bims-auttor Biomed News
on Autophagy and mTOR
Issue of 2020‒05‒31
thirty-four papers selected by
Viktor Korolchuk
Newcastle University


  1. Sci Rep. 2020 May 26. 10(1): 8663
    Singhal A, Krystofiak ES, Jerome WG, Song B.
      Niemann-Pick type C (NPC) disease is a fatal neurodegenerative disorder caused by mutations in NPC1 and NPC2 genes that result in an accumulation of cholesterol in lysosomes. The majority of children with NPC die in adolescence. Currently, no FDA-approved therapies exist for NPC and the mechanisms of NPC disease are not fully understood. Our recent study and the reports from other laboratories showed that 2-hydroxypropyl-γ-cyclodextrin (HPγCD) alleviates cholesterol accumulation in NPC1-deficient cells in spite of its low binding affinity for cholesterol. In this study, we explored the cellular changes that are induced upon HPγCD treatment in NPC1 patient-derived fibroblasts. We show that HPγCD treatment increases lysosome-ER association and enhances autophagic activity. Our study indicates that HPγCD induces an activation of the transcription factor EB (TFEB), a master regulator of lysosomal functions and autophagy. Lysosome-ER association could potentially function as conduits for cholesterol transport from lysosomes to the ER. Accumulating evidence suggests a role for autophagy in rescuing the cholesterol accumulation in NPC and other degenerative diseases. Collectively, our findings suggest that HPγCD restores cellular homeostasis in NPC1-deficient cells via enhancing lysosomal dynamics and functions. Understanding the mechanisms of HPγCD-induced cellular pathways could contribute to effective NPC therapies.
    DOI:  https://doi.org/10.1038/s41598-020-65627-4
  2. Autophagy. 2020 May 26. 1-2
    Puri C, Rubinsztein DC.
      Elucidation of the membranes contributing to autophagosomes has been a critical question in the field, and an area of active research. Recently, we showed that key events in autophagosome formation, from PtdIns3P formation/WIPI2 recruitment to LC3-GABARAP membrane conjugation, occur on the RAB11A-positive compartment (recycling endosomes). This observation raised the question of how the LC3-positive autophagosome precursors detach from the recycling endosome. We recently observed that DNM2 (dynamin 2) mediates this step, and described how the DNM2R465W mutation that causes centronuclear myopathy (CNM) leads to the accumulation of autophagic structures on recycling endosomes, thereby stalling macroautophagy/autophagy. This physiologically important step highlights the importance of understanding release of nascent autophagosomes from the recycling endosomes as part of the autophagy itinerary.
    Keywords:  Autophagy; RAB11; centronuclear myopathy; dynamin; muscle disease; phagophore; recycling endosome
    DOI:  https://doi.org/10.1080/15548627.2020.1764210
  3. J Cell Biol. 2020 Jul 06. pii: e201910063. [Epub ahead of print]219(7):
    Tomioka Y, Kotani T, Kirisako H, Oikawa Y, Kimura Y, Hirano H, Ohsumi Y, Nakatogawa H.
      The mechanisms underlying turnover of the nuclear pore complex (NPC) and the component nucleoporins (Nups) are still poorly understood. In this study, we found that the budding yeast Saccharomyces cerevisiae triggers NPC degradation by autophagy upon the inactivation of Tor kinase complex 1. This degradation largely depends on the selective autophagy-specific factor Atg11 and the autophagy receptor-binding ability of Atg8, suggesting that the NPC is degraded via receptor-dependent selective autophagy. Immunoelectron microscopy revealed that NPCs embedded in nuclear envelope-derived double-membrane vesicles are sequestered within autophagosomes. At least two pathways are involved in NPC degradation: Atg39-dependent nucleophagy (selective autophagy of the nucleus) and a pathway involving an unknown receptor. In addition, we found the interaction between Nup159 and Atg8 via the Atg8-family interacting motif is important for degradation of this nucleoporin not assembled into the NPC. Thus, this study provides the first evidence for autophagic degradation of the NPC and Nups, which we term "NPC-phagy" and "nucleoporinophagy."
    DOI:  https://doi.org/10.1083/jcb.201910063
  4. Autophagy. 2020 May 26. 1-12
    Tian S, Jin S, Wu Y, Liu T, Luo M, Ou J, Xie W, Cui J.
      Macroautophagy/autophagy, a eukaryotic homeostatic process that sequesters cytoplasmic constituents for lysosomal degradation, is orchestrated by a number of autophagy-related (ATG) proteins tightly controlled by post-translational modifications. However, the involvement of reversible ubiquitination in the regulation of autophagy remains largely unclear. Here, we performed a single-guide RNA-based screening assay to investigate the functions of deubiquitinating enzymes (DUBs) in regulating autophagy. We identified previously unrecognized roles of several DUBs in modulating autophagy at multiple levels by targeting various ATG proteins. Mechanistically, we demonstrated that STAMBP/AMSH (STAM-binding protein) promotes the stabilization of ULK1 by removing its lysine 48 (K48)-linked ubiquitination, whereas OTUD7B mediates the degradation of PIK3 C3 by enhancing its K48-linked ubiquitination, thus positively or negatively affects autophagy flux, respectively. Together, our study elaborated on the broad involvement of DUBs in regulating autophagy and uncovered the critical roles of the reversible ubiquitination in the modification of ATG proteins.ABBREVIATIONS: ATG: autophagy-related; Baf A1: bafilomycin A1; DUB: deubiquitinating enzyme; EBSS: Earle's balanced salt solution; KO: knockout; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; OTUD7B: OTU domain-containing protein 7B; PIK3C3: phosphatidylinositol 3-kinase catalytic subunit type 3; sgRNA: single-guide RNA; SQSTM1/p62: sequestosome 1; STAMBP/AMSH: STAM-binding protein; ULK1: unc-51 like autophagy activating kinase 1; USP: ubiquitin specific peptidase.
    Keywords:  Autophagy; OTUD7B; PIK3C3; STAMBP; ULK1; deubiquitinating enzymes; ubiquitination
    DOI:  https://doi.org/10.1080/15548627.2020.1761652
  5. Cell Rep. 2020 May 26. pii: S2211-1247(20)30648-3. [Epub ahead of print]31(8): 107695
    Jacomin AC, Petridi S, Di Monaco M, Bhujabal Z, Jain A, Mulakkal NC, Palara A, Powell EL, Chung B, Zampronio C, Jones A, Cameron A, Johansen T, Nezis IP.
      Autophagy is the degradation of cytoplasmic material through the lysosomal pathway. One of the most studied autophagy-related proteins is LC3. Despite growing evidence that LC3 is enriched in the nucleus, its nuclear role is poorly understood. Here, we show that Drosophila Atg8a protein, homologous to mammalian LC3, interacts with the transcription factor Sequoia in a LIR motif-dependent manner. We show that Sequoia depletion induces autophagy in nutrient-rich conditions through the enhanced expression of autophagy genes. We show that Atg8a interacts with YL-1, a component of a nuclear acetyltransferase complex, and that it is acetylated in nutrient-rich conditions. We also show that Atg8a interacts with the deacetylase Sir2, which deacetylates Atg8a during starvation to activate autophagy. Our results suggest a mechanism of regulation of the expression of autophagy genes by Atg8a, which is linked to its acetylation status and its interaction with Sequoia, YL-1, and Sir2.
    Keywords:  LC3/Atg8; LIR motif; acetylation; autophagy; nucleus; transcription
    DOI:  https://doi.org/10.1016/j.celrep.2020.107695
  6. Autophagy. 2020 May 28. 1-16
    Li W, Li Y, Guan Y, Du Y, Zhao M, Chen X, Zhu F, Guo C, Jia Y, Li Y, Wang X, Wang X, Shi Y, Wang Q, Li Y, Zhang L.
      Macroautophagy/autophagy is an evolutionarily conserved process that involves the selective degradation of cytoplasmic components within lysosomes in response to starvation. Autophagy is an ancient defense mechanism that has been closely integrated with the immune system and has multiple effects on innate and adaptive immunity. The pro-inflammatory and anti-inflammatory cytokines can activate and inhibit autophagy, respectively. TNFAIP8L2/TIPE2 (tumor necrosis factor, alpha-induced protein 8-like 2) is a newly identified immune negative regulator of innate and adaptive immunity that plays an important role in immune homeostasis. However, whether and how TNFAIP8L2 controls autophagy is still unknown. Murine TNFAIP8L2 can directly bind to and block the RAC1 GTPase activity to regulate innate immunity. RAC1 can also bind to MTOR and regulate MTORC1 cellular localization and activity. Here, we find that TNFAIP8L2 can compete with MTOR for binding to the GTP-bound state of RAC1 and negatively regulate MTORC1 activity. Interestingly, TNFAIP8L2 overexpression fails to induce autophagy flux by the suppression of the MTOR activity under glutamine and serum starvation. Instead, TNFAIP8L2 appears to impair autophagic lysosome reformation (ALR) during prolonged starvation. Finally, we demonstrate that TNFAIP8L2 overexpression leads to a defect in MTOR reactivation and disrupts autophagy flux, thereby leading to cell death. Furthermore, TNFAIP8L2 deficiency can exacerbate the inflammatory response and lung injury by controlling the MTOR activity in an LPS-induced mouse endotoxemia model. Our study reveals a novel role of TNFAIP8L2 in autophagy by regulating the RAC1-MTORC1 axis that supports its potential as a target for therapeutic treatment.Abbreviations: ALR: autophagic lysosome reformation; BafA1: bafilomycin A1; BMDMs: bone marrow-derived macrophages; Co-IP: Co-Immunoprecipitation; LAMP1: lysosomal associated membrane protein 1; MAP1LC3B/LC3B: microtubule associated protein 1 light chain 3 beta; MTORC1: mechanistic target of rapamycin kinase complex 1; RAPA: rapamycin; RPS6: ribosomal protein S6; SQSTM1/p62: sequestosome 1; Starv: Starvation; TNFAIP8L2/TIPE2: tumor necrosis factor-alpha-induced protein-8 like-2.
    Keywords:  Autophagy; MTORC1; RAC1; TNFAIP8L2/TIPE2; autophagic lysosome reformation (ALR); cell death
    DOI:  https://doi.org/10.1080/15548627.2020.1761748
  7. Autophagy. 2020 May 26.
    Choi I, Seegobin SP, Liang D, Yue Z.
      SNCA/α-synuclein is a major component in the Lewy body (LB), a pathological hallmark of Parkinson disease (PD) and dementia with Lewy body (DLB), collectively known as synucleinopathies. SNCA/α-synuclein can be secreted from neurons and transmitted to neighboring cells including neurons and glia, which underlie the spreading of LB pathology as described by Braak and colleagues. We recently have investigated the mechanism and significance for microglia, a prototypic phagocyte in the brain, in engulfing and controlling SNCA/α-synuclein homeostasis in the brain. Using microglia-specific autophagy-deficient mice, we demonstrated that microglia ingest and degrade neuron-released SNCA/α-synuclein through SQSTM1/p62-mediated selective autophagy both in vivo and in vitro. This process requires the presence of TLR4 (toll like receptor 4), which interacts with SNCA/α-synuclein to induce the transcriptional upregulation of Sqstm1/p62 through the NFKB/NF-κB pathway. We term the selective autophagy of SNCA/α-synuclein as "synucleinphagy". We showed that the disruption of microglial autophagy causes accumulation of misfolded SNCA/α-synuclein and loss of dopaminergic neurons, two hallmarks of PD. Hence, our study reveals a neuroprotective role of microglia through an autophagy-mediated "community cleanup program".
    Keywords:  Autophagy; Microglia; α-synuclein
    DOI:  https://doi.org/10.1080/15548627.2020.1774149
  8. J Physiol Biochem. 2020 May 25.
    Al-Bari MAA.
      Macroautophagy (hereafter called autophagy) is a highly conserved lysosomal pathway for catabolism of intracellular material in eukaryotic cells. Autophagy is also an essential homeostatic process through which intracellular components are recycled for reuse or energy production. The extremely regulated autophagy process begins with the formation of hallmarked double membrane bound organelles called autophagosomes which in turn fuse with lysosomes called autolysosomes and finally degrade the autophagic cargos. The multistages molecular machinery of autophagy is critically orchestrated by the action of a set of the autophagy proteins (Atg) and a supreme regulator, mTOR (mechanistic target of rapamycin). However, individual stages of autophagy are mechanistically complex and partially understood. In this review, the individual stages of autophagy are dissected, and the corresponding molecular regulation is discussed in view of current scientific knowledge of autophagy. This understanding of sequential events of autophagy machinery through this review may lead to great interest in the therapeutic potential for manipulating of autophagy in established diseases.
    Keywords:  ALR cycle; Beclin 1; Connexins; Molecular dissection
    DOI:  https://doi.org/10.1007/s13105-020-00746-0
  9. Autophagy. 2020 May 25. 1-19
    Hossain MI, Marcus JM, Lee JH, Garcia PL, Singh V, Shacka JJ, Zhang J, Gropen TI, Falany CN, Andrabi SA.
      Stroke is a leading cause of death and disability. The pathophysiological mechanisms associated with stroke are very complex and not fully understood. Lysosomal function has a vital physiological function in the maintenance of cellular homeostasis. In neurons, CTSD (cathepsin D) is an essential protease involved in the regulation of proteolytic activity of the lysosomes. Loss of CTSD leads to lysosomal dysfunction and accumulation of different cellular proteins implicated in neurodegenerative diseases. In cerebral ischemia, the role of CTSD and lysosomal function is not clearly defined. We used oxygen-glucose deprivation (OGD) in mouse cortical neurons and the middle cerebral artery occlusion (MCAO) model of stroke to assess the role of CTSD in stroke pathophysiology. Our results show a time-dependent decrease in CTSD protein levels and activity in the mouse brain after stroke and neurons following OGD, with concurrent defects in lysosomal function. We found that shRNA-mediated knockdown of CTSD in neurons is sufficient to cause lysosomal dysfunction. CTSD knockdown further aggravates lysosomal dysfunction and cell death in OGD-exposed neurons. Restoration of CTSD protein levels via lentiviral transduction increases CTSD activity in neurons and, thus, renders resistance to OGD-mediated defects in lysosomal function and cell death. This study indicates that CTSD-dependent lysosomal function is critical for maintaining neuronal survival in cerebral ischemia; thus, strategies focused on maintaining CTSD function in neurons are potentially novel therapeutic approaches to prevent neuronal death in stroke.ABBREVIATIONS: 3-MA: 3-methyladenine; ACTB: actin beta; AD: Alzheimer disease; ALS: amyotrophic lateral sclerosis; CQ: chloroquine; CTSB: cathepsin B; CTSD: cathepsin D; CTSL: cathepsin L; FTD: frontotemporal dementia, HD: Huntington disease; LAMP1: lysosomal associated membrane protein 1; LSD: lysosomal storage disease; MCAO: middle cerebral artery occlusion; OGD: oxygen glucose deprivation; OGR: oxygen glucose resupply; PD: Parkinson disease; SQSMT1: sequestosome 1; TCA: trichloroacetic acid; TTC: triphenyl tetrazolium chloride.
    Keywords:  Autophagic flux; cathepsin D; lysosome; protein aggregation; proteolysis; stroke
    DOI:  https://doi.org/10.1080/15548627.2020.1761219
  10. Stem Cell Reports. 2020 May 14. pii: S2213-6711(20)30151-X. [Epub ahead of print]
    Yamaguchi A, Ishikawa KI, Inoshita T, Shiba-Fukushima K, Saiki S, Hatano T, Mori A, Oji Y, Okuzumi A, Li Y, Funayama M, Imai Y, Hattori N, Akamatsu W.
      Parkinson disease (PD) is a neurodegenerative disorder caused by the progressive loss of midbrain dopaminergic neurons, and mitochondrial dysfunction is involved in its pathogenesis. This study aimed to establish an imaging-based, semi-automatic, high-throughput system for the quantitative detection of disease-specific phenotypes in dopaminergic neurons from induced pluripotent stem cells (iPSCs) derived from patients with familial PD having Parkin or PINK1 mutations, which exhibit abnormal mitochondrial homeostasis. The proposed system recapitulates the deficiency of mitochondrial clearance, ROS accumulation, and increasing apoptosis in these familial PD-derived neurons. We screened 320 compounds for their ability to ameliorate multiple phenotypes and identified four candidate drugs. Some of these drugs improved the locomotion defects and reduced ATP production caused by PINK1 inactivation in Drosophila and were effective for idiopathic PD-derived neurons with impaired mitochondrial clearance. Our findings suggest that the proposed high-throughput system has potential for identifying effective drugs for familial and idiopathic PD.
    Keywords:  PARK2; PARK6; Parkinson disease; drug screening; iPS cells; mitochondria; mitophagy
    DOI:  https://doi.org/10.1016/j.stemcr.2020.04.011
  11. Mech Ageing Dev. 2020 May 23. pii: S0047-6374(20)30062-2. [Epub ahead of print] 111266
    Leboutet R, Chen Y, Legouis R, Culetto E.
      Mitochondria is a key cellular organelle, which is tightly supervised by multiple oversight cellular mechanisms regulating mitochondrial biogenesis and mitochondria maintenance and/or elimination. Selective autophagy of mitochondria, id est mitophagy, is one of the cellular mechanisms controlling mitochondria homeostasis. The nematode Caenorhabditis elegans has recently emerged as a powerful model organism to study the roles and functions of mitophagy. We present here the current knowledge on cellular and molecular mechanisms underlying the selective elimination of mitochondria by autophagy in C. elegans in the context of developmental processes, aging and adaptive responses to various stresses.
    Keywords:  Caenorhabditis elegans; aging; heteroplasmy; mitochondria; mitochondria homeostasis; mitophagy; paternal mitochondria elimination; stress
    DOI:  https://doi.org/10.1016/j.mad.2020.111266
  12. Front Biosci (Landmark Ed). 2020 Jun 01. 25 1925-1973
    Maiese K.
      Metabolic disorders, such as diabetes mellitus (DM), are increasingly becoming significant risk factors for the health of the global population and consume substantial portions of the gross domestic product of all nations. Although conventional therapies that include early diagnosis, nutritional modification of diet, and pharmacological treatments may limit disease progression, tight serum glucose control cannot prevent the onset of future disease complications. With these concerns, novel strategies for the treatment of metabolic disorders that involve the vitamin nicotinamide, the mechanistic target of rapamycin (mTOR), mTOR Complex 1 (mTORC1), mTOR Complex 2 (mTORC2), AMP activated protein kinase (AMPK), and the cellular pathways of autophagy and apoptosis offer exceptional promise to provide new avenues of treatment. Oversight of these pathways can promote cellular energy homeostasis, maintain mitochondrial function, improve glucose utilization, and preserve pancreatic beta-cell function. Yet, the interplay among mTOR, AMPK, and autophagy pathways can be complex and affect desired clinical outcomes, necessitating further investigations to provide efficacious treatment strategies for metabolic dysfunction and DM.
  13. Sci Rep. 2020 May 29. 10(1): 8755
    Adriaenssens E, Tedesco B, Mediani L, Asselbergh B, Crippa V, Antoniani F, Carra S, Poletti A, Timmerman V.
      Three missense mutations targeting the same proline 209 (Pro209) codon in the co-chaperone Bcl2-associated athanogene 3 (BAG3) have been reported to cause distal myopathy, dilated cardiomyopathy or Charcot-Marie-Tooth type 2 neuropathy. Yet, it is unclear whether distinct molecular mechanisms underlie the variable clinical spectrum of the rare patients carrying these three heterozygous Pro209 mutations in BAG3. Here, we studied all three variants and compared them to the BAG3_Glu455Lys mutant, which causes dilated cardiomyopathy. We found that all BAG3_Pro209 mutants have acquired a toxic gain-of-function, which causes these variants to accumulate in the form of insoluble HDAC6- and vimentin-positive aggresomes. The aggresomes formed by mutant BAG3 led to a relocation of other chaperones such as HSPB8 and Hsp70, which, together with BAG3, promote the so-called chaperone-assisted selective autophagy (CASA). As a consequence of their increased aggregation-proneness, mutant BAG3 trapped ubiquitinylated client proteins at the aggresome, preventing their efficient clearance. Combined, these data show that all BAG3_Pro209 mutants, irrespective of their different clinical phenotypes, are characterized by a gain-of-function that contributes to the gradual loss of protein homeostasis.
    DOI:  https://doi.org/10.1038/s41598-020-65664-z
  14. J Cell Sci. 2020 May 27. pii: jcs240440. [Epub ahead of print]133(10):
    Cong Y, Dinesh Kumar N, Mauthe M, Verlhac P, Reggiori F.
      Macroautophagy (hereafter autophagy) is a highly conserved catabolic pathway, which mediates the delivery of unwanted cytoplasmic structures and organelles to lysosomes for degradation. In numerous situations, autophagy is highly selective and exclusively targets specific intracellular components. Selective types of autophagy are a central element of our cell-autonomous innate immunity as they can mediate the turnover of viruses or bacteria, that gain access to the cytoplasm of the cell. Selective autophagy also modulates other aspects of our immunity by turning over specific immunoregulators. Throughout evolution, however, the continuous interaction between this fundamental cellular pathway and pathogens has led several pathogens to develop exquisite mechanisms to inhibit or subvert selective types of autophagy, to promote their intracellular multiplication. This Cell Science at a Glance article and the accompanying poster provides an overview of the selective autophagy of both pathogens, known as xenophagy, and of immunoregulators, and highlights a few archetypal examples that illustrate molecular strategies developed by viruses and bacteria to manipulate selective autophagy for their own benefit.
    Keywords:  Autophagy; Autophagy receptor; Bacteria; Immunity; Subversion; Viruses
    DOI:  https://doi.org/10.1242/jcs.240440
  15. Int J Mol Sci. 2020 May 23. pii: E3691. [Epub ahead of print]21(10):
    Deval C, Calonne J, Coudy-Gandilhon C, Vazeille E, Bechet D, Polge C, Taillandier D, Attaix D, Combaret L.
      Mitochondria alterations are a classical feature of muscle immobilization, and autophagy is required for the elimination of deficient mitochondria (mitophagy) and the maintenance of muscle mass. We focused on the regulation of mitochondrial quality control during immobilization and remobilization in rat gastrocnemius (GA) and tibialis anterior (TA) muscles, which have very different atrophy and recovery kinetics. We studied mitochondrial biogenesis, dynamic, movement along microtubules, and addressing to autophagy. Our data indicated that mitochondria quality control adapted differently to immobilization and remobilization in GA and TA muscles. Data showed i) a disruption of mitochondria dynamic that occurred earlier in the immobilized TA, ii) an overriding role of mitophagy that involved Parkin-dependent and/or independent processes during immobilization in the GA and during remobilization in the TA, and iii) increased mitochondria biogenesis during remobilization in both muscles. This strongly emphasized the need to consider several muscle groups to study the mechanisms involved in muscle atrophy and their ability to recover, in order to provide broad and/or specific clues for the development of strategies to maintain muscle mass and improve the health and quality of life of patients.
    Keywords:  immobilization; microtubules; mitophagy; physical inactivity; recovery; skeletal muscle
    DOI:  https://doi.org/10.3390/ijms21103691
  16. Cancer Gene Ther. 2020 May 27.
    Liu Y, Wang Y, Liu J, Kang R, Tang D.
      Ferroptosis has become a topic of rapidly growing interest in recent years, and has possible therapy implications in cancer therapy. Although excessive autophagy may contribute to ferroptosis, its underlying molecular mechanism remains largely unknown. Here, we provide novel evidence that the interplay between the signals of mechanistic target of rapamycin kinase (MTOR) and glutathione peroxidase 4 (GPX4) modulates autophagy-dependent ferroptosis in human pancreatic cancer cells. Both the classical autophagy inducer rapamycin and the classical ferroptosis activator RSL3 can block MTOR activation and cause GPX4 protein degradation in human pancreatic cancer cells. Moreover, GPX4 plays an essential role in the inhibition of autophagy-dependent ferroptosis induced by rapamycin and RSL3. Consequently, GPX4 depletion by RNAi enhances the anticancer activity of rapamycin and RSL3 in vitro or in vivo. These findings not only increase our understanding of stress responses in cell death, but may also raise the possibility of developing new antitumor therapy targeting autophagy-dependent cell death.
    DOI:  https://doi.org/10.1038/s41417-020-0182-y
  17. Adv Exp Med Biol. 2020 ;1195 33
    Tavernarakis N.
      Autophagy is crucial for neuronal integrity. Loss of key autophagic components leads to progressive neurodegeneration and structural defects in neuronal synapses. However, the molecular mechanisms regulating autophagy in the brain remain elusive. Similarly, while it is widely accepted that protein turnover is required for synaptic plasticity, the contribution of autophagy to the degradation of synaptic proteins is unknown. We find that BDNF signaling via the tropomyosin receptor kinase B (TrkB) and the phosphatidylinositol 3-kinase (PI3K)/Akt pathway suppresses autophagy in vivo. Autophagy is differentially regulated by fasting, in different brain regions. Suppression of autophagy is required for BDNF-induced synaptic plasticity and for memory enhancement, under conditions of nutritional stress. BDNF signaling suppresses autophagy in the forebrain of adult mice. Indeed, BDNF ablation in the neural lineage causes uncontrolled increase in autophagy. In turn, increased autophagy mediates the synaptic defects caused by BDNF deficiency. Thus, fasting suppresses autophagy in regions of the mouse forebrain, thereby promoting synaptic remodeling and memory through a BDNF-regulated mechanism. We identify three key remodelers of postsynaptic densities as cargo of autophagy. Our results establish autophagy as a pivotal component of BDNF signaling, which is essential for BDNF-induced synaptic plasticity. This molecular mechanism underlies behavioral adaptations that increase fitness in times of scarcity.
    Keywords:  Autophagy; BDNF; Synaptic plasticity
    DOI:  https://doi.org/10.1007/978-3-030-32633-3_5
  18. Cells. 2020 May 22. pii: E1296. [Epub ahead of print]9(5):
    Dobner J, Simons IM, Rufinatscha K, Hänsch S, Schwarten M, Weiergräber OH, Abdollahzadeh I, Gensch T, Bode JG, Hoffmann S, Willbold D.
      The γ-aminobutyric acid type A receptor-associated protein (GABARAP) and its close paralogs GABARAPL1 and GABARAPL2 constitute a subfamily of the autophagy-related 8 (Atg8) protein family. Being associated with a variety of dynamic membranous structures of autophagic and non-autophagic origin, Atg8 proteins functionalize membranes by either serving as docking sites for other proteins or by acting as membrane tethers or adhesion factors. In this study, we describe that deficiency for GABARAP alone, but not for its close paralogs, is sufficient for accelerated EGF receptor (EGFR) degradation in response to EGF, which is accompanied by the downregulation of EGFR-mediated MAPK signaling, altered target gene expression, EGF uptake, and EGF vesicle composition over time. We further show that GABARAP and EGFR converge in the same distinct compartments at endogenous GABARAP expression levels in response to EGF stimulation. Furthermore, GABARAP associates with EGFR in living cells and binds to synthetic peptides that are derived from the EGFR cytoplasmic tail in vitro. Thus, our data strongly indicate a unique and novel role for GABARAP during EGFR trafficking.
    Keywords:  Atg8; EGFR; GABARAP; degradation; genome editing; receptor trafficking
    DOI:  https://doi.org/10.3390/cells9051296
  19. Mol Neurobiol. 2020 May 27.
    Rai SN, Singh C, Singh A, Singh MP, Singh BK.
      Mitochondrial dysfunction plays a very vital role in the pathogenesis of Alzheimer's disease (AD). Several shreds of evidence have indicated that the mitochondrial function is severely compromised under AD pathogenesis. Most of the recent therapeutic strategies have been conversed to treat AD by pinpointing the pathways involved in the pathophysiology of AD. In AD, mitochondria progressively lose their proper functions that are ultimately responsible for their accumulation and removal via the autophagic process, which is called mitophagy that further worsens the progression of this incapacitating disease. Preclinical and clinical studies have suggested that mitochondrial dysfunction along with mitophagy significantly contributes to the accumulation of amyloid-beta (Aβ) fibrils and hyperphosphorylated tau protein tangles which lead to synaptic dysfunctions and cognitive impairments such as memory loss through reactive oxygen species (ROS)-mediated pathway. The present review is intended to discuss the recent advancements in the frontiers of mitochondrial dysfunction and consequent therapeutic strategies that have been employed to treat AD.
    Keywords:  Alzheimer’s disease; Autophagy; Mitochondrial dysfunction; Mitophagy
    DOI:  https://doi.org/10.1007/s12035-020-01945-y
  20. Life Sci. 2020 May 21. pii: S0024-3205(20)30566-X. [Epub ahead of print]255 117817
    Zhou R, Li J, Zhang L, Cheng Y, Yan J, Sun Y, Wang J, Jiang H.
      Glucocorticoids can promote cardiomyocyte maturation. However, the mechanism underlying glucocorticoid-mediated cardiomyocyte maturation is still unclear. Mitophagy plays a key role in cardiomyocyte maturation. Based on current knowledge, our study evaluated the effects of the glucocorticoid dexamethasone (100 nM) on the maturation of mouse embryonic stem cell-derived cardiomyocytes and the role of mitophagy in this maturation. The results showed that dexamethasone can promote embryonic stem cell-derived cardiomyocyte maturation, inhibit cardiomyocyte proliferation, and promote myocardial fiber arrangement. However, dexamethasone did not affect mitochondrial morphology in cardiomyocytes. Glucocorticoid receptor inhibitors (RU486, 1 nM) can inhibit dexamethasone-mediated cardiomyocyte maturation. Additionally, dexamethasone can promote mitophagy in embryonic stem cell-derived cardiomyocytes and induce LC3 and lysosomal aggregation in mitochondria. The inhibition of mitophagy can inhibit the cardiomyocyte maturation effect of dexamethasone. Furthermore, our research found that dexamethasone may mediate the occurrence of mitophagy in cardiomyocytes through Parkin. The siRNA-mediated inhibition of Parkin expression can inhibit mitochondrial autophagy caused by dexamethasone, thus inhibiting cardiomyocyte maturation. Overall, our study found that dexamethasone can promote embryonic stem cell-derived cardiomyocyte maturation through Parkin-mediated mitophagy.
    Keywords:  Cardiomyocyte maturation; Glucocorticoids; Mitophagy
    DOI:  https://doi.org/10.1016/j.lfs.2020.117817
  21. Neurosci Res. 2020 May 22. pii: S0168-0102(20)30078-X. [Epub ahead of print]
    Mizuno Y.
      Discovery of Park2 is our finding of a family of young onset parkinsonism, in which this family was thought to be associated with a polymorphism of the manganese superoxide gene. The gene locus of the manganese superoxide dismutase has been known. We were able to pick up a gene for this family and related families in the close approximate position at the long arm of chromosome 6. The gene for this disease has a ubiquitin-like motif in the N-terminus and two RING finger structures. It was shown that this gene had a ubiquitin-protein ligase activity. But it is not elucidated the substrate of this enzyme. Meanwhile, it has become clear that PINK1 and Parkin work together to remove the mitochondria of the lowered membrane potential in the autophagosomes (mitophagy). Now that the molecular mechanisms of mitophagy is under investigation. In addition, many hot topics are going on such as Lewy body in Park2, single heterozygotes, rare clinical manifestations, and so on.
    Keywords:  Clinical aspects; Function of Parkin; Park2; Parkinson’s disease; Single heterozygotes
    DOI:  https://doi.org/10.1016/j.neures.2020.02.002
  22. J Neural Transm (Vienna). 2020 May 25.
    Pompili E, Fabrizi C, Fumagalli L, Fornai F.
      Autophagy is a degradative process playing an important role in removing misfolded or aggregated proteins, clearing damaged organelles, such as mitochondria and endoplasmic reticulum, as well as eliminating intracellular pathogens. The autophagic process is important for balancing sources of energy at critical developmental stages and in response to nutrient stress. Recently, autophagy has been involved in the pathophysiology of neurodegenerative diseases although its beneficial (pro-survival) or detrimental (pro-death) role remains controversial. In the present review, we discuss the role of autophagy following intoxication with trimethyltin (TMT), an organotin compound that induces severe hippocampal neurodegeneration associated with astrocyte and microglia activation. TMT is considered a useful tool to study the molecular mechanisms occurring in human neurodegenerative diseases such as Alzheimer's disease and temporal lobe epilepsy. This is also relevant in the field of environmental safety, since organotin compounds are used as heat stabilizers in polyvinyl chloride polymers, industrial and agricultural biocides, and as industrial chemical catalysts.
    Keywords:  Autophagy; Lithium; Neurodegeneration; TMT; Trimethyltin
    DOI:  https://doi.org/10.1007/s00702-020-02210-1
  23. Mol Cell. 2020 May 20. pii: S1097-2765(20)30275-6. [Epub ahead of print]
    Toda G, Soeda K, Okazaki Y, Kobayashi N, Masuda Y, Arakawa N, Suwanai H, Masamoto Y, Izumida Y, Kamei N, Sasako T, Suzuki R, Kubota T, Kubota N, Kurokawa M, Tobe K, Noda T, Honda K, Accili D, Yamauchi T, Kadowaki T, Ueki K.
      The physiological role of immune cells in the regulation of postprandial glucose metabolism has not been fully elucidated. We have found that adipose tissue macrophages produce interleukin-10 (IL-10) upon feeding, which suppresses hepatic glucose production in cooperation with insulin. Both elevated insulin and gut-microbiome-derived lipopolysaccharide in response to feeding are required for IL-10 production via the Akt/mammalian target of rapamycin (mTOR) pathway. Indeed, myeloid-specific knockout of the insulin receptor or bone marrow transplantation of mutant TLR4 marrow cells results in increased expression of gluconeogenic genes and impaired glucose tolerance. Furthermore, myeloid-specific Akt1 and Akt2 knockout results in similar phenotypes that are rescued by additional knockout of TSC2, an inhibitor of mTOR. In obesity, IL-10 production is impaired due to insulin resistance in macrophages, whereas adenovirus-mediated expression of IL-10 ameliorates postprandial hyperglycemia. Thus, the orchestrated response of the endogenous hormone and gut environment to feeding is a key regulator of postprandial glycemia.
    Keywords:  Akt; gut microbiome; insulin; interleukin-10; lipopolysaccharide; macrophages; mammalian target of rapamycin; postprandial gluconeogenesis
    DOI:  https://doi.org/10.1016/j.molcel.2020.04.033
  24. Biol Cell. 2020 May 23.
    Tong X, Ganta RR, Liu Z.
      Osteoclasts are multinucleated giant cells, responsible for bone resorption. Osteoclast differentiation and function requires a series of cytokines to remove the old bone, which coordinates with the induction of bone remodeling by osteoblast-mediated bone formation. Studies have demonstrated that adenosine 5'-phosphate (AMP)-activated protein kinase (AMPK) play a negative regulatory role in osteoclast differentiation and function. Research involving AMPK, a nutrient and energy sensor, has primarily focused on osteoclast differentiation and function; thus, its role in autophagy, inflammation, and immunity remains poorly understood. Autophagy, is a conservative homeostatic mechanism of eukaryotic cells, and response to osteoclast differentiation and function; however, how it interactswith inflammation remains unclear. Additionally, based on theregulatory function of different AMPK subunits for osteoclast differentiation and function, its activationis regulated by upstream factors to performbone metabolism. This review summarizes the critical role of AMPK-mediated autophagy, inflammation, and immunityby upstream and downstream signalingduring receptor activator of nuclear factor kappa-B ligand (RANKL)-induced osteoclast differentiation and function. This pathway mayprovide therapeutic targets for bone-related diseases, as well as functionas abiomarkerfor bone homeostasis. This article is protected by copyright. All rights reserved.
    Keywords:  AMPK; RANKL; autophagy; inflammation; osteoclast differentiation and function
    DOI:  https://doi.org/10.1111/boc.202000008
  25. Cells. 2020 May 24. pii: E1308. [Epub ahead of print]9(5):
    Margulis B, Tsimokha A, Zubova S, Guzhova I.
      Throughout their life cycles, cells are subject to a variety of stresses that lead to a compromise between cell death and survival. Survival is partially provided by the cell proteostasis network, which consists of molecular chaperones, a ubiquitin-proteasome system of degradation and autophagy. The cooperation of these systems impacts the correct function of protein synthesis/modification/transport machinery starting from the adaption of nascent polypeptides to cellular overcrowding until the utilization of damaged or needless proteins. Eventually, aging cells, in parallel to the accumulation of flawed proteins, gradually lose their proteostasis mechanisms, and this loss leads to the degeneration of large cellular masses and to number of age-associated pathologies and ultimately death. In this review, we describe the function of proteostasis mechanisms with an emphasis on the possible associations between them.
    Keywords:  aging; autophagy; molecular chaperones; ubiquitin-proteasomal system
    DOI:  https://doi.org/10.3390/cells9051308
  26. Int J Mol Sci. 2020 May 23. pii: E3689. [Epub ahead of print]21(10):
    Ren S, Ding C, Sun Y.
      Viruses have evolved different strategies to hijack subcellular organelles during their life cycle to produce robust infectious progeny. Successful viral reproduction requires the precise assembly of progeny virions from viral genomes, structural proteins, and membrane components. Such spatial and temporal separation of assembly reactions depends on accurate coordination among intracellular compartmentalization in multiple organelles. Here, we overview the rearrangement and morphology remodeling of virus-triggered intracellular organelles. Focus is given to the quality control of intracellular organelles, the hijacking of the modified organelle membranes by viruses, morphology remodeling for viral replication, and degradation of intracellular organelles by virus-triggered selective autophagy. Understanding the functional reprogram and morphological remodeling in the virus-organelle interplay can provide new insights into the development of broad-spectrum antiviral strategies.
    Keywords:  intracellular organelles; rearrangement; remodeling; selective autophagy; virus
    DOI:  https://doi.org/10.3390/ijms21103689
  27. J Cell Mol Med. 2020 May 29.
    Huang ML, Qi CL, Zou Y, Yang R, Jiang Y, Sheng JF, Kong YG, Tao ZZ, Chen SM.
      To explore the relationship between autophagy and cell function, we investigated how PLAC8-mediated autophagy influences proliferation, apoptosis and epithelial-mesenchymal transition (EMT) in NPC. Colony formation analyses and CCK8 assays were used to assess the proliferative capacity of NPC cells. Transmission electron microscopy (TEM) was used to identify autophagosomes. Autophagic flux was monitored using the tandem monomeric RFP-GFP-tagged LC3 (tfLC3) assay. The rate of apoptosis in NPC cells was analysed by flow cytometry. Western blot analysis was used to evaluate the activation of autophagy and the signalling status of the AKT/mTOR pathway. Our study reveals that knocking out PLAC8 (koPLAC8) induces autophagy and apoptosis, while suppressing NPC cell proliferation and EMT. However, inhibition of autophagy with 3-methyladenine or by knocking down Beclin-1 reverses the cell proliferation, apoptosis and EMT influenced by koPLAC8. We find that koPLAC8 inhibits the phosphorylation of AKT and its downstream target, mTOR. Moreover, immunofluorescence and co-immunoprecipitation reveal complete PLAC8/AKT colocalization and PLAC8/AKT interaction, respectively. Furthermore, knockout of PLAC8 induced autophagy and inactivated AKT/mTOR signalling pathway of NPC xenografts. Overall, our findings demonstrate that koPLAC8 induces autophagy via the AKT/mTOR pathway, thereby inhibiting cell proliferation and EMT, and promoting apoptosis in NPC cells.
    Keywords:  AKT/mTOR pathway; apoptosis; autophagy; epithelial-mesenchymal transition; nasopharyngeal carcinoma; placenta specific 8 gene knockout
    DOI:  https://doi.org/10.1111/jcmm.15409
  28. Cancer Cell. 2020 May 25. pii: S1535-6108(20)30259-2. [Epub ahead of print]
    Kroemer G, Zitvogel L.
      In a recent Nature paper, Yamamoto et al. demonstrate that, in the particular context of pancreatic carcinoma, autophagy causes the continuous destruction of major histocompatibility complex class I (MHC-I) proteins. Suppression of autophagy favors MHC-I re-appearance on the surface of malignant cells, facilitating their clearance by cytotoxic T lymphocytes.
    DOI:  https://doi.org/10.1016/j.ccell.2020.05.009
  29. Autophagy. 2020 May 27. 1-2
    Yamamoto K, Venida A, Perera RM, Kimmelman AC.
      Major histocompatibility complex class I (MHC-I) is a key molecule in anti-tumor adaptive immunity. MHC-I is essential for endogenous antigen presentation by cancer cells and subsequent recognition and clearance by CD8+ T cells. Defects in MHC-I expression occur frequently in several cancers, leading to impaired antigen presentation, immune evasion and/or resistance to immune checkpoint blockade (ICB) therapy. Pancreatic ductal adenocarcinoma (PDAC), a deadly malignancy with dismal patient prognosis, is resistant to ICB and shows frequent downregulation of MHC-I independent of genetic mutations abrogating MHC-I expression. Previously, we showed that PDAC cells exhibit elevated levels of autophagy and lysosomal biogenesis, which together support the survival and growth of PDAC tumors via both cell-autonomous and non-cell-autonomous mechanisms. In our recent study, we have identified NBR1-mediated selective macroautophagy/autophagy of MHC-I as a novel mechanism that facilitates immune evasion by PDAC cells. Importantly, autophagy or lysosome inhibition restores MHC-I expression, leading to enhanced anti-tumor T cell immunity and improved response to ICB in transplanted tumor models in syngeneic host mice. Our results highlight a previously unknown function of autophagy and the lysosome in regulation of immunogenicity in PDAC, and provide a novel therapeutic strategy for targeting this deadly disease.
    Keywords:  MHC-I; Pancreatic cancer; anti-tumor immunity; autophagy; immune checkpoint blockade; lysosome
    DOI:  https://doi.org/10.1080/15548627.2020.1769973
  30. Biochem Biophys Res Commun. 2020 May 22. pii: S0006-291X(20)31001-9. [Epub ahead of print]
    Kim J, Chee WY, Yabuta N, Kajiwara K, Nada S, Okada M.
      The naked mole-rat (NMR, Heterocephalus glaber) is the longest-living known rodent species, with a maximum lifespan of over 30 years. NMRs exhibit negligible senescence, exceptional resistance to cancer, and high basal autophagy activity compared with mouse. The molecular mechanisms and physiological roles underlying the high basal autophagy activity in NMRs remain to be elucidated. We identified that the Atg12-Atg5 conjugate, a critical component of autophagosome formation, was highly expressed in NMR skin fibroblasts (NSFs) compared with that in mouse skin fibroblasts. Phenotypic analysis of Atg5 knockdown NSFs revealed that high basal autophagy activity in NSFs was associated with abundant expression of the Atg12-Atg5 conjugate. Atg5 knockdown in NSFs led to accumulation of dysfunctional mitochondria, and suppressed cell proliferation and cell adhesion ability, promoting apoptosis/anoikis accompanied by upregulation of the apoptosis-related genes, Bax and Noxa. Furthermore, inhibition of the p53/Rb pro-apoptotic pathway with SV40 large T antigen abolished Atg5 knockdown-induced increases in apoptosis/anoikis. Taken together, these findings suggest that high basal autophagy activity in NMR cells, mediated by Atg5, contributes to suppression of p53/Rb-induced apoptosis, which could benefit the longevity of NMR cells.
    Keywords:  Aging; Anoikis; Apoptosis; Atg5; Autophagy; Longevity; Naked mole-rat
    DOI:  https://doi.org/10.1016/j.bbrc.2020.05.083
  31. Arch Pharm Res. 2020 May 26.
    Tilija Pun N, Jang WJ, Jeong CH.
      Autophagy is a self-degradation process in which the cytoplasmic cargoes are delivered to the lysosomes for degradation. As the cargoes are degraded/recycled, the autophagy process maintains the cellular homeostasis. Anti-cancer therapies induce apoptosis and autophagy concomitantly, and the induced autophagy normally prevents stress responses that are being induced. In such cases, the inhibition of autophagy can be a reasonable strategy to enhance the efficacy of anti-cancer therapies. However, recent studies have shown that autophagy induced by anti-cancer drugs causes cell death/apoptosis induction, indicating a controversial role of autophagy in cancer cell survival or death/apoptosis. Therefore, in the present review, we aimed to assess the signaling mechanisms involved in autophagy and cell death/apoptosis induction during anti-cancer therapies. This review summarizes the process of autophagy, autophagy flux and its blockade, and measurement and interpretation of autophagy flux. Further, it describes the signaling pathways involved in the blockade of autophagy flux and the role of signaling molecules accumulated by autophagy blockade in cell death/apoptosis in various cancer cells during anti-cancer therapies. Altogether, it implies that factors such as types of cancer, drug therapies, and characteristics of autophagy should be evaluated before targeting autophagy for cancer treatment.
    Keywords:  Anti-cancer therapy; Apoptosis; Autophagy; Autophagy flux; Lysosomal dysfunction
    DOI:  https://doi.org/10.1007/s12272-020-01239-w
  32. Biochem Biophys Res Commun. 2020 Jun 18. pii: S0006-291X(20)30755-5. [Epub ahead of print]527(1): 64-70
    Yamai T, Hikita H, Fukuoka M, Fukutomi K, Murai K, Nakabori T, Yamada R, Miyakawa K, Watashi K, Ryo A, Kodama T, Sakamori R, Tatsumi T, Takehara T.
      Autophagy is an intracellular process that can lead to the degradation of malfunctioned proteins and damaged organelles to maintain homeostasis during cellular stress. Here, we evaluated the change in hepatitis B virus (HBV) production by regulating hepatic autophagy in HBV-producing cells. We examined focusing on a relation with a positive autophagy regulator, sirtuin1 (SIRT1). Starvation and rapamycin treatment induced autophagy with increasing SIRT1 protein, HBc protein and pregenomic RNA (pgRNA) levels in HBV- producing cells. Knockdown of Atg7 or Atg13 suppressed hepatic autophagy, and it did not change SIRT1 protein, HBc protein or pgRNA levels in HBV- producing cells. Resveratrol, which increases SIRT1 expression and activity, promoted autophagy and increased HBc protein and pgRNA levels. siRNA-mediated knockdown of SIRT1 inhibited autophagy and decreased HBc protein and pgRNA levels. In SIRT1-knockdown cells, starvation promoted autophagy but did not increase HBc protein and pgRNA levels. In conclusion, HBc protein and pgRNA levels are upregulated not by the autophagic process itself but by the SIRT1 expression level.
    Keywords:  Autophagy; HBV; Replication; Sirtuin1 (SIRT1); Transcription
    DOI:  https://doi.org/10.1016/j.bbrc.2020.04.031
  33. Cells. 2020 May 25. pii: E1321. [Epub ahead of print]9(5):
    Bonam SR, Jagadeesh B, Tschan MP, Muller S.
      Tremendous efforts have been made these last decades to increase our knowledge of intracellular degradative systems, especially in the field of autophagy. The role of autophagy in the maintenance of cell homeostasis is well documented and the existence of defects in the autophagic machinery has been largely described in diseases and aging. Determining the alterations occurring in the many forms of autophagy that coexist in cells and tissues remains complicated, as this cellular process is highly dynamic in nature and can vary from organ to organ in the same individual. Although autophagy is extensively studied, its functioning in different tissues and its links with other biological processes is still poorly understood. Several assays have been developed to monitor autophagy activity in vitro, ex vivo, and in vivo, based on different markers, the use of various inhibitors and activators, and distinct techniques. This review emphasizes the methods applied to measure (macro-)autophagy in tissue samples and in vivo via a protein, which centrally intervenes in the autophagy pathway, the microtubule-associated protein 1A/1B-light chain 3 (MAP1LC3), which is the most widely used marker and the first identified to associate with autophagosomal structures. These approaches are presented and discussed in terms of pros and cons. Some recommendations are provided to improve the reliability of the interpretation of results.
    Keywords:  MAP1LC3; autophagic flux; autophagy; in vivo autophagy assays; lysosome
    DOI:  https://doi.org/10.3390/cells9051321
  34. Br J Pharmacol. 2020 May 30.
    Chen L, Liao F, Jiang Z, Zhang C, Wang Z, Luo P, Jiang Q, Wu J, Wang Q, Luo M, Li X, Leng Y, Ma L, Shen G, Chen Z, Wang Y, Tan X, Gan Y, Liu D, Liu Y, Shi C.
      BACKGROUND AND PURPOSE: There is an urgent but unmet need for mitigating radiation-induced intestinal toxicity while radio sensitising tumours for abdominal radiotherapy. We aimed to investigate the effects of metformin on radiation-induced intestinal toxicity and radiosensitivity of colorectal tumours.EXPERIMENTAL APPROACH: Acute and chronic histological injuries of the intestine from mice were used to assess radioprotection, and IEC-6 cell line was used to investigate the mechanisms in vitro. The fractionated abdominal radiation model of HCT116 and HT29 tumour grafts was used to determine the effects on colorectal cancer.
    KEY RESULTS: Metformin alleviated radiation-induced acute and chronic intestinal toxicity by optimising mitophagy which was AMPK-dependent. In addition, our data indicated that metformin increased the radiosensitivity of colorectal tumours with P53 mutation both in vitro and in vivo.
    CONCLUSION AND IMPLICATIONS: Metformin may be a radiotherapy adjuvant agent for colorectal cancers especially those carrying P53 mutation. Our findings provide a new strategy for further precise clinical trials for metformin on radiotherapy.
    Keywords:  AMPK; Colorectal cancer; Intestinal injury; Metformin; Nrf2; Radiotherapy
    DOI:  https://doi.org/10.1111/bph.15149