Two primary types of ROS have been described: 1) superoxide radicals (O2*-) and hydroxyl radicals (OH-) and other oxygen free radicals, and 2) non-radical derivatives of oxygen, such as hydrogen peroxide (H2O2) (Lushchak, 2014). One of the primary cellular sources of ROS is the mitochondrial inner membrane respiratory chain. Ninety percent of ROS are generated when electrons escape from the mitochondrial electron transport chain (ETC). The ETC is composed of transmembrane protein complexes (I-IV) and ubiquinone and cytochrome c (electron transfer carriers); when these complexes are assembled, together with complex V (F₁F₀ATP synthase), the oxidative phosphorylation occurs resulting in ATP production (Villalpando-Rodriguez and Gibson, 2021). Mitochondrial superoxide dismutase 2 (SOD2) is the main component of the ROS processing mechanism in the mitochondrial matrix (Palma et al., 2020). SOD2 is a ubiquitous enzyme in organisms that live in the presence of oxygen, catalyzing the conversion of superoxide into oxygen and H2O2 (Wang et al., 2018), thereby antagonizing and detoxifying ROS. Other proteins that antagonize and detoxify ROS include catalase (Nishikawa et al., 2009), peroxidase (Kidwai et al., 2020), glutathione (Zhang et al., 2020a), and thioredoxin (Ren et al., 2017). Studies have shown that mitochondrial uncoupling plays a role in protecting cells under oxidative stress conditions, including obesity, aging, drug resistance in tumor cells, neurodegeneration, and ischemia-reperfusion damage (Rai et al., 2021). These enzymes are abundant in phagocytes and transfer electrons through protons to generate ROS (Gobert and Wilson, 2017). More importantly, ROS can also be produced in the endoplasmic reticulum and peroxisomes and through the activity of xanthine and endothelial oxidase (Sarniak et al., 2016).

Another important cell source is nicotinamide adenine dinucleotide phosphate oxidase (NOX). NOX is a transmembrane protein specifically involved in generating ROS. Seven subtypes of the NOX family have been identified and characterized: NOX-1, NOX-2 (also known as gp91phox), NOX-3, NOX-4, NOX-5, dual oxidase (DUOX)-1, and DUOX-2 (Chocry and Leloup, 2020). Numerous studies have shown that ROS induce autophagy through NOX. Under different cellular conditions, NOX-derived ROS can regulate autophagy. For example, NOX-2 stimulates autophagy in human macrophages, and the subsequently generated ROS increase the level of LC3-II bound to phagosomes through a toll-like receptor–mediated pathway (Fatima et al., 2021). During Aspergillus fumigatus infection, NOX induces macrophage autophagy by generating ROS (Hu et al., 2015). NOX also regulates autophagy in vascular endothelial cells. NOX inhibition was shown to inhibit autophagy in pulmonary artery endothelial cells, resulting in decreased binding of gp91^phox-p47^phox and NOX activity, thus improving pulmonary artery angiogenesis (Teng et al., 2012). NOX-4 is highly expressed in the adventitia and endothelial layers of blood vessels, where it induces ROS-mediated vascular remodeling by regulating autophagy (Barman et al., 2014). In addition, silencing of NOX-2 or NOX-4 has a similar effect on endothelial cell apoptosis and proliferation, possibly due to the common P22^phox subunit of both NOX isoforms in ROS-dependent regulation (Djordjevic et al., 2005).

NOX-derived ROS also regulate autophagy in different organs, such as the heart, liver, and lung. NOX is the primary mediator of ROS production in the heart, and NOX-2 deficiency leads to dysplasia of the endocardial cushion and valvoseptal, which is associated with decreased expression of Gata4, Tgfβ2, Bmp2, Bmp4, and Snail1 (Moazzen et al., 2020). NOX-4 plays a major role in mediating autophagy and survival of cardiomyocytes during energy stress by triggering ROS production in the endoplasmic reticulum and stimulating the protein kinase RNA-activated–like endoplasmic reticulum kinase/eukaryotic initiation factor 2α/activating transcription factor 4 (PERK/eIF-2α/ATF4) signaling pathway (Sciarretta et al., 2013). Alamandine induces the migration of hepatic stellate cells and diminishes collagen synthesis by regulating autophagy induced by NOX4-dependent ROS, thereby attenuating hepatic fibrosis (Huang et al., 2020). Moreover, NOX-4 activity correlated with an increase in superoxide formation. NOX-4 forms a complex with p22phox after injury, forming free radicals at neuronal membranes. Alpha-lipoic acid is an oxidative stress inhibitor, which can acutely prevent the development of superoxide, significantly reduce the content of NOX4 and p22phox, and increase the antiapoptotic markers B-cell lymphoma 2 (Bcl-2) and heme oxygenase 1(HO-1) after single blast (Lucke-Wold et al., 2015). Furthermore, the Nox4 pathway produces hydrogen peroxide and superoxide radicals that damage mitochondria, cellular DNA, proteins, and lipid membranes (Lobo et al., 2010). An increase in NOX activity contributes to the increased autophagy in persistent pulmonary hypertension (PPHN)–pulmonary artery endothelial cells (PAECs), whereas the increase of autophagy can reciprocally promote the formation of ROS through NOX-2, leading to impaired angiogenesis involving PPHN-PAECs (Teng et al., 2012). NOX-1/NOX-4 inhibitors significantly reduce levels interferon-α (IFN-α) and IFN-β, thereby attenuating acute lung injury induced by ischemia-reperfusion in mice (Cui et al., 2018).

There is also a significant body of literature indicating that NOS is involved in the regulation of autophagy. In mammals, three distinct genes encode NOS isoenzymes, neuronal (nNOS or NOS-1), inducible (iNOS or NOS-2), and endothelial (eNOS or NOS-3) (Thomas et al., 2008). nNOS is present only in peripheral nerves (Gao et al., 2008), whereas eNOS is present in vascular endothelial cells (Siragusa and Fleming, 2016). iNOS is primarily produced by mesenchymal and parenchymal cells under pathologic conditions following stimulation by endotoxin and cytokines and expressed under transcriptional and post-transcriptional control (Carnovale and Ronco, 2012). NOS-1 prevents excessive autophagy and promotes the survival of nasopharyngeal carcinoma cells through S-nitrosylation of phosphatase and tensin homolog (PTEN) and activation of the AKT/mTOR signaling pathway (Zhu et al., 2017). Overexpression of eNOS and iNOS (but not nNOS) in liver cancer tissues contributes to increase NO levels in liver cancer patients, which in turn inhibit autophagy by disrupting the binding of BECN1/VPS34 and promoting apoptosis by increasing the B cell lymphoma-2 (BCL-2)/BECN1 interaction in liver cancer cells (Zhang et al., 2019a). Silica nanoparticles interfere with the NO/NOS system by inhibiting the PI3K/AKT/mTOR pathway, thereby reducing the activity of eNOS and significantly increasing the activity of iNOS, inducing inflammation and activating autophagy, which ultimately leads to endothelial dysfunction (Duan et al., 2014).

Existing research data show that ROS plays a crucial role in autophagy activation. Whether it is the result of autophagy survival or death, or the initiation conditions starvation, pathogen or death receptor, ROS is invariably involved in it. However, the nature of such participation remains unclear. Interestingly, although the link between ROS and autophagy has been observed under different pathological conditions, the activation mode of autophagy and its potential protective effect are still not completely clear. This part summarizes the latest internal mechanism of ROS regulating autophagy under relevant pathological conditions.

In the response to ROS damage, autophagy is essential for the clearance of damaged mitochondria. In particular, the ubiquitin (Ub)-proteasome system (UPS), which eliminates oxidized proteins, is considered the primary mechanism. The majority of misfolded proteins are degraded by the UPS, in which Ub-conjugated substrates are deubiquitinated, unfolded and cleaved into small peptides when passing through the narrow chamber of the proteasome. The substrates that expose a specific degradation signal, the KFERQ sequence motif, can be delivered to and degraded in lysosomes via the CMA. Aggregation-prone substrates resistant to both the UPS and the CMA can be degraded by macroautophagy, in which cargoes are segregated into autophagosomes before degradation by lysosomal hydrolases (Ciechanover and Kwon, 2015).

Known players or pathways that link the UPS to autophagy include the N-terminal arginylation of the N-end rule pathway, the unfolded protein response, and p53. The N-end rule pathway mediates autophagic proteolysis and modulates the activity of the autophagic adaptor p62 as well as autophagosome biogenesis. In this crosstalk, the accumulation of autophagic cargoes such as ubiquitinated proteins and aggregates induces the N-terminal arginylation of ER-resident chaperones such as BiP/GRP78, calreticulin, and protein disulfide isomerase, resulting in the cytosolic accumulation of their arginylated species. Amongst these, arginylated BiP, R-BiP, is associated with misfolded proteins, and its N-terminal Arg binds the ZZ domain of p62. This N-end rule interaction induces a conformational change of p62, exposing its PB1 and LC3-interacting domains, which accelerates its self-oligomerization and targeting to autophagosomes (Cha-Molstad et al., 2017). In the UPS-ER-autophagy circuit, proteasome inhibition causes the excessive accumulation of misfolded proteins in the ER lumen, which in turn leads to the dissociation of the molecular chaperone BiP from the ER membrane receptors PERK, IRE1α and ATF6α. The activated receptors initiate the UPR to temporarily halt global translation and facilitate the synthesis of ER-residing molecular chaperones. These recruited chaperones assist the folding of incoming ER clients and the degradation of terminally misfolded clients through ERAD. In parallel, each of PERK, IRE1α and ATF6α on the ER cytosolic surface activates its own downstream pathways toward autophagy (Hetz et al., 2015). Proteasome inhibition can induce autophagy via the metabolic stabilization of the transcriptional factor p53 (Lagunas-Martínez et al., 2017).

In fact, chaperone-mediated autophagy (CMA) can only degrade cytosolic proteins which contain a CMA-targeting motif, biochemically related to the pentapeptide KFERQ. The CMA pathway degrades and thus regulates specific cell proteins in a timely manner. In the case of chronic nutrient deficiency, the CMA pathway is responsible for the degradation of 30% of cytoplasmic proteins (Dice, 2007). The chaperone heat shock cognate 70 kDa (HSC70) and LAMP-2A are key components of CMA (Losmanová et al., 2020). CMA was the first lysosomal process discovered by which cellular proteins are selectively degraded. The process involves the identification, folding, translocation, and degradation of substrate proteins (Yang et al., 2019). Proteins are the only substrate degraded by this pathway, but not all proteins can be degraded by CMA. To be a CMA substrate, the amino acid sequence of a protein must include a specific targeting motif. This motif binds to HSC70 to bring the substrate protein into the lysosome for degradation (Kaushik and Cuervo, 2018). HSC70 recognizes pentapeptide motifs (KFERQ-like motifs) that are essential for the degradation of CMA substrate proteins in lysosomes (Kirchner et al., 2019). To date, HSC70 remains the only molecular chaperone that has been shown to directly bind to KFERQ-like motifs. KFERQ-like motifs serve a “bait” role to attract HSC70 as a cytoplasmic partner. After HSC70 binds to this region, the protein is targeted for lysosomal degradation (Chiang et al., 1989). In general, HSC70 participates in a variety of cellular functions by promoting the folding of unfolded or misfolded proteins. However, HSC70 also binds KFERQ-like motifs to target proteins for degradation via CMA. HSC70 also participates in the selective degradation of ubiquitin-positive protein aggregates (Arndt et al., 2010). Direct interaction between gastrokine-2 and HSC70 promotes ROS-induced mitochondrial dysfunction, inhibits the NF-κB signaling pathway, and activates the JNK signaling pathway, ultimately leading to an increase in the level of apoptosis (Zhang et al., 2019b). HSC70-based autophagy peptides effectively reduce the levels of Aβ oligomers in cortical neurons in AD patients and protect cortical neurons from Aβ oligomer–induced neurotoxicity (Dou et al., 2020). Dynamic interaction between USP14 (a de-ubiquitination enzyme) and the chaperone HSC70 mediates crosstalk between ERS and autophagy. Regulation of the USP14-HSC70 axis is therefore a potential therapeutic approach for Huntington disease, as it would beneficially affect a variety of protein homeostasis pathways (Srinivasan et al., 2020).

Interestingly, the induction of oxidative stress by CMA can also occur via an increase in the transcription of the LAMP-2A gene (Kiffin et al., 2004), whereas activation of CMA by nutrient deprivation depends on a reduction in LAMP-2A degradation and relocation of the receptor on the lysosomal membrane (Cuervo and Dice, 2000). LAMP-2A was the first lysosomal component required for CMA to be identified (Majeski and Dice, 2004). The level of LAMP-2A in the lysosomal membrane can be increased by reducing its degradation and/or promoting its redistribution from the lysosomal cavity to the lysosomal membrane. LAMP-2A is a transmembrane protein that can translocate proteins to lysosomes (Rout et al., 2014). Transport of LAMP-2A is regulated by cystine, Rab11, and Rab-interacting lysosomal protein, and the treatment of cells in cystinosin deficiency with CMA activator increases the localization of LAMP-2A in lysosomes and increases the survival rate of the cells (Zhang et al., 2017). Expression of DYNC1LI2 (LIC2) preserves LAMP-2A and CMA activity, cell homeostasis, and the localization of LRP2/megalin in cystinotic proximal tubular cells in cystine disease. Decreased expression levels of chaperone heat shock protein A5 (HSPA5)/glucose-regulated protein 78 (GRP78) and the transcription factors ATF4 and DDIT3–C/EBP homologous protein (CHOP) are related to ERS (Rahman et al., 2021). LAMP-2A activation of CMA requires the involvement of p38 MAPK. ER stressors induce PERK-dependent activation and mitogen-activated protein kinase 4 recruitment to lysosomes, in turn activating p38 MAPK at lysosomes. The p38 MAPK directly phosphorylates the CMA receptor LAMP-2A at T211 and T213, resulting in accumulation in the membrane and the induction of active conformational changes that ultimately activate CMA (Li et al., 2017).

The two-domain architecture of the lumenal domains of LAMP-2A mediates the interaction with HSC70 at the cytoplasmic surface of the lysosome (Ikami et al., 2022). In addition to HSC70, other co-molecular chaperones along with LAMP-2A are involved in mediating the translocation of CMA substrates across the lysosomal membrane, such as HSP90 and HSP40 (Agarraberes and Dice, 2001). An increase in sirtuin 3 (an NAD⁺-dependent deacetylase) expression stimulates formation of the LAMP-2A/HSC70/perilipin-2 complex and promotes CMA (Zhang et al., 2020b). A lack of sorting nexin 10 inhibits cathepsin-A maturation and increases the stability of LAMP-2A, leading to increased CMA activity (You et al., 2018). LAMP-2A effectively prevents the accumulation of the autophagy defect marker Ref (Levine and Klionsky, 2017)P/p62 in adult brain under acute oxidative stress. Studies have shown that LAMP-2A can also enhance autophagic flux in the Drosophila brain PD model, thereby enhancing anti-stress responses and exerting neuroprotective effects (Issa et al., 2018). In a PD model, geniposide exhibited neuroprotective effects by inhibiting α-synuclein (α-Syn) expression via the miR-21/LAMP-2A axis (Su et al., 2016). The transcription factor NFE2L2/NRF2 (nuclear factor, erythroid 2 like 2) modulates CMA via the regulation of LAMP-2A (Pajares et al., 2018). In addition, as NFE2L2/NRF2 also regulates macroautophagy genes (Pajares et al., 2016), it functions as a regulatory node in the proteolytic network involving macroautophagy and CMA.

The X-box binding protein 1 (XBP1) is not only an important component of the UPR, but also an important nuclear transcription factor. XBP1 is a member of the CEB/P family of transcription factors that bind to UPR elements (UPRE) on their target genes (Lee et al., 2003). XBP1 is the downstream target of IRE1α. Under the ERS, XBP1 is spliced by IRE1α, resulting in functional spliced XBP1 (XBP1s). XBP1s initiates transcription programs by translocation into the nucleus, which regulate UPR involved in the pathophysiology of various diseases (Wu et al., 2015). XBP1s occupies the −743 to −523 site of the promoter of TFEB, a major regulator of autophagy and lysosomal biogenesis. In mouse liver, the deletion of XBP1 inhibited the transcription of TFEB and autophagy, while the overexpression of XBP1s enhanced TFEB transcription as well as autophagy (Zhang et al., 2021). Furthermore, XBP1 regulated autophagy through the interaction of FOXO1 under ERS condition (Kishino et al, 2017). In the low oxidative stress group, FOXO1 was exported from the nucleus, modified into Ac-FOXO1 and bound to ATG7 in the cytoplasm, thereby promoting autophagy to protect cells. In the high oxidative stress group, FOXO1 was located in the nucleus, promoted the transcription of proapoptotic proteins, and then induced apoptosis (Lu et al., 2021). Therefore, FOXO1 can be used as a redox sensor switch to regulate the transformation of autophagy and apoptosis.

The cytoplasmic part of IRE1 bound tumor necrosis factor receptor-associated factor 2 (TRAF2), an adaptor protein that couples plasma membrane receptors to JNK activation (Urano et al., 2000). IRE1 participates in the activation of JNK by forming TRAF2-ASK1 complex, thereby inducing the formation of autophagy, which required BECN1 transcription by c-Jun (Liu et al., 2020b). High glucose and bupivacaine induced cytotoxicity in SH-SY5Y cells through enhancing cell apoptosis and inhibiting autophagy via the IRE1-TRAF2 signaling pathways (Liu et al., 2019a). CHIP (carboxyl terminus of HSC70-interacting protein) mediated ubiquitination of IRE1 contributes to the dynamic regulation of UPR, which requires the involvement of IRE1/TRAF2/JNK pathway (Zhu et al., 2014). The ketamine enhanced autophagy and ERS in vivo and in vitro via restraining IRE1/TRAF2/ASK1/JNK pathway (Yu et al., 2021). In addition, Wogonoside exhibited antitumor activity by inducing ERS-associated autophagy and cell death by the IRE1α/TRAF2/ASK1 pathway (Gu et al., 2021).

When ERS triggers the UPR, the PERK signaling pathway is activated upon PERK dimerization and autophosphorylation (Walter et al., 2015). The nuclear factor-erythroid-2-related factor 2 (NRF2) is the main regulator of HIF-1α and can be activated as a downstream effector of PERK signaling (Ma et al., 2018). In addition, HIF-1α is considered to be the key effector of the cellular response to hypoxia, while NRF2 is the main antioxidant transcription factor and the key factor essential for HIF-1α-mediated hypoxia response (Mahajan and Sitasawad, 2021). Autophagy induced by HIF-1α/BNIP3 signaling pathway plays a protective role in myocardial ischemia-reperfusion injury (Zhang et al., 2019c). In the neonatal corpus luteum, HIF-1α up regulates BNIP3 expression, promotes the initiation of autophagy by disrupting BECN1 of BCL-2/BECN1 complex, and protects cells from apoptosis by curbing the skew of mitochondria balance under avascular niche (Tang et al., 2020). Berberine mediates BNIP3 expression by enhancing the binding of HIF-1α to the BNIP3 promoter, thereby inducing cardiomyocyte proliferation and autophagosome formation to inhibit cardiomyocyte apoptosis and myocardial ischemia/reperfusion injury (Zhu et al., 2020).

The ATF6α branch of the UPR is the least understood aspect of ERS and autophagy. ATF6α primarily regulates the expression of ER resident proteins involved in the maturation and degradation of ER client proteins (Bommiasamy et al., 2009). The binding immunoglobulin protein (BIP)/glucose-regulated protein 78 (GRP78) through ATF6α regulation increases the folding capacity of ER protein, contributing to resolution of the stress, and helps reverse UPR activation (Yu et al., 2016). ATF6α participates in the initiation of autophagy by up-regulating the expression of the BIP and inhibiting the expression of PKB of the AKT1/AKT pathway (Yung et al., 2011). Interestingly, crosstalk between apoptosis and autophagy can be regulated by the arginylated BIP/BECN1/p62 complex. Dephosphorylation of BECN1 increases the cleavage of BECN1 and destroys the BIP/BECN1/p62 complex, which leads to autophagy to the synergistic induction of apoptosis (Song et al., 2018).

With the discovery of UPR components and the elucidation of its mechanism, many studies have shown that its signaling pathway plays an important role in the process of nervous system diseases. By establishing blast-induced traumatic brain injury, administration of the ERS inhibitor salubrinal (SAL) found that SAL reduced CHOP protein expression and diminished Caspase-3 cleavage, suggesting attenuation of apoptosis. Interestingly, SAL also ameliorated impulsive-like behavior indicative of head trauma (Logsdon et al., 2014). The Neuro-2a cells and primary cultured mice cortical neurons were subjected to oxygen-glucose deprivation and reperfusion (OGD/R). Urolithin A (Uro-A) alleviated OGD/R-induced injury in Neuro-2a cells and neurons and reduced ischemic brain injury in mice. And Uro-A reinforced ischemia-induced autophagy. Furthermore, Uro-A-conferred protection was abolished by 3-methyladenine, suggesting the requirement of autophagy for neuroprotection (Ahsan et al., 2019). Exposure of Neuro-2a cells to MeHg also triggered ERS. Pretreatment with the antioxidant N-acetylcysteine (NAC) effectively prevented MeHg-induced neuronal cell ROS generation, apoptotic and ERS-related signals, and Akt inactivation (Chung et al., 2019a). These observations provide evidence UPR is a promising molecular target for the attenuation of neuronal injury.

In type 1 mitophagy, PI3K, BECN1, ULK1, and other proteins for phagocytes along with LC3, the mammalian ortholog of yeast ATG8 (Li et al., 2011; Lemasters, 2014; Yamano and Youle, 2020). Phagocyte formation involves the growth of small pre-autophagic structures (PAS) containing LC3 associated with mitochondria. PAS expand to surround and sequester individual mitochondria into mitophagosomes, which requires the participation of PI3K and usually occurs in conjunction with mitochondrial fission (Lemasters and Zhong, 2018). After sequestration is completed, the outer compartment of the mitophagosome (the space between the inner and outer autophagosomal membranes) is acidified, followed by depolarization of the mitochondria. Finally, mitochondrial phagosomes containing depolarized mitochondria fuse with lysosomes (or late endosomes) to form autolysosomes, in which hydrolytic digestion of mitochondria occurs (Lemasters, 2014). Mitophagy induced by nutrient deprivation is a typical representative of type 1 mitophagy, and this is independent of PTEN-induced putative kinase 1 (PINK1) and parkin (an E3 ligase). The formation of autophagosomes during type 1 mitophagy is inhibited by PI3K inhibitors, such as wortmannin and 3-methyladenine (Dombi et al., 2018).

Mitophagy induced by photodamage is called type 2 mitophagy and involves PINK1 and parkin but does not require PI3K (Dombi et al., 2018). PINK1 requires depolarization of the mitochondrial membrane potential to pass through the inner mitochondrial membrane. Under normal conditions, PINK1 is cleaved in the transmembrane segment by the mitochondrial intramembrane protease presenilin-associated rhomboid-like and then released into the cytosol for proteasomal degradation (Jin et al., 2010). Parkin assembles the ubiquitin chain on the MOM and also recruits more parkin from MOM to amplify the “phospho-ubiquitin” mitophagy signal produced by PINK1 (Kane et al., 2014). Mitofusin 2 (MFN2) phosphorylated by PINK1 also promotes parkin recruitment to generate ubiquitin chains on the MOM and initiate mitophagy (McLelland et al., 2018). MFN2 is required for adaptation of mitochondrial respiration to stress conditions and produce ROS. In the absence of MFN2, insufficient ROS production can affect cytokine activity and induce the production of NO, which is related to dysfunctions in autophagy, apoptosis, phagocytosis, and antigen processing (Lloberas et al., 2020).

Unlike type 1 mitochondrial autophagy, goblet phagocytes are not formed during type 2 mitochondrial autophagy, and mitochondrial fission is not obvious (Lemasters, 2014). Damaged mitochondria accumulate PINK1 and then recruit parkin, leading to ubiquitination of mitochondrial proteins (Ivankovic et al., 2016). These ubiquitinated proteins are then sequestered by the autophagy proteins sequestosome 1 (SQSTM1)/p62 and LC3, ultimately leading to the degradation of the mitochondria via mitophagy (Lamark et al., 2017). SQSTM1/p62 is a stress-induced cellular protein complex that functions as a selective autophagy receptor to degrade ubiquitinated substrates (Katsuragi et al., 2015). The ZZ-type zinc finger domain of SQSTM1/p62 (SQSTM1/p62_ZZ) recognizes a degradation signal in proteins (an N-terminal arginine residue). Recognizing the internal SQSTM1/p62 sequence through SQSTM1/p62_ZZ modulates the activity of SQSTM1/p62 through post-translational modification (Zhang et al., 2019d). In addition, PI3K inhibitors cannot block the type 2 mitophagy (Lemasters and Zhong, 2018).

The third type of mitochondrial autophagy involves the formation of MDVs that germinate from mitochondria and then transfer to lysosomes (Soubannier et al., 2012a). MDVs are cargo-selective vesicles released from mitochondria independent of the mitochondrial fission mechanism. Oxidative stress stimulates the formation of MDVs, which are rich in oxidized mitochondrial proteins (Vasam et al., 2021). MDVs respond early to oxidative stress, the degree of which is determined by the respiratory state of the mitochondria. Unlike type 2 mitophagy, the formation of MDVs does not require mitochondrial depolarization. Although the formation and transport of MDVs to lysosomes do not involve the autophagy proteins ATG5 and LC3 (Soubannier et al., 2012b), PINK1 and parkin are required (Roberts et al., 2016). During the import process, PINK1 is first cleaved by matrix processing peptidase and presenilin associated rhomboid-like protein, and then cleaved PINK1 is released from the input channel and degraded in the cytoplasm via the N-end rule proteolytic pathway. After the mitochondria are depolarized, the import mechanism is inactivated, and PINK1 is trapped in the import channel or anchored on the MOM near the import channel. This exposes the kinase domain to the cytosol, where it phosphorylates ubiquitinates parkin, resulting in the recruitment and activation of stable parkin on the mitochondrial surface. Parkin then ubiquitinates a series of proteins on the surface of mitochondria, which are then recognized by autophagy adaptor proteins and delivered to autophagosomes (Greene et al., 2012; Lazarou et al., 2012; Sarraf et al., 2013; Yamano and Youle, 2013; Kazlauskaite et al., 2014).

In addition to the above signaling pathways, NIX/BNIP3L, FUNDC1 and p62/SQSTM1 can also mediate mitophagy. The most well-accepted mechanism underlying BNIP3L-mediated mitophagy is through an interaction with proteins from the Atg8 family, by which BNIP3L recruits autophagosomes to targeted mitochondria (Novak et al., 2010). Moreover, BNIP3L disrupts the BCL2-BECN1 complex and releases BECN1, which promotes the formation of autophagosomes (Mazure and Pouysségur, 2009). FUNDC1 is a mammalian mitophagy receptor that interacts with and recruits LC3 to mitochondria for mitophagy (Liu et al., 2012). FUNDC1 is phosphorylated at tyrosine 18 (Y18) and serine 13 (S13) by SRC kinase and CK2, respectively. The phosphorylation prevents the interaction between FUNDC1 and LC3 for subsequent mitophagy in a mammalian system (Chen et al., 2016). p62/SQSTM1, a cytoprotein, containing a cytosolute LIR motif protein, was the first mammalian selective autophagy receptor to be described. In addition to the above receptor proteins, other cytoplasmic receptor proteins, such as NBR1, NDP52, TANK-Binding Kinase1, and Optineurin, have also been confirmed to mediate the mitophagy (Heo et al., 2015; Shi et al., 2015; Vargas et al., 2019; Di Rita et al., 2021).

The results of in vivo and in vitro studies suggest that autophagy adversely affects the outcome of IS. Astrocyte-derived exosomes ameliorate neuronal injury after IS by modulating autophagy to inhibit the expression of caspase-3 and Bax and modulate the levels of TNF-α, IL-6, and IL-1β (Pei et al., 2019). In a rat model of IS, activation of autophagy in brain endothelial cells was shown to mediate occludin degradation, resulting in disruption of the blood-brain barrier, ultimately leading to cellular dysfunction and death (Kim et al., 2020). IL-17A mediates hyperautophagy, which aggravates the ischemic injury of neurons after IS via the Src-PP2B-mTOR pathway, whereas cyclosporine A blocks the induction of hyperautophagy (Liu et al., 2019b). FK506-binding protein 5 induces autophagy by regulating the AKT/FOXO3 signaling pathway, which aggravates nerve damage after IS (Yu et al., 2020b). Vitexin inhibits the induction of autophagy in the middle cerebral artery via mechanistic targeting of the mTOR/ULK1 pathway, decreasing the expression of ULK1 and BECN1 and the expression rate of LC3II/LC3I, reversing autophagy-related dysfunction and alleviating the symptoms of IS (Jiang et al, 2018). Exogenous netrin-1 inhibits autophagy via the PI3K/mTOR pathway, alleviating ischemic brain tissue damage and enhancing hypoxic neuron activity (Tang et al., 2019). Moreover, multiple agents, including melatonin (Feng et al., 2017) and homocysteine (Wang et al., 2019), reportedly either exert neuroprotective effects by inhibiting autophagy or aggravate ischemic brain injury by activating autophagy.

Considerable evidence also indicates that autophagy may exert a neuroprotective effect in IS. Ginaton induces autophagy by activating the AMPK pathway, thereby maintaining mitochondrial homeostasis and inhibiting apoptosis, which protects against ischemic neuronal injury (Li et al., 2019a). Overexpression of PINK1 was able to activate the PINK1-Parkin pathway and rescue IS-induced behavioral disorders. PINK1 protects IS‐induced brain injury by promoting mitochondrial autophagy in microglia, which may be of value as a therapeutic target for IS treatment (Li et al., 2021). The mTOR inhibitor rapamycin significantly reduces mTOR activation and infarct volume by activating autophagy and inhibiting neuronal apoptosis, improving neural function after IS (Wu et al., 2018a). Inducing autophagy flux of astrocytes in vitro enhances neuronal viability and reduces neuronal apoptosis, which contributes to the endogenous post-IS neuroprotection and neural recovery mechanism (Liu et al., 2018). Various agents, including compound C (Jiang et al., 2015) and eugenol (Sun et al., 2020), reportedly exert either neuroprotective effects by activating autophagy or harmful effects by inhibiting autophagy.

Aβ is produced by the beta-amyloid precursor protein (APP) during autophagic renewal of APP-rich organelles generated by autophagy and endocytosis (Nixon, 2007). In the early stage of AD, Aβ activates autophagy and is then degraded after transport from autophagosomes to lysosomes (Han et al., 2018), which in turn regulates Aβ accumulation and aggregate formation (Bharadwaj and Martins, 2020). Autophagy is the major cellular pathway for protein aggregate removal, and upregulation of autophagy represents a rational therapeutic strategy against neurotoxic Aβ overproduction. Activation of PPARA-mediated autophagy reduces AD-like pathology and cognitive decline in mouse models (Luo et al., 2020). Crocetin induces autophagy in neuronal cells via serine/threonine kinase 11/LKB1(live kinase B1)-mediated activation of the AMPK pathway, significantly reducing Aβ levels and neuroinflammation in the brain and improving memory function in mice (Wani et al., 2021). Reportedly, AMPK activation does not induce autophagy in cerebellar granule neurons or cultured SH-SY5Y cells or affect Aβ secretion (Benito-Cuesta et al., 2021). In addition, alborixin (an ionophore) also exhibits strong neuroprotective effects, inhibiting the PI3K/AKT pathway to induce autophagy, upregulating the levels of BECN1, ATG5, and ATG7 to increase lysosomal activity, and significantly reducing the levels of Aβ in microglia and primary neurons, thus preventing or slowing the progression of AD (Wani et al., 2019).

Phosphorylation of tau is another pathological feature of AD. Although the ubiquitin-proteasome system is considered the main pathway for Tau degradation, autophagy is thought to be another efficient degradation pathway (Chung et al., 2019b). It has been demonstrated that p300/CBP (CREB binding protein) overactivation blocks autophagic flux and increases tau secretion in neurons. Conversely, inhibition of p300/CBP promotes increased autophagic flux, decreases tau secretion, and reduces tau transmission (Chen et al., 2020). Polyethylene glycol (PEG)-ceramide nanomicelles increase the ratio of LC3-II/LC3-I and reduce the level of p62 protein, thereby increasing autophagic flux in N2a cells and the degradation of overexpressed human tau protein. PEG-ceramide nanomicelles thus hold tremendous potential as AD drugs to induce autophagy and degrade tau protein (Gao et al., 2020b). Dihydrotanshinone I (DTSI) significantly inhibits mTOR phosphorylation via the AMPK/mTOR signaling pathway, thereby enhancing autophagy, increasing Aβ clearance, and reducing tau phosphorylation, which highlights the potential of DTSI in the treatment of AD (Bao et al., 2020). Neuroprotectin D1 (NPD1) was shown to enhance autophagic activity, downregulate Aβ expression, and control tau hyperphosphorylation in AD cell models, thereby reducing apoptosis and promoting neuronal survival. The neuroprotective effect of NPD1 may be due to inhibition of GSK-3β activation in neuronal cells (Dai et al., 2021). In an AD cell model, galangin treatment was shown to inhibit the expression of BECN1 and p-GSK3β but promote autophagy and decrease levels of Aβ and p-Tau by regulating the AKT/GSK3β/mTOR pathway. Thus galangin is considered a potential drug candidate for the treatment of AD (Huang et al., 2019).