This study supports four major findings. First, galantamine treatment leads to survival of RGC soma and axons in experimental glaucoma. Second, galantamine-mediated RGC structural protection is independent of IOP-induced damage, as evidenced by the neuroprotective action of this drug after optic nerve axotomy. Third, functional deficits caused by high IOP are markedly improved by galantamine. Fourth, galantamine-mediated neuroprotection occurs primarily through activation of retinal mAChR M1, and is independent of nAChR.
Several recent clinical studies have suggested a correlation between glaucoma and Alzheimer's disease,14
but the most compelling evidence supporting such link stems from laboratory work. For example, neuronal loss in both glaucoma and Alzheimer's disease occurs by apoptosis,15
caspases are activated both in Alzheimer's disease and in injured RGCs,16
and intraocular injection of β
-amyloid has been shown to induce RGC degeneration.17
More recently, β
-amyloid deposition was associated with RGC death in experimental glaucoma and blockade of the β
-amyloid pathway reduced glaucomatous damage.18
Although the etiology of glaucoma and Alzheimer's disease may differ, their common features raise the provocative idea that drugs currently used to treat Alzheimer's disease may also have utility in glaucoma. Here, we show that one such drug, galantamine, is a powerful neuroprotectant for injured RGCs. Daily galantamine treatment promoted the survival of RGC soma and axons in glaucoma. Importantly, administration of galantamine by intravitreal injection also led to robust RGC protection after axotomy of the optic nerve. These data highlight several important properties of galantamine: it is effective when administered systemically or by intraocular injection, it promotes structural protection of RGCs in an IOP-independent manner and it delays RGC loss in different models, both acute and chronic, of optic nerve damage.
The neuroprotective effect of galantamine was superior to that conferred by memantine or donepezil. Galantamine has been shown to be a weaker AChE inhibitor than donepezil,19
therefore other factors likely account for this difference in neuroprotective efficacy. First, donepezil is a non-competitive inhibitor of AChE, which may result in the development of tolerance to donepezil and consequent downregulation of ACh receptors.20
In contrast, galantamine is a competitive AChE inhibitor and the galantamine–AChE complex follows the typical kinetics of reversible inhibitors, dissociating readily in the presence of excess ACh, with a reduced potential for tolerance.21
Second, galantamine acts more broadly on other neurotransmitter systems and has been shown to regulate the release of glutamate, serotonin and γ
-aminobutyric acid,22, 23
thus potentially modulating neural activity and delaying neurodegeneration. Third, mAChR are amenable to modulation at allosteric sites;24
hence, it is possible that galantamine may activate mAChR directly, although this possibility presently remains unknown.
Patients with glaucoma experience diminished visual function and poor quality of life; therefore, an ideal neuroprotective drug should preserve the structural viability of RGCs while retaining their ability to respond to visual stimulation. In this study, we aimed at providing a structure–function link based on the neuroprotective effect of galantamine. Our results show that there are major visual deficits in glaucomatous eyes treated with PBS, whereas galantamine treatment led to substantial preservation of the VEP amplitude at 3 weeks after OHT. Of interest, following longer periods of OHT (5 weeks) galantamine-protected RGCs (70%) did not respond to light stimulation unless IOP was also reduced. An IOP decrease of just a few mm
Hg was sufficient to restore almost 50% of the VEP response in galantamine-treated eyes, but not in PBS-treated controls. The observation that the majority of RGCs exposed to galantamine remained alive at 5 weeks of OHT, but did not respond to light stimulation, suggests that sustained high IOP has additional deleterious effects on RGC function. We conclude that, in the long-term, structural protection alone is not sufficient to restore visual function unless IOP is also controlled.
Galantamine increases the availability of ACh through its inhibitory action on AChE, the enzyme responsible for ACh breakdown, and it is also an allosteric modulator of nAChR enhancing their sensitivity to ACh.25
ACh in the retina is released by starburst cholinergic amacrine cells onto RGC dendrites and has a crucial role in visual information processing.26
Therefore, we postulated that galantamine-induced neuroprotection might result from stimulation of ACh receptors. As galantamine is an allosteric modulator of nAChR, its neuroprotective effect has been compared with that of nicotine. In fact, nicotine has been shown to promote neuronal survival in different models of neurodegeneration through nAChR and downstream activation of survival pathways.27
Previous in vitro
studies showed that galantamine promoted the survival of cortical neurons or neuroblastoma cells by α
nAChR and stimulation of phosphatidylinositol-3-kinase (PI3K).3, 7
As RGCs express several nAChR subtypes including α
we initially postulated that nAChR activation would contribute to galantamine-mediated neuroprotection. Surprisingly, our data show that the blockade of nAChR had no effect, whereas inhibition of mAChR completely curtailed the neuroprotective effect of galantamine in vivo
. The total blockade of galantamine-induced neuroprotection in the presence of mAChR inhibitors indicates that these receptors are the primary locus of the specific action of galantamine in the retina.
Immunocytochemical studies on the localization of mAChR subtypes in primate, rat and chick retinas showed that M2 and M4 are expressed by amacrine cells, and M3 is expressed primarily by bipolar cells.29, 30, 31
In addition, Müller cells, the most abundant glial cell type in the mammalian retina, express M1 and M4 mAChR types.32
Muscarine was shown to increase intracellular Ca+2
in rabbit RGCs;33
however, this effect was thought to be indirect because expression of mAChR has not been detected in isolated rat or cat RGCs, and muscarine did not elicit membrane currents measured in whole-cell patch clamp preparations.34
Our results indicate that galantamine-mediated RGC neuroprotection in vivo
occurs primarily by activation of M1, a mAChR subtype expressed by Müller cells. The M4 mAChR subtype, expressed by both Müller glia and amacrine cells, also contributes to this effect but to a lesser extent than M1
mAChR. Collectively, these data support a model in which non-cell-autonomous signaling events downstream of mAChR have a major role in galantamine-induced RGC neuroprotection. Activation of M1/M4 mAChR on neighboring Müller glia and amacrine cells may lead to stimulation of signaling pathways and production of prosurvival factors that protect injured RGCs. Other retinal cell types that express these mAChR subtypes, including endothelial cells,35
may also participate in galantamine-mediated RGC survival.
M1 and M4 mAChR are G-protein-coupled receptors linked to different signal-transduction pathways. M1
mAChR are preferentially coupled to pertussis toxin (PTX)-insensitive Gq
proteins that stimulate phospholipase C (PLC) and phosphatidylinositol hydrolysis with subsequent Ca+2
mobilization from intracellular stores. M4 mAChR, on the other hand, are preferentially coupled to PTX-sensitive Gi/o
proteins that inhibit adenylate cyclase and regulate intracellular cAMP levels. It has become increasingly clear that mAChR downstream signaling pathways converge or intersect with mediators of cell survival. For example, M4 mAChR interacts with the nerve growth factor receptor, through Gβγ
complexes, to enhance PI3K/Akt activation and neuronal survival.36
Of interest, M1 mAChR through Gα
q and PLC leads to activation of Nrf2, a transcription factor involved in redox homeostasis, which may increase the cellular antioxidant defenses and confer neuroprotection against oxidative stress.37
Moreover, M1 mAChR activation also regulates the activity of the hypoxia-inducible factor-1, a transcription factor involved in the cellular response to hypoxia.38
Oxidative stress and ischemia/hypoxia have been proposed to be major contributors to glaucomatous neurodegeneration. An important priority in future studies will be to determine the M1- and M4-coupled signaling pathways underlying galantamine-induced RGC neuroprotection. The precise delineation of these molecular events should be useful for the design of novel therapeutic interventions applicable to glaucoma.
In summary, our study reveals the potent role of galantamine in the protection of RGC structure and function in glaucoma, which could be used in conjunction with standard pressure controlling drugs. Our data also identify retinal mAChR as a novel therapeutic target for prevention of neuronal death and vision loss in optic neuropathies.