Oxidative stress induces autophagy in response to multiple noxious stimuli in retinal ganglion cells

Retinal ganglion cells (RGCs) are the only afferent neurons that can transmit visual information to the brain. The death of RGCs occurs in the early stages of glaucoma, diabetic retinopathy, and many other retinal diseases. Autophagy is a highly conserved lysosomal pathway, which is crucial for maintaining cellular homeostasis and cell survival under stressful conditions. Research has established that autophagy exists in RGCs after increasing intraocular pressure (IOP), retinal ischemia, optic nerve transection (ONT), axotomy, or optic nerve crush. However, the mechanism responsible for defining how autophagy is induced in RGCs has not been elucidated. Accumulating data has pointed to an essential role of reactive oxygen species (ROS) in the activation of autophagy. RGCs have long axons with comparatively high densities of mitochondria. This makes them more sensitive to energy deficiency and vulnerable to oxidative stress. In this review, we explore the role of oxidative stress in the activation of autophagy in RGCs, and discuss the possible mechanisms that are involved in this process. We aim to provide a more theoretical basis of oxidative stress-induced autophagy, and provide innovative targets for therapeutic intervention in retinopathy.     

Autophagy promotes survival of retinal ganglion cells after optic nerve axotomy in mice

Autophagy is an essential recycling pathway implicated in neurodegeneration either as a pro-survival or a pro-death mechanism. Its role after axonal injury is still uncertain. Axotomy of the optic nerve is a classical model of neurodegeneration. It induces retinal ganglion cell death, a process also occurring in glaucoma and other optic neuropathies. We analyzed autophagy induction and cell survival following optic nerve transection (ONT) in mice. Our results demonstrate activation of autophagy shortly after axotomy with autophagosome formation, upregulation of the autophagy regulator Atg5 and apoptotic death of 50% of the retinal ganglion cells (RGCs) after 5 days. Genetic downregulation of autophagy using knockout mice for Atg4B (another regulator of autophagy) or with specific deletion of Atg5 in retinal ganglion cells, using the Atg5(flox/flox) mice reduces cell survival after ONT, whereas pharmacological induction of autophagy in vivo increases the number of surviving cells. In conclusion, our data support that autophagy has a cytoprotective role in RGCs after traumatic injury and may provide a new therapeutic strategy to ameliorate retinal diseases.     

Axonal damage, autophagy and neuronal survival

In recent years autophagy modulation has been shown to reduce or increase neuronal cell death in several models of neurodegeneration. How autophagy exerts these dual effects is currently unknown. Here we review recent evidence from our laboratory demonstrating that autophagy can protect the cell soma after axonal traumatic injury. Damage in the optic nerve induces retinal ganglion cell (RGC) death in glaucoma and other retinal diseases and is often modeled by axotomy of the optic nerve in laboratory animals. Using this well-known model of RGC degeneration we show that autophagy is strongly upregulated following the insult and before cell death. Enhancement of autophagy by pharmacological treatment with rapamycin decreases the number of degenerating neurons. Conversely, axotomy in Atg4B (-/-) mice increases the number of dying cells in the retinal ganglion cell layer. Similar findings were observed in Atg5 (flox/flox) mice following specific downregulation of the autophagy regulator ATG5 in RGCs, by intravitreal injection of a cre-expressing vector. Taken together, these findings point to a cytoprotective role of autophagy following axonal damage in vivo.     

Lycium Barbarum (Wolfberry) Reduces Secondary Degeneration and Oxidative Stress,and Inhibits JNK Pathway in Retina after Partial Optic Nerve Transection

Our group has shown that the polysaccharides extracted from Lycium barbarum (LBP) are neuroprotective for retinal ganglion cells (RGCs) in different animal models. Protecting RGCs from secondary degeneration is a promising direction for therapy in glaucoma management. The complete optic nerve transection (CONT) model can be used to study primary degeneration of RGCs, while the partial optic nerve transection (PONT) model can be used to study secondary degeneration of RGCs because primary degeneration of RGCs and secondary degeneration can be separated in location in the same retina in this model; in other situations, these types of degeneration can be difficult to distinguish. In order to examine which kind of degeneration LBP could delay, both CONT and PONT models were used in this study. Rats were fed with LBP or vehicle daily from 7 days before surgery until sacrifice at different time-points and the surviving numbers of RGCs were evaluated. The expression of several proteins related to inflammation, oxidative stress, and the c-jun N-terminal kinase (JNK) pathways were detected with Western-blot analysis. LBP did not delay primary degeneration of RGCs after either CONT or PONT, but it did delay secondary degeneration of RGCs after PONT. We found that LBP appeared to exert these protective effects by inhibiting oxidative stress and the JNK/c-jun pathway and by transiently increasing production of insulin-like growth factor-1 (IGF-1). This study suggests that LBP can delay secondary degeneration of RGCs and this effect may be linked to inhibition of oxidative stress and the JNK/c-jun pathway in the retina.     

Retrograde Labeling of Retinal Ganglion Cells in Adult Zebrafish with Fluorescent Dyes

As retrograde labeling retinal ganglion cells (RGCs) can isolate RGCs somata from dying sites, it has become the gold standard for counting RGCs in RGCs survival and regeneration experiments. Many studies have been performed in mammalian animals to research RGCs survival after optic nerve injury. However, retrograde labeling of RGCs in adult zebrafish has not yet been reported, though some alternative methods can count cell numbers in retinal ganglion cell layers (RGCL). Considering the small size of the adult zebrafish skull and the high risk of death after drilling on the skull, we open the skull with the help of acid-etching and seal the hole with a light curing bond, which could significantly improve the survival rate. After absorbing the dyes for 5 days, almost all the RGCs are labeled. As this method does not need to transect the optic nerve, it is irreplaceable in the research of RGCs survival after optic nerve crush in adult zebrafish. Here, we introduce this method step by step and provide representative results.     

The Role of Peroxisome Proliferator-Activated Receptor and Effects of Its Agonist,Pioglitazone, on a Rat Model of Optic Nerve Crush: PPARγ in Retinal Neuroprotection

It has been shown that peroxisome proliferators-activated receptor gamma (PPARγ) is beneficial for central nervous system injury. However its role on optic nerve injury remains unknown. In the present study, we examined the change of PPARγ expression in rat retina following optic nerve injury and investigated the effect of pioglitazone (Pio), a PPARγ agonist, on retinal ganglion cells (RGCs) neuroprotection using a rat optic nerve crush (ONC) model. Our results showed that PPARγ mRNA and protein levels were increased after ONC, and most of PPARγ-immunoreactive cells colocalized with Müller cells. Pio treatment significantly enhanced the number of surviving RGCs and inhibited RGCs apoptosis induced by ONC. However, when PPARγ antagonist GW9662 was used, these neuroprotective effects were abolished. In addition, pio attenuated Müller cell activation after ONC. These results indicate that PPARγ appears to protect RGCs from ONC possibly via the reduction of Müller glial activation. It provides evidence that activation of PPARγ may be a potential alternative treatment for RGCs neuroprotection.     

Neuronal STAT3 activation is essential for CNTF- and inflammatory stimulation-induced CNS axon regeneration

CNS neurons, such as retinal ganglion cells (RGCs), do not normally regenerate injured axons, but instead undergo apoptotic cell death. Regenerative failure is due to inhibitory factors in the myelin and forming glial scar as well as due to an insufficient intrinsic capability of mature neurons to regrow axons. Nevertheless, RGCs can be transformed into an active regenerative state upon inflammatory stimulation (IS) in the inner eye, for instance by lens injury, enabling these RGCs to survive axotomy and to regenerate axons into the lesioned optic nerve. The beneficial effects of IS are mediated by various factors, including CNTF, LIF and IL-6. Consistently, IS activates various signaling pathways, such as JAK/STAT3 and PI3K/AKT/mTOR, in several retinal cell types. Using a conditional knockdown approach to specifically delete STAT3 in adult RGCs, we investigated the role of STAT3 in IS-induced neuroprotection and axon regeneration. Conditional STAT3 knockdown in RGCs did not affect the survival of RGCs after optic nerve injury compared with controls, but significantly reduced the neuroprotective effects of IS. STAT3 depletion significantly compromised CNTF-stimulated neurite growth in culture and IS-induced transformation of RGCs into an active regenerative state in vivo. As a consequence, IS-mediated axonal regeneration into the injured optic nerve was almost completely abolished in mice with STAT3 depleted in RGCs. In conclusion, STAT3 activation in RGCs is involved in neuroprotection and is a necessary prerequisite for optic nerve regeneration upon IS.     

Optic nerve degeneration and mitochondrial dysfunction: genetic and acquired optic neuropathies

Selective degeneration of the smallest fibers (papillo-macular bundle) of the human optic nerve occurs in a large number of optic neuropathies characterized primarily by loss of central vision. The pathophysiology that underlies this peculiar pattern of cell involvement probably reflects different forms of genetic and acquired mitochondrial dysfunction.Maternally inherited Leber's hereditary optic neuropathy (LHON), dominant optic atrophy (Kjer disease), the optic atrophy of Leigh's syndrome, Friedreich ataxia and a variety of other conditions are examples of inherited mitochondrial disorders with different etiologies. Tobacco-alcohol amblyopia (TAA), the Cuban epidemic of optic neuropathy (CEON) and other dietary (Vitamins B, folate deficiencies) optic neuropathies, as well as toxic optic neuropathies such as due to chloramphenicol, ethambutol, or more rarely to carbon monoxide, methanol and cyanide are probably all related forms of acquired mitochondrial dysfunction.Biochemical and cellular studies in LHON point to a partial defect of respiratory chain function that may generate either an ATP synthesis defect and/or a chronic increase of oxidative stress. Histopathological studies in LHON cases and a rat model mimicking CEON revealed a selective loss of retinal ganglion cells (RGCs) and the corresponding axons, particularly in the temporal-central part of the optic nerve. Anatomical peculiarities of optic nerve axons, such as the asymmetric pattern of myelination, may have functional implications on energy dependence and distribution of mitochondrial populations in the different sections of the nerve. Histological evidence suggests impaired axonal transport of mitochondria in LHON and in the CEON-like rat model, indicating a possible common pathophysiology for this category of optic neuropathies. Histological evidence of myelin pathology in LHON also suggests a role for oxidative stress, possibly affecting the oligodendrocytes of the optic nerves.     

Early downregulation of IGF-I decides the fate of rat retinal ganglion cells after optic nerve injury

Retinal ganglion cells (RGCs) die by apoptosis after optic nerve injury. A number of reports have separately shown changes in pro-apoptotic proteins such as the Bcl-2 family members following optic nerve injury. However, induction time of these apoptotic signals has not been identified due to different treatments of the optic nerve, and insufficient time intervals for measurements. Therefore, the stream of cell death signals is not well understood. In the present study, we systematically reinvestigated a detailed time course of these cell death/survival signals in the rat retina after optic nerve crush, to determine the signal cascade leading to RGC apoptosis. The most conspicuous changes detected in the retina were the rapid inactivation of phospho-Akt and phospho-Bad proteins 2-3 days after optic nerve damage, and the subsequent gradual activation of Bax protein and caspase-3 activity accompanied by cell loss of RGCs 6 days after nerve injury. Cellular localization of these molecular changes was limited to RGCs. Furthermore, amount of insulin-like growth factor-I (IGF-I), an activator of the phosphatidyl inositol-3-kinase (PI3K)/Akt system, was initially decreased from RGCs 1-2 days just prior to the inactivation of phospho-Akt by optic nerve crush. Conversely, supplementation with IGF-I into the rat retina induced upregulation of phospho-Akt expression and cell survival of RGCs both in vitro and in vivo. Thus, injury to the optic nerve might induce early changes in cellular homeostasis with a plausible loss of trophic support for injured RGCs. Actually, IGF-I drastically enhanced neurite outgrowth from adult rat RGCs via a wortmannin-dependent mechanism in a retinal explant culture. Our data strongly indicate that IGF-I is a key molecule that induces RGC apoptosis or RGC survival and regeneration in the retina during the early stage of optic nerve injury.     

Activation of autophagy induces retinal ganglion cell death in a chronic hypertensive glaucoma model

Autophagy is reported to have important roles in relation to regulated cell death pathways and neurodegeneration. This study used chronic hypertensive glaucoma rat model to investigate whether the autophagy pathway has a role in the apoptosis of retinal ganglion cells (RGCs) after chronic intraocular pressure (IOP) elevation. Under electron microscopy, autophagosomes were markedly accumulated in the dendrites and cytoplasm of RGCs after IOP elevation. Western blot analysis showed that LC3-II/LC3-I and beclin-1 were upregulated throughout the 8-weeks period after IOP elevation. The pattern of LC3 immunostaining showed autophagy activation in the cytoplasm of RGCs to increase and peak at 4 weeks after IOP elevation. Most of these LC3B-positive RGCs underwent apoptosis by terminal deoxynucleotidyltransferase-mediated biotinylated UTP nick end labeling, and inhibition of autophagy with 3-methyladenine decreased RGC apoptosis. The activated pattern shows that autophagy is initially activated in the dendrites of the RGCs, but, thereafter autophagy is mainly activated in the cytoplasm of RGCs. This may show that autophagy is differently regulated in different compartments of the neuron. This present study showed that autophgy is activated in RGCs and has a role in autophagic cell death after chronic IOP elevation.     

Endothelin B receptors contribute to retinal ganglion cell loss in a rat model of glaucoma

Glaucoma is an optic neuropathy, commonly associated with elevated intraocular pressure (IOP) characterized by optic nerve degeneration, cupping of the optic disc, and loss of retinal ganglion cells which could lead to loss of vision. Endothelin-1 (ET-1) is a 21-amino acid vasoactive peptide that plays a key role in the pathogenesis of glaucoma; however, the receptors mediating these effects have not been defined. In the current study, endothelin B (ET(B)) receptor expression was assessed in vivo, in the Morrison's ocular hypertension model of glaucoma in rats. Elevation of IOP in Brown Norway rats produced increased expression of ET(B) receptors in the retina, mainly in retinal ganglion cells (RGCs), nerve fiber layer (NFL), and also in the inner plexiform layer (IPL) and inner nuclear layer (INL). To determine the role of ET(B) receptors in neurodegeneration, Wistar-Kyoto wild type (WT) and ET(B) receptor-deficient (KO) rats were subjected to retrograde labeling with Fluoro-Gold (FG), following which IOP was elevated in one eye while the contralateral eye served as control. IOP elevation for 4 weeks in WT rats caused an appreciable loss of RGCs, which was significantly attenuated in KO rats. In addition, degenerative changes in the optic nerve were greatly reduced in KO rats compared to those in WT rats. Taken together, elevated intraocular pressure mediated increase in ET(B) receptor expression and its activation may contribute to a decrease in RGC survival as seen in glaucoma. These findings raise the possibility of using endothelin receptor antagonists as neuroprotective agents for the treatment of glaucoma.     

Differential effects of unfolded protein response pathways on axon injury-induced death of retinal ganglion cells

Loss of retinal ganglion cells (RGCs) accounts for visual function deficits after optic nerve injury, but how axonal insults lead to neuronal death remains elusive. By using an optic nerve crush model that results in the death of the majority of RGCs, we demonstrate that axotomy induces differential activation of distinct pathways of the unfolded protein response in axotomized RGCs. Optic nerve injury provokes a sustained CCAAT/enhancer binding homologous protein (CHOP) upregulation, and deletion of CHOP promotes RGC survival. In contrast, IRE/XBP-1 is only transiently activated, and forced XBP-1 activation dramatically protects RGCs from axon injury-induced death. Importantly, such differential activations of CHOP and XBP-1 and their distinct effects on neuronal cell death are also observed in RGCs with other types of axonal insults, such as vincristine treatment and intraocular pressure elevation, suggesting a new protective strategy for neurodegeneration associated with axonal damage.     

Nitric oxide-cGMP signaling regulates axonal elongation during optic nerve regeneration in the goldfish in vitro and in vivo

Nitric oxide (NO) signaling results in both neurotoxic and neuroprotective effects in CNS and PNS neurons, respectively, after nerve lesioning. We investigated the role of NO signaling on optic nerve regeneration in the goldfish ( Carassius auratus ). NADPH diaphorase staining revealed that nitric oxide synthase (NOS) activity was up-regulated primarily in the retinal ganglion cells (RGCs) 5–40 days after axotomy. Levels of neuronal NOS (nNOS) mRNA and protein also increased in the RGCs alone during this period. This period (5–40 days) overlapped with the process of axonal elongation during regeneration of the goldfish optic nerve. Therefore, we evaluated the effect of NO signaling molecules upon neurite outgrowth from adult goldfish axotomized RGCs in culture. NO donors and dibutyryl cGMP increased neurite outgrowth dose-dependently. In contrast, a nNOS inhibitor and small interfering RNA, specific for the nNOS gene, suppressed neurite outgrowth from the injured RGCs. Intra-ocular dibutyryl cGMP promoted the axonal regeneration from injured RGCs in vivo . None of these molecules had an effect on cell death/survival in this culture system. This is the first report showing that NO-cGMP signaling pathway through nNOS activation is involved in neuroregeneration in fish CNS neurons after nerve lesioning.     

Near complete loss of retinal ganglion cells in the math5/brn3b double knockout elicits severe reductions of other cell types during retinal development

Retinal ganglion cells (RGCs) are the first cell type to differentiate during retinal histogenesis. It has been postulated that specified RGCs subsequently influence the number and fate of the remaining progenitors to produce the rest of the retinal cell types. However, several genetic knockout models have argued against this developmental role for RGCs. Although it is known that RGCs secrete cellular factors implicated in cell proliferation, survival, and differentiation, until now, limited publications have shown that reductions in the RGC number cause significant changes in these processes. In this study, we observed that Math5 and Brn3b double null mice exhibited over a 99% reduction in the number of RGCs during development. This severe reduction of RGCs is accompanied by a drastic loss in the number of all other retinal cell types that was never seen before. Unlike Brn3b null or Math5 null animals, mice null for both alleles lack an optic nerve and have severe retinal dysfunction. Results of this study support the hypothesis that RGCs play a pivotal role in the late phase of mammalian retina development.     

Cytochrome <Emphasis Type="Italic">c</Emphasis> Release and Caspase-3 Activation in Retinal Ganglion Cells Following Different Distance of Axotomy of the Optic Nerve in Adult Hamsters

This study examined the relationship between the distance of axotomy and the death of injured retinal ganglion cells (RGCs) in adult hamsters and the relationship of cytochrome c and caspase-3 on the death pathway of RGCs. The left optic nerve (ON) of adult hamsters was transected either at 1 or 3 mm away from the optic disc, and retrogradely labeled with Flurogold on the ON stump. After a predetermined period of postoperative time, the surviving RGCs were counted by retina flat-mount, and the activation of cytochrome c and caspase-3 were investigated by immunohistochemistry. Cell loss was found to be much faster (P < 0.01), more cells with cytochrome c were observed (P < 0.05) and the activation of caspase-3 was earlier when ON was transected 1 mm away from the optic disc than when was transected 3 mm away from the optic disc. Distance of axotomy affects the axotomized cell death rate where more RGCs died when the ON transection was applied closer to the eye. The timing of activation of caspase-3 in the RGCs may be linked to the distance of axotomy.Special issue dedicated to Dr. Lawrence F. Eng     

A new vicious cycle involving glutamate excitotoxicity, oxidative stress and mitochondrial dynamics

Glutamate excitotoxicity leads to fragmented mitochondria in neurodegenerative diseases, mediated by nitric oxide and S-nitrosylation of dynamin-related protein 1, a mitochondrial outer membrane fission protein. Optic atrophy gene 1 (OPA1) is an inner membrane protein important for mitochondrial fusion. Autosomal dominant optic atrophy (ADOA), caused by mutations in OPA1, is a neurodegenerative disease affecting mainly retinal ganglion cells (RGCs). Here, we showed that OPA1 deficiency in an ADOA model influences N-methyl-D-aspartate (NMDA) receptor expression, which is involved in glutamate excitotoxicity and oxidative stress. Opa1^enu/+ mice show a slow progressive loss of RGCs, activation of astroglia and microglia, and pronounced mitochondrial fission in optic nerve heads as found by electron tomography. Expression of NMDA receptors (NR1, 2A, and 2B) in the retina of Opa1^enu/+ mice was significantly increased as determined by western blot and immunohistochemistry. Superoxide dismutase 2 (SOD2) expression was significantly decreased, the apoptotic pathway was activated as Bax was increased, and phosphorylated Bad and BcL-xL were decreased. Our results conclusively demonstrate that not only glutamate excitotoxicity and/or oxidative stress alters mitochondrial fission/fusion, but that an imbalance in mitochondrial fission/fusion in turn leads to NMDA receptor upregulation and oxidative stress. Therefore, we propose a new vicious cycle involved in neurodegeneration that includes glutamate excitotoxicity, oxidative stress, and mitochondrial dynamics.     

Differential expression of calretinin in the developing and regenerating zebrafish visual system

Calretinin is a calcium-binding protein which participates in a variety of functions including calcium buffering and neuronal protection. It also serves as a developmental marker of retinal ganglion cells (RGCs). In order to study the role of calretinin in the development and regeneration of RGCs, we have studied its pattern of expression in the retina at different developmental stages, as well as during optic nerve regeneration by means of immunohistochemistry. During development, calretinin is found for the first time in RGCs when they connect with the optic tectum. Optic nerves from adult zebrafish were crushed and after different survival times, calretinin expression in the retina, optic nerve tract and optic tectum was studied. From the day of crushing to 10 days later, calretinin expression was found to be downregulated within RGCs and their axons, as was also observed during the early developmental stages of RGCs, when they are not committed to a definite cell phenotype. Moreover, 13 days after lesion, when the regenerating axons arrived at the optic tectum, a recovery of calretinin immunoreactivity within the RGCs was observed. These results indicate that calretinin may play an important role during optic nerve regeneration, Thus, the down-regulation of Calretinin during the growth of the RGC axons towards the target during development as well as during their regeneration after injury, indicates that an increase the availability of cytosolic calcium is integral to axon outgrowth thus recapitulating the pattern observed during development.