N-Acetylcysteine Protects Against Hypoxia Mimetic-Induced Autophagy by Targeting the HIF-1α Pathway in Retinal Ganglion Cells

Hypoxia-induced retinal ganglion cell (RGC) death has been proposed to be the critical event in the pathophysiology of glaucoma. Therefore, delaying or halting RGC degeneration, known as neuroprotection, is a novel and promising approach with potential clinical applications for treating glaucoma. In this study, we investigate hypoxia-induced cell death of RGCs and the underlying mechanisms of N-acetylcysteine (NAC) as a neuroprotectant. To establish a model for chemical hypoxia-induced cell death, RGC-5 cells were treated with the hypoxia mimetic cobalt chloride (CoCl₂). Following CoCl₂ exposure, significant levels of apoptotic and autophagic cell death were observed in RGC-5 cells, evidenced by lysosome dysfunction and autophagosome formation. Pretreating RGC-5 cells with NAC significantly counteracted the autophagic cell death. NAC-mediated neuroprotection was attributed to the direct scavenging of reactive oxygen species and was mediated by targeting the hypoxia-inducible factor-1?? pathway via the BNIP3 and PI3K/Akt/mTOR pathways. These results provide insights into the degeneration of RGCs and present a potential clinical application for NAC as a neuroprotectant.     

Annexin 1 protects against apoptosis induced by serum deprivation in transformed rat retinal ganglion cells

To investigate whether Annexin 1 can protect a retinal ganglion cells line (RGC-5) from apoptosis as induced by serum deprivation. Annexin 1 location in RGC-5 cells was determined using an indirect immunofluorescent assay. Expression of Annexin 1 in RGC-5 cultures deprived of serum for 0, 2 days was semi-quantified by western blot and RT-PCR. Effects of varying concentrations of the Annexin 1 peptide fragment, Ac2-26, on the survival of the RGC-5 cells was determined, and apoptotic cells were quantified by flow cytometry. Immunoblot and RT-PCR analysis was preformed to identify caspase 3, bax and bcl-2 in RGC extracts. Annexin 1 was localized in the cytoplasm of RGC-5 cells and the expression of Annexin 1, caspase 3 and bax was upregulated in serum-deprived RGC-5 cells. Ac2-26 attenuated the apoptosis resulting from serum deprivation of RGC-5 in a concentration-dependent manner, decreased caspase 3 and bax levels and produced an increase of bcl-2 in cell lysates. Annexin 1, in specific the peptide fragment Ac2-26, may play an important role in decreasing apoptosis in serum-deprived RGC-5 cells.     

Long-term blue light exposure induces RGC-5 cell death in vitro: involvement of mitochondria-dependent apoptosis,oxidative stress,and MAPK signaling pathways

The mechanism of blue light-induced retinal ganglion cell (RGC) injury is poorly understood. In this study, we established a patented light-emitting diode-based system to study the effects of long-term blue light exposure under culture conditions on RGC-5 cells. Long-term blue light exposure significantly reduced cell viability in a time-dependent manner and induced apoptosis and necrosis in RGC-5 cells. Long-term blue light exposure marked an increase in the expression of Bax and active Caspase-3 (p17), which was accompanied by Bcl-2 down-regulation, and displayed features of the mitochondria-dependent apoptosis pathway. Blue light exposure also increased the generation of reactive oxygen species (ROS), and was a strong inducer of ROS-sensitive protein nuclear factor erythroid 2-related factor 2 (Nrf2) and heme oxygenase-1 (HO-1) expression. Moreover, blue light exposure constitutively activated p38 mitogen-activated protein kinases and c-Jun NH2-terminal kinase (JNK), as well as induced the phosphorylation of extracellular signal-regulated kinase in the early phase, in blue light-exposed RGC-5 cells. The protein expression of c-jun and c-fos was further enhanced after RGC-5 cells were exposed to blue light. Taken together, these findings indicated that blue light induced RGC-5 cell line death in dependence upon exposure duration. The potential mechanisms for this phenomenon might be via activated mitochondria-dependent apoptosis, increased ROS production and protein expressions of Nrf2 and HO-1, and activated JNK/p38 MAPK signaling pathways.     

Expression of novel opsins and intrinsic light responses in the mammalian retinal ganglion cell line RGC-5. Presence of OPN5 in the rat retina

The vertebrate retina is known to contain three classes of photoreceptor cells: cones and rods responsible for vision, and intrinsically photoresponsive retinal ganglion cells (RGCs) involved in diverse non-visual functions such as photic entrainment of daily rhythms and pupillary light responses. In this paper we investigated the potential intrinsic photoresponsiveness of the rat RGC line, RGC-5, by testing for the presence of visual and non-visual opsins and assessing expression of the immediate-early gene protein c-Fos and changes in intracellular Ca(2+) mobilization in response to brief light pulses. Cultured RGC-5 cells express a number of photopigment mRNAs such as retinal G protein coupled receptor (RGR), encephalopsin/panopsin (Opn3), neuropsin (Opn5) and cone opsin (Opn1mw) but not melanopsin (Opn4) or rhodopsin. Opn5 immunoreactivity was observed in RGC-5 cells and in the inner retina of rat, mainly localized in the ganglion cell layer (GCL). Furthermore, white light pulses of different intensities and durations elicited changes both in intracellular Ca(2+) levels and in the induction of c-Fos protein in RGC-5 cell cultures. The results demonstrate that RGC-5 cells expressing diverse putative functional photopigments display intrinsic photosensitivity which accounts for the photic induction of c-Fos protein and changes in intracellular Ca(2+) mobilization. The presence of Opn5 in the GCL of the rat retina suggests the existence of a novel type of photoreceptor cell.     

Oxidative stress upregulates Wnt signaling in human retinal microvascular endothelial cells through activation of disheveled

Abnormal retinal neovascularization associated with various retinopathies can result in irreversible vision loss. Although the mechanisms involved in this occurrence is unclear, increasing evidence suggests that aberrant Wnt signaling participates in the pathogenesis of abnormal neovascularization. Because Wnt signaling upregulation can be induced by oxidative stress through the activation of disheveled (DVL), a key molecule in the Wnt signaling pathway, we investigated whether oxidative stress can activate Wnt signaling and induce angiogenic phenotypes in human retinal microvascular endothelial cells (HRMECs). We found that increased Wnt signaling activity, as well as enhanced angiogenic phenotypes, such as tube formation and cell migration, were detected in the hydrogen peroxide-treated HRMECs. Moreover, these effects were effectively suppressed by a small-molecule Wnt inhibitor targeting the PDZ domain of DVL. Therefore, we propose that targeting abnormal Wnt signaling at the DVL level with a small-molecule inhibitor may represent a novel approach in retinal neovascularization treatment and prevention.     

Stimulation of Prostaglandin EP2 Receptors on RGC-5 Cells in Culture Blunts the Negative Effect of Serum Withdrawal

Reduced neurotrophic support is one possible cause for retinal ganglion cells dying in glaucoma. Experiments were designed to investigate the effect of EP2 receptor agonist butaprost on transformed retinal ganglion (RGC-5) cells where reduced neurotrophic support was simulated by serum withdrawal. Cultures were analysed for cell viability, flow cytometry, reactive oxygen species and apoptosis. Western blot and immunohistochemistry were used to provide information for the occurrence of PGE₂ receptor-types. We demonstrated the existence of all four types of PGE₂ receptors in RGC-5 cells and exposure of cultures to butaprost resulted in an elevation of cAMP. Serum deprivation induced RGC-5 cell death was significantly attenuated by butaprost as well as by rolipram and forskolin where intracellular cAMP levels were increased. These data are of value in relation to the possible use of EP2 receptor agonists to reduce both elevated intraocular pressure and retinal ganglion cell death as occurs in glaucoma.     

Diabetes and Overexpression of proNGF Cause Retinal Neurodegeneration via Activation of RhoA Pathway

Our previous studies showed positive correlation between accumulation of proNGF, activation of RhoA and neuronal death in diabetic models. Here, we examined the neuroprotective effects of selective inhibition of RhoA kinase in the diabetic rat retina and in a model that stably overexpressed the cleavage-resistance proNGF plasmid in the retina. Male Sprague-Dawley rats were rendered diabetic using streptozotosin or stably express cleavage-resistant proNGF plasmid. The neuroprotective effects of the intravitreal injection of RhoA kinase inhibitor Y27632 were examined in vivo. Effects of proNGF were examined in freshly isolated primary retinal ganglion cell (RGC) cultures and RGC-5 cell line. Retinal neurodegeneration was assessed by counting TUNEL-positive and Brn-3a positive retinal ganglion cells. Expression of proNGF, p75^NTR, cleaved-PARP, caspase-3 and p38MAPK/JNK were examined by Western-blot. Activation of RhoA was assessed by pull-down assay and G-LISA. Diabetes and overexpression of proNGF resulted in retinal neurodegeneration as indicated by 9- and 6-fold increase in TUNEL-positive cells, respectively. In vitro, proNGF induced 5-fold cell death in RGC-5 cell line, and it induced >10-fold cell death in primary RGC cultures. These effects were associated with significant upregulation of p75^NTR and activation of RhoA. While proNGF induced TNF-α expression in vivo, it selectively activated RhoA in primary RGC cultures and RGC-5 cell line. Inhibiting RhoA kinase with Y27632 significantly reduced diabetes- and proNGF-induced activation of proapoptotic p38MAPK/JNK, expression of cleaved-PARP and caspase-3 and prevented retinal neurodegeneration in vivo and in vitro. Taken together, these results provide compelling evidence for a causal role of proNGF in diabetes-induced retinal neurodegeneration through enhancing p75^NTR expression and direct activation of RhoA and p38MAPK/JNK apoptotic pathways.     

Protection of exenatide for retinal ganglion cells with different glucose concentrations

Exendin-4 is a peptide resembling glucagon-like peptide-1 (GLP-1), which has protective effects on nerve cells. However, the effects of Exendin-4 on retinal ganglion cells (RGC) are still under clear. The purpose of the present study is to demonstrate that exenatide prevents high- or low-glucose-induced retinal ganglion cell impairment. We observed the expression of GLP-1R in RGC-5 cells by immunofluorescence and Western blot. To investigate the effect of exenatide on RGC-5 cells incubated different glucose concentrations, CCK-8 measured the survival rates and electron microscopy detected cellular injury. The expression levels of Bcl-2 and Bax were analyzed by immunocytochemistry and Western blot. Exenatide protects RGC-5 from high- or low-glucose-induced cellular injury and the optimum concentration was 0.5μg/ml. Exenatide can inhibit high- or low-glucose-induced mitochondrial changes. Exenatide protects RGC-5 from high- or low-glucose-induced Bax increased and Bcl-2 decreased. Furthermore, the protective effect of exenatide could be inhibited by Exendin (9-39). These findings indicate that exenatide shows a neuroprotective effect for different glucose concentrations-induced RGC-5 cells injury. Exenatide could protect RGC-5 cells from degeneration or death, which may protect retinal function and have a potential value for patients with diabetic retinopathy.     

Eriodictyol inhibits high glucose-induced oxidative stress and inflammation in retinal ganglial cells

Diabetic retinopathy (DR) is one of the most common microvascular complications of diabetes mellitus and is considered as a leading cause of blindness. Oxidative stress and inflammation are significant drivers for the development of DR. Eriodictyol, a flavonoid compound, was proved to possess anti-inflammatory, antioxidative, and antidiabetic activities. However, the role of eriodictyol in DR has not been unveiled. In the current study, we explored the protective effects of eriodictyol on high glucose (HG)-induced rat retinal ganglial cells (RGCs). The results suggested that eriodictyol improved cell viability of HG-induced rat RGC-5 cells in a dose-dependent manner. Eriodictyol reduced the reactive oxygen species production and increased the activities of superoxide dismutase, glutathione peroxidase and catalase in rat RGC-5 cells in response to HG stimulation. The production of proinflammatory cytokines including tumor necrosis factor alpha and interleukin-8 was diminished after eriodictyol treatment. Eriodictyol also suppressed cell apoptosis induced HG in rat RGC-5 cells. Furthermore, eriodictyol enhanced the nuclear translocation of nuclear factor erythroid-2 (E2)-related factor 2 (Nrf2) and elevated the expression of antioxidant enzyme heme-oxygenase-1 (HO-1). These findings suggested that eriodictyol protects the RGC-5 cells from HG-induced oxidative stress, inflammation, and cell apoptosis through regulating the activation of Nrf2/HO-1 pathway.     

Neurotrophin-4/5 (NT-4/5) increases adult rat retinal ganglion cell survival and neurite outgrowth in vitro

Retinal ganglion cell (RGC) survival and neurite outgrowth were investigated in retinal explants from adult rats. Neutrotrophin-4/5 (NT-4/5) caused dose-dependent increases in neurite outgrowth with one-half maximal effects at approximately 0.5 ng/ml and maximal effects at 5 ng/ml. In explants treated for 7 days, the actions of NT-4/5 were similar to those of brain-derived neurotrophic factor (BDNF); with either neurotrophin, nearly twice as many RGCs survived and there was a two- to threefold increase in the number of neurites formed by RGCs. Combinations of saturating concentrations of NT-4/5 and BDNF did not enhance these in vitro effects, implying that both neurotrophins share a common signaling pathway. In contrast, nerve growth factor (NGF), neurotrophin-3 (NT-3), or ciliary nuerotrophic factor (CNTF) appeared to exert minimal influences on RGC survival or neurite outgrowth. 1994 John Wiley & Sons, Inc.     

Role of nonfeminizing estrogen analogues in neuroprotection of rat retinal ganglion cells against glutamate-induced cytotoxicity

Glaucoma is a family of eye disorders whose ultimate cause of vision loss is apoptosis of retinal ganglion cells. Although several etiologies of glaucoma exist, oxidative stress is thought to be a key mechanism by which ganglion cells die. From this perspective, the work presented here was designed to examine the efficacy of 17beta-estradiol and three synthetic estrogen analogues (ZYC-1, ZYC-3, ZYC-10) as retinal ganglion cell neuroprotectants. Compound ZYC-1 and its enantiomer ZYC-10, containing an additional double bond in the steroid C ring of 17beta-estradiol, had similar (ZYC-1) or modestly reduced (ZYC-10) affinity for estrogen receptors compared to the parent estrogen. In the case of ZYC-3, the addition of an adamantyl group to the C2 position of the A ring of estrone abolished its binding to the estrogen receptors. RGC-5 cells (an established clonal rat retinal ganglion cell line) and rat retinas were shown to predominantly express estrogen receptor alpha, with minimal detectable levels of estrogen receptor beta. The affinity of the synthetic compounds for estrogen receptors was as follows: ZYC-3 < ZYC-10 < ZYC-1. An in vitro model of glutamate-induced RGC-5 cell death was used. Glutamate treatment resulted in 50-60% RGC-5 cell death with respect to control untreated cells. 17beta-estradiol and the three estrogen analogues (0.5 to 1.0 microM) protected the RGC-5 cells against glutamate cytotoxicity. The efficacy of neuroprotection by the estrogen analogues was as follows: ZYC-3 > ZYC-1 > ZYC-10. EC(50) values for inhibition of TBARS levels were as follows: ZYC-3 > ZYC-10 > ZYC-1. Furthermore, these compounds worked independent of estrogen receptors, as inclusion of 100 nM ICI 182,780 did not reverse their neuroprotective properties against glutamate insult. These compounds seem to affect neuroprotection via pathways independent of the classical estrogen receptors. The data support the hypothesis that estrogen analogues may be useful in the treatment of neurodegenerative diseases, particularly in neuroprotection of retinal ganglion cells in ocular pathologies such as glaucoma.     

Hypoxia induces beta-amyloid in association with death of RGC-5 cells in culture

Beta-amyloid (Aβ) derived from amyloid precursor protein (APP) has been associated with retinal degeneration in Alzheimer’s disease (AD) and glaucoma. This study examined whether hypoxia exposure induces Aβ accumulation in RGC-5 cells. While levels of APP mRNA and protein significantly increased in the cells, elevated abundance of Aβ was also observed in cells and culture medium between 12 or 24 and 48 h after 5% O₂ hypoxia treatment. Additionally, there is a close relationship between induction of APP and Aβ and intracellular accumulation of ROS along with loss of mitochondrial membrane potential followed by the death of RGC-5 cells in culture under hypoxia. These results suggest a possible involvement of APP and Aβ in the death of RGCs challenged by hypoxia.     

The effect of extrinsic Wnt/β‐catenin signaling in Muller glia on retinal ganglion cell neurite growth

Muller glia are the predominant glial cell type in the retina, and they structurally and metabolically support retinal neurons. Wnt/β‐catenin signaling pathways play essential roles in the central nervous system, including glial and neuronal differentiation, axonal growth, and neuronal regeneration. We previously demonstrated that Wnt signaling activation in retinal ganglion cells (RGC) induces axonal regeneration after injury. However, whether Wnt signaling within the adjacent Muller glia plays an axongenic role is not known. In this study, we characterized the effect of Wnt signaling in Muller glia on RGC neurite growth. Primary Muller glia and RGC cells were grown in transwell co‐cultures and adenoviral constructs driving Wnt regulatory genes were used to activate and inhibit Wnt signaling specifically in primary Muller glia. Our results demonstrated that activation of Wnt signaling in Muller glia significantly increased RGC average neurite length and branch site number. In addition, the secretome of Muller glia after induction or inhibition of Wnt signaling was characterized using protein profiling of conditioned media by Q Exactive mass spectrometry. The Muller glia secretome after activation of Wnt signaling had distinct and more numerous proteins involved in regulation of axon extension, axon projection and cell adhesion. Furthermore, we showed highly redundant expression of Wnt signaling ligands in Muller glia and Frizzled receptors in RGCs and Muller glia. Therefore, this study provides new information about potential neurite growth promoting molecules in the Muller glia secretome, and identified Wnt‐dependent target proteins that may mediate the axonal growth.     

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.     

Latanoprost Promotes Neurite Outgrowth in Differentiated RGC-5 Cells via the PI3K-Akt-mTOR Signaling Pathway

Latanoprost, a synthetic derivative of the natural prostaglandin F_2a (PGF_2a), is a powerful antiglaucoma agent with ocular hypotensive and neuroprotective effects. However, the neuroregenerative effect and signaling pathway of latanoprost in retinal ganglion cells (RGCs) are still unknown. The purpose of this study is to investigate the regenerative effect of latanoprost in differentiated RGC-5 cells and its underlying mechanisms. Cell viability was determined by Cell Counting Kit-8 (CCK-8) assay and neurite length was examined by ArrayScan HCS Reader and Neurite outgrowth BioApplication. Expressions of Akt phosphorylation (p-Akt) and mammalian target of rapamycin phosphorylation (p-mTOR) were investigated by Western blot analysis. The results indicated that 0.1 μM latanoprost (at a clinically therapeutic concentration) significantly increased cell viability as compared with control. Meanwhile, 0.1 μM latanoprost resulted in the obvious promotion of neurite outgrowth similar to ciliary neurotrophic factor (CNTF) and simultaneously increased the levels of p-Akt and p-mTOR expression. The effects of latanoprost were blocked by the Prostaglandin F receptor (FP receptor) inhibitor AL8810, the phosphoinositide 3-kinase (PI3K) inhibitor LY294002 and the mTOR inhibitor rapamycin. This study presents novel in vitro evidence that latanoprost could promote neurite outgrowth through an FP receptor-mediated modulation of the PI3K-Akt-mTOR signaling pathway. This finding may provide insight into a better understanding of a new mechanism of latanoprost for glaucoma therapy and into the physiological-modulating activities of prostaglandins.     

Neuroprotection of a Novel Cyclopeptide C*HSDGIC* from the Cyclization of PACAP (1–5) in Cellular and Rodent Models of Retinal Ganglion Cell Apoptosis

Purpose

To investigate the protective effects of a novel cyclopeptide C*HSDGIC* (CHC) from the cyclization of Pituitary adenylate cyclase-activating polypeptide (PACAP) (1–5) in cellular and rodent models of retinal ganglion cell apoptosis.

Methodology/Principal Findings

Double-labeling immunohistochemistry was used to detect the expression of Thy-1 and PACAP receptor type 1 in a retinal ganglion cell line RGC-5. The apoptosis of RGC-5 cells was induced by 0.02 J/cm² Ultraviolet B irradiation. MTT assay, flow cytometry, fluorescence microscopy were used to investigate the viability, the level of reactive oxygen species (ROS) and apoptosis of RGC-5 cells respectively. CHC attenuated apoptotic cell death induced by Ultraviolet B irradiation and inhibited the excessive generation of ROS. Moreover, CHC treatment resulted in decreased expression of Bax and concomitant increase of Bcl-2, as was revealed by western-blot analysis. The in vivo apoptosis of retinal ganglion cells was induced by injecting 50 mM N-methyl-D-aspartate (NMDA) (100 nmol in a 2 µL saline solution) intravitreally, and different dosages of CHC were administered. At day 7, rats in CHC+ NMDA-treated groups showed obvious aversion to light when compared to NMDA rats. Electroretinogram recordings revealed a marked decrease in the amplitudes of a-wave, b-wave, and photopic negative response due to NMDA damage. In retina receiving intravitreal NMDA and CHC co-treatment, these values were significantly increased. CHC treatment also resulted in less NMDA-induced cell loss and a decrease in the proportion of dUTP end-labeling-positive cells in ganglion cell line.

Conclusions

C*HSDGIC*, a novel cyclopeptide from PACAP (1–5) attenuates apoptosis in RGC-5 cells and inhibits NMDA-induced retinal neuronal death. The beneficial effects may occur via the mitochondria pathway. PACAP derivatives like CHC may serve as a promising candidate for neuroprotection in glaucoma.     

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.