and c.452C>G;p.(Ser151Ter), as pathogenic; the former never previously described in the literature.
    Keywords:  COX15; Cytochrome c oxidase; complex IV; hyperlacticaemia; hypertrophic cardiomyopathy
  51. Biochim Biophys Acta Gen Subj. 2021 Mar 19. pii: S0304-4165(21)00052-0. [Epub ahead of print] 129894
      Parkin and phosphatase and tensin homolog (PTEN)-induced kinase 1 (PINK1) constitute a feed-forward signalling pathway that mediates autophagic removal of damaged mitochondria (mitophagy). With over 130 mutations identified to date in over 1000 patients with early onset parkinsonism, Parkin is considered a hot spot of signalling pathways involved in PD aetiology. Parkin is an E3 ligase and how its activity is regulated has been extensively studied: inter-domain interactions exert a tight inhibition on Parkin activity; binding to phospho-ubiquitin relieves this auto-inhibition; and phosphorylation of Parkin shifts the equilibrium towards maximal Parkin activation. This review focusses on recent, structural findings on the regulation of Parkin activity. What follows is a mechanistic introduction to the family of E3 ligases that includes Parkin, followed by a brief description of structural elements unique to Parkin that lock the enzyme in an autoinhibited state, contrasted with emerging models that have shed light on possible mechanisms of Parkin activation.
    Keywords:  Autoinhibition; E3; Parkin; RBR; Ubiquitylation
  52. PLoS One. 2021 ;16(3): e0248554
      Mitochondrial dynamics is a possible modulator of myocardial ischemia/reperfusion injuries (IRI). We previously reported that mice partially deficient in the fusion protein OPA1 exhibited higher IRI. Therefore, we investigated whether deficiency in the fission protein DRP1 encoded by Dnm1l gene would affect IRI in Dnm1l+/- mouse. After baseline characterization of the Dnm1l+/- mice heart, using echocardiography, electron microscopy, and oxygraphy, 3-month-old Dnm1l+/- and wild type (WT) mice were exposed to myocardial ischemia/reperfusion (I/R). The ischemic area-at-risk (AAR) and area of necrosis (AN) were delimited, and the infarct size was expressed by AN/AAR. Proteins involved in mitochondrial dynamics and autophagy were analyzed before and after I/R. Mitochondrial permeability transition pore (mPTP) opening sensitivity was assessed after I/R. Heart weight and left ventricular function were not significantly different in 3-, 6- and 12-month-old Dnm1l+/- mice than in WT. The cardiac DRP1 protein expression levels were 60% lower, whereas mitochondrial area and lipid degradation were significantly higher in Dnm1l+/- mice than in WT, though mitochondrial respiratory parameters and mPTP opening did not significantly differ. Following I/R, the infarct size was significantly smaller in Dnm1l+/- mice than in WT (34.6±3.1% vs. 44.5±3.3%, respectively; p<0.05) and the autophagic markers, LC3 II and P62 were significantly increased compared to baseline condition in Dnm1l+/- mice only. Altogether, data indicates that increasing fusion by means of Dnm1l deficiency was associated with protection against IRI, without alteration in cardiac or mitochondrial functions at basal conditions. This protection mechanism due to DRP1 haploinsufficiency increases the expression of autophagic markers.