Clinical and experimental evidence has revealed a rapidly initiated, inflammatory (TNF-α-mediated RGC apoptosis) and IOP-independent glaucoma pathway induced by acute anterior segment trauma or surgery, suggesting that cell apoptosis promotes the development of glaucoma (36,37). Administration of infliximab (a TNF-α antibody) or a TNF-α inhibitor has been revealed to protect against cell apoptosis, ameliorating neuroglial remodelling and inhibiting monocyte infiltration (36,37,45,46). Fas signalling reportedly contributes to the pathogenesis of glaucoma by activating both apoptotic and inflammatory pathways and the small peptide inhibitor of the Fas receptor, ONL1204, provides potent neuroprotection (35,47). In addition, several studies have shown that the apoptosis pathway is an essential mediator of RGC death in glaucoma (48–52). Moreover, in glaucoma, TM cells also undergo death through apoptotic pathway, such as the Fas/Fas ligand (FasL) pathway and ERS (51,53–55). Notably, benzalkonium chloride, the most common preservative used in glaucoma treatment also induces toxic changes in the TM and cell apoptosis, which further leads to impaired function of the TM and may worsen any glaucomatous process within the TM if used for a long time (38,56,57). Glaucoma-related cell death typically occurs through apoptosis, triggered by oxidative stress through mitochondrial damage, inflammation, endothelial dysregulation and dysfunction, hypoxia and other factors (39,58).

TM cell apoptosis in the anterior chamber is the most significant concern in glaucoma. It has been suggested that mechanical stress, oxidative stress and intense phagocytic activity of TM cells likely cause cell apoptosis (59). The extent of mechanical stress on the TM depends on the IOP. Thus, glaucoma itself can also induce TM cell apoptosis via mechanical stress or trabecular hypoperfusion (59,60). TM cells die due to apoptosis, loss of barrier function, alteration of aqueous humour outflow and increased IOP (61,62) (Fig. 1).

Programmed cell death can be detected in the TM of patients with glaucoma by using TUNEL (33). Moreover, the experimental results regarding Fas monoclonal antibody-induced apoptosis in cultured human TM cells demonstrated that human TM cells were stimulated to undergo apoptosis through the Fas/FasL pathway (63). Upregulation of activating transcription factor-4 (ATF4) and C/EBP homologous protein (CHOP) and colocalization of ATF4 with endothelial leukocyte adhesion molecule (ELAM-1) were found in the TM of patients with POAG and inhibition of ATF4 reduced tunicamycin-induced caspase-3 activation, ROS production, ELAM-1 expression and human TM cell phagocytosis impairment (53). An in vivo study in mice revealed that overexpression of ATF4 in the TM induces CHOP expression and TM cell apoptosis, leading to the production of inflammatory cytokines and possibly increased IOP (53). The aforementioned study demonstrated that the eukaryotic initiation factor 2 alpha (eIF2α)/ATF4/CHOP branch of the unfolded protein response (UPR) was activated in human TM cells from patients with glaucoma patients following tert-butyl hydroperoxide exposure (53). In addition, alteration of the TM cell extracellular matrix (ECM) is also considered to be one of the mechanisms that induces IOP elevation by increasing TM resistance. Furthermore, the effects of oxidative stress and latent transforming growth factor beta-binding protein 2 knockdown on ECM and TM cell apoptosis may be mediated by activation of the transforming growth factor-beta (TGF-β)/bone morphogenetic protein signalling pathway (64). CD9 was found to be involved in a wide range of biological processes, such as cell migration and differentiation and cell adhesion and motility, by regulating the phosphatidylinositol 3-kinase (PI3K)-protein kinase B (Akt) signalling pathway (65,66). Yan et al (67) reported that CD9 was downregulated in glaucoma and the overexpression of CD9 could activate integrin α4 (ITGA4), PI3K and Akt, leading to reduced TM cell apoptosis and alleviating glaucoma (Fig. 2). MicroRNAs (miRNAs) are single-stranded noncoding RNAs that regulate cellular processes in human TM cells and the effect of miRNAs on TM cell apoptosis in glaucoma has been reported in previous studies. The expression of miR-93 was significantly upregulated in human TM cells in glaucoma and miR-93 induced human TM cells and inhibited their viability by suppressing the expression of nuclear factor erythroid 2-like 2, thus indicating that miR-93 is a vital regulator in glaucoma (68). However, the effect of miR-200c-3p on TM cell is opposite to that of miR-93. A different study revealed that overexpression of miR-200c-3p negatively regulates the expression of phosphatase and tensin homologue (PTEN) to inhibit cleaved caspase-3, reduce Bax expression and activate the PTEN/Akt/mammalian target of rapamycin (mTOR) signalling pathway, thereby promoting cell proliferation and inhibiting TM cell apoptosis (69). Furthermore, the effects of miR-17-5p on the human TM cell apoptosis and proliferation are similar to those of miR-200c-3p (70). Moreover, miR-181a enhances the survival rate of TM cells by blocking the nuclear factor kappa-B and c-Jun amino-terminal kinase (JNK) signalling pathway (71).

In addition, increased TM stiffness leads to increased IOP (72). TM stiffness is affected by lysophospholipids, rho-associated kinase inhibitors (ROCKis), cytoskeletal disrupting agents, dexamethasone (DEX), TGF-β2, nitric oxide and cellular senescence (72–74).

Therefore, elevated IOP resulting from TM dysfunction caused by the apoptosis signalling pathway and changes in stiffness, inflammation and oxidative stress, aggravates TM cell damage and causes TM cell death. Moreover, TM-derived molecules from damaged TM cells and harmful signals released from stressed TM cells act as apoptotic stimulators, such as neuronal-like cell apoptosis (55,75) (Fig. 1).

The exact mechanisms of RGC apoptosis are not fully understood and increasing evidence suggests that RGC apoptosis may involve blocking anterograde and retrograde axonal transport resulting in the deprivation of neurotrophic signals (4). Moreover, this process, which is accompanied by microglial activation, neuroinflammation and ECM remodelling, was enhanced by high IOP (76–79).

Microglia are activated before RGC loss, and early changes in the retina and optic nerve were detected in the DBA/2J mouse model of glaucoma (80,81). Moreover, the degree of microglial activation in the optic nerve head (ONH) is proportional to the severity of optic nerve degeneration (81). Numerous studies have revealed that microglia may be essential modulators involved in peripheral monocyte infiltration and retinal pigment epithelial migration (36,45,82,83). The depletion of these proteins leads to abnormal neuroglial remodelling, exacerbating neuroretinal tissue damage (45). Furthermore, infiltrating monocytes may amplify the inflammatory cascade response and contribute to the activation of retinal microglia (36). Inflammatory cytokines, such as TNF-α, lead to permanent changes in the immune function of the retina, called ‘permanent neuroglial remodelling’ and the anti-inflammatory agents have significant neuroprotective effects on RGCs (36,37).

Furthermore, RGC apoptosis is associated with the level of IOP. Elevated IOP causes axonal degeneration at the ONH in the region of lamina cribriform, a process that occurs in parallel to RGC apoptosis (84). Elevated IOP is considered to damage the lamina cribrosa by mechanical stress, resulting in loss of normal structural and metabolic support of RGC axons and impaired axoplasmic transport. A reduction in neurotrophic signals in RGCs may lead to the initiation of apoptosis and ultimately to the RGC death (5,39). Another novel mechanism of IOP-induced RGC apoptosis is that IOP-induced changes in specific ECM components or cytokines in the retina may increase the activity of matrix metalloproteinases (MMPs), such as MMP-9 (79). One of the reasons for the increase in MMP-9 may be that the mechanical effects of elevated IOP may lead to the RGC axonal damage in the ONH region, which further results in retrograde damage to the RGC body and, in turn, may induce increased MMP-9 activity, causing changes in the ECM (79). Another explanation for the MMP-9 increase induced by elevated IOP is an indirect effect mediated by glutamate (79). Glutamate is a major excitatory neurotransmitter that is increased by stimuli, such as IOP, ischaemia and injury (85). Glutamate excitotoxicity has been reported to be one of the critical pathophysiological causes of RGC injury in glaucoma (86). Thus, glutamate-mediated activation of MMP-9 may lead to RGC apoptosis.

An increase in MMP-9 activity during RGC apoptosis parallels to a decrease in deposition of laminin in the RGC layer, which may lead to disruption of the cell-ECM and cell-cell interactions, increasing susceptibility to apoptosis (79). Tissue inhibitors of matrix metalloproteinase-1 (TIMP-1) are generally considered to be inhibitors of MMPs, particularly MMP-9, that maintain ECM homeostasis (87). TIMP-1 activity in the RGC layer increases with increasing MMP-9 activity and is correlated with IOP exposure (79). An increase in retinal TIMP-1 may contribute to its neuroprotective effects on RGCs by inhibiting MMP-9 and antiapoptotic effects. This finding is consistent with the theory that the ECM is continually remodelled after retinal exposure to elevated IOP.

Molecules associated with ECM changes at the glaucoma ONH site, such as TGF-β2 and collagen 1, are also involved in glaucomatous RGC loss. The experimental results revealed that TGF-β2 and collagen 1 deposition was significantly associated with increased IOP at the ONH (73,79,88). A possible mechanism for increased TGF-β expression in the ONH is the stress response to elevated IOP, as TGF-β is a crucial molecule stimulated by mechanical stress (89,90). However, with increased IOP exposure, TGF-β2 deposition in the retina significantly decreases, whereas MMP-9 increases. TGF-β1 regulates the mRNA and protein levels of MMP-9, similar to its inhibitor TIMP-2 (91). Furthermore, the effect of TGF-β2 depends on its concentration and specificity in targeting cells (92). The biphasic behaviour of TGF-β explains why both the low levels of TGF-β2 found in the RGC layer and the high levels of TGF-β2 in the ONH may be involved in RGC apoptosis (79).

Although several extrinsic DR pathways have been described, the best-known pathway is triggered by death signals including TNF-α and FasL (Fig. 3). A previous study has shown that Fas signal contributes to glaucoma pathogenesis by activating apoptotic and inflammatory pathways (35). At the same time, a marginal single nucleotide polymorphism association of TNF-α was also found in human glaucoma and the assessment of the expression levels of TNF-α may serve as a promising biomarker for POAG in African Americans (96). In this pathway, corresponding DRs of TNFα and FasL are TNF receptor1 (TNFR1) and Fas, respectively, which help transmit death signals from the cell surface to intracellular pathways through their death domain (DD). DD recruits adaptor proteins, such as Fas-associated DD proteins (FADD), TNFR1-associated DD protein and cysteine proteases such as caspase-8 (97). Subsequently, the death-inducing signalling complex (DISC), a ligand-receptor-adaptor protein complex, is formed by a sequential process. The death ligand of DISC binds to DR to recruit an adaptor protein in order (98,99). The DISC activates caspase-8, which initiates apoptosis through cleavage of the downstream caspases (97).

Previous studies have revealed that stimuli (toxic substances, ROS and aging) and inherent DNA deficiencies can impair mitochondrial structure and function, triggering the intrinsic mitochondrial pathway in glaucoma (Fig. 3) (100,101). Meanwhile, these stimuli induce the opening of the mitochondrial permeability transition pore and hinder the mitochondrial transmembrane potential, thus accelerating the release of proapoptotic proteins, such as cytochrome c (Cytc) and apoptosis-inducing factors, such as apoptosis-inducing factor (AIF) from mitochondria into the cytoplasm (102,103). Apoptotic protease activating factor 1 (Apaf-1, homologous to cell death protein 4) with an N-terminal caspase recruitment domain (CARD) consists of a six-helix bundle, is an activator of procaspase-9 and is considered to be a junction protein of the mitochondrial pathway (104). Apaf-1 oligomerizes in response to the release of Cytc and forms a disc-shaped heptamer (Cytc-Apaf-1)₇ called apoptosome (104). Subsequently, the apoptosome recruits and activates procaspase-9, an initiator of caspase in the mitochondrial pathway, leading to downstream caspase-3 processing (105).

Mitochondrial-initiated processes are mediated by B-cell lymphoma 2 (Bcl-2) family proteins. Bcl-2 family proteins are in the outer mitochondrial membrane that regulates mitochondrial outer membrane permeabilization (MOMP) and the release of the Cytc. The conformations of members of the Bcl-2 family all have one to four homology BH domains (BH1 to BH4), of which the homology BH3 domain of the proapoptotic proteins (Bax, Bak) is required for MOMP and the execution of intrinsic apoptosis (106). Based on their function in regulating MOMP and their domains, members of the Bcl-2 family are classified into three categories. The first category is antiapoptotic members, including Bcl-2, Bcl-extra-large, induced myeloid leukemia cell differentiation protein Mcl-1, Bcl-w and Bcl-2 related protein A1, characterized by all having four BH domains (107). These proteins inhibit Bax homo-oligomerization in MOMP by competing with Bax to bind to the BH3 helix of Bax through the groove BH1-3 (108). The second category is proapoptotic proteins comprising Bax, Bak and Bok, containing four BH domains (107). Activated by interaction with the BH3 domain of Bim or BH3 interacting-domain death agonist (Bid), oligomerization of Bak and Bax results in the formation of MOMP and induces Cytc release into the cytoplasm (107,109). The BH3-only proteins are the third subfamily, comprised of Bid, Bim, Bcl-2-interacting killer, Bcl2 modifying factor, p53-upregulated modulator of apoptosis and Noxa. Compared with the sequence homology of other subfamily members, these proteins only have the BH3 domain (107,110). BH-3 only proteins act as apoptotic signal receptors and Bax-like proteins contribute to MOMP, which results in the release of proapoptotic proteins (Cytc, AIF) from mitochondria (110). In the late stages of apoptosis, AIF nuclear translocation induces chromatin condensation or DNA fragmentation in a caspase-independent manner (111). In the apoptosis signalling pathway, Bcl-2 and Bax with negative or positive p53 response elements are located downstream of p53 (112). Oxidative stress-induced DNA damage upregulates Bax by phosphorylating p53 (113). Moreover, Bcl-2 inhibits p53-mediated apoptosis and transcriptional activation (114,115).

The third apoptosis pathway is mediated by ERS, which is critical for cell survival (Fig. 3). ERS is a condition in which some physiological and pathological impairments impede the ability of the cell to properly fold and post-translationally modify secretory and transmembrane proteins in the ER, resulting in the accumulation of misfolded proteins in the ER lumen (116). When protein misfolding persists or is excessive, ERS triggers cell death, usually apoptosis. ER proteostasis surveillance is sensed by the UPR, the highly conserved signal transduction pathway in the ER lumen that senses the fidelity of protein folding and determines cell fate in response to ERS (117).

UPR is activated by three transmembrane ER proteins, namely ATF6, inositol-requiring enzyme 1α (IRE1α) and protein kinase RNA-like ER kinase (PERK) (118,119). These ERS sensors have a common domain, namely the ER-luminal domain, which senses high concentrations of misfolded proteins to alter the oligomerization state of each senor and activates their downstream molecular signal (116). The phosphorylated form of IRE1α (p-IRE1α) activates chaperone genes and also activates JNK by binding to TNF receptor associated factor-2 (TRAF2) (120). In a rat model of experimental glaucoma, p-JNK is increased and may play a role in RGC death (121). Thus, it is assumed that IRE1α-JNK signalling pathway is involved in ERS of glaucoma. Besides, p-IRE1α initiates mRNA splicing of the X-box binding protein (XBP1) to produce a frameshift that code encodes a potent transcription factor, XBP1s. XBP1s enters the nucleus and initiates the transcription of a subset of UPR-related genes associated with protein folding, secretion, ER-associated degradation and lipid synthesis (122). In addition, p-IRE1α is involved in the regulated IRE1-dependent decay process to promote the degradation of mRNA and slow down the synthesis of new polypeptide chains, thereby alleviating ERS (123). Thus, IRE1α is a crucial protein that regulates cell survival or induces apoptosis in response to surrounding ERS.

PERK is activated in a similar way to IRE1α. Activation of PERK induces translational attenuation of p-eIF2α. Under excessive or prolonged ERS, p-eIF2α translates the transcription factor ATF4, which further induces the expression of other transcription factors, CHOP and ATF3, thus participating in the proapoptotic process (124,125). The expression of ATF4 and CHOP significantly increased in human glaucomatous TM cells (126). However, in the DBA/2J mouse model of glaucoma, an age-related, naturally occurring ocular hypertensive mouse model of glaucoma, though CHOP plays a minor role in contributing to RGC somal apoptosis, it does not lead to axonal degeneration (127). Additional studies have confirmed that the eIF2α/ATF4/CHOP pathway induces TM cell apoptosis in experimental glaucoma (53,128,129).

Similarly, ATF6 activates the target transcription factor, XBP1, to perform a pro-survival effect. XBP1 mRNA is induced by ATF6 and spliced by IRE1α to form the spliced form of XBP1 (130). TM cells treated with DEX showed that IRE1, ATF6 and GRP78 were downregulated (131). A recent study suggested that loss of ATF6 exacerbates retinal degeneration (132). However, the specific mechanism of ATF6-associated ERS in glaucoma has not been revealed.

The cysteine protease family is the core of the caspase-dependent apoptosis pathway. A total of 13 caspases encoded by the human genome belong to the peptidase C14A family and are orthologous to CED-3 in C. elegans (133,134). Caspases are divided into apoptotic and inflammatory caspases based on their biological function; apoptotic caspase includes initiator caspase (caspase-2, −8, −9 and −10) and effector caspase (caspase-3, −6 and −7). The initial caspase is the first caspase to be activated by induced self-cleavage. The primary structure of the initial caspase is characterized by the presence of a long N-terminal propeptide and two death effector domains (DED) or CARD and it mainly activates the effector caspases (135). The effector caspase, characterized by a short N-terminal propeptide without DED and CARD, performs apoptosis by shearing various target proteins (136). Cell pyroptosis is a form of inflammatory programmed cell death pathway activated by human and mouse caspase-1, human caspase-4 and caspase-5, or mouse caspase-11 (137). Inflammatory caspases (caspase-1, −4, −5, or caspase-11) with CARD followed by the catalytic domain mediate cell pyroptosis by the effector protein gasdermin D (137). A recent study reported that melatonin reduced the expression of cleaved caspase-1, cleaved gasdermin D and decreased the number of IL-1β-positive RGC cells after acute ocular hypertension injury (138).

The initiator caspase of the intrinsic pathway is caspase-9, while the extrinsic pathway is caspase-8 and both converge to caspase-3. In the caspase-8/-9 cleavage process of Bid in tBid to remodel mitochondria, favourable conditions are created for ROS production, inhibited by caspase-3 and enhanced by caspase-7 (105). Moreover, caspase-3 is the primary executor of apoptosis.

In a rat model of chronic hypertensive glaucoma induced by episcleral vein cauterization, activation of the PI3K/Akt/mTOR pathway was shown to be neuroprotective against glaucoma (139). Apoptosis crosstalk with autophagy remains limited in glaucoma and the balance between autophagy and apoptosis is crucial for the survival of glaucoma cells (22). In recent years, additional regulators have been found to be involved in the development of glaucoma through the PI3K/Akt pathway. For example, overexpression of CD9 decreases human TM cell apoptosis and attenuates symptoms of human glaucoma by activating ITGA4, PI3K and Akt (67). miR-145-5p induces RGC apoptosis by suppressing tripartite motif-containing 2 (TRIM2)-mediated activation of the PI3K/Akt signalling pathway in a rat model of glaucoma induced by intraocular injection of N-methyl-D-aspartate (NMDA) (140). Using the same animal model of glaucoma, miR-93-5p was revealed to negatively regulate phosphatase and PTEN to promote the survival of RGCs by activating the Akt/mTOR pathway (141). Moreover, p21/Ras integrates survival signals and induces the activation of PI3K to phosphorylate Akt (p-Akt) (142). p-Akt increases Bcl-2/Bcl-xL synthesis and phosphorylates Bad, preventing the heterodimerization of Bad with Bcl-2/Bcl-xL (142). Subsequently, it facilitates the closure of the permeability transition pore complex and prevents the release of mitochondrial factors into the cytoplasm (142). In addition, Yan et al (62) reported that accumulation of the Asn450Tyr mutant myocilin gene (Myoc-N450Y) promotes apoptosis of primary human TM cell through the ERS-induced apoptosis pathway, with the PI3K/Akt signalling pathway playing a crucial role. This evidence suggested that survival signals promote cell survival through the PI3K/Akt signalling pathway in glaucoma (Fig. 2).

Microglial activation is an early change in the retina and optic nerve in the DBA/2J mouse model of chronic inherited glaucoma and may contribute to the onset or progression of glaucoma (80,81). Detection of microglial activation may be valuable for early glaucoma diagnosis, while modulation of microglial responses may change disease progression (80). Increased Fas/FasL immunoreactivity was found in microglia and FADD immunoreactivity was found in Müller glial cells and RGCs (47). FasL (CD95-L/APO-1L) is a signal membrane-bound type II transmembrane protein that belongs to the TNF family, a central pathway in the regulation of apoptosis by the immune system (143,144). Membrane-bound FasL (mFasL) induces apoptosis and promotes inflammation upon binding to Fas. By contrast, the soluble Fas-ligand (sFasL), which is formed by cleavage of the 103–137 amino acid region of mFasL by MMPs, blocks the apoptosis and inhibits inflammation (145–148). Previous research has revealed that the sFas concentration in the aqueous humour is lower in patients with POAG than in control individuals, suggesting that a low level of sFas may provide an appropriate microenvironment for increased apoptosis of TM cells in glaucoma (149).

In the C57BL/6J ^ΔCS mouse model of glaucoma, a mouse model of a membrane-only FasL gene-targeted mouse in which the FasL metalloproteinase cleavage site was mutated in exon two, mFasL, which is expressed mainly in retinal microglia, had a neurotoxic effect on the retina, causing retinal degeneration and inducing RGC death (150). Injection of exogenous sFasL into a mouse model of glaucoma induced by intravitreal TNF-α led to a reduction in RGC loss (150). O'Reilly et al (151) also confirmed that mFasL toxicity induces apoptosis, while sFasL is non-apoptotic. In addition, a different study reported that heat shock protein-induced RGC apoptosis results in microglial activation and upregulation of Fas and suggested that RGC apoptosis is mediated by the inflammatory cytokine FasL (152).

Currently, for the antagonistic effect of mFasL and sFasL in glaucoma, Gregory-Ksande and Marshak-Rothstein (148) suggested that sFasL competes with mFasL for binding to Fas, resulting in steric hindrance. However, except for the Met12 small-molecule inhibitor, the steric hindrance mechanism by which Met12 binds to Fas results in fewer receptors available for FasL binding, directly interfering with FasL binding to Fas (153). There is no better answer to how the sFasL monomer effectively blocks the binding of mFasL multimers to Fas. Nevertheless, other studies have reported that the proapoptotic activity of trimeric sFasL in the ciliary body is enhanced when sFasL binds to corneal ECM proteins (154). A specific cytokine in the ECM, such as TGF-β, increases the local concentration of sFasL (154). Thus, the antagonistic effects of mFasL/sFasL may be a treatment target in glaucoma in the future.

Full-length tropomyosin receptor kinase C (TrkC) is the primary receptor for neurotrophin-3. TrkCT1 is a truncated receptor isoform of TrkC, that lacks the kinase domain and has a unique short intracellular domain. TrkCT1 has been reported to control the production of TNF-α in glial cells, leading to the death of RGCs (155). TNFα is a type II single-transmembrane protein (N-terminal at the cytoplasmic face), that is expressed mainly by macrophages, natural killer cells, T and B cells and plays a role in inflammation, cell proliferation, apoptosis and morphology (155–157). TNFα binds to two types of TNF-α receptors (TNFR1 and TNFR2) to perform its multiple biological functions (158,159). Soluble TNF-α (sTNFα) preferentially binds to TNFR1, which results in neuroinflammation and cell death (160). In addition, Cueva Vargas et al (161) reported that glia-derived sTNFα modulates neuronal death in glaucoma via calcium-permeable AMPA receptor activation. Conversely, transmembrane TNF-α (tTNFα) mainly binds to TNFR2 by activating the prosurvival PI3K-Akt/PKB signalling pathway which mediates neuroprotective effects (159,162,163). A reduction in the number of activated microglia shifts the balance towards antiapoptotic effects in glaucoma (164). A mouse model of glaucoma generated by ocular surgery or trauma has demonstrated that rapid inhibition of TNFα or IL-1β significantly inhibits monocyte infiltration and RGC apoptosis caused by surgical injury and global trauma (36). Additionally, the expression levels of IL-1β and TNFα in the TM of patients with POAG were significantly greater than those in the control group and TNFα induced TM cell death (165,166). TNFα stimulates mitochondria to form a stress response, which sequentially releases ROS, Cytc and Bax to activate caspase-9 and a downstream caspase cascade to initiate apoptosis (167). Moreover, neutralizing the action of TNFα and IL-1β prevents the loss of RGCs induced by elevated hydrostatic pressure or lipopolysaccharide (168). Previous studies demonstrated that patients with the TNFα-308 G/A polymorphism may have increased susceptibility to glaucoma (169,170). However, the relationship between the TNFα-308 G/A polymorphism and glaucoma has not yet been verified.

DR, which belongs to the TNFR superfamily, is a type I signal transmembrane receptor (97). All members of the DR family are characterized by the presence of a DD consisting of an ~80 amino-acid-long motif (171). DD recruits various junction proteins to form a DR platform to mediate cell death. In addition to TNFR1 (known as DR1) and Fas (known as DR2, CD95, or APO-1), DR includes DR-3 (APO-3), DR4, known as TNF-related apoptosis-inducing ligand receptor1 (TRAIL-R1), DR5 (TRAIL-R2), DR6 (CD358), nerve growth factor receptor and ectodysplasin A receptor (172). However, death ligands bind to decoy receptors without DDs, such as TRAIL-R3, which cannot transform apoptotic signals, thus producing antiapoptotic effect (172). The level of DR plays a role in transmitting an apoptotic signal to balance cell life or die. Regardless of the mechanism underlying the upregulation of DR or enhancement of DR function, cell death occurs.

For example, upregulation of the Fas receptor is a marker of human glaucomatous neuropathy (173). In glaucoma, inhibiting Fas expression via an inhibitor provides protection to the retinal nerve (35,174). The glial production of TNFα is increased in the glaucomatous retina and ONH which caused RGCs death through its direct or indirect neurotoxicity (175). Increased immunostaining for TNFα and TNFR1 is observed mainly in glial cells and in glial cells processed around axons and blood vessels in the ONH (176). Under normal circumstances, only TNFR1 is constitutively expressed in the vasculature of the ONH. In human glaucoma, the expression of TNFα and TNFR1 in astrocytes and microglia is increased (177). In severe glaucomatous damage, RGC axons expresses TNFR1, which may be a direct target for TNFα mediated optic nerve degeneration (177). The mRNA and protein expression levels of TNFα and TNFR1 are greater in the inner retinal layers in glaucomatous eyes and TNFR1 is expressed at high levels in RGCs, whereas TNFα, is expressed mainly in glial cells (178). In rat model of experimental glaucoma, TNFα strengthened the excitability of RGCs by activating TNFR1 to upregulate the current density of Nav1.6, namely, the voltage-gated Na⁺ channel, thereby promoting RGC apoptosis, while choosing a suitable sodium channel blocker to block Nav1.6 may be a useful strategy for treating glaucoma (179). An explanation for the cause of TNFR2-mediated RGC death, was that TNFα simulated ocular hypertension, leading to the release of cytotoxic agents followed by the activation of microglia and the loss of oligodendrocytes that encapsulate RGCs, which in turn led to slow RGC death (180).

The caspase family plays a significant role in the execution of cell apoptosis. Hence, caspase activation is likely related to cell apoptosis in glaucoma.

Activation of caspase-9, an initiator of the intrinsic caspase cascade, was found to be involved in RGC death in a rat model of experimental glaucoma (181). In addition, the downregulation of caspase-8 significantly alleviated RGC death in a mouse model of acute glaucoma by inhibiting the processing of IL-1β (182). Caspase-8 has various functions in both RGCs and astroglia in glaucoma. In the C57BL/6J mouse model of experimental glaucoma, caspase-8^−/− astroglia protected RGCs from glial-driven inflammatory impairment, while an inhibitor of caspase-8 cleavage rescued RGCs against from apoptosis, as Yang et al (183) reported that caspase-8 plays a crucial role in RGC apoptosis and astroglia-induced neuroinflammation in glaucoma. Additionally, in caspase-7^−/− mice, caspase-7^−/− ameliorated RGC death in optic nerve crush (ONC) injury and improved the functional response of RGCs (184).

Combined inhibition of caspase activity contributes to the survival of RGCs (185). In a mouse model of glaucoma induced by ischaemia/reperfusion injury, RGC death was reduced by the downregulation of caspase-8 and caspase-3 following resveratrol treatment (186). Inflammatory pyroptotic death, a non-apoptotic process that involves caspases, is essential in death of RGCs (187).

The Bcl-2 gene encodes Bcl-2, which has antiapoptotic effects. Bcl-2, the proto-oncogene of chromosomal t(14;18) in mammalian B-cell lymphomas, has been implicated in apoptosis (188). The Bcl-2 family of proteins comprises proapoptotic and antiapoptotic proteins that regulate MOMP formation and RGC apoptosis after optic nerve injury (189). The imbalance between the proapoptotic and antiapoptotic effects of Bcl-2 family proteins in health and disease determines the fate of cell death or survival (190). Dysregulation of Bcl-2 family members results in an imbalance in the ratio of antiapoptotic (Bcl-2) to proapoptotic (Bax) proteins (191). Other findings suggested that Bak and Bax were highly expressed, while Bcl-2 was expressed at low levels in glaucomatous optic axons in primary angle-closure glaucoma (PACG) (192). Bcl-2 overexpression protects mitochondria from oxidative stress-induced pH acidification, whereas the physiological pH is normal (193). Additionally, high-level Bcl-2 decreases MOMP to reduce mitochondrial-dependent apoptosis by binding to the megapore to facilitate megapore closure, which improves RGC survival in glaucoma (142,194,195). Moreover, high-level Bcl-xL protects neurons from death in the absence of trophic factors, preventing the degeneration of somas and proximal axon segments after degenerating after axotomy (196–198). In the DBA/2J mouse model of inherited glaucoma, Bcl-xL gene therapy prevents Bax translocation and RGC degeneration and reduces cell loss in the RGC layer after ONC (199). Moreover, overexpression of Bim and Bax promoted the RGC death, whereas elevation of Bcl-2 contributed to slowing RGC apoptosis (200). By contrast, decreasing the concentration of Bax in RGCs had a neuroprotective effect after ONC, which indicated that damaged RGCs in a quiescent state did not respond to apoptotic stimulation (201). These findings are consistent with the results of Libby et al (49), who reported that Bax ablation prevents RGC death.

IAPs regulate the cell cycle, signal transduction, cell apoptosis, cytokinesis and are composed of a group of proteins similar in structure and function. IAPs are characterized by the baculovirus IAP repeat (BIR) protein domain at the N-terminus; some possess a new, interesting gene finger domain at the C-terminus (202). To date, eight human IAPs have been identified, including X-chromosome-linked IAP (XIAP/BIRC4), cellular IAP1 (c-IAP1/BIRC2), cellular IAP2 (c-IAP2/BIRC3), IAP-like protein 2 (BIRC8), melanoma IAP (Livin/BIRC7), neuronal apoptosis inhibitory protein (BIRC1), survivin (BIRC5) and the BIR repeat-containing ubiquitin-conjugating enzyme system (BIRC6, Apollo) (202). Although not all proteins with a BIR domain antiapoptotic functions, the BIR domain is necessary for the antiapoptotic effects of IAP family proteins.

As an endogenous inhibitor, IAP inhibits caspase activity by binding its conserved BIR domain to the active sites of caspases (202). By promoting the degradation of caspases or separating caspases from their substrates, IAPs inhibit caspases (202). Recently, dysregulated expression of IAPs has attracted increased amounts of attention in eye disease research. c-IAP1 is upregulated early in experimental glaucoma, as part of the intrinsic neuroprotective mechanism (203). A reduction in the level of c-IAP1 and XIAP secreted by RGCs and the accumulation of TRAF2 result in increased susceptibility to death in cells in the mature RGC layer being more susceptible to death (204,205). Previous studies concluded that ageing impaired the endogenous neuroprotective mechanism of RGCs evoked by elevated IOP in glaucoma patients, namely, a reduction in survival signals mediated by IAPs and TRAF (204,205).

Another IAP, the T allele of rs2754511 in the BIRC6 gene, reportedly protects against glaucoma by alleviating ERS (206,207). On the other hand, overexpression of XIAP and survivin can protect TM cells and ONH astrocytes from apoptosis induced by oxidative stress (208). Besides, a number of studies have identified that Rho/ROCK is involved in the pathogenesis of glaucoma (74,209). In patients with glaucoma, Rho/ROCK activation alters the cytoskeleton and increases TM cell adhesion, reducing aqueous humour outflow and increasing IOP (74). Additionally, Liu et al (209) reported that the long non-coding RNA small nucleolar RNA host gene 11 regulates Wnt/β-catenin signalling through Rho/ROCK via β-catenin phosphorylation at Ser675 or through GSK-3β-mediated phosphorylation at Ser33/37/Thr41, affecting TM cell proliferation, migration, apoptosis and autophagy. Therefore, the absence of activation of prosurvival genes in patients with glaucoma is a potential explanation for the increased vulnerability of the optic nerve to elevated IOP (210).

The tumour suppressor gene p53 encodes the p53 protein. p53, located on chromosome 17p13.1, is a crucial transcription factor with a wide range of target gene repertoires. p53 with one promoter and three mRNA splice variants encodes full-length p53 and different isoforms of the p53 protein, respectively (211). The structure of the p53 protein contains transcriptional activation, DNA binding and oligomerization domains, the molecular weight of p53 is 53 kDa (211). The p53 protein responds to DNA damage or directs damaged cells to the apoptotic pathway to protect the organism from abnormal development (212). The functional polymorphism of the p53 gene that affects the activity of the p53 protein may be related to the induction of apoptosis and the reduction in the ability to induce cell cycle arrest and DNA repair (213,214). Thus, p53 activation is one of the critical steps of apoptosis and upregulates the expression of the proapoptotic gene Bax and downregulates the expression of the antiapoptotic gene Bcl-2 (112,215).

There is a link between polymorphisms in the p53 gene and POAG and PACG in some ethnic populations (216). The p53 codon 72 polymorphism has recently attracted widespread attention in glaucomatous neuropathy in patients with POAG. Due to the change in CGC to CCC, amino acid residue 72 (the fourth exon) was changed from arginine (Arg) to proline (Pro) (217). Lin et al (214) reported that the homozygous Pro/Pro form of p53 codon 72 led to an increased risk of POAG in the Chinese population. Fan et al (218) confirmed the role of p53 variants in POAG, at least in the Chinese population. Several studies have indicated that p53 codon 72 (Pro/Pro vs. Arg/Pro + Pro/Pro) polymorphisms and the introductions of three 16-bp insertions (insertion vs. deletion) possibly contribute to individual susceptibility to POAG, at least in Spain and Iran, and to an increased risk of developing PACG in North India (219–223). Additionally, Wiggs et al (224) demonstrated that the p53 codon 72 Pro/Pro genotype was a potential risk factor for early paracentral visual field defects in Caucasians with POAG. The pro-form of p53 codon 72 results in RGC instability but does not protect RGCs from apoptosis in glaucoma (214). More in vitro studies have revealed that the apoptotic activity of p32-Arg greater than that of p53-Pro due to the p53 inhibitor, which inhibits the activity of p53-Pro and that the apoptotic activity of p53-Pro may be enhanced by certain conditions (low oxygen tension) (224,225).

However, there are conflicting studies about p53 codon 72 polymorphisms increasing the risk of POAG. Dimasi et al (226) noted out that the Arg/Pro polymorphism of p53 codon 72 was irrelevant to the age of onset or severity of glaucoma in Australia. In addition, p53 polymorphisms were not related to POAG in the Japanese, Turkish, or Brazilian populations (227–229). Whether p53 increases individual susceptibility to POAG appears to vary by ethnic subgroup.

Blocking caspase activation is one of the targets for the treatment of glaucoma. Tahzib et al (231) proposed that if caspase activation results in prolonged apoptosis in RGCs, then the use of caspase inhibitors (such as XIAP) to inhibit apoptosis extends the therapeutic window. The pharmacological inhibitor Z-VDVAD-fmk (Z-VDVAD) is a small molecular inhibitor and an available agent that will enter clinical trials. Vigneswara et al (232) reported that Z-VDVAD protected RGCs from apoptosis after ONC by specifically inhibiting caspase-2 activation but did not promote regeneration of RGC axons. Moreover, these results resemble those reported by Monnier et al (233), who used Z-VEID-FMK, a selective inhibitor of caspase-6, to achieve similar effects on RGC survival at 14 days after axotomy and promoted axonal regeneration after ONC. In addition, the caspase-3 inhibitor, Z-DEVD-FMK has neuroprotective effects and significantly promotes visual recovery by inhibiting RGC apoptosis when injected 30 min after optic nerve injury (234). Similarly, the loss of RGCs was delayed by brain-derived neurotrophic factor, an inhibitor of caspase-3 (235,236).

More RGCs were retained in caspase-7^−/− mice than in wild-type mice after ONC, suggesting that blocking caspases may have neuroprotective effects (184). Small interfering RNAs (siRNAs) against caspases have been used in several studies. Tawfik et al (237) reported that non-viral gene therapy with siRNA-nanoparticles selectively silenced the expression of caspase-3 and blocked apoptosis in post-mitotic neurons. Furthermore, in a rat model of optic nerve transection, siRNA inhibited the expression of caspase-2 and provided neuroprotection to RGCs for at least 30 days (238).

Minocycline, which has anti-inflammatory and antiapoptotic properties is a second-generation tetracycline with protease inhibitory properties (239). It is considered a candidate neuroprotective drug for experimental glaucoma and other neurodegenerative diseases (239). Minocycline has been reported to reduce the number of activated microglia and improve RGC axon transport in glaucoma (240). Minocycline upregulates Bcl-2 expression and downregulates Bax and Tp53bp2 expression, shifting the balance to the antiapoptotic direction in experimental glaucoma, and is ready for clinical trials of acute neurological injury (164,239). A similar drug, Asiatic acid, ameliorates retinal dysfunction and protects RGCs from ocular hypertension (241). The antiapoptotic effect of a small molecule inhibitor [2,6-diaminopyridine-3,5-bis(thiocyanate) (PR-619)] on RGCs is mediated by modulation of parkin function and interaction with Bax and Bcl-2 (242). PR-619 protects RGCs from glutamate excitotoxicity-induced apoptosis by increasing the levels of Bcl-2 in RGCs (242). PR-619 regulates neurodegeneration-related stress by stabilizing the mitochondrial membrane potential of RGCs, reducing cytotoxicity and apoptosis, as well as Bax expression. Thus, PR-619 protects RGCs from glutamate excitotoxicity by enhancing parkin-mediated mitochondrial autophagy rather than the apoptotic pathway.

Injection of Szeto-Schiller peptide 31 (SS-31) into a Sprague Dawley rat model of experimental glaucoma had neuroprotective effects. SS-31 improved the a-wave and b-wave amplitudes of the ERG and F-VEP amplitude in the eye, upregulated the level of Bcl-2, downregulated the level of Bax, and inhibited the release of Cytc (243). Numerous experimental drugs have demonstrated that upregulating the expression of Bcl-2 and downregulating the expression of Bax and caspase-3 are potential methods for protecting RGCs (195,241,244).

IAPs, especially XIAP, are potent caspase inhibitors and are attractive molecular targets. IAPs significantly inhibit apoptosis by inhibiting the activity of caspase-9, −3 and −7 (245). A number of novel treatments include gene therapy, such as with XIAP, which offers a new direction in glaucoma treatment. In a rat model of glaucoma, recombinant adeno-associated viral (AAV) loaded with XIAP promoted the survival of optic nerve axons (246). Moreover, the expression of the XIAP prevents IOP elevation by regulating the production of aqueous humour (246). Other findings indicated that AAV-loaded XIAP protected both the structure and function of the axons of RGCs and decreased glial cell infiltration in a mouse model of glaucoma (247).

In addition, it has been revealed that tacrolimus inhibits the expression of survivin to reduce cell proliferation and induce cell apoptosis (248). A small-molecule survivin inhibitor decreased TGF-β-induced cell proliferation and migration during the epithelial mesenchymal transition (249). Thus, survivin depletion results in TGF-β1 induced cell cycle arrest and apoptosis and reduces the phosphorylation of retinoblastoma proteins (249). Therefore, a strategy to improve survivin expression may be a useful for treating glaucoma. Recently, ROCK inhibitors have been identified as a new class of drugs that directly target TM cells to reduce IOP (250,251). In a transgenic mouse model, ROCKi was reported to reduce IOP by promoting cell proliferation and phagocytosis of TM cell, reducing actin cross-linking and cell adhesion interactions (74,250,252). Currently, two types of ROCKis, namely, ripasudil (K-115) in Japan and netarsudil (known as AR-13324z), are approved for the clinical treatment of glaucoma in the United States (253–256). These findings indicated that the ROCKi may be a new first-line drug for glaucoma treatment.

The use of p53 as a therapeutic target has been studied in glaucoma. The function the transcription factor p53 depends on its phosphorylation and dephosphorylation. Serine/threonine-protein phosphatase-1 has been reported to directly dephosphorylate p53 to negatively regulate its transcriptional and apoptotic activities, thus promoting cell survival (257). Vitamin B1 has been revealed to significantly reduce p53 expression (258). Johnson et al (259) demonstrated that the adenoviral p53 gene replaced the role of mitomycin C and 5-fluorouracil in glaucoma surgery due to its antiproliferative properties.

The PI3K/Akt pathway is a pivotal intracellular signalling pathway that promotes cell proliferation, inhibits apoptosis and induces angiogenesis by activating multiple downstream regulatory elements. A sustained decrease in Akt activation was observed in the ocular-hypertensive retina and optic nerve of a rat model of glaucoma induced by injecting hypertonic saline into the limbal veins (260). Factors that activate the PI3K/Akt pathway constitute a novel molecular therapy for glaucoma (Fig. 2). Yan et al (67) demonstrated that CD9-knockdown significantly reduced the expression of ITGA4, p-PI3K and p-Akt and increased the apoptotic activity of TM cells, which was increased dramatically in the CD9-overexpressing group. Knockdown of ITGA4 rescued p-PI3K and Akt expression, suggesting that overexpression of CD9 activates the ITGA4/PI3K/Akt axis to attenuate TM cell apoptosis in human glaucoma (67).

Moreover, several traditional Chinese medicines may suppress glaucoma pathogenesis by activating PI3K/Akt signalling both in vitro and in vivo. Husain et al (260) reported that the δ-opioid receptor agonist SNC-121 significantly increased the ERG amplitude and RGC number in a rat model of chronic glaucoma by activating the PI3K/Akt pathway. Moreover, baicalein inhibits NMDA-induced apoptosis, autophagy and oxidative stress in RGCs by activating the PI3K/Akt signalling pathway in vitro and in vivo (261). A mouse model of glaucoma induced by injection of NMDA confirmed that baicalin inhibited autophagy and subsequently attenuated pathological changes in retinal tissues by activating PI3K/Akt signalling (261). Furthermore, ligustrazine increased protein levels of p-PI3K, p-Akt and p-mTOR in a rat model of chronic hypertensive glaucoma, while rapamycin or Ly294002 attenuated these changes (139). A recent study revealed that acteoside may be a potential protective drug for maintaining RGC homeostasis and preventing glaucoma-related blindness because of its therapeutic effects, such as antioxidative stress, anti-inflammatory, anti-aging and proliferative effects (262).

In addition to drugs, several microRNAs mediate apoptosis in glaucoma through the PI3K/Akt pathway. miR-145-5p suppresses TRIM2 expression by targeting the 3′-untranslated region of TRIM2. Inhibition of miR-145-5p promotes cell survival and suppresses RGCs apoptosis by activating the TRIM2/PI3K/Akt signalling pathway (140). miR-149 inhibition also promoted the viability of RGCs and inhibited RGC apoptosis in a mouse model of glaucoma by increasing the levels of BTC, p-PI3K and p-Akt (263).