bims-mikwok Biomed News
on Mitochondrial quality control
Issue of 2020‒11‒29
thirty-one papers selected by
Avinash N. Mukkala
University of Toronto

  1. Free Radic Biol Med. 2020 Nov 18. pii: S0891-5849(20)31607-5. [Epub ahead of print]
      Mitochondrial permeability transition pore (mPTP) is an important regulator in cell apoptosis and necrosis. However, its role in hepatic steatosis, especially its electrophysiological properties transformation remains elusive. Herein, using diabetes mice, we investigated the role of mPTP in hepatic steatosis triggered by diabetes and the mechanisms involved. We found that hepatic steatosis altered mitochondrial morphology, generating mega mitochondria, mitochondria swelling, calcein fluorescence quenching and mitochondrial membrane potential depolarization. At the same time, we confirmed an augmented mPTP opening with patch clamping in liver mitoplasts in db/db mice and a similar transformation with arachidonic acid (AA) simulating liquid deposition. We also found mPTP opening was significantly attenuated in wt mice after removing mitochondrial matrix, while that in db/db mice remained active. In addition, we observed that AA could directly activate mPTP in inside-out mode, independent of matrix calcium. In conclusion, we for the first time provided a physiological evidence of mPTP opening in lipid deposition, which could be directly induced by AA without Ca2+ and can be inhibited by cyclosporine A. As a result, it led to mitochondria morphology and function transformation. This might provide insights into potential therapeutic target for future treatment of mitochondrial liver disease.
    Keywords:  Arochidonic acid; Electrophysiological morphology; Mitochondrial permeability transition pore; Mouse; Non-alcoholic fatty liver disease
  2. J Am Heart Assoc. 2020 Nov 23. e017820
      Background The blood-brain barrier (BBB) is critical for cerebrovascular health. Although aging impairs the integrity of the BBB, the mechanisms behind this phenomenon are not clear. As mitochondrial components activate inflammation as mitochondria become dysfunctional, we examined how aging impacts cerebrovascular mitochondrial function, mitophagy, and inflammatory signaling; and whether any alterations correlate with BBB function. Methods and Results We isolated cerebral vessels from young (2-3 months of age) and aged (18-19 months of age) mice and found that aging led to increases in the cyclin-dependent kinase inhibitor 1 senescence marker with impaired mitochondrial function, which correlated with aged mice exhibiting increased BBB leak compared with young mice. Cerebral vessels also exhibited increased expression of mitophagy proteins Parkin and Nix with aging. Using mitophagy reporter (mtKeima) mice, we found that the capacity to increase mitophagy from baseline within the cerebral vessels on rotenone treatment was reduced with aging. Aging within the cerebral vessels also led to the upregulation of the stimulator of interferon genes and increased interleukin 6 (IL-6), a cytokine that alters mitochondrial function. Importantly, exogenous IL-6 treatment of young cerebral vessels upregulated mitophagy and Parkin and impaired mitochondrial function; whereas inhibiting IL-6 in aged cerebral vessels reduced Parkin expression and increased mitochondrial function. Furthermore, treating cerebral vessels of young mice with mitochondrial N-formyl peptides upregulated IL-6, increased Parkin, and reduced Claudin-5, a tight junction protein integral to BBB integrity. Conclusions Aging alters the cerebral vasculature to impair mitochondrial function and mitophagy and increase IL-6 levels. These alterations may impair BBB integrity and potentially reduce cerebrovascular health with aging.
    Keywords:  aging; cerebrovascular inflammation; interleukin 6; mitophagy
  3. Nat Rev Nephrol. 2020 Nov 24.
      Mitochondria are essential for the activity, function and viability of eukaryotic cells and mitochondrial dysfunction is involved in the pathogenesis of acute kidney injury (AKI) and chronic kidney disease, as well as in abnormal kidney repair after AKI. Multiple quality control mechanisms, including antioxidant defence, protein quality control, mitochondrial DNA repair, mitochondrial dynamics, mitophagy and mitochondrial biogenesis, have evolved to preserve mitochondrial homeostasis under physiological and pathological conditions. Loss of these mechanisms may induce mitochondrial damage and dysfunction, leading to cell death, tissue injury and, potentially, organ failure. Accumulating evidence suggests a role of disturbances in mitochondrial quality control in the pathogenesis of AKI, incomplete or maladaptive kidney repair and chronic kidney disease. Moreover, specific interventions that target mitochondrial quality control mechanisms to preserve and restore mitochondrial function have emerged as promising therapeutic strategies to prevent and treat kidney injury and accelerate kidney repair. However, clinical translation of these findings is challenging owing to potential adverse effects, unclear mechanisms of action and a lack of knowledge of the specific roles and regulation of mitochondrial quality control mechanisms in kidney resident and circulating cell types during injury and repair of the kidney.
  4. Methods Mol Biol. 2021 ;2192 147-158
      Even though the mammalian mitochondrial genome (mtDNA) is very small and only codes for 13 proteins, all being subunits of the oxidative phosphorylation system, it requires several hundred nuclear encoded proteins for its maintenance and expression. These include replication and transcription factors, approximately 80 mitoribosomal proteins and many proteins involved in the posttranscriptional modification, processing, and stability of mitochondrial RNAs. In recent years, many of these factors have been identified and functionally characterized, but the complete mtRNA-interacting proteome is not firmly established. Shotgun proteomics has been used successfully to define whole-cell polyadenylated RNA (poly(A)-RNA) interacting proteomes using the nucleotide analogue 4-thiouridine (4SU) combined with UV crosslinking, poly(A)-RNA isolation and mass spectrometry to identify all poly(A)-RNA bound proteins. Although in this case also a considerable number of mitochondrial proteins were identified, the method was not specifically directed at the mitochondrial poly(A)-RNA bound proteome. Here we describe a method for enrichment of the mitochondrial poly(A)-RNA bound proteome based on 4SU labeling and UV crosslinking. The method can be applied either for isolated mitochondria prior to UV crosslinking or for whole-cell crosslinking followed by mitochondrial isolation.
    Keywords:  4-Thiouridine; Crosslinking; Mass spectrometry; Mitochondrial RNA; mtDNA
  5. Methods Mol Biol. 2021 ;2192 69-73
      The incorporation of nucleoside analogs is a useful tool to study the various functions of DNA and RNA. These analogs can be detected directly by fluorescence or by immunolabeling, allowing to visualize, track, or measure the nucleic acid molecules in which they have been incorporated. In this chapter, methodologies to measure human mitochondrial transcription are described. The nascent RNA that is transcribed from mitochondrial DNA (mtDNA) has been shown to assemble into large ribonucleoprotein complexes that form discrete foci. These structures were called mitochondrial RNA granules (MRGs) and can be observed in vitro by the incorporation of a 5-Bromouridine (BrU), which is subsequently visualized by fluorescent immunolabeling. Here, a combined protocol for the MRGs detection is detailed, consisting of BrU labeling and visualization of one of their bona fide protein components, Fas-activated serine-threonine kinase domain 2 (FASTKD2). Based on immunodetection, the half-life and kinetics of the MRGs under various experimental conditions can further be determined by chasing the BrU pulse with an excess of Uridine.
    Keywords:  5-Bromouridine; Half-life; Immunofluorescence; Mitochondrial DNA; Mitochondrial RNA Granules (MRGs); Mitochondrial transcription; Nascent RNA; Pulse-chase; Uridine
  6. Methods Mol Biol. 2021 ;2192 159-181
      Human mitochondria contain their own DNA (mtDNA) that encodes 13 proteins all of which are core subunits of oxidative phosphorylation (OXPHOS) complexes. To form functional complexes, these 13 components need to be correctly assembled with approximately 70 nuclear-encoded subunits that are imported following synthesis in the cytosol. How this complicated coordinated translation and assembly is choreographed is still not clear. Methods are being developed to determine whether all members of a particular complex are translated in close proximity, whether protein synthesis is clustered in submitochondrial factories, whether these align with incoming polypeptides, and if there is evidence for co-translational translation that is regulated and limited by the interaction of the incoming proteins with synthesis of their mtDNA-encoded partners. Two methods are described in this chapter to visualize the distribution of mitochondrial ribosomal RNAs in conjunction with newly synthesized mitochondrial proteins. The first combines RNA Fluorescent In Situ Hybridization (FISH) and super-resolution immunocytochemistry to pinpoint mitochondrial ribosomal RNA. The second localizes nascent translation within the mitochondrial network through non-canonical amino acid labeling, click chemistry and fluorescent microscopy.
    Keywords:  Click chemistry; Fluorescence microscopy; Mitochondria; Mitochondrial RNA; Mitoribosome; Single-molecule RNA FISH; Stimulated emission depletion microscopy; Super-resolution microscopy; Translation
  7. Mol Biol Cell. 2020 Nov 25. mbcE20090605
      OPA1, a large GTPase of the dynamin superfamily, mediates fusion of the mitochondrial inner membranes, regulates cristae morphology, and maintains respiratory chain function. Inner-membrane-anchored long forms of OPA1 (l-OPA1) are proteolytically processed by the OMA1 or YME1L proteases, acting at cleavage sites S1 and S2 respectively, to produce short forms (s-OPA1). In both mouse and human, half of the mRNA splice forms of Opa1 are constitutively processed to yield exclusively s-OPA1. However, the function of s-OPA1 in mitochondrial fusion has been debated, because in some stress conditions, s-OPA1 is dispensable for fusion. By constructing cells in which the Opa1 locus no longer produces transcripts with S2 cleavage sites, we generated a simplified system to identify the new YME1L-dependent site S3 that mediates constitutive and complete cleavage of OPA1. We show that mitochondrial morphology is highly sensitive to the ratio of l-OPA1 to s-OPA1, indicating that s-OPA1 regulates mitochondrial fusion.
  8. Cell Rep. 2020 Nov 24. pii: S2211-1247(20)31400-5. [Epub ahead of print]33(8): 108411
      Phagocytes reallocate metabolic resources to kill engulfed pathogens, but the intracellular signals that rapidly switch the immunometabolic program necessary to fuel microbial killing are not understood. We report that macrophages use a fast two-step Ca2+ relay to meet the bioenergetic demands of phagosomal killing. Upon detection of a fungal pathogen, macrophages rapidly elevate cytosolic Ca2+ (phase 1), and by concurrently activating the mitochondrial Ca2+ (mCa2+) uniporter (MCU), they trigger a rapid influx of Ca2+ into the mitochondria (phase 2). mCa2+ signaling reprograms mitochondrial metabolism, at least in part, through the activation of pyruvate dehydrogenase (PDH). Deprived of mCa2+ signaling, Mcu-/- macrophages are deficient in phagosomal reactive oxygen species (ROS) production and defective at killing fungi. Mice lacking MCU in their myeloid cells are highly susceptible to disseminated candidiasis. In essence, this study reveals an elegant design principle that MCU-dependent Ca2+ signaling is an electrometabolic switch to fuel phagosome killing.
    Keywords:  MCU; NADPH; calcium; citrate; electrometabolic; immunometabolism; mitochondria, Ca(2+); phagosome; pyruvate dehydrogenase
  9. Methods Mol Biol. 2021 ;2192 43-57
      Transcriptomic technologies have revolutionized the study of gene expression and RNA biology. Different RNA sequencing methods enable the analyses of diverse species of transcripts, including their abundance, processing, stability, and other specific features. Mitochondrial transcriptomics has benefited from these technologies that have revealed the surprising complexity of its RNAs. Here we describe a method based upon cyclization of mitochondrial RNAs and next generation sequencing to analyze the steady-state levels and sizes of mitochondrial RNAs, their degradation products, as well as their processing intermediates by capturing both 5' and 3' ends of transcripts.
    Keywords:  Mitochondria; Next generation sequencing; RNA processing; RNA-Seq
  10. Methods Mol Biol. 2021 ;2192 269-285
      Complexome profiling combines blue native gel electrophoresis (BNE) and quantitative mass spectrometry to define an entire protein interactome of a cell, an organelle, or a biological membrane preparation. The method allows the identification of protein assemblies with low abundance and detects dynamic processes of protein complex assembly. Applications of complexome profiling range from the determination of complex subunit compositions, assembly of single protein complexes, and supercomplexes to comprehensive differential studies between patients or disease models. This chapter describes the workflow of complexome profiling from sample preparation, mass spectrometry to data analysis with a bioinformatics tool.
    Keywords:  Assembly; Blue native electrophoresis; Complexome profiling; Mass spectrometry; Membrane protein complexes; Mitochondria
  11. Cell Death Dis. 2020 Nov 23. 11(11): 1004
      Neuronal stress-adaptation combines multiple molecular responses. We have previously reported that thorax trauma induces a transient loss of hippocampal excitatory synapses mediated by the local release of the stress-related hormone corticotropin-releasing hormone (CRH). Since a physiological synaptic activity relies also on mitochondrial functionality, we investigated the direct involvement of mitochondria in the (mal)-adaptive changes induced by the activation of neuronal CRH receptors 1 (CRHR1). We observed, in vivo and in vitro, a significant shift of mitochondrial dynamics towards fission, which correlated with increased swollen mitochondria and aberrant cristae. These morphological changes, which are associated with increased NF-kB activity and nitric oxide concentrations, correlated with a pronounced reduction of mitochondrial activity. However, ATP availability was unaltered, suggesting that neurons maintain a physiological energy metabolism to preserve them from apoptosis under CRH exposure. Our findings demonstrate that stress-induced CRHR1 activation leads to strong, but reversible, modifications of mitochondrial dynamics and morphology. These alterations are accompanied by bioenergetic defects and the reduction of neuronal activity, which are linked to increased intracellular oxidative stress, and to the activation of the NF-kB/c-Abl/DRP1 axis.
  12. JCI Insight. 2020 Nov 24. pii: 141138. [Epub ahead of print]
      Inflammatory damage contributes to β-cell failure in type 1 and 2 diabetes (T1D and T2D). Mitochondria are damaged by inflammatory signaling in β-cells, resulting in impaired bioenergetics and initiation of pro-apoptotic machinery. Hence, the identification of protective responses to inflammation could lead to new therapeutic targets. Here we report that mitophagy serves as a protective response to inflammatory stress in both human and rodent β-cells. Utilizing in vivo mitophagy reporters, we observed that diabetogenic pro-inflammatory cytokines induced mitophagy in response to nitrosative/oxidative mitochondrial damage. Mitophagy-deficient β-cells were sensitized to inflammatory stress, leading to the accumulation of fragmented dysfunctional mitochondria, increased β-cell death, and hyperglycemia. Overexpression of CLEC16A, a T1D gene and mitophagy regulator whose expression in islets is protective against T1D, ameliorated cytokine-induced human β-cell apoptosis. Thus, mitophagy promotes β-cell survival and prevents diabetes by countering inflammatory injury. Targeting this pathway has the potential to prevent β-cell failure in diabetes and may be beneficial in other inflammatory conditions.
    Keywords:  Apoptosis survival pathways; Diabetes; Endocrinology; Mitochondria
  13. EMBO Rep. 2020 Nov 27. e50500
      The denitrosylase S-nitrosoglutathione reductase (GSNOR) has been suggested to sustain mitochondrial removal by autophagy (mitophagy), functionally linking S-nitrosylation to cell senescence and aging. In this study, we provide evidence that GSNOR is induced at the translational level in response to hydrogen peroxide and mitochondrial ROS. The use of selective pharmacological inhibitors and siRNA demonstrates that GSNOR induction is an event downstream of the redox-mediated activation of ATM, which in turn phosphorylates and activates CHK2 and p53 as intermediate players of this signaling cascade. The modulation of ATM/GSNOR axis, or the expression of a redox-insensitive ATM mutant influences cell sensitivity to nitrosative and oxidative stress, impairs mitophagy and affects cell survival. Remarkably, this interplay modulates T-cell activation, supporting the conclusion that GSNOR is a key molecular effector of the antioxidant function of ATM and providing new clues to comprehend the pleiotropic effects of ATM in the context of immune function.
    Keywords:  ATM; GSNOR; ROS; T cell; mitophagy
  14. Front Neurosci. 2020 ;14 536682
      In the brain, mitochondrial metabolism has been largely associated with energy production, and its dysfunction is linked to neuronal cell loss. However, the functional role of mitochondria in glial cells has been poorly studied. Recent reports have demonstrated unequivocally that astrocytes do not require mitochondria to meet their bioenergetics demands. Then, the question remaining is, what is the functional role of mitochondria in astrocytes? In this work, we review current evidence demonstrating that mitochondrial central carbon metabolism in astrocytes regulates overall brain bioenergetics, neurotransmitter homeostasis and redox balance. Emphasis is placed in detailing carbon source utilization (glucose and fatty acids), anaplerotic inputs and cataplerotic outputs, as well as carbon shuttles to neurons, which highlight the metabolic specialization of astrocytic mitochondria and its relevance to brain function.
    Keywords:  anaplerotic; astrocytes; bioenergetics; cataplerotic; fatty acid oxidation; glycolysis; mitochondria; redox
  15. Cell Rep. 2020 Nov 24. pii: S2211-1247(20)31412-1. [Epub ahead of print]33(8): 108423
      In many tissues, stem cell (SC) proliferation is dynamically adjusted to regenerative needs. How SCs adapt their metabolism to meet the demands of proliferation and how changes in such adaptive mechanisms contribute to age-related dysfunction remain poorly understood. Here, we identify mitochondrial Ca2+ uptake as a central coordinator of SC metabolism. Live imaging of genetically encoded metabolite sensors in intestinal SCs (ISCs) of Drosophila reveals that mitochondrial Ca2+ uptake transiently adapts electron transport chain flux to match energetic demand upon proliferative activation. This tight metabolic adaptation is lost in ISCs of old flies, as declines in mitochondrial Ca2+ uptake promote a "Warburg-like" metabolic reprogramming toward aerobic glycolysis. This switch mimics metabolic reprogramming by the oncogene RasV12 and enhances ISC hyperplasia. Our data identify a critical mechanism for metabolic adaptation of tissue SCs and reveal how its decline sets aging SCs on a metabolic trajectory reminiscent of that seen upon oncogenic transformation.
    Keywords:  Drosophila; Warburg; aging; calcium; cancer; intestine; metabolism; mitochondria; stem cell; tissue homeostasis
  16. Methods Mol Biol. 2021 ;2192 133-146
      RNA turnover is an essential part of the gene expression pathway, and there are several experimental approaches for its determination. High-throughput measurement of global RNA turnover rates can provide valuable information about conditions or proteins that impact gene expression. Here, we present a protocol for mitochondrial RNA turnover analysis which involves metabolic labeling of RNA coupled with quantitative high-throughput fluorescent microscopy. This approach gives an excellent opportunity to discover new factors involved in mitochondrial gene regulation when combined with loss-of-function screening strategy.
    Keywords:  Bromouridine (BrU); Metabolic labeling; Mitochondrial RNA; RNA turnover
  17. Methods Mol Biol. 2021 ;2192 287-311
      Blue-native polyacrylamide gel electrophoresis (BN-PAGE) is a technique optimized for the analysis of the five components of the mitochondrial oxidative phosphorylation (OXPHOS) system. BN-PAGE is based on the preservation of the interactions between the individual subunits within the integral complexes. To achieve this, the complexes are extracted from the mitochondrial inner membrane using mild detergents and separated by electrophoresis in the absence of denaturing agents. The electrophoretic procedures can then be combined with a variety of downstream detection techniques. Since its development in the 1990s, BN-PAGE has been applied in the study of mitochondria from all kinds of organisms and extensive amounts of data have been produced using this technique, being key for the understanding of many aspects of OXPHOS physiopathology.
    Keywords:  Blue-native gel electrophoresis; First-dimension BN-PAGE; In gel activity assays; Mitochondrial complexes I, II, III, IV, and V; Oxidative phosphorylation system; Second-dimension BN-PAGE
  18. Methods Mol Biol. 2021 ;2192 1-20
      Human mitochondrial DNA is a small circular double-stranded molecule that is essential for cellular energy production. A specialized protein machinery replicates the mitochondrial genome, with DNA polymerase γ carrying out synthesis of both strands. According to the prevailing mitochondrial DNA replication model, the two strands are replicated asynchronously, with the leading heavy-strand initiating first, followed by the lagging light-strand. By using purified recombinant forms of the replication proteins and synthetic DNA templates, it is possible to reconstitute mitochondrial DNA replication in vitro. Here we provide details on how to differentially reconstitute replication of the leading- and lagging-strands.
    Keywords:  DNA polymerase; In vitro; Mitochondria; Replication; mtDNA
  19. Mol Med Rep. 2021 Jan;pii: 73. [Epub ahead of print]23(1):
      Resveratrol confers neuroprotective effects in cerebral ischemia; however, the involvement of mitophagy in the neuroprotective function of resveratrol remains unclear. The aim of the present study was to investigate whether resveratrol exerts neuroprotective effects on primary cortical neurons subjected to oxygen/glucose deprivation/reoxygenation (OGD/R) via modulating mitophagy. The data demonstrated that resveratrol at 1‑10 µM during reoxygenation improved cell viability and suppressed apoptosis following OGD/R in a concentration‑dependent manner. Moreover, resveratrol alleviated OGD/R‑induced loss of mitochondrial membrane potential and excessive oxidative stress. Confocal imaging of LC3 and TOM20 antibody‑labeled mitochondria, as well as western blot analysis, demonstrated that mitophagy was further enhanced following resveratrol treatment. In addition, resveratrol was revealed to stimulate the phosphatase and tensin homolog‑induced kinase 1/Parkin pathway. Mitophagy inhibition then inhibited the protective effects of resveratrol. These results indicated that resveratrol exerts its protective effects against OGD/R damage, at least in part, by promoting mitophagy.
  20. Mitochondrion. 2020 Nov 18. pii: S1567-7249(20)30202-6. [Epub ahead of print]
      The efficient production of energy via oxidative phosphorylation is essential to the growth, survival, and reproduction of eukaryotes. The behavior (position of, and communication between, mitochondria) and morphology of mitochondria play key roles in efficient energy production and are influenced by oxidative stressors such as ultraviolet (UV) radiation. We tested the hypothesis that mitochondria change their behavior and morphology to meet energetic demands of responding to changes in oxidative stress. Specifically, we predicted that UV irradiation would increase the density of inner mitochondrial membrane and proportion of inter-mitochondrial junctions to influence whole-animal metabolic rate. Using transmission electron microscopy, we found that both three and six hours of UV-A/B irradiation (0.5 W/m2) increased the proportion of inter-mitochondrial junctions (with increasing mitochondrial aspect ratio) and the density of inner mitochondrial membrane in myocytes of Tigriopus californicus copepods. Mitochondrial density increased following both irradiation treatments, but mitochondrial size decreased under the six hour treatment. Metabolic rate was maintained under three hours of irradiation but decreased following six hours of exposure. These observations demonstrate that the density of inner mitochondrial membrane and proportion of inter-mitochondrial junctions can play formative roles in maintaining whole-animal metabolic rate, and ultimately organismal performance, under exposure to an oxidative stressor.
    Keywords:  Tigriopus californicus; behavior; fission; metabolic rate; morphology; transmission electron microscopy
  21. Biochem Soc Trans. 2020 Nov 27. pii: BST20200250. [Epub ahead of print]
      In fluctuating environmental conditions, organisms must modulate their bioenergetic production in order to maintain cellular homeostasis for optimal fitness. Mitochondria are hubs for metabolite and energy generation. Mitochondria are also highly dynamic in their function: modulating their composition, size, density, and the network-like architecture in relation to the metabolic demands of the cell. Here, we review the recent research on the post-transcriptional regulation of mitochondrial composition focusing on mRNA localization, mRNA translation, protein import, and the role that dynamic mitochondrial structure may have on these gene expression processes. As mitochondrial structure and function has been shown to be very important for age-related processes, including cancer, metabolic disorders, and neurodegeneration, understanding how mitochondrial composition can be affected in fluctuating conditions can lead to new therapeutic directions to pursue.
    Keywords:  mRNA; mRNA localization; mRNA translation; mitochondria; mitochondrial morphology; protein import
  22. Mitochondrion. 2020 Nov 18. pii: S1567-7249(20)30218-X. [Epub ahead of print]
      Mitochondrial dysfunction is known to be associated with neurodegenerative diseases (NDDs), which is a major burden on the society. Therefore, understanding the regulation of mitochondrial dysfunctions and its implication in neurodegeneration has been major goal for exploiting these mechanisms to rescue neuronal death. The crosstalk between mitochondria and nucleus is important for different neuronal functions including axonal branching, energy homeostasis, neuroinflammation and neuronal survival. The decreased mitochondria capacity during progressive neurodegeneration leads to the altered OXPHOS activity and generation of ROS. The ROS levels in narrow physiological range can reprogram nuclear gene expression to enhance the cellular survival by phenomenon called mitohormesis. Here, we have systematically reviewed the existing reports of mitochondrial dysfunctions causing altered ROS levels in NDDs. We further discussed the role of ROS in regulating mitohormesis and emphasized the importance of mitohormesis in neuronal homeostasis. The emerging role of mitohormesis highlights its importance in future studies on intracellular ROS mediated rescue of mitochondrial dysfunction along with other prevailing mechanisms to alleviate neurodegeneration.
    Keywords:  Mitochondria; Mitohormesis; Neurodegeneration; ROS; UPR(mt)
  23. Redox Biol. 2020 Sep 29. pii: S2213-2317(20)30944-7. [Epub ahead of print]37 101739
      Redox homeostasis regulates key cellular signaling in both physiology and pathology. While perturbations result in shifting the redox homeostasis towards oxidative stress are well documented, the influence of reductive stress (RS) in neurodegenerative diseases and its mechanisms are unknown. Here, we postulate that a redox shift towards the reductive arm (through the activation of Nrf2 signaling) will damage neurons and impair neurogenesis. In proliferating and differentiating neuroblastoma (Neuro 2a/N2a) cells, sulforaphane-mediated Nrf2 activation resulted in increased transcription/translation of antioxidants and glutathione (GSH) production along with significantly declined ROS in a dose-dependent manner leading to a reductive-redox state (i.e. RS). Interestingly, this resulted in endoplasmic reticulum (ER) stress leading to subsequent protein aggregation/proteotoxicity in neuroblastoma cells. Under RS, we also observed elevated Tau/α-synuclein and their co-localization with other protein aggregates in these cells. Surprisingly, we noticed that acute RS impaired neurogenesis as evidenced from reduced neurite outgrowth/length. Furthermore, maintaining the cells in a sustained RS condition (for five consecutive generations) dramatically reduced their differentiation and prevented the formation of axons (p < 0.05). This impairment in RS mediated neurogenesis occurs through the alteration of Tau dynamics i.e. RS activates the pathogenic GSK3β/Tau cascade thereby promoting the phosphorylation of Tau leading to proteotoxicity. Of note, intermittent withdrawal of sulforaphane from these cells suppressed the proteotoxic insult and re-activated the differentiation process. Overall, this results suggest that either acute or chronic RS could hamper neurogenesis through GSK3β/TAU signaling and proteotoxicity. Therefore, investigations identifying novel redox mechanisms impacting proteostasis are crucial to preserve neuronal health.
    Keywords:  ER stress; Glutathione; Neurogenesis; Nrf2; Proteotoxicity; Reductive stress
  24. Methods Mol Biol. 2021 ;2192 183-196
      Ribosome profiling (Ribo-Seq) is a technique that allows genome-wide, quantitative analysis of translation. In recent years, it has found multiple applications in studies of translation in diverse organisms, tracking protein synthesis with single codon resolution. Traditional protocols applied for generating Ribo-Seq libraries from mammalian cell cultures are not suitable to study mitochondrial translation due to differences between eukaryotic cytosolic and mitochondrial ribosomes. Here, we present an adapted protocol enriching for mitoribosome footprints. In addition, we describe the preparation of small RNA sequencing libraries from the resultant mitochondrial ribosomal protected fragments (mtRPFs).
    Keywords:  MitoRibo-Seq; Mitochondria; Mitoribosome; Ribosome profiling
  25. Sci Rep. 2020 Nov 26. 10(1): 20653
      Human embryonic and induced pluripotent stem cells (hESCs and hiPSCs) are self-renewing human pluripotent stem cells (hPSCs) that can differentiate to a wide range of specialized cells. Notably, hPSCs enhance their undifferentiated state and self-renewal properties in hypoxia (5% O2). Although thoroughly analyzed, hypoxia implication in hPSCs death is not fully determined. In order to evaluate the effect of chemically mimicked hypoxia on hPSCs cell survival, we analyzed changes in cell viability and several aspects of apoptosis triggered by CoCl2 and dimethyloxalylglycine (DMOG). Mitochondrial function assays revealed a decrease in cell viability at 24 h post-treatments. Moreover, we detected chromatin condensation, DNA fragmentation and CASPASE-9 and 3 cleavages. In this context, we observed that P53, BNIP-3, and NOXA protein expression levels were significantly up-regulated at different time points upon chemical hypoxia induction. However, only siRNA-mediated downregulation of NOXA but not HIF-1α, HIF-2α, BNIP-3, and P53 did significantly affect the extent of cell death triggered by CoCl2 and DMOG in hPSCs. In conclusion, chemically mimicked hypoxia induces hPSCs cell death by a NOXA-mediated HIF-1α and HIF-2α independent mechanism.
  26. Methods Mol Biol. 2021 ;2233 115-129
      Constitutive secretion is predominantly measured by collecting the media from cells and performing plate-based assays. This approach is particularly sensitive to changes in cell number, and a significant amount of effort has to be spent to overcome this. We have developed a panel of quantitative flow cytometry-based assays and reporter cell lines that can be used to measure constitutive secretion. These assays are insensitive to changes in cell number making them very robust and well suited to functional genomic and chemical screens. Here, we outline the key steps involved in generating and using these assays for studying constitutive secretion.
    Keywords:  Assay; Biosynthetic transport; Constitutive secretion; Flow cytometry; GPI; Trafficking; Transport; VSV-G; hGH
  27. Redox Biol. 2020 Nov 12. pii: S2213-2317(20)30990-3. [Epub ahead of print]38 101785
      Mechanical ventilation is a life-saving intervention in critically ill patients with respiratory failure due to acute respiratory distress syndrome (ARDS), a refractory lung disease with an unacceptable high mortality rate. Paradoxically, mechanical ventilation also creates excessive mechanical stress that directly augments lung injury, a syndrome known as ventilator-induced lung injury (VILI). The specific mechanisms involved in VILI-induced pulmonary capillary leakage, a key pathologic feature of VILI are still far from resolved. The mechanoreceptor, transient receptor potential cation channel subfamily V member 4, TRPV4 plays a key role in the development of VILI through unresolved mechanism. Endothelial nitric oxide synthase (eNOS) uncoupling plays an important role in sepsis-mediated ARDS so in this study we investigated whether there is a role for eNOS uncoupling in the barrier disruption associated with TRPV4 activation during VILI. Our data indicate that the TRPV4 agonist, 4α-Phorbol 12,13-didecanoate (4αPDD) induces pulmonary arterial endothelial cell (EC) barrier disruption through the disruption of mitochondrial bioenergetics. Mechanistically, this occurs via the mitochondrial redistribution of uncoupled eNOS secondary to a PKC-dependent phosphorylation of eNOS at Threonine 495 (T495). A specific decoy peptide to prevent T495 phosphorylation reduced eNOS uncoupling and mitochondrial redistribution and preserved PAEC barrier function under 4αPDD challenge. Further, our eNOS decoy peptide was able to preserve lung vascular integrity in a mouse model of VILI. Thus, we have revealed a functional link between TRPV4 activation, PKC-dependent eNOS phosphorylation at T495, and EC barrier permeability. Reducing pT495-eNOS could be a new therapeutic approach for the prevention of VILI.
    Keywords:  Barrier permeability; Mitochondrial bioenergetics; PKCα; TRPV4; VILI; eNOS
  28. Science. 2020 Nov 27. 370(6520): 1105-1110
      The human mitochondrial ribosome (mitoribosome) and associated proteins regulate the synthesis of 13 essential subunits of the oxidative phosphorylation complexes. We report the discovery of a mitoribosome-associated quality control pathway that responds to interruptions during elongation, and we present structures at 3.1- to 3.3-angstrom resolution of mitoribosomal large subunits trapped during ribosome rescue. Release factor homolog C12orf65 (mtRF-R) and RNA binding protein C6orf203 (MTRES1) eject the nascent chain and peptidyl transfer RNA (tRNA), respectively, from stalled ribosomes. Recruitment of mitoribosome biogenesis factors to these quality control intermediates suggests additional roles for these factors during mitoribosome rescue. We also report related cryo-electron microscopy structures (3.7 to 4.4 angstrom resolution) of elongating mitoribosomes bound to tRNAs, nascent polypeptides, the guanosine triphosphatase elongation factors mtEF-Tu and mtEF-G1, and the Oxa1L translocase.
  29. Methods Mol Biol. 2021 ;2192 197-210
      Mitochondrial ribosomes (mitoribosomes) are specialized machineries that carry out the synthesis of a limited number of proteins encoded in the mitochondrial genome, including components of the oxidative phosphorylation pathway. They have incorporated several structural features distinguishing them from bacterial and eukaryotic cytosolic counterparts. Our current understanding of the assembly and functioning of mitoribosomes is limited, and recent developments in cryo-EM provide promising directions for detailed investigation. Here we describe methods to purify mitoribosomes from human embryonic kidney cells for cryo-EM studies.
    Keywords:  Cryo-EM; Mitochondria; Mitoribosome; Translation
  30. Redox Biol. 2020 Nov 18. pii: S2213-2317(20)31008-9. [Epub ahead of print]38 101803
      Imbalances in redox homeostasis can result in oxidative stress, which is implicated in various pathological conditions including the fatal neuromuscular disease Duchenne Muscular Dystrophy (DMD). DMD is a complicated disease, with many druggable targets at the cellular and molecular level including calcium-mediated muscle degeneration; mitochondrial dysfunction; oxidative stress; inflammation; insufficient muscle regeneration and dysregulated protein and organelle maintenance. Previous investigative therapeutics tended to isolate and focus on just one of these targets and, consequently, therapeutic activity has been limited. Nuclear erythroid 2-related factor 2 (Nrf2) is a transcription factor that upregulates many cytoprotective gene products in response to oxidants and other toxic stressors. Unlike other strategies, targeted Nrf2 activation has the potential to simultaneously modulate separate pathological features of DMD to amplify therapeutic benefits. Here, we review the literature providing theoretical context for targeting Nrf2 as a disease modifying treatment against DMD.
    Keywords:  Duchenne muscular dystrophy; Hormesis; Nrf2; Oxidative stress; Reactive oxygen species; Skeletal muscle
  31. Cell. 2020 Nov 25. pii: S0092-8674(20)31461-6. [Epub ahead of print]183(5): 1185-1201.e20
      Spaceflight is known to impose changes on human physiology with unknown molecular etiologies. To reveal these causes, we used a multi-omics, systems biology analytical approach using biomedical profiles from fifty-nine astronauts and data from NASA's GeneLab derived from hundreds of samples flown in space to determine transcriptomic, proteomic, metabolomic, and epigenetic responses to spaceflight. Overall pathway analyses on the multi-omics datasets showed significant enrichment for mitochondrial processes, as well as innate immunity, chronic inflammation, cell cycle, circadian rhythm, and olfactory functions. Importantly, NASA's Twin Study provided a platform to confirm several of our principal findings. Evidence of altered mitochondrial function and DNA damage was also found in the urine and blood metabolic data compiled from the astronaut cohort and NASA Twin Study data, indicating mitochondrial stress as a consistent phenotype of spaceflight.
    Keywords:  GeneLab; NASA; NASA Twin Study; Rodent Research Missions; lipids; microgravity; mitochondria; space radiation; spaceflight; transcriptomic