The word “autophagy” is derived from Greek, meaning “self-eating” and refers to the metabolic process of cellular degradation and the recycling of cellular components within lysosomes (Galluzzi et al., 2017). There are three subtypes of autophagy, macroautophagy, chaperone-mediated autophagy, and microautophagy, which are divided depending on the mechanism by which the material destined for degradation enters the lysosome. Macroautophagy is accompanied by the emergence of a phagophore (a small bilayer of membranes) in the cytoplasm. Proteins and organelles are then encapsulated in a bilayer membrane structure that closes to form an organelle referred to as the autophagosome, which later fuses with the lysosome to enable the degradation of the enclosed material by lysosomal hydrolases (Figure 1). In chaperone-mediated autophagy, heat shock cognate 70 and several accessory chaperone proteins specifically recognize the KFERQ-like region and are transported to the lysosome for degradation (Kaushik and Cuervo, 2018). In microautophagy, the least characterized form of autophagy, the cytoplasmic cargo is destined for degradation through lysosomal or late endosomal membranes that directly engulfs cytoplasmic cargo (Schuck, 2020). Macroautophagy (hereafter referred to as autophagy) is the most widely studied of the three forms.

Although autophagy was originally thought to be a nonselective process, recent evidence suggests that it is highly selective (Villarejo-Zori et al., 2021). This selectivity means that specific types of cytoplasmic cargos have been identified (Johansen and Lamark, 2020) that are transported to the autophagosome for degradation, where they bind LC3 (light chain 3 protein) via LC3-interacting regions. Depending on the nature of the specific cargo, selective autophagy can be classified as heterogeneous phagocytosis (pathogen degradation), lipophagy (lipid droplet degradation), or aggregative phagocytosis (protein aggregate degradation) (Johansen and Lamark, 2020). Similarly, organelle-specific autophagy can be divided into endoplasmic reticulum, peroxisomal, mitochondrial and lysosomal autophagy (Johansen and Lamark, 2020). Recent studies have shown that the Golgi apparatus may degrade proteins by retrograde trafficking to the ER, suggesting Golgi might be a target for selective autophagy degradation (Benyair et al., 2022) (Nthiga et al., 2021).

Autophagy is primarily a cellular stress response and plays an important role in biological cells. Depending on the autophagy state, theprocess can be divided into several sequential stages: induction, initiation/vesicle nucleation, phagophore formation, elongation, autophagosome formation, maturation/fusion, and degradation (Kiriyama and Nochi, 2015).

Autophagy regulation is dependent on all phases. Autophagy occurs at a low basal level under physiological conditions, but it can be rapidly induced in certain states of cellular stress, such as hypoxia, glucose deficiency (Kim et al., 2011), starvation (Kuma et al., 2004) protein aggregates (Kraft et al., 2010), damaged organelles (e.g. mitochondria, endoplasmic reticulum, and peroxisomes) and intracellular pathogens infections (Glick et al., 2010). Autophagy is induced by these stresses and is regulated extensively by posttranslational mechanisms. Among all stresses, a lack of nutrients (especially amino acids) is the most important factor in the induction of autophagy. This process is mediated by upstream regulators of autophagy including mTOR and AMPK, which are responsible for monitoring nutritional status (Wong et al., 2013; Parzych and Klionsky, 2014). Phagophore formation is a key step that follows autophagy induction and is important for all phases. Autophagy initiation is controlled in part by the ULK complex (autophagy related gene (ATG)13, ULK1, FIP200), which is regulated by cellular energy sensors (AMPK and mTOR) (Bressan and Saghatelyan, 2020). After the autophagy-initiating kinase ULK1 is phosphorylated, a phagophore forms via phosphatidylinositol 3-phosphate (PI3P) generation (Metaxakis et al., 2018). Elongation is a crucial step in the complete autophagosomes (Ebrahimi-Fakhari et al., 2012). Tow ATG 7-catalyzed ubiquitin-like reactions control the elongation, one is to form autophagy elongation complex (ATG5-12/16L1), and the other is phosphatidylethanolamine (PE) -LC3I conjugated to form LC3-II, (Ebrahimi-Fakhari et al., 2012). Membrane elongation closure to maturation, which fuse with lysosomes to form autolysosomes and degrade proteins and organelles (Yim and Mizushima, 2020). Molecular mechanisms during macroautophagic processes are shown in Figure 1.

Autophagy is primarily regulated at the posttranslational level; various stressors can induce autophagy, and stress can also stimulate the upregulation of autophagy genes (Feng et al., 2015). The transcription factor EB (TFEB) is the main regulator of autophagy genes (Puertollano et al., 2018). Phosphorylation retains TFEB in the cytoplasm, but when dephosphorylated by stress, TFEB translocates to the nucleus where it promotes the transcription of its target genes (Puertollano et al., 2018). Optineurin (OPTN) is a gene linked to glaucoma (Shen et al., 2011), and its encoded protein is an autophagy receptor, which targets specific cargo for degradation and is itself degraded by autophagy (Mijaljica et al., 2012; Klionsky et al., 2021). Recently, at least 42 genes associated with the autophagy pathway (Mizushima, 2020), and more genes will be identified as research progresses.

Autophagy levels decline during normal human aging in all tissues examined to date, including the brain and retina, which contributes to the pathogenesis of several neurodegenerative diseases, such as glaucoma (Boya, 2017; Wong et al., 2020). Altered selective autophagy also has important pathological implications in the pathogenesis of neurodegenerative diseases. For example, inadequate removal of pathogenic protein aggregates is associated with neurodegenerative diseases (Conway et al., 2020), and dysregulation of mitochondrial autophagy, i.e., the selective removal of unnecessary or damaged mitochondria is impaired, constitutes a severe redox imbalance that is detrimental to neurons (Teodorof-Diedrich and Spector, 2018) and is common in neurodegenerative diseases.

Autophagy activation in HIOP (acute or chronic) has been observed in different experimental models of glaucoma (Piras et al., 2011; Hirt et al., 2018). IOP is exerted by the aqueous humor (AH) on the inner surface of the anterior segment of the eye; normal is approximately 16 mmHg (Carreon et al., 2017). AH is a clear fluid produced by filtering blood through the ciliary body, which enters the anterior chamber of the eye at a relatively constant rate. AH nourishes the cornea, lens, and TM in the anterior chamber and exits mainly through the TM/Schlemm’s canal (SC). Elevated IOP is caused by the failure of the TM/SC, a conventional outflow tract, leading to an inability to maintain an adequate level of AH outflow resistance (i.e., impaired outflow) (Stamer and Acott, 2012). Thus, the TM is therefore particularly important for maintaining normal IOP as it is the main outflow pathway for AH. Dysregulation of autophagy in the retina and TM has been demonstrated in the HIOP model of glaucoma (Nettesheim et al., 2020).

Numerous studies have demonstrated that cells in the outflow pathway are subjected and responsive to fluctuating IOP and mechanical loads (Hirt and Liton, 2017). Eleven known mechanotransduction channel proteins were detected in TM tissue and isolated TM cell cultures (Tran et al., 2014). Experiments conducted in in vitro perfusion eyes have demonstrated the presence of a mechanism regulating IOP homeostasis in TM/SC outflow pathway tissues (Acott et al., 2014). After the perfusion rate increased, resulting in a short IOP increase, even if the inflow was maintained at high pressure, the IOP gradually returned to the baseline level in a few days (Bradley et al., 2001). This observation has been confirmed by various researchers, suggesting that a compensatory mechanism helps the maintenance of IOP homeostasis (Borrás, 2003; Baetz et al., 2009; Porter et al., 2014). Cells in the outflow channel sense pressure imbalance in the form of mechanical stretching, which is thought to trigger a response (Hirt and Liton, 2017). TM cells are prone to stretch injury after long-term exposure to tension; thus, they must be guided to adapt to this mechanical force and repair the damage caused by potential stretching. One study suggested that autophagy may be a possible cellular adaptation mechanism in response to this stretch (Shim et al., 2020). A recent study has shown that primary cilia (PC) are mechanosensory for stretch-induced autophagy in TM cells, and PC-mediated autophagy is critical for IOP homeostasis (Shim et al., 2021). Stretch-induced autophagy under normal physiological forces is helpful to eliminate damaged components and accelerate the renewal of proteins and organelles, resulting in increased cytoskeletal and cortical stiffness in TM cells (Liton, 2016), maintaining cellular homeostasis, and inhibiting cell death (Porter et al., 2014). Conversely, autophagy dysregulation may trigger cell death and lead to disease if the mechanical forces exceed the physiological limits or if the conditions are pathological (Hirt and Liton, 2017). Overactivation of autophagy is a potential cellular mechanism leading to optic nerve degeneration in DBA/2J mice (Hirt et al., 2018).

Mitochondria are exquisitely dynamic organelles that move along axons and dendrites in neurons and constantly respond to endogenous and exogenous stimuli, to satisfy the energy requirements of neuronal networks and to maintain neuronal Ca2+ homeostasis and synaptic plasticity (Mattson et al., 2008). The highly coordinated regulation of mitochondria is essential for maintaining the energy in RGC dendrites and axon terminals for optimal neurotransmission (Ito and Di Polo, 2017). To date, several studies have shown that oxidative stress damage and mitochondrial dysfunction are involved in the pathogenesis of glaucoma and are tightly linked to autophagy pathways (Chang et al., 2022).

Studies have shown that overexpression of oxidative stress markers including lipid peroxidation, and oxidative DNA damage is present in various ocular tissues in glaucoma patients (Fernández-Durango et al., 2008). Oxidative stress results from the overproduction of reactive oxygen species (ROS) (⋅O₂−, ⋅OH, and H₂O₂), which are byproducts of cellular respiration, particularly during mitochondrial oxidative phosphorylation, and are produced by the electron transport chain in the inner mitochondrial membrane (Shadel and Horvath, 2015). Although low ROS at physiological functions in redox signaling, excessive ROS produced by mitochondrial malfunction can lead to the oxidative damage of cellular components, comprising lipids, proteins, and DNA (Shim and Liton, 2022). One is in the mitochondria ROS in the form of superoxide and H₂O₂ mediates the sustained activation of nuclear factor kappa-B (NF-κB) and the upregulation of inflammatory markers like interleukin-1α (IL-1α) and endothelial leukocyte adhesion molecule-1 (ELAM-1) (Li et al., 2007). Another crucial source is the production of ROS through the iron-catalyzed Fenton/Haber-Weiss reaction (Kruszewski, 2003; Yauger et al., 2019). The Fenton/Haber-Weiss reaction results in the production of highly oxidized hydroxyl radicals, which induce lipid and protein peroxidation and cause oxidative damage to DNA (Kruszewski, 2003). Among other things, the increase in lipid peroxidation may be attributed to a deficiency in the antioxidant system (Fernández-Durango et al., 2008). ROS exert two regulatory effects on autophagy, inducing autophagy through the activation of transcription factors (HIF-1α, NRF2, P53, and FOXO3) and by driving the expression of genes required for autophagy induction (including BECN1, LC3, SQSTM1) via posttranslational regulation (Chang et al., 2022). Autophagy can also be inhibited by the oxidation of ATG proteins (ATG7 and ATG10) or by the inactivation of autophagy regulators (TFEB and PTEN) (Su et al., 2018; Chang et al., 2022). In turn, autophagy can reduce ROS levels and alleviate oxidative damage by eliminating damaged molecules (e.g., protein aggregates) and damaged organelles (e.g., mitochondrial cartilage), thus maintaining proper redox signaling and DNA damage repair systems (Li et al., 2015). These findings suggests that mitochondrial autophagy induction may improve the pathogenesis of glaucoma.

The main neuroglia present in the retina are microglia, astrocytes, and Müller cells (Vecino et al., 2016). Of these, microglia play a crucial role in neuroinflammation (Zhang W. et al., 2021), which is a critical process in glaucoma (Guzman-Martinez et al., 2019). Microglia in the retina are resident innate immune cells in neural tissue that perform as neuropathological receptors and are also the primary immune defense against retinal damage (Chen et al., 2022). Under physiological conditions, glial cells secrete neurotrophic factors to promote nerve cell survival and tissue regeneration and perform phagocytosis to eliminate foreign components (Campagno et al., 2021). Microglia activated by stimulation [such as HIOP (Campagno et al., 2021)] secrete inflammatory cytokines to induce neuronal apoptosis, which further contributes to neuroinflammation. Activated microglia can be polarized into either an M1 proinflammatory phenotype or an M2 anti-inflammatory phenotype, and the two can switch between each other in response to different microenvironmental disorders, known as classical activation and alternative activation respectively (Lan et al., 2017). M1 polarized microglia activate the neuroinflammatory response and exacerbate neuronal damage via the synthesis and release of injury-mediating cytokines (TNFα, IL-1β, IL-6, IL-12), protein hydrolases (MMPs), and the expression of MHC-II (Ramirez et al., 2017). M2 polarized microglia, secrete anti-inflammatory cytokines and growth factors and play the opposite role of M1 microglia (Lan et al., 2017), promoting phagocytosis and waste removal, neurogenesis, axonal remodeling, or myelin regeneration after injury (Liu et al., 2018). Activated neuroinflammatory pathways may initially restore homeostasis between RGCs and the extracellular environment, with protective effects. However, the overproduction of chemokines, and cytokines and the excessive activation of retina-resident microglia and other innate immune cells, may prompt RGC degeneration (Quaranta et al., 2021). Neuroinflammation may be an important cause of RGC degeneration and targeting M2 microglial polarization could be a potential therapeutic option to treat glaucoma (Cherry et al., 2014; Reboussin et al., 2022).

The proinflammatory cytokine TNF-α produced by M1 microglia was demonstrated to inhibit autophagic flux in neurons and microglia, which further drives M1 polarization in microglia via activation of the Akt/mTOR pathway (Jin et al., 2018). Conversely, IL-4, which promotes microglial polarization to the M2 subtype, activates autophagic flux and exerts a protective effect (Tang et al., 2019). Autophagy has been reported to direct microglial activation to the M2 subtype, and consistently, autophagy inhibition promotes polarization toward the proinflammatory M1 subtype (Jin et al., 2018). The M2 subtype is maintained by high autophagic flux, and disruption of autophagic flux results in a shift in the M1-related subtype (Zubova et al., 2022). In conclusion, autophagy degrades proinflammatory proteins, limits the inflammatory process, and exerts a protective effect.

In most tissues, autophagy declines with age, which can lead to the accumulation of certain types of cellular components at potentially toxic levels that can be harmful to cells (Hansen et al., 2018). Dysregulation of autophagy, a cellular catabolic mechanism that removes misfolded proteins, is responsible for a range of neurodegenerative diseases (Lipinski et al., 2010). Autophagy activity is impaired as the brain ages in different species (Hansen et al., 2018). For example, autophagy is reduced in mouse hypothalamic neurons (Kaushik et al., 2012) and the mRNA levels of many ATG are reduced in the aging human brain (Lipinski et al., 2010). Studies on the retina have determined that glaucoma is also a neurodegenerative disease, and the mRNA expression of autophagy regulators (e.g., ATG and Beclin) is reduced (Rodríguez-Muela et al., 2013). In an optic nerve crush model, autophagy-deficiency in aged mice (Ambra1+/gt) increases axonal damage susceptibility (Bell et al., 2020). Autophagy in the retina decreases with age and activation of autophagy shows neuroprotective effects in vivo experimental of glaucoma (Bell et al., 2020). Dysregulation of TM autophagy associated with elevated IOP also occurs during aging (Hirt et al., 2018). Overall, there is growing evidence to support the beneficial role of autophagy in the fight against aging and age-related diseases.

OPTN, TANK binding kinase 1 (TBK1), and Myocilin (MYOC) are genes linked to glaucoma (Sears et al., 2019). Mutation in each of these genes may cause this major blinding disease (Sears et al., 2019). E50K and M98K mutants induced excess cell death than wild-type OPTN (Sirohi et al., 2013; Sirohi and Swarup, 2016). E50K mutation causes retinal cell death in the cell culture model and transgenic mouse model by inhibition of autophagy (Chalasani et al., 2014; Zhang S. et al., 2021). M98K is a gain-of-function mutation, overexpression of M98K shows enhanced autophagic signaling leading to endocytic recycling of transferrin receptor (TFRC) degradation and causing cell death (Sirohi et al., 2013). Uncontrolled activation of TBK1 by the mutation that A 2-bp insertion in OPTN leads to impaired autophagy that results in the accumulation of LC3-positive aggregates, which induces ER stress and a retinal cell line, 661 W, cell death (Medchalmi et al., 2021). In addition, duplication of TBK1 can cause glaucoma, for the reason that increased transcription of TBK1 encodes a kinase that phosphorylates OPTN, which recruits LC3B and initiates autophagy (Tucker et al., 2014). The mutant MYOC-induced impaired autophagy leads to TM cell death and HIOP, which is associated with the induction of transcription factor, CCAAT/enhancer-binding protein homologous protein (Stothert et al., 2016; Kasetti et al., 2021; Yan et al., 2022). These evidences indicated that some glaucoma caused by gene mutation is related to impaired autophagy.

In recent years, Chinese medicine has been effective for treating glaucoma in combination with Western medicine or surgery (Wen-bo et al., 2020; Xirui et al., 2021). In Chinese medicine, it is considered that the pathogenesis of glaucoma is based on “stasis” and “deficiency”, and treatment should be based on activating blood circulation, removing blood stasis, moving qi, and opening depression, which corresponds to the “clearing” effect of autophagy. Autophagy, a current research hotspot, is closely related to glaucomatous optic neuropathy in many ways and has led to initial research progress in using Chinese medicine therapies to regulate autophagy to protect the optic nerve.

In research on Chinese medicine/Chinese herbal extracts, in a rat model of chronic hypertensive glaucoma established by the extrascleral vein cautery (EVC) method, autophagy was significantly activated in retinal cells and the number of autophagic vesicles increased, while chuanxiongzin completely reversed this change in rats by activating the PI3K-Akt/mTOR pathway to inhibit autophagy, acting as a protective agent (Du et al., 2021). The possible signaling pathways through which each herbal medicine/Chinese medicine extract exerts its protective effect by regulating autophagy are shown in Table 2.

Clinical data show that acupuncture combined with conventional Western medicine can effectively reduce intraocular pressure and significantly improve the degree of optic nerve atrophy in patients with glaucoma (Wang, 2017). Acupuncture also reduces visual field defects and improves ocular artery hemodynamics and visual acuity (Leszczynska et al., 2018; Lan and Xiaojie, 2020). At present, studies involving the optic neuroprotective effects of acupuncture therapy have been limited to clinical data analysis, but in the neuroprotective effect of cranial nerves, acupuncture can play a protective role by inhibiting autophagy through the mTORC1–ULK complex–Beclin1pathway (Liu et al., 2016).

Enhanced autophagy efficacy may reduce the number of toxic protein aggregates, provide an effective stress response, and prevent or reduce cell death by degrading inessential components for energy and supporting adaptive protein synthesis. Allogeneic progesterone (Allop) increases autophagy vesicles, activates autophagy, and protects RGCs via GABR/GABAA receptors (Ishikawa et al., 2021). Rapamycin inhibits mTOR and therefore induces autophagy in peripheral organs and the CNS. Using an optic nerve transection model, rapamycin-treated mice showed a 40% improvement in RGC survival 10 days after axotomy, exerting a beneficial effect (Rodríguez-Muela et al., 2012). Rapamycin can also increase the RGC number and visual function, and reduce apoptosis of E50K-OPTN mice (Shen et al., 2011; Zhang S. et al., 2021). Stimulation of autophagy via tat-beclin 1 peptide or Torin 2 rescued MYOC-associated transgenic mice model (Kasetti et al., 2021). In various in vivo experimental models of glaucoma, the induction of autophagy by pharmacological or genetic manipulation can improve RGC survival (Table 2).

Evidence suggests that autophagy induction has a neuroprotective effect on glaucoma-damaged RGCs; however, autophagy induction cannot be directly said to be a panacea for neurodegenerative disease, and the induction of autophagosome biogenesis may be harmful if autophagosome accumulation is not effectively cleared. It has been shown that autophagy inhibition also has neuroprotective effect on glaucoma-damaged RGCs. In a rat model of chronic HIOP glaucoma, autophagy was significantly activated in RGCs (Deng et al., 2013). The number of TUNEL-positive cells was reduced in the GCL since autophagy inhibition by the autophagy inhibitor 3-Methyladenine (3-MA), indicating that apoptosis occurred in RGCs after autophagy-induced chronic IOP elevation (Park et al., 2012). M98K-OPTN potentiates the degradation of TFRC by autophagy and contributes to the death of RGCs; chloroquine (CQ) or the knockdown of Atg5 by shRNA significantly reduced cell death mediated by M98K (Sirohi et al., 2013; Swarup and Sayyad, 2018). The possible signaling pathways through which each Western drug exerts its protective effects by modulating autophagy are shown in Table 2.

Currently the role of autophagy in glaucoma is still controversial; it relies on the type of glaucoma model, the target of pharmacological treatment, and the period of drug administration (Wang et al., 2015; Russo et al., 2018). Studies have shown that is chemic/reperfusion can trigger autophagy in the first few hours; although RGC loss at this time, autophagy as the mechanism of self-adaptation under stress conditions might maintain cellular homeostasis and reduce the harm (Russo et al., 2018). The subsequent protective effect of rapamycin also confirmed this point (Russo et al., 2018). However, it is demonstrated that rapamycin aggravated RGC loss and axon demyelination at day 14 of reperfusion, as mTOR activity plays a crucial role in determining neuronal survival (Chen et al., 2016). The role of autophagy in glaucoma is multifunctional and dynamic. Consequently, a better comprehension of the underlying mechanisms of autophagy dysfunction in glaucoma is essential for developing therapeutic approaches.

RGCs degenerate in glaucoma, resulting in permanent vision loss. Cell replacement strategies are considered potential therapies for RGC loss. Researchers have recently explored a potential replacement therapy based on human stem cells that can differentiate into RGC cells (Nuzzi and Tridico, 2017; Chang et al., 2019). However, scaling up donor cells, advancing cell differentiation, and promoting cell durability remain challenges in replacement therapies.

Elaborate mechanisms render mesenchymal stem cells (MSCs) neuroprotective, including modulating neuroinflammation, enhancing cell survival, increasing neurogenesis, and regulating ubiquitinated proteins (Shin et al., 2014). It has been documented that hUC-MSCs have a protective effect on retinal neurons (Wang et al., 2021), and MSC-EVs also exert a neuroprotective effect on the retina by reducing neuroinflammation and neuronal apoptosis (Mathew et al., 2019). At present, studies involving the optic neuroprotective effects of MSCs and MSC-EVs are more limited to phenotypic analysis, and in the brain, MSCs can improve neuronal survival by activating autophagy (Shin et al., 2014; Ceccariglia et al., 2020).