bims-auttor Biomed News
on Autophagy and mTOR
Issue of 2020–02–09
twenty-two papers selected by
Viktor Korolchuk, Newcastle University
  1. Glutamine and asparagine activate mTORC1 independently of Rag GTPases.
  2. PP2A promotes ESCRT-0 complex formation on vacuolar membranes and microautophagy induction after TORC1 inactivation.
  3. Long-Lived Mice with Reduced Growth Hormone Signaling Have a Constitutive Upregulation of Hepatic Chaperone-Mediated Autophagy.
  4. The pROS of autophagy in neuronal health.
  5. Phase separation organizes the site of autophagosome formation.
  6. A conserved ATG2-GABARAP family interaction is critical for phagophore formation.
  7. Selective autophagy degrades nuclear pore complexes.
  8. TBC1D9 regulates TBK1 activation through Ca2+ signaling in selective autophagy.
  9. Localized de novo phospholipid synthesis drives autophagosome biogenesis.
  10. Yorkie Growth-Promoting Activity Is Limited by Atg1-Mediated Phosphorylation.
  11. Astroglial biotin deprivation under endoplasmic reticulum stress uncouples BCAA-mTORC1 role in lipid synthesis to prolong autophagy inhibition in the aging brain.
  12. Cholesterol derivatives induce dephosphorylation of the histone deacetylases Rpd3/HDAC1 to upregulate autophagy.
  13. Genome-wide siRNA screening reveals that DCAF4-mediated ubiquitination of optineurin stimulates autophagic degradation of Cu/Zn superoxide dismutase.
  14. Dysregulated iron metabolism in C. elegans catp-6/ATP13A2 mutant impairs mitochondrial function.
  15. Differential Effects of 2-Hydroxypropyl-Cyclodextrins on Lipid Accumulation in Npc1-Null Cells.
  16. Culprit or Bystander: Defective Mitophagy in Alzheimer's Disease.
  17. The Identification and characterisation of autophagy inhibitors from the published kinase inhibitor sets.
  18. Autophagy-based unconventional secretion of HMGB1 by keratinocytes plays a pivotal role in psoriatic skin inflammation.
  19. Mitochondrial adaptations to exercise do not require Bcl2-mediated autophagy but occur with BNIP3/Parkin activation.
  20. LATS suppresses mTORC1 activity to directly coordinate Hippo and mTORC1 pathways in growth control.
  21. Histone demethylase JMJD1C is phosphorylated by mTOR to activate de novo lipogenesis.
  22. Energy-stress-mediated AMPK activation inhibits ferroptosis.
  1. J Biol Chem. 2020 Feb 04. pii: jbc.AC119.011578. [Epub ahead of print]
    Glutamine and asparagine activate mTORC1 independently of Rag GTPases.
    Delong Meng, Qianmei Yang, Huanyu Wang, Chase H Melick, Rishika Navlani, Anderson R Frank, Jenna L Jewell.
      Nutrient sensing by cells is crucial, and when this sensing mechanism is disturbed human disease can occur. mTOR complex 1 (mTORC1) senses amino acids to control cell growth, metabolism and autophagy. Leucine, arginine, and methionine signal to mTORC1 through the well-characterized Rag GTPase signaling pathway. In contrast, glutamine activates mTORC1 through a Rag GTPase-independent mechanism that requires ADP-ribosylation factor 1 (Arf1). Here, using several biochemical and genetic approaches, we show that eight amino acids filter through the Rag GTPase pathway. Like glutamine, asparagine signals to mTORC1 through Arf1 in the absence of the Rag GTPases. Both the Rag-dependent and Rag-independent pathways required the lysosome and lysosomal function for mTORC1 activation. Our results show that mTORC1 is differentially regulated by amino acids through two distinct pathways.
    Keywords:  Arf1; Rag GTPase; amino acid; asparagine; glutamine; mTOR complex (mTORC); mTORC1; metabolism; signal transduction
    DOI:  https://doi.org/10.1074/jbc.AC119.011578
  2. Biochem Biophys Res Commun. 2020 Feb 03. pii: S0006-291X(20)30212-6. [Epub ahead of print]
    PP2A promotes ESCRT-0 complex formation on vacuolar membranes and microautophagy induction after TORC1 inactivation.
    Tasnuva Sharmin, Shamsul Morshed, Takashi Ushimaru.
      Deformation of vacuolar membranes mediated by endosomal sorting complex required for transport (ESCRT) is necessary for microautophagy. Target of rapamycin complex 1 (TORC1) protein kinase negatively regulates ESCRT-0 (Vps27-Hse1) recruitment onto vacuolar membranes and microautophagy induction. However, whether and how protein phosphatase regulates these events is unknown. Here, we show that the TORC1-downstream protein phosphatase PP2A-Cdc55 is important for these events after TORC1 inactivation in budding yeast. Loss of PP2A-Cdc55 compromised vacuolar localization of Hse1, but not Vps27. This study revealed that the orchestrated action of PP2A induces microautophagy upon TORC1 inactivation.
    Keywords:  ESCRT-0; Hse1; Microautophagy; PP2A; TORC1; Vps27
    DOI:  https://doi.org/10.1016/j.bbrc.2020.01.129
  3. Autophagy. 2020 Feb 03.
    Long-Lived Mice with Reduced Growth Hormone Signaling Have a Constitutive Upregulation of Hepatic Chaperone-Mediated Autophagy.
    S Joseph Endicott, Dennis N Boynton, Logan J Beckmann, Richard A Miller.
      Chaperone-mediated autophagy (CMA) is a selective, vesicle-independent, lysosomal degradation pathway for intracellular proteins. CMA modulates proteomic organization through selective protein degradation, with targets including metabolic enzymes, regulators of cell growth, and neurodegeneration-related proteins. CMA activity is low in ad libitum-fed rodents but is increased by prolonged fasting (>16 h). AKT negatively regulates CMA activity at the lysosomal membrane by phosphorylating and inhibiting the CMA regulator GFAP. We have previously reported that long-lived Pou1f1/Pit1 mutant (Snell) mice and ghr (growth hormone receptor) knockout mice (ghr KO) have lower AKT activity when fed compared to littermate controls, suggesting the hypothesis that these mice have increased baseline CMA activity. Here, we report that liver lysosomes from fed Snell dwarf mice and ghr KO mice have decreased GFAP phosphorylation and increased CMA substrate uptake activity. Liver lysosomes isolated from fed Snell dwarf mice and ghr KO mice injected with the protease inhibitor leupeptin had increased accumulation of endogenous CMA substrates, compared to littermate controls, suggesting an increase in CMA in vivo. Mice with liver-specific ablation of GH (growth hormone) signaling did not have increased liver CMA, suggesting that a signaling effect resulting from a loss of growth hormone in another tissue causes enhanced CMA in Snell dwarf and ghr KO mice. Finally, we find Snell dwarf mice have decreased protein levels (in liver and kidney) of CIP2A, a well-characterized CMA target protein, without an associated change in Cip2a mRNA. Collectively, these data suggest that CMA is enhanced downstream of an endocrine change resulting from whole-body ablation of GH signaling.
    Keywords:  Aging; chaperone-mediated autophagy; endocrine control of autophagy; endocrine signaling; growth hormone
    DOI:  https://doi.org/10.1080/15548627.2020.1725378
  4. J Mol Biol. 2020 Jan 29. pii: S0022-2836(20)30072-3. [Epub ahead of print]
    The pROS of autophagy in neuronal health.
    Lucia Sedlackova, George Kelly, Viktor I Korolchuk.
      Autophagy refers to a set of catabolic pathways that together facilitate degradation of superfluous, damaged and toxic cellular components. The most studied type of autophagy, called macroautophagy, involves membrane mobilization, cargo engulfment and trafficking of the newly formed autophagic vesicle to the recycling organelle, the lysosome. Macroautophagy responds to a variety of intra- and extra-cellular stress conditions including, but not limited to, pathogen intrusion, oxygen or nutrient starvation, proteotoxic and organelle stress, and elevation of reactive oxygen species (ROS). ROS are highly reactive oxygen molecules that can interact with cellular macromolecules (proteins, lipids, nucleic acids) to either modify their activity or, when released in excess, inflict irreversible damage. Although increased ROS release has long been recognized for its involvement in macroautophagy activation, the underlying mechanisms and the wider impact of ROS-mediated macroautophagy stimulation remain incompletely understood. We therefore discuss the growing body of evidence that describes the variety of mechanisms modulated by ROS that trigger cytoprotective detoxification via macroautophagy. We outline the role of ROS in signalling upstream of autophagy initiation, by increased gene expression and post-translational modifications of transcription factors, and in the formation and nucleation of autophagic vesicles by cysteine modification of conserved autophagy proteins including ATG4B, ATG7 and ATG3. Furthermore, we review the effect of ROS on selective forms of macroautophagy, specifically on cargo recognition by autophagy receptor proteins p62 and NBR1 (neighbour of BRCA1) and the recycling of mitochondria (mitophagy), and peroxisomes (pexophagy). Finally, we highlight both, the standalone and mutual contributions of abnormal ROS signalling and macroautophagy to the development and progression of neurodegenerative diseases.
    Keywords:  ROS; autophagy; cysteine modification; neurodegeneration; oxidation
    DOI:  https://doi.org/10.1016/j.jmb.2020.01.020
  5. Nature. 2020 Feb 05.
    Phase separation organizes the site of autophagosome formation.
    Yuko Fujioka, Jahangir Md Alam, Daisuke Noshiro, Kazunari Mouri, Toshio Ando, Yasushi Okada, Alexander I May, Roland L Knorr, Kuninori Suzuki, Yoshinori Ohsumi, Nobuo N Noda.
      Many biomolecules undergo liquid-liquid phase separation to form liquid-like condensates that mediate diverse cellular functions1,2. Autophagy is able to degrade such condensates using autophagosomes-double-membrane structures that are synthesized de novo at the pre-autophagosomal structure (PAS) in yeast3-5. Whereas Atg proteins that associate with the PAS have been characterized, the physicochemical and functional properties of the PAS remain unclear owing to its small size and fragility. Here we show that the PAS is in fact a liquid-like condensate of Atg proteins. The autophagy-initiating Atg1 complex undergoes phase separation to form liquid droplets in vitro, and point mutations or phosphorylation that inhibit phase separation impair PAS formation in vivo. In vitro experiments show that Atg1-complex droplets can be tethered to membranes via specific protein-protein interactions, explaining the vacuolar membrane localization of the PAS in vivo. We propose that phase separation has a critical, active role in autophagy, whereby it organizes the autophagy machinery at the PAS.
    DOI:  https://doi.org/10.1038/s41586-020-1977-6
  6. EMBO Rep. 2020 Feb 03. e201948412
    A conserved ATG2-GABARAP family interaction is critical for phagophore formation.
    Mihaela Bozic, Luuk van den Bekerom, Beth A Milne, Nicola Goodman, Lisa Roberston, Alan R Prescott, Thomas J Macartney, Nina Dawe, David G McEwan.
      The intracellular trafficking pathway, macroautophagy, is a recycling and disposal service that can be upregulated during periods of stress to maintain cellular homeostasis. An essential phase is the elongation and closure of the phagophore to seal and isolate unwanted cargo prior to lysosomal degradation. Human ATG2A and ATG2B proteins, through their interaction with WIPI proteins, are thought to be key players during phagophore elongation and closure, but little mechanistic detail is known about their function. We have identified a highly conserved motif driving the interaction between human ATG2 and GABARAP proteins that is in close proximity to the ATG2-WIPI4 interaction site. We show that the ATG2A-GABARAP interaction mutants are unable to form and close phagophores resulting in blocked autophagy, similar to ATG2A/ATG2B double-knockout cells. In contrast, the ATG2A-WIPI4 interaction mutant fully restored phagophore formation and autophagy flux, similar to wild-type ATG2A. Taken together, we provide new mechanistic insights into the requirements for ATG2 function at the phagophore and suggest that an ATG2-GABARAP/GABARAP-L1 interaction is essential for phagophore formation, whereas ATG2-WIPI4 interaction is dispensable.
    Keywords:   GABARAP ; ATG2; autophagosome; autophagy; phagophore
    DOI:  https://doi.org/10.15252/embr.201948412
  7. Nat Cell Biol. 2020 Feb;22(2): 159-166
    Selective autophagy degrades nuclear pore complexes.
    Chia-Wei Lee, Florian Wilfling, Paolo Ronchi, Matteo Allegretti, Shyamal Mosalaganti, Stefan Jentsch, Martin Beck, Boris Pfander.
      Nuclear pore complexes (NPCs) are very large proteinaceous assemblies that consist of more than 500 individual proteins1,2. NPCs are essential for nucleocytoplasmic transport of different cellular components, and disruption of the integrity of NPCs has been linked to aging, cancer and neurodegenerative diseases3-7. However, the mechanism by which membrane-embedded NPCs are turned over is currently unknown. Here we show that, after nitrogen starvation or genetic interference with the architecture of NPCs, nucleoporins are rapidly degraded in the budding yeast Saccharomyces cerevisiae. We demonstrate that NPC turnover involves vacuolar proteases and the core autophagy machinery. Autophagic degradation is mediated by the cytoplasmically exposed Nup159, which serves as intrinsic cargo receptor and directly binds to the autophagy marker protein Atg8. Autophagic degradation of NPCs is therefore inducible, enabling the removal of individual NPCs from the nuclear envelope.
    DOI:  https://doi.org/10.1038/s41556-019-0459-2
  8. Nat Commun. 2020 Feb 07. 11(1): 770
    TBC1D9 regulates TBK1 activation through Ca2+ signaling in selective autophagy.
    Takashi Nozawa, Shunsuke Sano, Atsuko Minowa-Nozawa, Hirotaka Toh, Shintaro Nakajima, Kazunori Murase, Chihiro Aikawa, Ichiro Nakagawa.
      Invading microbial pathogens can be eliminated selectively by xenophagy. Ubiquitin-mediated autophagy receptors are phosphorylated by TANK-binding kinase 1 (TBK1) and recruited to ubiquitinated bacteria to facilitate autophagosome formation during xenophagy, but the molecular mechanism underlying TBK1 activation in response to microbial infection is not clear. Here, we show that bacterial infection increases Ca2+ levels to activate TBK1 for xenophagy via the Ca2+-binding protein TBC1 domain family member 9 (TBC1D9). Mechanistically, the ubiquitin-binding region (UBR) and Ca2+-binding motif of TBC1D9 mediate its binding with ubiquitin-positive bacteria, and TBC1D9 knockout suppresses TBK1 activation and subsequent recruitment of the ULK1 complex. Treatment with a Ca2+ chelator impairs TBC1D9-ubiquitin interactions and TBK1 activation during xenophagy. TBC1D9 is also recruited to damaged mitochondria through its UBR and Ca2+-binding motif, and is required for TBK1 activation during mitophagy. These results indicate that TBC1D9 controls TBK1 activation during xenophagy and mitophagy through Ca2+-dependent ubiquitin-recognition.
    DOI:  https://doi.org/10.1038/s41467-020-14533-4
  9. Autophagy. 2020 Feb 06. 1-2
    Localized de novo phospholipid synthesis drives autophagosome biogenesis.
    Maximilian Schütter, Martin Graef.
      During (macro)autophagy, cells form transient organelles, termed autophagosomes, to target a broad spectrum of substrates for degradation critical to cellular and organismal health. Driven by rapid membrane assembly, an initially small vesicle (phagophore) elongates into a large cup-shaped structure to engulf substrates within a few minutes in a double-membrane autophagosome. In particular, how autophagic membranes expand has been a longstanding question. Here, we summarize our recent work that delineates a pathway that drives phagophore expansion by localized de novo phospholipid synthesis. Specifically, we found that the conserved acyl-CoA synthetase Faa1 localizes to nucleated phagophores to locally activate fatty acids for de novo phospholipid synthesis in the neighboring ER. These newly synthesized phospholipids are then preferentially incorporated into autophagic membranes and drive the expansion of the phagophore into a functional autophagosome. In summary, our work uncovers molecular principles of how cells coordinate phospholipid synthesis and flux with autophagic membrane formation during autophagy.Abbreviations: ACS: acyl-CoA synthestases; CoA: coenzyme A; ER: endoplasmic reticulum.
    Keywords:  Acyl-CoA synthetase; autophagosome biogenesis; autophagy; fatty acid channeling; fatty acid synthase; phospholipid synthesis
    DOI:  https://doi.org/10.1080/15548627.2020.1725379
  10. Dev Cell. 2020 Feb 04. pii: S1534-5807(20)30012-5. [Epub ahead of print]
    Yorkie Growth-Promoting Activity Is Limited by Atg1-Mediated Phosphorylation.
    Lauren K Tyra, Nilay Nandi, Charles Tracy, Helmut Krämer.
      The expression of multiple growth-promoting genes is coordinated by the transcriptional co-activator Yorkie with its major regulatory input provided by the Hippo-Warts kinase cascade. Here, we identify Atg1/ULK1-mediated phosphorylation of Yorkie as an additional inhibitory input independent of the Hippo-Warts pathway. Two serine residues in Yorkie, S74 and S97, are Atg1/ULK1 consensus target sites and are phosphorylated by ULK1 in vitro, thereby preventing its binding to Scalloped. In vivo, gain of function of Atg1, or its activator Acinus, caused elevated Yorkie phosphorylation and inhibited Yorkie's growth-promoting activity. Loss of function of Atg1 or Acinus raised expression of Yorkie target genes and increased tissue size. Unlike Atg1's role in autophagy, Atg1-mediated phosphorylation of Yorkie does not require Atg13. Atg1 is activated by starvation and other cellular stressors and therefore can impose temporary stress-induced constraints on the growth-promoting gene networks under the control of Hippo-Yorkie signaling.
    Keywords:  Acinus; autophagy; growth control; scalloped
    DOI:  https://doi.org/10.1016/j.devcel.2020.01.011
  11. J Neurochem. 2020 Feb 07. e14979
    Astroglial biotin deprivation under endoplasmic reticulum stress uncouples BCAA-mTORC1 role in lipid synthesis to prolong autophagy inhibition in the aging brain.
    Dhasarathan Ganesan, Anand Santhaseela Ramaian, Sudarshana Rajasekaran, Senthilraja Selvam, Tamilselvan Jayavelu.
      Autophagy delays the onset of endoplasmic reticulum (ER) stress by recycling cellular debris. However, the cues that elicit autophagy under the emergence of ER stress and their dysregulation during aging remains obscure. Amino acids, notably branched-chain amino acids (BCAA), get accumulated in cells once protein synthesis is inhibited by ER stress. The BCAA mimic satiety to inhibit autophagy via mechanistic targets of rapamycin complex 1 (mTORC1) activation and, in contrast, their catabolism supplements de novo lipogenesis for the formation of autophagosome membranes. Thus promoting BCAA utilization is hypothesized to induce autophagy to alleviate ER stress. Nevertheless, except protein synthesis, the rest of BCAA utilization and lipogenesis depends on the co-enzyme biotin. Hence, the levels of biotinylated carboxylases and lipids were assessed in the aging brain of Wistar rats. Despite the increased levels of biotinylated carboxylases and lipids, the aging brain accumulates BCAA. Since astrocytes are the primary site of BCAA and lipid metabolism and the increased expression of glial fibrillary acidic protein (GFAP) denotes astroglial ER stress, co-localization studies were performed to determine the extent of biotinylation in GFAP positive cells. Though total biotin intensity was higher in aged brain slices, the astrocytes specific decrease in biotinylation is attributed to BCAA accumulation, mTORC1 overactivation, autophagy inhibition and ER stress in the aging brain. The ER stress in primary astrocytes using tunicamycin also mimic the in vivo phenotype. Biotin supplementation ameliorated the changes observed in vitro, corroborating the significance of astrocytes biotin availability to promote autophagy under ER stress in aging.
    Keywords:  Aging; Autophagy; BCAA; ER Stress; Lipogenesis; mTORC1
    DOI:  https://doi.org/10.1111/jnc.14979
  12. Autophagy. 2020 Feb 04.
    Cholesterol derivatives induce dephosphorylation of the histone deacetylases Rpd3/HDAC1 to upregulate autophagy.
    Wenmei Wu, Man Luo, Kang Li, Yichen Dai, Huiyu Yi, Yangjin Zhong, Yang Cao, Gianluca Tettamanti, Ling Tian.
      Histone deacetylases (HDACs) are important for global gene expression and contribute to numerous physiological events. Deacetylase Rpd3 in yeast and its conserved homolog HDAC1 in mammals oppositely regulate autophagy; however, how Rpd3/HDAC1 is regulated to mediate autophagy remains unclear. Here, we showed autophagy occurrence in silkworm (Bombyx mori) required BmRpd3, wherein steroid hormone 20-hydroxyecdysone (20E) signaling regulated its protein level and nuclear localization negatively. Inhibition of MTOR led to dephosphorylation and nuclear-cytoplasmic translocation of BmRpd3/HsHDAC1. Besides, cholesterol, 20E, and 27-hydroxycholesterol could all induce massive dephosphorylation and cytoplasmic localization of BmRpd3/HsHDAC1, and thus autophagy by affecting MTORC1 activity. In addition, three phosphorylation sites (Ser392, Ser421, and Ser423) identified in BmRpd3 were conserved in HsHDAC1. Single or triple phosphorylation-site mutation attenuated the phosphorylation levels of BmRpd3/HsHDAC1, leading to their cytoplasmic localization and autophagy activation. In general, cholesterol derivatives, especially hydroxylated cholesterol, caused dephosphorylation and nucleo-cytoplasmic shuttling of BmRpd3/HsHDAC1 through inhibition of MTOR signaling to facilitate autophagy in B. mori and mammals. These findings improve our understandings of BmRpd3/HsHDAC1-mediated autophagy induced by cholesterol derivatives and shed light on their potential as a therapeutic target for neurodegenerative diseases and autophagy-related studies.
    Keywords:  Autophagy; BmRpd3/HsHDAC1; MTOR; cholesterol derivatives; dephosphorylation; nucleo-cytoplasmic translocation
    DOI:  https://doi.org/10.1080/15548627.2020.1725376
  13. J Biol Chem. 2020 Feb 03. pii: jbc.RA119.010239. [Epub ahead of print]
    Genome-wide siRNA screening reveals that DCAF4-mediated ubiquitination of optineurin stimulates autophagic degradation of Cu/Zn superoxide dismutase.
    Kengo Homma, Hiromitsu Takahashi, Naomi Tsuburaya, Isao Naguro, Takao Fujisawa, Hidenori Ichijo.
      Cu/Zn superoxide dismutase (SOD1) is one of the genes implicated in the devastating neurodegenerative disorder amyotrophic lateral sclerosis (ALS). Although the precise mechanisms of SOD1 mutant (SOD1mut)-induced motoneuron toxicity are still unclear, defects in SOD1 proteostasis are known to have a critical role in ALS pathogenesis. We previously reported that SOD1mut adopts a conformation that exposes a Derlin-1-binding region (DBR) and that DBR-exposed SOD1 interacts with Derlin-1, leading to motoneuron death. We also found that an environmental change, i.e. zinc depletion, induces a conformational change in wild-type SOD1 (SOD1WT) to the DBR-exposed conformation, suggesting the presence of an equilibrium state between the DBR-masked and DBR-exposed states even with SOD1WT Here, we conducted a high-throughput screening based on time-resolved FRET to further investigate the SOD1WT conformational change and used a genome-wide siRNA screen to search for regulators of SOD1 proteostasis. This screen yielded 30 candidate genes that maintained an absence of the DBR-exposed SOD1WT conformation. Among these genes was one encoding DDB1- and CUL4-associated factor 4 (DCAF4), a substrate receptor of the E3 ubiquitin-protein ligase complex. Of note, we found that DCAF4 mediates the ubiquitination of an ALS-associated protein and autophagy receptor, optineurin (OPTN), and facilitates autophagic degradation of DBR-exposed SOD1. In summary, our screen identifies DCAF4 as being required for proper proteostasis of DBR-exposed SOD1, which may have potential relevance for the development of therapies for managing ALS.
    Keywords:  DDB1 and CUL4 associated factor 4 (DCAF4); amyotrophic lateral sclerosis (ALS) (Lou Gehrig disease); autophagy; genome-wide siRNA screen; protein aggregation; protein misfolding; proteostasis; superoxide dismutase 1 (SOD1)
    DOI:  https://doi.org/10.1074/jbc.RA119.010239
  14. Neurobiol Dis. 2020 Feb 04. pii: S0969-9961(20)30061-9. [Epub ahead of print] 104786
    Dysregulated iron metabolism in C. elegans catp-6/ATP13A2 mutant impairs mitochondrial function.
    Nikhita Anand, Angelina Holcom, Michael Broussalian, Minna Schmidt, Shankar J Chinta, Gordon J Lithgow, Julie K Andersen, Manish Chamoli.
      Mutations in the human ATP13A2 gene are associated with an early-onset form of Parkinson's disease (PD) known as Kufor Rakeb Syndrome (KRS). Patients with KRS show increased iron deposition in the basal ganglia, suggesting iron toxicity-induced neurodegeneration as a potential pathogenesis associated with the ATP13A2 mutation. Previously we demonstrated that functional losses of ATP13A2 disrupt the lysosome's ability to store excess iron, leading to reduce survival of dopaminergic neuronal cells. To understand the possible mechanisms involved, we studied a Caenorhabditis elegans mutant defective in catp-6 function, an ortholog of human ATP13A2 gene. Here we show that catp-6 mutant worms have defective autophagy and lysosomal function, demonstrate characteristic PD phenotypes including reduced motor function and dysregulated iron metabolism. Additionally, these mutants have defective mitochondrial health, which is rescuable via iron chelation or mitophagy induction.
    Keywords:  ATP13A2; C. elegans; Catp-6; Iron chelation; Iron metabolism; Lysosomes; Mitochondrial function; Parkinson's disease; TFEB; Urolithin A
    DOI:  https://doi.org/10.1016/j.nbd.2020.104786
  15. Int J Mol Sci. 2020 Jan 30. pii: E898. [Epub ahead of print]21(3):
    Differential Effects of 2-Hydroxypropyl-Cyclodextrins on Lipid Accumulation in Npc1-Null Cells.
    Sanzana Hoque, Yuki Kondo, Nodoka Sakata, Yusei Yamada, Madoka Fukaura, Taishi Higashi, Keiichi Motoyama, Hidetoshi Arima, Katsumi Higaki, Akio Hayashi, Takaki Komiya, Yoichi Ishitsuka, Tetsumi Irie.
      Niemann-Pick disease type C (NPC) is an autosomal recessive disorder characterized by abnormal accumulation of free cholesterol and sphingolipids in lysosomes. The iminosugar miglustat, which inhibits hexosylceramide synthesis, is used for NPC treatment, and 2-hydroxypropyl-β-cyclodextrin (HP-β-CD), a cyclic oligosaccharide derivative, is being developed to treat NPC. Moreover, therapeutic potential of 2-hydroxypropyl-γ-cyclodextrin (HP-γ-CD) was shown in NPC models, although its mechanism of action remains unclear. Here, we investigated the effects of HP-β-CD, HP-γ-CD, and their homolog 2-hydroxypropyl-α-cyclodextrin (HP-α-CD) on lipid accumulation in Npc1-null Chinese hamster ovary (CHO) cells compared with those of miglustat. HP-β-CD and HP-γ-CD, unlike HP-α-CD, reduced intracellular free cholesterol levels and normalized the lysosome changes in Npc1-null cells but not in wild-type CHO cells. In contrast, miglustat did not normalize intracellular free cholesterol accumulation or lysosome changes in Npc1-null cells. However, miglustat decreased the levels of hexosylceramide and tended to increase those of sphingomyelins in line with its action as a glucosylceramide synthase inhibitor in both Npc1-null and wild-type CHO cells. Interestingly, HP-β-CD and HP-γ-CD, unlike HP-α-CD, reduced sphingomyelins in Npc1-null, but not wild-type, cells. In conclusion, HP-β-CD and HP-γ-CD reduce the accumulation of sphingolipids, mainly sphingomyelins, and free cholesterol as well as lysosome changes in Npc1-null, but not in wild-type, CHO cells.
    Keywords:  2-hydroxypropyl-β-cyclodextrin; 2-hydroxypropyl-γ-cyclodextrin; Niemann–Pick disease type C; cholesterol; cyclodextrin; lysosome; miglustat; sphingolipid; sphingomyelin
    DOI:  https://doi.org/10.3390/ijms21030898
  16. Front Cell Dev Biol. 2019 ;7 391
    Culprit or Bystander: Defective Mitophagy in Alzheimer's Disease.
    Chenglong Xie, Yahyah Aman, Bryan A Adriaanse, M Zameel Cader, Hélène Plun-Favreau, Jian Xiao, Evandro F Fang.
      Mitophagy is a selective engulfment and degradation of damaged mitochondria through the cellular autophagy machinery, a major mechanism responsible for mitochondrial quality control. Increased accumulation of damaged mitochondria in the Alzheimer's disease (AD) human brain are evident, although underlying mechanisms largely elusive. Recent studies indicate impaired mitophagy may contribute to the accumulation of damaged mitochondria in cross-species AD animal models and in AD patient iPSC-derived neurons. Studies from AD highlight feed-forward vicious cycles between defective mitophagy, and the principal AD pathological hallmarks, including amyloid-β plaques, tau tangles, and inflammation. The concomitant and intertwined connections among those hallmarks of AD and the absence of a real humanized AD rodent model present a challenge on how to determine if defective mitophagy is an early event preceding and causal of Tau/Aβ proteinopathies. Whilst further studies are required to understand these relationships, targeting defective mitophagy holds promise as a new therapeutic strategy for AD.
    Keywords:  Alzheimer’s disease; aging; memory; mitophagy; neuroprotection
    DOI:  https://doi.org/10.3389/fcell.2019.00391
  17. Biochem J. 2020 Feb 03. pii: BCJ20190846. [Epub ahead of print]
    The Identification and characterisation of autophagy inhibitors from the published kinase inhibitor sets.
    Maria Zachari, Julie Marie Rainard, George Cherian Pandarakalam, Lindsay Robinson, Jonathan Gillespie, Muralikrishnan Rajamanickam, Veronique Hamon, Angus Morrison, Ian G Ganley, Stuart Patrick McElroy.
      Autophagy is a critical cellular homeostatic mechanism, the dysfunction of which has been linked to a wide variety of disease states. It is regulated through the activity of specific kinases, in particular Unc-51 like autophagy activating kinase 1 (ULK1) and Phosphatidylinositol 3-kinase vacuolar protein sorting 34 (VPS34), which have both been suggested as potential targets for drug development. To identify new chemical compounds that might provide useful chemical tools or act as starting points for drug development, we screened each protein against the Published Kinase Inhibitor Set (PKIS), a library of known kinase inhibitors. In vitro screening and analysis of the published selectivity profiles of the hits informed the selection of three relatively potent ATP-competitive inhibitors against each target that presented the least number of off-target kinases in common. Cellular assays confirmed potent inhibition of autophagy in response to two of the ULK1 inhibitors and all three of the VPS34 inhibitors. These compounds represent not only a new resource for the study of autophagy but also potential chemical starting points for the validation or invalidation of these two centrally important autophagy kinases in disease models.
    Keywords:  autophagy; chemical biology; high-throughput screening; kinases
    DOI:  https://doi.org/10.1042/BCJ20190846
  18. Autophagy. 2020 Feb 04.
    Autophagy-based unconventional secretion of HMGB1 by keratinocytes plays a pivotal role in psoriatic skin inflammation.
    Zhen Wang, Hong Zhou, Huaping Zheng, Xikun Zhou, Guobo Shen, Xiu Teng, Xiao Liu, Jun Zhang, Xiaoqiong Wei, Zhonglan Hu, Fanlian Zeng, Yawen Hu, Jing Hu, Xiaoyan Wang, Shuwen Chen, Juan Cheng, Chen Zhang, Yiyue Gui, Song Zou, Yan Hao, Qixiang Zhao, Wenling Wu, Yifan Zhou, Kaijun Cui, Nongyu Huang, Yuquan Wei, Wei Li, Jiong Li.
      The precise mechanism through which macroautophagy/autophagy affects psoriasis is poorly understood. Here, we found that keratinocyte (KC) autophagy, which was positively correlated with psoriatic severity in patients and mouse models and could be inhibited by mitogen-activated protein kinase (MAPK) family inactivation. The impairment of autophagic flux alleviated psoriasisform inflammation. We also found that an autophagy-based unconventional secretory pathway (autosecretion) dependent on ATG5 (autophagy related 5) and GORASP2 (golgi reassembly stacking protein 2) promoted psoriasiform KC inflammation. Moreover, the alarmin HMGB1 (high mobility group box 1) was more effective than other autosecretory proteins in regulating psoriasiform cutaneous inflammation. HMGB1 neutralization in autophagy-efficient KCs eliminated the differences in psoriasiform inflammation between Krt14+/+-Atg5f/f KCs and Krt14Cre/+-atg5f/f KCs, and conversely, recombinant HMGB1 almost completely restored psoriasiform inflammation in Krt14Cre/+-atg5f/f KCs in vivo. These results suggest that HMGB1-associated autosecretion plays a pivotal role in cutaneous inflammation. Finally, we demonstrated that Krt14Cre/+-hmgb1f/f mice displayed attenuated psoriatic inflammation due to the essential crosstalk between KC-specific HMGB1-associated autosecretion and γδT cells. Thus, this study uncovered a novel autophagy mechanism in psoriasis pathogenesis, and the findings imply the clinical significance of investigating and treating psoriasis.
    Keywords:  Alarmin; autophagy; keratinocytes; psoriasis; secretion
    DOI:  https://doi.org/10.1080/15548627.2020.1725381
  19. FASEB J. 2020 Feb 06.
    Mitochondrial adaptations to exercise do not require Bcl2-mediated autophagy but occur with BNIP3/Parkin activation.
    Sarah E Ehrlicher, Harrison D Stierwalt, Benjamin F Miller, Sean A Newsom, Matthew M Robinson.
      Understanding the mechanisms regulating mitochondrial respiratory function and adaptations to metabolic challenges, such as exercise and high dietary fat, is necessary to promote skeletal muscle health and attenuate metabolic disease. Autophagy is a constitutively active degradation pathway that promotes mitochondrial turnover and transiently increases postexercise. Recent evidence indicates Bcl2 mediates exercise-induced autophagy and skeletal muscle adaptions to training during high-fat diet. We determined if improvements in mitochondrial respiration due to exercise training required Bcl2-mediated autophagy using a transgenic mouse model of impaired inducible autophagy (Bcl2AAA ). Mitochondrial adaptations to a treadmill exercise training protocol, in either low-fat or high-fat diet fed mice, did not require Bcl2-mediated autophagy activation. Instead, training increased protein synthesis rates and basal autophagy in the Bcl2AAA mice, while acute exercise activated BNIP3 and Parkin autophagy. High-fat diet stimulated lipid-specific mitochondrial adaptations. These data demonstrate increases in basal mitochondrial turnover, not transient activation with exercise, mediate adaptations to exercise and high-fat diet.
    Keywords:  mitochondrial respiration; protein turnover; skeletal muscle
    DOI:  https://doi.org/10.1096/fj.201902594RR
  20. Nat Cell Biol. 2020 Feb;22(2): 246-256
    LATS suppresses mTORC1 activity to directly coordinate Hippo and mTORC1 pathways in growth control.
    Wenjian Gan, Xiaoming Dai, Xiangpeng Dai, Jun Xie, Shasha Yin, Junjie Zhu, Chen Wang, Yuchen Liu, Jianping Guo, Min Wang, Jing Liu, Jia Hu, Ryan J Quinton, Neil J Ganem, Pengda Liu, John M Asara, Pier Paolo Pandolfi, Yingzi Yang, Zhigang He, Guangping Gao, Wenyi Wei.
      The Hippo and mammalian target of rapamycin complex 1 (mTORC1) pathways are the two predominant growth-control pathways that dictate proper organ development. We therefore explored potential crosstalk between these two functionally relevant pathways to coordinate their growth-control functions. We found that the LATS1 and LATS2 kinases, the core components of the Hippo pathway, phosphorylate S606 of Raptor, an essential component of mTORC1, to attenuate mTORC1 activation by impairing the interaction of Raptor with Rheb. The phosphomimetic Raptor-S606D knock-in mutant led to a reduction in cell size and proliferation. Compared with Raptor+/+ mice, RaptorD/D knock-in mice exhibited smaller livers and hearts, and a significant inhibition of elevation in mTORC1 signalling induced by Nf2 or Lats1 and Lats2 loss. Thus, our study reveals a direct link between the Hippo and mTORC1 pathways to fine-tune organ growth.
    DOI:  https://doi.org/10.1038/s41556-020-0463-6
  21. Nat Commun. 2020 Feb 07. 11(1): 796
    Histone demethylase JMJD1C is phosphorylated by mTOR to activate de novo lipogenesis.
    Jose A Viscarra, Yuhui Wang, Hai P Nguyen, Yoon Gi Choi, Hei Sook Sul.
      Fatty acid and triglyceride synthesis increases greatly in response to feeding and insulin. This lipogenic induction involves coordinate transcriptional activation of various enzymes in lipogenic pathway, including fatty acid synthase and glycerol-3-phosphate acyltransferase. Here, we show that JMJD1C is a specific histone demethylase for lipogenic gene transcription in liver. In response to feeding/insulin, JMJD1C is phosphorylated at T505 by mTOR complex to allow direct interaction with USF-1 for recruitment to lipogenic promoter regions. Thus, by demethylating H3K9me2, JMJD1C alters chromatin accessibility to allow transcription. Consequently, JMJD1C promotes lipogenesis in vivo to increase hepatic and plasma triglyceride levels, showing its role in metabolic adaption for activation of the lipogenic program in response to feeding/insulin, and its contribution to development of hepatosteatosis resulting in insulin resistance.
    DOI:  https://doi.org/10.1038/s41467-020-14617-1
  22. Nat Cell Biol. 2020 Feb;22(2): 225-234
    Energy-stress-mediated AMPK activation inhibits ferroptosis.
    Hyemin Lee, Fereshteh Zandkarimi, Yilei Zhang, Jitendra Kumar Meena, Jongchan Kim, Li Zhuang, Siddhartha Tyagi, Li Ma, Thomas F Westbrook, Gregory R Steinberg, Daisuke Nakada, Brent R Stockwell, Boyi Gan.
      Energy stress depletes ATP and induces cell death. Here we identify an unexpected inhibitory role of energy stress on ferroptosis, a form of regulated cell death induced by iron-dependent lipid peroxidation. We found that ferroptotic cell death and lipid peroxidation can be inhibited by treatments that induce or mimic energy stress. Inactivation of AMP-activated protein kinase (AMPK), a sensor of cellular energy status, largely abolishes the protective effects of energy stress on ferroptosis in vitro and on ferroptosis-associated renal ischaemia-reperfusion injury in vivo. Cancer cells with high basal AMPK activation are resistant to ferroptosis and AMPK inactivation sensitizes these cells to ferroptosis. Functional and lipidomic analyses further link AMPK regulation of ferroptosis to AMPK-mediated phosphorylation of acetyl-CoA carboxylase and polyunsaturated fatty acid biosynthesis. Our study demonstrates that energy stress inhibits ferroptosis partly through AMPK and reveals an unexpected coupling between ferroptosis and AMPK-mediated energy-stress signalling.
    DOI:  https://doi.org/10.1038/s41556-020-0461-8
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