PMC

(A) ROS/RNS production and signalling of autophagy. ROS and RNS production can be induced by protein aggregates, generated by the mitochondrial respiratory activities and by other cellular oxidases. These ROS/RNS species can modify signalling molecules to stimulate or inhibit autophagy. Atg4 Cys⁸¹ thiol modification is involved in starvation and H₂O₂-induced autophagy. S-nitrosation of IKKβ and JNK1 are involved in NO-induced inhibition of autophagy. Although many ROS/RNS have effects on autophagy, besides a handful of known ROS/RNS-targeted autophagy regulators, the exact mechanisms for the effect of ROS/RNS on autophagy are largely unknown. (B) Mitochondrial production of ROS/RNS. Regardless of their sources, H₂O₂ and NO can target to the mitochondria. Mitochondrially produced superoxide (O₂^•−) can react with NO to produce peroxynitrite (ONOOH). O₂^•−, H₂O₂ and ONOOH can further damage the mitochondria in combination with iron and produce lipid peroxidation (LPO) products, including HNE. MnSOD, aconitase, GPX (glutathione peroxidase) and catalase are involved in generation and reduction of these products. HNE, NO and H₂O₂ can modify proteins to initiate signalling mechanisms or damage. The most sensitive moieties to oxidative modification are the thiols on cysteine residues. Redox reactions in protein modification are involved in signalling of autophagy.

Mitophagy is the specific degradation of the mitochondria in response to global signals, including starvation and oxidative stress, or specific signals including mitochondrial targeting of signalling proteins or modification of mitochondrial proteins. In yeast, Atg32 is specific for mitophagy by targeting the mitochondria to the autophagosome. In mammalian cells, Nix is involved in mitochondrial clearance during erythrocyte maturation. In response to reduced cellular ATP, AMPK is activated and phosphorylates ULK1 and ULK2 (two Atg1 homologues) to activate both general macroautophagy and mitophagy. Parkinson's disease genes encoding α-synuclein, parkin, PINK1 and DJ-1 are all involved in mitophagy. A decrease in mitochondrial membrane potential (ψ_m) can be induced by ROS, and by targeting α-synuclein to the mitochondria. This decrease in mitochondrial membrane potential serves as a signal for mitophagy. In addition to a decrease in mitochondrial membrane potential, mitochondrial fission is another signal for mitophagy. PINK1 facilitates parkin targeting to the mitochondria, and ubiquitinates the mitochondrial outer membrane protein VDAC. Ubiquitinated VDAC can be recognized by p62 to initiate mitophagy. DJ-1 senses oxidative stress and serves a parallel pathway to maintain mitochondrial membrane potential and preserve mitochondria from fragmentation. Many of the regulators of mitophagy can be regulated by ROS. For example, α-synuclein is nitrated and, as a consequence, increases its aggregation propensity. Parkin can be sulfonated and S-nitrosated. Drp1 S-nitrosation is also involved in regulation of mitochondrial fission and associated induction of mitophagy.

There are three different mechanisms of autophagy: (i) macroautophagy, the bulk degradation of cargo; (ii) microautophagy, involution of the lysosome around cargo; and (iii) chaperone-mediated autophagy (CMA), the degradation of the tagged cargo. The autophagosome and the lysosome fuse to degrade the cargo by lysosomal acidic hydrolases, including cathepsins D, B and L. Macroautophagy is regulated by starvation through the Atg1–Atg13 complex, which is inhibited by mTOR activation. Rapamycin or nutrient starvation causes mTOR inactivation, subsequent Atg1–Atg13 activation and activates macroautophagy. The beclin-1–PI3K complex is involved in autophagosomal expansion. 3-MA inhibits PI3K activity. Atg8–pro-LC3 is cleaved by Atg4, modified by PE to become LC3-II and inserted into the autophagosomes. The puncta formed by LC3-II and the electrophoretic separation of LC3-I and LC3-II are used as a marker for autophagosomal accumulation. RFP–GFP–LC3 (tfLC3) form puncta with both red and green fluorescence in the autophagosomes, and only red puncta when the autophagosomes fuse with lysosomes where GFP is inactivated, therefore tfLC3 is used to monitor autophagic flux. The degradation rate of long-lived proteins is another method for measurement of autophagic flux. p62 binds both LC3 and ubiquitin and therefore plays a role in targeting ubiquitinated proteins to the autophagosomes. Red arrows are indicative of negative regulators of autophagy, and green arrows are indicative of positive regulators of autophagy. Atg4 Cys⁸¹ thiol modification is involved in starvation and H₂O₂-induced autophagy. S-nitrosation of IKKβ and JNK1 are involved in NO-induced inhibition of autophagy.