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.     

A distinct effect of transient and sustained upregulation of cellular factor XIII in the goldfish retina and optic nerve on optic nerve regeneration

Unlike in mammals, fish retinal ganglion cells (RGCs) have a capacity to repair their axons even after optic nerve transection. In our previous study, we isolated a tissue type transglutaminase (TG) from axotomized goldfish retina. The levels of retinal TG (TG(R)) mRNA increased in RGCs 1-6weeks after nerve injury to promote optic nerve regeneration both in vitro and in vivo. In the present study, we screened other types of TG using specific FITC-labeled substrate peptides to elucidate the implications for optic nerve regeneration. This screening showed that the activity of only cellular coagulation factor XIII (cFXIII) was increased in goldfish optic nerves just after nerve injury. We therefore cloned a full-length cDNA clone of FXIII A subunit (FXIII-A) and studied temporal changes of FXIII-A expression in goldfish optic nerve and retina during regeneration. FXIII-A mRNA was initially detected at the crush site of the optic nerve 1h after injury; it was further observed in the optic nerve and achieved sustained long-term expression (1-40days after nerve injury). The cells producing FXIII-A were astrocytes/microglial cells in the optic nerve. By contrast, the expression of FXIII-A mRNA and protein was upregulated in RGCs for a shorter time (3-10days after nerve injury). Overexpression of FXIII-A in RGCs achieved by lipofection induced significant neurite outgrowth from unprimed retina, but not from primed retina with pretreatment of nerve injury. Addition of extracts of optic nerves with injury induced significant neurite outgrowth from primed retina, but not from unprimed retina without pretreatment of nerve injury. The transient increase of cFXIII in RGCs promotes neurite sprouting from injured RGCs, whereas the sustained increase of cFXIII in optic nerves facilitates neurite elongation from regrowing axons.     

Wortmannin Blocks Goldfish Retinal Phosphatidylinositol 3-Kinase and Neurite Outgrowth

The goldfish retina has been used extensively for the study of nerve regeneration. A role for phosphatidylinositol 3-kinase (PI3K) in neurite outgrowth from goldfish retinal explants has been examined by means of wortmannin (WT), a selective inhibitor of the enzyme. The presence of PI3K in retinal extracts was determined by means of immunoprecipitation as well as by an in vitro assay system for catalytic activity. The relative amount of the p85 subunit of PI3K detected by western blot in the retina following optic nerve crush was unchanged. WT inhibited goldfish brain PI3K activity at concentrations as low as 10^–9 M, approximating that reported for inhibition of mammalian PI3K's. Daily addition of 10^–8 M WT to retinal explants, activated by prior crush of the optic nerve, significantly inhibited neurite outgrowth during a 7 day in vitro culture period, while a single addition of WT to freshly explanted retina had no effect on neurite outgrowth. These results suggest that a PI3K-mediated process may be critical for nerve regrowth.     

Serotonin transporter-mediated molecular axis regulates regional retinal ganglion cell vulnerability and axon regeneration after nerve injury

Molecular insights into the selective vulnerability of retinal ganglion cells (RGCs) in optic neuropathies and after ocular trauma can lead to the development of novel therapeutic strategies aimed at preserving RGCs. However, little is known about what molecular contexts determine RGC susceptibility. In this study, we show the molecular mechanisms underlying the regional differential vulnerability of RGCs after optic nerve injury. We identified RGCs in the mouse peripheral ventrotemporal (VT) retina as the earliest population of RGCs susceptible to optic nerve injury. Mechanistically, the serotonin transporter (SERT) is upregulated on VT axons after injury. Utilizing SERT-deficient mice, loss of SERT attenuated VT RGC death and led to robust retinal axon regeneration. Integrin β3, a factor mediating SERT-induced functions in other systems, is also upregulated in RGCs and axons after injury, and loss of integrin β3 led to VT RGC protection and axon regeneration. Finally, RNA sequencing analyses revealed that loss of SERT significantly altered molecular signatures in the VT retina after optic nerve injury, including expression of the transmembrane protein, Gpnmb. GPNMB is rapidly downregulated in wild-type, but not SERT- or integrin β3-deficient VT RGCs after injury, and maintaining expression of GPNMB in RGCs via AAV2 viruses even after injury promoted VT RGC survival and axon regeneration. Taken together, our findings demonstrate that the SERT-integrin β3-GPNMB molecular axis mediates selective RGC vulnerability and axon regeneration after optic nerve injury.     

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.     

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.     

Na,K-ATPase alpha3 subunit in the goldfish retina during optic nerve regeneration

The goldfish optic nerve can regenerate after injury. To understand the molecular mechanism of optic nerve regrowth, we identified genes whose expression is specifically up-regulated during the early stage of optic nerve regeneration. A cDNA library constructed from goldfish retina 5 days after transection was screened by differential hybridization with cDNA probes derived from axotomized or normal retina. Of six cDNA clones isolated, one clone was identified as the Na,K-ATPase catalytic subunit alpha3 isoform by high- sequence homology. In northern hybridization, the expression level of the mRNA was significantly increased at 2 days and peaked at 5-10 days, and then gradually decreased and returned to control level by 45 days after optic nerve transection. Both in situ hybridization and immunohistochemical staining have revealed the location of this transient retinal change after optic nerve transection. The increased expression was observed only in the ganglion cell layer and optic nerve fiber layer at 5-20 days after optic nerve transection. In an explant culture system, neurite outgrowth from the retina 7 days after optic nerve transection was spontaneously promoted. A low concentration of ouabain (50-100 nm ) completely blocked the spontaneous neurite outgrowth from the lesioned retina. Together, these data indicate that up-regulation of the Na,K-ATPase alpha3 subunit is involved in the regrowth of ganglion cell axons after axotomy.     

Quantitative iTRAQ analysis of retinal ganglion cell degeneration after optic nerve crush

Retinal ganglion cells (RGCs) are central nervous system (CNS) neurons that transmit visual information from the retina to the brain. Apoptotic RGC degeneration causes visual impairment that can be modeled by optic nerve crush. Neuronal apoptosis is also a salient feature of CNS trauma, ischemia (stroke), and diseases of the CNS such as Alzheimer's, Parkinson's, multiple sclerosis, and amyotrophic lateral sclerosis. Optic nerve crush induces the apoptotic cell death of ～ 70% of RGCs within the first 14 days after injury. This model is particularly attractive for studying adult neuron apoptosis because the time-course of RGC death is well established and axon regeneration within the myelinated optic nerve can be concurrently evaluated. Here, we performed a large scale iTRAQ proteomic study to identify and quantify proteins of the rat retina at 1, 3, 4, 7, 14, and 21 days after optic nerve crush. In total, 337 proteins were identified, and 110 were differentially regulated after injury. Of these, 58 proteins were upregulated (>1.3 ×), 46 were downregulated (<0.7 ×), and 6 showed both positive and negative regulation over 21 days, relative to normal retinas. Among the differentially expressed proteins, Thymosin-β4 showed an early upregulation at 3 days, the time-point that immediately precedes the induction of RGC apoptosis after injury. We examined the effect of exogenous Thymosin-β4 administration on RGC death after optic nerve injury. Intraocular injections of Thymosin-β4 significantly increased RGC survival by ～ 3-fold compared to controls and enhanced axon regeneration after crush, demonstrating therapeutic potential for CNS insults. Overall, our study identified numerous proteins that are differentially regulated at key time-points after optic nerve crush, and how the temporal profiles of their expression parallel RGC death. This data will aid in the future development of novel therapeutics to promote neuronal survival and regeneration in the adult CNS.     

Enhanced Survival of Melanopsin-expressing Retinal Ganglion Cells After Injury is Associated with the PI3 K/Akt Pathway

In the present study, we studied the factors that contribute to the injury-resistant property of melanopsin-expressing retinal ganglion cells (mRGCs). Since phosphatidylinositol-3 kinase (PI3 K)/Akt signaling pathway is one of the well-known pathways for neuronal cell survival, we investigated the survival of mRGCs by applying the PI3 K/Akt specific inhibitors after injury. Two injury models, unilateral optic nerve transection and ocular hypertension, were adopted using Sprague-Dawley rats. Inhibitors of PI3 K/Akt were injected intravitreally following injuries to inhibit the PI3 K/Akt signaling pathway. Retinas were dissected after designated survival time, immunohistochemistry was carried out to visualize the mRGCs using melanopsin antibody and the number of mRGCs was counted. Co-expression of melanopsin and phospho-Akt (pAkt) was also examined. Compared to the survival of non-melanopsin-expressing RGCs, mRGCs showed a marked resistance to injury and co-expressed pAkt. Application of PI3 K/Akt inhibitors decreased the survival of mRGCs after injury. Our previous study has shown that mRGC are less susceptible to injury following the induction of ocular hypertension. In this study, we report that mRGCs were injury-resistant to a more severe type of injury, the optic nerve transection. More importantly, the PI3 K/Akt pathway was found to play a role in maintaining the survival of mRGCs after injury.     

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.     

Requirement of Retinoic Acid Receptor β for Genipin Derivative-Induced Optic Nerve Regeneration in Adult Rat Retina

Like other CNS neurons, mature retinal ganglion cells (RGCs) are unable to regenerate their axons after nerve injury due to a diminished intrinsic regenerative capacity. One of the reasons why they lose the capacity for axon regeneration seems to be associated with a dramatic shift in RGCs’ program of gene expression by epigenetic modulation. We recently reported that (1R)-isoPropyloxygenipin (IPRG001), a genipin derivative, has both neuroprotective and neurite outgrowth activities in murine RGC-5 retinal precursor cells. These effects were both mediated by nitric oxide (NO)/S-nitrosylation signaling. Neuritogenic activity was mediated by S-nitrosylation of histone deacetylase-2 (HDAC2), which subsequently induced retinoic acid receptor β (RARβ) expression via chromatin remodeling in vitro. RARβ plays important roles of neural growth and differentiation in development. However, the role of RARβ expression during adult rat optic nerve regeneration is not clear. In the present study, we extended this hypothesis to examine optic nerve regeneration by IPRG001 in adult rat RGCs in vivo. We found a correlation between RARβ expression and neurite outgrowth with age in the developing rat retina. Moreover, we found that IPRG001 significantly induced RARβ expression in adult rat RGCs through the S-nitrosylation of HDAC2 processing mechanism. Concomitant with RARβ expression, adult rat RGCs displayed a regenerative capacity for optic axons in vivo by IPRG001 treatment. These neuritogenic effects of IPRG001 were specifically suppressed by siRNA for RARβ. Thus, the dual neuroprotective and neuritogenic actions of genipin via S-nitrosylation might offer a powerful therapeutic tool for the treatment of RGC degenerative disorders.     

Hepatocyte growth factor protects retinal ganglion cells by increasing neuronal survival and axonal regeneration in vitro and in vivo

Hepatocyte growth factor (HGF) is known to promote the survival and foster neuritic outgrowth of different subpopulations of CNS neurons during development. Together with its corresponding receptor c-mesenchymal-epithelial transition factor (Met), it is expressed in the developing and the adult murine, rat and human CNS. We have studied the role of HGF in paradigms of retinal ganglion cell (RGC) regeneration and cell death in vitro and in vivo. After application of recombinant HGF in vitro, survival of serum-deprived RGC-5 cells and of growth factor-deprived primary RGC was significantly increased. This was shown to be correlated to the phosphorylation of c-Met and subsequent activation of serine/threonine protein kinase Akt and MAPK downstream signalling pathways involved in neuronal survival. Furthermore, neurite outgrowth of primary RGC was stimulated by HGF. In vivo, c-Met expression in RGC was up-regulated after optic nerve axotomy lesion. Here, treatment with HGF significantly improved survival of axotomized RGC and enhanced axonal regeneration after optic nerve crush. Our data demonstrates that exogenously applied HGF has a neuroprotective and regeneration-promoting function for lesioned CNS neurons. We provide strong evidence that HGF may represent a trophic factor for adult CNS neurons, which may play a role as therapeutic target in the treatment of neurotraumatic and neurodegenerative CNS disorders.     

Cyclic AMP and the regeneration of retinal ganglion cell axons

In this paper we present a brief review of studies that have reported therapeutic benefits of elevated cAMP on plasticity and regeneration after injury to the central nervous system (CNS). We also provide new data on the cellular mechanisms by which elevation of cyclic adenosine monophosphate (cAMP) promotes cytokine driven regeneration of adult CNS axons, using the visual system as the experimental model. cAMP is a second messenger for many intracellular signalling pathways. Elevation of cAMP in the eye by intravitreal injection of the cell permeant analogue (8-(4-chlorophenylthio)-adenosine-3′,5′-cyclic monophosphate; CPT-cAMP), when added to recombinant ciliary neurotrophic factor (rCNTF), significantly enhances rCNTF-induced regeneration of adult rat retinal ganglion cell (RGC) axons into peripheral nerve (PN) grafted onto transected optic nerve. This effect is mediated to some extent by protein kinase A (PKA) signalling, but CPT-cAMP also acts via PI3K/Akt signalling to reduce suppressor of cytokine signalling protein 3 (SOCS3) activity in RGCs. Another target for cAMP is the exchange protein activated by cAMP (Epac), which can also mediate cAMP-induced axonal growth. Here we describe some novel results and discuss to what extent the pro-regenerative effects of CPT-cAMP on adult RGCs are mediated via Epac as well as via PKA-dependent pathways. We used the established PN–optic nerve graft model and quantified the survival and regenerative growth of adult rat RGCs after intravitreal injection of rCNTF in combination with a selective activator of PKA and/or a specific activator of Epac. Viable RGCs were identified by βIII-tubulin immunohistochemistry and regenerating RGCs retrogradely labelled and quantified after an injection of fluorogold into the distal end of the PN grafts, 4 weeks post-transplantation. The specific agonists of either PKA or Epac were both effective in enhancing the effects of rCNTF on RGC axonal regeneration, but interestingly, injections that combined rCNTF with both agonists were significantly less effective. The results are discussed in relation to previous CPT-cAMP studies on RGCs, and we also consider the need to modulate cAMP levels in order to obtain the most functionally effective regenerative response after CNS trauma.This article is part of a directed issue entitled: Regenerative Medicine: the challenge of translation.     

Neuroprotective effects and intracellular signaling pathways of erythropoietin in a rat model of multiple sclerosis

In multiple sclerosis (MS), long-term disability is primarily caused by axonal and neuronal damage. We demonstrated in a previous study that neuronal apoptosis occurs early during experimental autoimmune encephalomyelitis, a common animal model of MS. In the present study, we show that, in rats suffering from myelin oligodendrocyte glycoprotein (MOG)-induced optic neuritis, systemic application of erythropoietin (Epo) significantly increased survival and function of retinal ganglion cells (RGCs), the neurons that form the axons of the optic nerve. We identified three independent intracellular signaling pathways involved in Epo-induced neuroprotection in vivo: Protein levels of phospho-Akt, phospho-MAPK 1 and 2, and Bcl-2 were increased under Epo application. Using a combined treatment of Epo together with a selective inhibitor of phosphatidylinositol 3-kinase (PI3-K) prevented upregulation of phospho-Akt and consecutive RGC rescue. We conclude that in MOG-EAE the PI3-K/Akt pathway has an important influence on RGC survival under systemic treatment with Epo.     

Neuronal Intermediate Filament Expression During Neurite Outgrowth from Explanted Goldfish Retina: Effect of Retinoic Acid

Regulation of the goldfish neuronal intermediate filament proteins ON1 and ON2 was investigated in a retinal explant system. The synthesis of these proteins in explanted retina decreased with increasing time in culture, despite continuing neurite outgrowth. Thus, ON1/ON2 neurofilament expression is regulated independently from neurite outgrowth. During regeneration of the goldfish optic nerve in vivo, the expression of these proteins increased during the later phase of the process, when growing axons make contact with the optic tectum. The declining synthesis of ON1 and ON2 during neurite outgrowth in culture suggests that factors extrinsic to the retina are necessary to support synthesis of these proteins. Treating retinal explants with retinoic acid stimulated the synthesis of the ON1/ON2 proteins in a dose-dependent manner. This stimulation was effective during a period of declining synthesis of the ON1/ON2 proteins, restoring their synthesis towards initial levels of expression. These results show that retinoic acid serves as a modulator of neurofilament expression in this in vitro model of nerve regeneration.     

Neuronal Injury External to the Retina Rapidly Activates Retinal Glia,Followed by Elevation of Markers for Cell Cycle Re-Entry and Death in Retinal Ganglion Cells

Retinal ganglion cells (RGCs) are neurons that relay visual signals from the retina to the brain. The RGC cell bodies reside in the retina and their fibers form the optic nerve. Full transection (axotomy) of the optic nerve is an extra-retinal injury model of RGC degeneration. Optic nerve transection permits time-kinetic studies of neurodegenerative mechanisms in neurons and resident glia of the retina, the early events of which are reported here. One day after injury, and before atrophy of RGC cell bodies was apparent, glia had increased levels of phospho-Akt, phospho-S6, and phospho-ERK1/2; however, these signals were not detected in injured RGCs. Three days after injury there were increased levels of phospho-Rb and cyclin A proteins detected in RGCs, whereas these signals were not detected in glia. DNA hyperploidy was also detected in RGCs, indicative of cell cycle re-entry by these post-mitotic neurons. These events culminated in RGC death, which is delayed by pharmacological inhibition of the MAPK/ERK pathway. Our data show that a remote injury to RGC axons rapidly conveys a signal that activates retinal glia, followed by RGC cell cycle re-entry, DNA hyperploidy, and neuronal death that is delayed by preventing glial MAPK/ERK activation. These results demonstrate that complex and variable neuro-glia interactions regulate healthy and injured states in the adult mammalian retina.     

Hydrogen exerts neuroprotection by activation of the miR-21/PI3K/AKT/GSK-3β pathway in an in vitro model of traumatic brain injury

Few studies have explored the effect of hydrogen on neuronal apoptosis or impaired nerve regeneration after traumatic brain injury, and the mechanisms involved in these processes are unclear. In this study, we explored neuroprotection of hydrogen-rich medium through activation of the miR-21/PI3K/AKT/GSK-3β pathway in an in vitro model of traumatic brain injury. Such model adopted PC12 cells with manual scratching. Then, injured cells were cultured in hydrogen-rich medium for 48 hours. Expression of miR-21, p-PI3K, p-Akt, p-GSK-3β, Bax and Bcl-2 was measured using RT-qPCR, Western blot analysis and immunofluorescence staining. Rate of apoptosis was determined using TUNEL staining. Neuronal regeneration was assessed using immunofluorescence staining. The results showed that hydrogen-rich medium improved neurite regeneration and inhibited apoptosis in the injured cells. Scratch injury was accompanied by up-regulation of miR-21, p-PI3K, p-Akt and p-GSK-3β. A miR-21 antagomir inhibited the expression of these four molecules, while a PI3K blocker only affected the three proteins and not miR-21. Both the miR-21 antagomir and PI3K blocker reversed the protective effect of hydrogen. In conclusion, hydrogen exerted a neuroprotective effect against neuronal apoptosis and impaired nerve regeneration through activation of miR-21/PI3K/AKT/GSK-3β signalling in this in vitro model of traumatic brain injury.