Oxidative stress and autophagy: the clash between damage and metabolic needs

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. A growing amount of evidence in recent years argues for oxidative stress 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. This review aims at providing novel insight into the regulatory pathways of autophagy in response to glucose and amino acid deprivation, as well as their tight interconnection with metabolic networks and redox homeostasis. The role of oxidative and nitrosative stress in autophagy is also discussed in the light of its being harmful for both cellular biomolecules and signal mediator through reversible posttranslational modifications of thiol-containing proteins. The redox-independent relationship between autophagy and antioxidant response, occurring through the p62/Keap1/Nrf2 pathway, is also addressed in order to provide a wide perspective upon the interconnection between autophagy and oxidative stress. Herein, we also attempt to afford an overview of the complex crosstalk between autophagy and DNA damage response (DDR), focusing on the main pathways activated upon ROS and RNS overproduction. Along these lines, the direct and indirect role of autophagy in DDR is dissected in depth.

Figure 1

Main molecular pathways activated in the presence or absence of nutrients. (a) The synergic import of leucine (Leu) and glutamine (Gln) (top left) results in mTORC1 recruitment to the lysosomal membrane and its subsequent activation by at least two distinct pathways. The first one proposes that cytosolic amino acids enter the lysosome and signal their presence to RAG-A and RAG-C (or RAG-B and RAG-D, not shown in the figure) through the lysosome-located proton pump v-ATPase and the multimolecular complex called Ragulator. The second pathway provides for the double deamination of Gln catalysed by the enzymes glutaminase (GLN) and glutamate dehydrogenase (GDH). This sequence of reactions subsequently generates glutamate (Glu) and α-ketoglutarate (αKG) that, by acting as co-substrate for prolyl hydroxylases (PHD), finally leads to RAG activation. GTP-bound RAG-A (or B) and GDP-bound RAG-C (or D) can then recruit mTORC1 to the lysosome membrane where it is activated by RHEB (middle left). Once activated, mTORC1 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 glucose-6-phosphate (G6P) by hexokinase (the only mitochondrial isoform II, HKII, is shown in the top right side of the figure). G6P is then isomerized to fructose-6-phosphate (F6P), oxidized 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) through the oxidative phosphorylation (OXPHOS). G6P can also undergo oxidation via the glucose-6-phosphate dehydrogenase (G6PDH)-mediated catalysis along the pentose phosphate pathway (middle right). In this way, electrons required for NADP⁺-to-NADPH reduction, and sugars needed for DNA de novo synthesis (e.g., ribulose-5-phosphate, R5P) are also provided (bottom right). (b) Upon amino acid deprivation, RAGs exchange nucleotides located in their binding sites (GTP with GDP or vice versa), thus leading to mTORC1 release from the 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 (especially in regenerating the reduced thiol pool) (middle right) and in energetic stress that the cell attempts to counteract by the adenylate kinase (AK)-mediated conversion of ADP into ATP and AMP (centre). AMP increase induces to the activation of AMPK that inhibits protein synthesis by phosphorylating TSC2 and mTORC1 and activates autophagy by phospho-activating ULK1. Once activated, ULK1 phosphorylates its interactors (Atg13 and FIP200) 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.g., Atg9) contribute to phagophore elongation and autophagosome formation (bottom)

Figure 2

Crosstalk between autophagy and oxidative stress. Superoxide (O) and H₂O₂ are the main ROS produced by mitochondria upon nutrient deprivation. They positively regulate autophagy by means of at least three different mechanisms, including: (1) S-glutathionylation (SH → S-SG) of cysteines located in the α and β subunits of AMPK (top right); (2) oxidation of Cys81 (SH → Sox) of Atg4 that in turn leads to the inactivation of its ‘delipidating' activity on LC3 and to the accumulation of the pro-autophagic LC3-II isoform (top centre); and (3) wide alteration of thiol redox state (e.g., decrease of GSH/GSSG ratio and general increase of oxidized thiols, Sox) that is facilitated by the release of reduced glutathione (GSH) to the extracellular milieu through the multidrug resistance protein 1 (MRP1) (top left). In a redox-independent manner, it has also been demonstrated that p62, when bound to ubiquitylated protein aggregates, can undergo phosphorylation on Ser351, 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)

Figure 3

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. Cells were then subjected to two different autophagic stimuli: (1) they were treated for 6 h with 5 μM of the proteasome inhibitor MG132 or, alternatively, (2) allowed to grow for 6 h in a nutrient-deprived cell medium (stv). 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

Figure 4

Implication of autophagy in DNA damage repair. Endogenous (e.g., dysfunctional mitochondria, top right) or exogenous (e.g., radiations or genotoxic stimuli, bottom left) sources of ROS and RNS induce DNA damage, whose primary sensors are PARP1 and ATM. Once activated by DNA breaks, PARP1 catalyses poly-ADP ribosylation of itself, as well as of other nuclear proteins, thereby leading to a massive decrease of NAD⁺ and to a subsequent energetic stress (bottom left). Upon DNA damage, ATM can activate p53-mediated 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)