bims-mitdis Biomed News
on Mitochondrial Disorders
Issue of 2021‒03‒14
28 papers selected by
Catalina Vasilescu
University of Helsinki

  1. Cell Calcium. 2021 Feb 23. pii: S0143-4160(21)00037-3. [Epub ahead of print]96 102383
      Astrocytes govern critical aspects of brain function via spontaneous calcium signals in their soma and processes. A significant proportion of these spontaneous astrocytic calcium events are associated with mitochondria, however, the extent, sources, or kinetics of astrocytic mitochondrial calcium influx have not been studied in the adult mouse brain. To measure calcium influx into astrocytic mitochondria in situ, we generated an adeno-associated virus (AAV) with the astrocyte-specific GfaABC1D promoter driving expression of the genetically encoded calcium indicator, GCaMP6f tagged to mito7, a mitochondrial matrix targeted signal sequence. Using this construct, we observed AAV-mediated expression of GCaMP6f in adult mouse astrocytic mitochondria that co-localized with MitoTracker deep red (MTDR) in the dorsolateral striatum (DLS) and in the hippocampal stratum radiatum (HPC). Astrocytic mitochondria co-labeled with MTDR and GCaMP6f displayed robust, spontaneous calcium influx events in situ, with subcellular differences in calcium influx kinetics between somatic, branch, and branchlet mitochondria, and inter-regional differences between mitochondria in DLS and HPC astrocytes. Calcium influx into astrocytic mitochondria was strongly dependent on endoplasmic reticulum calcium stores, but did not require the mitochondrial calcium uniporter, MCU. Exposure to either glutamate, D1 or D2 dopamine receptor agonists increased calcium influx in some mitochondria, while simultaneously decreasing calcium influx in other mitochondria from the same astrocyte. These findings show that astrocytic mitochondria possess unique properties with regard to their subcellular morphology, mechanisms of calcium influx, and responses to neurotransmitter receptor agonists. Our results have important implications for understanding the role of astrocytic mitochondria during pathological processes.
    Keywords:  Astrocyte; Calcium influx; Hippocampus; MCU; Mitochondria; Striatum
  2. Ann Neurol. 2021 Mar 11.
      A rapidly expanding catalogue of neurogenetic disorders has encouraged a diagnostic shift towards early clinical whole exome sequencing (WES). Adult primary mitochondrial diseases (PMDs) frequently exhibit neurological manifestations that overlap with other nervous system disorders. However, mitochondrial DNA (mtDNA) is not routinely analyzed in standard clinical WES bioinformatic pipelines. We reanalyzed 11,424 exomes, enriched with neurological diseases, for pathogenic mtDNA variants. Twenty-four different mtDNA mutations were detected in 64 exomes, 11 of which were considered disease causing based on the associated clinical phenotypes. These findings highlight the diagnostic uplifts gained by analyzing mtDNA from WES data in neurological diseases. This article is protected by copyright. All rights reserved.
  3. Stem Cell Res Ther. 2021 Mar 12. 12(1): 177
      Current methods to differentiate cardiomyocytes from human pluripotent stem cells (PSCs) inadequately recapitulate complete development and result in PSC-derived cardiomyocytes (PSC-CMs) with an immature or fetal-like phenotype. Embryonic and fetal development are highly dynamic periods during which the developing embryo or fetus is exposed to changing nutrient, oxygen, and hormone levels until birth. It is becoming increasingly apparent that these metabolic changes initiate developmental processes to mature cardiomyocytes. Mitochondria are central to these changes, responding to these metabolic changes and transitioning from small, fragmented mitochondria to large organelles capable of producing enough ATP to support the contractile function of the heart. These changes in mitochondria may not simply be a response to cardiomyocyte maturation; the metabolic signals that occur throughout development may actually be central to the maturation process in cardiomyocytes. Here, we review methods to enhance maturation of PSC-CMs and highlight evidence from development indicating the key roles that mitochondria play during cardiomyocyte maturation. We evaluate metabolic transitions that occur during development and how these affect molecular nutrient sensors, discuss how regulation of nutrient sensing pathways affect mitochondrial dynamics and function, and explore how changes in mitochondrial function can affect metabolite production, the cell cycle, and epigenetics to influence maturation of cardiomyocytes.
    Keywords:  Cardiomyocytes; Maturation; Metabolic regulation; Mitochondria; Stem cells
  4. J Am Chem Soc. 2021 Mar 12.
      Mitochondria are the site of aerobic respiration, producing ATP via oxidative phosphorylation as protons flow down their electrochemical gradient through ATP synthase. This negative membrane potential across the inner mitochondrial membrane (ΔΨm) represents a fundamental biophysical parameter central to cellular life. Traditional, electrode-based methods for recording membrane potential are impossible to implement on mitochondria within intact cells. Fluorescent ΔΨm indicators based on cationic, lipophilic dyes are a common alternative, but these indicators are complicated by concentration-dependent artifacts and the requirement to maintain dye in the extracellular solution to visualize reversible ΔΨm dynamics. Here, we report the first example of a fluorescent ΔΨm reporter that does not rely on ΔΨm-dependent accumulation. We redirected the localization of a photoinduced electron transfer (PeT)-based indicator, Rhodamine Voltage Reporter (RhoVR), to mitochondria by masking the carboxylate of RhoVR 1 as an acetoxymethyl (AM) ester. Once within mitochondria, esterases remove the AM ester, trapping RhoVR inside of the mitochondrial matrix, where it can incorporate within the inner membrane and reversibly report on changes in ΔΨm. We show that this Small molecule, Permeable, Internally Redistributing for Inner membrane Targeting Rhodamine Voltage Reporter, or SPIRIT RhoVR, localizes to mitochondria across a number of different cell lines and responds reversibly to changes in ΔΨm induced by exceptionally low concentrations of the uncoupler FCCP without the need for exogenous pools of dye (unlike traditional, accumulation-based rhodamine esters). SPIRIT RhoVR is compatible with multi-color imaging, enabling simultaneous, real-time observation of cytosolic Ca2+, plasma membrane potential, and reversible ΔΨm dynamics.
  5. Front Immunol. 2021 ;12 564720
      Neutrophil granulocytes represent the first line of defense against invading pathogens. In addition to the production of Reactive Oxygen Species, degranulation, and phagocytosis, these specialized cells are able to extrude Neutrophil Extracellular Traps. Extensive work was done to elucidate the mechanism of this special form of cell death. However, the exact mechanisms are still not fully uncovered. Here we demonstrate that the small GTPase Cdc42 is a negative regulator of NET formation in primary human and murine neutrophils. We present a functional role for Cdc42 activity in NET formation that differs from the already described NETosis pathways. We show that Cdc42 deficiency induces NETs independent of the NADPH-oxidase but dependent on protein kinase C. Furthermore, we demonstrate that Cdc42 deficiency induces NETosis through activation of SK-channels and that mitochondria play a crucial role in this process. Our data therefore suggests a mechanistic role for Cdc42 activity in primary human neutrophils, and identify Cdc42 activity as a target to modulate the formation of Neutrophil Extracellular Traps.
    Keywords:  Cdc42; mitochondria; mitochondrial ROS; mitochondrial membrane potential; neutrophil extracellular traps; neutrophil granulocytes; small GTPases
  6. Redox Biol. 2021 Feb 28. pii: S2213-2317(21)00069-0. [Epub ahead of print]41 101921
      Mitochondria participate in various metabolic pathways, and their dysregulation results in multiple disorders, including aging-related diseases. However, the metabolic changes and mechanisms of mitochondrial disorders are not fully understood. Here, we found that induced pluripotent stem cells (iPSCs) from a patient with mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes (MELAS) showed attenuated proliferation and survival when glycolysis was inhibited. These deficits were rescued by taurine administration. Metabolomic analyses showed that the ratio of the reduced (GSH) to oxidized glutathione (GSSG) was decreased; whereas the levels of cysteine, a substrate of GSH, and oxidative stress markers were upregulated in MELAS iPSCs. Taurine normalized these changes, suggesting that MELAS iPSCs were affected by the oxidative stress and taurine reduced its influence. We also analyzed the retinal pigment epithelium (RPE) differentiated from MELAS iPSCs by using a three-dimensional culture system and found that it showed epithelial mesenchymal transition (EMT), which was suppressed by taurine. Therefore, mitochondrial dysfunction caused metabolic changes, accumulation of oxidative stress that depleted GSH, and EMT in the RPE that could be involved in retinal pathogenesis. Because all these phenomena were sensitive to taurine treatment, we conclude that administration of taurine may be a potential new therapeutic approach for mitochondria-related retinal diseases.
    Keywords:  Epithelial mesenchymal transition; Induced pluripotent stem cells; Metabolomics; Mitochondria; Retinal pigment epithelium; Taurine
  7. Free Radic Biol Med. 2021 Mar 03. pii: S0891-5849(21)00139-8. [Epub ahead of print]
      Mitochondria are essential signaling organelles that regulate a broad range of cellular processes and thereby heart function. Multiple mechanisms participate in the communication between mitochondria and the nucleus that maintain cardiomyocyte homeostasis, including mitochondrial reactive oxygen species (ROS) and metabolic shifts in TCA cycle metabolite availability. An increased rate of ROS generation can cause irreversible damage to the cell and proposed to be a leading cause of many pathologies, including accelerated aging and heart disease. Myocardial impairments are also characterised by specific coordinated metabolic changes and dysregulated inflammatory responses. Hence, the mitochondrial respiratory chain is an important mediator between health and disease in the heart. This review will first outline the sources of ROS in the heart, mitochondrial metabolite dynamics, and provide an overview of their implications for heart disease. In addition, we will concentrate our discussion around current cardioprotective strategies relevant to mitochondrial ROS. Thorough understanding of mitochondrial signaling and the complex interplay with vital signaling pathways in the heart might allow us to develop novel therapeutic approaches to cardiovascular disease.
    Keywords:  Cardiovascular disease; ROS; metabolism; mitochondria; redox signaling
  8. Biochim Biophys Acta Mol Cell Res. 2021 Mar 05. pii: S0167-4889(21)00052-5. [Epub ahead of print]1868(6): 118998
      Mitochondria are involved in a large number of essential roles related to neuronal function. Ca2+ handling by mitochondria is critical for many of these functions, including energy production and cellular fate. Conversely, mitochondrial Ca2+ mishandling has been related to a variety of neurodegenerative diseases. Investigating mitochondrial Ca2+ dynamics is essential for advancing our understanding of the role of intracellular mitochondrial Ca2+ signals in physiology and pathology. Improved Ca2+ indicators, and the ability to target them to different cells and compartments, have emerged as useful tools for analysis of Ca2+ signals in living organisms. Combined with state-of-the-art techniques such as multiphoton microscopy, they allow for the study of mitochondrial Ca2+ dynamics in vivo in mouse models of the disease. Here, we provide an overview of the Ca2+ transporters/ion channels in mitochondrial membranes, and the involvement of mitochondrial Ca2+ in neurodegenerative diseases followed by a summary of the main tools available to evaluate mitochondrial Ca2+ dynamics in vivo using the aforementioned technique.
    Keywords:  Alzheimer's disease; Calcium; Fluorescent proteins; GECIs; Mitochondria; Multiphoton microscopy
  9. Nat Protoc. 2021 Mar 08.
      The stable maintenance of DNA methylation patterns during mitotic cell division is crucial for cell identity. Precisely determining the maintenance kinetics and dissecting the exact contributions of relevant regulators requires a method to accurately measure parent and daughter strand DNA methylation at the same time, ideally at the single-molecule level. Recently, we developed a method referred to as Hammer-seq (hairpin-assisted mapping of methylation of replicated DNA) that fulfils the above criteria. This method integrates 5-ethynyl-2'-deoxyuridine (EdU) labeling of replicating DNA, biotin conjugation and streptavidin-based affinity purification, and whole-genome hairpin bisulfite sequencing technologies. Hammer-seq offers the unique advantage of simultaneously measuring the methylation status of parent and daughter strands within a single DNA molecule, which makes it possible to determine maintenance kinetics across various genomic regions without averaging effects from bulk measurements and to assess de novo methylation events that accompany methylation maintenance. Importantly, when combined with mutant cell lines in which mechanisms of interest are disrupted, Hammer-seq can be applied to determine the functional contributions of potential regulators to methylation maintenance, with accurate kinetics information that cannot be acquired with other currently available methods. Hammer-seq library preparation requires ~100 ug EdU-labeled genomic DNA as input (~15 million mammalian cells). The whole protocol, from pulse labeling to library construction, can be completed within 2-3 d, depending on the chasing time.
  10. Genome Med. 2021 Mar 10. 13(1): 41
      Genome-wide association studies (GWAS) have uncovered thousands of genetic variants that influence risk for human diseases and traits. Yet understanding the mechanisms by which these genetic variants, mainly noncoding, have an impact on associated diseases and traits remains a significant hurdle. In this review, we discuss emerging experimental approaches that are being applied for functional studies of causal variants and translational advances from GWAS findings to disease prevention and treatment. We highlight the use of genome editing technologies in GWAS functional studies to modify genomic sequences, with proof-of-principle examples. We discuss the challenges in interrogating causal variants, points for consideration in experimental design and interpretation of GWAS locus mechanisms, and the potential for novel therapeutic opportunities. With the accumulation of knowledge of functional genetics, therapeutic genome editing based on GWAS discoveries will become increasingly feasible.
    Keywords:  CRISPR/Cas; GWAS; Genome editing; High throughput
  11. Free Radic Biol Med. 2021 Mar 04. pii: S0891-5849(21)00144-1. [Epub ahead of print]
      Primary Coenzyme Q (CoQ) deficiencies are clinically heterogeneous conditions and lack clear genotype-phenotype correlations, complicating diagnosis and prognostic assessment. Here we present a compilation of all the symptoms and patients with primary CoQ deficiency described in the literature so far and analyse the most common clinical manifestations associated with pathogenic variants identified in the different COQ genes. In addition, we identified new associations between the age of onset of symptoms and different pathogenic variants, which could help to a better diagnosis and guided treatment. To make these results usable for clinicians, we created an online platform ( about clinical manifestations of primary CoQ deficiency that will be periodically updated to incorporate new information published in the literature. As CoQ primary deficiency is a rare disease, the available data are still limited, but as new patients are added over time, this tool could become a key resource for a more efficient diagnosis of primary CoQ deficiencies.
    Keywords:  Coenzyme Q primary deficiency; genotype-phenotype correlation; mitochondrial diseases; rare diseases; web-based live platform
  12. Cell Rep. 2021 Mar 09. pii: S2211-1247(21)00141-8. [Epub ahead of print]34(10): 108827
      Calcium transfer from the endoplasmic reticulum (ER) to mitochondria is a critical contributor to apoptosis. B cell lymphoma 2 (BCL-2) ovarian killer (BOK) localizes to the ER and binds the inositol 1,4,5-trisphosophate receptor (IP3R). Here, we show that BOK is necessary for baseline mitochondrial calcium levels and stimulus-induced calcium transfer from the ER to the mitochondria. Murine embryonic fibroblasts deficient for BOK have decreased proximity of the ER to the mitochondria and altered protein composition of mitochondria-associated membranes (MAMs), which form essential calcium microdomains. Rescue of the ER-mitochondrial juxtaposition with drug-inducible interorganelle linkers reveals a kinetic disruption, which when overcome in Bok-/- cells is still insufficient to rescue thapsigargin-induced calcium transfer and apoptosis. Likewise, a BOK mutant unable to interact with IP3R restores ER-mitochondrial proximity, but not ER-mitochondrial calcium transfer, MAM protein composition, or apoptosis. This work identifies the dynamic coordination of ER-mitochondrial contact by BOK as an important control point for apoptosis.
    Keywords:  BCL-2 family; BOK; IP3R; MAMs; MERCs; apoptosis; calcium; endoplasmic reticulum; mitochondria-ER contact sites; mitochondria-associated membranes
  13. Cell Calcium. 2021 Feb 22. pii: S0143-4160(21)00036-1. [Epub ahead of print]96 102382
      Mitochondrial Ca2+ transport is essential for regulating cell bioenergetics, Ca2+ signaling and cell death. Mitochondria accumulate Ca2+ via the mitochondrial Ca2+ uniporter (MCU), whereas Ca2+ is extruded by the mitochondrial Na+/Ca2+ (mtNCX) and H+/Ca2+ exchangers. The balance between these processes is essential for preventing toxic mitochondrial Ca2+ overload. Recent work demonstrated that MCU activity varies significantly among tissues, likely reflecting tissue-specific Ca2+ signaling and energy needs. It is less clear whether this diversity in MCU activity is matched by tissue-specific diversity in mitochondrial Ca2+ extrusion. Here we compared properties of mitochondrial Ca2+ extrusion in three tissues with prominent mitochondria function: brain, heart and liver. At the transcript level, expression of the Na+/Ca2+/Li+ exchanger (NCLX), which has been proposed to mediate mtNCX transport, was significantly greater in liver than in brain or heart. At the functional level, Na+ robustly activated Ca2+ efflux from brain and heart mitochondria, but not from liver mitochondria. The mtNCX inhibitor CGP37157 blocked Ca2+ efflux from brain and heart mitochondria but had no effect in liver mitochondria. Replacement of Na+ with Li+ to test the involvement of NCLX, resulted in a slowing of mitochondrial Ca2+ efflux by ∼70 %. Collectively, our findings suggest that mtNCX is responsible for Ca2+ extrusion from the mitochondria of the brain and heart, but plays only a small, if any, role in mitochondria of the liver. They also reveal that Li+ is significantly less effective than Na+ in driving mitochondrial Ca2+ efflux.
    Keywords:  Ca(2+) transport; Hippocampal neurons; Mitochondria; NCLX; NCX
  14. Redox Biol. 2021 Mar 03. pii: S2213-2317(21)00065-3. [Epub ahead of print]41 101917
      Cells naturally produce mitochondrial reactive oxygen species (mROS), but the in vivo pathophysiological significance has long remained controversial. Within the brain, astrocyte-derived mROS physiologically regulate behaviour and are produced at one order of magnitude faster than in neurons. However, whether neuronal mROS abundance differentially impacts on behaviour is unknown. To address this, we engineered genetically modified mice to down modulate mROS levels in neurons in vivo. Whilst no alterations in motor coordination were observed by down modulating mROS in neurons under healthy conditions, it prevented the motor discoordination caused by the pro-oxidant neurotoxin, 3-nitropropionic acid (3-NP). In contrast, abrogation of mROS in astrocytes showed no beneficial effect against the 3-NP insult. These data indicate that the impact of modifying mROS production on mouse behaviour critically depends on the specific cell-type where they are generated.
    Keywords:  Astrocyte; In vivo; Mitochondria; Neuron; ROS; Signallling
  15. J Neurol. 2021 Mar 11.
      COQ4 is a component of an enzyme complex involved in the biosynthesis of coenzyme Q10 (CoQ10), a molecule with primary importance in cell metabolism. Mutations in the COQ4 gene are responsible for mitochondrial diseases showing heterogeneous age at onset, clinical presentations and association with CoQ10 deficiency. We herein expand the phenotypic and genetic spectrum of COQ4-related diseases, by reporting two patients harboring bi-allelic variants but not showing CoQ10 deficiency. One patient was found to harbor compound heterozygous mutations (specifically, c.577C>T/p.Pro193Ser and the previously reported c.718C>T/p.Arg240Cys) associated with progressive spasticity, while the other harbored two novel missense (c.284G>A/p.Gly95Asp and c.305G>A/p.Arg102His) associated with a neurodevelopmental disorder. Both patients presented motor impairment and ataxia. To further understand the role of COQ4, we performed functional studies in patient-derived fibroblasts, yeast and "crispant" zebrafish larvae. Micro-oxygraphy showed impaired oxygen consumption rates in one patient, while yeast complementation assays showed that all the mutations were presumably disease related. Moreover, characterization of the coq4 F0 CRISPR zebrafish line showed motor defects and cell reduction in a specific area of the hindbrain, a region reminiscent of the human cerebellum. Our expanded phenotype associated with COQ4 mutations allowed us to investigate, for the first time, the role of COQ4 in brain development in vivo.
    Keywords:  Ataxia; COQ4; CRISPR; Cas9; Yeast complementation assay; Zebrafish
  16. Circulation. 2021 Mar 12.
      Background: Marfan syndrome (MFS) is an autosomal dominant disorder of the connective tissue caused by mutations in the FBN1 gene encoding a large glycoprotein in the extracellular matrix called fibrillin-1. The major complication of this connective disorder is the risk to develop thoracic aortic aneurysm (TAA). To date, no effective pharmacological therapies have been identified for the management of thoracic aortic disease and the only options capable of preventing aneurysm rupture are endovascular repair or open surgery. Here, we have studied the role of mitochondrial dysfunction in the progression of thoracic aortic aneurysm and mitochondrial boosting strategies as a potential treatment to managing aortic aneurysms. Methods: Combining transcriptomics and metabolic analysis of aortas from a Marfan mouse model (Fbn1c1039g/+) and MFS-patients, we have identified mitochondrial dysfunction alongside with mtDNA depletion as a new hallmark of aortic aneurysm disease in MFS. To demonstrate the importance of mitochondrial decline in the development of aneurysms, we generated a conditional mouse model with mitochondrial dysfunction specifically in vascular smooth muscle cells (VSMCs) by conditional depleting mitochondrial transcription factor A (Tfam) (Myh11-CreERT2Tfamflox/flox mice). We have used a mouse model of Marfan syndrome to test for drugs that can revert aortic disease by enhancing Tfam levels and mitochondrial respiration Results: The main canonical pathways highlighted in the transcriptomic analysis in aortas from Fbn1c1039g/+ mice were those related to metabolic function, such as mitochondrial dysfunction. Mitochondrial complexes, whose transcription depends on Tfam and mitochondrial-DNA content, were reduced in aortas from young Fbn1c1039g/+ mice. In vitro experiments in Fbn1-silenced VSMCs presented increased lactate production and decreased oxygen consumption. Similar results were found in MFS-patients. VSMCs seeded in matrices produced by Fbn1-deficient VSMCs undergo mitochondrial dysfunction. Conditional Tfam-deficient-VSMCs mice, lose their contractile capacity, showed aortic aneurysms and died prematurely. Restoring mitochondrial metabolism with the NAD precursor nicotinamide riboside (NR) rapidly reverses aortic aneurysm in Fbn1c1039g/+ mice. Conclusions: Mitochondrial function of VSMCs is controlled by the extracellular matrix and drives the development of aortic aneurysm in Marfan Syndrome. Targeting vascular metabolism is a new available therapeutic strategy for managing aortic aneurysms associated with genetic disorders.
    Keywords:  Tfam; aortopathies; genetic disorders
  17. JCI Insight. 2021 Mar 09. pii: 147193. [Epub ahead of print]
      Liver regeneration is critical to survival after traumatic injuries, exposure to hepatotoxins, or surgical interventions, yet the underlying signaling and metabolic pathways remain unclear. Here we show that hepatocyte-specific loss of the mitochondrial deacetylase SIRT3 drastically impairs regeneration and worsens mitochondrial function after partial hepatectomy. Sirtuins, including SIRT3, require nicotinamide adenine dinucleotide (NAD) as a cosubstrate. We previously showed that the NAD precursor nicotinamide riboside (NR) promotes liver regeneration, but whether this involves sirtuins has not been tested. Here we show that despite their NAD-dependence and critical roles in regeneration, neither SIRT3 nor its nuclear counterpart SIRT1 is required for NR to enhance liver regeneration. NR improves mitochondrial respiration in regenerating wild type or mutant livers and rapidly increases oxygen consumption and glucose output in cultured hepatocytes. Our data support a direct enhancement of mitochondrial redox metabolism as the mechanism mediating improved liver regeneration after NAD supplementation and exclude signaling via SIRT1 and SIRT3. Thus, we provide the first evidence for an essential role for a mitochondrial sirtuin during liver regeneration and insight into the beneficial effects of NR.
    Keywords:  Fatty acid oxidation; Hepatology; Metabolism; Mitochondria; Molecular pathology
  18. Autophagy. 2021 Mar 08. 1-10
      PINK1 and PRKN, which cause Parkinson disease when mutated, form a quality control mitophagy pathway that is well-characterized in cultured cells. The extent to which the PINK1-PRKN pathway contributes to mitophagy in vivo, however, is controversial. This is due in large part to conflicting results from studies using one of two mitophagy reporters: mt-Keima or mito-QC. Studies using mt-Keima have generally detected PINK1-PRKN mitophagy in vivo, whereas those using mito-QC generally have not. Here, we directly compared the performance of mito-QC and mt-Keima in cell culture and in mice subjected to a PINK1-PRKN activating stress. We found that mito-QC was less sensitive than mt-Keima for mitophagy, and that this difference was more pronounced for PINK1-PRKN mitophagy. These findings suggest that mito-QC's poor sensitivity may account for conflicting reports of PINK1-PRKN mitophagy in vivo and caution against using mito-QC as a reporter for PINK1-PRKN mitophagy.
    Keywords:  Autophagy; PARK2; PARKIN; Parkinson; degradation; disease; mitochondria; neurodegeneration; organelle
  19. Blood Adv. 2021 Mar 23. 5(6): 1605-1616
      Hematopoietic cell transplantation is a critical curative approach for many blood disorders. However, obtaining grafts with sufficient numbers of hematopoietic stem cells (HSCs) that maintain long-term engraftment remains challenging; this is due partly to metabolic modulations that restrict the potency of HSCs outside of their native environment. To address this, we focused on mitochondria. We found that human HSCs are heterogeneous in their mitochondrial activity as measured by mitochondrial membrane potential (MMP) even within the highly purified CD34+CD38-CD45RA-CD90+CD49f+ HSC population. We further found that the most potent HSCs exhibit the lowest mitochondrial activity in the population. We showed that the frequency of long-term culture initiating cells in MMP-low is significantly greater than in MMP-high CD34+CD38-CD45RA-CD90+ (CD90+) HSCs. Notably, these 2 populations were distinct in their long-term repopulating capacity when transplanted into immunodeficient mice. The level of chimerism 7 months posttransplantation was >50-fold higher in the blood of MMP-low relative to MMP-high CD90+ HSC recipients. Although more than 90% of both HSC subsets were in G0, MMP-low CD90+ HSCs exhibited delayed cell-cycle priming profile relative to MMP-high HSCs. These functional differences were associated with distinct mitochondrial morphology; MMP-low in contrast to MMP-high HSCs contained fragmented mitochondria. Our findings suggest that the lowest MMP level selects for the most potent, likely dormant, stem cells within the highly purified HSC population. These results identify a new approach for isolating highly potent human HSCs for further clinical applications. They also implicate mitochondria in the intrinsic regulation of human HSC quiescence and potency.
  20. Proc Natl Acad Sci U S A. 2021 Mar 16. pii: e2025022118. [Epub ahead of print]118(11):
      Human High temperature requirement A2 (HtrA2) is a mitochondrial protease chaperone that plays an important role in cellular proteostasis and in regulating cell-signaling events, with aberrant HtrA2 function leading to neurodegeneration and parkinsonian phenotypes. Structural studies of the enzyme have established a trimeric architecture, comprising three identical protomers in which the active sites of each protease domain are sequestered to form a catalytically inactive complex. The mechanism by which enzyme function is regulated is not well understood. Using methyl transverse relaxation optimized spectroscopy (TROSY)-based solution NMR in concert with biochemical assays, a functional HtrA2 oligomerization/binding cycle has been established. In the absence of substrates, HtrA2 exchanges between a heretofore unobserved hexameric conformation and the canonical trimeric structure, with the hexamer showing much weaker affinity toward substrates. Both structures are substrate inaccessible, explaining their low basal activity in the absence of the binding of activator peptide. The binding of the activator peptide to each of the protomers of the trimer occurs with positive cooperativity and induces intrasubunit domain reorientations to expose the catalytic center, leading to increased proteolytic activity. Our data paint a picture of HtrA2 as a finely tuned, stress-protective enzyme whose activity can be modulated both by oligomerization and domain reorientation, with basal levels of catalysis kept low to avoid proteolysis of nontarget proteins.
    Keywords:  Human High temperature requirement A2; cooperativity; ligand-binding thermodynamics and kinetics; methyl transverse relaxation optimized spectroscopy; trimer–hexamer equilibrium
  21. Elife. 2021 Mar 09. pii: e64351. [Epub ahead of print]10
      The hypothalamic orexigenic Agouti-related peptide (AgRP)-expressing neurons are crucial for the regulation of whole-body energy homeostasis. Here, we show that fasting-induced AgRP neuronal activation is associated with dynamin-related peptide 1 (DRP1)-mediated mitochondrial fission and mitochondrial fatty acid utilization in AgRP neurons. In line with this, mice lacking Dnm1l in adult AgRP neurons (Drp1 cKO) show decreased fasting- or ghrelin-induced AgRP neuronal activity and feeding and exhibited a significant decrease in body weight, fat mass, and feeding accompanied by a significant increase in energy expenditure. In support of the role for mitochondrial fission and fatty acids oxidation, Drp1 cKO mice showed attenuated palmitic acid-induced mitochondrial respiration. Altogether, our data revealed that mitochondrial dynamics and fatty acids oxidation in hypothalamic AgRP neurons is a critical mechanism for AgRP neuronal function and body-weight regulation.
    Keywords:  AgRP; feeding; metabolism; mitochondria; mouse; neuroscience
  22. Am J Med Genet A. 2021 Mar 08.
      THG1L-associated autosomal recessive ataxia belongs to a group of disorders that occur due to abnormal mitochondrial tRNA modification. The product of THG1L is the tRNA-histidine guanylyltransferase 1-like enzyme that catalyzes the 3'-5"addition of guanine to the 5"-end of tRNA-histidine in the mitochondrion. To date, five individuals with homozygosity for p.(Val55Ala) in THG1L have been reported and presented with mild delays or normal development and cerebellar dysfunction. We present seven individuals with biallelic variants in THG1L. Three individuals were compound heterozygous for the p.(Cys51Trp) and p.(Val55Ala) variants and presented with profound developmental delays, microcephaly, intractable epilepsy, and cerebellar hypoplasia. Four siblings were homozygous for the p.(Val55Ala) variant and presented with cerebellar ataxia with cerebellar vermis hypoplasia, dysarthria, mild developmental delays, and normal/near-normal cognition. All seven patients were of Ashkenazi Jewish descent. Carrier rates for the two variants were calculated in a cohort of 26,731 Ashkenazi Jewish individuals tested by the Dor Yeshorim screening program. The p.(Cys51Trp) variant is novel and was found in 40 of the Ashkenazi Jewish individuals tested, with a carrier rate of 1 in 668 (0.15%). The p.(Val55Ala) variant was found in 229 of the Ashkenazi Jewish individuals tested, with a carrier rate of 1 in 117 (0.85%). The individuals with compound heterozygosity of the p.(Val55Ala) and p.(Cys51Trp) variants expand the phenotypic spectrum of THG1L-related disorders to include severe epileptic encephalopathy. The individuals with homozygosity of the p.(V55A) variant further establish the associated mild and slowly progressive or nonprogressive neurodevelopmental phenotype.
    Keywords:  THG1L; ataxia; epileptic encephalopathy; genotype phenotype; mitochondria; tRNA modification
  23. J Mol Biol. 2021 Mar 09. pii: S0022-2836(21)00123-6. [Epub ahead of print] 166924
      The late-acting steps of the pathway responsible for the maturation of mitochondrial [4Fe-4S] proteins are still elusive. Three proteins ISCA1, ISCA2 and NFU1 were shown to be implicated in the assembly of [4Fe-4S] clusters and their transfer into mitochondrial apo proteins. We present here a NMR-based study showing a detailed molecular model of the succession of events performed in a coordinated manner by ISCA1, ISCA2 and NFU1 to make [4Fe-4S] clusters available to mitochondrial apo proteins. We show that ISCA1 is the key player of the [4Fe-4S] protein maturation process because of its ability to interact with both NFU1 and ISCA2. On the contrary, the two latter proteins do not interact each other. ISCA1 works as the promoter of the interaction between ISCA2 and NFU1 being indeed able to determine the formation of a transient ISCA1-ISCA2-NFU1 ternary complex. We also show that ISCA1, through its specific recognition with the C-domain of NFU1 that contains the cluster binding CXXC motif, mediates a [4Fe-4S] cluster transfer from the site where the cluster is assembled on the ISCA1-ISCA2 complex to a cluster binding site formed by ISCA1 and NFU1 in the ternary ISCA1-ISCA2-NFU1 complex. Such mechanism guarantees that the [4Fe-4S] cluster can be safely moved from the ISCA1-ISCA2 complex, where it is assembled, to NFU1, thus making the [4Fe-4S] cluster available for mitochondrial apo proteins specifically requiring NFU1 for their maturation.
    Keywords:  NMR; iron sulfur cluster assembly machinery; iron-sulfur protein; mitochondria; protein-protein interaction
  24. Nat Commun. 2021 03 08. 12(1): 1504
      Elucidating functionality in non-coding regions is a key challenge in human genomics. It has been shown that intolerance to variation of coding and proximal non-coding sequence is a strong predictor of human disease relevance. Here, we integrate intolerance to variation, functional genomic annotations and primary genomic sequence to build JARVIS: a comprehensive deep learning model to prioritize non-coding regions, outperforming other human lineage-specific scores. Despite being agnostic to evolutionary conservation, JARVIS performs comparably or outperforms conservation-based scores in classifying pathogenic single-nucleotide and structural variants. In constructing JARVIS, we introduce the genome-wide residual variation intolerance score (gwRVIS), applying a sliding-window approach to whole genome sequencing data from 62,784 individuals. gwRVIS distinguishes Mendelian disease genes from more tolerant CCDS regions and highlights ultra-conserved non-coding elements as the most intolerant regions in the human genome. Both JARVIS and gwRVIS capture previously inaccessible human-lineage constraint information and will enhance our understanding of the non-coding genome.
  25. Hum Mol Genet. 2021 Mar 09. pii: ddab065. [Epub ahead of print]
      Spinal Muscular Atrophy (SMA) is an autosomal recessive degenerative motor neuron disease characterized by symmetrical muscle weakness and atrophy of limb and trunk muscles being the most severe genetic disease in children. In SMA mouse models, motor nerve terminals display neurotransmitter release reduction, endocytosis decrease, and mitochondria alterations. The relationship between these changes is, however, not well understood. In the present study, we investigated whether the endocytosis impairment could be related to the functional alteration of the presynaptic mitochondria during action potential (AP) firing. To this aim, we generated a Synaptophysin-pHluorin (SypHy) transgenic mouse, crossed it with Taiwanese SMA mice, and recorded exo- and endocytosis and mitochondria Ca2+ signaling in real-time at ex vivo motor nerve terminals of Taiwanese-SypHy mice. The experiments were performed at the beginning of the motor symptoms to get an integrated view of the nerve terminal's functional state before degeneration. Our electrophysiological and live imaging results demonstrated that the mitochondria's capacity to increase matrix-free Ca2+ in SMA mice was significantly limited during nerve AP firing, except when the rate of Ca2+ entry to the cytosol was considerably reduced. These results indicate that both the mitochondrial Ca2+ signaling alterations and the secretion machinery defects are significant players in the dysfunction of the presynaptic terminal in SMA.
  26. Am J Clin Nutr. 2021 Mar 10. pii: nqaa432. [Epub ahead of print]
      BACKGROUND: The association of moderate hyperhomocysteinemia (HHcy) (15-30 μmol/L) with cardiovascular diseases (CVD) has been challenged by the lack of benefit of vitamin supplementation to lowering homocysteine. Consequently, the results of interventional studies have confused the debate regarding the management of patients with intermediate/severe HHcy.OBJECTIVE: We sought to evaluate the association of intermediate (30-50 and 50-100 μmol/L) and severe (>100 μmol/L) HHcy related to vitamin deficiencies and/or inherited disorders with CVD outcomes.
    METHODS: We performed a retrospective cross-sectional study on consecutive patients who underwent a homocysteine assay in a French University Regional Hospital Center. Patients with CVD outcomes were assessed for vitamin B12, folate, Hcy, methylmalonic acid, and next-generation clinical exome sequencing.
    RESULTS: We evaluated 165 patients hospitalized for thromboembolic and other cardiovascular (CV) manifestations among 1006 patients consecutively recruited. Among them, 84% (138/165) had Hcy >30 μmol/L, 27% Hcy >50 μmol/L (44/165) and 3% Hcy >100 μmol/L (5/165). HHcy was related to vitamin B12 and/or folate deficiency in 55% (87/165), mutations in one or more genes of one-carbon and/or vitamin B12 metabolisms in 11% (19/165), and severe renal failure in 15% (21/141) of the studied patients. HHcy was the single vascular risk retrieved in almost 9% (15/165) of patients. Sixty % (101/165) of patients received a supplementation to treat HHcy, with a significant decrease in median Hcy from 41 to 17 µmol/L (IQR: 33.6-60.4 compared with 12.1-28). No recurrence of thromboembolic manifestations was observed after supplementation and antithrombotic treatment of patients who had HHcy as a single risk, after ∼4 y of follow-up.
    CONCLUSION: The high frequency of intermediate/severe HHcy differs from the frequent moderate HHcy reported in previous observational studies of patients with pre-existing CVD. Our study points out the importance of diagnosing and treating nutritional deficiencies and inherited disorders to reverse intermediate/severe HHcy associated with CVD outcomes.
    Keywords:  1-carbon metabolism; cardiovascular disease risk; folate; homocysteine; inborn errors of metabolism; thromboembolic manifestations; vitamin B12
  27. Mol Cell. 2021 Mar 05. pii: S1097-2765(21)00100-3. [Epub ahead of print]
      Enhancers harbor binding motifs that recruit transcription factors (TFs) for gene activation. While cooperative binding of TFs at enhancers is known to be critical for transcriptional activation of a handful of developmental enhancers, the extent of TF cooperativity genome-wide is unknown. Here, we couple high-resolution nuclease footprinting with single-molecule methylation profiling to characterize TF cooperativity at active enhancers in the Drosophila genome. Enrichment of short micrococcal nuclease (MNase)-protected DNA segments indicates that the majority of enhancers harbor two or more TF-binding sites, and we uncover protected fragments that correspond to co-bound sites in thousands of enhancers. From the analysis of co-binding, we find that cooperativity dominates TF binding in vivo at the majority of active enhancers. Cooperativity is highest between sites spaced 50 bp apart, indicating that cooperativity occurs without apparent protein-protein interactions. Our findings suggest nucleosomes promoting cooperativity because co-binding may effectively clear nucleosomes and promote enhancer function.
    Keywords:  chromatin dynamics; enhancer; enhancer cooperativity; nucleosome; transcription factor
  28. Artif Intell Med. 2021 Mar;pii: S0933-3657(21)00027-0. [Epub ahead of print]113 102034
      Identification of RNA-binding proteins (RBPs) that bind to ribonucleic acid molecules is an important problem in Computational Biology and Bioinformatics. It becomes indispensable to identify RBPs as they play crucial roles in post-transcriptional control of RNAs and RNA metabolism as well as have diverse roles in various biological processes such as splicing, mRNA stabilization, mRNA localization, and translation, RNA synthesis, folding-unfolding, modification, processing, and degradation. The existing experimental techniques for identifying RBPs are time-consuming and expensive. Therefore, identifying RBPs directly from the sequence using computational methods can be useful to annotate RBPs and assist the experimental design efficiently. In this work, we present a method called AIRBP, which is designed using an advanced machine learning technique, called stacking, to effectively predict RBPs by utilizing features extracted from evolutionary information, physiochemical properties, and disordered properties. Moreover, our method, AIRBP, use the majority vote from RBPPred, DeepRBPPred, and the stacking model for the prediction for RBPs. The results show that AIRBP attains Accuracy (ACC), Balanced Accuracy (BACC), F1-score, and Mathews Correlation Coefficient (MCC) of 95.84 %, 94.71 %, 0.928, and 0.899, respectively, based on the training dataset, using 10-fold cross-validation (CV). Further evaluation of AIRBP on independent test set reveals that it achieves ACC, BACC, F1-score, and MCC of 94.36 %, 94.28 %, 0.897, and 0.860, for Human test set; 91.25 %, 93.00 %, 0.896, and 0.835 for S. cerevisiae test set; and 90.60 %, 90.41 %, 0.934, and 0.775 for A. thaliana test set, respectively. These results indicate that the AIRBP outperforms the existing Deep- and TriPepSVM methods. Therefore, the proposed better-performing AIRBP can be useful for accurate identification and annotation of RBPs directly from the sequence and help gain valuable insight to treat critical diseases. Availability: Code-data is available here:∼tamjid/Software/AIRBP/
    Keywords:  Machine learning; Protein sequence; RNA-binding prediction; RNA-binding proteins; Stacking