bims-auttor 2020-08-09 papers

bims-auttor

Biomed News

on Autophagy and mTOR

Issue of 2020‒08‒09
34 papers selected by
Viktor Korolchuk, Newcastle University

Mammalian Atg8 proteins and the autophagy factor IRGM control mTOR and TFEB at a regulatory node critical for responses to pathogens.
TBK1 phosphorylates mutant Huntingtin and suppresses its aggregation and toxicity in Huntington's disease models.
The Neurodevelopmental Pathogenesis of Tuberous Sclerosis Complex (TSC).
A chemical genomics-aggrephagy integrated method studying functional analysis of autophagy inducers.
HFE inhibits type I IFNs signaling by targeting the SQSTM1-mediated MAVS autophagic degradation.
Mislocalization of TORC1 to Lysosomes Caused by KIF11 Inhibition Leads to Aberrant TORC1 Activity.
Mitochondria (cross)talk with proteostatic mechanisms; focusing on ageing and neurodegenerative diseases.
Ferritinophagy is not required for colon cancer cell growth.
Exosomal cargos modulate autophagy in recipient cells via different signaling pathways.
Autophagy in Osteosarcoma.
ATM loss disrupts the autophagy-lysosomal pathway.
RAB39B's role in membrane traffic, autophagy, and associated neuropathology.
Endosomal-lysosomal dysfunction in metabolic diseases and Alzheimer's disease.
TLR7 Stimulation With Imiquimod Induces Selective Autophagy and Controls Mycobacterium tuberculosis Growth in Mouse Macrophages.
Nuclear miR-30b-5p suppresses TFEB-mediated lysosomal biogenesis and autophagy.
AtNBR1 is a selective autophagic receptor for AtExo70E2 in Arabidopsis.
LC3B upregulation by NANOG promotes immune resistance and stem-like property through hyperactivation of EGFR signaling in immune-refractory tumor cells.
Garciesculenxanthone B induces PINK1-Parkin-mediated mitophagy and prevents ischemia-reperfusion brain injury in mice.
Mitochondria-Associated ER Membranes - The Origin Site of Autophagy.
Pathological Consequences of Hepatic mTORC1 Dysregulation.
Antioxidant Defense Mechanisms and its Dysfunctional Regulation in the Mitochondrial Disease, Friedreich's Ataxia.
Investigating mitochondrial autophagy by routine transmission electron microscopy: seeing is believing?
Loss of Tmem106b exacerbates FTLD pathologies and causes motor deficits in progranulin-deficient mice.
Regulation of immune checkpoint blockade efficacy in breast cancer by FIP200: A canonical-autophagy-independent function.
Lysosome-targeted ratiometric fluorescent sensor for monitoring pH in living cells based on one-pot-synthesized carbon dots.
LDL receptor-related protein LRP6 senses nutrient levels and regulates Hippo signaling.
Cyclopamine and Rapamycin Synergistically Inhibit mTOR Signalling in Mouse Hepatocytes, Revealing an Interaction of Hedgehog and mTor Signalling in the Liver.
Structural Insights into TOR Signaling.
Rational design and implementation of a chemically inducible heterotrimerization system.
Elevating sestrin2 attenuates endoplasmic reticulum stress and improves functional recovery through autophagy activation after spinal cord injury.
Huntingtin-lowering strategies for Huntington's disease.
Mice deficient in UXT exhibit retinitis pigmentosa-like features via aberrant autophagy activation.
Parkinson's: A Disease of Aberrant Vesicle Trafficking.
Molecular mechanisms of interplay between autophagy and metabolism in cancer.

Nat Cell Biol. 2020 Aug;22(8): 973-985

Mammalian Atg8 proteins and the autophagy factor IRGM control mTOR and TFEB at a regulatory node critical for responses to pathogens.

Suresh Kumar, Ashish Jain, Seong Won Choi, Gustavo Peixoto Duarte da Silva, Lee Allers, Michal H Mudd, Ryan Scott Peters, Jan Haug Anonsen, Tor-Erik Rusten, Michael Lazarou, Vojo Deretic.

Autophagy is a homeostatic process with multiple functions in mammalian cells. Here, we show that mammalian Atg8 proteins (mAtg8s) and the autophagy regulator IRGM control TFEB, a transcriptional activator of the lysosomal system. IRGM directly interacted with TFEB and promoted the nuclear translocation of TFEB. An mAtg8 partner of IRGM, GABARAP, interacted with TFEB. Deletion of all mAtg8s or GABARAPs affected the global transcriptional response to starvation and downregulated subsets of TFEB targets. IRGM and GABARAPs countered the action of mTOR as a negative regulator of TFEB. This was suppressed by constitutively active RagB, an activator of mTOR. Infection of macrophages with the membrane-permeabilizing microbe Mycobacterium tuberculosis or infection of target cells by HIV elicited TFEB activation in an IRGM-dependent manner. Thus, IRGM and its interactors mAtg8s close a loop between the autophagosomal pathway and the control of lysosomal biogenesis by TFEB, thus ensuring coordinated activation of the two systems that eventually merge during autophagy.

DOI: https://doi.org/10.1038/s41556-020-0549-1
EMBO J. 2020 Aug 05. e104671

TBK1 phosphorylates mutant Huntingtin and suppresses its aggregation and toxicity in Huntington's disease models.

Ramanath Narayana Hegde, Anass Chiki, Lara Petricca, Paola Martufi, Nicolas Arbez, Laurent Mouchiroud, Johan Auwerx, Christian Landles, Gillian P Bates, Malvindar K Singh-Bains, Mike Dragunow, Maurice A Curtis, Richard Lm Faull, Christopher A Ross, Andrea Caricasole, Hilal A Lashuel.

  Phosphorylation of the N-terminal domain of the huntingtin (HTT) protein has emerged as an important regulator of its localization, structure, aggregation, clearance and toxicity. However, validation of the effect of bona fide phosphorylation in vivo and assessing the therapeutic potential of targeting phosphorylation for the treatment of Huntington's disease (HD) require the identification of the enzymes that regulate HTT phosphorylation. Herein, we report the discovery and validation of a kinase, TANK-binding kinase 1 (TBK1), that efficiently phosphorylates full-length and N-terminal HTT fragments in vitro (at S13/S16), in cells (at S13) and in vivo. TBK1 expression in HD models (cells, primary neurons, and Caenorhabditis elegans) increases mutant HTT exon 1 phosphorylation and reduces its aggregation and cytotoxicity. We demonstrate that the TBK1-mediated neuroprotective effects are due to phosphorylation-dependent inhibition of mutant HTT exon 1 aggregation and an increase in autophagic clearance of mutant HTT. These findings suggest that upregulation and/or activation of TBK1 represents a viable strategy for the treatment of HD by simultaneously lowering mutant HTT levels and blocking its aggregation.

Keywords:  Huntington's disease; TBK1; autophagy; huntingtin phosphorylation; reducing aggregation

DOI:  https://doi.org/10.15252/embj.2020104671
Front Neuroanat. 2020 ;14 39

The Neurodevelopmental Pathogenesis of Tuberous Sclerosis Complex (TSC).

David M Feliciano.

  Tuberous sclerosis complex (TSC) is a model disorder for understanding brain development because the genes that cause TSC are known, many downstream molecular pathways have been identified, and the resulting perturbations of cellular events are established. TSC, therefore, provides an intellectual framework to understand the molecular and biochemical pathways that orchestrate normal brain development. The TSC1 and TSC2 genes encode Hamartin and Tuberin which form a GTPase activating protein (GAP) complex. Inactivating mutations in TSC genes (TSC1/TSC2) cause sustained Ras homologue enriched in brain (RHEB) activation of the mammalian isoform of the target of rapamycin complex 1 (mTORC1). TOR is a protein kinase that regulates cell size in many organisms throughout nature. mTORC1 inhibits catabolic processes including autophagy and activates anabolic processes including mRNA translation. mTORC1 regulation is achieved through two main upstream mechanisms. The first mechanism is regulation by growth factor signaling. The second mechanism is regulation by amino acids. Gene mutations that cause too much or too little mTORC1 activity lead to a spectrum of neuroanatomical changes ranging from altered brain size (micro and macrocephaly) to cortical malformations to Type I neoplasias. Because somatic mutations often underlie these changes, the timing, and location of mutation results in focal brain malformations. These mutations, therefore, provide gain-of-function and loss-of-function changes that are a powerful tool to assess the events that have gone awry during development and to determine their functional physiological consequences. Knowledge about the TSC-mTORC1 pathway has allowed scientists to predict which upstream and downstream mutations should cause commensurate neuroanatomical changes. Indeed, many of these predictions have now been clinically validated. A description of clinical imaging and histochemical findings is provided in relation to laboratory models of TSC that will allow the reader to appreciate how human pathology can provide an understanding of the fundamental mechanisms of development.

Keywords:  SEGA; TSC1; TSC2; Tuber; mTOR; subependymal giant cell astrocytoma

DOI:  https://doi.org/10.3389/fnana.2020.00039
Autophagy. 2020 Aug 07. 1-17

A chemical genomics-aggrephagy integrated method studying functional analysis of autophagy inducers.

Tetsushi Kataura, Etsu Tashiro, Shota Nishikawa, Kensuke Shibahara, Yoshihito Muraoka, Masahiro Miura, Shun Sakai, Naohiro Katoh, Misato Totsuka, Masafumi Onodera, Kazuo Shin-Ya, Kengo Miyamoto, Yukiko Sasazawa, Nobutaka Hattori, Shinji Saiki, Masaya Imoto.

  Macroautophagy/autophagy plays a critical role in the pathogenesis of various human diseases including neurodegenerative disorders such as Parkinson disease (PD) and Huntington disease (HD). Chemical autophagy inducers are expected to serve as disease-modifying agents by eliminating cytotoxic/damaged proteins. Although many autophagy inducers have been identified, their precise molecular mechanisms are not fully understood because of the complicated crosstalk among signaling pathways. To address this issue, we performed several chemical genomic analyses enabling us to comprehend the dominancy among the autophagy-associated pathways followed by an aggresome-clearance assay. In a first step, more than 400 target-established small molecules were assessed for their ability to activate autophagic flux in neuronal PC12D cells, and we identified 39 compounds as autophagy inducers. We then profiled the autophagy inducers by testing their effect on the induction of autophagy by 200 well-established signal transduction modulators. Our principal component analysis (PCA) and clustering analysis using a dataset of "autophagy profiles" revealed that two Food and Drug Administration (FDA)-approved drugs, memantine and clemastine, activate endoplasmic reticulum (ER) stress responses, which could lead to autophagy induction. We also confirmed that SMK-17, a recently identified autophagy inducer, induced autophagy via the PRKC/PKC-TFEB pathway, as had been predicted from PCA. Finally, we showed that almost all of the autophagy inducers tested in this present work significantly enhanced the clearance of the protein aggregates observed in cellular models of PD and HD. These results, with the combined approach, suggested that autophagy-activating small molecules may improve proteinopathies by eliminating nonfunctional protein aggregates.ABBREVIATIONS: ADK: adenosine kinase; AMPK: AMP-activated protein kinase; ATF4: activating transcription factor 4; BECN1: beclin-1; DDIT3/CHOP: DNA damage inducible transcript 3; EIF2AK3/PERK: eukaryotic translation initiation factor 2 alpha kinase 3; EIF2S1/eIF2α: eukaryotic translation initiation factor 2 subunit alpha; ER: endoplasmic reticulum; ERN1/IRE1α: endoplasmic reticulum to nucleus signaling 1; FDA: Food and Drug Administration; GSH: glutathione; HD: Huntington disease; HSPA5/GRP78: heat shock protein family A (Hsp70) member 5; HTT: huntingtin; JAK: Janus kinase, MAP1LC3B/LC3: microtubule associated protein 1 light chain 3 beta; MAP2K/MEK: mitogen-activated protein kinase kinase; MAP3K8/Tpl2: mitogen-activated protein kinase kinase kinase 8; MAPK: mitogen-activated protein kinase; MPP+: 1-methyl-4-phenylpyridinium; MTOR: mechanistic target of rapamycin kinase; MTORC: MTOR complex; NAC: N-acetylcysteine; NGF: nerve growth factor 2; NMDA: N-methyl-D-aspartate; PCA: principal component analysis; PD: Parkinson disease; PDA: pancreatic ductal adenocarcinoma; PIK3C3: phosphatidylinositol 3-kinase catalytic subunit type 3; PMA: phorbol 12-myristate 13-acetate; PRKC/PKC: protein kinase C; ROCK: Rho-associated coiled-coil protein kinase; RR: ribonucleotide reductase; SIGMAR1: sigma non-opioid intracellular receptor 1; SQSTM1/p62: sequestosome 1; STK11/LKB1: serine/threonine kinase 11; TFEB: Transcription factor EB; TGFB/TGF-β: Transforming growth factor beta; ULK1: unc-51 like autophagy activating kinase 1; XBP1: X-box binding protein 1.

Keywords:  Aggrephagy; PRKC/PKC; TFEB; chemical genomics; neurodegenerative disease

DOI:  https://doi.org/10.1080/15548627.2020.1794590
Autophagy. 2020 Aug 04.

HFE inhibits type I IFNs signaling by targeting the SQSTM1-mediated MAVS autophagic degradation.

Juan Liu, Xiaopeng Wu, Hailong Wang, Jiayu Wei, Qian Wu, Xingbo Wang, Yan Yan, Jun Cui, Junxia Min, Fudi Wang, Jiyong Zhou.

  Iron metabolism is involved in numerous physiological processes such as erythropoiesis, oxidative metabolism. However, the in vivo physiological functions of the iron metabolism-related gene Hfe in immune response during viral infection remain poorly understood. Here, we identified 5 iron metabolism-associated genes specifically affected during RNA virus infection by a high-throughput assay and further found that HFE was a key negative regulator of RIG-I-like receptors (RLR)-mediated type I interferons (IFNs) signaling. RNA virus infection inhibited the binding of HFE to MAVS (mitochondrial antiviral signaling protein) and blocked MAVS degradation via selective autophagy. HFE mediated MAVS autophagic degradation by binding to SQSTM1/p62. Depletion of Hfe abrogated the autophagic degradation of MAVS, leading to the stronger antiviral immune response. These findings established a novel regulatory role of selective autophagy in innate antiviral immune response by the iron metabolism-related gene Hfe. These data further provided insights into the crosstalk among iron metabolism, autophagy, and innate immune response.

Keywords:  HFE; MAVS degradation; RNA virus; autophagy; innate immune; iron metabolism

DOI:  https://doi.org/10.1080/15548627.2020.1804683
Mol Cells. 2020 Aug 07.

Mislocalization of TORC1 to Lysosomes Caused by KIF11 Inhibition Leads to Aberrant TORC1 Activity.

Yoon-Gu Jang, Yujin Choi, Kyoungho Jun, Jongkyeong Chung.

  While the growth factors like insulin initiate a signaling cascade to induce conformational changes in the mechanistic target of rapamycin complex 1 (mTORC1), amino acids cause the complex to localize to the site of activation, the lysosome. The precise mechanism of how mTORC1 moves in and out of the lysosome is yet to be elucidated in detail. Here we report that microtubules and the motor protein KIF11 are required for the proper dissociation of mTORC1 from the lysosome upon amino acid scarcity. When microtubules are disrupted or KIF11 is knocked down, we observe that mTORC1 localizes to the lysosome even in the amino acid-starved situation where it should be dispersed in the cytosol, causing an elevated mTORC1 activity. Moreover, in the mechanistic perspective, we discover that mTORC1 interacts with KIF11 on the motor domain of KIF11, enabling the complex to move out of the lysosome along microtubules. Our results suggest not only a novel way of the regulation regarding amino acid availability for mTORC1, but also a new role of KIF11 and microtubules in mTOR signaling.

Keywords:  Drosophila; KIF11; lysosome; mTORC1; microtubule

DOI:  https://doi.org/10.14348/molcells.2020.0089
Mech Ageing Dev. 2020 Aug 03. pii: S0047-6374(20)30120-2. [Epub ahead of print] 111324

Mitochondria (cross)talk with proteostatic mechanisms; focusing on ageing and neurodegenerative diseases.

Anna Gioran, Niki Chondrogianni.

  Perturbations of proteostatic mechanisms and mitochondrial decline during ageing and neurodegenerative diseases are well-established. Nevertheless, only a handful of interventions boosting proteostasis and mitochondrial function have been shown to delay ageing while therapies against neurodegeneration are still unavailable. Increasing evidence links the function of proteostatic mechanisms with each other and with the mitochondrial network. Tracing of this complex crosstalking network might lead to effective anti-ageing or neurodegenerative disease-modifying approaches. In this review we present evidence on the crosstalk of proteostatic mechanisms with mitochondria and discuss how incorporating this knowledge in future studies may help us develop more efficacious interventions against ageing and neurodegeneration.

Keywords:  Ubiquitin-Proteasome System (UPS); ageing; autophagy-lysosomal pathway (ALP); crosstalk; mitochondria; neurodegeneration

DOI:  https://doi.org/10.1016/j.mad.2020.111324
Cell Biol Int. 2020 Aug 07.

Ferritinophagy is not required for colon cancer cell growth.

Mazen Hasan, Sreedhar M Reddy, Nupur K Das.

  Ferritinophagy is a form of selective autophagy responsible for degrading intracellular ferritin, mediated by nuclear receptor coactivator 4 (NCOA4). NCOA4 plays significant roles in systemic iron homeostasis, and its disruption leads to simultaneous anemia and susceptibility to iron overload. The importance of iron colorectal cancer pathogenesis is well studied, however the role of ferritinophagy in colon cancer cell growth has not been assessed. Disruption of ferritinophagy via NCOA4 knockout leads to only marginal differences in growth under basal and iron-restricted conditions. Moreover, NCOA4 played no significant role in cell death induced by 5-fluorouracil and erastin. Western blot analysis for ferritin and transferrin receptor 1 found a dose-dependent effect on expression in both proteins in wild-type and NCOA4 knockout cell lines, but further investigation revealed no difference in growth response when treated at both high and low doses. Our data demonstrate a marginal role for ferritinophagy in growth both under normal and cytotoxic conditions in colon cancer cells, as well as a possible compensatory mechanism in colon cancer cells in response to ferroptosis induction. This article is protected by copyright. All rights reserved.

Keywords:  colon cancer; ferritin; ferritinophagy; iron homeostasis

DOI:  https://doi.org/10.1002/cbin.11439
Cell Biosci. 2020 ;10 92

Exosomal cargos modulate autophagy in recipient cells via different signaling pathways.

Mehdi Hassanpour, Aysa Rezabakhsh, Jafar Rezaie, Mohammad Nouri, Reza Rahbarghazi.

  Vesicular system of mammalian cells is composed of two intracellular and extracellular vesicles systems, which contributes to the intra/intercellular communication and cellular homeostasis. These systems mediate transferring of biological molecules like proteins, nucleic acids, and lipids inside the cytoplasm, and between the cells. By the present study, authors describe molecular crosslink between exosome biogenesis and autophagy and take a certain focus on the autophagic cargos of exosomes and signaling pathways involved in exosome-induced autophagy in target cells and vice versa. Autophagy the generation of double-phospholipid vesicles, is a process that engulfs damaged proteins and organelles, share molecular similarity and function synergy with exosomes biogenesis for degradation or exocytosis of certain cargo. Exosomes, the smallest subtype of extracellular vesicles, originating from the membrane of the multivesicular body located inside cells demonstrate key roles in the intracellular and intercellular communication. Growing evidence demonstrates the interaction between exosome biogenesis and autophagy both at intertwined molecular pathways and crossbred vesicles known as amphisomes. Crosstalk between exosome biogenesis and autophagy contributes to maintain cellular homeostasis under external and internal stresses. Moreover, these processes can modulate each other via different signaling pathways. Exosomes contain autophagic cargos that induce autophagy via the cascade of molecular events in target cells, which called here exosome-induced autophagy. Taken together, crosstalk between exosome biogenesis and autophagy plays pivotal roles in cell homeostasis. Shedding light on the interaction between endomembrane systems may promote our knowledge about the relation between exosome and autophagy pathways in lysosome-related disorders against treatments; proposing a theoretical approach for therapy.

Keywords:  Autophagy; Exosome-induced autophagy; Exosomes; Extracellular vesicles

DOI:  https://doi.org/10.1186/s13578-020-00455-7
Adv Exp Med Biol. 2020 ;1258 167-175

Autophagy in Osteosarcoma.

Grace Nehme, Nancy Gordon.

  Osteosarcoma (OS) remains a difficult disease to treat. The standard chemotherapy regimen has not improved survival for the past three decades. Resistance to chemotherapy remains a challenge and constitutes a major concern to clinical investigators. Autophagy has been recognized as a survival mechanism implicated in resistance to chemotherapy. We previously demonstrated chemotherapy to induce autophagy in OS. However, whether induction of autophagy will lead to survival or death has been the focus of many laboratories. Autophagy is a very context-dependent process, and no specific biomarker has been identified to define whether the process will lead to survival or death. In the present chapter, we present some of the mechanisms involved in the process of autophagy and summarize some of the most recent work related to autophagy in OS and the challenges encountered with the use of old and new autophagy inhibitors.

Keywords:  Autophagy; Chemotherapy; Death; Osteosarcoma; Survival

DOI:  https://doi.org/10.1007/978-3-030-43085-6_11
Autophagy. 2020 Aug 05.

ATM loss disrupts the autophagy-lysosomal pathway.

Aifang Cheng, Kai-Hei Tse, Hei-Man Chow, Yunqiao Gan, Xuan Song, Fulin Ma, Yi Xuan Yvonne Qian, Weiyi She, Karl Herrup.

  ATM (ataxia telangiectasia mutated) protein is found associated with multiple organelles including synaptic vesicles, endosomes and lysosomes, often in cooperation with ATR (ataxia telangiectasia and Rad3 related). Mutation of the ATM gene results in ataxia-telangiectasia (A-T), an autosomal recessive disorder with defects in multiple organs including the nervous system. Precisely how ATM deficiency leads to the complex phenotypes of A-T, however, remains elusive. Here, we reported that part of the connection may lie in autophagy and lysosomal abnormalities. We found that ATM was degraded through the autophagy pathway, while ATR was processed by the proteasome. Autophagy and lysosomal trafficking were both abnormal in atm-/- neurons and the deficits impacted cellular functions such as synapse maintenance, neuronal survival and glucose uptake. Upregulated autophagic flux was observed in atm-/- lysosomes, associated with a more acidic pH. Significantly, we found that the ATP6V1A (ATPase, H+ transporting, lysosomal V1 subunit A) proton pump was an ATM kinase target. In atm-/- neurons, lysosomes showed enhanced retrograde transport and accumulated in the perinuclear regions. We attributed this change to an unexpected physical interaction between ATM and the retrograde transport motor protein, dynein. As a consequence, SLC2A4/GLUT4 (solute carrier family 4 [facilitated glucose transporter], member 4) translocation to the plasma membrane was inhibited and trafficking to the lysosomes was increased, leading to impaired glucose uptake capacity. Together, these data underscored the involvement of ATM in a variety of neuronal vesicular trafficking processes, offering new and therapeutically useful insights into the pathogenesis of A-T.

Keywords:  SLC2A4/GLUT4; ataxia-telangiectasia; autophagy; lysosome; neurodegeneration; protein degradation; trafficking

DOI:  https://doi.org/10.1080/15548627.2020.1805860
J Cell Physiol. 2020 Aug 06.

RAB39B's role in membrane traffic, autophagy, and associated neuropathology.

Bor Luen Tang.

  Neuropathological disorders are increasingly associated with dysfunctions in neuronal membrane traffic and autophagy, with defects among members of the Rab family of small GTPases implicated. Mutations in the human Xq28 localized gene RAB39B have been associated with X-linked neurodevelopmental defects including macrocephaly, intellectual disability, autism spectrum disorder (ASD), as well as rare cases of early-onset Parkinson's disease (PD). Despite the finding that RAB39B regulates GluA2 trafficking and could thus influence synaptic α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor subunit composition, reasons for the wide-ranging neuropathological consequences associated with RAB39B defects have been unclear. Recent studies have now unraveled possible mechanisms underlying the neuropathological roles of this brain-enriched small GTPase. Studies in RAB39B knockout mice showed that RAB39B interacts with components of Class I phosphatidylinositol-3-kinase (PI3K) signaling. In its absence, the PI3K-AKT-mechanistic target of rapamycin signaling pathway in neural progenitor cells (NPCs) is hyperactivated, which promotes NPC proliferation, leading to macrocephaly and ASD. Pertaining to early-onset PD, a complex of C9orf72, Smith-Magenis syndrome chromosome region candidate 8 and WD repeat domain 41 that functions in autophagy has been identified as a guanine nucleotide exchange factor of RAB39B. Here, recent findings that have shed light on our mechanistic understanding of RAB39B's role in neurodevelopmental and neurodegenerative pathologies are reviewed. Caveats and unanswered questions are also discussed, and future perspectives outlined.

Keywords:  Parkinson's disease; RAB39B; Rabs; X-linked intellectual disability; autophagy; membrane traffic

DOI:  https://doi.org/10.1002/jcp.29962
Int Rev Neurobiol. 2020 ;pii: S0074-7742(20)30021-0. [Epub ahead of print]154 303-324

Endosomal-lysosomal dysfunction in metabolic diseases and Alzheimer's disease.

Michael F Almeida, Ben A Bahr, Stephen T Kinsey.

  The endosomal-lysosomal pathways and related autophagic processes are responsible for proteostasis, involving complexes between lysosomes and autophagosomes. Lysosomes are a key component of homeostasis, involved in cell signaling, metabolism, and quality control, and they experience functional compromise in metabolic diseases, aging, and neurodegenerative diseases. Many genetic mutations and risk factor genes associated with proteinopathies, as well as with metabolic diseases like diabetes, negatively influence endocytic trafficking and autophagic clearance. In contrast, health-improving exercise induces autophagy-lysosomal degradation, perhaps promoting efficient digestion of injured organelles so that undamaged organelles ensure cellular healthiness. Reductions in lysosomal hydrolases are implicated in Alzheimer's, Parkinson's, and lysosomal storage diseases, as well as obesity-related pathology, and members of the cathepsin enzyme family are involved in clearing both Aβ42 and α-synuclein. Upregulation of cathepsin hydrolases improves synaptic and memory functions in models of dementia and in exercising humans, thus identifying lysosomal-related systems as vital for healthy cognitive aging.

Keywords:  Age-related neurodegenerative disorders; Autophagic-lysosomal pathway; Autophagy; Cathepsin B; Endocytic trafficking; Protein accumulation disorders; Proteinopathy; Quality control; Synaptic pathology; Trafficking

DOI:  https://doi.org/10.1016/bs.irn.2020.02.012
Front Microbiol. 2020 ;11 1684

TLR7 Stimulation With Imiquimod Induces Selective Autophagy and Controls Mycobacterium tuberculosis Growth in Mouse Macrophages.

Hyo-Ji Lee, Su-Jin Kang, Yunseo Woo, Tae-Wook Hahn, Hyun-Jeong Ko, Yu-Jin Jung.

  Autophagy is a lysosomal self-digestion pathway that maintains internal homeostasis inside cells and critical process by which the innate immune system eliminates intracellular bacteria. In this study, we showed that stimulation of toll-like receptor 7 (TLR7) with imiquimod (IMQ) triggered autophagic cell death in macrophages by enhancing the generation of reactive oxygen species (ROS) via the p38- or MEK/ERK1/2-mediated signaling pathway in the early phase. IMQ significantly increased mitochondrial ROS and targeted autophagosomes to the mitochondria. Stimulation of TLR7 with IMQ enhanced the expression of BNIP3, which was localized to mitochondria and interacted with beclin-1, leading to mitophagy. In addition, IMQ substantially induced NO production through the GSK-3β-mediated signaling pathway, which led to autophagy in the late stage. We further examined whether the induction of autophagy by IMQ effectively eliminated intracellular microbes. Macrophages were infected with a virulent Mycobacterium tuberculosis (Mtb) strain, H37Rv, and then treated with IMQ. IMQ suppressed intracellular Mtb growth by inducing autophagy in a dose-dependent manner and increased NO production. Inhibition of autophagy using 3-methyladenine (3-MA) prevented autophagosome formation and control of intracellular Mtb growth in macrophages. These findings revealed a novel mechanism by which IMQ induces selective autophagy to promote intracellular killing machinery against Mtb infection in macrophages.

Keywords:  Mycobacterium tuberculosis; autophagosome; imiquimod (IMQ); mitophagy; mycobactericidal activity; toll-like receptor 7 (TLR7)

DOI:  https://doi.org/10.3389/fmicb.2020.01684
Cell Death Differ. 2020 Aug 06.

Nuclear miR-30b-5p suppresses TFEB-mediated lysosomal biogenesis and autophagy.

Huijie Guo, Mei Pu, Yusi Tai, Yuxiang Chen, Henglei Lu, Junwen Qiao, Guanghui Wang, Jing Chen, Xinming Qi, Ruimin Huang, Zhouteng Tao, Jin Ren.

Lysosome is a crucial organelle in charge of degrading proteins and damaged organelles to maintain cellular homeostasis. Transcription factor EB (TFEB) is the master transcription factor regulating lysosomal biogenesis and autophagy. Under external stimuli such as starvation, dephosphorylated TFEB transports into the nucleus to specifically recognize and bind to the coordinated lysosomal expression and regulation (CLEAR) elements at the promotors of autophagy and lysosomal biogenesis-related genes. The function of TFEB in the nucleus is fine regulated but the molecular mechanism is not fully elucidated. In this study, we discovered that miR-30b-5p, a small RNA which is known to regulate a series of genes through posttranscriptional regulation in the cytoplasm, was translocated into the nucleus, bound to the CLEAR elements, suppressed the transcription of TFEB-dependent downstream genes, and further inhibited the lysosomal biogenesis and the autophagic flux; meanwhile, knocking out the endogenous miR-30b-5p by CRISPR/Cas9 technique significantly increased the TFEB-mediated transactivation, resulting in the increased expression of autophagy and lysosomal biogenesis-related genes. Overexpressing miR-30b-5p in mice livers showed a decrease in lysosomal biogenesis and autophagy. These in vitro and in vivo data indicate that miR-30b-5p may inhibit the TFEB-dependent transactivation by binding to the CLEAR elements in the nucleus to regulate the lysosomal biogenesis and autophagy. This novel mechanism of nuclear miRNA regulating gene transcription is conducive to further elucidating the roles of miRNAs in the lysosomal physiological functions and helps to understand the pathogenesis of abnormal autophagy-related diseases.

DOI: https://doi.org/10.1038/s41418-020-0602-4
Plant Physiol. 2020 Aug 05. pii: pp.00470.2020. [Epub ahead of print]

AtNBR1 is a selective autophagic receptor for AtExo70E2 in Arabidopsis.

Chang-Yang Ji, Jun Zhou, Rongfang Guo, Youshun Lin, Chun-Hong Kung, Shuai Hu, Wing Yin Ng, Xiao-Hong Zhuang, Liwen Jiang.

Selective autophagy is a subcellular process whereby cytoplasmic materials are selectively sequestered into autophagosomes for subsequent delivery to the vacuole for degradation and recycling. Arabidopsis (Arabidoposis thaliana) next to BRCA1 gene 1 protein (AtNBR1) has been proposed to function as a selective autophagy receptor in plants, whereby AtNBR1 anchors the ubiquitinated targets to autophagosomes for degradation. However, the specific cargos of AtNBR1 remain elusive. We previously showed that Arabidopsis exocyst subunit EXO70 family protein E2 (AtExo70E2), a marker for Exocyst-positive organelle (EXPO), colocalized with the autophagosome marker Arabidopsis autophagy-related protein 8 (AtATG8) and was delivered to the vacuole for degradation upon autophagic induction. Here, through multiple analyses, we demonstrate that AtNBR1 is a selective receptor for AtExo70E2 during autophagy in Arabidopsis. First, two novel loss-of-function nbr1 CRISPR mutants (nbr1-c1 and nbr1-c2) showed an early-senescence phenotype under short-day growth conditions. Second, during autophagic induction, the vacuolar delivery of AtExo70E2 or EXPO was significantly reduced in nbr1 mutants compared to wild-type plants. Third, biochemical and recruitment assays demonstrated that AtNBR1 specifically interacted and recruited AtExo70E2 or its EXPO to AtATG8-positive autophagosomes in a UBA-independent manner during autophagy. Taken together, our data indicate that AtNBR1 functions as a selective receptor in mediating vacuolar delivery of AtExo70E2 or EXPO in a UBA-independent manner in plant autophagy.

DOI: https://doi.org/10.1104/pp.20.00470
Autophagy. 2020 Aug 07.

LC3B upregulation by NANOG promotes immune resistance and stem-like property through hyperactivation of EGFR signaling in immune-refractory tumor cells.

Suyeon Kim, Hanbyoul Cho, Soon-Oh Hong, Se Jin Oh, Hyo-Jung Lee, Eunho Cho, Seon Rang Woo, Joon Seon Song, Joon-Yong Chung, Sung Wook Son, Sang Min Yoon, Yu-Min Jeon, Seunghyun Jeon, Cassian Yee, Kyung-Mi Lee, Stephen M Hewitt, Jae-Hoon Kim, Kwon-Ho Song, Tae Woo Kim.

  Immune selection drives tumor cells to acquire refractory phenotypes. We previously demonstrated that cytotoxic T lymphocyte (CTL)-mediated immune pressure enriches NANOG+ tumor cells with stem-like and immune-refractory properties that make them resistant to CTLs. Here, we report that the emergence of refractory phenotypes is highly associated with an aberrant macroautophagic/autophagic state of the NANOG+ tumor cells and that the autophagic phenotype arises through transcriptional induction of MAP1LC3B/LC3B by NANOG. Furthermore, we found that upregulation of LC3B expression contributes to an increase in EGF secretion. The subsequent hyperactivation of EGFR-AKT signaling rendered NANOG+ tumor cells resistant to CTL killing. The NANOG-LC3B-p-EGFR axis was preserved across various types of human cancer and correlated negatively with the overall survival of cervical cancer patients. Inhibition of LC3B in immune-refractory tumor models rendered tumors susceptible to adoptive T-cell transfer, as well as PDCD1/PD-1 blockade, and led to successful, long-term control of the disease. Thus, our findings demonstrate a novel link among immune-resistance, stem-like phenotypes, and LC3B-mediated autophagic secretion in immune-refractory tumor cells, and implicate the LC3B-p-EGFR axis as a central molecular target for controlling NANOG+ immune-refractory cancer.

Keywords:  EGFR; LC3B; MAP1LC3B; NANOG; cancer immunoediting; immune resistance; immunotherapy

DOI:  https://doi.org/10.1080/15548627.2020.1805214
Acta Pharmacol Sin. 2020 Aug 05.

Garciesculenxanthone B induces PINK1-Parkin-mediated mitophagy and prevents ischemia-reperfusion brain injury in mice.

Man Wu, Guang Lu, Yuan-Zhi Lao, Hong Zhang, Dan Zheng, Zhao-Qing Zheng, Juan Yi, Qian Xiang, Li-Ming Wang, Hong-Sheng Tan, Hua Zhou, Han-Ming Shen, Hong-Xi Xu.

  Mitophagy is a selective form of autophagy involving the removal of damaged mitochondria via the autophagy-lysosome pathway. PINK1-Parkin-mediated mitophagy is one of the most important mechanisms in cardiovascular disease, cerebral ischemia-reperfusion (I/R) injury, and neurodegenerative diseases. In this study we conducted an image-based screening in YFP-Parkin HeLa cells to discover new mitophagy regulators from natural xanthone compounds. We found that garciesculenxanthone B (GeB), a new xanthone compound from Garcinia esculenta, induced the formation of YFP-Parkin puncta, a well known mitophagy marker. Furthermore, treatment with GeB dose-dependently promoted the degradation of mitochondrial proteins Tom20, Tim23, and MFN1 in YFP-Parkin HeLa cells and SH-SY5Y cells. We revealed that GeB stabilized PINK1 and triggered Parkin translocation to the impaired mitochondria to induce mitophagy, and these effects were abolished by knockdown of PINK1. Finally, in vivo experiments demonstrated that GeB partially rescued ischemia-reperfusion-induced brain injury in mice. Taken together, our findings demonstrate that the natural compound GeB can promote the PINK1-Parkin-mediated mitophagy pathway, which may be implicated in protection against I/R brain injury.

Keywords:  PINK1; Parkin; garciesculenxanthone B; ischemia-reperfusion injury; mitophagy

DOI:  https://doi.org/10.1038/s41401-020-0480-9
Front Cell Dev Biol. 2020 ;8 595

Mitochondria-Associated ER Membranes - The Origin Site of Autophagy.

Ming Yang, Chenrui Li, Shikun Yang, Ying Xiao, Xiaofen Xiong, Wei Chen, Hao Zhao, Qin Zhang, Yachun Han, Lin Sun.

  Autophagy is a process of intracellular self-recycling and degradation that plays an important role in maintaining cell homeostasis. However, the molecular mechanism of autophagy remains to be further studied. Mitochondria-associated endoplasmic reticulum membranes (MAMs) are the region of the ER that mediate communication between the ER and mitochondria. MAMs have been demonstrated to be involved in autophagy, Ca2+ transport and lipid metabolism. Here, we discuss the composition and function of MAMs, more specifically, to emphasize the role of MAMs in regulating autophagy. Finally, some key information that may be useful for future research is summarized.

Keywords:  autophagy; endoplasmic reticulum; mitochondria; mitochondria-associated endoplasmic reticulum membranes (MAMs); mitophagy

DOI:  https://doi.org/10.3389/fcell.2020.00595
Genes (Basel). 2020 Aug 05. pii: E896. [Epub ahead of print]11(8):

Pathological Consequences of Hepatic mTORC1 Dysregulation.

Chun-Seok Cho, Allison Ho Kowalsky, Jun Hee Lee.

  The mammalian target of rapamycin complex 1 (mTORC1) is a central regulator of metabolism that integrates environmental inputs, including nutrients, growth factors, and stress signals. mTORC1 activation upregulates anabolism of diverse macromolecules, such as proteins, lipids, and nucleic acids, while downregulating autolysosomal catabolism. mTORC1 dysregulation is often found in various diseases, including cancer, cardiovascular and neurodegenerative diseases, as well as metabolic syndromes involving obesity and type II diabetes. As an essential metabolic organ, the liver requires proper regulation of mTORC1 for maintaining homeostasis and preventing pathologies. For instance, aberrant hyper- or hypoactivation of mTORC1 disrupts hepatocellular homeostasis and damages the structural and functional integrity of the tissue, leading to prominent liver injury and the development of hepatocellular carcinogenesis. Proper regulation of mTORC1 during liver diseases may be beneficial for restoring liver function and ameliorating the detrimental consequences of liver failure.

Keywords:  liver; mTORC1; metabolism

DOI:  https://doi.org/10.3390/genes11080896
Free Radic Biol Med. 2020 Jul 30. pii: S0891-5849(20)31155-2. [Epub ahead of print]

Antioxidant Defense Mechanisms and its Dysfunctional Regulation in the Mitochondrial Disease, Friedreich's Ataxia.

S Chiang, M L H Huang, K C Park, D R Richardson.

Redox stress is associated with the pathogenesis of a wide variety of disease states. This can be amplified potentially through redox active iron deposits in oxidatively active organelles such as the mitochondrion. There are a number of disease states, including Friedreich's ataxia (FA) and sideroblastic anemia, where iron metabolism is dysregulated and leads to mitochondrial iron accumulation. Considering FA, which is due to the decreased expression of the mitochondrial protein, frataxin, this iron accumulation does not occur within protective storage proteins such as mitochondrial ferritin. Instead, it forms unbound biomineral aggregates composed of high spin iron(III), phosphorous and sulfur, which probably contributes to the observed redox stress. There is also a dysregulated response to the ensuing redox assault, as the master regulator of oxidative stress, nuclear factor erythroid 2-related factor-2 (Nrf2), demonstrates marked down-regulation. The dysfunctional response of Nrf2 in FA is due to multiple mechanisms including: (1) up-regulation of Keap1 that is involved in Nrf2 degradation; (2) activation of the nuclear Nrf2 export/degradation machinery via glycogen synthase kinase-3β (Gsk3β) signaling; and (3) inhibited nuclear translocation of Nrf2. More recently, increased microRNA (miRNA) 144 expression has been demonstrated to down-regulate Nrf2 in several disease states, including an animal model of FA. Other miRNAs have also demonstrated to be dysregulated upon frataxin depletion in vivo in humans and animal models of FA. Collectively, frataxin depletion results in multiple, complex responses that lead to detrimental redox effects that could contribute to the mechanisms involved in the pathogenesis of FA.

DOI: https://doi.org/10.1016/j.freeradbiomed.2020.07.019
Pharmacol Res. 2020 Jul 30. pii: S1043-6618(20)31405-5. [Epub ahead of print] 105097

Investigating mitochondrial autophagy by routine transmission electron microscopy: seeing is believing?

Joy Chakraborty, Federico Caicci, Moumita Roy, Elena Ziviani.

  Mitochondrial autophagy is affected in many diseases. In the past few years, the multiple-steps process of selective degradation of mitochondria has been dissected in details by combining outcomes from different approaches. Perhaps one of the most rigorous methods to clearly visualise mitochondria undergoing autophagic engulfment and degradation, is transmission electron microscopy (TEM). In this opinion paper, we want to give a brief summary of the mitophagic process, and by which means mitophagy can be addressed, including TEM analysis. We will report examples of autophagy and mitophagy-related TEM images, and discuss how to decipher the different steps of the mitophagic process by routine TEM. In our opinion, this technique can be used as a powerful confirmatory approach for mitochondrial autophagy, and can provide details of the organelle fate throughout the course of mitophagy with no substantial sample manipulation.

Keywords:  Autophagy; Mitophagy; Transmission Electron Microscopy (TEM)

DOI:  https://doi.org/10.1016/j.phrs.2020.105097
EMBO Rep. 2020 Aug 05. e50197

Loss of Tmem106b exacerbates FTLD pathologies and causes motor deficits in progranulin-deficient mice.

Xiaolai Zhou, Mieu Brooks, Peizhou Jiang, Shunsuke Koga, Aamir R Zuberi, Matthew C Baker, Tammee M Parsons, Monica Castanedes-Casey, Virginia Phillips, Ariston L Librero, Aishe Kurti, John D Fryer, Guojun Bu, Cathleen Lutz, Dennis W Dickson, Rosa Rademakers.

  Progranulin (PGRN) and transmembrane protein 106B (TMEM106B) are important lysosomal proteins implicated in frontotemporal lobar degeneration (FTLD) and other neurodegenerative disorders. Loss-of-function mutations in progranulin (GRN) are a common cause of FTLD, while TMEM106B variants have been shown to act as disease modifiers in FTLD. Overexpression of TMEM106B leads to lysosomal dysfunction, while loss of Tmem106b ameliorates lysosomal and FTLD-related pathologies in young Grn-/- mice, suggesting that lowering TMEM106B might be an attractive strategy for therapeutic treatment of FTLD-GRN. Here, we generate and characterize older Tmem106b-/- Grn-/- double knockout mice, which unexpectedly show severe motor deficits and spinal cord motor neuron and myelin loss, leading to paralysis and premature death at 11-12 months. Compared to Grn-/- , Tmem106b-/- Grn-/- mice have exacerbated FTLD-related pathologies, including microgliosis, astrogliosis, ubiquitin, and phospho-Tdp43 inclusions, as well as worsening of lysosomal and autophagic deficits. Our findings confirm a functional interaction between Tmem106b and Pgrn and underscore the need to rethink whether modulating TMEM106B levels is a viable therapeutic strategy.

Keywords:  Tdp-43; Tmem106b; frontotemporal lobar degeneration; lysosomes; progranulin

DOI:  https://doi.org/10.15252/embr.202050197
Cell Stress. 2020 Jul 02. 4(8): 216-217

Regulation of immune checkpoint blockade efficacy in breast cancer by FIP200: A canonical-autophagy-independent function.

Syn Kok Yeo, Jun-Lin Guan.

  Immune checkpoint blockade (ICB) has emerged as a promising therapeutic strategy because of its potential to induce durable therapeutic responses in cancer patients. However, in the case of breast cancer, its application and efficacy has been limited. As such, combinatorial therapeutic strategies that can unlock the potential of ICB in breast cancer are of urgent need. In view of that, autophagy-related proteins that play a role in the autophagic cell recycling process have been implicated in the regulation of inflammatory and anti-tumor immune responses. Accordingly, autophagy-related proteins represent a group of prospective therapeutic targets in conjunction with ICB. In our recent study (Okamoto T et al. (2020), Cancer Res), we developed immune-competent mouse models of breast cancer which were deficient for the autophagic function of FIP200 or had FIP200 completely ablated to test the efficacy of ICB. We showed that although FIP200's autophagy function was required for progression of PyMT-driven mammary tumors, FIP200's canonical-autophagy-independent function was responsible for increased T-cell infiltration, IFN-signaling and ICB efficacy. These findings provide genetic proof of principle for a combinatorial therapeutic strategy that involves ablation of FIP200 to improve ICB efficacy in non-responsive breast cancers.

Keywords:  FIP200/RB1CC1; TBK1; autophagy; checkpoint blockade; immuno-therapy

DOI:  https://doi.org/10.15698/cst2020.08.229
Mikrochim Acta. 2020 Aug 01. 187(8): 478

Lysosome-targeted ratiometric fluorescent sensor for monitoring pH in living cells based on one-pot-synthesized carbon dots.

Shaomei Xu, Xu He, Yibing Huang, Xin Liu, Lihe Zhao, Xinghua Wang, Ying Sun, Pinyi Ma, Daqian Song.

  A hydrothermal method has been employed to synthesize a green and one-pot carbon dots-based sensor for ratiometric monitoring and imaging lysosomal pH in living cells. The carbon dots were directly functionalized by abundant amino groups during synthesis and exhibited dual emission bands at 439 and 550 nm under single-wavelength excitation of 380 nm without any additional modification. In addition to its small size, the established sensor had good biocompatibility. Owing to its abundant amino groups and good hydrophilicity, the sensor is able to target lysosome with high Pearson's colocalization coefficients (0.935 and 0.924) and responds to change of lysosomal pH in living cells. It also had excellent pH sensitivity and reversibility, and anti-interference capability, thus enabling sensing pH change in intracellular environment in real time, as demonstrated by successful monitoring of lysosomal pH changes during lysosomal alkalization, dexamethasone-induced stimulation, and stress in Michigan Cancer Foundation-7 cells (blue channel, excitation = 405 nm and emission = 419-459 nm bandpass; and yellow channel, excitation = 405 nm and emission = 530-570 nm bandpass). Graphical abstract.

Keywords:  Carbon dots; Intracellular pH probe; Living cell imaging; Lysosome; Ratiometric fluorescence sensor

DOI:  https://doi.org/10.1007/s00604-020-04462-w
EMBO Rep. 2020 Aug 07. e50103

LDL receptor-related protein LRP6 senses nutrient levels and regulates Hippo signaling.

Wonyoung Jeong, Soyoung Kim, Ukjin Lee, Zhendong A Zhong, Mikhail Savitsky, Hyeryun Kwon, Jiyoung Kim, Taebok Lee, Jin Won Cho, Bart O Williams, Vladimir L Katanaev, Eek-Hoon Jho.

  Controlled cell growth and proliferation are essential for tissue homeostasis and development. Wnt and Hippo signaling are well known as positive and negative regulators of cell proliferation, respectively. The regulation of Hippo signaling by the Wnt pathway has been shown, but how and which components of Wnt signaling are involved in the activation of Hippo signaling during nutrient starvation are unknown. Here, we report that a reduction in the level of low-density lipoprotein receptor-related protein 6 (LRP6) during nutrient starvation induces phosphorylation and cytoplasmic localization of YAP, inhibiting YAP-dependent transcription. Phosphorylation of YAP via loss of LRP6 is mediated by large tumor suppressor kinases 1/2 (LATS1/2) and Merlin. We found that O-GlcNAcylation of LRP6 was reduced, and the overall amount of LRP6 was decreased via endocytosis-mediated lysosomal degradation during nutrient starvation. Merlin binds to LRP6; when LRP6 is less O-GlcNAcylated, Merlin dissociates from it and becomes capable of interacting with LATS1 to induce phosphorylation of YAP. Our data suggest that LRP6 has unexpected roles as a nutrient sensor and Hippo signaling regulator.

Keywords:   YAP ; Hippo signaling; LRP6; O-GlcNAcylation; Starvation

DOI:  https://doi.org/10.15252/embr.202050103
Cells. 2020 Jul 31. pii: E1817. [Epub ahead of print]9(8):

Cyclopamine and Rapamycin Synergistically Inhibit mTOR Signalling in Mouse Hepatocytes, Revealing an Interaction of Hedgehog and mTor Signalling in the Liver.

Luise Spormann, Christiane Rennert, Erik Kolbe, Fritzi Ott, Carolin Lossius, Robert Lehmann, Rolf Gebhardt, Thomas Berg, Madlen Matz-Soja.

  In the liver, energy homeostasis is mainly regulated by mechanistic target of rapamycin (mTOR) signalling, which influences relevant metabolic pathways, including lipid metabolism. However, the Hedgehog (Hh) pathway is one of the newly identified drivers of hepatic lipid metabolism. Although the link between mTOR and Hh signalling was previously demonstrated in cancer development and progression, knowledge of their molecular crosstalk in healthy liver is lacking. To close this information gap, we used a transgenic mouse model, which allows hepatocyte-specific deletion of the Hh pathway, and in vitro studies to reveal interactions between Hh and mTOR signalling. The study was conducted in male and female mice to investigate sexual differences in the crosstalk of these signalling pathways. Our results reveal that the conditional Hh knockout reduces mitochondrial adenosine triphosphate (ATP) production in primary hepatocytes from female mice and inhibits autophagy in hepatocytes from both sexes. Furthermore, in vitro studies show a synergistic effect of cyclopamine and rapamycin on the inhibition of mTor signalling and oxidative respiration in primary hepatocytes from male and female C57BL/6N mice. Overall, our results demonstrate that the impairment of Hh signalling influences mTOR signalling and therefore represses oxidative phosphorylation and autophagy.

Keywords:  cyclopamine; hedgehog; hepatocytes; liver; mTOR; mTORC2; rapamycin

DOI:  https://doi.org/10.3390/cells9081817
Genes (Basel). 2020 Aug 04. pii: E885. [Epub ahead of print]11(8):

Structural Insights into TOR Signaling.

Lucas Tafur, Jennifer Kefauver, Robbie Loewith.

  The Target of Rapamycin (TOR) is a highly conserved serine/threonine protein kinase that performs essential roles in the control of cellular growth and metabolism. TOR acts in two distinct multiprotein complexes, TORC1 and TORC2 (mTORC1 and mTORC2 in humans), which maintain different aspects of cellular homeostasis and orchestrate the cellular responses to diverse environmental challenges. Interest in understanding TOR signaling is further motivated by observations that link aberrant TOR signaling to a variety of diseases, ranging from epilepsy to cancer. In the last few years, driven in large part by recent advances in cryo-electron microscopy, there has been an explosion of available structures of (m)TORC1 and its regulators, as well as several (m)TORC2 structures, derived from both yeast and mammals. In this review, we highlight and summarize the main findings from these reports and discuss both the fascinating and unexpected molecular biology revealed and how this knowledge will potentially contribute to new therapeutic strategies to manipulate signaling through these clinically relevant pathways.

Keywords:  cell growth homeostasis; structural biology; target of rapamycin

DOI:  https://doi.org/10.3390/genes11080885
Nat Methods. 2020 Aug 03.

Rational design and implementation of a chemically inducible heterotrimerization system.

Helen D Wu, Masaki Kikuchi, Onur Dagliyan, Adam K Aragaki, Hideki Nakamura, Nikolay V Dokholyan, Takashi Umehara, Takanari Inoue.

Chemically inducible dimerization (CID) uses a small molecule to induce binding of two different proteins. CID tools such as the FK506-binding protein-FKBP-rapamycin-binding- (FKBP-FRB)-rapamycin system have been widely used to probe molecular events inside and outside cells. While various CID tools are available, chemically inducible trimerization (CIT) does not exist, due to inherent challenges in designing a chemical that simultaneously binds three proteins with high affinity and specificity. Here, we developed CIT by rationally splitting FRB and FKBP. Cellular and structural datasets showed efficient trimerization of split pairs of FRB or FKBP with full-length FKBP or FRB, respectively, by rapamycin. CIT rapidly induced tri-organellar junctions and perturbed intended membrane lipids exclusively at select membrane contact sites. By conferring one additional condition to what is achievable with CID, CIT expands the types of manipulation in single live cells to address cell biology questions otherwise intractable and engineer cell functions for future synthetic biology applications.

DOI: https://doi.org/10.1038/s41592-020-0913-x
Cell Biol Toxicol. 2020 Aug 01.

Elevating sestrin2 attenuates endoplasmic reticulum stress and improves functional recovery through autophagy activation after spinal cord injury.

Yao Li, Jing Zhang, Kailiang Zhou, Ling Xie, Guangheng Xiang, Mingqiao Fang, Wen Han, Xiangyang Wang, Jian Xiao.

  Spinal cord injury (SCI) is a devastating neurological trauma that causes losses of motor and sensory function. Sestrin2, also known as hypoxia inducible gene 95, is emerging as a critical determinant of cell homeostasis in response to cellular stress. However, the role of sestrin2 in the neuronal response to endoplasmic reticulum (ER) stress and the potential mechanism remain undefined. In this study, we investigated the effects of sestrin2 on ER stress and delineated an underlying molecular mechanism after SCI. Here, we found that elevated sestrin2 is a protective process in neurons against chemical ER stress induced by tunicamycin (TM) or traumatic invasion, while treatment with PERK inhibitor or knockdown of ATF4 reduces sestrin2 expression upon ER stress. In addition, we demonstrated that overexpression of sestrin2 limits ER stress, promoting neuronal survival and improving functional recovery after SCI, which is associated with activation of autophagy and restoration of autophagic flux mediated by sestrin2. Moreover, we also found that sestrin2 activates autophagy dependent on the AMPK-mTOR signaling pathway. Consistently, inhibition of AMPK abrogates the effect of sestrin2 on the activation of autophagy, and blockage of autophagic flux abolishes the effect of sestrin2 on limiting ER stress and neural death. Together, our data reveal that upregulation of sestrin2 is an important resistance mechanism of neurons to ER stress and the potential role of sestrin2 as a therapeutic target for SCI. Graphical abstract.

Keywords:  Apoptosis; Autophagy; Endoplasmic reticulum stress; Spinal cord injury; sestrin2

DOI:  https://doi.org/10.1007/s10565-020-09550-4
Expert Opin Investig Drugs. 2020 Aug 03.

Huntingtin-lowering strategies for Huntington's disease.

Roger A Barker, Motoki Fujimaki, Priya Rogers, David C Rubinsztein.

  INTRODUCTION: Huntington's disease (HD) is an incurable, autosomal dominant neurodegenerative disease caused by an abnormally long polyglutamine tract in the huntingtin protein. Because this mutation causes disease via gain-of-function, lowering huntingtin levels represents a rational therapeutic strategy.AREAS COVERED: We searched MEDLINE, CENTRAL and other trial databases, and relevant company and HD funding websites for press releases until April 2020 to review strategies for huntingtin lowering, including autophagy and PROTACs, which have been studied in preclinical models. We focussed our analyses on oligonucleotide (ASOs) and miRNA approaches, which have entered or are about to enter clinical trials.
EXPERT OPINION: ASO and mRNA approaches for lowering mutant huntingtin protein production and strategies for increasing mutant huntingtin clearance are attractive because they target the cause of disease. However, questions concerning the optimal mode of delivery and associated safety issues remain. It is unclear if the human CNS coverage with intrathecal or intraparenchymal delivery will be sufficient for efficacy. The extent that one must lower mutant huntingtin levels for it to be therapeutic is uncertain and the extent to which CNS lowering of wild-type huntingtin is safe is unclear. Polypharmacy may be an effective approach for ameliorating signs and symptoms and for preventing/delaying onset and progression.

Keywords:  Allele specific oligonucleotide; Autophagy; Huntington’s disease; PROTAC; huntingtin; miRNA; neurodegeneration; polyglutamine

DOI:  https://doi.org/10.1080/13543784.2020.1804552
Autophagy. 2020 Aug 02. 1-16

Mice deficient in UXT exhibit retinitis pigmentosa-like features via aberrant autophagy activation.

Mingyu Pan, Yue Yin, Xinxia Wang, Quanyi Wang, Lele Zhang, Haiyang Hu, Chen Wang.

  UXT (ubiquitously expressed prefoldin like chaperone), a small chaperone-like protein, is widely expressed in diverse human and mouse tissues and is more abundant in retina and kidney. However, the functional characterization of UXT at tissue level was largely unknown. Here, we reported that mice deficient in UXT exhibited salient features of retinal degenerative disease, similar to retinitis pigmentosa. Conditional knockout (CKO) of Uxt led to retinal degeneration and pigmentation in mice retina along with significant alterations of retinitis pigmentosa-related genes, which indicated UXT might be associated with retinal degenerative disease sharing key features to retinitis pigmentosa. Consistently, the electroretinogram (ERG) responses were dramatically impaired in uxt CKO retinas. Strong degenerative features were observed in uxt CKO retinas, including specific and progressive reduction of photoreceptor cells and increased numbers of apoptotic cells. Intriguingly, macroautophagic/autophagic ﬂux was enhanced in uxt CKO retina. Mechanistically, we found UXT was indispensable to suppress photoreceptor apoptotic cell death by inhibiting autophagy through regulating the activity of MTOR (mechanistic target of rapamycin kinase), a key negative regulator of autophagy. Conversely, knockdown of UXT induced the robust expression of the canonical autophagy-related genes and boosted autophagic ﬂux and apoptosis, finally resulting in severe retina degeneration in uxt CKO mice. Taken together, our study reveals a vital role of UXT in preventing retina from degeneration. The loss of UXT results in a hyper-autophagic state leading to massive retinal degeneration. Therefore, UXT may be a crucial target for retinal degenerative disease.ABBREVIATIONS: 3-ma: 3-methyladenine; casp3: caspase 3; cko: conditional knockout; erg: electroretinogram; gapdh: glyceraldehyde-3-phosphate dehydrogenase; map1lc3b/lc3b: microtubule-associated protein 1 light chain 3; mtor: mechanistic target of rapamycin kinase; parp: poly (adp-ribose) polymerase family; rna-seq: rna sequencing; rp: retinitis pigmentosa; rps6kb1/s6k: ribosomal protein s6 kinase b1; sqstm1: sequestosome 1; tunel: terminal deoxynucleotidyl transferase mediated dutp nick-end labeling; uxt: ubiquitously expressed prefoldin like chaperone.

Keywords:  Apoptosis; MTOR; UXT; autophagy; degeneration; photoreceptor; retinitis pigmentosa

DOI:  https://doi.org/10.1080/15548627.2020.1796015
Annu Rev Cell Dev Biol. 2020 Aug 04.

Parkinson's: A Disease of Aberrant Vesicle Trafficking.

Pawan Kishor Singh, Miratul M K Muqit.

Parkinson's disease (PD) is a leading cause of neurodegeneration that is defined by the selective loss of dopaminergic neurons and the accumulation of protein aggregates called Lewy bodies (LBs). The unequivocal identification of Mendelian inherited mutations in 13 genes in PD has provided transforming insights into the pathogenesis of this disease. The mechanistic analysis of several PD genes, including α-synuclein (α-syn), leucine-rich repeat kinase 2 (LRRK2), PTEN-induced kinase 1 (PINK1), and Parkin, has defined central roles for protein aggregation, mitochondrial damage, and defects in endolysosomal trafficking in PD neurodegeneration. In this review, we outline recent advances in our understanding of these gene pathways with a focus on the emergent role of Rab (Ras analog in brain) GTPases and vesicular trafficking as a common mechanism that underpins how mutations in PD genes lead to neuronal loss. These advances have led to previously distinct genes such as vacuolar protein-sorting-associated protein 35 (VPS35) and LRRK2 being implicated in common signaling pathways. A greater understanding of these common nodes of vesicular trafficking will be crucial for linking other PD genes and improving patient stratification in clinical trials underway against α-syn and LRRK2 targets. Expected final online publication date for the Annual Review of Cell and Developmental Biology, Volume 36 is October 6, 2020. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

DOI: https://doi.org/10.1146/annurev-cellbio-100818-125512
Life Sci. 2020 Aug 04. pii: S0024-3205(20)30936-X. [Epub ahead of print] 118184

Molecular mechanisms of interplay between autophagy and metabolism in cancer.

Sibi Raj, Vaishali Chandel, Arun Kumar, Kavindra Kumar Kesari, Shailendra Asthana, Janne Ruokolainen, Mohammad Amjad Kamal, Dhruv Kumar.

  Autophagy is an essential mechanism of cellular degradation, a way to protect the cells under stress conditions, such as deprivation of nutrients, growth factors and cellular damage. However, in normal physiology autophagy plays a significant role in cancer cells. Current research is in progress to understand how autophagy and cancer cells go hand in hand to support cancer cell progression. The important aspect in cancer and autophagy is the interdependence of autophagy in the survival and progression of cancer cells. Autophagy is known to be a major cause of chemotherapeutic resistance in various cancer cell types. Therefore, inhibition of autophagy as an effective therapeutic approach is being actively studied and tested in clinical studies. Multiple metabolic pathways are linked with autophagy that could potentially be a significant target for chemotherapeutic strategy. The comprehension of the interconnection of autophagy with cancer metabolism can pave a novel findings for effective combinatorial therapeutic strategies.

Keywords:  Autophagy; Cancer; Glycolysis; Metabolism

DOI:  https://doi.org/10.1016/j.lfs.2020.118184