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Introduction

Glaucoma is a group of progressive optic neuropathies and is currently the most prevalent ocular disease worldwide, second only to cataracts in terms of blindness (1). Current estimates indicate a global prevalence of 3.5% among individuals aged 40 to 80 years, with projections suggesting a staggering 111.8 million individuals will suffer from blindness attributable to glaucoma by the year 2040 (2,3). This condition, characterized by its prevalence and impact on vision, primarily stems from the deterioration of retinal ganglion cell (RGC) axons as they traverse the optic nerve head (ONH) upon exiting the eye (4). This degenerative process results in persistent impairment of the retinal nerve fiber layer (5,6), with exacerbation often observed due to deformation of the lamina cribrosa (LC) (7). Previous investigations have established a positive correlation between elevated intraocular pressure (IOP) and high-pressure glaucoma (8), underscoring its pivotal role in inducing damage to RGC axons and subsequent cell demise (9), thereby emerging as a significant risk factor for the development of glaucoma (10). Consequently, interventions aimed at reducing IOP, through pharmacological, laser, or surgical modalities, have proven efficacious in attenuating glaucoma progression and preserving visual function (3). Nonetheless, certain presentations of glaucoma, such as those featuring normal IOP, pose challenges as they diverge from the typical paradigm (11,12). Additionally, the heterogeneity of glaucomatous subtypes, coupled with the limited effectiveness of conventional therapies, underscores the complexity inherent to managing this multifaceted condition (13). Glaucoma poses a significant challenge in clinical management, which is exacerbated by the limited efficacy of conventional treatments in reversing the optic nerve damage associated with the condition (3). Thus, a comprehensive understanding of the underlying mechanisms and pathophysiology governing glaucoma pathogenesis is required. This entails elucidating the complex signaling pathways implicated in pathogenesis to identify novel targets for interventions aimed at optic nerve protection and regeneration at the molecular level. Previous studies have highlighted the degeneration of RGCs and loss of axons, along with damage and remodeling of the LC, as pivotal events in glaucoma pathogenesis (Fig. 1) (5). Moreover, in our research (data not shown), it was found that there appears to be a certain correlation among the above-mentioned pivotal events-Caveolin-1 (Cav-1). Cav-1 cannot only prevent the degeneration of RGCs and optic nerve damage in glaucoma by positively or negatively mediating cell signaling pathways and neurotrophic factors, but also regulate the physiological and pathological changes of the LC by influencing the metabolism of the extracellular matrix (ECM), thus playing an important role in the occurrence and prevention of glaucoma (14-16). The signaling pathways activated by genetic and environmental factors constitute a complex and multifaceted system characterized by extensive crosstalk between downstream signaling molecules. Consequently, these pathways are interlinked and overlapping, thereby preventing their complete isolation. Nevertheless, the regulation of proapoptotic genes, neuroprotective factors and regenerative factors are subject to complex modulation within these pathways, ultimately culminating in glaucoma development (13). The regulatory effects within these pathways are not unidimensional but emerge from the complex interplay and mutual influence of multiple factors. In light of the structural framework of this discourse, the present review delves into the pathophysiological mechanisms of RGCs apoptosis, optic nerve safeguarding and regeneration, and LC damage and remodeling; the specific molecular mechanisms and the interrelationships of pathways are shown in Fig. 2. This categorization is based on the most extensively researched segments of pathways, with the aim of offering deeper insights into cellular processes and furnishing vital clues and targets for the treatment and pharmacological management of glaucoma.

Figure 1 Summary of the role of signaling pathways in the mechanisms of glaucoma in retinal ganglion cells apoptosis, optical nerve protection and regeneration, and the LC damage and remodeling. TNF-α, tumor necrosis factor-alpha; IL, interleukin; ROS, reactive oxygen species; cyt-c, cytochrome c; caspase, cysteinyl aspartate-specific proteinase; LC, lamina cribrosa; ECM, extracellular matrix; IOP, intraocular pressure.

Figure 2 Review of glaucoma related signaling pathways and their mechanisms. BDNF, brain-derived neurotrophic factor; TrkB, tropomyosin receptor kinase B; PLCγ1, 1-phosphatidylinositol 4,5-bisphosphate phosphodiesterase γ-1; CaMKII, Ca2+-calmodulin protein kinase II; DAG, diacylglycerol; PKC, protein kinase C; CREB, cAMP-response element binding protein; Shp2, tyrosine phosphatase 2; Cav-1, caveolin-1; RTKs, receptor tyrosine kinases; IRS1, insulin receptor substrate 1; PI3K, phosphoinositide 3-kinase; PKB, protein kinase B; GSK-3, glycogen synthase kinase 3; BAD, Bcl-2-associated death promoter; BAX, Bcl-2 Associated X Protein; cyt-c, cytochrome c; Apaf-1, apoptotic protease activating factor 1; caspase, cysteinyl aspartate specific proteinase; PTEN, phosphatase and tensin homolog; PDK1, 3-phosphoinositide-dependent protein kinase 1; TNF, tumor necrosis factor; TNFR, TNF receptor; TRADD, TNFR type 1-associated death domain protein; TRAF2, TNFR associated factor 2; Trx, thioredoxin; MAPK, mitogen-activated protein kinase; MAPKKK, MAPK kinase kinase; ASK1, apoptosis signal-regulating kinase 1; MAPKK, MAPK kinase; MKK4/7, MAPK kinase 4/7; MKK3/6, MAPKkinase 3/6; ROCK1, Rho-associated coiled-coil forming protein kinase 1; JIP1/2, JNK-interacting protein 1; JNK, c-Jun N-terminal kinase; TGF-β, Transforming growth factor-beta; TβRI, TGF-β type I receptor; TβRII, TGF-β type II receptor; RhoA, Ras homolog family member A; ECM, extracellular matrix; Src, non-receptor tyrosine kinase; CASL, Src scaffolding protein; PIP2, phosphatidylinositol-4,5-bisphosphate; PIP3, phosphatidylinositol-3,4,5-bisphosphate.

Molecular signaling pathways and apoptosis in RGCs

Apoptosis, also known as programmed cell death, is widely considered the principal contributor to the loss of RGCs in glaucoma (17). Numerous mechanisms capable of triggering apoptosis in RGCs have been identified, including increased IOP, ischemia and reperfusion events, oxidative stress and neuroinflammatory responses (18). The complicated involvement of multiple molecular and signaling pathways in these pathological changes is of paramount importance, with particular emphasis on the phosphoinositide 3-kinase (PI3K)/AKT, mitogen-activated protein kinase (MAPK) and Bcl-2 family pathways (19,20). A comprehensive understanding of these complex signaling pathways holds great promise for intervention at the molecular level during the early stages of glaucoma, with the goal of impeding apoptotic pathways of RGCs. These interventions have the potential to significantly slow or even halt glaucoma progression.

PI3K/AKT signaling pathway

The PI3K/AKT signaling pathway represents a pivotal intracellular cascade that governs various cellular processes, such as inhibition of apoptosis, facilitation of proliferation (19) and safeguarding against injury-induced loss of RGCs in the context of glaucoma (21,22). Its complex orchestration involves several key molecular components, including receptor tyrosine kinases (RTKs), PI3K, phosphatidylinositol-4,5-bisphosphate (PIP2), phosphatidylinositol-3,4,5-bisphosphate (PIP3) and protein kinase B (PKB)/AKT (23-25). RTKs serve as a principal upstream regulator of the PI3K/AKT signaling cascade, instigated by extracellular growth factors to initiate signaling events and activate PI3K (25,26). Within the cell membrane, PIP2 and PIP3 are minor phospholipid constituents. In PI3K/AKT-mediated apoptosis, PIP2 is converted to PIP3, which accumulates as a pivotal docking phospholipid within the plasma membrane. Subsequently, PIP3 binds to specific structural domains, facilitating the recruitment of AKT to its kinase PDK1, thereby activating PKB/AKT (27). This activation event culminates in the attenuation of RGCs apoptosis, thereby impeding glaucoma (28). Ultimately, this complex regulatory network contributes to a reduction in RGCs apoptosis and a subsequent delay in the onset and progression of glaucoma pathology (Fig. 2).

In addition, previous studies have shown that Caveolins play a significant role in regulating several signal transduction pathways in ocular diseases including glaucoma (29,30). There are three subtypes of Caveolins in vertebrates, namely Cav-1, Cav-2 and Cav-3 (31). Among them, Cav-1 exhibits strong immunoreactivity in the RGC layer, and has been identified as a gene locus associated with glaucoma in genome-wide association studies (15). Experiments have found that the expression of Cav-1 is downregulated in the induced glaucoma mouse model, and the mice showed ocular hypertension, further indicating the protective role of Cav-1 in glaucoma (32). It has been found that Cav-1 has been proven to play a positive role in the PI3K/AKT signaling pathway, and can inhibit apoptosis, inflammation and oxidative stress by upregulating the PI3K/AKT signal transduction pathway (33-35), thereby preventing the induced degeneration of RGCs in glaucoma. Moreover, studies have suggested that acteoside, the main ingredient of Yunnan purple taro, and baicalin, the dry root of the Chinese herbal medicine Scutellaria baicalensis, have significant effects in preventing and treating glaucoma, reducing the loss, autophagy and oxidative stress of RGCs, and thus preventing the occurrence and development of glaucoma (36-38). Experiments have demonstrated that Acteoside can prevent the autophagic apoptosis of RGCs and delay the optic nerve atrophy induced by glaucoma by activating the PI3K/AKT signaling pathway (16,38). A recent study found that the protective effect of Acteoside on glaucoma is affected by Cav-1 (16). Cav-1-deficient mice not only reverse the effects of Acteoside on the oxidative stress and autophagy of RGCs, but also reverse the inhibitory effect of Acteoside on RGCs loss and its activation effect on the PI3K/AKT signaling pathway. This indicates that Acteoside relies on the positive regulation of Cav-1 to activate the PI3K/AKT pro-survival signaling pathway, thereby preventing the deterioration of glaucoma (16). Furthermore, in a study by Zhao et al (39), RGCs were stimulated with N-methyl-D-aspartate (NMDA) to establish an in vitro glaucoma model. Methyl-thiazolyl-tetrazolium assay indicated a decline in RGCs' viability and an increase in apoptotic rates with increasing NMDA concentrations. Concurrently, there was a decrease in the expression of phosphorylated (p-)AKT and p-PI3K in RGCs. Subsequent administration of baicalin, a compound found to counteract the promoting effect of NMDA on RGCs apoptosis, restored the diminished levels of p-AKT and p-PI3K proteins, further corroborating the involvement of the PI3K/AKT pathway in alleviating apoptosis and injury in NMDA-treated RGCs. Additionally, treatment with LY294002, a PI3K inhibitor, reversed the beneficial effects of baicalein on viability and expression of RGCs, underscoring the crucial role of the PI3K/AKT signaling pathway in modulating survival and function of RGCs. Collectively, these findings highlight the significant association between the PI3K/AKT pathway and RGCs, highlighting its potential as a therapeutic target for impeding RGCs' apoptosis and delaying glaucoma progression.

MAPK signaling pathway

MAPK constitutes a group of serine-threonine kinases that serve as pivotal elements in intracellular signal transduction mechanisms and orchestrate a diverse array of cellular processes, including differentiation, proliferation, apoptosis, inflammation and responses to various stress stimuli (40). The MAPK signaling cascade delineates a sequential series of kinase events involving MAP3K, MAP2K and MAPK. Initially, MAP3K, a Ser/Thr protein kinase, is activated through phosphorylation, which subsequently catalyzes the activation of MAP2K at its activation site. Consequently, MAP2K increases MAPK activity by affecting dual phosphorylation events at Thr and Tyr residues situated within specific motifs (41). The MAPK pathway, in its complex network, diverges into three principal routes: Extracellular signal-regulated kinase (ERK), p38 MAPK and c-Jun N-terminal kinase (JNK) (40) (Fig. 2). Activation of ERK promotes cell survival, whereas the activation of p38 MAPK and JNK plays a critical role in mediating apoptosis in RGCs (41,42).

p38 MAPK signaling pathway

The apoptosis of RGCs has been associated with the activation of microglia and the subsequent release of inflammatory mediators such as tumor necrosis factor-alpha (TNF-α), interleukin (IL)-16 and IL-6 (5,43). Moreover, canonical inducers of p38 MAPK activation encompass inflammatory cytokines such as TNF-α, IL-6 and IL-1β (44). Osteopontin (OPN) serves not only as a phosphorylated glycoprotein but also as a pro-inflammatory cytokine in various tissues and cellular contexts (45-47). Its involvement extends to the pathogenesis of numerous neurodegenerative disorders and is closely associated with the regulation of autophagy and its ramifications on neuronal function (48). In the retina, knockout of OPN in mice revealed that microglia showed no signs of activation, such as migration from the inner retina to the subretinal space, or morphological changes in the amoeboid microglia (49). It was deduced that OPN may promote the transition of microglia to the amoeboid phenotype and activate microglia (50). A study conducted by Yu et al (51) established a rat model characterized by chronic ocular hypertension (COH), revealing microglial activation, upregulated expression of microglia-derived OPN and changes in inflammatory cytokine levels (TNF-α, IL-1β and IL-6). Treatment with an inhibitor targeting the p38 MAPK pathway in activated microglia led to diminished production of TNF-α, IL-1β and IL-6. Additionally, intravitreal administration of anti-OPN antibodies in COH-afflicted rats resulted in a significant reduction in p38 expression compared with that in the untreated COH cohort. These findings highlight the potential of OPN to induce apoptosis of RGCs via microglial activation, with the p38 MAPK pathway playing a pivotal role in this complex cascade of events.

JNK signaling pathway

The JNK pathway plays a pivotal role in the pathogenesis of diverse diseases, including Alzheimer's disease, Parkinson's disease and glaucoma (52-54). Its significance as a prospective target for neuroprotective intervention has attracted considerable attention (55). Among the proteins implicated in JNK activation and the regulation of neuronal apoptosis, JNK interacting protein 1 (JIP1) emerges as a noteworthy scaffolding protein (42). In the specific context of glaucoma, the complex interplay between mitochondrial dysfunction, oxidative stress and apoptosis in RGCs is well-established (56). Notably, Rotenone, a lipid-soluble environmental toxin, induces RGCs' apoptosis by impeding mitochondrial complex I (57). In a study conducted by Liu et al (42), the suppression of JIP1 in mice exposed to rotenone led to diminished JNK activation, reduced caspase-3 cleavage, and a decrease in TUNEL-positive RGCs within the retina. This attenuation of rotenone-induced RGCs electrophysiological dysfunction highlights the potential therapeutic relevance of the JIP1-JNK signaling axis in alleviating RGCs degeneration. Furthermore, thioredoxin 1 (Trx1), a 12 kDa oxidoreductase, assumes a critical role in antioxidant and anti-apoptotic processes during periods of oxidative stress (58,59). Melatonin, renowned for its protective effects against H2O2-induced apoptosis and oxidative stress, functions by preserving the expression of Trx1 and thioredoxin reductase 1 (TrxR1), and the activity of TrxR1 in RGC-5 cells. The significance of Trx1 in melatonin-mediated protection against oxidative stress-induced apoptosis is highlighted by observations indicating compromised protective effects of Trx1 knockdown. Intriguingly, the alleviating effect of Trx1 silencing in RGC-5 cells was partially counteracted by the administration of a JNK signaling inhibitor (60). This suggests a complex interaction between JNK signaling and Trx1 in modulating apoptosis and oxidative damage in RGC-5 cells, implicating the JNK signaling pathway in safeguarding RGC-5 cells against detrimental effects.

Indirect traumatic optic neuropathy (ITON) shares a pathological resemblance with glaucoma, characterized by apoptosis of RGCs and subsequent optic nerve atrophy (61). Experimental induction of an ITON model coupled with the activation of JNK/c-Jun signaling revealed concurrent microglial and NLRP3 inflammasome activation. Conversely, inhibition of JNK/c-Jun signaling was found to forestall NLRP3 inflammasome activation in microglia, thereby protecting against RGCs' death and axonal degeneration (62). This elucidated the complex interaction among JNK signaling, microglial activation and RGCs' survival pathways, suggesting a potential approach for therapeutic intervention in conditions characterized by optic nerve damage.

Bcl-2 family/caspase

Apoptosis typically occurs via two distinct pathways: The intrinsic pathway, which is mediated by mitochondria, and the extrinsic pathway, which is mediated by death receptors. The Bcl-2 family of proteins plays a critical role (63). In RGCs, mitochondrial dysfunction and oxidative stress are closely associated with apoptosis, suggesting the involvement of the Bcl-2 family of proteins in RGCs' death (64). This family is comprised of three main types: Pro-apoptotic BH3 proteins (BIM, BID, PUMA, BMF, NOXA, BIK, BAD and HRK), pro-survival proteins (BCL-2, BCL-XL, BCL-W, MCL-1 and A1/BFL-1), and apoptotic effectors (BAX, BAK and BOK) (65-67). Notably, BAX and BAK play pivotal roles in triggering apoptosis and exhibit a strong affinity for BH3 structural domains (67). Under conditions such as nutrient deprivation, lack of growth factors, oxidative stress, exposure to γ-irradiation, or treatment with cytotoxic drugs, BH3 activator proteins selectively bind to the BH3 structural domain-binding groove in BAX/BAK, prompting their activation through structural changes. This triggers the formation of BAX and BAK oligomers in the outer mitochondrial membrane, leading to the creation of pores that permeabilize the membrane. Consequently, cytochrome, an apoptotic factor residing within the mitochondria, is released, which subsequently activates caspase-9 and caspase-3. Activated caspases initiate the breakdown and elimination of RGCs (66-68) (Fig. 2).

In cases of glaucoma resulting from damage to the optic nerve, there is a significant increase in the levels of genes that promote cell death, such as BAX and BAD, in both the affected retina and optic nerve (20). This increase led to the widespread death of RGCs. Extensive research has revealed that activating the cAMP-response element binding protein (CREB)/BCL-2 pathway can prevent mitochondrial cell death. Conversely, the disruption of CREB function tends to worsen cell death, resulting in a decreased BCL-2/BAX ratio, ultimately leading to the loss of mitochondrial function (69). Pituitary adenylate cyclase-activating polypeptide (PACAP) has emerged as a powerful protector of nerve cells, exhibiting significant neuroprotective properties (70). PACAP is important in preventing RGCs' death, as well documented in various animal models of retinal damage (71). After optic nerve injury, there is a significant increase in PACAP and its receptor PAC1R in the layer of RGCs, indicating the prevention of cell death mediated by caspase-3 in RGCs. This series of events is further characterized by an increase in the activation of cAMP-responsive CREB and an elevation in the levels of BCL-2 (70). Altogether, these findings highlight the crucial role of the CREB/BCL-2 pathway in reducing cell death in RGCs and emphasize its importance in maintaining retinal health and function.

Molecular signaling pathways and optic nerve protection and regeneration

Degeneration of RGC axons is a pivotal element in the pathogenesis of glaucomatous neurodegeneration, as postulated in scholarly discourse (6). Ocular hypertension, which is frequently correlated with elevated IOP, poses a significant risk of structural impairment of the ONH, manifesting as retinal rim thinning, augmented cup/disc ratio, and optic disc cupping in severe manifestations. Such pathological alterations can cause irreversible damage to the optic nerve in patients with glaucoma (72). Although reduction of IOP can mitigate disease progression, it does not adequately address the fundamental issue of optic nerve degeneration (73). Hence, there is a compelling impetus to explore the complexities of the signaling pathways associated with the safeguarding and rejuvenation of the optic nerve, presenting promising broad for impeding or reversing the process of optic nerve damage in glaucoma.

Brain-derived neurotrophic factor (BDNF) signaling pathway

BDNF constitutes a critical neurotrophic element pivotal in the processes of neuronal development, differentiation and survival (74). Its principal synthesis occurs in the brain and retina, where it exerts considerable neuroprotective effects in conjunction with other neurotrophic factors. This protective mechanism operates by alleviating the loss of RGCs following optic nerve trauma through its interaction with receptor tyrosine kinases (Trk family) located in the ONH (75). Specifically, the binding of nerve growth factor to TrkA, BDNF to TrkB, and neurotrophic factor-3 to TrkC has been established (74). Among them, furthermore, the BDNF/TrkB signaling pathway plays a crucial role in the health of RGCs and the prevention of apoptosis (14). BDNF administration has been proven to delay the apoptosis of RGCs and extend the survival of neurons under various stress conditions (76). As aforementioned, Cav-1 participates in signaling pathways, and its deficiency will impair retinal function. However, a recent study proposed that Cav-1 negatively regulates the BDNF/TrkB signal transduction in RGCs through its interaction with the tyrosine phosphatase 2 (Shp2) (14). It has been reported that the dysregulation of Shp2 is related to neurodegenerative diseases in the brain and eyes (77,78). Abbasi et al (15) demonstrated through experiments that the silencing of Shp2 has a protective effect on retinal function under chronic experimental glaucoma conditions, and this protective effect depends on Cav-1 in the retina. Cav-1 may use raft microdomains as a platform to recruit Shp2, and then transfer Shp2 to the vicinity of its target TrkB receptor through this platform for interaction (79). TrkB is a high-affinity receptor for BDNF and can support the long-term survival of RGCs (75). Under stress conditions, Cav-1 will be hyperphosphorylated in RGCs, and the number of its bindings with Shp2 will increase significantly, leading to the activation of Shp2 and then the negative phosphorylation of the TrkB receptor (80). The long-term dephosphorylation of TrkB will result in the loss of axonal integrity and hinder the axonal regeneration and other neuroprotective effects of BDNF and neurotrophic factor-4 (NT-4) (14). Therefore, in-depth exploration of the complex interactions among BDNF, TrkB, Shp2 and Cav-1 is of great significance for the treatment of glaucoma. Targeting this pathway is expected to increase the survival probability of neurons, protect the optic nerve and relieve vision problems, bringing new hope for the clinical treatment of glaucoma.

Activation of the PI3K/AKT signaling cascade and ERK, accompanied by CREB phosphorylation (Fig. 2), plays a pivotal role in fostering cellular survival and thwarting mitochondrial apoptosis, thereby conferring neuroprotection (81). In glaucoma, noteworthy deviations in the functional dynamics and expression patterns of BDNF and TrkB have been observed, particularly within the retinal milieu. In this context, the interaction between BDNF and TrkB elicits the activation of the PI3K/AKT and ERK pathways, culminating in CREB phosphorylation and consequently enhancing retinal resilience (75,82). Furthermore, engagement of the p75 neurotrophic factor receptor (p75NTR), acting as a BDNF receptor, with the brain-derived neurotrophic factor precursor (pro-BDNF) induces apoptosis, thus presenting a counterpoint to the effects induced by TrkB binding (75,83). Consequently, strategic interventions targeting the proBDNF/p75NTR signaling axis have emerged as a promising approach for increasing neuronal sustenance and proliferation.

A previous study has revealed the capacity of lithium to increase the population density of RGCs in a dose-dependent manner, concomitant with the upregulation of BDNF observed at both the mRNA and protein tiers (84). The protein Dock3, belonging to the Dock family and known for its involvement in cellular adhesion and neurite elongation, has been delineated as a facilitator of axonal regeneration and neuroprotection in vivo (85,86). Notably, the signaling cascade mediated by Dock3 is activated by BDNF, and conversely, Dock3 reciprocally enhances the stimulatory influence of BDNF on neurite extension (74).

Furthermore, BDNF levels consistently decreased in patients with glaucoma having ONH damage (81). Patients with primary open-angle glaucoma (POAG) and normotensive glaucoma exhibit significantly lower serum BDNF levels than controls, with even lower levels observed in early-stage glaucoma than in moderate glaucoma. These findings suggest that BDNF can potentially serve as a biomarker for glaucoma.

Phosphatase and tensin homolog (PTEN) signaling pathway

PTEN is a phosphatase that acts on lipids and proteins and has been implicated in the pathogenesis of neurodegenerative diseases. Notably, in murine models with a PTEN knockout, substantial enhancement of axonal regeneration following optic nerve injury has been observed (87). This phenomenon underscores the pivotal role of PTEN in the modulation of cellular responses to injury. Mechanistically, PTEN exerts its inhibitory effect on cell proliferation by impeding the phosphorylation of PIP2 to PIP3, thereby thwarting the activation of the PI3K pathway and its downstream effectors, including AKT and the mTOR cascade (88) (Fig. 2). Conversely, the downregulation of PTEN promotes the activation of the PI3K/AKT/mTOR axis, which promotes axon regeneration in the optic nerve and augments the survival of RGCs post-injury (89). Furthermore, the suppressor of cytokine signaling 3 (SOCS3) is a pivotal regulator of signaling pathways associated with fundamental cellular processes such as proliferation, survival, migration and genomic stability (90). Comparative analyses revealed markedly reduced RGCs' survival in murine models featuring either pure optic nerve injury or SOCS3 knockout, compared with those with PTEN deficiency alone or in combination with SOCS3 (91). Additionally, investigations of optic nerve compression have revealed dendrite and axon retention and regeneration (91).

The miR-200 family plays a critical role in regulating cellular proliferation and apoptosis (92). PTEN has been identified as a co-target of the miR-200 family, indicating a regulatory interplay between them (93,94). Current investigations on POAG have shifted attention toward the involvement of trabecular meshwork (TM) cells and apoptosis of RGCs. Shen et al (93) elucidated the interaction between PTEN and miR-200c in TM cells, with oxidative stress-inducing decreased expression of miR-200c-3p, consequently leading to elevated PTEN levels and heightened cellular apoptosis (95). Additionally, miR-200c-3p has been shown to negatively regulate PTEN expression, cleaved caspase-3 and Bax, while concurrently enhancing the expression of p-AKT, AKT and mTOR, thereby promoting cell proliferation and inhibiting apoptosis, which can be reversed by PTEN (93).

Furthermore, genes associated with mitochondrial function, such as Dynlt1a and Lars2, have been implicated in facilitating axon regeneration, and PTEN inhibition upregulates their expression (96). Moreover, PTEN inhibition was found to induce the dedifferentiation of intrinsically light-sensitive RGCs, thereby activating the intrinsic peripheral axon regeneration capacity (96).

In summary, PTEN has emerged as a pivotal regulator in both the upstream and downstream pathways governing optic nerve axons and survival and regeneration of RGCs. Consequently, PTEN inhibition has emerged as a promising therapeutic target for optic nerve protection and regeneration in patients with glaucoma.

Rho/ROCK signaling pathways

Rho, a constituent of the cytoplasmic Rho family of small GTP-binding proteins, belongs to the Ras superfamily of GTPases and encompasses three distinct isoforms: RhoA, RhoB and RhoC (97). Notably, RhoA exhibits a marked increase in the ONH of individuals with glaucoma (98). Acting as a pivotal downstream effector of Rho GTPases, ROCK (Rho-associated protein kinase) represents a serine/threonine kinase, existing in two isoforms, namely ROCK1 and ROCK2 (98,99). Pertinently, microglia, acknowledged for their significance in the pathogenesis of glaucoma, are implicated in neurotoxicity under the influence of activated ROCK, thereby contributing to optic neuropathy (100,101). Significantly, the administration of ROCK inhibitors, including Y-27632, Y-39983, netarsudil and ripasudil, inhibits ROCK activation, consequently facilitating the axonal regeneration of RGCs (102-105). Hence, the use of ROCK inhibitors has emerged as a promising approach for the advancement of optical neuroprotective strategies in glaucoma.

ROCK inhibitors have been proposed to slow down optic nerve damage by lowering IOP in glaucoma (99), ROCK can improve cell proliferation, inhibit apoptosis, and lower IOP by blocking the Rho kinase cascade activation response (106). Moreover, ROCK inhibitors can provide optic neuroprotection by targeting the cytoskeleton in the TM and Schlemm's canal (SC) cells, increasing aqueous humor (AH) outflow and reducing IOP (99). In glaucoma, ischemia leads to progressive damage to retinal tissue and optic nerves (107). The Rho/ROCK signaling pathway is present in vascular smooth muscle and promotes relaxation, suggesting that ROCK inhibitors could offer neuroprotection by inducing vasodilation to improve blood flow to the retina and optic nerve, thereby supporting axonal regeneration and survival of RGCs (108). Shaw et al (104) discovered that RGCs survival and optic nerve axon regeneration were significantly higher in mice treated topically with a Rock/norepinephrine transporter protein (Net) inhibitor than in mice administered a placebo. This topical treatment also resulted in reduced ROCK target protein phosphorylation in the retina and proximal optic nerve. RGCs play a crucial role in optic nerve damage in glaucoma, with the expression of Rho-kinase significantly increased in apoptotic RGCs. Liu et al (109) conducted in vitro experiments by treating RGCs with siRhoA, and found that this treatment effectively downregulated RhoA expression, protecting cells from H2O2-induced damage. They also observed a reduction in the expression of ROCK1, the ROCK2 receptor and Casepase-3, along with an elevation in Bcl-2 expression at both the mRNA and protein levels. In conclusion, blocking the Rho/ROCK signaling pathway could be a promising approach for developing new strategies for optic nerve protection and axon regeneration in glaucoma treatment.

Molecular signaling pathways and LC damage and remodeling

The LC is a reticular sieve-like structure located in the sclera, from which the axons of RGCs converge for the optic nerve to penetrate (110); this is the initial site where damage to ganglion cells and axons occurs in glaucoma (111). The aforementioned study found that RGCs are the initial sites of damage to ganglion cells and axons in glaucoma. It has been found that the LC depth is generally more posterior, and the LC morphology is more distorted in patients with glaucoma than in healthy eyes, which blocks axonal transport and reduces the diffusion of nutrients from capillaries within the laminar bundles to neighboring axons, promoting damage to axons and their cell bodies (112,113). In addition, LC cells can pass through laminar bundles to adjacent axons. In addition, LC cells can exhibit negative effects on the environment in which RGC axons traverse through ECM remodeling (114). The current study of the signaling pathways involved in the damage and remodeling of the LC could help identify potential therapeutic targets to stop the damage and remodeling of the LC and restore the normal morphology and function of the LC to slow down the glaucomatous process.

Calcium ions (Ca2+) signaling pathways

Ca2+ enter cells and interact with various Ca2+-binding proteins, serving as pivotal signaling entities in the regulation of numerous physiological processes (115). Perturbation of Ca2+ homeostasis has been implicated in various pathological conditions, including oxidative stress-induced cell death in glaucoma (116). Notably, cytoplasmic Ca2+ levels are aberrantly elevated in LC cells in models of oxidative stress-induced glaucoma (117). This elevation comprises two distinct components: The direct influx of extracellular calcium into the cytoplasm in response to the stress stimulus, and release from intracellular stores, namely the endoplasmic reticulum and sarcoplasmic reticulum, mediated by the Inositol 1,4,5-trisphosphate receptor (IP3R) and ryanodine receptor (118,119). The increase in intracellular Ca2+ in glaucomatous LC cells promotes the expression of ECM genes, thereby instigating excessive ECM deposition, as shown in Fig. 3. Consequently, fibroblasts undergo phenotypic alterations and differentiate into myofibroblasts, driving connective tissue fibrosis, and exacerbating glaucomatous optic neuropathy (120). Additionally, other studies have also demonstrated that the synthesis and secretion processes of ECM-related molecules are also regulated by Cav-1 (121,122). The decrease in Cav-1 expression contributes to the disruption of ECM remodeling, thus protecting the microenvironment around RGCs and maintaining the integrity of the structure and normal physiological functions of ocular tissues (122).

Figure 3 Ca2+ signaling pathway of lamina cribrosa remodeling in glaucoma. IP3R, inositol 1,4,5-trisphosphate receptor; mito, mitochondria; ER, endoplasmic reticulum; RYR, ryanodine receptor; n, nucleus; TF, transcription factor.

In the context of fibrosis, protein kinase C (PKC), p38, p42/44-MAPK and the PI3K/mTOR axis were proposed as pivotal signaling transduction mediators downstream of Ca2+ signaling (120). Experimental simulation of glaucoma through hypo-osmotic-induced cell swelling has revealed notable upregulation in the expression and activity of PKCα, p38 and p42/p44-MAPKs (120). Additionally, increased mRNA expression of PI3K, IP3R, mTOR and Ca2+-calmodulin protein kinase II was observed in LC under such conditions. The calcium-modulated phosphatase-NFAT signaling pathway plays a significant role in promoting fibroblast proliferation, activation and contraction. Notably, Cyclosporin A, an inhibitor of NFAT activity, has shown significant suppression of NFATc3 expression induced by oxidative stress, as well as elevation of pro-fibrotic ECM genes in both normal and glaucomatous LC cells (123). Hence, targeting PKCα, p38, p42/p44-MAPKs, PI3K/mTOR and the calmodulin phosphatase-NFATc3 signaling pathways emerges as a potential therapeutic strategy for addressing glaucoma-associated LC fibrosis.

Besides, L-type Ca2+ channels are increased in glaucomatous LC cells, and the administration of verapamil, a blocker of L-type Ca2+ channels, reduces the mechanical strain-induced ECM gene response in human LC cells (124). Blocking Ca2+ signaling has been hypothesized to attenuate the fibrotic response in glaucomatous optic neuropathy. It has been found that voltage-independent, stretch-activated cation channels and transient receptor potentials typical of TRPC1 and TRPC6 are highly expressed in glaucomatous LC cells and are also involved in abnormally high levels of Ca2+ in glaucomatous LC cells (125). Hu et al (126) established a mouse model of COH and found that the increase in ATP levels and Ca2+ endocytosis in response to hydrostatic pressure was accompanied by upregulation of Transient Receptor Potential Vanilloid 1 (TRPV1, encoded by the TRPV1 gene) and 1-phosphatidylinositol 4,5-bisphosphate phosphodiesterase γ-1 (PLCγ1, encoded by the PLCG1 gene) expression. In the same study, knockdown of TRPV1 and PLCG1 genes resulted in a significant reduction in ATP levels and intracellular Ca2+, as well as a significant attenuation of apoptosis and autophagy in RGCs. In addition, the expression and activity of the Ca2+-dependent potassium channel maxi-K were significantly elevated in glaucomatous LC cells (127). In summary, Ca2+ homeostasis is a critical factor for cell function and survival, and the maintenance of Ca2+ homeostasis helps attenuate LC cell injury and ECM remodeling, providing a potential target for the treatment of persistent optic neuropathy (128).

Transforming growth factor-beta (TGF-β) signaling pathway

TGF-β belongs to the dimeric peptide growth factor family and plays pivotal roles in various signaling cascades associated with cellular processes such as differentiation, proliferation, chemotaxis and fibrosis. It encompasses three primary isoforms, namely TGF-β1, TGF-β2 and TGF-β3, among which TGF-β2 exhibits a particularly close association with glaucoma (88,129). TGF-β orchestrates ECM remodeling, which contributes to the deposition of fibrotic material in the retina, consequently instigating glaucomatous conditions (130). Elevated levels of TGF-β have been detected in glaucomatous AH (131), underscoring its involvement in the pathogenesis of glaucoma. Additionally, TGF-β2 has been shown to activate Src signaling by upregulating the Src scaffolding protein CasL, thereby impeding the expression of tissue plasminogen activator and attenuating ECM degradation (132). It has been proposed that TGF-β exerts its influence on ECM generation and remodeling in tandem with the classical Smad pathway (88). Within this pathway, TGF-β binds to TGF-β type II receptor (TβRII), initiating a cascade wherein the TβRII phosphorylates TGF-β type I receptor (133,134), leading to downstream phosphorylation of Smad2 and Smad3. These phosphorylated Smads form a complex with Smad4, translocate to the nucleus, and modulate gene transcription, thereby promoting ECM production within the ONH and precipitating glaucoma (88,135). Moreover, members of the TGF-β superfamily can induce glaucoma development by activating PI3K/AKT and MAPK signaling pathways (134). The above content is clearly presented in Fig. 2.

Recent investigations have revealed a significant elevation of TGF-β2 levels in glaucomatous ONH and LC, as evidenced in a non-human primate glaucoma model, highlighting the involvement of TGF-β2 in glaucoma pathogenesis (136). TGF-β signaling exerts a direct influence on the expression of microRNAs (miRNAs), which in turn regulate protein translation. Specifically, dysregulation of miRNA-29 family expression, attributed to TGF-β2 signaling, may contribute to glaucomatous pathology by modulating ECM deposition and remodeling within the LC (137). Furthermore, TGF-β signaling has been implicated in the differentiation of myoblasts into myofibroblasts through Smad3-mediated downregulation of miRNA-29 expression (138). Aberrant miRNA-29 expression, which results in dysregulated TGF-β2 signaling, may promote glaucoma by exacerbating ECM deposition and remodeling within the LC (137). Additionally, upregulation of the long non-coding RNA (lncRNA) series NR_003923 has been observed in glaucomatous tissues, with knockdown experiments revealing inhibition of TGF-β signaling and reduced cell migration, fibrosis and autophagy (139). To summarize, targeting TGF-β signaling emerges as a promising therapeutic approach for the treatment of glaucoma.

Other mechanisms related to signaling pathways

In recent years, notable advances in molecular biology and cytogenetics have unveiled novel mechanisms, along with conventional molecular signal transduction pathways. Oxidative stress, inflammation and glutamate have emerged as pivotal factors in the pathogenesis of glaucoma (140-142). Previous studies have demonstrated that the dysregulation of the WNT/β-catenin pathway induces glutamate excitotoxicity, leading to increased inflammation and oxidative stress, and then participates in the occurrence and development of glaucoma (143). In the retina, Wnt/β-catenin signal transduction can maintain neuronal homeostasis and regulate neuronal regeneration as well as provide neuroprotection against mutations, oxidative stress, laser and light damage (144,145). The Wnt/β-catenin pathway has two states, namely 'on' and 'off' (146). In the off state, adenomatous polyposis coli, glycogen synthase kinase 3 (GSK3), axin and casein kinase form a β-catenin protein destruction complex, which mediates its phosphorylation and inhibits the transcription of Wnt/β-catenin target genes. When the pathway is turned on, Wnt binds to the Frizzled receptor and forms a complex with LRP5/6, phosphorylates and activates the Dishevelled (Dvl) protein. Dvl inhibits the β-catenin protein destruction complex, causing β-catenin to be retained in the cytoplasm and translocated to the nucleus. There, it forms a complex with the T cell factor family/lymphocyte enhancer-binding factor to activate the transcription of downstream target genes such as c-Myc, c-Jun and cyclin D1 (Fig. 4), thereby regulating the cell cycle and playing a regulatory role in cell survival and proliferation (147,148). In addition, experimental studies have found that the activation of the Wnt/β-catenin signal pathway can lead to axonal regeneration in a mouse model of optic nerve injury (149). In the same experimental context, it was also found that Wnt signal transduction is active in RGCs, and Wnt3a can also increase the survival rate of RGCs after injury. Moreover, Wnt signal transduction can induce the expression of genes with pro-regenerative properties (such as STAT3 and CNTF) (145,150,151), while being inhibited by genes that suppress regeneration (such as KLF4 and ephrin) (145,152,153). It has been previously shown that the WNT/β-catenin pathway is also involved in the pathophysiology of TM cells (154). The activation of this pathway can promote the survival of TM cells and may participate in the metabolic regulation of the ECM. When this signal pathway becomes abnormal, the structure and function of the TM will be affected, which may lead to the obstruction of AH outflow, increased IOP, and is closely related to the occurrence of ocular diseases such as glaucoma (143). In conclusion, the Wnt/β-catenin signaling pathway plays an important regulatory role in multiple aspects of the pathogenesis of glaucoma, including its impact on the survival of RGCs, axonal regeneration, and cell behaviors and tissue microenvironment related to the maintenance of IOP homeostasis. Therefore, it can be regarded as a highly promising target for the treatment of glaucoma.

Figure 4 Regulation of the Wnt/β-catenin signaling pathway. LRP5/6, low-density lipoprotein receptor-related protein 5/6; frizzled, frizzled receptor; axin, axis inhibition protein; Dvl, dishevelled protein; CK1, casein kinase 1; GSK-3, glycogen synthase kinase 3; βTrCP, beta-transducin repeat-containing protein; TCF, T cell factor; LEF, lymphocyte enhancer-binding factor; APC, adenomatous polyposis coli protein; c-Myc, cellular myelocytomatosis virus oncogene homolog; c-Jun, proto-oncogene c-Jun.

Besides, in the research on the pathogenesis of glaucoma, serine proteases and their inhibitors have also become relatively new focuses. In glaucoma, serine proteases (such as plasmin) can participate in the remodeling of the ECM and affect the survival of RGCs (155,156). Under pathological conditions, when excessive serine proteases act on the ECM in the LC, they may overly degrade the components in the ECM, making the structure of the LC loose or causing local defects, which will cause compression and damage to the optic nerve (124,157) (Fig. 5). When serine proteases act on the TM, the disorder of ECM homeostasis leads to an increase in the resistance to AH outflow and an increase in IOP (Fig. 5), thus contributing to the occurrence and development of glaucoma (158). Furthermore, it has been revealed that while the proteolytic activity of serine proteases in glaucoma increases, the protease inhibitory activity of nerve serine protease inhibitors decreases in chronic glaucoma animal models (156). SerpinA1 and SerpinA3 are important members of the serine protease inhibitor family. Elevated SerpinA1 levels have been proven to indirectly affect Wnt signaling by regulating the AKT pathway through increasing the synthesis of GSK-3β, resulting in a decrease in β-catenin levels and delaying glaucoma to a certain extent (159). Several research studies have put forward that gene therapy or the external application of nerve serine protease inhibitors, and likewise in transgenic mice with overexpressed nerve serine protease inhibitors, is capable of demonstrating a protective impact on the functions of RGCs (160-162). This protection encompasses the restoration of autophagy, microglial and synaptic functions in the context of glaucoma (163). In summary, serine proteases may damage intraocular tissues due to excessive hydrolysis in glaucoma, while their inhibitors can inhibit the harmful effects of proteases by regulating relevant signaling pathways and other means, thus playing an inhibitory role in the development of glaucoma. They are expected to become new targets for the treatment of glaucoma.

Figure 5 Diagram of the action of serine proteases and inhibitors in the extracellular matrix of the LC and trabecular meshwork. LC, lamina cribrosa; IOP, intraocular pressure.

Research findings show that insights into the role of the protease cysteinyl asparaginase in the apoptotic pathway have emerged, with evidence suggesting that the absence of caspase-8 in astrocytes can shield RGCs from inflammatory injury driven by glial cells (163). Conversely, the inhibition of caspase-8 cleavage suppressed apoptosis in RGCs. Moreover, mice lacking cysteine 7 exhibited greater retention of RGCs following an optic nerve crush than wild-type mice, indicating the potential neuroprotective effect of blocking cysteine (164). Although histone and DNA modifications, miRNAs and lncRNAs have been implicated in glaucoma development (88), their epigenetic nature has not been extensively examined.

Conclusions

Glaucoma has emerged as a significant threat to human vision and is characterized by complex and diverse pathogenesis. By investigating the signaling pathways involved in RGCs apoptosis, optic nerve protection and regeneration, as well as damage and remodeling of the LC, it was identified that these signaling pathway molecules can interact with various signal cascades. These factors cover signaling pathways including PI3K/AKT, p38 MAPK, JNK, Bcl-2 family/caspase, BDNF, PTEN, Rho/ROCK, Ca2+ and TGF-β. They regulate the activity and expression of transcription factors through interactive coupling, and then directly act on the level of gene expression, assuming a crucial regulatory function throughout the whole process from the onset to the progression of glaucoma and profoundly influencing the disease progression of glaucoma. Cav-1 also has a critical significance in the pathophysiological process of glaucoma. It has complex interactions with the numerous signaling pathways aforementioned, which is essential for in-depth exploration of the pathogenesis of glaucoma. In addition, the WNT/β-catenin pathway, caspase, together with the biological effects of serine proteases and their inhibitors, have all been confirmed to be closely associated with the occurrence and development of glaucoma and play an indispensable role in the disease evolution process of glaucoma. In recent years, increasing experimental evidence suggests that intervening or regulating interactions between signaling pathways could potentially treat or alleviate symptoms associated with glaucoma. At present, numerous drug experiments have developed targeted drugs associated with the aforementioned signaling pathways that exhibit promising effects on RGCs' apoptosis while promoting optic nerve regeneration and preventing LC remodeling; however, most of them remain at the stage of animal experimentation or clinical research. Further extensive and comprehensive studies are required to achieve large-scale clinical translation of these findings. Additionally, the present review did not adequately address some emerging mechanisms and non-classical signaling pathways, which will be explored further in future studies. Understanding and harnessing these signaling pathways presents immense potential for reversing RGCs apoptosis and alleviating optic nerve damage caused by glaucoma.

Availability of data and materials

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Authors' contributions

XW collected literature, created images, wrote and revised the manuscript. LS collected literature and wrote the manuscript. XH designed the study and edited the manuscript. ZL, XC and RX created images and revised the manuscript. YX and YS reviewed and edited the manuscript. GW designed the study, reviewed and edited the manuscript. PZ designed the study, reviewed and revised the manuscript. All authors read and approved the final version of the manuscript. Data authentication is not applicable.

Ethics approval and consent to participate

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Patient consent for publication

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Competing interests

The authors declare that they have no competing interests.

Acknowledgments

Not applicable.

Funding

The present review was supported by the National Natural Youth Science Foundation (grant no. 82205198), the China Postdoctoral Science Foundation project (grant no. 2022M711984), the Shandong Provincial Natural Science Foundation General Program (grant no. ZR2020MH393), the Postdoctoral Innovation Project of Shandong (grant no. 202101012) and the China Postdoctoral Foundation General Program (grant no. 2020M672127).

References