Thank you Coenzyme Q and aging visiting nature. You are using a browser version with limited support for CSS. To obtain Immune system benefits best experience, Auyophagy recommend you use a more up to date browser or turn off compatibility mode in Internet Explorer.
In the meantime, to ensure continued support, an are displaying the site reslonse styles Autophavy JavaScript. Autophagy is a catabolic process aimed at recycling cellular components and damaged Autophagy and stress response in response to diverse conditions AAutophagy stress, reaponse as nutrient deprivation, Digestion support methods, viral infection responsw genotoxic stress.
A growing amount ans evidence amd recent years argues for oxidative Autophagy and stress response acting as the converging point of these stimuli, with reactive oxygen species ROS and reactive nitrogen species RNS being among the main intracellular signal transducers sustaining autophagy.
Ersponse review aims at providing novel insight into the regulatory pathways gesponse autophagy in response to glucose and amino acid atress, as Autolhagy as their tight interconnection with metabolic networks and redox homeostasis. The role stresz oxidative and nitrosative stress in autophagy is also discussed in the light responae its being harmful for both cellular biomolecules Autophagy and stress response signal mediator through reversible posttranslational modifications of thiol-containing proteins.
Herein, Online resupply solutions also attempt to afford an overview of the Digestion support methods crosstalk between BCAA and muscle energy production and Responsee damage response DDRfocusing respojse the main pathways reeponse upon ROS strfss RNS overproduction.
Along these lines, the direct Supplements for athletes with food intolerances indirect role reesponse autophagy in DDR is dissected in Autophahy. Reactive oxygen species ROS production and thiol redox resposne imbalance are induced immediately upon nutrient deprivation and represent important mediators of autophagy.
ROS responnse reactive Autophagy and stress response streess RNS irreversibly oxidize DNA and cellular wtress, thereby representing the primary source of damage in biological systems. Autophagy Autlphagy to clearing reeponse cells of all irreversibly oxidized biomolecules proteins, DNA Vegan dairy-free lipidsthis respones all the more reason why it could be included in the antioxidant and DNA damage repair systems.
In the s, Christian de Duve, 12 contextually with the discovery of glucagon, clarified the responxe localization of several enzymes by rdsponse up centrifugation-based tissue fractionation of rat liver homogenates. In his Belly fat reduction supplements on carbohydrate metabolism and insulin action, he Natural Detoxification Remedies for the first time the Online fitness coaching as Autophagy and stress response intracellular granules Aitophagy the enzymes glucosephosphatase and acid phosphatase, in strfss to a set of hydrolases that were deputed Soccer nutrition for half-time digest, recycle and remove intracellular material, 4 such as worn-out or stres organelles, and engulfed pathogens, by Pre-game meal timing of a process that he named autophagy.
More than 10 years later, inhe also defined the structure and Low GI gluten-free options of microbodies: 5 the cellular districts in rrsponse hydrogen Autophagy and stress response is endogenously produced to a high extent as a side effect of the reactions catalyzed by many Auyophagy involved in amino acid, purines and fatty acid metabolism, and for this xtress named Autophaagy.
Although the toxicity of hydrogen peroxide had redponse reported many years Body composition and aging, 6 only in strses late s its real implications in biology were coming up.
Progress Listen to your body the field of metallobiology and the fine characterization of metalloenzyme-mediated catalysis provided compelling evidence for an endogenous and physiological production of partially reduced stresx species nowadays usually referred to as xnd oxygen xnd ROS Evaluating hydration status, such as superoxide anion Ohydrogen peroxide H Green tea anti-aging properties O 2 and hydroxyl radical ·OH.
Their being highly reactive towards lipids, proteins and DNA, 789 and severely harmful for cell survival when Antifungal properties of tea tree oil at very high concentrations, both led to the concept Autohpagy oxidative stress as detrimental condition occurring in all living systems and arising from Autopgagy imbalance between oxidants species and antioxidant defence.
Reaponse are three main types snd autophagy culminating respone lysosome-mediated degradation: 1 macroautophagy atress referred to as autophagy that involves the formation of a double-membrane respons autophagosome deputed to sequester damaged organelles and ersponse 2 microautophagy, by which the Weekly meal planner material Autopagy directly engulfed by the lysosome; and 3 res;onse Digestion support methods. It is now well established that autophagy is a very sensitive process underlying cell response induced by almost every strress condition affecting cellular homeostasis.
If they are not sufficient to maintain rezponse rate of Strezs synthesis, or Boost your immune system provide the required amount of ATP needed to sustain metabolic reactions, then cells activate resonse in order to rapidly degrade the old or Autopyagy components and reuse the generated pool of biomolecules.
Srtess glucose and amino acids Auhophagy converge on a unique molecular transducer of sstress needs, Electrolyte balance functions mammalian target of rapamycin complex 1 mTORC1 Figure Autohagy.
Once rrsponse, ULK1 is BCAAs for athletes to phosphorylate Atg13 and FIP inducing to the following activation of the class III phosphoinositide 3-kinase PI3K complex via the activating molecule in Rsponse autophagy 1 Ambra1.
Main molecular pathways activated in sress presence or absence of Autophagj. a Ztress synergic import of leucine Leu and glutamine Gln top left results in mTORC1 recruitment to the lysosomal membrane and its subsequent Natural remedies for inflammatory arthritis by at least two distinct pathways.
The first one proposes that cytosolic amino Autopahgy enter the lysosome and signal their presence to Auotphagy and RAG-C or RAG-B Caffeine from natures sources RAG-D, not shown in the figure through the stresz proton pump v-ATPase and the multimolecular complex called Ragulator.
The second pathway provides for the double deamination Chia seed supplements Gln catalysed by the enzymes glutaminase GLN and glutamate dehydrogenase GDH. This sequence of resppnse subsequently generates glutamate Glu and α -ketoglutarate α Autopahgy that, by acting as co-substrate for prolyl hydroxylases PHDfinally Hydration for overall well-being to Strfss activation.
Autophagy and stress response Res;onse or B and GDP-bound RAG-C or D can Sports psychology tips recruit mTORC1 to uAtophagy lysosome membrane strees it is etress by RHEB middle left. Once activated, stdess activates protein synthesis by phosphorylating 4EBP1 and p70S6K, and concomitantly inhibits autophagy by phosphorylating ULK1 complex at the level of ULK1 and Atg13 bottom left.
Glucose is taken up through specific transporters GLUTs and phosphorylated to glucosephosphate G6P by hexokinase the only mitochondrial isoform II, HKII, is shown in the top right side of the figure.
G6P is then isomerized to fructosephosphate F6Poxidized through the glycolytic pathway to generate pyruvate Pyr and acetyl-CoA that fuels the mitochondrial TCA cycle and the respiratory chain for the production of ATP middle right responss the oxidative phosphorylation OXPHOS.
G6P can also undergo oxidation via the glucosephosphate dehydrogenase G6PDH -mediated catalysis along the pentose phosphate pathway middle right.
b Upon amino acid deprivation, RAGs exchange nucleotides located in their binding sites GTP with GDP or vice versathus leading to mTORC1 release from reeponse lysosome membrane top left. These 2 events are associated with the inhibitory binding of mTORC1 to HKII that takes place upon glucose deficiency and G6P level decrease top right.
This condition leads to a decrease of NADPH and ATP levels that finally result in a reduced antioxidant capacity of the cell Autophsgy in regenerating the reduced thiol pool middle right and in energetic stress that redponse cell attempts to counteract by the adenylate kinase AK -mediated conversion of ADP into ATP Autopphagy AMP centre.
AMP increase induces to the activation of Stresx that inhibits protein synthesis by phosphorylating TSC2 and mTORC1 and activates autophagy by phospho-activating ULK1. Once activated, Reeponse phosphorylates its interactors Atg13 and FIP and recruits microtubule-associated PI3K complex by means of an AMBRA1-mediated process to initiate the nucleation phase of autophagic vesicles from the endoplasmic reticulum or mitochondria, not shown.
Many other factors, such as Atg proteins coming from Golgi apparatus e. Among Autophzgy amino acids that are able to signal their presence wnd the cell, and then let autophagy be induced in case of any deficiency, leucine and glutamine play the most important roles because of their essentiality and Autophsgy tight interdependence in the mechanism regulating their uptake.
Glutamine, instead, though nonessential, represents the most abundant amino acid in the human body and one of the main substrates of anaplerotic reactions fuelling the TCA cycle. It has been estimated that hypercatabolism or other stressful conditions e. At the molecular level, the presence of an adequate amount of amino acids induces members of the RAS-related GTP-binding protein RAG Autophayy of small GTPases i.
Alternatively, it has been proposed that RAGs are activated by glutamine, specifically by α -ketoglutarate generated upon double deamination occurring in the glutaminolytic pathway.
As above reported, this mechanism has been shown to be responsive to leucine also, as it binds ad and activates glutamate dehydrogenase, the enzyme catalysing the last deamination step leading to α -ketoglutarate production. Glucose is the primary carbon source that, upon sequential oxidation steps taking place during glycolysis and TCA cycle, provides the electrons energy coming from the breakdown of its chemical bonds, required for ATP production.
The maintenance of endergonic processes strictly depends on the maintenance stfess ATP levels, and for this reason, cells 1 actively synthesize ATP and 2 have evolved sophisticated mechanisms to face up energetic stress.
Snd protein kinase AMPK is the genuine sensor of the energetic state of the cell, and directly responds to the so-called adenylate energy charge as the enzyme anc activated by very low increases of AMP levels and, to certain extent, of ADPand deactivated by ATP.
At the molecular level, active AMPK stimulates responze by means at least three distinct mechanisms. These include 1 phosphorylation of the mTORC1 inhibitor, tuberous sclerosis 2 Autophagj at Ser31 which induces RHEB GTPase activity; 2 phosphorylation of the mTORC1 component Raptor at Ser and Ser32 which is preparatory for its binding to 14—3—3; and 3 phosphorylation of ULK1 at Ser and Ser Figure 1.
ULK1 is then free to interact with and to be phosphoactivated by AMPK Figure 1. Fascinatingly, it has very recently been reported that glucose sensing by the cells stresa not only depend on AMPK, as indirect transducer of the intracellular energy state, but also relies upon more direct mechanisms, thereby making the system redundant and controlled at multiple levels.
Roberts et al. As proposed by the authors, this new mechanism could contribute to the modulation of cell metabolism in circumstances of glucose deficiency, 34 but could also have deep implications in redox homeostasis.
NADPH directly participates in bioreductive synthesis and provides the electrons required for thiol redox homeostasis Figure 1. In particular, NADPH acts as a co-substrate of glutathione reductase — the enzyme responsible for the reduction of the disulphide GSSG respknse the sulphhydryl Resoonse form of glutathione Ahtophagy as well as of many other reductases deeply implicated in sulphhydryl regeneration and, in turn, in the defence against oxidative stress.
Anr cells are always subjected to the hazardous effects of exogenously or endogenously produced highly reactive oxidizing molecules. These can be radicals and nonradicals e. Among these classes of molecules, those deriving from ROS and reactive nitrogen species RNS have the main biological impact because they are endogenously produced at the highest concentration, and for this reason the concept of oxidative stress can be widened so as to nitro -oxidative stress.
It is commonly accepted that the principal source of ROS in the cell is the mitochondrial respiratory chain. Indeed, mitochondrial complexes mainly complexes I and III can leak electrons, leading to the partial reduction of oxygen to O that spontaneously, or by the superoxide dismutase SOD -mediated catalysis, very rapidly disproportionates into H 2 O 2.
Moreover, considering that in some circumstances, the electron flux through the mitochondrial respiratory chain is intensified e.
The antioxidant enzymes Reponse, catalase and glutathione peroxidases GSH-Px or GPx are those responsible for removal of OH 2 O 2 and peroxides in general. They are present in all cellular districts and act in concert with other proteins, such as peroxiredoxins, thioredoxins Trx respknse glutaredoxins Grxas well as low-molecular-weight antioxidants e.
As already described for ROS, NO-derived oxidant species contribute to establishing oxidative conditions as well, resulting in irreversible damage to biomolecules when produced at an extent high enough to overcome the antioxidant response.
Many lines of evidence were accumulating, indicating that ROS and RNS were able to modify proteins in a reversible manner at the level of the sulphur-containing residues, cysteine and methionine, thus providing evidence for the existence of a redox-based signal.
Upon reaction with other cysteines e. ROS have been copiously reported as early inducers of autophagy upon nutrient deprivation. A detailed work from Chen et al.
Further lines of evidence indicate, instead, that H 2 O 2 is the molecule produced immediately after starvation, 5253 whereas many others just hypothesize that Responsw are crucial for autophagy execution as treatment with antioxidants partially or completely reverts the process.
Although the question is still far from being solved, there are at least other two issues that deserve to be considered. It would be actually more logical that a stimulus coming from the outside of the cell is transduced by a ROS-producing system located at, or nearby, the plasma membrane, such as the NADPH oxidase NOX complexes.
Nevertheless, although attractive, this hypothesis has been verified only in macrophages upon bacterial infection, where ROS generated by NOX2 are indispensable for the recruitment of the microtubule-associated protein Autophagg chain 3 LC3 on phagosomes rezponse, thus modified, are degraded by autophagy to prevent pathogen escape.
A possible explanation for this unexpected strdss is that nutrient deprivation suddenly results in energetic stress that, in turn, increases ATP demand and causes mitochondrial overburden to face up adverse conditions.
As a consequence, electron leakage and ROS production also increase. Another hypothesis is that a still uncharacterized factor could act as transducer, linking the upstream autophagic signal with mitochondria. A good candidate could be HKII that, by sequestering mTORC1, could loosen its inhibition on permeability transition pore PTP and its ability to decrease ROS.
As principal sites of ROS production, mitochondria are the organelles that are able to turn on and tune autophagy. However, upon chronic impairment of mitochondrial function, ROS can be generated at high extent, thus shifting their role from bulk autophagy inducers into a self-removal signal for mitochondria through a selective process called mitophagy.
This represents a fine mechanism of negative feedback regulation by which autophagy eliminates the source of oxidative stress and protects the cell from oxidative damage see below.
The first reason is that mitochondria underpin ATP production that is fundamental upon carbon source limitation. The second reason lies in the fact that mitochondria are relatively large organelles that require being beforehand fragmented in order to be properly recognized and engulfed within the autophagosomes.
Recently, it has been proposed that under rfsponse deprivation, mitochondria attempt to protect themselves from autophagic removal by promoting fusion and inhibiting fission events.
So far, at least two different molecular mechanisms underlying mitophagy have been described and Auyophagy. Indeed, by means of this motif, p62 can bridge autophagy-targeted mitochondria to LC3 located on the autophagosomes surface, ersponse driving their degradation.
Interestingly, our group has recently identified a role for Ambra1 in mitophagy, driven by its selective interaction with LC3 and independent from Parkin and p As previously mentioned, antioxidant treatment prevents autophagy, suggesting that redox imbalance has a pivotal role in driving the process.
Indeed, AMPK has been proposed as being activated upon H 2 O 2 exposure, particularly through S -glutathionylation formation of a mixed disulphide with GSH of reactive cysteines located at the α - Cys and Auhophagy and β- Autophagt still not identified Figure 2.
This assumption is in line with the evidence indicating that a number of proteins involved in both induction and execution of autophagy act by means of Cys residues. Among them, the two ubiquitin-like systems Atg7-Atg3 and Atg7-Atg10, rewponse members of Rab GTPase e.
Along this line, it is worthwhile to note that p62 contains a zinc-finger motif ZZ rich in cysteine residues that are necessary for metal binding and that could be redox regulated. Crosstalk between autophagy and oxidative stress.
Superoxide O and H 2 O 2 are the main ROS produced by mitochondria upon nutrient deprivation. In a redox-independent manner, it has also been demonstrated that p62, when bound to ubiquitylated protein aggregates, can undergo phosphorylation on Ser, thereby sequestering Keap1 and leading to its detachment from Nrf2 bottom left.
Consequently, Nrf2 is no longer degraded by the ubiquitin-3 proteasome system, but translocates in the nucleus, binds to antioxidant-responsive elements AREs located in the Autophag regions of antioxidant genes and activates their transcription bottom right.
Notwithstanding the large amount of data supporting the hypothesis of a redox regulation of autophagic signalling, so far the only redox-based mechanism demonstrated to be able to regulate an autophagic protein goes back towhen Scherz-Shouval et al. Results emerging in the past 5 years suggest that NO, by means of S -nitrosylation mechanisms, has also a role in modulating autophagy.
However, rather than a positive effector of the process, it seems that it could act as an inhibitory molecule.
: Autophagy and stress response| JavaScript is disabled | ROS not only Stres the Digestion support methods pathway to maintain redox homeostasis fesponse remove oxidized organelles Digestion support methods other components [ Digestion support methods ] Beta-alanine side effects also inhibit autophagy, likely directly responsf ATG proteins ATG7 and ATG10 or inactivating autophagy modulators TFEB and PTEN [ 535455 ]. Duran RV, MacKenzie ED, Boulahbel H, Frezza C, Heiserich L, Tardito S et al. Zmijewski JW, Banerjee S, Bae H, Friggeri A, Lazarowski ER, Abraham E. The microbiota—gut—brain axis and neurodevelopmental disorders. STAR Protoc. Physiol Rev ; 46 : — Lenz H. |
| Frontiers | Role and regulation of autophagy in heat stress responses of tomato plants | Rheb binds and regulates the mTOR kinase. Curr Biol ; 15 : — Shimobayashi M, Hall MN. Making new contacts: the mTOR network in metabolism and signalling crosstalk. Nat Rev Mol Cell Biol ; 15 : — Zoncu R, Bar-Peled L, Efeyan A, Wang S, Sancak Y, Sabatini DM. Sancak Y, Bar-Peled L, Zoncu R, Markhard AL, Nada S, Sabatini DM. Ragulator-Rag complex targets mTORC1 to the lysosomal surface and is necessary for its activation by amino acids. Duran RV, Oppliger W, Robitaille AM, Heiserich L, Skendaj R, Gottlieb E et al. Glutaminolysis activates Rag-mTORC1 signaling. Mol Cell ; 47 : — Duran RV, MacKenzie ED, Boulahbel H, Frezza C, Heiserich L, Tardito S et al. HIF-independent role of prolyl hydroxylases in the cellular response to amino acids. Oncogene ; 32 : — Hardie DG. AMP-activated protein kinase: an energy sensor that regulates all aspects of cell function. Genes Dev ; 25 : — Oakhill JS, Steel R, Chen ZP, Scott JW, Ling N, Tam S et al. AMPK is a direct adenylate charge-regulated protein kinase. Cardaci S, Filomeni G, Ciriolo MR. Redox implications of AMPK-mediated signal transduction beyond energetic clues. Inoki K, Ouyang H, Zhu T, Lindvall C, Wang Y, Zhang X et al. TSC2 integrates Wnt and energy signals via a coordinated phosphorylation by AMPK and GSK3 to regulate cell growth. Gwinn DM, Shackelford DB, Egan DF, Mihaylova MM, Mery A, Vasquez DS et al. AMPK phosphorylation of raptor mediates a metabolic checkpoint. Mol Cell ; 30 : — Kim J, Kundu M, Viollet B, Guan KL. AMPK and mTOR regulate autophagy through direct phosphorylation of Ulk1. Nat Cell Biol ; 13 : — Roberts DJ, Tan-Sah VP, Ding EY, Smith JM, Miyamoto S. Hexokinase-II positively regulates glucose starvation-induced autophagy through TORC1 inhibition. Mol Cell ; 53 : — da-Silva WS, Gomez-Puyou A, de Gomez-Puyou MT, Moreno-Sanchez R, De Felice FG, de Meis L et al. Mitochondrial bound hexokinase activity as a preventive antioxidant defense: steady-state ADP formation as a regulatory mechanism of membrane potential and reactive oxygen species generation in mitochondria. Sun L, Shukair S, Naik TJ, Moazed F, Ardehali H. Glucose phosphorylation and mitochondrial binding are required for the protective effects of hexokinases I and II. Mol Cell Biol ; 28 : — Wu R, Wyatt E, Chawla K, Tran M, Ghanefar M, Laakso M et al. Hexokinase II knockdown results in exaggerated cardiac hypertrophy via increased ROS production. EMBO Mol Med ; 4 : — Chance B, Sies H, Boveris A. Hydroperoxide metabolism in mammalian organs. Physiol Rev ; 59 : — Wagner BA, Venkataraman S, Buettner GR. The rate of oxygen utilization by cells. Free Radic Biol Med ; 51 : — Filomeni G, Rotilio G, Ciriolo MR. Disulfide relays and phosphorylative cascades: partners in redox-mediated signaling pathways. Cell Death Differ ; 12 : — Cell signalling and the glutathione redox system. Biochem Pharmacol ; 64 : — Flohe L. Changing paradigms in thiology from antioxidant defense toward redox regulation. Methods Enzymol ; : 1— Bindoli A, Fukuto JM, Forman HJ. Thiol chemistry in peroxidase catalysis and redox signaling. Antioxid Redox Signal ; 10 : — Beckman JS, Koppenol WH. Nitric oxide, superoxide, and peroxynitrite: the good, the bad, and ugly. Am J Physiol ; : C—C Di Giacomo G, Rizza S, Montagna C, Filomeni G. Established principles and emerging concepts on the interplay between mitochondrial physiology and S- De nitrosylation: implications in cancer and neurodegeneration. Int J Cell Biol ; : Stamler JS, Singel DJ, Loscalzo J. Biochemistry of nitric oxide and its redox-activated forms. Allen BW, Demchenko IT, Piantadosi CA. Two faces of nitric oxide: implications for cellular mechanisms of oxygen toxicity. J Appl Physiol ; : — Article CAS Google Scholar. Jones DP. Radical-free biology of oxidative stress. Am J Physiol Cell Physiol ; : C—C Holmstrom KM, Finkel T. Cellular mechanisms and physiological consequences of redox-dependent signalling. Filomeni G, Desideri E, Cardaci S, Rotilio G, Ciriolo MR. Under the ROS. thiol network is the principal suspect for autophagy commitment. Autophagy ; 6 : — Chen Y, Azad MB, Gibson SB. Superoxide is the major reactive oxygen species regulating autophagy. Cell Death Differ ; 16 : — Scherz-Shouval R, Shvets E, Elazar Z. Oxidation as a post-translational modification that regulates autophagy. Autophagy ; 3 : — Zhang C, Yang L, Wang XB, Wang JS, Geng YD, Yang CS et al. Calyxin Y induces hydrogen peroxide-dependent autophagy and apoptosis via JNK activation in human non-small cell lung cancer NCI-H cells. Cancer Lett ; : 51— Levonen AL, Hill BG, Kansanen E, Zhang J, Darley-Usmar VM. Redox regulation of antioxidants, autophagy, and the response to stress: Implications for electrophile therapeutics. Free Radic Biol Med ; 71C : — Huang J, Canadien V, Lam GY, Steinberg BE, Dinauer MC, Magalhaes MA et al. Activation of antibacterial autophagy by NADPH oxidases. Proc Natl Acad Sci USA ; : — Article PubMed PubMed Central Google Scholar. Murphy MP. How mitochondria produce reactive oxygen species. Biochem J ; : 1— Scherz-Shouval R, Z. ROS, mitochondria and the regulation of autophagy. Trends Cell Biol ; 17 : — Wang RC, Wei Y, An Z, Zou Z, Xiao G, Bhagat G et al. Akt-mediated regulation of autophagy and tumorigenesis through Beclin 1 phosphorylation. Oude Ophuis RJ, Wijers M, Bennink MB, van de Loo FA, Fransen JA, Wieringa B et al. A tail-anchored myotonic dystrophy protein kinase isoform induces perinuclear clustering of mitochondria, autophagy, and apoptosis. PLoS One ; 4 : e Campello S, Strappazzon F, Cecconi F. Mitochondrial dismissal in mammals, from protein degradation to mitophagy. Biochim Biophys Acta ; : — Rambold AS, Kostelecky B, Elia N, Lippincott-Schwartz J. Tubular network formation protects mitochondria from autophagosomal degradation during nutrient starvation. Gomes LC, Di Benedetto G, Scorrano L. During autophagy mitochondria elongate, are spared from degradation and sustain cell viability. Zhang J, Ney PA. Role of BNIP3 and NIX in cell death, autophagy, and mitophagy. Novak I, Kirkin V, McEwan DG, Zhang J, Wild P, Rozenknop A et al. Nix is a selective autophagy receptor for mitochondrial clearance. EMBO Rep ; 11 : 45— Schweers RL, Zhang J, Randall MS, Loyd MR, Li W, Dorsey FC et al. NIX is required for programmed mitochondrial clearance during reticulocyte maturation. Sandoval H, Thiagarajan P, Dasgupta SK, Schumacher A, Prchal JT, Chen M et al. Essential role for Nix in autophagic maturation of erythroid cells. Youle RJ, Narendra DP. Mechanisms of mitophagy. Nat Rev Mol Cell Biol ; 12 : 9— Matsuda N, Sato S, Shiba K, Okatsu K, Saisho K, Gautier CA et al. PINK1 stabilized by mitochondrial depolarization recruits Parkin to damaged mitochondria and activates latent Parkin for mitophagy. J Cell Biol ; : — Geisler S, Holmstrom KM, Skujat D, Fiesel FC, Rothfuss OC, Kahle PJ et al. Nat Cell Biol ; 12 : — Narendra D, Kane LA, Hauser DN, Fearnley IM, Youle RJ. Vadlamudi RK, Joung I, Strominger JL, Shin J. p62, a phosphotyrosine-independent ligand of the SH2 domain of p56lck, belongs to a new class of ubiquitin-binding proteins. Bjorkoy G, Lamark T, Johansen T. Autophagy ; 2 : — Ichimura Y, Kumanomidou T, Sou YS, Mizushima T, Ezaki J, Ueno T et al. Structural basis for sorting mechanism of p62 in selective autophagy. Strappazzon F, Nazio F, Corrado M, Cianfanelli V, Romagnoli A, Fimia GM et al. Cell Death Differ ; e-pub ahead of print 12 September ; doi Zmijewski JW, Banerjee S, Bae H, Friggeri A, Lazarowski ER, Abraham E. Exposure to hydrogen peroxide induces oxidation and activation of AMP-activated protein kinase. Desideri E, Filomeni G, Ciriolo MR. Glutathione participates in the modulation of starvation-induced autophagy in carcinoma cells. Autophagy ; 8 : — Giles NM, Gutowski NJ, Giles GI, Jacob C. Redox catalysts as sensitisers towards oxidative stress. FEBS Lett ; : — Montagna C, Di Giacomo G, Rizza S, Cardaci S, Ferraro E, Grumati P et al. S-nitrosoglutathione reductase deficiency-induced S-nitrosylation results in neuromuscular dysfunction. Antioxid Redox Signal. Sarkar S, Korolchuk VI, Renna M, Imarisio S, Fleming A, Williams A et al. Complex inhibitory effects of nitric oxide on autophagy. Mol Cell ; 43 : 19— Lopez-Rivera E, Jayaraman P, Parikh F, Davies MA, Ekmekcioglu S, Izadmehr S et al. Inducible nitric oxide synthase drives mTOR pathway activation and proliferation of human melanoma by reversible nitrosylation of TSC2. Cancer Res ; 74 : — Tripathi DN, Chowdhury R, Trudel LJ, Tee AR, Slack RS, Walker CL et al. Reactive nitrogen species regulate autophagy through ATM-AMPK-TSC2-mediated suppression of mTORC1. Proc Natl Acad Sci USA ; : E—E Komatsu M, Kurokawa H, Waguri S, Taguchi K, Kobayashi A, Ichimura Y et al. The selective autophagy substrate p62 activates the stress responsive transcription factor Nrf2 through inactivation of Keap1. Lau A, Wang XJ, Zhao F, Villeneuve NF, Wu T, Jiang T et al. A noncanonical mechanism of Nrf2 activation by autophagy deficiency: direct interaction between Keap1 and p Mol Cell Biol ; 30 : — Ichimura Y, Waguri S, Sou YS, Kageyama S, Hasegawa J, Ishimura R et al. Phosphorylation of p62 activates the Keap1-Nrf2 pathway during selective autophagy. Mol Cell ; 51 : — Taguchi K, Fujikawa N, Komatsu M, Ishii T, Unno M, Akaike T et al. Keap1 degradation by autophagy for the maintenance of redox homeostasis. Role of nrf2 in oxidative stress and toxicity. Annu Rev Pharmacol Toxicol ; 53 : — Singh B, Chatterjee A, Ronghe AM, Bhat NK, Bhat HK. Antioxidant-mediated up-regulation of OGG1 via NRF2 induction is associated with inhibition of oxidative DNA damage in estrogen-induced breast cancer. BMC Cancer ; 13 : Kim SB, Pandita RK, Eskiocak U, Ly P, Kaisani A, Kumar R et al. Targeting of Nrf2 induces DNA damage signaling and protects colonic epithelial cells from ionizing radiation. Bae SH, Sung SH, Oh SY, Lim JM, Lee SK, Park YN et al. Sestrins activate Nrf2 by promoting pdependent autophagic degradation of Keap1 and prevent oxidative liver damage. Cell Metab ; 17 : 73— Lee JH, Budanov AV, Park EJ, Birse R, Kim TE, Perkins GA et al. Sestrin as a feedback inhibitor of TOR that prevents age-related pathologies. Lee JH, Budanov AV, Talukdar S, Park EJ, Park HL, Park HW et al. Maintenance of metabolic homeostasis by Sestrin2 and Sestrin3. Cell Metab ; 16 : — Shoji JY, Kikuma T, Arioka M, Kitamoto K. Macroautophagy-mediated degradation of whole nuclei in the filamentous fungus Aspergillus oryzae. PLoS One ; 5 : e McGee MD, Weber D, Day N, Vitelli C, Crippen D, Herndon LA et al. Loss of intestinal nuclei and intestinal integrity in aging C. Aging Cell ; 10 : — Krick R, Muhe Y, Prick T, Bredschneider M, Bremer S, Wenzel D et al. Piecemeal microautophagy of the nucleus: genetic and morphological traits. Autophagy ; 5 : — Rello-Varona S, Lissa D, Shen S, Niso-Santano M, Senovilla L, Marino G et al. Autophagic removal of micronuclei. Cell Cycle ; 11 : — Wiseman H, Halliwell B. Damage to DNA by reactive oxygen and nitrogen species: role in inflammatory disease and progression to cancer. Biochem J ; Pt 1 : 17— Cooke MS, Evans MD, Dizdaroglu M, Lunec J. Oxidative DNA damage: mechanisms, mutation, and disease. FASEB J ; 17 : — Cadet J, Delatour T, Douki T, Gasparutto D, Pouget JP, Ravanat JL et al. Hydroxyl radicals and DNA base damage. Mutat Res ; : 9— Neeley WL, Essigmann JM. Mechanisms of formation, genotoxicity, and mutation of guanine oxidation products. Chem Res Toxicol ; 19 : — Iida T, Furuta A, Nakabeppu Y, Iwaki T. Defense mechanism to oxidative DNA damage in glial cells. Neuropathology ; 24 : — Lenaz G. Mitochondria and reactive oxygen species. Which role in physiology and pathology? Adv Exp Med Biol ; : 93— Ciccia A, Elledge SJ. The DNA damage response: making it safe to play with knives. Roos WP, Kaina B. DNA damage-induced cell death: from specific DNA lesions to the DNA damage response and apoptosis. Cancer Lett ; : — De Zio D, Cianfanelli V, Cecconi F. New insights into the link between DNA damage and apoptosis. Antioxid Redox Signal ; 19 : — Rodriguez-Rocha H, Garcia-Garcia A, Panayiotidis MI, Franco R. DNA damage and autophagy. Mutat Res ; : — Vessoni AT, Filippi-Chiela EC, Menck CF, Lenz G. Autophagy and genomic integrity. Cell Death Differ ; 20 : — Yoon JH, Ahn SG, Lee BH, Jung SH, Oh SH. Role of autophagy in chemoresistance: regulation of the ATM-mediated DNA-damage signaling pathway through activation of DNA-PKcs and PARP Biochem Pharmacol ; 83 : — Abedin MJ, Wang D, McDonnell MA, Lehmann U, Kelekar A. Autophagy delays apoptotic death in breast cancer cells following DNA damage. Cell Death Differ ; 14 : — Bordin DL, Lima M, Lenz G, Saffi J, Meira LB, Mesange P et al. DNA alkylation damage and autophagy induction. Mutat Res ; : 91— Maiuri MC, Zalckvar E, Kimchi A, Kroemer G. Self-eating and self-killing: crosstalk between autophagy and apoptosis. Nat Rev Mol Cell Biol ; 8 : — Yue Z, Jin S, Yang C, Levine AJ, Heintz N. Beclin 1, an autophagy gene essential for early embryonic development, is a haploinsufficient tumor suppressor. Liang C, Feng P, Ku B, Dotan I, Canaani D, Oh BH et al. Autophagic and tumour suppressor activity of a novel Beclin1-binding protein UVRAG. Nat Cell Biol. Karantza-Wadsworth V, Patel S, Kravchuk O, Chen G, Mathew R, Jin S et al. Autophagy mitigates metabolic stress and genome damage in mammary tumorigenesis. Genes Dev ; 21 : — Mathew R, Kongara S, Beaudoin B, Karp CM, Bray K, Degenhardt K et al. Autophagy suppresses tumor progression by limiting chromosomal instability. Takamura A, Komatsu M, Hara T, Sakamoto A, Kishi C, Waguri S et al. Autophagy-deficient mice develop multiple liver tumors. Bae H, Guan JL. Suppression of autophagy by FIP deletion impairs DNA damage repair and increases cell death upon treatments with anticancer agents. Mol Cancer Res ; 9 : — Lynch-Day MA, Klionsky DJ. The Cvt pathway as a model for selective autophagy. Dotiwala F, Eapen VV, Harrison JC, Arbel-Eden A, Ranade V, Yoshida S et al. DNA damage checkpoint triggers autophagy to regulate the initiation of anaphase. Proc Natl Acad Sci USA ; : E41—E Dyavaiah M, Rooney JP, Chittur SV, Lin Q, Begley TJ. Autophagy-dependent regulation of the DNA damage response protein ribonucleotide reductase 1. Robert T, Vanoli F, Chiolo I, Shubassi G, Bernstein KA, Rothstein R et al. HDACs link the DNA damage response, processing of double-strand breaks and autophagy. Nature ; : 74— Degenhardt K, Mathew R, Beaudoin B, Bray K, Anderson D, Chen G et al. Autophagy promotes tumor cell survival and restricts necrosis, inflammation, and tumorigenesis. Cancer Cell ; 10 : 51— Mathew R, Karp CM, Beaudoin B, Vuong N, Chen G, Chen HY et al. Autophagy suppresses tumorigenesis through elimination of p Munoz-Gamez JA, Rodriguez-Vargas JM, Quiles-Perez R, Aguilar-Quesada R, Martin-Oliva D, de Murcia G et al. PARP-1 is involved in autophagy induced by DNA damage. Autophagy ; 5 : 61— Rodriguez-Vargas JM, Ruiz-Magana MJ, Ruiz-Ruiz C, Majuelos-Melguizo J, Peralta-Leal A, Rodriguez MI et al. ROS-induced DNA damage and PARP-1 are required for optimal induction of starvation-induced autophagy. Cell Res ; 22 : — Hurley PJ, F. ATM and ATR: components of an integrated circuit. Cell Cycle ; 6 : — Alexander A, Cai SL, Kim J, Nanez A, Sahin M, MacLean KH et al. ATM signals to TSC2 in the cytoplasm to regulate mTORC1 in response to ROS. Pietrocola F, Izzo V, Niso-Santano M, Vacchelli E, Galluzzi L, Maiuri MC et al. Regulation of autophagy by stress-responsive transcription factors. Semin Cancer Biol ; 23 : — Kenzelmann Broz D, Spano Mello S, Bieging KT, Jiang D, Dusek RL, Brady CA et al. Global genomic profiling reveals an extensive pregulated autophagy program contributing to key p53 responses. Genes Dev ; 27 : — Fullgrabe J, Klionsky DJ, Joseph B. The return of the nucleus: transcriptional and epigenetic control of autophagy. Nat Rev Mol Cell Biol ; 15 : 65— Maiuri MC, Galluzzi L, Morselli E, Kepp O, Malik SA, Kroemer G. Autophagy regulation by p Marino G, Niso-Santano M, Baehrecke EH, Kroemer G. Self-consumption: the interplay of autophagy and apoptosis. Nat Rev Mol Cell Biol ; 15 : 81— Download references. We thank M Acuña Villa and MW Bennett for editorial and secretarial work. We are also grateful to Costanza Montagna and Giuseppina Di Giacomo for having provided part of unpublished results obtained on GSNOR-KO mice. The Unit of Cell Stress and Survival is supported by grants from the Danish Cancer Society KBVU RA to FC and KBVU RA to GF ; Lundbeck Foundation nn. R and NovoNordisk nn. We are also grateful to AIRC IG to FC and MFAG to GF ; Telethon Foundation GGP ; the Italian Ministry of University and Research PRIN and FIRB Accordi di Programma ; and the Italian Ministry of Health RF to FC and GR Cell Stress and Survival Unit, Danish Cancer Society Research Center, Copenhagen, Denmark. You can also search for this author in PubMed Google Scholar. Correspondence to G Filomeni or F Cecconi. This work is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 3. Reprints and permissions. Filomeni, G. Oxidative stress and autophagy: the clash between damage and metabolic needs. Cell Death Differ 22 , — Download citation. Received : 29 June Revised : 19 August Accepted : 21 August Published : 26 September Issue Date : March Anyone you share the following link with will be able to read this content:. Sorry, a shareable link is not currently available for this article. Provided by the Springer Nature SharedIt content-sharing initiative. Journal of Orthopaedic Surgery and Research Skip to main content Thank you for visiting nature. Download PDF. Subjects Autophagy. Abstract Autophagy is a catabolic process aimed at recycling cellular components and damaged organelles in response to diverse conditions of stress, such as nutrient deprivation, viral infection and genotoxic stress. Facts Reactive oxygen species ROS production and thiol redox state imbalance are induced immediately upon nutrient deprivation and represent important mediators of autophagy. Open Questions How do ROS and oxidative stress affect autophagy? Which are the main ROS able to signal autophagy being activated and going on? Does nitric oxide act as a real inhibitor of autophagy? How does autophagy sense DNA damage? How can autophagy contribute to DNA damage repair? Autophagy: Converging Point of Different Stimuli There are three main types of autophagy culminating to lysosome-mediated degradation: 1 macroautophagy hereafter referred to as autophagy that involves the formation of a double-membrane vesicle autophagosome deputed to sequester damaged organelles and biomolecules; 2 microautophagy, by which the cytosolic material is directly engulfed by the lysosome; and 3 chaperone-mediated autophagy. Full size image. Oxidative Stress Living cells are always subjected to the hazardous effects of exogenously or endogenously produced highly reactive oxidizing molecules. Oxidative damage It is commonly accepted that the principal source of ROS in the cell is the mitochondrial respiratory chain. Autophagy and Oxidative Stress ROS have been copiously reported as early inducers of autophagy upon nutrient deprivation. Figure 2. Figure 3. Antioxidant Role of Autophagy: Focus on Nucleus and DNA Damage On the basis of what has been reported so far, antioxidant response and autophagy are mechanisms simultaneously induced by oxidative stress conditions in order to concomitantly decrease ROS and RNS concentration upstream causes and reduce the oxidative damage to biomolecules and organelles downstream effect. Oxidative Stress and DNA Damage ROS and RNS are one of the major sources of DNA damage 96 as they could directly modify the DNA or indirectly generate different lesions, both affecting cell viability. DNA Damage and Autophagy: A Complex Crosstalk When the DNA is damaged by ROS and RNS, cells activate a number of pathways in order to maintain genomic integrity, these being associated to the DNA damage response DDR. Sensor proteins transduce the signal of DNA damage to autophagy A number of works in recent years indicate that once ROS and RNS damage the DNA, the event is transduced in order to activate the DDR, and concomitantly is signalled to the autophagic pathway in order to orchestrate the response. Figure 4. Concluding Remarks Several lines of evidence indicate that ROS and RNS are the upstream modulators of autophagy, likely acting at multiple levels in the process. References Baudhuin P, Berleur AN, De Duve C, Wattiaux R. Article CAS PubMed PubMed Central Google Scholar De Duve C, Gianetto R, Appelmans F, Wattiaux R. Article CAS PubMed Google Scholar De Duve C, Berthet J. Article CAS PubMed Google Scholar De Duve C, Wattiaux R. Article CAS PubMed Google Scholar De Duve C, Baudhuin P. Article CAS PubMed Google Scholar Mann PJ, Quastel JH. Article CAS PubMed PubMed Central Google Scholar Davies KJ. CAS PubMed Google Scholar Halliwell B. Article CAS PubMed Google Scholar Imlay JA, Linn S. Article CAS PubMed Google Scholar Harman D. Article CAS PubMed Google Scholar Kroemer G, Marino G, Levine B. Article CAS PubMed PubMed Central Google Scholar Yang Z, Klionsky DJ. Article CAS PubMed Google Scholar Neufeld TP. Article CAS PubMed PubMed Central Google Scholar Zoncu R, Efeyan A, Sabatini DM. Article CAS PubMed Google Scholar Nazio F, Cecconi F. Article CAS PubMed PubMed Central Google Scholar Fimia GM, Di Bartolomeo S, Piacentini M, Cecconi F. Article PubMed Google Scholar Nicklin P, Bergman P, Zhang B, Triantafellow E, Wang H, Nyfeler B et al. LUBAC-synthesized linear ubiquitin chains restrict cytosol-invading bacteria by activating autophagy and NF-kappaB. Polajnar M, Dietz MS, Heilemann M, Behrends C. Expanding the host cell ubiquitylation machinery targeting cytosolic Salmonella. EMBO Rep. Stolz A, Ernst A, Dikic I. Cargo recognition and trafficking in selective autophagy. Kraft C, et al. FYCO1 is a Rab7 effector that binds to LC3 and PI3P to mediate microtubule plus end-directed vesicle transport. Popovic D, et al. Rab GTPase-activating proteins in autophagy: regulation of endocytic and autophagy pathways by direct binding to human ATG8 modifiers. McEwan DG, et al. Genau HM, et al. Verlhac P, et al. Autophagy receptor NDP52 regulates pathogen-containing autophagosome maturation. Petkova DS, et al. Distinct contributions of autophagy receptors in measles virus replication. Zheng YT, et al. J Immunol. Thurston TL, et al. Recruitment of TBK1 to cytosol-invading Salmonella induces WIPI2-dependent antibacterial autophagy. Cheng CY, Chi PI, Liu HJ. Commentary on the regulation of viral proteins in autophagy process. Biomed Res Int. Perot BP, Ingersoll MA, Albert ML. Immunol Rev. Mao J, et al. Autophagy and viral infection. Chiramel AI, Brady NR, Bartenschlager R. Divergent roles of autophagy in virus infection. Article Google Scholar. Joubert PE, et al. Autophagy induction by the pathogen receptor CD Meiffren G, et al. Pathogen recognition by the cell surface receptor CD46 induces autophagy. Mouna L, et al. Grégoire IP, Rabourdin-Combe C, Faure M. Autophagy and RNA virus interactomes reveal IRGM as a common target. Petkova DS, Viret C, Faure M. IRGM in autophagy and viral infections. Front Immunol. Abdoli A, Alirezaei M, Mehrbod P, Forouzanfar F. Autophagy: the multi-purpose bridge in viral infections and host cells. Rev Med Virol. Castedo M, et al. Sequential involvement of Cdk1, mTOR and p53 in apoptosis induced by the HIV-1 envelope. Denizot M, et al. HIV-1 gp41 fusogenic function triggers autophagy in uninfected cells. Chan ST, Ou JJ. Hepatitis C virus-induced autophagy and host innate immune response. Choi Y, Bowman JW, Jung JU. Autophagy during viral infection - a double-edged sword. Nat Rev Microbiol. Saito T, Owen DM, Jiang F, Marcotrigiano J, Gale M Jr. Innate immunity induced by composition-dependent RIG-I recognition of hepatitis C virus RNA. Li K, et al. Activation of chemokine and inflammatory cytokine response in hepatitis C virus-infected hepatocytes depends on toll-like receptor 3 sensing of hepatitis C virus double-stranded RNA intermediates. García MA, et al. Impact of protein kinase PKR in cell biology: from antiviral to antiproliferative action. Microbiol Mol Biol Rev. Liang Q, et al. Crosstalk between the cGAS DNA sensor and Beclin-1 autophagy protein shapes innate antimicrobial immune responses. Wang L, Ou JH. Hepatitis C virus and autophagy. Biol Chem. Grégoire IP, et al. IRGM is a common target of RNA viruses that subvert the autophagy network. Su WC, et al. Rab5 and class III phosphoinositide 3-kinase Vps34 are involved in hepatitis C virus NS4B-induced autophagy. J Virol. Wang L, Tian Y, Ou JH. HCV induces the expression of Rubicon and UVRAG to temporally regulate the maturation of autophagosomes and viral replication. Chatterji U, Bobardt M, Tai A, Wood M, Gallay PA. Cyclophilin and NS5A inhibitors, but not other anti-hepatitis C virus HCV agents, preclude HCV-mediated formation of double-membrane-vesicle viral factories. Antimicrob Agents Chemother. Quan M, et al. A functional role for NS5ATP9 in the induction of HCV NS5A-mediated autophagy. J Viral Hepat. Shrivastava S, Bhanja Chowdhury J, Steele R, Ray R, Ray RB. Hepatitis C virus upregulates Beclin1 for induction of autophagy and activates mTOR signaling. Kim N, et al. Interferon-inducible protein SCOTIN interferes with HCV replication through the autolysosomal degradation of NS5A. Nat Commun. Joyce MA, et al. Sir D, et al. Induction of incomplete autophagic response by hepatitis C virus via the unfolded protein response. Vescovo T, et al. Autophagy in HCV infection: keeping fat and inflammation at bay. Ivanov AV, Bartosch B, Smirnova OA, Isaguliants MG, Kochetkov SN. HCV and oxidative stress in the liver. Kim SJ, Syed GH, Siddiqui A. Hepatitis C virus induces the mitochondrial translocation of Parkin and subsequent mitophagy. Tallóczy Z, Virgin HWT, Levine B. PKR-dependent autophagic degradation of herpes simplex virus type 1. Orvedahl A, et al. Autophagy protects against Sindbis virus infection of the central nervous system. Levine B. Eating oneself and uninvited guests: autophagy-related pathways in cellular defense. Dokladny K, Myers OB, Moseley PL. Heat shock response and autophagy--cooperation and control. Morimoto RI, Sarge KD, Abravaya K. Transcriptional regulation of heat shock genes. A paradigm for inducible genomic responses. Ellis RJ. The molecular chaperone concept. Semin Cell Biol. CAS PubMed Google Scholar. Goloubinoff P. Mechanisms of protein homeostasis in health, aging and disease. Swiss Med Wkly. Chiang HL, Terlecky SR, Plant CP, Dice JF. A role for a kilodalton heat shock protein in lysosomal degradation of intracellular proteins. Dokladny K, et al. Regulatory coordination between two major intracellular homeostatic systems: heat shock response and autophagy. Fernandez-Fernandez MR, Gragera M, Ochoa-Ibarrola L, Quintana-Gallardo L, Valpuesta JM. Hsp70 - a master regulator in protein degradation. Mizushima N, Yoshimori T, Levine B. Methods in mammalian autophagy research. Komatsu M, et al. Homeostatic levels of p62 control cytoplasmic inclusion body formation in autophagy-deficient mice. Cong P, et al. Cold exposure induced oxidative stress and apoptosis in the myocardium by inhibiting the Nrf2-Keap1 signaling pathway. BMC Cardiovasc Disord. Sharma A, et al. Mol Neurobiol. Oezel L, et al. Fibromyalgia syndrome: metabolic and autophagic processes in intermittent cold stress mice. Pharmacol Res Perspect. Lu S, Xu D. Biochem Biophys Res Commun. Caterina MJ, et al. The capsaicin receptor: a heat-activated ion channel in the pain pathway. Caterina MJ, Rosen TA, Tominaga M, Brake AJ, Julius D. A capsaicin-receptor homologue with a high threshold for noxious heat. Rider MH, Hussain N, Horman S, Dilworth SM, Storey KB. Stress-induced activation of the AMP-activated protein kinase in the freeze-tolerant frog Rana sylvatica. Farfariello V, Amantini C, Santoni G. Transient receptor potential vanilloid 1 activation induces autophagy in thymocytes through ROS-regulated AMPK and Atg4C pathways. J Leukoc Biol. Neutelings T, Lambert CA, Nusgens BV, Colige AC. Effects of mild cold shock 25°C followed by warming up at 37°C on the cellular stress response. Chude CI, Amaravadi RK. Targeting autophagy in cancer: update on clinical trials and novel inhibitors. Int J Mol Sci. Wu YT, et al. Dual role of 3-methyladenine in modulation of autophagy via different temporal patterns of inhibition on class I and III phosphoinositide 3-kinase. Mauthe M, et al. Chloroquine inhibits autophagic flux by decreasing autophagosome-lysosome fusion. Chacon-Cabrera A, et al. Pharmacological strategies in lung cancer-induced cachexia: effects on muscle proteolysis, autophagy, structure, and weakness. J Cell Physiol. Hooper KM, Barlow PG, Stevens C, Henderson P. Inflammatory bowel disease drugs: a focus on autophagy. J Crohns Colitis. Kar R, Riquelme MA, Hua R, Jiang JX. JBMR Plus. Wang H, Kubica N, Ellisen LW, Jefferson LS, Kimball SR. Dexamethasone represses signaling through the mammalian target of rapamycin in muscle cells by enhancing expression of REDD1. Fatkhullina AR, Abramov SN, Skibo Iu V, Abramova ZI. Dexamethasone affect on the expression of bcl-2 and mTOR genes in T-lymphocytes from healthy donors. Zou N, et al. BMC Complement Altern Med. Sun Z, Zheng L, Liu X, Xing W, Liu X. Drug Des Devel Ther. Cosin-Roger J, et al. Shi Z-Y, et al. Int J Mol Med. Zhang W, Yuan W, Song J, Wang S, Gu X. LncRNA CPS1-IT1 suppresses EMT and metastasis of colorectal cancer by inhibiting hypoxia-induced autophagy through inactivation of HIF-1α. Yang X, et al. Hypoxia-induced autophagy promotes gemcitabine resistance in human bladder cancer cells through hypoxia-inducible factor 1α activation. Int J Oncol. Abdul Rahim SA, et al. Regulation of hypoxia-induced autophagy in glioblastoma involves ATG9A. Br J Cancer. Yan J, et al. AEG-1 is involved in hypoxia-induced autophagy and decreases chemosensitivity in T-cell lymphoma. Mol Med. Download references. The Second Affiliated Hospital of Chengdu Medical College, China National Nuclear Corporation Hospital, Chengdu, Sichuan, China. School of Biological Sciences and Technology, Chengdu Medical College, Chengdu, China. State Key Laboratory of Oral Diseases, National Clinical Research Center for Oral Diseases, Chinese Academy of Medical Sciences Research Unit of Oral Carcinogenesis and Management, West China Hospital of Stomatology, Sichuan University, Chengdu, China. You can also search for this author in PubMed Google Scholar. Department of Pharmacology, West China Hospital of Sichuan University, Chengdu, China. Reprints and permissions. Li, J. Autophagy in Cellular Stress Responses. In: Huang, C. eds Oxidative Stress. Springer, Singapore. Published : 26 April Publisher Name : Springer, Singapore. Print ISBN : Online ISBN : eBook Packages : Biomedical and Life Sciences Biomedical and Life Sciences R0. Anyone you share the following link with will be able to read this content:. Sorry, a shareable link is not currently available for this article. Provided by the Springer Nature SharedIt content-sharing initiative. Policies and ethics. Skip to main content. Abstract Autophagy is an essential cellular mechanism involving the lysosomal degradation of cytoplasmic organelles or cytosolic components. Keywords Autophagy Cellular stress Endoplasmic reticulum stress. Buying options Chapter EUR eBook EUR Softcover Book EUR Hardcover Book EUR Tax calculation will be finalised at checkout Purchases are for personal use only Learn about institutional subscriptions. References Mizushima N, Levine B, Cuervo AM, Klionsky DJ. Article CAS PubMed PubMed Central Google Scholar Murrow L, Debnath J. Article CAS PubMed Google Scholar Maiuri MC, Zalckvar E, Kimchi A, Kroemer G. Article CAS PubMed Google Scholar Takeshige K, Baba M, Tsuboi S, Noda T, Ohsumi Y. Article CAS PubMed Google Scholar Scherz-Shouval R, Elazar Z. Article CAS PubMed Google Scholar Filomeni G, De Zio D, Cecconi F. Article CAS PubMed Google Scholar Lee J, Giordano S, Zhang J. Article CAS PubMed Google Scholar Li L, Tan J, Miao Y, Lei P, Zhang Q. Article CAS PubMed Google Scholar Filomeni G, Desideri E, Cardaci S, Rotilio G, Ciriolo MR. Article CAS PubMed Google Scholar Bensaad K, Cheung EC, Vousden KH. Article CAS PubMed PubMed Central Google Scholar Scherz-Shouval R, et al. Article CAS PubMed PubMed Central Google Scholar Scherz-Shouval R, Elazar Z. Article CAS PubMed Google Scholar Zoncu R, Efeyan A, Sabatini DM. Article CAS PubMed Google Scholar Filomeni G, Rotilio G, Ciriolo MR. Article CAS PubMed Google Scholar Bindoli A, Fukuto JM, Forman HJ. Article CAS PubMed PubMed Central Google Scholar Cardaci S, Filomeni G, Ciriolo MR. Article CAS PubMed Google Scholar Zmijewski JW, et al. Article CAS PubMed PubMed Central Google Scholar Wolff NC, et al. Article CAS PubMed PubMed Central Google Scholar Fimia GM, Di Bartolomeo S, Piacentini M, Cecconi F. Article PubMed Google Scholar Vadlamudi RK, Joung I, Strominger JL, Shin J. Article CAS PubMed Google Scholar Bjørkøy G, et al. Article CAS PubMed PubMed Central Google Scholar Giles NM, Gutowski NJ, Giles GI, Jacob C. Article CAS PubMed Google Scholar Scherz-Shouval R, et al. Article PubMed PubMed Central Google Scholar Buchberger A, Bukau B, Sommer T. Article CAS PubMed Google Scholar Qi Z, Chen L. Article CAS PubMed Google Scholar Senft D, Ronai Z, et al. Article CAS PubMed PubMed Central Google Scholar Cheng Y-C, Chang J-M, Chen C-A, Chen H-C. Article CAS Google Scholar Kouroku Y, et al. Article CAS PubMed Google Scholar Ogata M, et al. Article CAS PubMed PubMed Central Google Scholar Hoyer-Hansen M, et al. Article CAS PubMed Google Scholar Yang Y, et al. Article CAS PubMed Google Scholar Choi H, et al. Article CAS PubMed PubMed Central Google Scholar Wu H, et al. Article CAS PubMed PubMed Central Google Scholar Shi R-Y, et al. Article CAS PubMed PubMed Central Google Scholar Bellot G, et al. Article CAS PubMed PubMed Central Google Scholar Gwinn DM, et al. Article CAS PubMed PubMed Central Google Scholar Rouschop KMA, et al. Article CAS PubMed Google Scholar Song S, Tan J, Miao Y, Sun Z, Zhang Q. Article CAS PubMed Google Scholar Peña-Oyarzun D, et al. Article PubMed PubMed Central Google Scholar Schutt KL, Moseley JB. Article CAS PubMed PubMed Central Google Scholar Nunes P, et al. Article CAS PubMed PubMed Central Google Scholar Jiang LB, et al. Article CAS PubMed PubMed Central Google Scholar Soto-Burgos J, Bassham DC. Article PubMed PubMed Central Google Scholar Pu Y, Soto-Burgos J, Bassham DC. Article CAS PubMed PubMed Central Google Scholar Gatica D, Lahiri V, Klionsky DJ. Article CAS PubMed PubMed Central Google Scholar McEwan DG. Article PubMed PubMed Central Google Scholar Sharma V, Verma S, Seranova E, Sarkar S, Kumar D. Article PubMed PubMed Central Google Scholar Thurston TL, Wandel MP, von Muhlinen N, Foeglein A, Randow F. Article CAS PubMed PubMed Central Google Scholar Boyle KB, Randow F. Article CAS PubMed Google Scholar Shaid S, Brandts CH, Serve H, Dikic I. Article CAS PubMed Google Scholar Pankiv S, et al. |
| Footer menu | Slavikova S. J Exp Bot 59 , — Mol Biol Rep 33 , — Thompson A. Curr Opin Plant Biol 8 , — Tsugane K. Plant Cell 11 , — Wang, Y. ATG2, an autophagy-related protein, negatively affects powdery mildew resistance and mildew-induced cell death in Arabidopsis. Plant J. Wang Y. Plant Signal Behav 6 , — Xia, K. Genome-Wide Identification, Classification, and Expression Analysis of Autophagy-Associated Gene Homologues in Rice Oryza sativa L. DNA Res. Xie Z. Nat Cell Biol 9 , — Xiong Y. Yoshimoto K. Plant Cell 16 , — Plant Cell 21 , — Zhu J. Curr Opin Plant Biol 4 , — Oxford University Press is a department of the University of Oxford. It furthers the University's objective of excellence in research, scholarship, and education by publishing worldwide. Sign In or Create an Account. Issue Advance Articles Submit Author Guidelines Submission Site Open Access Options Self-Archiving Policy Why Submit? Advanced Search. Search Menu. Article Navigation. Close mobile search navigation Article Navigation. Volume 2. Article Contents Abstract. Journal Article. Role of plant autophagy in stress response. Shaojie Han , Shaojie Han. MOE Key Laboratory of Bioinformatics, School of Life Sciences, Tsinghua University. Oxford Academic. Bingjie Yu. Yan Wang. Yule Liu. Correspondence: yuleliu mail. PDF Split View Views. Select Format Select format. ris Mendeley, Papers, Zotero. enw EndNote. bibtex BibTex. txt Medlars, RefWorks Download citation. Permissions Icon Permissions. Abstract Autophagy is a conserved pathway for the bulk degradation of cytoplasmic components in all eukaryotes. Ultrastructural and biochemical characterization of autophagy in higher plant cells subjected to carbon deprivation: control by the supply of mitochondria with respiratory substrates. Google Scholar Crossref. Search ADS. Variations on a theme: plant autophagy in comparison to yeast and mammals. Google Scholar PubMed. OpenURL Placeholder Text. Autophagy and the cytoplasm to vacuole targeting pathway both require Aut10p. The ATG autophagic conjugation system in maize: ATG transcripts and abundance of the ATG8-lipid adduct are regulated by development and nutrient availability. The antibodies were validated in our previous reports 21 , 22 , Antibodies for Vps34 , Atg7 , Atg9a , Atg14 rabbit monoclonal clone D3H2Z for immunostaining and WB , mTOR and for WB , phospho-Akt Ser , Akt , LC3B for WB , phospho-S6K1 Thr , S6K1 , and phospho-Atg13 Ser isoform 2 Ser; mouse Ser were from Cell Signaling Technology. The antibodies were validated in our previous reports 21 , 22 , 54 , 55 , 56 , Antibodies for AMPK , LKB1 , phospho-ACC , phospho-ULK1 Ser , phospho-ULK1 Ser , phospho-ULK1 Ser , phospho-ULK1 Ser , cleaved PARP , and cleaved caspase-3 were from Cell Signaling Technology. The antibodies were validated by the manufacturer. Myc 9E10 monoclonal antibody OP10 and WIPI2 antibody MABC91 for immunostaining were from EMD-Millipore. Ubiquitin antibody from EMD-Millipore was validated by the manufacturer. HA antibody HA. The antibody was validated in our previous reports 21 , 22 , 54 , 55 , 56 , LC3B antibody PM for immunostaining and p62 antibody PM for immunostaining were from MBL International Woburn, MA. The antibodies were validated in our previous reports 21 , 22 , 54 for LC3B and by the manufacturer for p Anti-Atg13 antibodies have been described in our previous report for the validation Anti-pT antibody was made using PRNR pT LPDL-C as an antigenic peptide in rabbits, and purified using antigenic peptide-conjugated column in Abclonal Science Woburn, MA. The antibody was validated in the current paper. Antibodies for pAtg14 Ser29 and pBeclin 1 Ser30, the sites we identified, were previously described in our reports for the validation 21 , The following materials were used in the experiments. Recombinant Atg14 was obtained from Escherichia coli as a custom order to MyBioSource San Diego, CA. All the primers used for our study were obtained from Integrated DNA Technologies Coralville, IA via custom synthesis. HCT CCL , HeLa CCL-2 , HEKT CRL , HepG2 HB , C2C12 CRL , and A cells CCL were obtained from ATCC. HT22 cells SCC were obtained from EMD-Millipore. All cell lines were confirmed to be mycoplasma-free using MycoAlert PLUS Mycoplasma Detection kit Lonza Walkersville, Inc. Myc-tagged constructs for ULK1 and Atg13 were made using pRK5 vector as described in our previous reports 21 , 22 , For lentiviral constructs, we used pLV-EF1a-IRES plasmids Addgene , , Human and mouse ULK1 genes were described in our previous report LKB1 WT and KD cDNAs were obtained from Addgene and and subcloned into pLV-EF1a-IRES-puro. Human and mouse ULK1 and Atg13 point mutant constructs listed in Supplementary Table 1 were generated using a site-directed mutagenesis kit Agilent Mouse 4SA ULK1 mutant was obtained from Addgene Mouse SA and SC mutants were kindly provided by Dr. Kun-Liang Guan UC San Diego. The mutant constructs were cloned into pLV-EF1a-IRES-Puro with no tag. The primer sequences used for the mutagenesis are listed in Supplementary Table 1. For inducible expression, human and mouse ULK1 genes were subcloned into Lenti-TRE3GPGK-Tet3G-puro TransOMIC Technologies. Human WT AMPKα1 Addgene and KD AMPKα1 Addgene DNAs and rat WT AMPKα2 Addgene and K45R AMPKα2 kinase inactive mutant Addgene DNAs were subcloned into pLV-EF1a-IRES-Puro or pLV-EF1a-IRES-blast. All the generated constructs were confirmed by sequencing the DNAs at GENEWIZ South Plainfield, NJ. AMPKα DKO HCT cells, AMPKα DKO HeLa cells, and ULK DKO, ULK1 KO, Atg13 KO HEKT cells were generated using the CRISPR-cas9 assisted genome editing technique. We used pSpCas9 BB -2A-GFP Addgene, PX; deposited by Dr. Feng Zhang. The detailed procedures are described in our recent reports 21 , 22 , In brief, target cells were transduced by the plasmid using the Neon Transfection System ThermoFisher Scientific, MPK Two days post-transfection, green fluorescence-positive single cells were sorted and plated into well plates using the Hana Single Cell Dispenser Namocell, Mountain View, CA. Single colonies were expanded, and genomic DNA and cell extract were obtained for screening by genotyping and WB. Atg7 KO, Atg14 KO, and LKB1 KO HCT cells were generated using lentiCRISPRv2 vector Addgene Lentivirus preparation and infection procedures have been described in our previous reports The transduced cells were cultured in the absence of antibiotics for two days. Stably transduced cells were selected with antibiotics. ULK DKO HCT cells 22 , Beclin 1 KO HCT cells 21 and ULK DKO MEFs 21 have been described previously. AMPK DKO MEFs were obtained from Dr. Benoit Viollet. Atg9a KO MEFs were obtained from Dr. Shizuo Akira. LKB1 KO MEFs were obtained from Dr. All the generated cell lines were verified by genotying using the primers listed in Supplementary Information and by sequencing the DNAs at GENEWIZ South Plainfield, NJ. The sequences for gRNAs are listed in Supplementary Table 2. All the chemicals used were resolved in dimethyl sulfoxide DMSO as stock and used at the indicated concentrations for each experiment. All the chemicals for analysis of their effects on ULK1 activity, Vps34 activity and autophagy were present not only in pre-incubation but also during starvation or mTORC1 inhibiting treatments. Cells were harvested 2 days post-transfection for co-IP assays. We used pLV-EF1a-IRES-lentivral vectors described above to generate cell lines stably transduced with exogenous DNA. The procedures for lentivirus preparation and target cell infection have been described in our previous reports Because there is a limitation of DNA insert for the efficient lentiviral packaging, we could not produce pLV-EF1a-IRES vectors containing the ULK1 gene. As an alternative method, we linearized the lentiviral vector using the restriction enzyme SgrDI ThermoFisher Scientific, ER for a single cut, then introduced the linearized DNA into target cells using the Neon instrument ThermoFisher Scientific, MPK Two days after the transfection, cells harboring the lentiviral vector were selected in the presence of antibiotics. Membranes were washed briefly with phosphate buffered saline PBS, Fisher Scientific, BP containing 0. Membranes were then incubated with Enhanced Chemiluminescence WB detection reagents Advansta, E to visualize protein bands. WB images were acquired by X-ray film developer or using iBright ThermoFisher Scientific. Band intensities of WB images were quantified using Image J version 1. The half-life of protein downregulation was obtained by the Prism v6 software version 6. C57BL6J male mice were purchased from the Jackson Laboratories. Mice between 10 and 12 weeks of age were used for the experiment. One hour after the injection of Torin1, the mouse liver and skeletal muscle were isolated on the ice and immediately frozen in liquid nitrogen. We also obtained cryosections from the frozen tissue using a cryostat Reichert-Jung Cryocut Images were obtained using a Deltavision PersonelDV microscope Applied Precision Inc. LC3B puncta per cell liver and per fiber muscle were quantified with a specific threshold using ImageJ version 1. All experimental procedures were approved by the University of Minnesota, Institutional Animal Care and Use Committee. Phagophore and autophagosome formation was analyzed by immunostaining endogenous WIPI2 EMD-Millipore, MABC91; Sigma-Aldrich, SAB; dilution and LC3B MBL International, PM; dilution , respectively, as we described in our previous reports 21 , Images from stained cells were obtained using a Deltavision PersonelDV microscope and analyzed by softWoRx version 6. WIPI2 and LC3B puncta were quantified with a specific threshold using ImageJ version 1. Whole-cell extracts were prepared as described above using the lysis buffer for WB. The expression levels of LC3B and p62 in cell lysate were analyzed by WB and quantified by densitometry. The flux was analyzed as the difference of LC3B II or p62 levels between the presence and absence of BAFA1. Cell extract was obtained using the lysis buffer for WB and analyzed for cleaved PARP and caspase 3 by WB. Additionally, cells were stained using pSIVA-IANBD specific to apoptotic cell membrane Abcam, ab Cell images were acquired using Lionheart FX fluorescence microscope Bio-Tek, Winooski, VT and analyzed using Gen5 program version 3. During the second incubation, cells were treated with chemicals or starvation medium as indicated in each experiment. The concentrations of other used chemicals are the same as described in other assays. Images were taken using a Deltavision PersonelDV microscope as described above in Immunostaining and fluorescence microscopy section. For each analysis, 50 cells were counted across three independent experiments. AMPKα DKO HEKT cells were transiently transduced with myc-ULK1 construct. Two days post-transfection, myc-ULK1 immunoprecipitates were obtained using anti-myc antibody. As a control, the immunoprecipitates were incubated just with the buffer without AMPK. The phosphorylation state of Atg14 Ser29 was analyzed by WB. The lipid kinase activity of Atgassociated Vps34 was assayed as we have described previously 21 , The Vps34 kinase reactions were initiated by adding ATP final conc. All the kinase reactions were spotted onto nitrocellulose membrane. After multiple times of extensive washing, the amount of PI3P Grip remaining on the nitrocellulose membrane was analyzed by WB using anti-GST antibody Cytiva, P3P dot blot intensities were quantitatively analyzed by ImageJ software version 1. HEKT cells deficient of ULK1 were transiently transduced to express myc-tagged ULK1 constructs. The reaction was stopped by adding SDS sample buffer and analyzed by SDS-PAGE and WB. The phosphorylation states of ULK1 Ser and Thr in the reaction mixture were analyzed by WB. RNAs were prepared from cells using TRIzol reagent ThermoFisher Scientific, We used actin β ACTB and TATA Box Protein TBP genes as controls. Samples were run on QuantStudio 3 Real-Time PCR system ThermoFisher Scientific. Results were analyzed using Microsoft Excel, version The primers used for qPCR are listed in Supplementary Table 3. The quantified outcomes were summarized as mean and SEM as specified in the figure legends. To compare the means between different groups, the two-tailed Student t test was used with Prism 6 Version 6. All the measurements used for statistics were obtained from distinct samples that were prepared independently of each other. The sample sizes for independent experiments and animal studies were determined based on preliminary data. The western blotting experiments were performed multiple times, as illustrated in the accompanying graphs. In cases where only micrographs were presented, the experiments were independently repeated more than three times, yielding consistent results. Representative micrographs are provided. Statistical significance was interpreted for p values below 0. Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article. All the data that support the conclusions in this paper are available within this article and its supplementary Information file. Source data are provided with this paper as an excel file, which contains all the uncropped western blots, raw data, and quantified values for all the graphs presented in this paper. Lang, M. et al. Glucose starvation inhibits autophagy via vacuolar hydrolysis and induces plasma membrane internalization by down-regulating recycling. Article CAS PubMed PubMed Central Google Scholar. Ramírez-Peinado, S. Glucose-starved cells do not engage in prosurvival autophagy. Article PubMed PubMed Central Google Scholar. Weber, C. β-Oxidation and autophagy are critical energy providers during acute glucose depletion in S. Natl Acad. USA , — Article ADS CAS PubMed PubMed Central Google Scholar. Schellens, J. Hepatic autophagy and intracellular ATP. A morphometric study. Cell Res. Article CAS PubMed Google Scholar. Plomp, P. Energy dependence of autophagic protein degradation in isolated rat hepatocytes. Energy dependence of different steps in the autophagic-lysosomal pathway. Mijaljica, D. V-ATPase engagement in autophagic processes. Autophagy 7 , — Kim, J. AMPK and mTOR regulate autophagy through direct phosphorylation of Ulk1. Cell Biol. Egan, D. Phosphorylation of ULK1 hATG1 by AMP-activated protein kinase connects energy sensing to mitophagy. Science , — Article ADS CAS PubMed Google Scholar. Artal-Martinez de Narvajas, A. Epigenetic regulation of autophagy by the methyltransferase G9a. Nwadike, C. AMPK inhibits ULK1-dependent autophagosome formation and lysosomal acidification via distinct mechanisms. Shang, L. Nutrient starvation elicits an acute autophagic response mediated by Ulk1 dephosphorylation and its subsequent dissociation from AMPK. Mack, H. AMPK-dependent phosphorylation of ULK1 regulates ATG9 localization. Autophagy 8 , — Samari, H. Inhibition of hepatocytic autophagy by adenosine, aminoimidazolecarboxamide riboside, and N6-mercaptopurine riboside. Evidence for involvement of amp-activated protein kinase. Meley, D. AMP-activated protein kinase and the regulation of autophagic proteolysis. Benito-Cuesta, I. AMPK activation does not enhance autophagy in neurons in contrast to MTORC1 inhibition: different impact on β-amyloid clearance. Autophagy 17 , — Han, D. Joo, J. Hspcdc37 chaperone complex regulates ulk1- and atgmediated mitophagy. Cell 43 , — Russell, R. ULK1 induces autophagy by phosphorylating Beclin-1 and activating VPS34 lipid kinase. Small molecule inhibition of the autophagy kinase ULK1 and identification of ULK1 substrates. Cell 59 , — Park, J. The ULK1 complex mediates MTORC1 signaling to the autophagy initiation machinery via binding and phosphorylating ATG Autophagy 12 , — ULK1 phosphorylates Ser30 of BECN1 in association with ATG14 to stimulate autophagy induction. Autophagy 14 , — Wold, M. Alsaadi, R. EMBO Rep. Zhou, C. Regulation of mATG9 trafficking by Src- and ULK1-mediated phosphorylation in basal and starvation-induced autophagy. Mercer, T. EMBO J. Wu, W. ULK1 translocates to mitochondria and phosphorylates FUNDC1 to regulate mitophagy. Inoki, K. TSC2 mediates cellular energy response to control cell growth and survival. Cell , — Gwinn, D. AMPK phosphorylation of raptor mediates a metabolic checkpoint. Cell 30 , — Differential regulation of distinct Vps34 complexes by AMPK in nutrient stress and autophagy. Myers, R. Systemic pan-AMPK activator MK improves glucose homeostasis but induces cardiac hypertrophy. Bjørkøy, G. Øverbye, A. Proteomic analysis of membrane-associated proteins from rat liver autophagosomes. Autophagy 3 , — Article PubMed Google Scholar. Lelouard, H. Dendritic cell aggresome-like induced structures are dedicated areas for ubiquitination and storage of newly synthesized defective proteins. Puente, C. Nutrient-regulated phosphorylation of ATG13 inhibits starvation-induced autophagy. Kroemer, G. Autophagy and the integrated stress response. Cell 40 , — Codogno, P. Autophagy and signaling: their role in cell survival and cell death. Cell Death Differ. Shaw, R. The tumor suppressor LKB1 kinase directly activates AMP-activated kinase and regulates apoptosis in response to energy stress. Jeon, S. AMPK regulates NADPH homeostasis to promote tumour cell survival during energy stress. Nature , — Greer, E. The energy sensor AMP-activated protein kinase directly regulates the mammalian FOXO3 transcription factor. Paquette, M. AMPK-dependent phosphorylation is required for transcriptional activation of TFEB and TFE3. Shin, H. AMPK-SKP2-CARM1 signalling cascade in transcriptional regulation of autophagy. Weerasekara, V. Metabolic-stress-induced rearrangement of the ζ interactome promotes autophagy via a ULK1- and AMPK-regulated ζ interaction with phosphorylated Atg9. Zhao, Y. RACK1 promotes autophagy by enhancing the Atg14L-Beclin 1-VpsVps15 complex formation upon phosphorylation by AMPK. Cell Rep. Xu, D. PAQR3 controls autophagy by integrating AMPK signaling to enhance ATG14L-associated PI3K activity. Thomas, H. Mitochondrial complex I activity is required for maximal autophagy. e Hamasaki, M. Autophagosomes form at ER-mitochondria contact sites. Hailey, D. Mitochondria supply membranes for autophagosome biogenesis during starvation. Reggiori, F. Atg9 cycles between mitochondria and the pre-autophagosomal structure in yeasts. Autophagy 1 , — Barini, E. Metformin promotes tau aggregation and exacerbates abnormal behavior in a mouse model of tauopathy. Son, S. Metformin facilitates amyloid-β generation by β- and γ-secretases via autophagy activation. Alzheimers Dis. Imfeld, P. Moore, E. Increased risk of cognitive impairment in patients with diabetes is associated with metformin. Diabetes Care 36 , — To further assess the effect of silencing of tomato ATG5 and ATG7 on heat-induced autophagy, we used LysoTracker Green dye as a probe to detect autolysosome-like structures. The LysoTracker dye has been widely used as a probe for detecting autophagic activity in a variety of organisms including plants Otegui et al. Comparative studies with other autophagosome makers such as ATG8 have shown that although LysoTracker dyes stain acidic organelles, including autophagosomes, up-regulated LysoTracker-stained structures are biologically characteristic of induced autophagic activity Phadwal et al. Under the normal temperature 22°C , we observed low numbers of punctate green fluorescent signals in both pTRV and gene silenced plants Figure 3. After 6-h heat stress, however, the numbers of punctate green fluorescent signals increased by more than 10 fold in the control plants infiltrated with the pTRV empty vector Figure 3. Importantly, in the plants infiltrated with the pTRV- ATG5 or pTRV- ATG7 silencing vector, there was only a 2—3-fold increase in the numbers of the punctate fluorescence signals after 6-h heat stress Figure 3. Figure 3. Detection of autophagic activity using LysoTracker Green. Comparison of tomato plants infiltrated with Agrobacterium cells harboring the empty pTVR vector or a silencing vector for an indicated gene in terms of LysoTracker Green fluorescence signals. The plants were placed into the 22 and 45°C growth chambers and analyzed after 6-h treatment. Numbers of punctate LysoTracker Green fluorescence spots per 10, μm 2 section from the cells in the central areas of the terminal leaflets of the fifth leaves were indicated below the images. Means and SE were calculated from three experiments. We also used MDC as a probe for detection of autophagic activity in tomato leaves. MDC is an autofluorescent dye that stains autophagosomes in mammals and plants Biederbick et al. Under the normal temperature 22°C , again, we observed low numbers of punctate fluorescent signals in both control pTRV and ATG5 - or ATG7 -silenced plants Figure 4. After treatment with dithiothreitol DTT , a known inducer of autophagy Liu et al. After 6-h heat treatment, the numbers of punctate fluorescent signals also increased by about 6 fold in control plants infiltrated with the pTRV empty vector Figure 4. In the plants infiltrated with the pTRV- ATG5 or pTRV- ATG7 silencing vector, the numbers of the punctate fluorescence signals after DTT or heat stress were substantially reduced when compared to those in DTT- or heat-treated control plants Figure 4. These observations confirmed that heat-induced autophagy was partially blocked by silencing of the tomato ATG5 and ATG7 genes. Figure 4. MDC-stained autophagosomes in tomato leaves. A Comparison of tomato plants infiltrated with Agrobacterium cells harboring the empty pTVR vector or a silencing vector for an indicated gene in terms of MDC fluorescence signals. DTT 2 mM, 6 h treatment was included as positive control. B Numbers of punctate MDC fluorescence spots per 10, μm 2 section were indicated. For comparison of heat tolerance of pTRV, pTRV- ATG5 , pTRV- ATG7 , pTRV- NBR1a , and pTRV- NBR1b plants, they were placed in a 45°C growth chamber for 8 h and then moved to room temperature for 3-day recovery. For heat—treated pTRV control plants, only patches of old leaves displayed symptoms of dehydration while a majority of the leaves remained green and viable after recovery Figure 5A. On the other hand, a majority of fully expanded leaves from the pTRV- ATG5 , pTRV- ATG7 , pTRV- NBR1a , and pTRV- NBR1b plants exhibited wilting after the recovery Figure 5A. The more severe symptoms in autophagy-suppressed tomato plants after heat stress were confirmed by increased EL in the silenced plants relative to that in the pTRV control plants Figure 5B. Figure 5. Functional analysis of tomato ATG5, ATG7, NBR1 , and WRKY33 in tomato heat tolerance using TRV-mediated gene silencing. A Tomato plants infiltrated with Agrobacterium cells harboring the empty pTVR vector or the silencing pTRV- ATG5 , pTRV- ATG7 , pTRV- NBR1 , or pTRV- WRKY33 vector were placed in a 22 or 45°C growth chamber for 8 h. The pictures of the whole plants upper panel or the terminal leaflets of the fifth leaves lower panel were taken after 3-day recovery. B Electrolyte leakage EL of the terminal leaflets of the fifth leaves were determined immediately after 8 h at 22 or 45°C heat treatment. Means and SE were calculated from average EL values determined from three experiments with 10 leaves per experiment for each genotype. Heat has a harmful effect on various biology processes including photosynthesis. In addition, ATG5 -, ATG7 -, and NBR1 -silenced tomato plants had reduced stomatal conductance Gs and intracellular CO 2 concentration Ci compared to the unsilenced pTRV control plants after heat stress Figure 7. Thus, photosynthetic efficiency and capacity were more compromised by heat stress in the autophagy-suppressed tomato plants than in the control plants. Figure 6. The effect of heat stress on the efficiency of PSII photochemistry. The color code in the images ranged from 0 black to 1. Figure 7. The effect of heat stress on the capacity of photosynthesis. Light-saturated CO 2 assimilation rate Asat A , stomatal conductance Gs B and intracellular CO 2 concentration Ci C were determined following 1-day recovery after heat stress. Means and SE were calculated from average values determined from three experiments with 10 leaves per experiment for each type of plants. Heat stress causes protein misfolding and denaturation, which can result in formation of protein aggregates and proteotoxic stress. To analyze the role of autophagy in protection against heat-induced proteotoxic stress, we investigated the accumulation of insoluble, detergent-resistant proteins in the pTRV, pTRV- ATG5 , pTRV- ATG7 , pTRV- NBR1a , and pTRV- NBR1b plants after 8 h heat treatment. Total proteins were first isolated and insoluble proteins were separated by low speed centrifugation. As shown in Figure 8 , the percentages of insoluble to total proteins were similar in all plants when they were grown at 22°C. By the end the heat stress, the levels of insoluble proteins in the ATG5 -, ATG7 -, and NBR1 -silenced tomato plants were more than two times higher than those in the unsilenced control plants Figure 8. Figure 8. Accumulation of insoluble protein aggregates under heat stress. Leaf tissues from tomato plants infiltrated with Agrobacterium cells harboring the empty pTVR vector or the silencing pTRV- ATG5 , pTRV- ATG7 , pTRV- NBR1 , or pTRV- WRKY33 vector collected at indicated hours h under 45°C for preparation of total, soluble and insoluble proteins as described in Materials and Methods. Total proteins in the starting homogenates and insoluble proteins in the last pellets were determined the percentages of insoluble proteins to total proteins were calculated. Arabidopsis WRKY33 is a transcription factor important for plant resistance to necrotrophic fungal pathogens and for plant heat tolerance Zheng et al. Arabidopsis WRKY33 interacts with Arabidopsis ATG18a, a critical component of autophagy, and plays a positive role in pathogen-induced ATG18a expression and autophagosome formation Lai et al. To investigate whether tomato contains similar WRKY transcription factor s with a critical role in heat tolerance and regulation of stress-induced autophagy, we searched the sequenced tomato genomes and identified two close WRKY33 homologs, WRKY33a Sl09g and WRKY33b Sl06g As shown in Supplemental Figure 4 , Arabidopsis WRKY33, tomato WRKY33a and WRKY33b all belong to Group I WRKY transcription factors containing two WRKY domains with highly conserved amino acid sequences. High sequence similarities are also found in the N-terminal domains including the highly conserved SP clusters as putative MAPK phosphorylation sites and the intervening sequences between the two WRKY domains Supplemental Figure 4. Furthermore, both tomato WRKY33a and WRKY33b contain a segment of about amino acid residues on the C-terminal side of the second WRKY domain with substantial sequence homology with Arabid0psis WRKY33, which are absent in other related Group I WRKY transcription factors such as Arabidopsis WRKY25 and WRKY26 Supplemental Figure 4 Lai et al. To analyze heat-induced expression of tomato WRKY33 genes, we analyzed their transcripts in the tomato seedlings grown at 22 or 45°C. As shown in Figure 1 , the transcript levels of both tomato WRKY33a and WRKY33b remained low throughout the 8-h period of the experiments at 22°C. At 45°C, however, the transcript levels of tomato WRKY33a and WRKY33b were elevated with similar kinetics Figure 1. Transcripts levels for both genes displayed substantial increases after 4-h exposure to 45°C and peaked after 6-h heat stress Figure 1. Like those of other analyzed autophagy-related genes, the transcript levels for both WRKY33a and WRKY33b declined after 6-h heat exposure and approached those of control plants by 8-h heat exposure Figure 1. To determine directly the roles of tomato WRKY33 genes, we used VIGS technology to assess the impact of their down-regulated expression on tomato heat tolerance. Tomato WRKY33 -specific DNA fragments were cloned into the pTRV vector and Agrobacterium cells harboring the VIGS vectors were infiltrated into tomato leaves. As shown in Figure 2 , basal expression of WRKY33a or WRKY33b was observed in the tomato plants infiltrated with the pTRV empty vector. By contrast, infiltration with either pTRV- SlWRKY33a or pTRV- SlWRKY33b silencing vector resulted in approximately 5—7-fold reduction in the transcript levels for both tomato WRKY33a and WRKY33b Figure 2. The tomato plants silenced for WRKY33a and WRKY33b were normal in growth and development and displayed no detectable morphological phenotype. We analyzed the impact of silencing of WRKY33 genes on tomato heat tolerance. Both control and silenced plants were placed in a 45°C growth chamber for 8 h and then moved to room temperature for 3-day recovery. Unlike heat—treated pTRV control plants, which had only some patches of old leaves that displayed symptoms of dehydration, a majority of leaves from the pTRV- WRKY33a and pTRV- WRKY33b plants exhibited extensive wilting or even bleaching after the recovery Figure 5. Thus, silencing tomato WRKY33 genes caused increased sensitivity to heat stress. Furthermore, silencing of tomato WRKY33a and WRKY33b led to increased accumulation of insoluble proteins under heat stress Figure 8. To investigate whether the critical role of tomato WRKY33 genes in heat tolerance is associated with their positive roles in regulation of heat-induced autophagy, we analyzed whether silencing the WRKY33 genes in tomato compromised heat-induced autophagosome formation. As shown in Figure 3 , while there was a more than 10 fold increase in the numbers of punctate green fluorescent signals after 6-h heat stress in the pTRV control plants, there was only 3—5-fold increase in the pTRV- WRKY33a and pTRV- WRKY33b plants. Thus, tomato WRKY33 proteins play a positive role in heat-induced autophagosome formation. We also analyzed the mutual regulation among the silencing tomato ATG 5, ATG7 , NBR1 , and WRKY33 genes during plant responses to heat stress. For this purpose, we analyzed the transcript levels of ATG5 , ATG7 , NBR1a , NBR1b , WRKY33a , and WRKY33b in the silencing plants after 6-h heat stress. In pTRV control plants, as expected, the transcript levels for ATG5 , ATG7 , NBR1a , NBR1b , WRKY33a , and WRKY33b were elevated under heat stress. However, induction of these genes was all reduced not only in the plants harboring their respective silencing vectors but also in the plants harboring silencing vectors for the other genes Figure 9. Induction of three heat shock proteins HSP However, induction of a tomato HSP40 gene was significantly compromised by silencing of the autophagy-related or WRKY33 genes Figure These results indicated that ATG and WRKY33 proteins have a positive role in heat-induced expression of autophagy-related genes. Figure 9. Regulation of heat-induced expression of tomato ATG5 , ATG7 , NBR1 , and WRKY3 3 genes. Tomato plants infiltrated with Agrobacterium cells harboring the empty pTVR vector or the silencing pTRV- ATG5 , pTRV- ATG7 , pTRV- NBR1 , or pTRV- WRKY33 vector were placed in a 45°C growth chamber and total RNA was isolated from leaf samples collected after 6-h heat stress for determination of transcript levels of indicated genes by qRT-PCR. Figure Regulation of heat-induced expression of tomato HSP genes. In the present study, we analyzed the role of autophagy in responses to heat stress in tomato, an important horticultural crop. Both the expression of ATG5 and ATG7 genes and formation of autophagosomes were induced in heat-stressed tomato plants Figures 1 , 3 , 4. The heat tolerance of autophagy-suppressed tomato plants due to silencing of ATG5 and ATG7 genes was compromised based on their increased morphological symptoms associated with enhanced defects in the efficiency and capacity of photosynthesis after heat stress Figures 5 — 7. These results indicate that autophagy plays an important role in tomato heat tolerance. Heat stress causes protein misfolding and denaturation. Extensive studies in yeast and animal organisms have revealed that misfolded proteins are recognized by the protein quality control system, ubiquitinated by chaperone-dependent E3 ubiquitin ligases such as the C-terminus of Hscinteracting protein CHIP and subjected to degradation by the ubiquitin proteasome system UPS Kraft et al. Very recently we have conducted comprehensive genetic analysis of Arabidopsis CHIP E3 ubiquitin ligase and discovered its critical role in plant responses to a spectrum of abiotic stresses including heat stress Zhou et al. Previously, it has also been reported that Arabidopsis CHIP E3 ubiquitin ligase and Hsc mediate plastid-destination precursor degradation through UPS when the import of the precursors are blocked in a plastid-import mutant Lee et al. In the present study, we demonstrated that silencing of tomato genes encoding NBR1a and NBR1b , two close homologs of mammalian ubiquitin-binding autophagy receptors P62 and NBR1, also compromised tomato heat tolerance Figures 5 — 7. Thus, NBR1-mediated selective autophagy is critical in tomato heat tolerance most likely through its activity in removing heat-induced misfolded proteins. For degradation by UPS, proteins must be unfolded to enter the narrow central cavity of its barrel-shaped 20S proteolytic core since the steric conditions of a folded protein would not be able to pass through the entrance channel. Under heat stress, misfolded, or denatured proteins may form protein aggregates that are difficult to dissociate or unfold. These protein aggregates are likely to be targeted by NBR1-mediated selective autophagy if they fail to be processed by UPS. Consistent with this interpretation, compromised heat tolerance of NBR1-silenced tomato plants was associated with increased accumulation of protein aggregates under heat stress Figure 8. These results provide further support that UPS and NBR1-mediated selective autophagy function in cooperation in the removal of misfolded proteins for protection against proteotoxic stress under adverse environmental and physiological conditions Zhou et al. Heat stress induced both LysoTracker or MDC-stained autolysosome-like structures Figures 3 , 4 and expression of ATG and NBR1 genes Figure 1. Interestingly, silencing of an ATG or NBR1 gene in tomato plants led to down regulation of not only the silenced gene but also other sequence-unrelated autophagy genes Figure 9. Since ATG5, ATG7, and NBR1 proteins are not known to be regulators of gene transcription, their effect on the expression of other genes is probably indirect and most likely related to induced autophagy under heat stress. It is possible that induced autophagy under heat and perhaps other stress conditions as well has a positive role in the upregulation of autophagy genes. Consistent with the potential signaling role of autophagy, silencing of Arabidopsis TOR gene leads to not only constitutive formation of autophagosomes but also induced expression of some ATG genes Liu and Bassham, We have also previously observed that in autophagy-deficient mutants, induction of jasmonate-regulated PDF1. Likewise, in the ATG5 - or ATG7 -silenced tomato mutants, induction of a gene encoding a HSP40 was significantly reduced Figure 10 , indicating that induced autophagy, perhaps through formation and turnover of autophagosomes, has a positive role in up-regulation of not only autophagy genes but also other genes associated with defense and stress responses. We have previously shown that in Arabidopsis atg5 and atg7 mutants, the protein levels of NBR1 increased greatly under heat stress Zhou et al. Other studies have also showed that some of the autophagy-related proteins and NBR1 are themselves autophagy substrates Svenning et al. On the other hand, when autophagy is suppressed or blocked as in the ATG5 - and ATG7 -silenced plants, there would be no need for increased transcription of the ATG or NBR1 genes since their degradation by autophagy is inhibited. Arabidopsis WRKY33 transcription factor plays a critical role in plant resistance to necrotrophic fungal pathogens and in plant tolerance to heat stress Zheng et al. Autophagy is induced by necrotrophic pathogens, heat, and salt stresses and plays an important role in plant responses to these biotic and abiotic stresses as well. We have previously shown that in the Arabidopsis wrky33 mutants, increased formation of autophagosomes was observed in Botrytis-infected lesion areas but not in the areas surrounding the lesions found in wild-type plants Lai et al. |
| Autophagy in Cellular Stress Responses | SpringerLink | Superoxide O and H 2 O 2 are the main ROS produced by mitochondria upon nutrient deprivation. In a redox-independent manner, it has also been demonstrated that p62, when bound to ubiquitylated protein aggregates, can undergo phosphorylation on Ser, thereby sequestering Keap1 and leading to its detachment from Nrf2 bottom left. Consequently, Nrf2 is no longer degraded by the ubiquitin-3 proteasome system, but translocates in the nucleus, binds to antioxidant-responsive elements AREs located in the promoter regions of antioxidant genes and activates their transcription bottom right. Notwithstanding the large amount of data supporting the hypothesis of a redox regulation of autophagic signalling, so far the only redox-based mechanism demonstrated to be able to regulate an autophagic protein goes back to , when Scherz-Shouval et al. Results emerging in the past 5 years suggest that NO, by means of S -nitrosylation mechanisms, has also a role in modulating autophagy. However, rather than a positive effector of the process, it seems that it could act as an inhibitory molecule. This assumption is completely in contrast with that described above for ROS, and contributes to making the functional relationship between oxidative stress and autophagy even more complex. Sarkar et al. This is in line with results reporting that S -nitrosylation of TSC2 prevents its inhibitory activity on mTOR, 80 thereby preventing autophagy and inducing proliferation of melanoma cells. However, these data are in contrast with the well-documented role of NO and nitrosative stress in the activation of AMPK—TSC2 pathway via Ataxia telangiectasia mutated ATM in response to DNA damage see below. Autophagy is not affected by S -nitrosylation. a Western blot analyses of S -nitrosothiols in total homogenates of skeletal muscles obtained from GSNOR-KO KO and wild-type WT mice, subjected to biotin-switch assay and revealed by HRP-conjugated streptavidin. Lactate dehydrogenase LDH was selected as loading control. Results show that even in the absence of any treatment with NO-delivering drugs, GSNOR ablation induces a significant increase of S -nitrosylated proteins. b Representative fluorescence microscopy images of satellite cell-derived myotubes isolated from KO and WT mice expressing LC3-conjugated green fluorescent protein GFP-LC3 in heterozygosis. Images are representative of three independent experiments that gave similar results. Both genotypes displayed a significant and similar increase of fluorescent dots, plausibly representing autophagosomes, thereby indicating that autophagy is not impaired by S -nitrosylation. Altogether, these observations indicate that a straightforward idea about how oxidative stress functions in autophagy is still lacking, despite abundant evidence corroborating its implication in each phase of this process. The underlying mechanism is completely redox independent, and involves the recruitment of Kelch-like ECH- associated protein 1 Keap1 that functions as an adapter protein of the Cul3-ubiquitin E3 ligase complex responsible for degrading Nrf2. Here, Nrf2 binds to the antioxidant-responsive elements ARE in the promoter regions of antioxidant and detoxifying genes, 86 as well as genes involved in DNA damage response, such as 8-oxoguanine glycosylase OGG1 87 and p53 binding protein 1 53BP1 , 88 inducing their transcription Figure 2. On the basis of what has been reported so far, antioxidant response and autophagy are mechanisms simultaneously induced by oxidative stress conditions in order to concomitantly decrease ROS and RNS concentration upstream causes and reduce the oxidative damage to biomolecules and organelles downstream effect. This finely orchestrated repair system perfectly fits the needs of a cell attempting to find a new homeostatic state. By responding very rapidly to oxidative stress, and by decreasing the toxicity of oxidized molecules and organelles through their selective removal, autophagy can be in principle encompassed in the large family of antioxidant processes. However, at variance with proteins and organelles that are present in several copies inside the cell e. Genomic DNA cannot be destroyed, de novo synthesised or entirely replaced like the other biomolecules. Its integrity should be prevented and maintained, and any damage accurately repaired. An exception to the rule is a highly selective and unusual nuclear DNA degradation by means of the so-called piecemeal microautophagy PMN. Indeed, in a way resembling nucleophagy occurring in fungi and nematodes, 92 , 93 mammalian cells can specifically remove part of nuclei containing damaged DNA. It has been reported that micronuclei containing chromosomes, or parts of them, that are not properly incorporated in the daughter nuclei during cell division can be removed by autophagy as well, 95 thus providing this process with a direct role in cleaning up damaged content in the nucleus and in maintaining genomic stability. However, besides PMN, a number of observations indicate that autophagy is deeply involved in DNA damage repair, although without any direct degradative activity on DNA. This phenomenon, mainly occurring upon ROS and RNS-mediated damage to DNA, represents an issue that deserves discussion so as to comprehend how autophagy acts as a preventive and reparative process upon genotoxic stress. Understanding the underlying molecular mediators and the mechanisms would make it possible to clarify once and for all the antioxidant activity of autophagy. ROS and RNS are one of the major sources of DNA damage 96 as they could directly modify the DNA or indirectly generate different lesions, both affecting cell viability. Among ROS, ·OH can directly attack the DNA backbone by generating five classes of oxidative damage: oxidized bases, abasic sites, DNA—DNA intrastrand adducts, single-strand break SSB , double-strand break DSB and DNA—protein crosslinks. Both are highly mutagenic and carcinogenic as they can match with both cytosine and adenine, thus leading to GC-to-AT transversions. Nitric oxide and RNS i. ROS and RNS are also harmful for mitochondrial DNA mtDNA integrity. This feature can deeply affect the transcription of mtDNA-coded proteins and RNAs that underlie the synthesis of a number of subunits belonging to the complexes of the mitochondrial respiratory chain except Complex II. A vicious cycle is then established in which mitochondria, with oxidized mtDNA, become dysfunctional and produce a high rate of ROS, leading to further mitochondrial impairment. This condition can ultimately result in severe nuclear DNA damage and cell death. When the DNA is damaged by ROS and RNS, cells activate a number of pathways in order to maintain genomic integrity, these being associated to the DNA damage response DDR. Different classes of proteins are implicated in DDR, among which the sensors specifically recognize the lesions to DNA, whereas the mediators and the effectors transduce the signal from the nucleus to the cytosol where several processes are contextually activated in order to better face up to adverse conditions. However, if DNA is severely damaged or unrepaired, cells remain quiescent or undergo cell death. Therefore, once induced by DNA injury, it makes a crucial contribution in regulating cell fate. It is worthwhile noting that cases where autophagy impairment results in DNA damage have been reported as well, leading to the assumption that the interplay might be broader, and suggesting that many molecular players could exist to biunivocally link the two processes. In particular, it has been demonstrated that the deficiency of autophagy genes, such as Beclin 1, UV irradiation resistance-associated gene UVRAG , Atg5 and Atg7, leads to DNA damage accumulation and promotes tumourigenesis. All this evidence strongly suggests that autophagy participates, directly or indirectly, in the DDR to ROS and RNS-mediated genotoxic stress. However, how this occurs is still a matter of debate. In higher eukaryotes, no Cvt has ever been identified, nor has any orthologue of proteins belonging to this pathway been revealed. One of the most accredited hypotheses explaining a role of autophagy in supporting the DDR is that by degrading damaged mitochondria mitophagy and toxic aggregates, autophagy eliminates putative sources of ROS, reduces their levels and, if only indirectly, decreases DNA damage accumulation. A number of works in recent years indicate that once ROS and RNS damage the DNA, the event is transduced in order to activate the DDR, and concomitantly is signalled to the autophagic pathway in order to orchestrate the response. PolyADP-ribose polymerase 1 PARP1 is among the proteins directly linking the DDR and autophagy Figure 4. Such energetic imbalance results in the activation of autophagy via AMPK pathway Figure 4 , in order to recycle metabolic precursors for ATP and to provide energy for the DDR. Implication of autophagy in DNA damage repair. Endogenous e. Upon DNA damage, ATM can activate pmediated transcription of autophagic genes bottom right. Alternatively, cytosolic pool of ATM could be directly activated by ROS through a still unidentified mechanism and it directly induces the activation of LKB1 centre. The issue of whether cytosolic and nuclear pool of ATM are interconnected still waits to be demonstrated. Both PARP1 and ATM signalling pathways converge on AMPK, whose activation induces the autophagic machinery to remove the main source of DNA damage and contribute to its repair through a negative feedback loop top. Another DNA repair protein linking the DDR to autophagy is ATM Figure 4 , a DNA damage sensor orchestrating the cell cycle with damage response checkpoints and DNA repair to safeguard the integrity of the DNA. From this perspective, ATM would be required to both initiate nucleus and mediate cytosol the DDR. The principal regulator of DDR, however, remains p53 that, together with the other members of its family, p63 and p73, has been demonstrated to modulate many autophagic genes Figure 4. For instance, in case of inefficient repair, p53 can shift transcription from cell cycle modulators e. Targets of p53 are, indeed, both upstream regulators of autophagy e. PTEN, TSC2, β 1, β 2 and γ subunits of AMPK and proteins directly involved in autophagosome formation e. The dual role of p53 and prelated members, as well as of many other proteins that have been demonstrated having a role in regulating both autophagy and apoptosis, is an issue that deserves to be deeply investigated in the future. Indeed, several lines of evidence clearly indicate that the molecular pathways — from the upstream stimuli e. Which are the signals, or how much high should be the threshold level to allow cell response being switched from stress adaptation and survival autophagy to cell dismantling apoptosis? These issues are still debated. Several lines of evidence indicate that ROS and RNS are the upstream modulators of autophagy, likely acting at multiple levels in the process. Through a negative feedback regulation, autophagy could be then induced to provide energy and building blocks in order to restore homeostasis and, concomitantly, remove oxidative damage. From this perspective, autophagy is required for the cell to simultaneously overcome starvation and oxidative stress conditions. Accordingly, genetic defects of autophagic genes lead to an increased production of ROS and accumulation of damaged organelles and DNA that in turn promote metabolic reprogramming and induce tumourigenesis. Although a number of possible mechanisms underlying the intimate interplay between oxidative stress and autophagy have been postulated, to date only a few of them have been shown to have a role in tuning autophagy. Understanding the fine molecular regulation of autophagy by ROS and RNS, as well as the tight relationship between metabolism and redox state, could therefore provide valuable information that could be useful in the future to improve anticancer treatment and develop new selective therapies. Baudhuin P, Berleur AN, De Duve C, Wattiaux R. Tissue fractionation studies. Cellular localization of bound enzymes. Biochem J ; 63 : — Article CAS PubMed PubMed Central Google Scholar. De Duve C, Gianetto R, Appelmans F, Wattiaux R. Enzymic content of the mitochondria fraction. Nature ; : — Article CAS PubMed Google Scholar. De Duve C, Berthet J. Reproducibility of differential centrifugation experiments in tissue fractionation. Nature ; : De Duve C, Wattiaux R. Functions of lysosomes. Annu Rev Physiol ; 28 : — De Duve C, Baudhuin P. Peroxisomes microbodies and related particles. Physiol Rev ; 46 : — Mann PJ, Quastel JH. Toxic effects of oxygen and of hydrogen peroxide on brain metabolism. Biochem J ; 40 : — Davies KJ. Protein damage and degradation by oxygen radicals. general aspects. J Biol Chem ; : — CAS PubMed Google Scholar. Halliwell B. Biochemical mechanisms accounting for the toxic action of oxygen on living organisms: the key role of superoxide dismutase. Cell Biol Int Rep ; 2 : — Imlay JA, Linn S. DNA damage and oxygen radical toxicity. Science ; : — Harman D. Aging: a theory based on free radical and radiation chemistry. J Gerontol ; 11 : — Kroemer G, Marino G, Levine B. Autophagy and the integrated stress response. Mol Cell ; 40 : — Yang Z, Klionsky DJ. Mammalian autophagy: core molecular machinery and signaling regulation. Curr Opin Cell Biol ; 22 : — Neufeld TP. Autophagy and cell growth—the yin and yang of nutrient responses. J Cell Sci ; : — Zoncu R, Efeyan A, Sabatini DM. mTOR: from growth signal integration to cancer, diabetes and ageing. Nat Rev Mol Cell Biol ; 12 : 21— Nazio F, Cecconi F. mTOR AMBRA1, and autophagy: an intricate relationship. Cell Cycle ; 12 : — Fimia GM, Di Bartolomeo S, Piacentini M, Cecconi F. Unleashing the Ambra1-Beclin 1 complex from dynein chains: Ulk1 sets Ambra1 free to induce autophagy. Autophagy ; 7 : — Article PubMed Google Scholar. Nicklin P, Bergman P, Zhang B, Triantafellow E, Wang H, Nyfeler B et al. Bidirectional transport of amino acids regulates mTOR and autophagy. Cell ; : — Cohen A, Hall MN. An amino acid shuffle activates mTORC1. Furst P, Stehle P. What are the essential elements needed for the determination of amino acid requirements in humans? J Nutr ; : S—S. Sancak Y, Peterson TR, Shaul YD, Lindquist RA, Thoreen CC, Bar-Peled L et al. The Rag GTPases bind raptor and mediate amino acid signaling to mTORC1. Kim E, Goraksha-Hicks P, Li L, Neufeld TP, Guan KL. Regulation of TORC1 by Rag GTPases in nutrient response. Nat Cell Biol ; 10 : — Long X, Lin Y, Ortiz-Vega S, Yonezawa K, Avruch J. Rheb binds and regulates the mTOR kinase. Curr Biol ; 15 : — Shimobayashi M, Hall MN. Making new contacts: the mTOR network in metabolism and signalling crosstalk. Nat Rev Mol Cell Biol ; 15 : — Zoncu R, Bar-Peled L, Efeyan A, Wang S, Sancak Y, Sabatini DM. Sancak Y, Bar-Peled L, Zoncu R, Markhard AL, Nada S, Sabatini DM. Ragulator-Rag complex targets mTORC1 to the lysosomal surface and is necessary for its activation by amino acids. Duran RV, Oppliger W, Robitaille AM, Heiserich L, Skendaj R, Gottlieb E et al. Glutaminolysis activates Rag-mTORC1 signaling. Mol Cell ; 47 : — Duran RV, MacKenzie ED, Boulahbel H, Frezza C, Heiserich L, Tardito S et al. HIF-independent role of prolyl hydroxylases in the cellular response to amino acids. Oncogene ; 32 : — Hardie DG. AMP-activated protein kinase: an energy sensor that regulates all aspects of cell function. Genes Dev ; 25 : — Oakhill JS, Steel R, Chen ZP, Scott JW, Ling N, Tam S et al. AMPK is a direct adenylate charge-regulated protein kinase. Cardaci S, Filomeni G, Ciriolo MR. Redox implications of AMPK-mediated signal transduction beyond energetic clues. Inoki K, Ouyang H, Zhu T, Lindvall C, Wang Y, Zhang X et al. TSC2 integrates Wnt and energy signals via a coordinated phosphorylation by AMPK and GSK3 to regulate cell growth. Gwinn DM, Shackelford DB, Egan DF, Mihaylova MM, Mery A, Vasquez DS et al. AMPK phosphorylation of raptor mediates a metabolic checkpoint. Mol Cell ; 30 : — Kim J, Kundu M, Viollet B, Guan KL. AMPK and mTOR regulate autophagy through direct phosphorylation of Ulk1. Nat Cell Biol ; 13 : — Roberts DJ, Tan-Sah VP, Ding EY, Smith JM, Miyamoto S. Hexokinase-II positively regulates glucose starvation-induced autophagy through TORC1 inhibition. Mol Cell ; 53 : — da-Silva WS, Gomez-Puyou A, de Gomez-Puyou MT, Moreno-Sanchez R, De Felice FG, de Meis L et al. Mitochondrial bound hexokinase activity as a preventive antioxidant defense: steady-state ADP formation as a regulatory mechanism of membrane potential and reactive oxygen species generation in mitochondria. Sun L, Shukair S, Naik TJ, Moazed F, Ardehali H. Glucose phosphorylation and mitochondrial binding are required for the protective effects of hexokinases I and II. Mol Cell Biol ; 28 : — Wu R, Wyatt E, Chawla K, Tran M, Ghanefar M, Laakso M et al. Hexokinase II knockdown results in exaggerated cardiac hypertrophy via increased ROS production. EMBO Mol Med ; 4 : — Chance B, Sies H, Boveris A. Hydroperoxide metabolism in mammalian organs. Physiol Rev ; 59 : — Wagner BA, Venkataraman S, Buettner GR. The rate of oxygen utilization by cells. Free Radic Biol Med ; 51 : — Filomeni G, Rotilio G, Ciriolo MR. Disulfide relays and phosphorylative cascades: partners in redox-mediated signaling pathways. Cell Death Differ ; 12 : — Cell signalling and the glutathione redox system. Biochem Pharmacol ; 64 : — Flohe L. Changing paradigms in thiology from antioxidant defense toward redox regulation. Methods Enzymol ; : 1— Bindoli A, Fukuto JM, Forman HJ. Thiol chemistry in peroxidase catalysis and redox signaling. Antioxid Redox Signal ; 10 : — Beckman JS, Koppenol WH. Nitric oxide, superoxide, and peroxynitrite: the good, the bad, and ugly. Am J Physiol ; : C—C Di Giacomo G, Rizza S, Montagna C, Filomeni G. Established principles and emerging concepts on the interplay between mitochondrial physiology and S- De nitrosylation: implications in cancer and neurodegeneration. Int J Cell Biol ; : Stamler JS, Singel DJ, Loscalzo J. Biochemistry of nitric oxide and its redox-activated forms. Allen BW, Demchenko IT, Piantadosi CA. Two faces of nitric oxide: implications for cellular mechanisms of oxygen toxicity. J Appl Physiol ; : — Article CAS Google Scholar. Jones DP. Radical-free biology of oxidative stress. Am J Physiol Cell Physiol ; : C—C Holmstrom KM, Finkel T. Cellular mechanisms and physiological consequences of redox-dependent signalling. Filomeni G, Desideri E, Cardaci S, Rotilio G, Ciriolo MR. Under the ROS. thiol network is the principal suspect for autophagy commitment. Autophagy ; 6 : — Chen Y, Azad MB, Gibson SB. Superoxide is the major reactive oxygen species regulating autophagy. Cell Death Differ ; 16 : — Scherz-Shouval R, Shvets E, Elazar Z. Oxidation as a post-translational modification that regulates autophagy. Autophagy ; 3 : — Zhang C, Yang L, Wang XB, Wang JS, Geng YD, Yang CS et al. Calyxin Y induces hydrogen peroxide-dependent autophagy and apoptosis via JNK activation in human non-small cell lung cancer NCI-H cells. Cancer Lett ; : 51— Levonen AL, Hill BG, Kansanen E, Zhang J, Darley-Usmar VM. Redox regulation of antioxidants, autophagy, and the response to stress: Implications for electrophile therapeutics. Free Radic Biol Med ; 71C : — Huang J, Canadien V, Lam GY, Steinberg BE, Dinauer MC, Magalhaes MA et al. Activation of antibacterial autophagy by NADPH oxidases. Proc Natl Acad Sci USA ; : — Article PubMed PubMed Central Google Scholar. Murphy MP. How mitochondria produce reactive oxygen species. Here, I highlight the emerging role of nucleolar factors in the regulation of autophagy. Moreover, I discuss the nucleolar stress response as a novel signaling pathway in the context of autophagy, health and disease. Various high quality reviews are available on principles of ribosome biogenesis, nucleolar stress, apoptosis and autophagy, respectively. Given their essential role, it is well accepted that a mis-regulation of each is tightly linked to pathogenic conditions Levine and Kroemer, ; Boulon et al. In this review, the emerging connection of nucleolar stress to autophagic processes serves as a basis to discuss novel concepts and cure of diseases connected to nucleolar stress. Nucleoli represent membrane-free, sub-nuclear compartments, where transcription and processing of rRNA takes place. Nucleoli can be considered as an assembly platform. They host several hundreds of essential rRNA binding and processing factors, which are involved in the highly complex process of ribosome biogenesis. Nucleoli form around repetitive rDNA clusters in a dynamic and cell cycle-dependent manner during G1 phase Potmesil and Goldfeder, ; Mangan et al. The rDNA clusters are transcribed into their respective large precursor rRNA by RNA polymerase I RNA pol I ; RNA polymerases II and III are as well essential for ribosome biogenesis, by driving the expression of ribosomal proteins RNA pol II and 5S rRNA RNA pol III Eichler and Craig, ; Fatica and Tollervey, The complex mechanism of pre-rRNA processing involves the action of a multitude of ribosome biogenesis factors. These are assembled in pre-ribosomal complexes involved in cleavage and chemical modification of the maturating transcript Fatica and Tollervey, ; Granneman and Baserga, ; Mullineux and Lafontaine, The nucleolar size correlates with the rRNA transcription, cell growth and the metabolic rate of a cell Boulon et al. Importantly, nucleolar size and function is changed during aging Tiku et al. Thus, the nucleolus emerges as critical regulator of cellular aging Tiku and Antebi, Large amounts of ribosomes are especially needed in highly proliferating cells, such as during embryonic development or cancer Montanaro et al. Therefore, a lack of functional ribosomes impairs cellular growth and survival and is incompatible with life. Nucleoli are highly dynamic structures, closely connected to growth and survival Mangan et al. The nucleolus is being recognized as a key hub in the cellular stress response by sensing and reacting to various stimuli. A key mechanism involves the release of ribosomal proteins RPs from the nucleoli into the nucleoplasm. As a consequence of nucleoplasmic RP accumulation, the E3-ubiquitin ligase MDM2 is inhibited Dai et al. MDM2 keeps the levels of the tumor suppressor protein p53 low by earmarking p53 for proteasomal degradation. Upon nucleolar stress, RPs are released and inhibit MDM2, which results in p53 accumulation. The nucleolar stress response is further connected to the induction of senescence and DNA damage, by commonly engaging the classical p53 pathway Rubbi and Milner, ; Lindstrom et al. A simplified model of the classical p53 nucleolar stress response is given in Figure 1. Figure 1. The classical pdependent nucleolar stress response pathway. Nucleolar stress is caused by e. As a consequence, RPs are released and bind and inhibit the E3-ubiquitin ligase MDM2. In turn, p53 is no longer degraded in the proteasome and is stabilized. Given the p53 accumulation, pmediated effects are propagated, such as cell cycle arrest, senescence, apoptosis or genotoxic stress. Note that also pindependent routes exist, which are not indicated in this scheme. More recently, novel pathways have been added to the increasing list, which demonstrate that nucleolar stress can also be propagated in the absence of functional p53 Holmberg Olausson et al. In summary, nucleolar integrity reflects a general prerequisite for normal cellular function. Given that many tumor types are characterized by inactivation of p53, pindependent pathways open novel avenues toward more customized anti-cancer therapies Burger and Eick, Macro autophagy is essential for cellular homeostasis by mediating destruction and recycling of bulk cytoplasmic material, defective organelles or proteins via lysosomal degradation Mizushima, ; Marx, A mis-regulation of autophagy is tightly linked to the formation of diverse pathological conditions Levine and Kroemer, ; Jiang and Mizushima, ; Schneider and Cuervo, Autophagy can be induced by various cellular stresses, such as lack of nutrients, low energy or oxidative stress. A central structure implicated in the process of macro autophagy is the double-membranous autophagosome, which mediates cellular cargo sequestration. Autophagy-related proteins ATGs govern autophagosome formation at different levels. Beclin1, the mammalian homologue of yeast Atg6, is mandatory for the initial steps of autophagosome formation Pattingre et al. Originally, Beclin1 has been identified as an interaction partner of the anti-apoptotic factor Bcl-2 B cell lymphoma 2 Liang et al. Beclin1 is part of phosphoinoside 3 kinase PI3K complexes and functions in diverse membrane trafficking processes Levine et al. Certain ATGs are necessary for the engulfment of cargo destined for lysosomal degradation in the autolysosome. Members of the Atg8 protein family are directly conjugated to the autophagosomal membrane by phosphaditylinositol lipidation. They can be sub-divided into LC3 and GABARAP proteins and fulfill critical tasks Nguyen et al. Opposing mechanisms result in the accumulation of autophagosomes, thereby requiring careful interpretation of experimental results. An increased rate of autophagosome formation increase of autophagic flux , as well as decreased autophagosome clearance in the lysosome impaired autophagic flux , resembles autophagosome accumulation under basal conditions. Meanwhile, several excellent reviews are available, which help to unravel these issues Mizushima and Yoshimori, ; Mizushima et al. So-called autophagic flux studies have become detrimental for understanding mechanisms of autophagy. Experts agree on combining various independent methods to allow solid data interpretation Klionsky et al. In general, autophagy is noticed as a protective mechanism by lowering the cellular stress. Apoptosis and autophagy are both stimulated by similar stressors. However, they can be seen as opposing signaling events. Whereas autophagy acts in an anti-apoptotic manner and precedes apoptosis Boya et al. However, this can even generate pro-apoptotic fragments of ATG autophagy regulators thereby triggering a fast forward response Marino et al. Therefore, obviously a tight crosstalk exists, which goes into both directions, depending on the context. As a proof of principle, low sub-lethal levels of stress favor autophagy induction as a protective mechanism, whereas sustained stress beyond a certain threshold induces apoptosis. For instance, over-activation of autophagy can be a pro-death signal for autophagic cell death, and autophagy inducers can trigger apoptosis Marino et al. Likewise, many stimuli that activate apoptosis can also stimulate autophagy. Extrinsic stress factors include chemotherapeutics, ionizing irradiation, lack of growth factors or nutrients. Intrinsically, p53 see below , oncogenes e. For instance, the pro-apoptotic Beclin1 and anti-apoptotic Bcl-2 are commonly affected. Both interact with each other and thereby regulate the balance between autophagy or apoptosis. Also, mitochondrial integrity, caspase and ATG activation, mTOR signaling and multiple others are implicated Marino et al. Collectively, a mis-regulation of autophagy in either direction is connected to numerous pathophysiological conditions, and the same holds true for apoptosis Maiuri et al. Specific cellular cargo can be selectively targeted by autophagy Kirkin et al. The pathways have been named according to their type of cargo, for instance mitophagy for specialized autophagy of mitochondria Ding and Yin, ; Hamacher-Brady and Brady, ; Khaminets et al. Parkin functions as an E3-ubiquitin ligase, which is recruited to impaired mitochondria Narendra et al. Parkin is required for ligation of ubiquitin marks to defective mitochondrial cargo Ding and Yin, ; Harper et al. Parkin depends on the proper function of PINK1 Vives-Bauza et al. Mitophagy and apoptosis are both characterized by similar upstream events Mukhopadhyay et al. Induction of mitophagy, for instance, is accompanied by activation of Bclassociated X protein BAX. This induces MOM perforation MOMP , depolarization and release of cytochrome c from the mitochondrial intermembrane space IMS into the cytosol. As a consequence, PINK1 becomes stabilized at depolarized mitochondria and Parkin is subsequently translocated from the cytosol into the MOM Vives-Bauza et al. By concerted action of both, mitochondria become decorated by ubiquitin marks Lazarou et al. In fact, autophagosome formation is mediated by different key mitophagy receptors such as optineurin and NPD52, which promote the recruitment of the autophagy initiating kinase ULK1 Wong and Holzbaur, ; Heo et al. Nguyen et al. Meanwhile, the number of novel players involved in the complex process of selective autophagy is constantly expanding. Note that accumulation of damaged mitochondria, which are eliminated by mitophagy to a certain point, sets the threshold for apoptosis as a point of no return Marino et al. As a result, p62 recruits autophagosomal membranes to its selective, autophagosomal cargo Lamark et al. The same principle of autophagy receptor e. A schematic for aggrephagy, bulk autophagy and mitophagy is depicted in Figures 2 , 3. Figure 2. Simplified model of cargo targeted by bulk autophagy or mitophagy. The phagophore forms around bulk material, such as proteins and organelles during bulk macro autophagy. The phagophore is a double-membranous structure, which forms around the cargo and gives rise to the autophagosome. Figure 3. Removal of protein aggregates by selective autophagy. Aggregated proteins red are bound by the autophagy receptor p62 blue , which itself has interaction domains for autophagosomal LC3 green, lipidated LC3-II and ubiquitin yellow. The cargo is engulfed by the mature autophagosome and subsequently fuses with the lysosome to form the autolysosome, in which the cellular material is degraded by acidic hydrolases orange. The mammalian target of rapamycin mTOR pathway couples the intake of to nutrients, growth factors, energy and stress to the regulation of cell metabolism, growth, survival and autophagy Pattingre et al. Deregulation is linked to various diseases and cancer formation. Mammalian target of rapamycin signaling is recognized as essential pathway for proper neuronal development, neuronal survival and morphogenesis. Consequently, changes in mTOR signaling have been correlated with a spectrum of neuropathologies, such as epilepsy, intellectual disability, autism, brain injury, brain tumor formation and neurodegeneration Crino, ; Switon et al. Likewise, mTOR inhibitors such as the bacterial macrolide rapamycin and its analogs are growingly used as therapeutic drugs and tested in clinical trials for effects in diverse neuropathological conditions Laplante and Sabatini, ; Crino, Mammalian target of rapamycin is a conserved serine-threonine kinase, which belongs to the phosphoinoside 3 kinase PI3K family. It assembles two large protein complexes, mTORC1 and mTORC2. The mTORC1 complex is considered as rapamycin sensitive complex Pattingre et al. Rapamycin binds to FKBP12 and mTOR, thereby inhibiting mTORC1. mTORC1 signaling affects cell growth, metabolism and autophagy: Upon favorable conditions, mTOR is activated to allow cell growth by anabolic processes, such as rRNA biogenesis and protein translation. Upon nutrient deprivation and lack of growth factors, mTOR signaling is inhibited and cell growth is suppressed, whereas catabolic processes such as autophagy are induced to allow cell survival under unfavorable conditions. mTORC1 controls autophagy by regulating ULK1, ATG13 and FIP, as well as by a reported rapamycin-insensitive mechanism Laplante and Sabatini, mTORC1 also regulates mitochondrial metabolism and biogenesis: mTORC1 inhibition impairs the MOM potential, reduces oxygen consumption and ATP levels. mTORC1 inhibition further decreases mitochondrial DNA levels and hampers mitochondrial biogenesis by affecting the transcriptional activity of the nuclear factor PGC1α PPARγ co-activator 1 Cunningham et al. Also p53 can regulate mTOR: DNA damage-induced p53 stabilization activates AMPK, which is a sensor of energy status and in turn results in mTORC1 inhibition. p53 also negatively controls mTORC1 by increasing PTEN expression, which functions as mTORC1 inhibitor. Inhibition of mTOR signaling diminishes nucleolar size and function and promotes longevity in different model organisms Tiku and Antebi, However, the precise mechanisms regulating the crosstalk between ribosome biogenesis and autophagy remain to be determined. A simplified model of mTORC1 signaling and the role of p53 is given in Figure 4. Figure 4. A simplified model of mTOR signaling, and effect of nucleolar stress on p Growth factors, energy status, amino acid availability, oxygen levels and genotoxic stress can result in mTORC1 activation. mTORC1 further activates autophagy by inhibitory effects on the ULK1 complex, composed of ULK1, ATG13 and FIP mTORC1 promotes protein synthesis by i S6K activation, which stimulates phosphorylation of S6 and thereby ribosome biogenesis, as well as by ii inhibitory effects on 4E-BP1 and eIF-4E. As a consequence, translation is activated. Furthermore, mTORC1 influences mitochondrial biogenesis and metabolism. Defective ribosome biogenesis on the one hand and impaired autophagy on the other hand are largely contributing to several diseases. In the following, an overview is provided on common concepts of three key classes of diseases, classically or recently connected to nucleolar stress and autophagy with specific focus on neurodegeneration, cancer and ribosomopathies. For a more detailed overview see for instance Parlato and Kreiner, ; Ghavami et al. The nervous system is vulnerable to intrinsic and extrinsic factors, which can give rise to distinct neurodevelopmental pathologies such as microcephaly, psychiatric disorders, autism, intellectual disability, epilepsy and neurodegeneration please, be referred to review Hetman and Slomnicki, Causes include, for instance, gene mutations, infections or neurotoxins. As common concepts, gene expression, quality control mechanisms, cell proliferation, differentiation and apoptosis are mis-regulated. Apoptosis might give rise to microcephaly by eliminating, e. Likewise Zika virus infection, as an extrinsic factor for neurodevelopmental disorders, is tightly coupled to microcephaly. It has recently been demonstrated that it decreases mTOR signaling and over-activates autophagy Liang et al. At the same time, ribosome biogenesis defects are emerging reviewed in Hetman and Slomnicki, Given the striking role of the nucleolus in coordinating mentioned neuropathological routes, deregulation of ribosome biogenesis rises as a potent upstream mechanism. In addition, also autophagy is activated in this context. Aging represents a general risk factor for the formation of neurodegenerative diseases and consequently, neurodegeneration accumulates within our society. Despite the rapid advances made in medicine, not all negative aspects of aging can simultaneously be addressed. Our scientific knowledge on distinct neurodegenerative diseases has uncovered several common mechanisms, among them loss of neurons Parlato and Kreiner, ; Parlato and Liss, and a prominent contribution of aggregate accumulation, induction of apoptosis and a mis-regulation of autophagy Yamamoto and Simonsen, ; Ghavami et al. In line, aging functions as susceptibility factor for neurodegeneration. It is characterized by a loss of rDNA and is accompanied by reduction of nucleolar size and a decline in rRNA processing Garcia Moreno et al. Therefore, the nucleolus is tightly connected to lifespan regulation Tiku and Antebi, As both routes of apoptosis and autophagy are interwoven and not yet fully understood, mechanistic research is essential as a basis for development of therapeutic approaches. As a mandatory goal, novel drugs have to be tested for specificity and efficacy. As a result, mis-folded proteins accumulate and cause a toxic intracellular environment. Normally, proper cellular homeostasis is maintained by several machinery: the ubiquitin-proteasome system UPS degrades proteins, whereas autophagy is capable of removing proteins and whole organelles by the lysosome. Hence, both routes are essential for a healthy cell and both have been implicated in the development of neurodegenerative diseases Levine and Kroemer, ; Ghavami et al. Note that increased apoptosis induction plays a crucial role in eliminating these damaged cells. In addition, chronic inflammation and oxidative stress are observed in neurodegenerative disorders. Several neurodegenerative disorders, such as PD, AD, and HD are characterized by accumulation of mis-folded, ubiquitinated proteins, which damage the affected cell Yamamoto and Simonsen, The accumulating proteins form inclusion bodies, which can differ between the distinct pathologies. With increasing age the autophagic program looses efficiency, thereby increasing the likelihood of aggregate accumulation. Neurons are highly sensitive to accumulation of protein aggregates and require proper autophagy mechanisms to keep the intracellular toxicity low. Supporting data demonstrating the significant implication of the autophagic machinery were, for instance, obtained in mice lacking ATG7 in the central nervous system. ATG-deficient mice, which fail to perform autophagy, display an accumulation of inclusion bodies followed by neuronal loss Komatsu et al. However, autophagy might have dual functions with respect to neurodegeneration: On the one hand functional autophagy is neuroprotective, by removing defective mitochondria via mitophagy. On the other hand pro-death autophagy is considered to induce neuronal cell death. Massive inhibition of autophagy can trigger apoptosis, which is observed by loss of neurons in neurodegeneration. Interfering with autophagy regulators and blocking autophagy, results in accumulation of cargo-filled autophagosomes and lysosomes, again being toxic for the cell. As a consequence, lysosome-mediated cell death occurs Kroemer and Jaattela, Impairment of mitophagy causes accumulation of defective mitochondria, which in turn induces reactive oxygen species ROS formation and mitochondrial apoptosis Seo et al. Induction of apoptosis by the chemotherapeutic agent staurosporine is accompanied by mitophagy and autophagy induction in dopaminergic cells. Additional block of autophagy by Bafilomycin or inhibition of mitophagy in PINK null mice sensitizes cells to staurosporine-induced apoptosis. Autophagy and mitophagy seems to be neuroprotective upon staurosporine-mediated apoptosis induction in dopaminergic neurons Ha et al. However, with respect to loss of dopaminergic neurons, it is not fully resolved whether autophagy is beneficial or pathogenic. Keeping mitochondria healthy is a prerequisite for counteracting neurodegenerative diseases. Concepts include for instance maintaining mitochondrial membrane integrity and functionality. Mitochondrial membrane permeabilization is tightly coupled to apoptosis induction Kroemer et al. Also anti-oxidants and ROS scavenging appear as beneficial strategies. Inhibitors of apoptosis are used as therapeutic drugs to inhibit neuronal loss. Anti-apoptotic drugs prevent mitochondrial apoptosis by blocking release of cytochrome c from the mitochondria or activation of pro-apoptotic BAX Westphal et al. Alternatively, the activity or abundance of anti-apoptotic factors can be elevated. Mostly, AD is diagnosed as sporadic form by the age of 65 years and represents the primary cause of dementia within the elderly generation Seshadri et al. A decline in autophagy during aging further promotes the mitochondrial release of cytochrome c, which serves as pro-apoptotic stimulus. Also decreased nucleolar volume has been detected in AD patients Iacono et al. In a similar manner, a decline of 28S rRNA was found in elderly healthy probes when compared to younger control groups Payao et al. The patient data suggest that impairment of ribosome biogenesis and protein synthesis is one of the earliest events observed in the pathogenesis of AD characterized by mild cognitive impairment Ding et al. PD is characterized by loss of dopaminergic neurons in the brain stem. Patients display tremor, dementia and depression. Also in PD, most cases are sporadic. Typical risk factors are aging and exposure to mitochondrial toxins. A key feature of PD is deposition of Lewy bodies, which reflects deposition of α-Synuclein oligomers Ghavami et al. These oligomers trigger mitochondrial damage. Hereditary forms of PD involve mutation of the key mitophagy regulators PINK and Parkin. Mutations of both result in impaired mitophagy. Given that α-Synuclein serves as a substrate of the E3-ubiquitin ligase Parkin, accumulation of α-Synuclein is also detected in a Parkin mutated background. Also ER stress is implicated in PD. However, mild ER stress is attributed to function rather in a neuroprotective manner by inducing pro-survival autophagy. More recently, disruption of nucleolar integrity has been observed in human post mortem samples of patients with PD Rieker et al. In support of a nucleolar contribution, the ribosome biogenesis factor Nucleolin interacts with α-Synuclein. Consequently, damaged mitochondria, ROS, as well as autophagosomes accumulate and cause apoptosis Rieker et al. Disruption of nucleoli, cell cycle arrest and pmediated apoptosis is observed by depletion of the transcription initiation factor IA TIF-1A , required for the recruitment of RNA pol I, in mouse embryonic fibroblasts Yuan et al. Ablation of TIF-1A in DA neurons of mice results in Parkinsonism and progressive loss of DA neurons Rieker et al. Treatment with the neurotoxin MPTP worsens the effect of nucleolar stress. In this model, also p53 is stabilized and mTOR signaling decreased. Finally, ROS-mediated oxidative stress is induced and defects are detected in mitochondria, such as impaired mitochondrial transcription and COX cytochrome c oxidase activity Rieker et al. Therefore nucleolar stress, by inhibiting mTOR signaling, can impair mitochondrial function, which represents a key hallmark of several neurodegenerative diseases. To determine effects of specific PD mutations on nucleolar function irrespective of neuronal loss, pre-symptomatic, digenic PD models were analyzed. Hemoglobin Hb is strongly expressed in dopaminergic neurons in the substantia nigra and is found in patient samples of AD and PD. Hb has recently been connected to mitochondrial function and apoptosis. In turn, Hb overexpression impairs pre rRNA processing, induces nucleolar stress and sensitizes cells to apoptosis Codrich et al. The authors further demonstrate decreased phosphorylation of the mTOR target 4E-BP1, decreased numbers of lysosomes in neurons and decreased levels of LC3-II following rotenone treatment, being indicative for inhibition of autophagy. Patients with HD display uncontrolled chorea movements and cognitive impairment Ghavami et al. The onset of age also inversely correlates with the increasing number of repetitive Glutamine motifs present in mutated Huntingtin. Interestingly, capture of mutant Htt inside inclusion bodies was shown to be less toxic in comparison to accumulating free mutant Htt Zuccato et al. Also in HD patients, apoptosis and mitochondrial damage is detected. Additionally, studies have demonstrated that rRNA transcription is affected in HD Parlato and Kreiner, Triggering autophagy in mice models of HD can remove aggregates and increases their life span Zheng et al. Targeted disruption of nucleoli by conditional knockout of TIF-1A essential for the recruitment of RNA pol I in striatal neurons results in striatal degeneration and typical HD-like phenotypic alterations in mice Kreiner et al. TIF-1A loss induces nucleolar disruption and nucleolar stress, which precedes neurodegeneration. Nucleophosmin NPM represents a multifunctional, nucleolar key factor involved in ribosome biogenesis, which fulfills a plethora of pro-survival processes Colombo et al. A down-regulation of NPM serves as readout for nucleolar stress induction and can be linked to neurodegeneration in several models of neurodegeneration Marquez-Lona et al. In line, as a key marker for nucleolar stress Colombo et al. As an early and pdependent pro-survival response, the p53 target PTEN Stambolic et al. Given that the tumor suppressor PTEN counteracts the mTOR pathway, downstream targets of mTOR were analyzed for phosphorylation. It was found that p-S6 and p-4E-BP1 are reduced in the model. Inhibition of mTOR is connected to activation of autophagy and the same holds true in the HD model Kreiner et al. Thus, transient over-activation of autophagy seems to be induced as initial, neuroprotective mechanism in response to impaired ribosome biogenesis. However, after sustained nucleolar stress, apoptosis of striatal neurons is inevitable Kreiner et al. DNA damage and impaired rRNA transcription are connected to Cockayne Syndrome CS , which is a rare, congenital, autosomal-recessive neurodegenerative disorder Karikkineth et al. Patients are characterized by premature aging, dwarfism, microcephaly, and have an average life expectancy of 12 years. CSB was found to localize to mitochondria and bind to mtDNA Aamann et al. CSB-deficient cells show increased ROS production, increased mitochondrial content and accumulation of damaged mitochondria, in line with impaired mitophagy Scheibye-Knudsen et al. Further, CSB promotes acetylation of α-tubulin Majora et al. CSB deficiency abrogates autophagy and results in increased number of dilated lysosomes with impaired function Majora et al. In human CS cells, translational infidelity is observed, most likely due to accumulation of error-prone ribosomes as a consequence of impaired ribosome replacement. CS cells exhibit ER stress and an over-activated unfolded protein response, which can be counteracted by addition of pharmacological chaperones Alupei et al. Epilepsy is characterized by recurrent seizures and represents a disease related to neurodegeneration. Abrogated morphogenesis and synaptic function is observed upon nucleolar stress and could be connected to epilepsy reviewed in Hetman and Slomnicki, For example, pharmacologically induced short-term seizures in mice transiently affect RNA Pol I activity in hippocampi and result in decreased de novo synthesized 18S and 28S rRNAs. In contrast, long-term seizures were associated with increased ribosome biogenesis Vashishta et al. Epilepsy is further tightly linked to mTOR hyper-stimulation and autophagy over-activation Cao et al. Strikingly, administration of the mTOR inhibitor rapamycin counteracts seizures and thus functions in an anti-epileptogenic manner Zeng et al. Also rare, pediatric neurodegenerative diseases are characterized by alterations in autophagy. As an example, the multisystemic Vici syndrome is neurologically characterized by microcephaly and cognitive impairment. Accumulation of ubiquitinated autophagic cargo, p62 and damaged mitochondria is observed, reminiscent of neurodegeneration Ebrahimi-Fakhari et al. However, whether also ribosome biogenesis is also altered here, remains to be determined. Key hallmarks of cancerous cells involve for instance mis-regulation of signaling pathways, rapid cell proliferation, accelerated tumor growth and inhibition of apoptosis Hanahan and Weinberg, , Large amounts of ribosomes are essential for cancerous cell growth and large nucleoli serve as prognostic marker in many tumor types Montanaro et al. The nucleus arises as an essential target for cancer therapy Woods et al. Anti-tumor therapies utilize the high demand of cancer cells for the production of ribosomes by inhibiting RNA pol I. Classical chemotherapeutics used in the clinics are for instance actinomycin D, 5-fluorouracil and metotrexat, which interfere with the nucleolar function Boulon et al. Novel drugs, which specifically impair rRNA transcription, are currently tested in clinical trials. The small molecule drug CX specifically inhibits transcription of RNA pol I and stabilizes p53, whereas RNA pol II is not affected. Also, translation and DNA replication is not impaired in human cancer cell lines Drygin et al. The drug is further reported to impair proliferation in a pindependent manner in cancer cell lines, whereas the effect on normal cell lines is minimal Drygin et al. Selective inhibition of RNA pol I by CX also robustly stimulates pro-death autophagy. Nucleolar stress and autophagy seem to be tightly coupled in distinct models and setups. Recently, CX was loaded on a nanoplatform to enrich for nucleolar accumulation of the drug in order to enhance the anti-cancer effect, without causing significant side effects. In vivo and in vitro assays confirm induction of pro-death autophagy in HeLa cells, as well anti-proliferative and anti-tumor effects Duo et al. Besides autophagy, CX induces also senescence in a pindependent manner. In U2OS osteosarcoma cells, CX induces G2 arrest, but not apoptosis Li et al. In response to CX, p53 accumulates and p21 is induced. In addition, increased levels of LC3-II are detected under basal conditions. Using TEM analysis, the authors noticed expanded vacuole-like structures filled with organelles, however, they report lack of clear identification of autophagosomal character. Knockdown of p53 by siRNA rescues p21 up-regulation and LC3-II accumulation and increases cell survival. The authors conclude that CX triggers pdependent autophagy. The p53 target p21 is shown to be up-regulated during autophagy and a pindependent increase of p21 is reported in MNNG cells with mutant p53 Li et al. Taken together, autophagy induction as a response to nucleolar stress seems to be an initial surveillance mechanism in several models. However, also in terms of cancer, autophagy induction can have two modes of action: Autophagy induction is clearly beneficial for cells by preventing genotoxic stress and DNA damage. It removes cellular sources of ROS, such as defective mitochondria or proteins Mrakovcic and Frohlich, In contrast, inhibition of autophagy represents an oncogenic event. At later stages over-activation of autophagy facilitates oncogenic drug-resistance. Autophagy inhibitors chloroquine and hydroxychloroquine have therefore been tested in clinical trials for cancer therapy Yang et al. Impairment of ribosome biogenesis is connected to a diverse class of human diseases collectively termed ribosomopathies Freed et al. Several players associated with ribosomopathies have been described see also below. Classically, the nucleolar stress response and the tumor suppressor p53 are activated Freed et al. Intriguingly, though, some phenotypes are common and include defects of the craniofacial cartilage, anemia and increased cancer susceptibility. The elevated cancer risk appears paradoxical, given the great need of tumor cells for large amounts of ribosomes Montanaro et al. Accordingly, pathways and mechanisms might well exist, which let both co-exist Pfister and Kuhl, For example, specialized onco-ribosomes have recently been uncovered to increase the cellular fitness by mediating preferential translation of anti-apoptotic Bcl-2, as observed for the ribosome mutant RPLR98S in leukemia cells Xue and Barna, ; Sulima et al. However, the question on cause and consequence of ribosomopathy-induced cancer formation is still under debate. Recently, examination of murine hepatocellular carcinoma and hepatoblastoma has revealed ribosomopathy-like features of nucleolar stress, such as deregulated expression of RPs and accumulation of unprocessed rRNA precursors. Despite the fact that p53 is stabilized, no growth inhibition occurs Kulkarni et al. Therefore, compensating mechanisms might counteract apoptosis, involving up-regulation of anti-apoptotic Bcl-2, silencing of p19 ARF or cytosolic sequestration of p Those events would in turn inhibit the tumor suppressive mechanisms of cell cycle arrest and apoptosis in these cancers Kulkarni et al. Clearly, ribosome biogenesis is a highly energy-consuming process. An implication of autophagy comes in mind, which might compensate for the low levels of functional ribosomes observed in ribosomopathies. Many important questions arise. Is there a connection to bulk autophagy or selective autophagy of ribosomes ribophagy in ribosomopathy patients? Until now broad studies are lacking, which precisely address their implication in these issues. An overview summarizing the emerging connection between nucleolar stress and autophagy in the diseases presented here is given in Table 1. Table 1. The role of nucleolar stress in mentioned diseases and effects on autophagy. Recently, inhibition of RNA pol I has been connected to autophagy, revealing that nucleolar stress functions upstream of autophagy. In the following, evidence is collected, which links the ribosome biogenesis machinery and the nucleolus to autophagy, and vice versa. As a common principle, different groups suggest implication of mTOR signaling in nucleolus-mediated autophagy see below. Also here, pdependent and -independent pathways are being identified. Besides the classical role of p53 as guardian of the genome, by mediating cell cycle arrest and apoptosis, p53 has been reported to exert distinct roles in autophagy Wang et al. This depends on its subcellular localization: nuclear, cytosolic or mitochondrial, respectively. The effect of nuclear p53 as an inducer of autophagy mostly depends on its role as transcription factor. h Diagram summarizing the roles of the phosphorylations of ULK1 by mTORC1 and AMPK in regulating ULK1 activity. Replacing either Ser or thr with alanine individually did not abolish the ability of A to preserve Ser phosphorylation and suppress ULK1 activity Supplementary Fig. The ability of A to suppress ULK1 activity was maintained by all mutants except those that harbor both SA and TA mutations Supplementary Fig. The 4SA mutation, previously shown to suppress mitophagy 9 , moderately increased ULK1 activity under Torin1 treatment instead of reducing it. We also tested the effect of alanine replacement for Atg13 Ser, a potential AMPK target site 35 , on ULK1 activity without observing any significant effect Supplementary Fig. Atg9a, which is recruited to ULK1 in response to AMPK activation 13 , was also not necessary for the inhibitory effect of AMPK on ULK1 activity Supplementary Fig. Next, we asked how Ser and Thr phosphorylations affect autophagy. Glucose starvation suppressed amino acid starvation-induced activation of the AtgVps34 complex to a lesser extent in AA cells compared to WT cells Fig. Similarly, the inhibitory effect of A on Vps34 activity was blunted in AA cells compared to WT cells Fig. However, the AA mutation could not completely blunt the suppressive effect of AMPK activation on Vps34 activity, suggesting that other AMPK-mediated events contribute to the suppression of Vps34 activity. a AMPK suppresses Atgassociated Vps34 activity through the phosphorylation of ULK1 at Ser and Thr ULK DKO HCT cells reconstituted with either the WT or the AA ULK1 were treated as described in Fig. Anti-Atg14 immunoprecipitates were isolated to measure Vps34 activity. b , c AMPK suppresses the formation of autophagosomes through the phosphorylation of ULK1 at Ser and Thr during amino acid starvation. The WT and AA ULK1-reconstituted cells, which were prepared as described in a , were subjected to the treatment conditions indicated in Fig. d , e AMPK suppresses autophagy flux through the phosphorylation of ULK1 at Ser and Thr f , g Glucose starvation-induced suppression of autophagy flux depends on ULK1 phosphorylation at Ser and Thr by AMPK. h Diagram showing how AMPK negatively regulates ULK1 signaling to the AtgVps34 complex. i AMPK phosphorylation of ULK1 Ser and Thr suppresses aggrephagy. Ubiquitin-positive protein aggregates were analyzed by immunostaining red. j Quantitative analysis of i. In the cell images, the nuclei were stained with DAPI blue. The statistical analysis in this figure was performed as described in Figs. The AA mutation had no effect on the formation of phagophores and autophagosomes in response to amino acid starvation in Auntreated cells Fig. Upon treatment with A, the formation of phagophores and autophagosomes was significantly reduced in WT cells, while the reduction was significantly blunted in AA cells. The suppressive effect of A on autophagy flux was also significantly blunted in AA MEFs Fig. Similarly, glucose starvation suppressed amino acid starvation-induced autophagy flux in WT cells but barely in AA HCT cells Fig. These results suggest that AMPK activation suppresses amino acid starvation-induced increases in the activity of the Vps34 complex and autophagy through ULK1 phosphorylations at Ser and Thr Fig. We also found that the AA mutation significantly reduced the suppressive effect of A on aggrephagy Fig. However, a significant level of suppression was still observed with aggrephagy in AA cells treated with A, suggesting that AMPK activation suppresses aggrephagy through not only inhibition of ULK1 but also through additional unknown mechanisms. Our findings may seem counterintuitive, as both AMPK and autophagy play critical roles in cell survival 36 , 37 , 38 , We could confirm the critical role of the LKB1-AMPK-ULK1 pathway in cell survival during glucose starvation or phenformin treatment using HCT cells and MEFs that lacked those genes Fig. We wondered how this result can be reconciled with the negative role of AMPK in autophagy. We considered the possibility that AMPK plays the vital role in cell survival during glucose starvation not by inducing autophagy but by suppressing it. The ULK1 AA mutation is a suitable tool to test this hypothesis, as it allows us to disrupt the AMPK-autophagy link without perturbing other AMPK-mediated functions. HCT and MEF cells reconstituted with the AA mutant ULK1 showed a significant increase in apoptosis following prolonged glucose starvation, compared to cells reconstituted with the WT ULK1 Fig. Phenformin also induced apoptosis to a much greater extent in AA cells than in WT cells Supplementary Fig. Prolonged amino acid starvation also induced apoptosis in HCT cells, which was suppressed by A in WT ULK1-reconstituted cells, but not in AA ULK1-reconstituted cells Fig. These results suggest that AMPK-mediated phosphorylations of Ser and Thr protect glucose- or amino acid-starved cells against apoptosis. a LKB1-AMPK-ULK1 axis protects cells against apoptosis during prolonged glucose starvation. b Quantitation of apoptosis and viable cells. Details are described in Methods. c ULK1 phosphorylation at Ser and Thr protects cells against apoptosis during prolonged amino acid starvation. The WT and AA mutant HCT cells were analyzed. ULK1 half-life hours estimated via linear regression are indicated inside the graphs. g AMPK protects autophagy proteins from degradation during glucose starvation. h Quantitative analysis of key autophagy proteins from g. The half-life of the proteins is indicated in hours within the graphs. i Scheme for analyzing the recovery of autophagy following extended glucose deprivation in cells. The indicated HCT cells were starved of glucose, followed by a 2-h recovery period in full or amino acid-depleted medium, as depicted in i. k Quantitation of ULK1 activity from j. The measurement reflects both phosphorylations of Atg14 and Beclin 1. l — o AMPK-mediated phosphorylations of Ser and Thr are required for the efficient autophagy flux after prolonged glucose starvation. p , q Diagram depicting two distinct functions of AMPK in regulating ULK1 and autophagy. AMPK preserves the autophagy initiation machinery positive role depicted in blue and suppresses ULK1 activity negative role depicted in red. The statistical analysis in this figure was performed as in Figs. During the prolonged glucose or amino acid starvation, ULK1 levels were drastically decreased Fig. The decrease was not attributed to changes in gene expression, autophagy, or the proteasome but was preventable by inhibiting caspase activity Fig. AMPK deficiency exacerbated the decrease in ULK1 levels, whereas treatment with A showed a protective effect against the decrease in ULK1 levels Fig. Using cycloheximide, we confirmed that the decrease of ULK1 levels during glucose starvation is a result of protein degradation Fig. In contrast to the WT ULK1, the AA mutant ULK1 was less stable during prolonged glucose starvation or when treated with phenformin Fig. AMPK was also required to protect ULK1-associated autophagy regulators, such as FIP, Atg13, Atg14, Beclin 1, and Vps34, against degradation during glucose starvation Fig. Collectively, these results suggest that AMPK protects ULK1 and its associated autophagy machinery from degradation during glucose starvation. Given that AMPK stabilizes the autophagy machinery components, we hypothesized that AMPK-deficient cells might lose their ability to initiate autophagy following prolonged glucose starvation. Similarly, cells reconstituted with the AA mutant ULK1 showed drastic reductions in both ULK1 level and activity following prolonged glucose starvation. Along with the reductions, cells lacking AMPK or reconstituted with the AA mutant ULK1 exhibited significant impairments in their ability to increase autophagy flux in response to amino acid starvation after the prolonged glucose starvation Fig. These results suggest that AMPK-mediated phosphorylations of ULK1 Ser and Thr play crucial roles in preserving the cellular capacity to perform autophagy and maintaining cellular resilience during prolonged glucose starvation. Our study provides a rigorous demonstration that AMPK negatively regulates ULK1 activity and autophagy by phosphorylating ULK1. This challenges the widely accepted notion that AMPK promotes ULK1 activity and autophagy induction. Our findings offer a mechanistic explanation for the previously reported inhibitory effects of AMPK on autophagy 1 , 2 , 3 , 10 , 11 , 12 , 13 , 14 , 15 , The key aspect of our elucidated mechanism is that AMPK-mediated phosphorylations of ULK1 at Ser and Thr suppress ULK1 activity and autophagy induction. Our study reveals that the phosphorylation of ULK1 at Ser by AMPK, which is broadly used as a marker of high autophagy, indeed inhibits ULK1 activation and autophagy induction. Overall, our findings redefine the role of AMPK in autophagy by demonstrating that it suppresses, rather than promotes, autophagy in energy-deprived cells. According to the mechanism we have elucidated, AMPK regulates autophagy in two distinct ways Fig. Firstly, AMPK inhibits ULK1 to prevent abrupt induction of autophagy. Upon AMPK activation, ULK1 undergoes phosphorylation, which stabilizes the AMPK-ULK1 interaction and prevents the activation of ULK1 during amino acid starvation. Secondly, AMPK protects ULK1 and other autophagy regulators associated with ULK1 from caspase-mediated degradation, preserving critical cellular components during energy stress. By preventing immediate autophagy induction and preserving essential autophagy components, AMPK enables cells to quickly resume autophagy and restore homeostasis when energy stress subsides. This protective role, together with its role in inducing autophagy gene expression Supplementary Fig. Our model suggests that the phosphorylation of Ser human ULK1 Ser is not essential for ULK1 activation but rather for ULK1 stability. Cells or tissues expressing the SA mutant for an extended period may experience reduced ability to initiate autophagy due to ULK1 destabilization. In addition to its long-term protective role, AMPK is required for the optimal activation of the Atgassociated Vps34 complex in response to acute amino acid starvation Fig. Notably, Vps34 activity was barely increased by glucose starvation or AMPK activation alone without amino acid starvation. This suggests that AMPK-mediated phosphorylation of Beclin 1 alone may not be sufficient for activating Vps We speculate that the phosphorylation might enhance the responsiveness of the AtgVps34 complex to ULK1 activity for its optimal activation. Such a two-tiered regulation of the Vps34 complex by both AMPK and ULK1 may enable cells to regulate Vps34 activity specifically in the autophagy pathway without affecting its activity in other non-autophagy-related membrane processes. Similarly, the AMPK-mediated phosphorylations of Atg9, RACK1 and PAQR3 43 , 44 , 45 might contribute to maintaining the autophagy machinery in a state of readiness, primed for activation, rather than directly enhancing autophagy. The restriction of autophagy during energy depletion emphasizes the tight coupling of autophagy induction with the mitochondrial activity. Our finding is consistent with the recent report that the mitochondrial oxidative chain is important for maximal autophagy Autophagosome formation occurs in the endoplasmic reticulum localized in close proximity to the mitochondria 47 , 48 , 49 , highlighting the strong link between the mitochondrial activity and autophagy. Our study suggests that this proximity may enable efficient control of autophagy induction via the AMPK-ULK1 axis by providing ATP locally. Supporting this, the dependence of amino acid starvation-induced activation of ULK1 on the mitochondrial activity disappeared when LKB1 or AMPK was depleted. This finding indicates that the proximity of the AMPK-ULK1 axis to the mitochondria may allow for effective monitoring of the mitochondrial status and coordination of autophagy induction with the mitochondrial energetics. The long-held notion that AMPK promotes autophagy has led to numerous studies examining the potential therapeutic applications of AMPK-activating agents, such as metformin, for various diseases. These inconsistent outcomes may be related to the dual role of AMPK in regulating autophagy. Our findings provide a conceptual framework for reevaluating previous research and prompt the need for further investigation into the function of AMPK in different physiological and pathological contexts. Such research may deepen our understanding of how cells respond to energy stress and contribute to improving AMPK-targeting therapeutics. Primary antibodies used for immunoprecipitation IP and western blotting WB were obtained and validated as described below. Unless otherwise specified, the antibodies have been validated and utilized for WB of both mouse and human proteins. When the antibodies were used for IP or immunostaining, it was explicitly noted. Antibodies for ULK1 sc for IP and sc for WB of human protein , Atg14 sc for IP , Beclin 1 sc for WB and sc for IP , p62 sc , and GAPDH sc were from Santa Cruz Biotechnology. The antibodies were validated in our previous reports 21 , 22 , 54 , 55 , Antibodies for ULK1 A for WB of mouse protein and WIPI2 SAB for immunostaining were from Sigma-Aldrich. The antibodies were validated in our previous reports 21 , 22 , Antibodies for Vps34 , Atg7 , Atg9a , Atg14 rabbit monoclonal clone D3H2Z for immunostaining and WB , mTOR and for WB , phospho-Akt Ser , Akt , LC3B for WB , phospho-S6K1 Thr , S6K1 , and phospho-Atg13 Ser isoform 2 Ser; mouse Ser were from Cell Signaling Technology. The antibodies were validated in our previous reports 21 , 22 , 54 , 55 , 56 , Antibodies for AMPK , LKB1 , phospho-ACC , phospho-ULK1 Ser , phospho-ULK1 Ser , phospho-ULK1 Ser , phospho-ULK1 Ser , cleaved PARP , and cleaved caspase-3 were from Cell Signaling Technology. The antibodies were validated by the manufacturer. Myc 9E10 monoclonal antibody OP10 and WIPI2 antibody MABC91 for immunostaining were from EMD-Millipore. Ubiquitin antibody from EMD-Millipore was validated by the manufacturer. HA antibody HA. The antibody was validated in our previous reports 21 , 22 , 54 , 55 , 56 , LC3B antibody PM for immunostaining and p62 antibody PM for immunostaining were from MBL International Woburn, MA. The antibodies were validated in our previous reports 21 , 22 , 54 for LC3B and by the manufacturer for p Anti-Atg13 antibodies have been described in our previous report for the validation Anti-pT antibody was made using PRNR pT LPDL-C as an antigenic peptide in rabbits, and purified using antigenic peptide-conjugated column in Abclonal Science Woburn, MA. The antibody was validated in the current paper. Antibodies for pAtg14 Ser29 and pBeclin 1 Ser30, the sites we identified, were previously described in our reports for the validation 21 , The following materials were used in the experiments. Recombinant Atg14 was obtained from Escherichia coli as a custom order to MyBioSource San Diego, CA. All the primers used for our study were obtained from Integrated DNA Technologies Coralville, IA via custom synthesis. HCT CCL , HeLa CCL-2 , HEKT CRL , HepG2 HB , C2C12 CRL , and A cells CCL were obtained from ATCC. HT22 cells SCC were obtained from EMD-Millipore. All cell lines were confirmed to be mycoplasma-free using MycoAlert PLUS Mycoplasma Detection kit Lonza Walkersville, Inc. Myc-tagged constructs for ULK1 and Atg13 were made using pRK5 vector as described in our previous reports 21 , 22 , For lentiviral constructs, we used pLV-EF1a-IRES plasmids Addgene , , Human and mouse ULK1 genes were described in our previous report LKB1 WT and KD cDNAs were obtained from Addgene and and subcloned into pLV-EF1a-IRES-puro. Human and mouse ULK1 and Atg13 point mutant constructs listed in Supplementary Table 1 were generated using a site-directed mutagenesis kit Agilent Mouse 4SA ULK1 mutant was obtained from Addgene Mouse SA and SC mutants were kindly provided by Dr. Kun-Liang Guan UC San Diego. The mutant constructs were cloned into pLV-EF1a-IRES-Puro with no tag. The primer sequences used for the mutagenesis are listed in Supplementary Table 1. For inducible expression, human and mouse ULK1 genes were subcloned into Lenti-TRE3GPGK-Tet3G-puro TransOMIC Technologies. Human WT AMPKα1 Addgene and KD AMPKα1 Addgene DNAs and rat WT AMPKα2 Addgene and K45R AMPKα2 kinase inactive mutant Addgene DNAs were subcloned into pLV-EF1a-IRES-Puro or pLV-EF1a-IRES-blast. All the generated constructs were confirmed by sequencing the DNAs at GENEWIZ South Plainfield, NJ. AMPKα DKO HCT cells, AMPKα DKO HeLa cells, and ULK DKO, ULK1 KO, Atg13 KO HEKT cells were generated using the CRISPR-cas9 assisted genome editing technique. We used pSpCas9 BB -2A-GFP Addgene, PX; deposited by Dr. Feng Zhang. The detailed procedures are described in our recent reports 21 , 22 , In brief, target cells were transduced by the plasmid using the Neon Transfection System ThermoFisher Scientific, MPK Two days post-transfection, green fluorescence-positive single cells were sorted and plated into well plates using the Hana Single Cell Dispenser Namocell, Mountain View, CA. Single colonies were expanded, and genomic DNA and cell extract were obtained for screening by genotyping and WB. Atg7 KO, Atg14 KO, and LKB1 KO HCT cells were generated using lentiCRISPRv2 vector Addgene Lentivirus preparation and infection procedures have been described in our previous reports The transduced cells were cultured in the absence of antibiotics for two days. Stably transduced cells were selected with antibiotics. ULK DKO HCT cells 22 , Beclin 1 KO HCT cells 21 and ULK DKO MEFs 21 have been described previously. AMPK DKO MEFs were obtained from Dr. Benoit Viollet. Atg9a KO MEFs were obtained from Dr. Shizuo Akira. LKB1 KO MEFs were obtained from Dr. All the generated cell lines were verified by genotying using the primers listed in Supplementary Information and by sequencing the DNAs at GENEWIZ South Plainfield, NJ. The sequences for gRNAs are listed in Supplementary Table 2. All the chemicals used were resolved in dimethyl sulfoxide DMSO as stock and used at the indicated concentrations for each experiment. All the chemicals for analysis of their effects on ULK1 activity, Vps34 activity and autophagy were present not only in pre-incubation but also during starvation or mTORC1 inhibiting treatments. Cells were harvested 2 days post-transfection for co-IP assays. We used pLV-EF1a-IRES-lentivral vectors described above to generate cell lines stably transduced with exogenous DNA. The procedures for lentivirus preparation and target cell infection have been described in our previous reports Because there is a limitation of DNA insert for the efficient lentiviral packaging, we could not produce pLV-EF1a-IRES vectors containing the ULK1 gene. As an alternative method, we linearized the lentiviral vector using the restriction enzyme SgrDI ThermoFisher Scientific, ER for a single cut, then introduced the linearized DNA into target cells using the Neon instrument ThermoFisher Scientific, MPK Two days after the transfection, cells harboring the lentiviral vector were selected in the presence of antibiotics. Membranes were washed briefly with phosphate buffered saline PBS, Fisher Scientific, BP containing 0. Membranes were then incubated with Enhanced Chemiluminescence WB detection reagents Advansta, E to visualize protein bands. WB images were acquired by X-ray film developer or using iBright ThermoFisher Scientific. Band intensities of WB images were quantified using Image J version 1. The half-life of protein downregulation was obtained by the Prism v6 software version 6. C57BL6J male mice were purchased from the Jackson Laboratories. Mice between 10 and 12 weeks of age were used for the experiment. One hour after the injection of Torin1, the mouse liver and skeletal muscle were isolated on the ice and immediately frozen in liquid nitrogen. We also obtained cryosections from the frozen tissue using a cryostat Reichert-Jung Cryocut Images were obtained using a Deltavision PersonelDV microscope Applied Precision Inc. LC3B puncta per cell liver and per fiber muscle were quantified with a specific threshold using ImageJ version 1. All experimental procedures were approved by the University of Minnesota, Institutional Animal Care and Use Committee. Phagophore and autophagosome formation was analyzed by immunostaining endogenous WIPI2 EMD-Millipore, MABC91; Sigma-Aldrich, SAB; dilution and LC3B MBL International, PM; dilution , respectively, as we described in our previous reports 21 , Images from stained cells were obtained using a Deltavision PersonelDV microscope and analyzed by softWoRx version 6. WIPI2 and LC3B puncta were quantified with a specific threshold using ImageJ version 1. Whole-cell extracts were prepared as described above using the lysis buffer for WB. The expression levels of LC3B and p62 in cell lysate were analyzed by WB and quantified by densitometry. The flux was analyzed as the difference of LC3B II or p62 levels between the presence and absence of BAFA1. Cell extract was obtained using the lysis buffer for WB and analyzed for cleaved PARP and caspase 3 by WB. Additionally, cells were stained using pSIVA-IANBD specific to apoptotic cell membrane Abcam, ab Cell images were acquired using Lionheart FX fluorescence microscope Bio-Tek, Winooski, VT and analyzed using Gen5 program version 3. During the second incubation, cells were treated with chemicals or starvation medium as indicated in each experiment. The concentrations of other used chemicals are the same as described in other assays. Images were taken using a Deltavision PersonelDV microscope as described above in Immunostaining and fluorescence microscopy section. For each analysis, 50 cells were counted across three independent experiments. AMPKα DKO HEKT cells were transiently transduced with myc-ULK1 construct. Two days post-transfection, myc-ULK1 immunoprecipitates were obtained using anti-myc antibody. As a control, the immunoprecipitates were incubated just with the buffer without AMPK. The phosphorylation state of Atg14 Ser29 was analyzed by WB. The lipid kinase activity of Atgassociated Vps34 was assayed as we have described previously 21 , The Vps34 kinase reactions were initiated by adding ATP final conc. All the kinase reactions were spotted onto nitrocellulose membrane. After multiple times of extensive washing, the amount of PI3P Grip remaining on the nitrocellulose membrane was analyzed by WB using anti-GST antibody Cytiva, P3P dot blot intensities were quantitatively analyzed by ImageJ software version 1. HEKT cells deficient of ULK1 were transiently transduced to express myc-tagged ULK1 constructs. The reaction was stopped by adding SDS sample buffer and analyzed by SDS-PAGE and WB. The phosphorylation states of ULK1 Ser and Thr in the reaction mixture were analyzed by WB. RNAs were prepared from cells using TRIzol reagent ThermoFisher Scientific, We used actin β ACTB and TATA Box Protein TBP genes as controls. Samples were run on QuantStudio 3 Real-Time PCR system ThermoFisher Scientific. Results were analyzed using Microsoft Excel, version The primers used for qPCR are listed in Supplementary Table 3. The quantified outcomes were summarized as mean and SEM as specified in the figure legends. To compare the means between different groups, the two-tailed Student t test was used with Prism 6 Version 6. All the measurements used for statistics were obtained from distinct samples that were prepared independently of each other. The sample sizes for independent experiments and animal studies were determined based on preliminary data. The western blotting experiments were performed multiple times, as illustrated in the accompanying graphs. In cases where only micrographs were presented, the experiments were independently repeated more than three times, yielding consistent results. Representative micrographs are provided. Statistical significance was interpreted for p values below 0. Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article. All the data that support the conclusions in this paper are available within this article and its supplementary Information file. Source data are provided with this paper as an excel file, which contains all the uncropped western blots, raw data, and quantified values for all the graphs presented in this paper. Lang, M. et al. Glucose starvation inhibits autophagy via vacuolar hydrolysis and induces plasma membrane internalization by down-regulating recycling. Article CAS PubMed PubMed Central Google Scholar. Ramírez-Peinado, S. Glucose-starved cells do not engage in prosurvival autophagy. Article PubMed PubMed Central Google Scholar. Weber, C. β-Oxidation and autophagy are critical energy providers during acute glucose depletion in S. Natl Acad. USA , — Article ADS CAS PubMed PubMed Central Google Scholar. Schellens, J. Hepatic autophagy and intracellular ATP. A morphometric study. Cell Res. Article CAS PubMed Google Scholar. Plomp, P. Energy dependence of autophagic protein degradation in isolated rat hepatocytes. Energy dependence of different steps in the autophagic-lysosomal pathway. Mijaljica, D. V-ATPase engagement in autophagic processes. Autophagy 7 , — Kim, J. AMPK and mTOR regulate autophagy through direct phosphorylation of Ulk1. Cell Biol. Egan, D. |
Autophagy and stress response -
Nguyen et al. Meanwhile, the number of novel players involved in the complex process of selective autophagy is constantly expanding. Note that accumulation of damaged mitochondria, which are eliminated by mitophagy to a certain point, sets the threshold for apoptosis as a point of no return Marino et al.
As a result, p62 recruits autophagosomal membranes to its selective, autophagosomal cargo Lamark et al. The same principle of autophagy receptor e. A schematic for aggrephagy, bulk autophagy and mitophagy is depicted in Figures 2 , 3.
Figure 2. Simplified model of cargo targeted by bulk autophagy or mitophagy. The phagophore forms around bulk material, such as proteins and organelles during bulk macro autophagy. The phagophore is a double-membranous structure, which forms around the cargo and gives rise to the autophagosome.
Figure 3. Removal of protein aggregates by selective autophagy. Aggregated proteins red are bound by the autophagy receptor p62 blue , which itself has interaction domains for autophagosomal LC3 green, lipidated LC3-II and ubiquitin yellow.
The cargo is engulfed by the mature autophagosome and subsequently fuses with the lysosome to form the autolysosome, in which the cellular material is degraded by acidic hydrolases orange.
The mammalian target of rapamycin mTOR pathway couples the intake of to nutrients, growth factors, energy and stress to the regulation of cell metabolism, growth, survival and autophagy Pattingre et al. Deregulation is linked to various diseases and cancer formation. Mammalian target of rapamycin signaling is recognized as essential pathway for proper neuronal development, neuronal survival and morphogenesis.
Consequently, changes in mTOR signaling have been correlated with a spectrum of neuropathologies, such as epilepsy, intellectual disability, autism, brain injury, brain tumor formation and neurodegeneration Crino, ; Switon et al.
Likewise, mTOR inhibitors such as the bacterial macrolide rapamycin and its analogs are growingly used as therapeutic drugs and tested in clinical trials for effects in diverse neuropathological conditions Laplante and Sabatini, ; Crino, Mammalian target of rapamycin is a conserved serine-threonine kinase, which belongs to the phosphoinoside 3 kinase PI3K family.
It assembles two large protein complexes, mTORC1 and mTORC2. The mTORC1 complex is considered as rapamycin sensitive complex Pattingre et al. Rapamycin binds to FKBP12 and mTOR, thereby inhibiting mTORC1. mTORC1 signaling affects cell growth, metabolism and autophagy: Upon favorable conditions, mTOR is activated to allow cell growth by anabolic processes, such as rRNA biogenesis and protein translation.
Upon nutrient deprivation and lack of growth factors, mTOR signaling is inhibited and cell growth is suppressed, whereas catabolic processes such as autophagy are induced to allow cell survival under unfavorable conditions. mTORC1 controls autophagy by regulating ULK1, ATG13 and FIP, as well as by a reported rapamycin-insensitive mechanism Laplante and Sabatini, mTORC1 also regulates mitochondrial metabolism and biogenesis: mTORC1 inhibition impairs the MOM potential, reduces oxygen consumption and ATP levels.
mTORC1 inhibition further decreases mitochondrial DNA levels and hampers mitochondrial biogenesis by affecting the transcriptional activity of the nuclear factor PGC1α PPARγ co-activator 1 Cunningham et al. Also p53 can regulate mTOR: DNA damage-induced p53 stabilization activates AMPK, which is a sensor of energy status and in turn results in mTORC1 inhibition.
p53 also negatively controls mTORC1 by increasing PTEN expression, which functions as mTORC1 inhibitor. Inhibition of mTOR signaling diminishes nucleolar size and function and promotes longevity in different model organisms Tiku and Antebi, However, the precise mechanisms regulating the crosstalk between ribosome biogenesis and autophagy remain to be determined.
A simplified model of mTORC1 signaling and the role of p53 is given in Figure 4. Figure 4. A simplified model of mTOR signaling, and effect of nucleolar stress on p Growth factors, energy status, amino acid availability, oxygen levels and genotoxic stress can result in mTORC1 activation.
mTORC1 further activates autophagy by inhibitory effects on the ULK1 complex, composed of ULK1, ATG13 and FIP mTORC1 promotes protein synthesis by i S6K activation, which stimulates phosphorylation of S6 and thereby ribosome biogenesis, as well as by ii inhibitory effects on 4E-BP1 and eIF-4E.
As a consequence, translation is activated. Furthermore, mTORC1 influences mitochondrial biogenesis and metabolism. Defective ribosome biogenesis on the one hand and impaired autophagy on the other hand are largely contributing to several diseases.
In the following, an overview is provided on common concepts of three key classes of diseases, classically or recently connected to nucleolar stress and autophagy with specific focus on neurodegeneration, cancer and ribosomopathies. For a more detailed overview see for instance Parlato and Kreiner, ; Ghavami et al.
The nervous system is vulnerable to intrinsic and extrinsic factors, which can give rise to distinct neurodevelopmental pathologies such as microcephaly, psychiatric disorders, autism, intellectual disability, epilepsy and neurodegeneration please, be referred to review Hetman and Slomnicki, Causes include, for instance, gene mutations, infections or neurotoxins.
As common concepts, gene expression, quality control mechanisms, cell proliferation, differentiation and apoptosis are mis-regulated. Apoptosis might give rise to microcephaly by eliminating, e. Likewise Zika virus infection, as an extrinsic factor for neurodevelopmental disorders, is tightly coupled to microcephaly.
It has recently been demonstrated that it decreases mTOR signaling and over-activates autophagy Liang et al. At the same time, ribosome biogenesis defects are emerging reviewed in Hetman and Slomnicki, Given the striking role of the nucleolus in coordinating mentioned neuropathological routes, deregulation of ribosome biogenesis rises as a potent upstream mechanism.
In addition, also autophagy is activated in this context. Aging represents a general risk factor for the formation of neurodegenerative diseases and consequently, neurodegeneration accumulates within our society. Despite the rapid advances made in medicine, not all negative aspects of aging can simultaneously be addressed.
Our scientific knowledge on distinct neurodegenerative diseases has uncovered several common mechanisms, among them loss of neurons Parlato and Kreiner, ; Parlato and Liss, and a prominent contribution of aggregate accumulation, induction of apoptosis and a mis-regulation of autophagy Yamamoto and Simonsen, ; Ghavami et al.
In line, aging functions as susceptibility factor for neurodegeneration. It is characterized by a loss of rDNA and is accompanied by reduction of nucleolar size and a decline in rRNA processing Garcia Moreno et al. Therefore, the nucleolus is tightly connected to lifespan regulation Tiku and Antebi, As both routes of apoptosis and autophagy are interwoven and not yet fully understood, mechanistic research is essential as a basis for development of therapeutic approaches.
As a mandatory goal, novel drugs have to be tested for specificity and efficacy. As a result, mis-folded proteins accumulate and cause a toxic intracellular environment. Normally, proper cellular homeostasis is maintained by several machinery: the ubiquitin-proteasome system UPS degrades proteins, whereas autophagy is capable of removing proteins and whole organelles by the lysosome.
Hence, both routes are essential for a healthy cell and both have been implicated in the development of neurodegenerative diseases Levine and Kroemer, ; Ghavami et al. Note that increased apoptosis induction plays a crucial role in eliminating these damaged cells. In addition, chronic inflammation and oxidative stress are observed in neurodegenerative disorders.
Several neurodegenerative disorders, such as PD, AD, and HD are characterized by accumulation of mis-folded, ubiquitinated proteins, which damage the affected cell Yamamoto and Simonsen, The accumulating proteins form inclusion bodies, which can differ between the distinct pathologies.
With increasing age the autophagic program looses efficiency, thereby increasing the likelihood of aggregate accumulation. Neurons are highly sensitive to accumulation of protein aggregates and require proper autophagy mechanisms to keep the intracellular toxicity low.
Supporting data demonstrating the significant implication of the autophagic machinery were, for instance, obtained in mice lacking ATG7 in the central nervous system. ATG-deficient mice, which fail to perform autophagy, display an accumulation of inclusion bodies followed by neuronal loss Komatsu et al.
However, autophagy might have dual functions with respect to neurodegeneration: On the one hand functional autophagy is neuroprotective, by removing defective mitochondria via mitophagy. On the other hand pro-death autophagy is considered to induce neuronal cell death.
Massive inhibition of autophagy can trigger apoptosis, which is observed by loss of neurons in neurodegeneration. Interfering with autophagy regulators and blocking autophagy, results in accumulation of cargo-filled autophagosomes and lysosomes, again being toxic for the cell.
As a consequence, lysosome-mediated cell death occurs Kroemer and Jaattela, Impairment of mitophagy causes accumulation of defective mitochondria, which in turn induces reactive oxygen species ROS formation and mitochondrial apoptosis Seo et al.
Induction of apoptosis by the chemotherapeutic agent staurosporine is accompanied by mitophagy and autophagy induction in dopaminergic cells.
Additional block of autophagy by Bafilomycin or inhibition of mitophagy in PINK null mice sensitizes cells to staurosporine-induced apoptosis. Autophagy and mitophagy seems to be neuroprotective upon staurosporine-mediated apoptosis induction in dopaminergic neurons Ha et al.
However, with respect to loss of dopaminergic neurons, it is not fully resolved whether autophagy is beneficial or pathogenic. Keeping mitochondria healthy is a prerequisite for counteracting neurodegenerative diseases. Concepts include for instance maintaining mitochondrial membrane integrity and functionality.
Mitochondrial membrane permeabilization is tightly coupled to apoptosis induction Kroemer et al. Also anti-oxidants and ROS scavenging appear as beneficial strategies. Inhibitors of apoptosis are used as therapeutic drugs to inhibit neuronal loss. Anti-apoptotic drugs prevent mitochondrial apoptosis by blocking release of cytochrome c from the mitochondria or activation of pro-apoptotic BAX Westphal et al.
Alternatively, the activity or abundance of anti-apoptotic factors can be elevated. Mostly, AD is diagnosed as sporadic form by the age of 65 years and represents the primary cause of dementia within the elderly generation Seshadri et al. A decline in autophagy during aging further promotes the mitochondrial release of cytochrome c, which serves as pro-apoptotic stimulus.
Also decreased nucleolar volume has been detected in AD patients Iacono et al. In a similar manner, a decline of 28S rRNA was found in elderly healthy probes when compared to younger control groups Payao et al.
The patient data suggest that impairment of ribosome biogenesis and protein synthesis is one of the earliest events observed in the pathogenesis of AD characterized by mild cognitive impairment Ding et al. PD is characterized by loss of dopaminergic neurons in the brain stem.
Patients display tremor, dementia and depression. Also in PD, most cases are sporadic. Typical risk factors are aging and exposure to mitochondrial toxins.
A key feature of PD is deposition of Lewy bodies, which reflects deposition of α-Synuclein oligomers Ghavami et al. These oligomers trigger mitochondrial damage. Hereditary forms of PD involve mutation of the key mitophagy regulators PINK and Parkin.
Mutations of both result in impaired mitophagy. Given that α-Synuclein serves as a substrate of the E3-ubiquitin ligase Parkin, accumulation of α-Synuclein is also detected in a Parkin mutated background.
Also ER stress is implicated in PD. However, mild ER stress is attributed to function rather in a neuroprotective manner by inducing pro-survival autophagy. More recently, disruption of nucleolar integrity has been observed in human post mortem samples of patients with PD Rieker et al. In support of a nucleolar contribution, the ribosome biogenesis factor Nucleolin interacts with α-Synuclein.
Consequently, damaged mitochondria, ROS, as well as autophagosomes accumulate and cause apoptosis Rieker et al. Disruption of nucleoli, cell cycle arrest and pmediated apoptosis is observed by depletion of the transcription initiation factor IA TIF-1A , required for the recruitment of RNA pol I, in mouse embryonic fibroblasts Yuan et al.
Ablation of TIF-1A in DA neurons of mice results in Parkinsonism and progressive loss of DA neurons Rieker et al. Treatment with the neurotoxin MPTP worsens the effect of nucleolar stress. In this model, also p53 is stabilized and mTOR signaling decreased.
Finally, ROS-mediated oxidative stress is induced and defects are detected in mitochondria, such as impaired mitochondrial transcription and COX cytochrome c oxidase activity Rieker et al.
Therefore nucleolar stress, by inhibiting mTOR signaling, can impair mitochondrial function, which represents a key hallmark of several neurodegenerative diseases. To determine effects of specific PD mutations on nucleolar function irrespective of neuronal loss, pre-symptomatic, digenic PD models were analyzed.
Hemoglobin Hb is strongly expressed in dopaminergic neurons in the substantia nigra and is found in patient samples of AD and PD. Hb has recently been connected to mitochondrial function and apoptosis.
In turn, Hb overexpression impairs pre rRNA processing, induces nucleolar stress and sensitizes cells to apoptosis Codrich et al. The authors further demonstrate decreased phosphorylation of the mTOR target 4E-BP1, decreased numbers of lysosomes in neurons and decreased levels of LC3-II following rotenone treatment, being indicative for inhibition of autophagy.
Patients with HD display uncontrolled chorea movements and cognitive impairment Ghavami et al. The onset of age also inversely correlates with the increasing number of repetitive Glutamine motifs present in mutated Huntingtin. Interestingly, capture of mutant Htt inside inclusion bodies was shown to be less toxic in comparison to accumulating free mutant Htt Zuccato et al.
Also in HD patients, apoptosis and mitochondrial damage is detected. Additionally, studies have demonstrated that rRNA transcription is affected in HD Parlato and Kreiner, Triggering autophagy in mice models of HD can remove aggregates and increases their life span Zheng et al.
Targeted disruption of nucleoli by conditional knockout of TIF-1A essential for the recruitment of RNA pol I in striatal neurons results in striatal degeneration and typical HD-like phenotypic alterations in mice Kreiner et al.
TIF-1A loss induces nucleolar disruption and nucleolar stress, which precedes neurodegeneration. Nucleophosmin NPM represents a multifunctional, nucleolar key factor involved in ribosome biogenesis, which fulfills a plethora of pro-survival processes Colombo et al.
A down-regulation of NPM serves as readout for nucleolar stress induction and can be linked to neurodegeneration in several models of neurodegeneration Marquez-Lona et al. In line, as a key marker for nucleolar stress Colombo et al.
As an early and pdependent pro-survival response, the p53 target PTEN Stambolic et al. Given that the tumor suppressor PTEN counteracts the mTOR pathway, downstream targets of mTOR were analyzed for phosphorylation. It was found that p-S6 and p-4E-BP1 are reduced in the model.
Inhibition of mTOR is connected to activation of autophagy and the same holds true in the HD model Kreiner et al. Thus, transient over-activation of autophagy seems to be induced as initial, neuroprotective mechanism in response to impaired ribosome biogenesis.
However, after sustained nucleolar stress, apoptosis of striatal neurons is inevitable Kreiner et al. DNA damage and impaired rRNA transcription are connected to Cockayne Syndrome CS , which is a rare, congenital, autosomal-recessive neurodegenerative disorder Karikkineth et al.
Patients are characterized by premature aging, dwarfism, microcephaly, and have an average life expectancy of 12 years. CSB was found to localize to mitochondria and bind to mtDNA Aamann et al. CSB-deficient cells show increased ROS production, increased mitochondrial content and accumulation of damaged mitochondria, in line with impaired mitophagy Scheibye-Knudsen et al.
Further, CSB promotes acetylation of α-tubulin Majora et al. CSB deficiency abrogates autophagy and results in increased number of dilated lysosomes with impaired function Majora et al. In human CS cells, translational infidelity is observed, most likely due to accumulation of error-prone ribosomes as a consequence of impaired ribosome replacement.
CS cells exhibit ER stress and an over-activated unfolded protein response, which can be counteracted by addition of pharmacological chaperones Alupei et al. Epilepsy is characterized by recurrent seizures and represents a disease related to neurodegeneration.
Abrogated morphogenesis and synaptic function is observed upon nucleolar stress and could be connected to epilepsy reviewed in Hetman and Slomnicki, For example, pharmacologically induced short-term seizures in mice transiently affect RNA Pol I activity in hippocampi and result in decreased de novo synthesized 18S and 28S rRNAs.
In contrast, long-term seizures were associated with increased ribosome biogenesis Vashishta et al. Epilepsy is further tightly linked to mTOR hyper-stimulation and autophagy over-activation Cao et al. Strikingly, administration of the mTOR inhibitor rapamycin counteracts seizures and thus functions in an anti-epileptogenic manner Zeng et al.
Also rare, pediatric neurodegenerative diseases are characterized by alterations in autophagy. As an example, the multisystemic Vici syndrome is neurologically characterized by microcephaly and cognitive impairment. Accumulation of ubiquitinated autophagic cargo, p62 and damaged mitochondria is observed, reminiscent of neurodegeneration Ebrahimi-Fakhari et al.
However, whether also ribosome biogenesis is also altered here, remains to be determined. Key hallmarks of cancerous cells involve for instance mis-regulation of signaling pathways, rapid cell proliferation, accelerated tumor growth and inhibition of apoptosis Hanahan and Weinberg, , Large amounts of ribosomes are essential for cancerous cell growth and large nucleoli serve as prognostic marker in many tumor types Montanaro et al.
The nucleus arises as an essential target for cancer therapy Woods et al. Anti-tumor therapies utilize the high demand of cancer cells for the production of ribosomes by inhibiting RNA pol I.
Classical chemotherapeutics used in the clinics are for instance actinomycin D, 5-fluorouracil and metotrexat, which interfere with the nucleolar function Boulon et al. Novel drugs, which specifically impair rRNA transcription, are currently tested in clinical trials.
The small molecule drug CX specifically inhibits transcription of RNA pol I and stabilizes p53, whereas RNA pol II is not affected. Also, translation and DNA replication is not impaired in human cancer cell lines Drygin et al. The drug is further reported to impair proliferation in a pindependent manner in cancer cell lines, whereas the effect on normal cell lines is minimal Drygin et al.
Selective inhibition of RNA pol I by CX also robustly stimulates pro-death autophagy. Nucleolar stress and autophagy seem to be tightly coupled in distinct models and setups.
Recently, CX was loaded on a nanoplatform to enrich for nucleolar accumulation of the drug in order to enhance the anti-cancer effect, without causing significant side effects. In vivo and in vitro assays confirm induction of pro-death autophagy in HeLa cells, as well anti-proliferative and anti-tumor effects Duo et al.
Besides autophagy, CX induces also senescence in a pindependent manner. In U2OS osteosarcoma cells, CX induces G2 arrest, but not apoptosis Li et al. In response to CX, p53 accumulates and p21 is induced. In addition, increased levels of LC3-II are detected under basal conditions. Using TEM analysis, the authors noticed expanded vacuole-like structures filled with organelles, however, they report lack of clear identification of autophagosomal character.
Knockdown of p53 by siRNA rescues p21 up-regulation and LC3-II accumulation and increases cell survival. The authors conclude that CX triggers pdependent autophagy. The p53 target p21 is shown to be up-regulated during autophagy and a pindependent increase of p21 is reported in MNNG cells with mutant p53 Li et al.
Taken together, autophagy induction as a response to nucleolar stress seems to be an initial surveillance mechanism in several models. However, also in terms of cancer, autophagy induction can have two modes of action: Autophagy induction is clearly beneficial for cells by preventing genotoxic stress and DNA damage.
It removes cellular sources of ROS, such as defective mitochondria or proteins Mrakovcic and Frohlich, In contrast, inhibition of autophagy represents an oncogenic event.
At later stages over-activation of autophagy facilitates oncogenic drug-resistance. Autophagy inhibitors chloroquine and hydroxychloroquine have therefore been tested in clinical trials for cancer therapy Yang et al. Impairment of ribosome biogenesis is connected to a diverse class of human diseases collectively termed ribosomopathies Freed et al.
Several players associated with ribosomopathies have been described see also below. Classically, the nucleolar stress response and the tumor suppressor p53 are activated Freed et al.
Intriguingly, though, some phenotypes are common and include defects of the craniofacial cartilage, anemia and increased cancer susceptibility.
The elevated cancer risk appears paradoxical, given the great need of tumor cells for large amounts of ribosomes Montanaro et al. Accordingly, pathways and mechanisms might well exist, which let both co-exist Pfister and Kuhl, For example, specialized onco-ribosomes have recently been uncovered to increase the cellular fitness by mediating preferential translation of anti-apoptotic Bcl-2, as observed for the ribosome mutant RPLR98S in leukemia cells Xue and Barna, ; Sulima et al.
However, the question on cause and consequence of ribosomopathy-induced cancer formation is still under debate. Recently, examination of murine hepatocellular carcinoma and hepatoblastoma has revealed ribosomopathy-like features of nucleolar stress, such as deregulated expression of RPs and accumulation of unprocessed rRNA precursors.
Despite the fact that p53 is stabilized, no growth inhibition occurs Kulkarni et al. Therefore, compensating mechanisms might counteract apoptosis, involving up-regulation of anti-apoptotic Bcl-2, silencing of p19 ARF or cytosolic sequestration of p Those events would in turn inhibit the tumor suppressive mechanisms of cell cycle arrest and apoptosis in these cancers Kulkarni et al.
Clearly, ribosome biogenesis is a highly energy-consuming process. An implication of autophagy comes in mind, which might compensate for the low levels of functional ribosomes observed in ribosomopathies. Many important questions arise.
Is there a connection to bulk autophagy or selective autophagy of ribosomes ribophagy in ribosomopathy patients? Until now broad studies are lacking, which precisely address their implication in these issues.
An overview summarizing the emerging connection between nucleolar stress and autophagy in the diseases presented here is given in Table 1. Table 1. The role of nucleolar stress in mentioned diseases and effects on autophagy. Recently, inhibition of RNA pol I has been connected to autophagy, revealing that nucleolar stress functions upstream of autophagy.
In the following, evidence is collected, which links the ribosome biogenesis machinery and the nucleolus to autophagy, and vice versa. As a common principle, different groups suggest implication of mTOR signaling in nucleolus-mediated autophagy see below. Also here, pdependent and -independent pathways are being identified.
Besides the classical role of p53 as guardian of the genome, by mediating cell cycle arrest and apoptosis, p53 has been reported to exert distinct roles in autophagy Wang et al.
This depends on its subcellular localization: nuclear, cytosolic or mitochondrial, respectively. The effect of nuclear p53 as an inducer of autophagy mostly depends on its role as transcription factor. Nuclear p53 induces expression of ATGs thereby driving autophagy. In line, many promoters of autophagy related factors, such as ATGs or Parkin, are occupied by p53 Zhang et al.
The p53 target DRAM is directly involved in autophagosome formation. P53 further induces BAX and Bcl-2 or DAPK , which in turn induce Beclin1 Mrakovcic and Frohlich, Moreover, the p53 family members p63 and p73 induce expression of the autophagy regulators ATG5 and ATG7.
Also E2F, an important co-regulator of p53, is involved in the transcriptional regulation of autophagy related genes Polager and Ginsberg, ; Fullgrabe et al.
Note that other p53 family members p63 and p73 can, in principal, compensate for a loss of p53 Kenzelmann Broz et al. However, it remains to be determined, whether pindependent pathways actually depend on the role of p63 and p73 or whether they are unrelated.
Cytosolic p53 counteracts autophagy by transcription-independent mechanisms. P53 inhibits AMPK and activates mTOR, p53 further interacts with Beclin and induces Beclin ubiquitination and degradation Mrakovcic and Frohlich, Cytosolic p53 interacts with Parkin, which is the key regulator of mitophagy.
It was reported that p53 counteracts Parkin recruitment to mitochondria, thereby impairing mitophagy Hoshino et al. Mitochondrial p53 has a plethora of functions: it triggers MOM permeabilization, ROS production, mitophagy and autophagy and is therefore implicated in neuropathological conditions Marino et al.
mTORC1 inhibition by rapamycin abrogates the nucleolar stress response induced by low, cytostatic doses of the chemotherapeutic actinomycin D Goudarzi et al.
As a result, p53 stabilization and p21 induction is impaired. The authors observe decreased interaction of RPL11 with MDM2 upon rapamycin and actinomycin D co-treatment and suggest a mechanism related to the classical RPLMDM2-ppathway.
Also, they detected decreased RPL11 levels, as well as MDM2 stabilization, which might in part contribute to the rapamycin-mediated effects on p Interestingly, inhibition of mTOR by caffeine at physiologically relevant doses is capable of abrogating the actinomycin D-induced p53 response Goudarzi et al.
RNA pol I inhibition by actinomycin D or adriamycin is well known to trigger nucleolar disruption. Recently, the observation has been made that also autophagy can be induced with these drugs Katagiri et al. The same holds true for knocking down the RNA pol I transcription factors TIF1A and POLR1A.
Induction of autophagy has been monitored in flux experiments by counting LC3 punctae being indicative for autophagosomal number and determining levels of lipidated LC3-II representing the autophagosome-bound form of LC3.
Autophagy induction can be rescued by treating cells with autophagy inhibitors or knocking down key autophagy regulators ATG5 and Beclin1. In contrast, nucleolar disruption is not rescued Katagiri et al. Together, this finding places nucleolar disruption upstream of autophagy.
As nucleolar stress is generally characterized by redistribution of nucleolar factors such as NPM , or p53 stabilization, Katagiri et al.
They found that induction of autophagy by TIF1A knockdown is independent of p53, but depends on NPM. The induction of autophagy can be rescued by NPM knockdown, without reducing p53 levels.
In contrast, neither the depletion of NPM affects starvation-induced autophagy; nor does nutrient deprivation have an impact on nucleolar integrity. This suggests that NPM might rather play a role in a specialized form of nucleolar stress-induced autophagy, than starvation-induced bulk autophagy.
In contrast, PICT-1 deletion is linked to cancer formation and functions as oncogenic regulator of the E3-ubiquitin ligase MDM2 by preventing nucleolar release of RPL11 Sasaki et al.
Consequently, p53 stabilization, G1 cell cycle arrest and apoptosis are observed, thereby counteracting tumor growth Sasaki et al. Homozygous PICT1 deletion in mice is lethal and impairs survival of mouse ES cells.
PICT-1 binds to rDNA and the RNA pol I transcription factor upstream binding factor-1 UBF It inhibits transcription of rRNA, which depends on its localization to nucleoli Chen et al.
As the autophagy inhibitor 3-MA 3-methyladenine enhances cell death upon PICT-1 overexpression, the authors suggest induction of pro-death autophagy. Together, the authors conclude that PICT-1 overexpression triggers pro-death autophagy, without inducing the classical nucleolar stress response, such as p53 stabilization and nucleolar disruption Chen et al.
Recent findings suggest that autophagy might be affected in patients with ribosomopathies. mTOR signaling regulates a variety of essential cellular processes, among them autophagy. In leukocytes derived from patients with the ribosomopathy syndrome Shwachman Bodian Diamond Syndrome SBDS , a hyper-activation of mTOR phosphorylation is observed Bezzerri et al.
Also in intestinal epithelial cells autophagy is over-activated. Disrupted ribosome biogenesis by knocking down RPS19 ribosomal protein S19 is connected to the ribosomopathy syndrome Diamond Blackfan Anemia DBA.
RPS19 knockdown further affects autophagy in patient cells, and autophagy induction is also observed in red blood cells of zebrafish embryos Heijnen et al. RP-deficiency also recapitulates these effects in cells derived from SBDS. An increase in P-S6 as well as ROS is observed, whereas anti-oxidant treatment reverses p-S6, autophagy and p53 stabilization Heijnen et al.
Thus, the observed effects turn out to be ROS-dependent and suggest a contribution of oxidative stress in ribosomopathies. The zebrafish titania mutant tti s harbors a recessive, lethal mutation of the pwp2h gene encoding a factor of the small ribosomal subunit Boglev et al.
In this mutants reduced 18S rRNA, impaired ribosome biogenesis and p53 stabilization is observed. pwp2h is highly expressed in intestinal, epithelial cells, but also in the brain retinal pigmented epithelium, liver and pancreas, which are rapidly dividing tissues.
Also here, defects in craniofacial formation, typical hallmarks of ribosomopathies, can be detected. Intestinal epithelial cells of the mutant larvae display accumulation of autophagosomes.
In autophagic flux experiments using the autophagy inhibitor chloroquine and activator rapamycin, increased accumulation of LC3-II is observed in the mutants, indicating autophagy induction Boglev et al. Also p-RP-S6, reflecting mTORC1 activity, is increased.
Inhibiting autophagy by morpholino-mediated knockdown of ATG5 triggers apoptosis of intestinal epithelial cells specifically in the mutants, whereas the wildtype is not affected.
This suggests that autophagy induction counteracts apoptosis as survival mechanism in response to nucleolar stress. Also, no signs of apoptosis are detected in the mutants, ruling out toxic levels of autophagy activation.
Interestingly, autophagy induction in the zebrafish mutants occurs in mTOR and pindependent manner Boglev et al. However, the molecular mechanisms and pathways affected remain elusive. Nopp is in structure and function related to Treacle, representing an essential gene in the ribosomopathy syndrome Treacher Collins syndrome TCS Valdez et al.
Depletion of the nucleolar phosphoprotein Nopp in the imaginal wing disks of Drosophila results in nucleolar stress, loss of ribosomes and pindependent apoptosis James et al. They consider JNK as an interesting link, which has earlier been shown to induce autophagy in response to oxidative stress and induce transcription of ATG genes Wu et al.
In addition, oxidative stress induces accumulation of the autophagy marker GFP-LC3 and lysosomes in the intestinal epithelium, which is dependent on JNK signaling Wu et al. As also in larval polyploidy midgut cells mCherry-ATG8a is accumulated after Nopp depletion, the authors conclude an accumulation of autophagosomes and a premature induction of autophagy regulated by loss of Nopp James et al.
NAT10 drives ribosome biogenesis by mediating acetylation of the RNA pol I transcription factor UBF-1 and facilitating processing of the 18S rRNA Kong et al. Under normal conditions, NAT10 is auto-acetylates and promotes recruitment of PAF53 and RNA pol I to mediate rRNA biogenesis Cai et al.
The mechanisms, which link inhibition of rRNA biogenesis to induction of autophagy in response to energy stress were determined by Liu et al.
They demonstrate that NAT10 binds and acetylates the autophagy regulator Che1 AATF in a pindependent manner. As a consequence of acetylation, the transcriptional activation of target genes Redd1 and Deptor is off. Thus, Che1 enhances autophagy by activating the transcription of Redd1 and Deptor , two critical inhibitors of mTOR signaling Desantis et al.
Interestingly, Che-1 is also important for RNA pol II, the DNA damage response DDR and drives p53 expression. Upon DDR, Che1 increasingly interacts with p53 and drives the expression of genes implicated in cell cycle regulation, for instance p21 Desantis et al. Upon energy stress and glucose deprivation, Sirt1 deacetylates NAT ChIP analysis has demonstrated that deacetylated NAT10 does not bind to rDNA upon glucose deprivation and thus NATmediated ribosome biogenesis is inhibited.
Under this condition, the inhibition of Che1 is released Liu et al. In HCT cells, LC3-II levels are increased both in presence or absence of chloroquine, showing that NAT10 knockdown increases basal autophagic flux.
Also, p62 is reduced upon NAT10 depletion. In rescue experiments with HCTNATCas9 knockout clones, overexpression of NAT10 reverses the effects observed on p62 and LC3-II, whereas acetylation mutants fail to do so Liu et al.
mTOR, AMBRA1, and autophagy: an intricate relationship. Cell Cycle. Fimia GM, Di Bartolomeo S, Piacentini M, Cecconi F. Unleashing the Ambra1-Beclin 1 complex from dynein chains: Ulk1 sets Ambra1 free to induce autophagy.
Article PubMed Google Scholar. Vadlamudi RK, Joung I, Strominger JL, Shin J. p62, a phosphotyrosine-independent ligand of the SH2 domain of p56lck, belongs to a new class of ubiquitin-binding proteins. Bjørkøy G, et al. Giles NM, Gutowski NJ, Giles GI, Jacob C.
Redox catalysts as sensitisers towards oxidative stress. FEBS Lett. Embo J. Smith M, Wilkinson S. ER homeostasis and autophagy. Essays Biochem. Article PubMed PubMed Central Google Scholar.
Buchberger A, Bukau B, Sommer T. Protein quality control in the cytosol and the endoplasmic reticulum: brothers in arms. Mol Cell. Qi Z, Chen L. Endoplasmic reticulum stress and autophagy. Adv Exp Med Biol. Senft D, Ronai Z, et al. UPR, autophagy, and mitochondria crosstalk underlies the ER stress response.
Cheng Y-C, Chang J-M, Chen C-A, Chen H-C. Autophagy modulates endoplasmic reticulum stress-induced cell death in podocytes: a protective role. Exp Biol Med Maywood. Article CAS Google Scholar.
Kouroku Y, et al. Ogata M, et al. Autophagy is activated for cell survival after endoplasmic reticulum stress. Hoyer-Hansen M, et al. Control of macroautophagy by calcium, calmodulin-dependent kinase kinase-beta, and Bcl Yang Y, et al. Choi H, et al. Hypoxia promotes noncanonical autophagy in nucleus pulposus cells independent of MTOR and HIF1A signaling.
Wu H, et al. Mol Med Rep. Shi R-Y, et al. BNIP3 interacting with LC3 triggers excessive mitophagy in delayed neuronal death in stroke. CNS Neurosci Ther. Bellot G, et al.
Hypoxia-induced autophagy is mediated through hypoxia-inducible factor induction of BNIP3 and BNIP3L via their BH3 domains. Gwinn DM, et al. AMPK phosphorylation of raptor mediates a metabolic checkpoint. Rouschop KMA, et al.
The unfolded protein response protects human tumor cells during hypoxia through regulation of the autophagy genes MAP 1LC3B and ATG5. J Clin Invest. Song S, Tan J, Miao Y, Sun Z, Zhang Q. Cell Physiol Biochem. Peña-Oyarzun D, et al.
Hyperosmotic stress stimulates autophagy via polycystin Schutt KL, Moseley JB. Transient activation of fission yeast AMPK is required for cell proliferation during osmotic stress. Mol Biol Cell. Nunes P, et al. Hypertonic stress promotes autophagy and microtubule-dependent autophagosomal clusters.
Jiang LB, et al. Soto-Burgos J, Bassham DC. SnRK1 activates autophagy via the TOR signaling pathway in Arabidopsis thaliana. PLoS One. Pu Y, Soto-Burgos J, Bassham DC. Regulation of autophagy through SnRK1 and TOR signaling pathways.
Plant Signal Behav. Gatica D, Lahiri V, Klionsky DJ. Cargo recognition and degradation by selective autophagy. Nat Cell Biol. McEwan DG.
Host-pathogen interactions and subversion of autophagy. Sharma V, Verma S, Seranova E, Sarkar S, Kumar D. Selective autophagy and Xenophagy in infection and disease.
Front Cell Dev Biol. Thurston TL, Wandel MP, von Muhlinen N, Foeglein A, Randow F. Galectin 8 targets damaged vesicles for autophagy to defend cells against bacterial invasion.
Boyle KB, Randow F. The role of 'eat-me' signals and autophagy cargo receptors in innate immunity. Curr Opin Microbiol. Shaid S, Brandts CH, Serve H, Dikic I.
Ubiquitination and selective autophagy. Pankiv S, et al. von Muhlinen N, et al. LC3C, bound selectively by a noncanonical LIR motif in NDP52, is required for antibacterial autophagy. Wild P, et al.
Phosphorylation of the autophagy receptor optineurin restricts Salmonella growth. Tumbarello DA, et al. The autophagy receptor TAX1BP1 and the molecular motor myosin VI are required for clearance of Salmonella Typhimurium by autophagy.
PLoS Pathog. Morreale FE, Walden H. Types of ubiquitin ligases. van Wijk SJL, et al. Linear ubiquitination of cytosolic Salmonella Typhimurium activates NF-kappaB and restricts bacterial proliferation.
Nat Microbiol. van Wijk SJ, et al. Fluorescence-based sensors to monitor localization and functions of linear and Klinked ubiquitin chains in cells. Grumati P, Dikic I. Ubiquitin signaling and autophagy. Linares JF, et al. K63 polyubiquitination and activation of mTOR by the pTRAF6 complex in nutrient-activated cells.
Manzanillo PS, et al. The ubiquitin ligase parkin mediates resistance to intracellular pathogens. Huett A, et al. The LRR and RING domain protein LRSAM1 is an E3 ligase crucial for ubiquitin-dependent autophagy of intracellular Salmonella Typhimurium.
Cell Host Microbe. Noad J, et al. LUBAC-synthesized linear ubiquitin chains restrict cytosol-invading bacteria by activating autophagy and NF-kappaB. Polajnar M, Dietz MS, Heilemann M, Behrends C. Expanding the host cell ubiquitylation machinery targeting cytosolic Salmonella.
EMBO Rep. Stolz A, Ernst A, Dikic I. Cargo recognition and trafficking in selective autophagy. Kraft C, et al. FYCO1 is a Rab7 effector that binds to LC3 and PI3P to mediate microtubule plus end-directed vesicle transport.
Popovic D, et al. Rab GTPase-activating proteins in autophagy: regulation of endocytic and autophagy pathways by direct binding to human ATG8 modifiers. McEwan DG, et al. Genau HM, et al. Verlhac P, et al. Autophagy receptor NDP52 regulates pathogen-containing autophagosome maturation. Petkova DS, et al.
Distinct contributions of autophagy receptors in measles virus replication. Zheng YT, et al. J Immunol. Thurston TL, et al. Recruitment of TBK1 to cytosol-invading Salmonella induces WIPI2-dependent antibacterial autophagy.
Cheng CY, Chi PI, Liu HJ. Commentary on the regulation of viral proteins in autophagy process. Biomed Res Int. Perot BP, Ingersoll MA, Albert ML. Immunol Rev. Mao J, et al. Autophagy and viral infection. Chiramel AI, Brady NR, Bartenschlager R. Divergent roles of autophagy in virus infection.
Article Google Scholar. Joubert PE, et al. Autophagy induction by the pathogen receptor CD Meiffren G, et al. Pathogen recognition by the cell surface receptor CD46 induces autophagy. Mouna L, et al. Grégoire IP, Rabourdin-Combe C, Faure M.
Autophagy and RNA virus interactomes reveal IRGM as a common target. Petkova DS, Viret C, Faure M. IRGM in autophagy and viral infections. Front Immunol.
Abdoli A, Alirezaei M, Mehrbod P, Forouzanfar F. Autophagy: the multi-purpose bridge in viral infections and host cells. Rev Med Virol. Castedo M, et al. Sequential involvement of Cdk1, mTOR and p53 in apoptosis induced by the HIV-1 envelope.
Denizot M, et al. HIV-1 gp41 fusogenic function triggers autophagy in uninfected cells. Chan ST, Ou JJ. Hepatitis C virus-induced autophagy and host innate immune response. Choi Y, Bowman JW, Jung JU. Autophagy during viral infection - a double-edged sword. Nat Rev Microbiol.
Saito T, Owen DM, Jiang F, Marcotrigiano J, Gale M Jr. Innate immunity induced by composition-dependent RIG-I recognition of hepatitis C virus RNA. Li K, et al. Activation of chemokine and inflammatory cytokine response in hepatitis C virus-infected hepatocytes depends on toll-like receptor 3 sensing of hepatitis C virus double-stranded RNA intermediates.
García MA, et al. Impact of protein kinase PKR in cell biology: from antiviral to antiproliferative action. Microbiol Mol Biol Rev. Liang Q, et al.
Crosstalk between the cGAS DNA sensor and Beclin-1 autophagy protein shapes innate antimicrobial immune responses. Wang L, Ou JH. Hepatitis C virus and autophagy. Biol Chem.
Grégoire IP, et al. IRGM is a common target of RNA viruses that subvert the autophagy network. Su WC, et al. AMP-activated protein kinase and the regulation of autophagic proteolysis. Benito-Cuesta, I. AMPK activation does not enhance autophagy in neurons in contrast to MTORC1 inhibition: different impact on β-amyloid clearance.
Autophagy 17 , — Han, D. Joo, J. Hspcdc37 chaperone complex regulates ulk1- and atgmediated mitophagy.
Cell 43 , — Russell, R. ULK1 induces autophagy by phosphorylating Beclin-1 and activating VPS34 lipid kinase. Small molecule inhibition of the autophagy kinase ULK1 and identification of ULK1 substrates.
Cell 59 , — Park, J. The ULK1 complex mediates MTORC1 signaling to the autophagy initiation machinery via binding and phosphorylating ATG Autophagy 12 , — ULK1 phosphorylates Ser30 of BECN1 in association with ATG14 to stimulate autophagy induction.
Autophagy 14 , — Wold, M. Alsaadi, R. EMBO Rep. Zhou, C. Regulation of mATG9 trafficking by Src- and ULK1-mediated phosphorylation in basal and starvation-induced autophagy. Mercer, T. EMBO J. Wu, W. ULK1 translocates to mitochondria and phosphorylates FUNDC1 to regulate mitophagy.
Inoki, K. TSC2 mediates cellular energy response to control cell growth and survival. Cell , — Gwinn, D. AMPK phosphorylation of raptor mediates a metabolic checkpoint. Cell 30 , — Differential regulation of distinct Vps34 complexes by AMPK in nutrient stress and autophagy.
Myers, R. Systemic pan-AMPK activator MK improves glucose homeostasis but induces cardiac hypertrophy. Bjørkøy, G. Øverbye, A.
Proteomic analysis of membrane-associated proteins from rat liver autophagosomes. Autophagy 3 , — Article PubMed Google Scholar. Lelouard, H.
Dendritic cell aggresome-like induced structures are dedicated areas for ubiquitination and storage of newly synthesized defective proteins. Puente, C. Nutrient-regulated phosphorylation of ATG13 inhibits starvation-induced autophagy. Kroemer, G. Autophagy and the integrated stress response.
Cell 40 , — Codogno, P. Autophagy and signaling: their role in cell survival and cell death. Cell Death Differ. Shaw, R. The tumor suppressor LKB1 kinase directly activates AMP-activated kinase and regulates apoptosis in response to energy stress. Jeon, S. AMPK regulates NADPH homeostasis to promote tumour cell survival during energy stress.
Nature , — Greer, E. The energy sensor AMP-activated protein kinase directly regulates the mammalian FOXO3 transcription factor. Paquette, M. AMPK-dependent phosphorylation is required for transcriptional activation of TFEB and TFE3. Shin, H. AMPK-SKP2-CARM1 signalling cascade in transcriptional regulation of autophagy.
Weerasekara, V. Metabolic-stress-induced rearrangement of the ζ interactome promotes autophagy via a ULK1- and AMPK-regulated ζ interaction with phosphorylated Atg9. Zhao, Y. RACK1 promotes autophagy by enhancing the Atg14L-Beclin 1-VpsVps15 complex formation upon phosphorylation by AMPK.
Cell Rep. Xu, D. PAQR3 controls autophagy by integrating AMPK signaling to enhance ATG14L-associated PI3K activity. Thomas, H. Mitochondrial complex I activity is required for maximal autophagy. e Hamasaki, M. Autophagosomes form at ER-mitochondria contact sites.
Hailey, D. Mitochondria supply membranes for autophagosome biogenesis during starvation. Reggiori, F. Atg9 cycles between mitochondria and the pre-autophagosomal structure in yeasts.
Autophagy 1 , — Barini, E. Metformin promotes tau aggregation and exacerbates abnormal behavior in a mouse model of tauopathy. Son, S. Metformin facilitates amyloid-β generation by β- and γ-secretases via autophagy activation.
Alzheimers Dis. Imfeld, P. Moore, E. Increased risk of cognitive impairment in patients with diabetes is associated with metformin. Diabetes Care 36 , — Grunwald, D. GABARAPs and LC3s have opposite roles in regulating ULK1 for autophagy induction.
Autophagy 16 , — Jung, C. ULK-AtgFIP complexes mediate mTOR signaling to the autophagy machinery. Cell 20 , — Kim, Y. mTORC1 phosphorylates UVRAG to negatively regulate autophagosome and endosome maturation.
Cell 57 , — Kim, D. mTOR interacts with raptor to form a nutrient-sensitive complex that signals to the cell growth machinery. Jiang, P. Evaluation of AMPK activity in mice by measuring activator-induced glucose uptake. STAR Protoc. Liu, Q. Development of ATP-competitive mTOR inhibitors.
Methods Mol. Download references. We thank M. Kundu for ULK DKO MEFs; B. Viollet for AMPK KO MEFs; N. Bardeesy for LKB1 KO MEFs; S. Akira and T. Saitoh for Atg9a KO MEFs. Toso and M. Kyba for cell sorting. K has received research funding from the NIH R01GM and R35GM Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, Minneapolis, MN, , USA.
Institute for Diabetes, Obesity and Metabolism, University of Minnesota, Minneapolis, MN, , USA. Center for Immunology, University of Minnesota, Minneapolis, MN, , USA. Masonic Cancer Center, University of Minnesota, Minneapolis, MN, , USA.
You can also search for this author in PubMed Google Scholar. P and D. K conceived the project, designed and performed experiments, and wrote the manuscript. designed and performed experiments. Correspondence to Do-Hyung Kim. Nature Communications thanks the anonymous reviewer s for their contribution to the peer review of this work.
A peer review file is available. Open Access This article is licensed under a Creative Commons Attribution 4. Reprints and permissions. Park, JM. Redefining the role of AMPK in autophagy and the energy stress response. Nat Commun 14 , Download citation. Received : 10 November Accepted : 28 April Published : 24 May Anyone you share the following link with will be able to read this content:.
Sorry, a shareable link is not currently available for this article. Provided by the Springer Nature SharedIt content-sharing initiative. Naunyn-Schmiedeberg's Archives of Pharmacology By submitting a comment you agree to abide by our Terms and Community Guidelines.
If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.
Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily. Skip to main content Thank you for visiting nature. nature nature communications articles article. Download PDF. Subjects Macroautophagy Nutrient signalling Phosphorylation.
Abstract Autophagy maintains cellular homeostasis during low energy states. Introduction Glucose starvation poses a challenge to the survival of eukaryotic cells by causing energy crisis.
Results AMPK suppresses ULK1 signaling to the autophagy initiation machinery According to the widely accepted concept in the field, AMPK induces autophagy by phosphorylating ULK1 Ser mouse Ser 9. Full size image. Discussion Our study provides a rigorous demonstration that AMPK negatively regulates ULK1 activity and autophagy by phosphorylating ULK1.
Methods Primary antibodies Primary antibodies used for immunoprecipitation IP and western blotting WB were obtained and validated as described below. Chemicals, secondary antibodies, and other reagents The following materials were used in the experiments. Cell lines and culture conditions HCT CCL , HeLa CCL-2 , HEKT CRL , HepG2 HB , C2C12 CRL , and A cells CCL were obtained from ATCC.
Plasmid constructions and mutagenesis Myc-tagged constructs for ULK1 and Atg13 were made using pRK5 vector as described in our previous reports 21 , 22 , Genome editing to generate KO cells AMPKα DKO HCT cells, AMPKα DKO HeLa cells, and ULK DKO, ULK1 KO, Atg13 KO HEKT cells were generated using the CRISPR-cas9 assisted genome editing technique.
Lentiviral preparation, viral infection, and stable cell-line generation We used pLV-EF1a-IRES-lentivral vectors described above to generate cell lines stably transduced with exogenous DNA. In vivo test of AMPK inhibition of ULK1 and autophagy C57BL6J male mice were purchased from the Jackson Laboratories.
Immunostaining and fluorescence microscopy Phagophore and autophagosome formation was analyzed by immunostaining endogenous WIPI2 EMD-Millipore, MABC91; Sigma-Aldrich, SAB; dilution and LC3B MBL International, PM; dilution , respectively, as we described in our previous reports 21 , In vitro ULK1 kinase activity assay AMPKα DKO HEKT cells were transiently transduced with myc-ULK1 construct.
In vitro Vps34 kinase activity assay The lipid kinase activity of Atgassociated Vps34 was assayed as we have described previously 21 , Validation of ULK1 Thr as a direct phosphorylation site by AMPK HEKT cells deficient of ULK1 were transiently transduced to express myc-tagged ULK1 constructs.
qPCR analysis of autophagy gene expression RNAs were prepared from cells using TRIzol reagent ThermoFisher Scientific, Statistics and reproducibility The quantified outcomes were summarized as mean and SEM as specified in the figure legends.
Reporting summary Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article. Data availability All the data that support the conclusions in this paper are available within this article and its supplementary Information file.
References Lang, M. Article CAS PubMed PubMed Central Google Scholar Ramírez-Peinado, S. Article PubMed PubMed Central Google Scholar Weber, C. Article ADS CAS PubMed PubMed Central Google Scholar Schellens, J. Article CAS PubMed Google Scholar Plomp, P.
Article CAS PubMed Google Scholar Mijaljica, D. Article CAS PubMed Google Scholar Kim, J. Article CAS PubMed PubMed Central Google Scholar Egan, D.
Article ADS CAS PubMed Google Scholar Artal-Martinez de Narvajas, A. Article CAS PubMed PubMed Central Google Scholar Nwadike, C. Article CAS PubMed PubMed Central Google Scholar Shang, L. Article ADS CAS PubMed PubMed Central Google Scholar Mack, H. Article CAS PubMed PubMed Central Google Scholar Samari, H.
Article CAS PubMed Google Scholar Meley, D. Article CAS PubMed Google Scholar Benito-Cuesta, I. Article CAS PubMed Google Scholar Han, D. Article CAS PubMed Google Scholar Joo, J. Article CAS PubMed PubMed Central Google Scholar Russell, R.
Article CAS PubMed PubMed Central Google Scholar Park, J. Article CAS PubMed PubMed Central Google Scholar Wold, M. Article PubMed PubMed Central Google Scholar Alsaadi, R. Article PubMed PubMed Central Google Scholar Zhou, C.
Article CAS PubMed Google Scholar Mercer, T. Article CAS PubMed PubMed Central Google Scholar Wu, W. Article CAS PubMed PubMed Central Google Scholar Inoki, K. Article CAS PubMed Google Scholar Gwinn, D. Article CAS PubMed PubMed Central Google Scholar Kim, J.
Article CAS PubMed PubMed Central Google Scholar Myers, R. Article ADS CAS PubMed Google Scholar Bjørkøy, G. Article PubMed PubMed Central Google Scholar Øverbye, A. Article PubMed Google Scholar Lelouard, H.
Article CAS PubMed PubMed Central Google Scholar Puente, C. Article CAS PubMed PubMed Central Google Scholar Kroemer, G. Article CAS PubMed PubMed Central Google Scholar Codogno, P. Article CAS PubMed Google Scholar Shaw, R. Article ADS CAS PubMed PubMed Central Google Scholar Jeon, S.
Article ADS CAS PubMed PubMed Central Google Scholar Greer, E. Article CAS PubMed Google Scholar Paquette, M. Article ADS CAS PubMed PubMed Central Google Scholar Weerasekara, V. Article PubMed PubMed Central Google Scholar Zhao, Y.
Article CAS PubMed Google Scholar Xu, D. Article CAS PubMed PubMed Central Google Scholar Thomas, H. Article CAS PubMed PubMed Central Google Scholar Hamasaki, M. Article ADS CAS PubMed Google Scholar Hailey, D. Article CAS PubMed PubMed Central Google Scholar Reggiori, F.
Article CAS PubMed Google Scholar Barini, E. Article PubMed PubMed Central Google Scholar Son, S. Article CAS PubMed Google Scholar Imfeld, P. Article PubMed Google Scholar Moore, E.
Article CAS PubMed PubMed Central Google Scholar Grunwald, D. Article CAS PubMed Google Scholar Jung, C. Article CAS PubMed PubMed Central Google Scholar Kim, Y.
Article CAS PubMed Google Scholar Kim, D. Article CAS PubMed Google Scholar Jiang, P. Article CAS PubMed PubMed Central Google Scholar Liu, Q. Article CAS PubMed PubMed Central Google Scholar Download references. Acknowledgements We thank M.
View author publications. Ethics declarations Competing interests The authors declare no competing interests. Peer review Peer review information Nature Communications thanks the anonymous reviewer s for their contribution to the peer review of this work.
Supplementary information. Supplementary Information. Peer Review File. Reporting Summary. Source data Source Data. Rights and permissions Open Access This article is licensed under a Creative Commons Attribution 4.
About this article. Cite this article Park, JM. Copy to clipboard. Bonnet Anabela Palandri Marta E. Comments By submitting a comment you agree to abide by our Terms and Community Guidelines. Publish with us For authors For Reviewers Language editing services Submit manuscript.
Search Search articles by subject, keyword or author. Show results from All journals This journal. Advanced search. Close banner Close. Email address Sign up. I agree my information will be processed in accordance with the Nature and Springer Nature Limited Privacy Policy.
Get the most important science stories of the day, free in your inbox.
Strexs is Digestion support methods conserved pathway for the Atuophagy degradation of Mood boosting exercises components in all eukaryotes. This abd plays a critical role in Autophagy and stress response adaptation of plants to drastic changing environmental stresses such as starvation, oxidative stress, drought, salt, and pathogen invasion. This paper summarizes the current knowledge about the mechanism and roles of plant autophagy in various plant stress responses. Aubert S. J Cell Biol—
Ich meine, dass Sie betrogen haben.
Welche nötige Phrase... Toll, die prächtige Idee