Glaucoma is characterized by a slow and progressive degeneration of the optic nerve, including retinal ganglion cell (RGC) axons in the optic nerve head (ONH), leading to visual impairment. Despite its high prevalence, the biological basis of glaucoma pathogenesis still is not yet fully understood, and the factors contributing to its progression are currently not well characterized. Intraocular pressure (IOP) is the only modifiable risk factor, and reduction of IOP is the standard treatment for glaucoma. However, lowering IOP itself is not always effective for preserving visual function in patients with primary open-angle glaucoma. The second messenger cyclic adenosine 3′,5′-monophosphate (cAMP) regulates numerous biological processes in the central nervous system including the retina and the optic nerve. Although recent studies revealed that cAMP generated by adenylyl cyclases (ACs) is important in regulating aqueous humor dynamics in ocular tissues, such as the ciliary body and trabecular meshwork, as well as cell death and growth in the retina and optic nerve, the functional role and significance of cAMP in glaucoma remain to be elucidated. In this review, we will discuss the functional role of cAMP in aqueous humor dynamics and IOP regulation, and review the current medications, which are related to the cAMP signaling pathway, for glaucoma treatment. Also, we will further focus on cAMP signaling in RGC growth and regeneration by soluble AC as well as ONH astrocytes by transmembrane ACs to understand its potential role in the pathogenesis of glaucoma neurodegeneration
Glaucoma is an optic neuropathy and the main cause of irreversible blindness worldwide (1–3). It has been estimated that glaucoma will affect more than 80 million individuals worldwide by 2020, with at least 6 to 8 million individuals becoming bilaterally blind (1, 2). Primary open-angle glaucoma (POAG), the most common form of open-angle glaucoma, is characterized by a slow and progressive degeneration of retinal ganglion cell (RGC) axons in the optic nerve head (ONH) and retinal nerve fiber layer, leading to an excavated appearance of the optic disc and visual impairment (1, 3). Regardless, the biological basis of glaucoma pathogenesis is not yet fully understood, and the factors contributing to its progression are currently not well characterized.
Cyclic adenosine 3′,5′-monophosphate (cAMP) is the first discovered second messenger for signal transduction (4). Its signaling pathway exists in all types of cells and contributes to numerous biological processes, such as cell growth, differentiation, death, gene expression, inflammatory cytokine secretion, and neurotransmission (5–7) in the central nervous system (CNS). Upon stimulation, cAMP synthesis and its degradation are tightly regulated by adenylyl cyclases (ACs) and cyclic nucleotide phosphodiesterases (PDEs), respectively (6). The activation of cAMP signaling causes opposite effects on cell survival in a cell-type-specific manner (8), because it exerts its effect through various effectors, such as cAMP-dependent protein kinase A (PKA) (9, 10), exchange protein directly activated by cAMP (Epac) (11, 12), and cyclic-nucleotide-gated ion channels (13, 14).
Among the key regulators of the cAMP signaling pathway, ACs are enzymes that catalyze the synthesis of cAMP from adenosine 5′-triphosphate (ATP). To date, ten distinct AC genes (AC1-10) have been identified by molecular cloning techniques, and these genes encode nine mammalian transmembrane ACs (tmACs; AC1-9), and a soluble AC (sAC; AC10), respectively (15–17). Each AC has various functional roles and distribution patterns in tissues (18, 19). The activity of tmACs is regulated by physical and functional interaction with G-protein coupled receptors (GPCRs) in the plasma membrane (19–21). In contrast, sAC does not have transmembrane domains and is localized in the cytoplasm compartments and within distinct organelles, such as nuclei and mitochondria (17, 22). While tmACs except AC9 are sensitive to forskolin but not to bicarbonate, sAC is sensitive to bicarbonate but not to forskolin, and requires a divalent cation such as Ca2+ for its activity (6, 17).
ACs have been thought of as potential drug targets in many neurodegenerative disorders, including glaucoma (23, 24). Since the activation of the cAMP signaling pathway by forskolin, a tmACs activator, has been reported to be involved in the reduction of intraocular pressure (IOP) (25, 26), a recent clinical trial for POAG treatment has demonstrated that 1% forskolin eye drops can be used as a safe alternative to β-adrenergic receptor blockers (β-blockers) and prostaglandin analogues (27), which are mostly used for glaucoma treatment although they have several side effects (2, 28). Since the evidence demonstrates that RGC survival and axon growth are enhanced via activation of the sAC-mediated cAMP signaling pathway (29–32), the therapeutic strategy for modulating the cAMP signaling pathway in glaucoma treatment is considered to rescue RGCs from glaucomatous insults. However, the effect of the cAMP pathway activation on IOP regulation, RGC, and ONH degeneration remains poorly understood. In this review, we will discuss recent literature on the role of cAMP in the eye, addressing its possible relationship to glaucoma protection or degeneration.
IOP is currently the only proven treatable risk factor in glaucoma (1, 28). As an aqueous humor that is secreted to the iris by the ciliary body in the posterior chamber, it not only regulates IOP by a balance between the secretion and drainage, but also provides nutrients to the iris, lens, and cornea by circulation in the anterior chamber (1). The outflow of the aqueous humor is controlled via a conventional pathway through a trabecular meshwork (TM) and Schlemm’s canal (SC), and via an independent uveoscleral outflow pathway through the ciliary body and iris root (33, 34). In this regard, the therapeutic strategies that reduce aqueous humor inflow and/or increase its outflow have been thought to be important in treating IOP-related glaucomatous optic neuropathy.
Lowering or stabilizing IOP is considered to be an effective approach to reducing glaucoma progression (2, 35). Previous clinical studies have reported that the adrenergic agents, such as epinephrine and phenylephrine, lower IOP in patients with POAG (36, 37). Variations of aqueous humor inflow in IOP changes are associated with the 24 h circadian IOP profile and body posture (35, 38). Since Neufeld
Because of the discrepancy between adrenergic agonists and blockers (e.g., epinephrine and timolol) on IOP regulation, however, it is difficult to conclude whether increasing cAMP level reduces IOP inflow. Using molecular and cellular biological techniques, recent evidence indicates that adrenergic receptors are GPCRs, which are classified into two main categories, α and β, and these are further grouped according to their isotypes (α1, α2, β1, β2, and β3), which are linked to different Gα subunits (Table 1). Epinephrine, also known as adrenaline, is a nonselective agonist of all adrenergic receptors, and timolol is a non-selective β-blocker. Currently, the agonists which are selectively targeted to the α2 subclass are most commonly prescribed to lower IOP in patients with glaucoma (52). The activation of α2 adrenergic receptor reduces cAMP production because it is linked to Gαi, the inhibitory Gα subunit. Indeed, adrenergic receptor agonists (e.g., apraclodine and brimonidine) decrease aqueous humor production (53–55). However, β adrenergic receptors are mainly linked to Gαs, a stimulatory Gα subunit (Table 1) and β2 adrenergic receptor is predominantly present in human ciliary processes from donor eyes (56). Also, timolol decreases the aqueous humor formation in the ciliary epithelium in a cAMP-dependent manner (57, 58). Together, these findings support the notion that reducing cAMP, not increasing cAMP, lowers aqueous humor formation and IOP. Although current studies do not provide a clear conclusion whether the increase or decrease of cAMP level reduces aqueous humor formation, it is possible that cAMP plays a critical role in the regulation of aqueous humor production and IOP inflow.
Aqueous humor outflow decreases with aging and glaucoma progression (59). Elevated IOPs in glaucoma result from the predominantly reduced capacity of outflow in the conventional pathway rather than disruption of IOP-maintaining strategies through decreasing both inflow and uveoscleral outflow without a change in the conventional outflow facility in healthy aging eyes (59, 60).
Increasing the outflow facility by elevating the cAMP level by adrenergic agents has also been reported (48, 61, 62); however, the precise effect of cAMP was not explained until sAC was found to play a role in the outflow control. Carbonic anhydrases are a family of enzymes that catalyze the rapid interconversion of carbon dioxide (CO2) and water (H2O) to bicarbonate (HCO3−) and hydrogen ion (H+), and its inhibition lowers IOP in patients with glaucoma (63). Since an HCO3-sensitive AC activity has been reported in the ciliary body of rabbit eyes (64), sAC expression was identified in the non-pigmented epithelium of the ciliary body and the sAC was characterized as an enzyme responsible for controlling the activity of cAMP in the ciliary body (65). Although carbonic anhydrase inhibitors, including acetazolamide, are known to lower IOP by diminishing the rate of aqueous humor formation in the ciliary epithelium (63, 66), the relationship between carbonic anhydrase-generated HCO3− and the cAMP signaling pathway has yet to be characterized in IOP regulation. Furthermore, it is not known whether sAC contributes to aqueous humor formation in the eye.
If so, how does sAC regulate IOP? Shahidullah
Cholinergic drugs, also known as cholinomimetics, miotics, parasympathomimetics, and acetylcholine receptor agonists, are the first class of drugs that are used to treat glaucoma (74). Cholinergic drugs, including pilocarpine and carbachol, have been used to increase outflow through the conventional pathway (75, 76). Cholinergic drugs can act directly by binding to muscarinic acetylcholine receptors, which are GPCRs (77). These receptors have five isoforms (M1–M5) and all types of these receptors are expressed in the eye (77). Although cholinergic drugs have been reported to increase the outflow facility of the aqueous humor via M3 that is linked to Gαq, a Gα subunit which activates the phospholipase/Ca2+ pathway (77), some types of these receptors (M1, M2 and M4) are also linked to Gαs or Gαi subunits that can stimulate or inhibit AC activity, respectively (Table 1). Interestingly, AC2 and 4 are expressed in the human outflow tissues, and carbachol treatment increases outflow facility that is mediated by cAMP (78).
Prostaglandin analogs are the newest class of drugs that are the most efficacious for lowering IOP in patients with POAG (28, 79). Prostaglandins are a group of physiologically active lipid compounds that act like the hormone and exert their effects by binding to ten known prostaglandin receptors, such as types I, E and F, which are GPCRs linking to various Gα subunits, including Gαs, Gαi, and Gαq. Since a Gαq-linked prostaglandin F receptor has been mostly targeted and used for glaucoma treatment, little is known about the effect of prostaglandin analogs through the cAMP signaling pathway in IOP regulation. However, several studies have intriguingly demonstrated that a Gαs-linked prostaglandin EP4 receptor is expressed in eye tissues, including the cornea, iris, ciliary body, TM/SC complex, and retina, and that activation of this receptor with its agonists (3,7-di-thia PGE1 and PF-04475270) reduces IOP in experimental animal models of glaucoma (80). Although there may be limited opportunity to develop EP4 agonists for clinical evaluation in patients, because of the risk of corneal neovascularization and persistent ocular hyperemia (80), these results also strongly support the notion that cAMP is a key regulator of IOP control in glaucoma. To date, there is no direct evidence that the sAC-mediated cAMP signaling pathway is involved in the IOP-lowering effect of cholinergic drugs and prostaglandin analogs. Considering the recent evidence that GPCR-mediated Ca2+ increment can also directly activate sAC (81), however, it is possible that the effect of IOP lowering by these drugs may result from sAC activation via Gαq-mediated Ca2+ signaling. Further studies to examine the relationship between sAC-mediated cAMP signaling and these drugs may provide important insight into the functional role of cAMP in IOP regulation.
RGCs communicate the information from visual processing in the retina to the brain. RGCs are the most predominant cell type in the ganglion cell layer, which is the innermost retinal layer. The cell body of the RGC extends an axon that runs along the nerve fiber layer of the optic disc (also known as ONH). In humans, RGC axons terminate mostly in the lateral geniculate nucleus and some in the superior colliculus to complete the visual system (1). Because RGC and its axon loss are a major pathological phenotype during visual impairment in glaucoma (1, 28), current studies focus on the direct or indirect prevention of the loss of RGC and its axon for glaucoma treatment. Currently, several studies have demonstrated that cAMP is involved in RGC survival (29, 82–86) and differentiation (87), as well as its axonal growth (82, 83) and regeneration (30).
Glutamate excitotoxicity has been implicated as an important pathophysiological mechanism underlying RGC death in glaucomatous neurodegeneration (88–91). Brimonidine, a selective α2 adrenergic receptor agonist, provides significant evidence that links the cAMP signaling pathway and glutamate excitotoxicity to protect RGCs directly against glaucomatous damage. The potential mechanisms for brimonidine-mediated RGCs protection are thought to be inhibition of glutamate release, upregulation of brain-derived neurotrophic factor expression, regulation of cytosolic Ca2+ signaling, and modulation of
Although the excessive Ca2+ influx in the excitotoxicity condition causes RGC death, Ca2+ homeostasis in a normal condition is essential for RGC function and survival. Furthermore, the elevated Ca2+ level has been reported to protect RGCs by activating the cAMP signaling pathway (82, 83, 86, 108–110). Surprisingly, a recent study has demonstrated that RGC death was not exacerbated by overstimulation of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor-mediated Ca2+ influx in purified RGCs
In the adult human ONH, approximately one million nerve fibers converge in and exit from the eye to the optic nerve through the lamina cribrosa (LC) region (1, 28). The LC preserves a pressure gradient between the intraocular and extraocular space, forming the cribriform plates with astrocytes and LC cells (114, 115). Elevated IOP triggers optic disc cupping in the LC region and remodels the extracellular matrix (ECM), and in turn, leads to RGC axonal degeneration in glaucoma (28). Astrocytes are predominant cells in the ONH (116, 117) and their processes ensheath axon bundles in the prelaminar and LC region (118). ONH astrocytes not only provide cellular support to unmyelinated RGC axons by interfacing between connective tissue surfaces and surrounding blood vessels, but also play a fundamental role in the mechanical stability of the LC by modulating ECM remodeling in most mammals (116, 117). Upon glaucomatous injuries, activated astrocytes in the ONH induce reactive astrogliosis, which is characterized by morphological alteration of astrocytes by hypertrophy with thickened, enlarged processes and by the increase of glial fibrillary acidic protein (GFAP) expression (115). Importantly, we and others have demonstrated that ONH astrocyte dysfunction that is accompanied by RGCs axon loss is closely associated with the pathogenesis of glaucomatous ONH degeneration in patients with glaucoma (116, 119–121) as well as in experimental animal models of glaucoma (116, 122–125).
Although ONH astrocytes play a critical role in RGC and its axon protection against glaucomatous damages, little is known about the relationship between cAMP and ONH astrocytes in glaucomatous neurodegeneration. Previous studies have demonstrated that the basal level of cAMP was significantly higher in the unstimulated glaucomatous ONH astrocytes from Caucasian American (CA) and African American (AA) donors with POAG compared with unstimulated ONH astrocytes from normal healthy counterparts (120). In addition, transcriptome analysis for cAMP-signaling-pathway related genes showed that, while regulators of G-protein signaling 5 (
Since the expression of α and β adrenergic receptors has been found in cultured astrocytes from the cerebral cortex of rats (128, 129), only α1 and β2 adrenergic receptors are found to be expressed in the astrocytes of the rabbit, rat, and human optic nerve
Oxidative stress has been thought to be an important pathophysiological mechanism in many neurodegenerative diseases, including glaucoma (116, 138–141). In the CNS, neurons are the cells most vulnerable to oxidative stress, because of their low reactive oxygen species detoxifying capacity; therefore its survival is highly dependent on the capacity of neighboring astrocytes during oxidative stress-induced neurodegeneration (142, 143). Furthermore, astrocytes are the responsible cell type that is mostly related to oxidative-stress-mediated glaucomatous ONH degeneration (116, 122, 138, 144). Indeed, we have demonstrated that oxidative-stress-mediated mitochondrial dysfunction or alteration could be an important pathophysiological mechanism in the dysfunction of ONH astrocytes (144). Further, we have found that coenzyme Q10, an essential cofactor of the electron transport chain and a potent antioxidant, protected cultured ONH astrocytes from H2O2-induced oxidative stress (144) as well as RGCs and their axons in experimental rodent models of retinal ischemia or glaucoma (145–147). However, the relationship between the cAMP signaling pathway and oxidative stress in ONH astrocyte dysfunction and degeneration remains unknown. Previous studies have demonstrated that tmAC5 knockout mice show resistance to oxidative stress (148) and activation of the tmACs-mediated cAMP/PKA signal pathway induced by forskolin is associated with increased vulnerability to H2O2-induced oxidative stress in rat neocortical astrocytes
Brimonidine protects not only RGC somas but also their axons in the optic nerve of rats with elevated IOP induced by laser cauterization of the episcleral veins (104). We also found that brimonidine prevents the increased GFAP expression in müller cells, the most predominant retinal glial cells, as well as protects RGCs in ischemic retina (105), suggesting the possibility that brimonidine-mediated protection may also be involved in modulation of glial responses against pressure-induced ischemic insults. Our previous report demonstrated that functional NMDARs are present in human ONH astrocytes, and its expression levels are increased in cultured ONH astrocytes from patients with glaucoma (122). Because brimonidine-mediated tmACs inhibition protects RGCs against NMDARs-mediated glutamate excitotoxicity (107), these findings suggest another possibility, that brimonidine may also protect astrocytes by inhibiting tmACs activation in glaucomatous ONH degeneration. Although future studies need to investigate the effect of brimonidine on ONH astrocytes, this idea is supported by the evidence that the activation of metabotropic glutamate receptors 3, a GPCR linked to Gαi subunit, protects cultured astrocytes against hypoxic/ischemic damage by tmACs inhibition (150, 151). Therefore, it would be important to know whether the tmACs activation contributes to ONH astrocyte dysfunction in glaucomatous neurodegeneration.
Glaucoma is the leading cause of irreversible blindness worldwide. Despite its high prevalence, the biological basis of POAG still is not yet fully understood. Since adrenergic agents such as brimonidine have beneficial effects on IOP lowering and RGC protection in POAG, the current understanding of the cAMP signaling pathway regulated by adrenergic agents may provide a therapeutic potential for glaucoma treatment. In this regards, inhibition of tmACs activation by adrenergic receptors reflects an important explanation for the utilization of adrenergic agents, such as α2 adrenergic receptor agonists and β blockers, in glaucoma treatment. On the other hand, activation of the cAMP signaling pathway by sAC has been shown to have dual action in IOP lowering and RGCs protection (Fig. 1). Therefore, it is possible that the cAMP signaling pathway by tmACs and sAC activation may have distinct roles in various cell types of the eye. Moreover, because the functional role of tmACs or sAC in ocular tissues is yet to be characterized, it would be important to investigate the functional role of the cAMP signaling pathway induced by tmACs or sAC activation, not only in these ocular tissues, but also in specific cell types of neurons and glial cells. Future studies into the pathogenic or protective mechanisms of the cAMP signaling pathway will provide new therapeutic strategies to understand aqueous humor dynamics and IOP regulation, and to enhance the survival of RGC and its axon, as well as ONH astrocytes in glaucoma and other optic neuropathies.
This work was supported, in part, by NIH grants EY018658 (WKJ).
cAMP signaling pathway-related IOP reducing drugs used in glaucoma treatment
|Drug target||Subtype||GPCR type||ACs type||Available drugs||Drug type||Mechanisms of action|
|Inflow||α-ARs (2, 52)||α1||Gq||-||Apraclonidine||Agonists||Decrease inflow|
|β-ARs (2, 152)||β1||Gs||tmAC||Timolol, betaxol, carteolol and levobunolol||Blockers||Decrease inflow|
|β2||Gs and Gi||tmAC|
|CA (2, 63)||-||sAC?||Dorzolamide, brinzolamide, acetazolamide and methazolamide||Inhibitors||Decrease inflow|
|Outflow||CRs (2, 77)||M1||Gq (153) and Gs (154, 155)||tmAC||Pilocarpine, carbachol||Agonists||Increase outflow|
|PGR (EP4) (80)||Gs||tmAC||-||Agonists||Increase outflow|
|PGR (F) (2, 79)||Gq||-||Latanoprost, travoprost, bimatoprost and tafluprost||PGF2α analogues||Increase outflow|
ARs, adrenergic receptors; CA, Carbonic anhydrase; CRs, Cholinergic receptors; PGR, Prostaglandin receptor; sAC, soluble adenylyl cyclase; tmACs, transmembrane adenylyl cyclases.