Pilocarpine Protects Cobalt Chloride-induced Apoptosis of RGC-5 Cells: Involvement of Muscarinic Receptors and HIF-1α Pathway

The retina is the most metabolically active tissue in the human body and hypoxia-induced retinal ganglion cell (RGC) death has been implicated in glaucomatous optic neuropathy. The aim of this study is to determine whether muscarinic receptor agonist pilocarpine, a classic antiglaucoma drug, possesses neuroprotection against cobalt chloride (CoCl₂)-mimetic hypoxia-induced apoptosis of rat retinal ganglion cells (RGC-5 cells) and its underlying mechanisms. Cell viability was determined by Cell Counting Kit-8 assay and apoptosis was examined by annexin V and mitochondrial membrane potential (MMP) assays. Expressions of hypoxia-induced factor-1α (HIF-1α), p53, and BNIP3 were investigated by quantitative real-time PCR and western blot analysis. After treatment of 200 μM CoCl₂ for 24 h, RGC-5 cells showed a marked decrease of cell viability by approximately 30%, increased apoptosis rate and obvious decline in MMP, which could largely be reversed by the pretreatment of 1 μM pilocarpine mainly via the activation of muscarinic receptors. Meanwhile, pretreatment of 1 μM pilocarpine could significantly prevent CoCl₂-induced HIF-1α translocation from cytoplasm to nucleus and down-regulate the expression of HIF-1α, p53, and BNIP3. These studies demonstrated that pilocarpine had effective protection against hypoxia-induced apoptosis in RGCs via muscarinic receptors and HIF-1α pathway. The findings suggest that HIF-1α pathway as a “master switch” may be used as a therapeutic target in the cholinergic treatment of glaucoma.     

Muscarinic activation attenuates abnormal processing of β-amyloid precursor protein induced by cobalt chloride-mimetic hypoxia in retinal ganglion cells

β-Amyloid peptide (Aβ), the major pathological factor in Alzheimer’s disease, has recently been reported to be implicated in the development of glaucoma. In this study, we explored the effect of muscarinic activation on abnormal processing of β-amyloid precursor protein (APP) induced by a risk factor hypoxia in retinal ganglion cells. Hypoxia mimetic compound cobalt chloride could increase the generation of Aβ via up-regulating the expression of APP as well as the expression of β-secretase and γ-secretase, whereas muscarinic receptor agonist pilocarpine could significantly attenuate this abnormal pathway, thereby resulting in a decreased amyloidogenic cleavage of APP. This finding may provide an insight into better understanding of pathophysiology for the retinal neurodegenerative disease and searching for its new modifying approach.     

A potential neuroprotective role of apolipoprotein E-containing lipoproteins through low density lipoprotein receptor-related protein 1 in normal tension glaucoma

Glaucoma is an optic neuropathy and the second major cause of blindness worldwide next to cataracts. The protection from retinal ganglion cell (RGC) loss, one of the main characteristics of glaucoma, would be a straightforward treatment for this disorder. However, the clinical application of neuroprotection has not, so far, been successful. Here, we report that apolipoprotein E-containing lipoproteins (E-LPs) protect primary cultured RGCs from Ca(2+)-dependent, and mitochondrion-mediated, apoptosis induced by glutamate. Binding of E-LPs to the low density lipoprotein receptor-related protein 1 recruited the N-methyl-d-aspartate receptor, blocked intracellular Ca(2+) elevation, and inactivated glycogen synthase kinase 3β, thereby inhibiting apoptosis. When compared with contralateral eyes treated with phosphate-buffered saline, intravitreal administration of E-LPs protected against RGC loss in glutamate aspartate transporter-deficient mice, a model of normal tension glaucoma that causes glaucomatous optic neuropathy without elevation of intraocular pressure. Although the presence of α2-macroglobulin, another ligand of the low density lipoprotein receptor-related protein 1, interfered with the neuroprotective effect of E-LPs against glutamate-induced neurotoxicity, the addition of E-LPs overcame the inhibitory effect of α2-macroglobulin. These findings may provide a potential therapeutic strategy for normal tension glaucoma by an LRP1-mediated pathway.     

Exploring the potential for subtype-selective muscarinic agonists in glaucoma

Pilocarpine has been used to lower intraocular pressure (IOP) in glaucoma patients for more than 100 years. Since the identification of five muscarinic receptor subtypes, there has been an interest in separating the IOP-lowering effects from the ocular side effects of pupil constriction and lens accommodation. However, all these actions seem to be mediated by the M3 receptor. A novel muscarinic receptor agonist, AGN 199170, that has no activity on the M3 subtype was compared to pilocarpine in a monkey glaucoma model. This compound lowered IOP suggesting that muscarinic agonists targeted at muscarinic receptors other than the M3 subtype may be able to selectively lower IOP.     

Effects of neurokinin receptor antagonists in virus-infected airways

We investigated the effects of a neurokinin-1 (NK(1)) receptor antagonist (SR-140333) and a NK(2) receptor antagonist (SR-48968) on airway responsiveness and on the function of neuronal M(2) muscarinic receptors, which normally inhibit vagal acetylcholine release, in guinea pigs infected with parainfluenza virus. Antagonists were given 1 h before infection and daily thereafter. Four days later, bronchoconstriction induced by either intravenous histamine (which is partly vagally mediated) or electrical stimulation of the vagus nerves was increased by viral infection compared with control. In addition, the ability of the muscarinic agonist pilocarpine to inhibit vagally induced bronchoconstriction was lost in virus-infected animals, demonstrating loss of neuronal M(2) receptor function. Macrophage influx into the lungs was inhibited by pretreatment with both antagonists. However, only the NK(1) receptor antagonist prevented M(2) receptor dysfunction and inhibited hyperresponsiveness (measured as an increase in either vagally induced or histamine-induced bronchoconstriction). Thus virus-induced M(2) receptor dysfunction and hyperresponsiveness are prevented by a NK(1) receptor antagonist, but not by a NK(2) receptor antagonist, whereas both antagonists had similar anti-inflammatory effects.     

Effect of selective muscarinic receptor agonists and antagonists on active-avoidance learning acquisition in rats

Effect of some selective muscarinic receptor agonists and antagonists was investigated on learning acquisition in an active-avoidance paradigm in rats which records an anticipatory conditioned avoidance apart from the classical conditioned avoidance response. The muscarinic M1 agonists, arecholine, pilocarpine and McN-A-343, facilitated learning acquisition, which was attenuated by the selective M1 antagonist, pirenzepine. On the other hand, M2 receptor agonist, carbachol, and physostigmine, induced a dose-related dual response, with lower doses retarding and higher doses facilitating the learning acquisition. The former effect was attenuated by gallamine, a muscarinic M2 antagonist, while the latter response was inhibited by pirenzepine, indicating that these putative M2 receptor agonist lose their receptor specificity on dose increment. The selective M2 receptor antagonists, gallamine and AF-DX 116, facilitated learning acquisition, which was inhibited by pirenzepine and the acetylcholine synthesis inhibitor hemicholinium. The results support the cholinergic hypothesis of learning and memory and indicate that M1 receptor agonists and M2 receptor antagonists are likely to prove beneficial in memory deficits. The data also indicates that the clinical dose of some drugs, like physostigmine, needs to be carefully established for optimum therapeutic benefit.     

Muscarinic receptor modulation of glucose-induced electrical activity in mouse pancreatic B-cells

Acetylcholine (1-10 microM) depolarized the membrane and stimulated glucose-induced bursts of electrical activity in mouse pancreatic B-cells. The acetylcholine effects were mimicked by muscarine while nicotine had no effect on membrane potential. Pirenzepine, an antagonist of the classical M1-type muscarinic receptors, but not gallamine (1-100 microM), an antagonist of the classical M2-type receptors, antagonized the acetylcholine action on glucose-induced electrical activity (IC50 = 0.25 microM). Bethanechol, an agonist of the classical M2-type muscarinic receptors, was approximately 100 times less effective than acetylcholine in stimulating the electrical activity. In addition, acetylcholine (1 microM) induced a marked increase (25%) in input resistance to the B-cell membrane. The results indicate that acetylcholine exerted its effects on the B-cell membrane by inhibiting K+ conductance via activation of a muscarinic receptor subtype distinct from the classical M2-type receptor.     

The nature of the acetylcholine and 5-hydroxytryptamine receptors in buccal smooth muscle of the pest slug Deroceras reticulatum

The characteristics of the acetylcholine (ACh) and 5-hydroxytryptamine (5-HT) receptors of Deroceras buccal muscle were examined using specific pharmacological probes and sucrose gap electrophysiological analysis. ACh induced concentration-dependent smooth tonic contractures coupled with considerable depolarisation from the normal resting membrane potential of -30.6 mV. The use of choline ester analogues such as carbachol, propionylcholine and butyrylcholine, specific cholinergic agonists such as nicotine, muscarine, bethanecol and pilocarpine and antagonists such as d-tubocurarine, succinylcholine, hexamethomium, atropine, gallamine, pirenzepine and scopolamine indicated that the ACh receptor showed both nicotinic and muscarinic characteristics; the muscarinic activity resembled that of a mammalian M(2)-like receptor. Alternatively, it can not be ruled out that both mammalian types of receptor may be present in this preparation since both nicotine and muscarine induced noticeable tension. 5-HT application induced characteristic dose-dependent phasic contractions accompanied by small but quite consistent depolarisations. Serotonergic agonist and antagonist experiments using 1-(3-chlorophenyl) piperazine, 1-(m-chlorophenyl) biguanide, methiothepin, methysergide and metoclopramide strongly suggested that the 5-HT receptor showed closest pharmacological affinity with the 5-HT(1) receptor class of mammals but with some 5-HT(2) activity. In view of the phylogenetic gap between molluscs and mammals it is not surprising that the ACh and 5-HT receptors of Deroceras can not be properly classified by conventional mammalian terminology.     

Gartanin Protects Neurons against Glutamate-Induced Cell Death in HT22 Cells: Independence of Nrf-2 but Involvement of HO-1 and AMPK

Oxidative stress mediates the pathogenesis of neurodegenerative disorders. Gartanin, a natural xanthone of mangosteen, possesses multipharmacological activities. Herein, the neuroprotection capacity of gartanin against glutamate-induced damage in HT22 cells and its possible mechanism(s) were investigated for the first time. Glutamate resulted in cell death in a dose-dependent manner and supplementation of 1–10 µM gartanin prevented the detrimental effects of glutamate on cell survival. Additional investigations on the underlying mechanisms suggested that gartanin could effectively reduce glutamate-induced intracellular ROS generation and mitochondrial depolarization. We further found that gartanin induced HO-1 expression independent of nuclear factor erythroid-derived 2-like 2 (Nrf2). Subsequent studies revealed that the inhibitory effects of gartanin on glutamate-induced apoptosis were partially blocked by small interfering RNA-mediated knockdown of HO-1. Finally, the protein expression of phosphorylation of AMP-activated protein kinase (AMPK) and its downstream signal molecules, Sirtuin activator (SIRT1) and peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α), increased after gartanin treatment. Taken together, these findings suggest gartanin is a potential neuroprotective agent against glutamate-induced oxidative injury partially through increasing Nrf-2-independed HO-1 and AMPK/SIRT1/PGC-1α signaling pathways.     

Role of the ETB receptor in retinal ganglion cell death in glaucoma

Recent observations suggest that the vasoactive peptide endothelin-1 (ET-1) may be an important contributor to the etiology of glaucoma. ET-1 administration has been shown to produce optic nerve axonal loss and apoptosis of retinal ganglion cells. Ocular ET-1 levels are elevated in aqueous humor in response to elevated intraocular pressure both in glaucoma patients and in animal models of glaucoma; however, the precise mechanisms by which ET-1 mediates glaucomatous optic neuropathy are not clear. Presently we report that ET-1-mediated apoptosis was markedly attenuated in ETB receptor-deficient rats, suggesting a key role for ETB receptors in apoptosis of retinal ganglion cells by ET-1 treatment. Using virally transformed rat retinal ganglion cells (RGC-5 cells), we found that ET-1 (100 nmol/L) treatment produced apoptotic changes in these cells that was determined by flow cytometric analyses, release of mitochondrial cytochrome c to the cytosol, and increased phosphorylation of c-Jun N-terminal kinase. Pretreatment with the ETB-receptor antagonist BQ788 (1 micromol/L) was able to significantly attenuate ET-1-mediated apoptosis in RGC-5 cells. ET-1-mediated apoptotic changes in RGC-5 cells were associated with ETB-receptor activation and were accompanied by a significant upregulation of ETB-receptor expression. These studies suggest that ocular ET-1 acts through ETB receptors to mediate apoptosis of retinal ganglion cells, a key event in glaucoma and related optic neuropathies.     

In vivo demonstration of M3 muscarinic receptor subtype selectivity of darifenacin in mice

A novel muscarinic receptor antagonist, darifenacin, inhibited specific binding of [N-methyl-(3)H]scopolamine ([(3)H]NMS) in the mouse bladder, submaxillary gland and heart in a concentration-dependent manner. The inhibitory effect was most potent in the submaxillary gland, followed by the bladder and heart. In addition, darifenacin inhibited specific [(3)H]NMS binding in the membranes of CHO-K1 cell lines expressing muscarinic M(2) and M(3) receptor subtypes, and the potency was significantly (22-fold) greater at the M(3) than at the M(2) subtype. At 0.5 to 12 h after oral administration of darifenacin, a significant increase in K(d) values for specific [(3)H]NMS binding was seen in the bladder, submaxillary gland and lung of mice, compared with control values. Also, there was a sustained decrease in the B(max) values in the submaxillary gland. These data suggest that muscarinic receptor binding of oral darifenacin is rapid in onset and of a long duration. On the other hand, oral darifenacin exerted only temporary or little binding of muscarinic receptors in the heart and colon. Pilocarpine-induced salivary secretion in mice was continuously suppressed by oral darifenacin. The time-course of suppression coincided well with that for the muscarinic receptor binding in the submaxillary gland. The antagonistic effect of darifenacin against the dose-response curves for pilocarpine appeared to be insurmountable. In conclusion, the present study has shown that oral darifenacin may exert a pronounced and long-lasting binding of muscarinic receptors in tissues expressing the M(3) subtype.     

The molecular mechanism underlying mitophagy-mediated hippocampal neuron apoptosis in diabetes-related depression

Diabetes-related depression (DD) is a major complication of diabetes mellitus. Our previous studies indicated that glutamate (Glu) and hippocampal neuron apoptosis are key signal and direct factor leading to diabetes-related depression, respectively. However, the accurate pathogenesis remains to be unclear. We hypothesized that diabetes-related depression might be associated with the mitophagy-mediated hippocampal neuron apoptosis, triggered by aberrant Glu-glutamate receptor2 (GluR2)-Parkin pathway. To testify this hypothesis, here the rat model of DD in vivo and in vitro were both established so as to uncover the potential mechanism of DD based on mitophagy and apoptosis. We found that DD rats exhibit an elevated glutamate levels followed by monoamine neurotransmitter deficiency and depressive-like behaviour, and DD modelling promoted autophagosome formation and caused mitochondrial impairment, eventually leading to hippocampal neuron apoptosis via aberrant Glu-GluR2-Parkin pathway. Further, in vitro study demonstrated that the simulated DD conditions resulted in an abnormal glutamate and monoamine neurotransmitter levels followed by autophagic flux increment, mitochondrial membrane potential reduction and mitochondrial reactive oxygen species and lactic dehydrogenase elevation. Interestingly, both GluR2 and mammalian target of rapamycin (mTOR) receptor blocker aggravated mitophagy-induced hippocampal neuron apoptosis and abnormal expression of apoptotic protein. In contrast, both GluR2 and mTOR receptor agonist ameliorated those apoptosis in simulated DD conditions. Our findings revealed that mitophagy-mediated hippocampal neuron apoptosis, triggered by aberrant Glu-GluR2-Parkin pathway, is responsible for depressive-like behaviour and monoamine neurotransmitter deficiency in DD rats. This work provides promising molecular targets and strategy for the treatment of DD.     

Retinal ganglion cell protection by 17-beta-estradiol in a mouse model of inherited glaucoma

Glaucoma is the second leading cause of blindness in the world. The ultimate cause of vision loss due to glaucoma is thought to be retinal ganglion cell (RGC) apoptosis. Neuroprotection of RGC is becoming an important approach of glaucoma therapy. Several lines of evidence suggest that estrogen has neurotrophic and neuroprotective properties. In this study, we examine the role of estrogen in preventing RGC loss in DBA/2J mouse, an in vivo model of an inherited (pigmentary) glaucoma. Two-month-old female DBA/2J mice were anesthetized and ovariectomized with or without subcutaneous 17beta-estradiol (betaE2) pellet implantation. RGC survival was evaluated from flat-mounted whole retinas by counting retrograde-labeled cells. The loss of nerve fibers and RGC were also evaluated in paraffin-fixed retinal cross sections. Biochemical alterations in the retinas of DBA/2J mice in response to systemic injection of betaE2 were also examined. We have made several important observations showing that: (1) betaE2 treatment reduced the loss of RGC and neurofibers through inhibition of ganglion cell apoptosis, (2) betaE2 activated Akt and cAMP-responsive-element-binding-protein (CREB), (3) betaE2 up-regulated thioredoxin-1 (Trx-1) expression, (4) betaE2 reduced the increased activations of mitogen-activated protein kinases (MAPK) and NF-kappaB, (5) betaE2 inhibited the increased interleukin-18 (IL-18) expression, and (6) treatment with tamoxifen, an estrogen receptor antagonist, blocked betaE2-mediated activation of Akt and inhibition of MAPK phosphorylation in the retinas of DBA/2J mice. These findings suggest the possible involvement of multiple biochemical events, including estrogen receptor/Akt/CREB/thioredoxin-1, and estrogen receptor/MAPK/NF-kappaB, in estrogen-mediated retinal ganglion cell protection.     

Identification of an N-Methyl-d-aspartate Receptor in Isolated Nervous System Mitochondria

NMDA ionotropic glutamate receptors gate the cytoplasmic influx of calcium, which may, depending on the intensity of the stimulus, subserve either normal synaptic communication or cell death. We demonstrate that when isolated mitochondria are exposed to calcium and NMDA agonists, there is a significant increase in mitochondrial calcium levels. The agonist/antagonist response studies on purified mitochondria suggest the presence of a receptor on mitochondria with features similar to plasma membrane NMDA receptors. Immunogold electron microscopy of hippocampal tissue sections revealed extensive localization of NR2a subunit immunoreactivity on mitochondria. Transient transfection of neuronal GT1-7 cells with an NR1-NR2a NMDA receptor subunit cassette specifically targeting mitochondria resulted in a significant increase in mitochondrial calcium and neuroprotection against glutamate-induced cell death. Mitochondria prepared from GT1-7 cells in which the NR1 subunit of NMDA receptors was silenced demonstrated a decrease in calcium uptake. Our findings are the first to demonstrate that mitochondria express a calcium transport protein that shares characteristics with the NMDA receptor and may play a neuroprotective role.     

Calcium influx through receptor-operated channel induces mitochondria-triggered paraptotic cell death

We address the specific role of cytoplasmic Ca(2+) overload as a cell death trigger by expressing a receptor-operated specific Ca(2+) channel, vanilloid receptor subtype 1 (VR1), in Jurkat cells. Ca(2+) uptake through the VR1 channel, but not capacitative Ca(2+) influx stimulated by the muscarinic type 1 receptor, induced sustained intracellular [Ca(2+)] rises, exposure of phosphatidylserine, and cell death. Ca(2+) influx was necessary and sufficient to induce mitochondrial damage, as assessed by opening of the permeability transition pore and collapse of the mitochondrial membrane potential. Ca(2+)-induced cell death was inhibited by ruthenium red, protonophore carbonyl cyanide m-chlorophenylhydrazone, or cyclosporin A treatment, as well as by Bcl-2 expression, indicating that this process requires mitochondrial calcium uptake and permeability transition pore opening. Cell death occurred without caspase activation, oligonucleosomal/50-kilobase pair DNA cleavage, or release of cytochrome c or apoptosis inducer factor from mitochondria, but it required oxidative/nitrative stress. Thus, Ca(2+) influx triggers a distinct program of mitochondrial dysfunction leading to paraptotic cell death, which does not fulfill the criteria for either apoptosis or necrosis.     

Neuroprotective Effects of Bis(7)-tacrine in a Rat Model of Pressure-Induced Retinal Ischemia

The retinal ischemia–reperfusion model has been studied extensively and is an ideal animal model for studying clinical situations such as acute glaucoma and optic neuropathy. Our previous reports showed that bis(7)-tacrine had neuroprotective effects against glutamate-induced retinal ganglion cells damage through the drug’s anti-NMDA receptor effects. Here, we investigated whether bis(7)-tacrine protects the retina from ischemic injury in a rat model. Retinal ischemia was induced by raising the intraocular pressure to 120 mmHg for 90 min. Rats received intraperitoneal injections of 0.2 mg/kg bis(7)-tacrine or saline at 30 min before ischemia, and then twice a day after retinal ischemia. Morphometric evaluation showed that bis(7)-tacrine dramatically reduced the retinal damage compared with the control group. Moreover, bis(7)-tacrine suppressed ischemia-induced reductions in a- and b-wave amplitudes of electroretinography. Protein levels of p53, the tumor suppressor gene known to induce apoptosis, were increased after ischemic injury, and treatment with bis(7)-tacrine reduced the expression of the protein. Our results suggest that bis(7)-tacrine has a neuroprotective effect against ischemic injury in the rat retina, possibly through the drug’s anti-apoptotic effects. Bis(7)-tacrine may potentially be useful as a therapeutic drug in the management of ischemic retinal diseases.     

Subtype-specific regulation of the expression and function of muscarinic acetylcholine receptors in embryonic chicken retinal cells

We examined the effect of long-term agonist exposure on muscarinic acetylcholine receptor expression and function in embryonic chicken retinal cells. Long-term carbachol exposure induced a time- and concentration-dependent decrease in M2, M3 and M4 muscarinic receptor numbers. Kinetic analyses revealed a first-order process with similar rate constants for all three subtypes. Both the maximal decrease and the agonist potency for regulation of M3 were significantly higher than those for M2 and M4. Upon agonist removal, M2 and M4 numbers returned to control values, but M3 recovery after 24 h was no higher than 40%. Agonist treatment did not alter the levels of receptor mRNAs. Receptor inactivation with a covalent alkylating antagonist demonstrated that the partial M3 protein recovery was not due to a decreased intrinsic basal rate of synthesis, suggesting that it is induced by agonist treatment. Prolonged carbachol exposure induced concomitant decreases in muscarinic-mediated inhibition of cyclic AMP accumulation which were completely reversed after agonist removal. Sustained receptor activation also promoted significant decreases in muscarinic receptor-stimulated phosphoinositide turnover, which were only partially reversed after agonist removal. These data demonstrate subtype-specific regulation of the expression and function of muscarinic receptors in the retina.     

Molecular and pharmacological characterization of muscarinic receptors in retinal pigment epithelium: role in light-adaptive pigment movements

Muscarinic receptors are the predominant cholinergic receptors in the central and peripheral nervous systems. Recently, activation of muscarinic receptors was found to elicit pigment granule dispersion in retinal pigment epithelium isolated from bluegill fish. Pigment granule movement in retinal pigment epithelium is a light-adaptive mechanism in fish. In the present study, we used pharmacological and molecular approaches to identify the muscarinic receptor subtype and the intracellular signaling pathway involved in the pigment granule dispersion in retinal pigment epithelium. Of the muscarinic receptor subtype-specific antagonists used, only antagonists specific for M1 and M3 muscarinic receptors were found to block carbamyl choline (carbachol)-induced pigment granule dispersion. A phospholipase C inhibitor also blocked carbachol-induced pigment granule dispersion, and a similar result was obtained when retinal pigment epithelium was incubated with an inositol trisphosphate receptor inhibitor. We isolated M2 and M5 receptor genes from bluegill and studied their expression. Only M5 was found to be expressed in retinal pigment epithelium. Taken together, pharmacological and molecular evidence suggest that activation of an odd subtype of muscarinic receptor, possibly M5, on fish retinal pigment epithelium induces pigment granule dispersion.     

Modulation of mitochondria in the axon and soma of retinal ganglion cells in a rat glaucoma model

Mitochondrial abnormality has been implicated in various models of retinal ganglion cell (RGC) degeneration. We investigated modulation of mitochondrial membrane permeability and apoptosis-inducing factor (AIF) translocation in a rat experimental glaucoma model. A decrease in MitoTracker-labeled mitochondria around the lamina area of the optic nerve was observed in the glaucomatous eye. Immunoblot analysis for axonal motor proteins showed that a significant decrease in kinesin 1 and myosin Va levels in the glaucomatous optic nerve. A significant decrease in mitochondrial thioredoxin 2 (Trx2) level was observed in the optic nerve after intraocular pressure (IOP) elevation. Translocation of AIF from the mitochondria to the axoplasm and nucleus was observed in the axon and cell body, respectively. Trx2 over-expression in the mitochondrial membrane of RGC-5 cells inhibited AIF translocation, resulting in cytoprotective effect against neurotoxicity induced by TNF-α/buthionine sulfoximine treatment. In vivo transfection was performed with EGFP-Trx2 plasmid and electroporation. Over-expression of Trx2 in the retina and optic nerve indicated the protective effect against high IOP induced axonal degeneration. Thus, the decreased mitochondrial membrane potential and subsequent AIF translocation were involved in the glaucomatous neurodegeneration. Furthermore, modulation of mitochondria through the inhibition of AIF translocation may become a new treatment strategy for neurodegenerative disease, such as glaucoma.