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Retinal ganglion cells (RGCs), neurons transmitting visual information via the optic nerve, fail to regenerate their axons after injury. The progressive loss of RGC function underlies the pathophysiology of glaucoma and other optic neuropathies, often leading to irreversible blindness. Therefore, there is an urgent need to identify the regulators of RGC survival and the regenerative program. In this study, we investigated the role of the family of transcription factors known as nuclear factor of activated T cells (NFAT), which are expressed in the retina; however, their role in RGC survival after injury is unknown. Using the optic nerve crush (ONC) model, widely employed to study optic neuropathies and central nervous system axon injury, we found that NFATc4 is specifically but transiently up-regulated in response to mechanical injury. In the injured retina, NFATc4 immunolocalized primarily to the ganglionic cell layer. Utilizing NFATc4 -/- and NFATc3 -/- mice, we demonstrated that NFATc4, but not NFATc3, knockout increased RGC survival, improved retina function, and delayed axonal degeneration. Microarray screening data, along with decreased immunostaining of cleaved caspase-3, revealed that NFATc4 knockout was protective against ONC-induced degeneration by suppressing pro-apoptotic signaling. Finally, we used lentiviral-mediated NFATc4 delivery to the retina of NFATc4 -/- mice and reversed the pro-survival effect of NFATc4 knockout, conclusively linking the enhanced survival of injured RGCs to NFATc4-dependent mechanisms. In summary, this study is the first to demonstrate that NFATc4 knockout may confer transient RGC neuroprotection and decelerate axonal degeneration after injury, providing a potent therapeutic strategy for optic neuropathies., (© 2024. The Author(s).)

As the primary connection between the eye and brain, the optic nerve plays a pivotal role in visual information transmission. Injuries to the optic nerve can occur for various reasons, including trauma, glaucoma, and neurodegenerative diseases. Retinal ganglion cells (RGCs), a type of neurons that extend axons through the optic nerve, can rapidly respond to injury and initiate cell death. Additionally, following optic nerve injury microglia, which serve as markers of neuroinflammation, transition from a resting state to an activated state. The phosphorylation of collapsin response mediator protein2 (CRMP2) in the semaphorin 3A (Sema3A) signalling pathway affects several processes, including axon guidance and neuron regeneration. In this study, we used an optic nerve crush (ONC) mouse model to investigate the effects of suppressing CRMP2 phosphorylation on microglia activation. We found that CRMP2 phosphorylation inhibitor suppressed RGCs loss and promoted neuronal regeneration following ONC. In addition, CRMP2 S522A mutant (CRMP2 KI) mice exhibited decreased microglial activation in both the retina and optic nerve following ONC. These results suggest that inhibiting the phosphorylation of CRMP2 can alleviate the loss of RGCs and microglial activation after optic nerve injury, providing insight into the development of treatments for optical neuropathies and neurodegenerative diseases., (© 2024. The Author(s).)

Background: Activated microglia release harmful substances to retinal ganglion cells (RGCs), but may also benefit by removing cellular debris and secreting neurotrophic factors. These paradoxical roles remain controversial because the nature and time-course of the injury that defines their role is unknown. The aim of this study was to determine if pharmacological manipulation of microglia to acquire a pro-inflammatory or pro-survival phenotype will exacerbate or enhance neuronal survival after injury. Material and methods: Treated HAP I (highly aggressively proliferating immortalized) microglia were injected into the vitreous or tail vein (T V) of female Sprague-Dawley rats. Retinas were examined at 4-14 days following optic nerve crush (ONC) and the number of surviving RGCs was determined. Results: Injection of untreated HAP I cells resulted in the greater loss of RGCs early after ONC when injected into the vitreous and later after ONC when injected into the T V. LP S activated HAP I cells injected into the vitreous resulted in greater RGC loss with and without injury. When injected into the T V with ONC there was no loss of RGCs 4 days after ONC but greater loss afterwards. Minocycline treated HAP I cells injected into the vitreous resulted in greater RGC survival than untreated HAP I cells. However, when injected into the T V with ONC there was greater loss of RGCs. These results suggest that optic nerve signals attract extrinsic microglia to the retina, resulting in a proinflammatory response. Conclusion: Neuroprotection or cytotoxicity of microglia depends on the type of activation, time course of the injury, and if they act on the axon or cell body.

Current clinical treatments have not yet effectively cured progressive retinal ganglion cell (RGC) death and axonal degeneration after optic nerve (ON) injury. We previously demonstrated green tea extract (GTE) can reduce RGC death in rats after ischemic injury. Here, we aim to determine the prophylactic and therapeutic effects and mechanisms of GTE on RGC survival and axonal regeneration in rats with ON injury. GTE (275 or 550 mg/kg) was administered intragastrically for 7 d before or 14 d post-ON crush surgery in adult Fischer 344 rats. Rats with pre- or post-operative treatment of 275 mg/kg GTE showed significantly higher numbers of RGCs and regenerated axons post-ON injury with improved pupillary light reflex as compared to saline-treated rats. Akt and Erk p42/44 activation was higher in the retina of rats given 275 mg/kg GTE pre-surgery, whereas Stat3 activation was higher in those with 275 mg/kg GTE post-operation. Less activated microglia were observed in rats with pre-treatment of 275 or 550 mg/kg GTE. RNA sequencing analysis identified the downregulation of inflammation, apoptosis, and microglia activation genes in the retina of rats with pre- or post-treatment with 275 mg/kg GTE as compared to the saline-treated rats. In summary, this study revealed the prophylactic and therapeutic treatment effects of GTE on RGC survival and axonal regeneration in rats with ON injury, indicating a potential alternative treatment for traumatic optic neuropathy., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Collapsin response mediator proteins (Crmps) play roles in neuronal development and axon growth. However, neuronal-specific roles of Crmp1, Crmp4, and Crmp5 in regeneration of injured central nervous system (CNS) axons in vivo are unclear. Here, we analyzed developmental and subtype-specific expression of Crmp genes in retinal ganglion cells (RGCs), tested whether overexpressing Crmp1, Crmp4, or Crmp5 in RGCs through localized intralocular AAV2 delivery promotes axon regeneration after optic nerve injury in vivo, and characterized developmental co-regulation of gene-concept networks associated with Crmps. We found that all Crmp genes are developmentally downregulated in RGCs during maturation. However, while Crmp1, Crmp2, and Crmp4 were expressed to a varying degree in most RGC subtypes, Crmp3 and Crmp5 were expressed only in a small subset of RGC subtypes. We then found that after optic nerve injury, Crmp1, Crmp4, and Crmp5 promote RGC axon regeneration to varying extents, with Crmp4 promoting the most axon regeneration and also localizing to axons. We also found that Crmp1 and Crmp4, but not Crmp5, promote RGC survival. Finally, we found that Crmp1, Crmp2, Crmp4, and Crmp5's ability to promote axon regeneration is associated with neurodevelopmental mechanisms, which control RGC's intrinsic axon growth capacity., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Introduction: Optic nerve injury is a leading cause of irreversible blindness worldwide. The retinal ganglion cells (RGCs) and their axons cannot be regenerated once damaged. Therefore, reducing RGC damage is crucial to prevent blindness. Accordingly, we aimed to investigate the potential influence of the gut microbiota on RGC survival, as well as the associated action mechanisms., Methods: We evaluated the effects of microbiota, specifically Bifidobacterium, on RGC. Optic nerve crush (ONC) was used as a model of optic nerve injury. Vancomycin and Bifidobacterium were orally administered to specific pathogen-free (SPF) mice., Results: Bifidobacterium promoted RGC survival and optic nerve regeneration. The administration of Bifidobacterium inhibited microglia activation but promoted Müller cell activation, which was accompanied by the downregulation of inflammatory cytokines and upregulation of neurotrophic factors and retinal ERK/Fos signaling pathway activation., Conclusions: Our study demonstrates that Bifidobacterium-induced changes in intestinal flora promote RGC survival. The protective effect of Bifidobacterium on RGC can be attributed to the inhibition of microglia activation and promotion of Müller cell activation and the secondary regulation of inflammatory and neurotrophic factors., (© 2023 The Authors. CNS Neuroscience & Therapeutics published by John Wiley & Sons Ltd.)

Activation of neurotrophic factor signaling is a promising therapy for neurodegeneration. However, the transient nature of ligand-dependent activation limits its effectiveness. In this study, we solved this problem by inventing a system that forces membrane localization of the intracellular domain of tropomyosin receptor kinase B (iTrkB), which results in constitutive activation without ligands. Our system overcomes the small size limitation of the genome packaging in adeno-associated virus (AAV) and allows high expression of the transgene. Using AAV-mediated gene therapy in the eyes, we demonstrate that iTrkB expression enhances neuroprotection in mouse models of glaucoma and stimulates robust axon regeneration after optic nerve injury. In addition, iTrkB expression in the retina was also effective in an optic tract transection model, in which the injury site is near the superior colliculus. Regenerating axons successfully formed pathways to their brain targets, resulting in partial recovery of visual behavior. Our system may also be applicable to other trophic factor signaling pathways and lead to a significant advance in the field of gene therapy for neurotrauma and neurodegenerative disorders, including glaucoma., Competing Interests: Declaration of interests A patent based on the results in this article was filed by the Tokyo Metropolitan Institute of Medical Science (K.N. and T.H. are co-inventors)., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

Increasing the level of cyclic adenosine 3, 5'-monophosphate is an important mechanism for axon outgrowth and recovery of central nervous system function. This study aimed to investigate the effects of papaverine, a non-specific phosphodiesterase inhibitor, on axon outgrowth of primary retinal ganglion cells from Sprague Dawley rats. Experiments were performed on primary retinal ganglion cells extracted from Sprague Dawley rat pups within 48-72 h of birth. At 24 h after seeding, immunofluorescence was used to identify and calculate the purity of retinal ganglion cells isolated by an improved two-step immunopanning method developed by author Sujia Ma. The effects of a range of papaverine concentrations on axon outgrowth of primary retinal ganglion cells cultures were observed by immunofluorescence and measured by ImageJ software at three different time points: 24, 48, and 72 h. The ability of papaverine to enable retinal ganglion cells to overcome the inhibitory effects of glial scar component chondroitin sulfate proteoglycans was examined using chondroitin sulfate proteoglycans-coated culture plates. Rp-adenosine 3',5'-cyclic monophosphorothioate triethylammonium salt, a blocking agent of cyclic adenosine 3, 5'-monophosphate, and dibutyryl cyclic adenosine 3, 5'-monophosphate, an analogue of cyclic adenosine 3, 5'-monophosphate, were used to explore the mechanism of papaverine in promoting retinal ganglion cells axon outgrowth. Our study shows 2 μg/mL papaverine concentration significantly promoted axon outgrowth in primary retinal ganglion cells and restored axon outgrowth of these cells on chondroitin sulfate proteoglycans. Axon outgrowth was blocked by Rp-adenosine 3',5'-cyclic monophosphorothioate triethylammonium salt and obviously promoted by dibutyryl cyclic adenosine 3, 5'-monophosphate. Our study is the first to describe the use of papaverine to promote axon outgrowth of retinal ganglion cells. These results may help to expand the application of papaverine, and they provide a cytological basis for papaverine in the treatment of optic nerve injury caused by glaucoma and other diseases., (Copyright © 2021 The Authors. Published by Elsevier Ltd.. All rights reserved.)

CNS neurons are generally incapable of regenerating their axons after injury due to several intrinsic and extrinsic factors, including the presence of axon growth inhibitory molecules. One such potent inhibitor of CNS axon regeneration is Reticulon (RTN) 4 or Nogo-A. Here, we focused on RTN3 as its contribution to CNS axon regeneration is currently unknown. We found that RTN3 expression correlated with an axon regenerative phenotype in dorsal root ganglion neurons (DRGN) after injury to the dorsal columns, a well-characterised model of spinal cord injury. Overexpression of RTN3 promoted disinhibited DRGN neurite outgrowth in vitro and dorsal column axon regeneration/sprouting and electrophysiological, sensory and locomotor functional recovery after injury in vivo. Knockdown of protrudin, however, ablated RTN3-enhanced neurite outgrowth/axon regeneration in vitro and in vivo. Moreover, overexpression of RTN3 in a second model of CNS injury, the optic nerve crush injury model, enhanced retinal ganglion cell (RGC) survival, disinhibited neurite outgrowth in vitro and survival and axon regeneration in vivo, an effect that was also dependent on protrudin. These results demonstrate that RTN3 enhances neurite outgrowth/axon regeneration in a protrudin-dependent manner after both spinal cord and optic nerve injury.

Optic neuropathies are leading causes of irreversible visual impairment and blindness, currently affecting more than 100 million people worldwide. Glaucoma is a group of optic neuropathies attributed to progressive degeneration of retinal ganglion cells (RGCs). We have previously demonstrated an increase in survival of RGCs by the activation of macrophages, whereas the inhibition of macrophages was involved in the alleviation on endotoxin-induced inflammation by antagonist of growth hormone-releasing hormone (GHRH). Herein, we hypothesized that GHRH receptor (GHRH-R) signaling could be involved in the survival of RGCs mediated by inflammation. We found the expression of GHRH-R in RGCs of adult rat retina. After optic nerve crush, subcutaneous application of GHRH agonist MR-409 or antagonist MIA-602 promoted the survival of RGCs. Both the GHRH agonist and antagonist increased the phosphorylation of Akt in the retina, but only agonist MR-409 promoted microglia activation in the retina. The antagonist MIA-602 reduced significantly the expression of inflammation-related genes Il1b , Il6 , and Tnf Moreover, agonist MR-409 further enhanced the promotion of RGC survival by lens injury or zymosan-induced macrophage activation, whereas antagonist MIA-602 attenuated the enhancement in RGC survival. Our findings reveal the protective effect of agonistic analogs of GHRH on RGCs in rats after optic nerve injury and its additive effect to macrophage activation, indicating a therapeutic potential of GHRH agonists for the protection of RGCs against optic neuropathies especially in glaucoma., Competing Interests: Competing interest statement: L.-P.C. and L.B. began collaborating on an upcoming research article after this article was accepted. Other authors declare no potential conflicts of interest.

We systematically reviewed published translational research on gene-based therapy for retinal ganglion cell (RGC) neuroprotection. A search was conducted on Entrez PubMed on 23 December 2020 using the keywords "gene therapy", "retinal ganglion cell" and "neuroprotection". The initial search yielded 82 relevant articles. After restricting publications to those with full text available and in the English language, and then curating for only original articles on gene-based therapy, the final yield was 18 relevant articles. From the 18 papers, 17 of the papers utilized an adeno-associated viral (AAV) vector for gene therapy encoding specific genes of interest. Specifically, six of the studies utilized an AAV vector encoding brain-derived neurotrophic factor (BDNF), two of the studies utilized an AAV vector encoding erythropoietin (EPO), the remaining 10 papers utilized AAV vectors encoding different genes and one microRNA study. Although the literature shows promising results in both in vivo and in vitro models, there is still a significant way to go before gene-based therapy for RGC neuroprotection can proceed to clinical trials. Namely, the models of injury in many of the studies were more acute in nature, unlike the more progressive and neurodegenerative pathophysiology of diseases, such as glaucoma. The regulation of gene expression is also highly unexplored despite the use of AAV vectors in the majority of the studies reviewed. It is also expected that with the successful launch of messenger ribonucleic acid (mRNA)-based vaccinations in 2020, we will see a shift towards this technology for gene-based therapy in glaucoma neuroprotection.

Background: Optic-nerve injury results in impaired transmission of visual signals to central targets and leads to the death of retinal ganglion cells (RGCs) and irreversible vision loss. Therapies with mesenchymal stem cells (MSCs) from different sources have been used experimentally to increase survival and regeneration of RGCs., Methods: We investigated the efficacy of human umbilical Wharton's jelly-derived MSCs (hWJ-MSCs) and their extracellular vesicles (EVs) in a rat model of optic nerve crush., Results: hWJ-MSCs had a sustained neuroprotective effect on RGCs for 14, 60, and 120 days after optic nerve crush. The same effect was obtained using serum-deprived hWJ-MSCs, whereas transplantation of EVs obtained from those cells was ineffective. Treatment with hWJ-MSCs also promoted axonal regeneration along the optic nerve and reinnervation of visual targets 120 days after crush., Conclusions: The observations showed that this treatment with human-derived MSCs promoted sustained neuroprotection and regeneration of RGCs after optic nerve injury. These findings highlight the possibility to use cell therapy to preserve neurons and to promote axon regeneration, using a reliable source of human MSCs.

Aims: Neurodegeneration of the optic nerve and retinal ganglion cells (RGCs) leads to progressive vision loss. As part of the central nervous system, RGCs show limited ability to regenerate and there is extensive search for neuroprotective agents for optic nerve damage. Methylene blue (MB) exhibits beneficial effects against various neurodegenerative diseases of the central nervous system. However, the mechanisms associated with its putative protection on neuronal survival and regeneration remain obscure. This study used the optic nerve transection model to examine the effects of MB on RGC survival, the expression of regenerative marker GAP-43 in RGCs and microglial activation., Main Methods: Axons of RGCs were injured by cutting the optic nerve. MB was injected intravitreally either immediately post-injury or delayed to 3 days post-injury. Using immunohistochemical staining, surviving RGCs, GAP-43-positive RGCs and microglial cells were quantified in wholemount retinas 7 days post-injury., Key Findings: Both immediate and delayed (a more clinically realistic situation) intravitreal injection of MB promoted RGC survival. MB also increased the number of GAP-43-positive RGCs, suggesting an enhanced ability of RGCs to regenerate. This was exemplified by the regenerative sprouting of axon-like processes from injured RGCs after MB treatment. The increase in RGC survival and GAP-43 expression correlated with an increase in the number of microglial cells., Significance: These results reveal that MB has survival-promoting and growth-promoting effects on RGCs after optic nerve injury. Together with the established safety profile of MB in humans, MB is a promising treatment for neurodegeneration and injury of the optic nerve., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Traumatic optic neuropathy is a common clinical problem. Damage to the optic nerve leads to shear stress and triggers secondary swelling within the optic canal. The study aims to explore the role of the inflammatory response following optic nerve injury (ONI) in toll-like receptor-9 knockout mice (TLR-9 -/- ) compared to wild-type mice (WT). At first, TLR-9 -/- and WT mice were subjected to ONI. We then found that ONI significantly up-regulated TLR-9 expression levels in retinal tissues of WT mice. The retinal degeneration after ONI was alleviated in TLR-9 -/- mice, as evidenced by the increased number of retinal ganglion cells (RGCs) and thickness of inner retinal layer (IRL). TUNEL staining and immunofluorescence staining of BRN3A indicated that TLR-9 knockout effectively improved the survival of RGCs. ONI-enhanced expression of Iba-1 and TMEM119 was markedly reduced in TLR-9 -/- mice, indicating the suppression of microglial activation. Moreover, production of pro-inflammatory regulators, including inducible nitric oxide synthase (iNOS), macrophage chemo-attractant protein (MCP)-1, cyclooxygenase-2 (COX-2), interleukin (IL)-1β, IL-18 and tumor necrosis factor-α (TNF-α), was significantly decreased in TLR-9 -/- mice following ONI. TLR-9 knockout-attenuated inflammation was mainly through repressing myeloid differentiation factor 88 (MyD88) and IL-1 receptor-associated kinase 4 (IRAK4). Furthermore, ONI greatly up-regulated the protein expression levels of phosphorylated (p)-IKKα, p-IκBα and p-nuclear factor (NF)-κB, whereas being repressed in TLR-9 -/- mice. The effects of TLR-9 on ONI were verified in lipopolysaccharide (LPS)-stimulated retinal microglial cells transfected with small interfering RNA TLR-9 (siTLR-9). As expected, promoting TLR-9 with its agonist markedly restored inflammation in TLR-9 knockdown cells stimulated by LPS. Therefore, all findings above suggested that suppressing TLR-9 showed neuroprotective effects against ONI through reducing inflammatory response, and TILR-9 might be a promising therapeutic target to develop effective strategies for the treatment of optic neuropathies., (Copyright © 2020. Published by Elsevier Inc.)

Backgrounds: Insults to the axons in the optic nerve head are the primary cause of loss of retinal ganglion cells (RGCs) in traumatic, ischemic nerve injury or degenerative ocular diseases. The central nervous system-specific leucine-rich repeat protein, LINGO-1, negatively regulates axon regeneration and neuronal survival after injury. However, the upstream molecular mechanisms that regulate LINGO-1 signaling and contribute to LINGO-1-mediated death of RGCs are unclear., Methods: The expression of SP1 was profiled in optic nerve crush (ONC)-injured RGCs. LINGO-1 level was examined after SP1 overexpression by qRT-PCR. Luciferase assay was used to examine the binding of SP1 to the promoter regions of LINGO-1. Primary RGCs from rat retina were isolated by immunopanning and RGCs apoptosis were determined by Tunnel. SP1 and LINGO-1 expression was investigated using immunohistochemistry and Western bolting. Neuroprotection was assessed by RGC counts, RNFL thickness, and VEP tests after inhibition of SP1 shRNA., Results: We demonstrate that SP1 was upregulated in ONC-injured RGCs. SP1 was bound to the LINGO-1 promoter, which led to increased expression of LINGO-1. Treatment with recombinant Nogo-66 or LINGO-1 promoted apoptosis of RGCs cultured under serum-deprivation conditions, while silencing of SP1 promoted the survival of RGCs. SP1 and LINGO-1 colocalized and were upregulated in ONC-injured retinas. Silencing of SP1 in vivo reduced LINGO-1 expression and protected the structure of RGCs from ONC-induced injury, but there was no sign of recovery in VEP., Conclusions: Our findings imply that SP1 regulates LINGO-1 expression in RGCs in the injured retina and provide insight into mechanisms underlying LINGO-1-mediated RGC death in optic nerve injury., (© 2020 The Authors. CNS Neuroscience & Therapeutics published by John Wiley & Sons Ltd.)

Retinal ganglion cell (RGC) degeneration, leading to irreversible blindness in chronic optic neuropathies, commonly begins with dendritic shrinkage followed by axon degeneration. Although limited axon regeneration in the optic nerve is possible with a genetic deletion of PTEN/SOCS3 after optic nerve injury, the roles of PTEN/SOCS3 on dendritic preservation and regeneration remain unclear. This study investigated the effect of PTEN/SOCS3 genetic deletion on the structural integrity of RGC dendrites and axons in the retina following optic nerve crush. Using time-lapse, in vivo confocal scanning laser ophthalmoscopy to serially image dendritic and axonal arborizations of RGCs over six months after injury, RGC dendrites and axons were only preserved in Thy-1-YFP/PTEN -/ - and Thy-1-YFP/PTEN -/- SOCS3 -/- mice, and axons in the retina regenerated at a rate of 21.1 μm/day and 15.5 μm/day, respectively. By contrast, dendritic complexity significantly decreased in Thy-1-YFP-SOCS3 -/- and control mice at a rate of 7.0 %/day and 7.1 %/day, respectively, and no axon regeneration in the retina was observed. RGC survival probability was higher in Thy-1-YFP/PTEN -/ - and Thy-1-YFP/PTEN -/- SOCS3 -/- mice compared with Thy-1-YFP-SOCS3 -/- and control mice. The differential responses between the transgenic mice demonstrate that although a genetic deletion of PTEN, SOCS3, or PTEN/SOCS3 allows partial axon regeneration in the optic nerve after optic nerve crush, a deletion of PTEN, but not SOCS3, ameliorates RGC dendritic shrinkage. This shows that the signaling pathways involved in promoting axon regeneration do not equally contribute to the preservation of dendrites, which is crucial to the translational application of neuroregenerative therapies for visual restoration., Competing Interests: Declaration of competing interest The authors declare that they have no competing interests., (Copyright © 2020. Published by Elsevier Ltd.)

There are contradictory reports on the role of the serine/threonine kinase isoform glycogen synthase kinase-3β (GSK3β) after injury to the central nervous system (CNS). Some report that GSK3 activity promotes axonal growth or myelin disinhibition, whilst others report that GSK3 activity prevents axon regeneration. In this study, we sought to clarify if suppression of GSK3β alone and in combination with the cellular-stress-induced factor RTP801 (also known as REDD1: regulated in development and DNA damage response protein), using translationally relevant siRNAs, promotes retinal ganglion cell (RGC) survival and neurite outgrowth/axon regeneration. Adult mixed retinal cell cultures, prepared from rats at five days after optic nerve crush (ONC) to activate retinal glia, were treated with siRNA to GSK3β (siGSK3β) alone or in combination with siRTP801 and RGC survival and neurite outgrowth were quantified in the presence and absence of Rapamycin or inhibitory Nogo-A peptides. In in vivo experiments, either siGSK3β alone or in combination with siRTP801 were intravitreally injected every eight days after ONC and RGC survival and axon regeneration was assessed at 24 days. Optimal doses of siGSK3β alone promoted significant RGC survival, increasing the number of RGC with neurites without affecting neurite length, an effect that was sensitive to Rapamycin. In addition, knockdown of GSK3β overcame Nogo-A-mediated neurite growth inhibition. Knockdown of GSK3β after ONC in vivo enhanced RGC survival but not axon number or length, without potentiating glial activation. Knockdown of RTP801 increased both RGC survival and axon regeneration, whilst the combined knockdown of GSK3β and RTP801 significantly increased RGC survival, neurite outgrowth, and axon regeneration over and above that observed for siGSK3β or siRTP801 alone. These results suggest that GSK3β suppression promotes RGC survival and axon initiation whilst, when in combination with RTP801, it also enhanced disinhibited axon elongation., Competing Interests: The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

This article has been retracted: please see Elsevier Policy on Article Withdrawal (https://www.elsevier.com/about/our-business/policies/article-withdrawal). This article has been retracted at the request of the Editor-in-Chief as panels from Figures 1D, 2E and 4E appear similar to each other. Given the comments of Dr Elisabeth Bik regarding this article “As previously described by Christopher …, the Western blot bands in all 400+ papers are all very regularly spaced and have a smooth appearance in the shape of a dumbbell or tadpole, without any of the usual smudges or stains”, the journal requested the authors to provide the raw data. However, the authors were not able to fulfil this request., (Copyright © 2019 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

Background: Carvedilol, which is considered as a nonselective β-adrenoreceptor blocker, has many pleiotropic activities. It also causes great impact on neuroprotection because of its antioxidant ability, which suggested that carvedilol may be effective in protecting RGCs from increased oxidative stress., Objective: To examine the effects of carvedilol on preventing Retinal Ganglion Cell (RGC) death in a mouse model of Optic Nerve Injury (ONI)., Methods: C57BL/6J mice were subjected to Optic Nerve Injury (ONI) model and treated with carvedilol or placebo. Histological and morphometric studies were performed; the RGC number, the amount of neurons in the ganglion cell layer and the thickness of the Inner Retinal Layer (IRL) was quantified. The average thickness of Ganglion Cell Complex (GCC) was determined by the Spectral- Domain OCT (SD-OCT) assay. Immunohistochemistry, western blot and quantitative real-time PCR analysis were also applied., Results: Daily treatment of carvedilol reduced RGC death following ONI, and in vivo retinal imaging revealed that carvedilol can effectively prevent retinal degeneration. The expression of chemokines important for micorglia recruitment was deceased with carvedilol ingestion and the accumulation of retinal microglia is reduced consequently. In addition, the ONI-induced expression of inducible nitric oxide synthase in the retina was inhibited with carvedilol treatment in the retina. We also discovered that carvedilol suppressed ONI-induced activation of Apoptosis Signal-regulating Kinase-1 (ASK1) and p38 Mitogen-Activated Protein Kinase (MAPK) pathway., Conclusion: The results of this study indicate that carvedilol can stimulate neuroprotection and neuroregeneration, and may be useful for treatment of various neurodegenerative diseases., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Optic nerve injury is a common disease. The present study aimed to examine the possible role of microRNA‑204 (miR‑204) in optic nerve injury through the regulation of growth‑associated protein-43 (GAP‑43). Initially, optic nerve injury models were established in Sprague‑Dawley (SD) rats, and the function of miR‑204 was either enhanced or inhibited through injection of miR‑204 mimic and inhibitor, respectively. Subsequently, the mRNA and protein levels of miR‑204, GAP‑43, toll‑like receptor 4 (TLR4), myeloid differentiation factor 88 (MyD88) and nuclear factor‑κB (NF‑κB) were examined in retinal tissues using reverse transcription‑quantitative polymerase chain reaction and western blot analyses. The apoptosis of retinal tissue cells was also detected using a terminal deoxynucleotidyl transferase mediated dUTP nick end labeling assay. There was a significant increase in the level of miR‑204 in retinal blood vessels of the model SD rats, compared with that in the normal SD rats (P<0.05), and the expression of GAP‑43 was significantly decreased (P<0.05). The results confirmed that the expression of GAP‑43 was significantly reduced, compared with that in the normal control group when the rats were treated with miR‑204 mimic (P<0.05), which was similar to the result in the model group. By contrast, its expression of GAP‑43 was significantly increased when treated with the miR‑204 inhibitor (P<0.05). Compared with the normal control group, the expression levels of TLR4, MyD88 and NF‑κB were significantly increased in the miR‑204 mimic group and model group (P<0.05), whereas the same three factors in the miR‑204 inhibitor group were effectively inhibited, compared with those in the model group, and showed similar results to the normal control group. The apoptotic rates of retinal cells in the miR‑204 mimic group and model group were significantly increased, compared with that in the normal control group (P<0.05), whereas miR‑204 inhibitor effectively reversed the effects on apoptotic rate observed in the model group, showing similar results to those in the normal control group. Taken together, miR‑204 promoted the apoptosis of retinal cells through inhibiting GAP‑43, providing theoretical guidance for the function of GAP‑43 in retinal injury.

The ability of light to cause pain is paradoxical. The retina detects light but is devoid of nociceptors while the trigeminal sensory ganglia (TG) contain nociceptors but not photoreceptors. Melanopsin-expressing intrinsically photosensitive retinal ganglion cells (ipRGCs) are thought to mediate light-induced pain but recent evidence raises the possibility of an alternative light responsive pathway independent of the retina and optic nerve. Here, we show that melanopsin is expressed in both human and mouse TG neurons. In mice, they represent 3% of small TG neurons that are preferentially localized in the ophthalmic branch of the trigeminal nerve and are likely nociceptive C fibers and high-threshold mechanoreceptor Aδ fibers based on a strong size-function association. These isolated neurons respond to blue light stimuli with a delayed onset and sustained firing, similar to the melanopsin-dependent intrinsic photosensitivity observed in ipRGCs. Mice with severe bilateral optic nerve crush exhibit no light-induced responses including behavioral light aversion until treated with nitroglycerin, an inducer of migraine in people and migraine-like symptoms in mice. With nitroglycerin, these same mice with optic nerve crush exhibit significant light aversion. Furthermore, this retained light aversion remains dependent on melanopsin-expressing neurons. Our results demonstrate a novel light-responsive neural function independent of the optic nerve that may originate in the peripheral nervous system to provide the first direct mechanism for an alternative light detection pathway that influences motivated behavior.

Injury to the central nervous system causes progressive degeneration of injured axons, leading to loss of the neuronal bodies. Neuronal survival after injury is a prerequisite for successful regeneration of injured axons. In this study, we investigated the effects of increased production of omega-3 fatty acids and elevation of cAMP on retinal ganglion cell (RGC) survival and axonal regeneration after optic nerve (ON) crush injury in adult mice. We found that increased production of omega-3 fatty acids in mice enhanced RGC survival, but not axonal regeneration, over a period of 3 weeks after ON injury. cAMP elevation promoted RGC survival in wild type mice, but no significant difference in cell survival was seen in mice over-producing omega-3 fatty acids and receiving intravitreal injections of CPT-cAMP, suggesting that cAMP elevation protects RGCs after injury but does not potentiate the actions of the omega-3 fatty acids. The observed omega-3 fatty acid-mediated neuroprotection is likely achieved partially through ERK1/2 signaling as inhibition of this pathway by PD98059 hindered, but did not completely block, RGC protection. Our study thus enhances our current understanding of neural repair after CNS injury, including the visual system., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Optic neuropathies are characterized by retinal ganglion cell (RGC) death, resulting in the loss of vision. In glaucoma, the most common optic neuropathy, RGC death is initiated by axonal damage, and can be modeled by inducing acute axonal trauma through procedures such as optic nerve crush (ONC) or optic nerve axotomy. One of the early events of RGC death is nuclear atrophy, and is comprised of RGC-specific gene silencing, histone deacetylation, heterochromatin formation, and nuclear shrinkage. These early events appear to be principally regulated by epigenetic mechanisms involving histone deacetylation. Class I histone deacetylases HDACs 1, 2, and 3 are known to play important roles in the process of early nuclear atrophy in RGCs, and studies using both inhibitors and genetic ablation of Hdacs also reveal a critical role in the cell death process. Select inhibitors, such as those being developed for cancer therapy, may also provide a viable secondary treatment option for optic neuropathies., (Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.)

Parvalbumin (PARV) is a Ca(2+)-binding protein that may offer resistance to cell death as it primarily functions to maintain Ca(2+) homeostasis. The purpose of this study was to investigate whether PARV expressing retinal ganglion cells (RGCs) would be more resistant to cell death than RGCs that do not express PARV. RGCs in Sprague-Dawley rats were retrogradely labeled with Fluorogold (FG). After 2-28 days following an optic nerve crush (ONC) injury immunohistochemistry was performed on the sections using antibodies against PARV and markers of RGCs. The proportion of retinal ganglion cell layer cells labeled with PARV colocalized with FG or Brn3a and labeled only with PARV (displaced amacrine cells; dACs) were analyzed. PARV staining intensity was measured in ACs, dACs, and RGCs. Double labeling studies revealed that 49% of RGCs and 22% of dACs expressed PARV. There was an immediate reduction in RGC PARV staining after ONC but the overall rate of cell death after 28 days was similar in PARV and non-PARV expressing RGCs. There was no change in PARV AC or dAC number or staining intensity. Although this study suggests that there is no selective survival of the subpopulation of RGCs that contain PARV, there is down-regulation of PARV expression by these RGCs. This suggests that down-regulation of PARV may contribute to RGC death due to a compromised Ca(2+) buffering capacity. Maintaining PARV expression after injury could be an important neuroprotective strategy to improve RGC survival after injury., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Purpose: Vision loss after traumatic optic nerve injury is considered irreversible because of the retrograde loss of retinal ganglion cells (RGCs) which undergo apoptosis. Because the second messenger caspase-3 plays a major role in apoptosis, we now evaluated the efficacy of the specific caspase-3 inhibitor, Z-DEVD-FMK, in a rabbit model of fluid percussion injury (FPI) which mimics traumatic optic nerve injury in humans to enhance cell survival and improve vision., Methods: Survival of RGCs and recovery of vision were studied using retinal morphological markers and visual evoked potentials (VEP), respectively. The FPI traumatized animals were treated in their right eye with a single intravitreal or peribulbar injection of Z-DEVD-FMK 30 min post-injury compared to 2% DMSO control injections in their left eye., Results: Intravitreal Z-DEVD-FMK, but not control injections, led to down-regulation of capase-3 and reduced, in a dose-dependent manner, RGCs apoptosis from 7 to 21 days post-injury. These morphological improvements were accompanied by vision restoration as documented by VEP. The neuroprotection after intravitreal injection of Z-DEVD-FMK was more effective than the peribulbar application., Conclusions: The caspase-3 inhibitor Z-DEVD-FMK is neuroprotective by inhibiting RGCs apoptosis when injected 30 min after optic nerve damage and significantly promotes restoration of vision. A controlled clinical trial is now needed to evaluate the efficacy and safety of Z-DEVD-FMK in humans.

The dedicator of cytokinesis 3 (Dock3) is an atypical guanine nucleotide exchange factor that is predominantly expressed in the CNS. Dock3 exerts neuroprotective effects and stimulates optic nerve regeneration. The p38 mitogen-activated protein kinase acts downstream of apoptosis signal-regulating kinase 1 (ASK1) signaling and plays an important role in neural cell death. We assessed a therapeutic efficacy of Dock3 stimulation and p38 inhibition in retinal degeneration induced by optic nerve injury (ONI). In vivo retinal imaging using optical coherence tomography revealed that ONI-induced retinal degeneration was ameliorated in SB203580 (a p38 inhibitor)-treated WT mice and PBS-treated Dock3 overexpressing (Dock3 Tg) mice, and SB203580 further stimulated retinal protection in Dock3 Tg mice. In addition, SB203580 increased the number of regenerating axons after ONI in both WT and Dock3 Tg mice. ONI-induced phosphorylation of ASK1, p38 and the N-methyl-d-aspartate receptor 2B subunit were suppressed in the retina of Dock3 Tg mice. Inhibition of the ASK1 pathway in Dock3 Tg mice suggests that Dock3 may have an antioxidant-like property. These results indicate that overexpression of Dock3 and pharmacological interruption of p38 have synergistic effects for both neuroprotection and axon regeneration, thus combined application may be beneficial for the treatment of ONI., (Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.)

Purpose: BM88 is a cell-cycle exit and neuronal differentiation protein that has been used as a marker of surviving retinal ganglion cells (RGCs) after optic nerve injury. Thy1.1 has also been used as a marker for RGC loss, but after optic nerve crush (ONC) a decrease in Thy1.1 expression precedes the loss of RGCs. The purpose of this study was to determine if BM88 expression was correlated with RGC loss after ONC and optic nerve transection (ONT) injuries., Methods: Rats were injected with Fluorogold (FG) into the superior colliculus to label RGCs and received ONC or ONT 7 days later. Eyes were collected 2 to 28 days after injury. Retinas were labeled with BM88 and intensity of the BM88 cell labeling was measured., Results: In control retinas, 98.9% of RGCs were immunoreactive (-IR) for BM88. There was a significant downregulation of BM88 by 52% to 80% of RGCs 7 days after ONC or ONT. The staining intensity of the remaining labeled cells was reduced to 41% to 51% of the control after 28 days of optic nerve injury. However, early in the injury there was a significant increase in the staining intensity of BM88., Conclusions: Nearly all BM88-IR RGCs colocalized with FG-labeled RGCs in control retinas. However, both the number of BM88-IR RGCs and their intensity decreased gradually between 4 and 28 days, preceding the loss of FG-labeled cells. These findings indicate that BM88 is not a good marker of surviving RGCs but may indicate abnormal RGC functioning, which precedes cell death.

Aims: Activation of retinal microglial cells (RMCs) is known to contribute to retinal ganglion cell (RGC) death after optic nerve injury. The purpose of this study was to investigate the effects of intravenous injection of α-crystallin on RGC survival and RMC activation in a rat model of optic nerve crush., Main Methods: RGCs were retrogradely labeled with fluorogold. Rats were intravenously injected with normal saline or α-crystallin (0.05g/kg, 0.5g/kg, and 5 g/kg) at 2, 4, 6, 8, 10, and 12 days after the optic nerve crush. Activated RMCs were characterized using immunofluorescence labeling with CD11b, and TNF-α and iNOS expression was detected using immunoblot analyses. We analyzed the morphology and numbers of RGC and RMC 2 and 4 weeks after injury using fluorescence and confocal microscopy., Key Findings: The number of RGCs decreased after optic nerve injury, accompanied by significantly increased numbers of activated RMCs. Intravenous injection of α-crystallin decreased the number of RMCs, and enhanced the number of RGCs compared to saline injection. α-Crystallin administration inhibited TNF-α and iNOS protein expression induced by optic nerve injury., Significance: Our results suggest that α-crystallin promotes RGC survival and inhibits RMC activation. Intravenous injection of α-crystallin could be a possible strategy for the treatment of optic nerve injury., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Purpose: In adult rats, intravitreal injections of the Rho-GTPase inhibitor C3 transferase (BA-210), or a cocktail of recombinant ciliary neurotrophic factor (CNTF) and a cyclic AMP analogue (CPTcAMP), increase retinal ganglion cell (RGC) survival and axonal regeneration. Here we examined whether these treatments also affect the dendritic architecture of regrowing RGCs., Methods: In Fischer F344 rats, one optic nerve was cut and an autologous peripheral nerve graft was sutured onto it. Rats then received intravitreal injections (4 μl) of saline, BA-210 or CNTF + CPTcAMP four and eleven days after the PN graft surgery. After 5 weeks, regenerating RGCs were retrogradely labelled with fluorogold (FG) and in living wholemounts the dendritic trees of FG labelled RGCs were visualised by intracellular injection of 2% Lucifer Yellow., Results: Injection of BA-210 or CNTF + CPTcAMP resulted in significantly more regenerating RGCs with abnormal dendritic morphologies, including abnormally long looping processes. Compared to saline-injected regenerating controls, RGCs in BA-210 injected eyes had significantly smaller dendritic field areas and sparser dendrites, while in CNTF + CPTcAMP injected eyes there was increased branching of more distal dendrites., Conclusions: While both intraocular treatments enhance RGC axonal regrowth, they also induce significant changes in RGC dendritic morphology. It remains to be determined if such changes alter the function of the regenerating neuronal population.

Progressive death of retinal ganglion cells (RGCs) is a major cause of irreversible visual impairment after optic nerve injury. Clinically, there are still no effective treatments for recovering the visual function at present. The probable approaches to maintain the vision and RGCs function involve in preventing RGCs from death and/or promoting the regeneration of damaged RGCs. Previous studies have shown that mesenchymal stem cells (MSCs) take neuroprotective effects on ischemia-induced cortical and spinal cord injury, however, whether MSCs have a beneficial effect on the optical nerve injury is not clearly determined. In present study, we transplanted MSCs derived from human umbilical cord blood (hUCB-MSCs) into the vitreous cavity of adult rats and investigated the probable capacity of anti-apoptosis and pro-neuroprotective effects on RGCs. RGCs were retrogradely traced by fluorescent gold particles (FG); cellular apoptosis was investigated by caspase-3 immunohistochemistry and terminal dUTP nick end labeling (TUNEL) staining. Hematoxylin-eosin (HE) staining was used to observe the morphological changes of the retina. Growth associated protein 43 (GAP-43), an established marker for axonal regeneration, was used to visualize the regenerative process over time. Expression of P2X7 receptors (P2X7R), which are responsible for inflammatory and immune responses, was also monitored in our experiments. We found that the hUCB-MSC transplantation significantly decreased cellular apoptosis and promoted the survival of RGCs in early phase. However, this protection was transient and the RGCs could not be protected from death in the end. Consistent with apoptosis detection, P2X7R was also significantly decreased in hUCB-MSC transplanted rats in the early time but without obvious difference to the rats from control group in the end. Thus, our results imply that hUCB-MSCs take anti-apoptotic, pro-neuroregenerative and anti-inflammatory effects in the early time for acute optic nerve injury in adult rats but could not prevent RGCs from death eventually., (© 2013 Elsevier B.V. All rights reserved.)

The retinal ganglion cells (RGCs) are the sole output neurons that connect information from the retina to the brain. Optic neuropathies such as glaucoma, trauma, inflammation, ischemia and hereditary optic neuropathy can cause RGC loss and axon damage, and lead to partial or total loss of vision, which is an irreversible process in mammals. The accurate diagnoses of optic neuropathies are crucial for timely treatments to prevent irrevocable RGCs loss. After severe ON damage in optic neuropathies, promoting RGC axon regeneration is vital for restoring vision. Clearance of neuronal debris, decreased intrinsic growth capacity, and the presence of inhibitory factors have been shown to contribute to the failure of post‐traumatic CNS regeneration. Here, we review the current understanding of manifestations and treatments of various common optic neuropathies. We also summarise the current known mechanisms of RGC survival and axon regeneration in mammals, including specific intrinsic signalling pathways, key transcription factors, reprogramming genes, inflammation‐related regeneration factors, stem cell therapy, and combination therapies. Significant differences in RGC subtypes in survival and regenerative capacity after injury have also been found. Finally, we highlight the developmental states and non‐mammalian species that are capable of regenerating RGC axons after injury, and cellular state reprogramming for neural repair. [ABSTRACT FROM AUTHOR]

Sphingosine-1-phosphate receptor (S1PR) signaling regulates diverse pathophysiological processes in the central nervous system. The role of S1PR signaling in neurodegenerative conditions is still largely unidentified. Siponimod is a specific modulator of S1P1 and S1P5 receptors, an immunosuppressant drug for managing secondary progressive multiple sclerosis. We investigated its neuroprotective properties in vivo on the retina and the brain in an optic nerve injury model induced by a chronic increase in intraocular pressure or acute N-methyl-D-aspartate excitotoxicity. Neuronal-specific deletion of sphingosine-1-phosphate receptor (S1PR1) was carried out by expressing AAV-PHP.eB-Cre recombinase under Syn1 promoter in S1PR1flox/flox mice to define the role of S1PR1 in neurons. Inner retinal electrophysiological responses, along with histological and immunofluorescence analysis of the retina and optic nerve tissues, indicated significant neuroprotective effects of siponimod when administered orally via diet in chronic and acute optic nerve injury models. Further, siponimod treatment showed significant protection against trans-neuronal degenerative changes in the higher visual center of the brain induced by optic nerve injury. Siponimod treatment also reduced microglial activation and reactive gliosis along the visual pathway. Our results showed that siponimod markedly upregulated neuroprotective Akt and Erk1/2 activation in the retina and the brain. Neuronal-specific deletion of S1PR1 enhanced retinal and dorsolateral geniculate nucleus degenerative changes in a chronic optic nerve injury condition and attenuated protective effects of siponimod. In summary, our data demonstrated that S1PR1 signaling plays a vital role in the retinal ganglion cell and dorsolateral geniculate nucleus neuronal survival in experimental glaucoma, and siponimod exerts direct neuroprotective effects through S1PR1 in neurons in the central nervous system independent of its peripheral immuno-modulatory effects. Our findings suggest that neuronal S1PR1 is a neuroprotective therapeutic target and its modulation by siponimod has positive implications in glaucoma conditions.

Optic nerve damage is a common cause of blindness. Optic nerve injury is often accompanied by fundus vascular disease, retinal ganglion cell apoptosis, and changes in retinal thickness. These changes can cause alterations in protein expression within neurons in the retina. Proteomics analysis offers conclusive evidence to decode a biological system. Furthermore, animal models of optic nerve injury made it possible to gain insight into pathological mechanisms, therapeutic targets, and effective treatment of such injuries. Proteomics takes the proteome as the research object and studies protein changes in cells and tissues. At present, a variety of proteomic analysis methods have been widely used in the research of optic nerve injury diseases. This review summarizes the application of proteomic research in optic nerve injury diseases and animal models of optic nerve injury. Additionally, differentially expressed proteins are summarized and analyzed. Various optic nerve injuries, including those associated with different etiologies, are discussed along with their potential therapeutic targets and future directions. [ABSTRACT FROM AUTHOR]

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Both inflammation and anti-inflammation are involved in the protection of retinal cells. Antagonists of the hypothalamic growth hormone-releasing hormone receptor (GHRHR) have been shown to possess potent anti-inflammatory properties in experimental disease models of various organs, some with systemic complications. Such effects are also found in ocular inflammatory and neurologic injury studies. In experimental models of mice and rats, both growth hormone-releasing hormone receptor agonists and antagonists may alleviate death of ocular neural cells under certain experimental conditions. This review explores the properties of growth hormone-releasing hormone receptor agonists and antagonists that lead to its protection against inflammatory responses induced by extrinsic agents or neurologic injures in ocular animal models.

Qianxue Mou,1 Ke Yao,1 Meng Ye,1 Bowen Zhao,1 Yuanyuan Hu,1 Xiaotong Lou,1 Huixia Li,2 Hong Zhang,1 Yin Zhao1 1Department of Ophthalmology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430030, People’s Republic of China; 2Department of Physiology and Pathophysiology, School of Basic Medical Sciences, Xi’an Jiaotong University Health Science Center, Xi’an, Shaanxi, People’s Republic of ChinaCorrespondence: Hong ZhangDepartment of Ophthalmology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430030, People’s Republic of ChinaEmail tjyksys@163.comCorrespondence: Yin ZhaoDepartment of Ophthalmology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430030, People’s Republic of ChinaEmail zhaoyin85@hust.edu.cnPurpose: Optic nerve injury (ONI) causes neuroinflammation and neurodegeneration leading to visual deficits. The response of microglia has emerged as an impactful component of etiology in neurodegeneration. This study aimed to investigate the effect of SIRT1-mTORC1 signaling pathway in microglia regulation after ONI.Methods: Cx3Cr1-CreERT2/RaptorF/F and Cx3Cr1-CreERT2/Sirt1F/F mice were used to delete Raptor and Sirt1 in microglia, respectively. Optic nerve crush (ONC) model was established to mimic ONI. PLX5622, a highly specific inhibitor of the colony-stimulating factor 1 receptor (CSF1R), is used to eliminate microglia in optic nerve. Ionized calcium binding adaptor molecule 1 (Iba1) immunostaining was used to detect microglial activation. Retinal ganglion cells (RGCs) were quantified by Nissl staining and retinal whole-mount immunostaining with RNA-binding protein with multiple splicing (RBPMS). Axonal damage was valued by transmission electron microscopy (TEM).Results: Microglial activation emerged on day 3 post ONC and was earlier than RGCs loss which occurred at day 5 after injury. Depleting microglia with PLX5622 could attenuate the loss of RGCs and axon damage after ONC. Gain- and loss-of-function studies revealed that SIRT1 determined the activation of microglia in optic nerve. In addition, microglia-specific deletion of Raptor resulted in decreased microglial activation. Interestingly, activating mTORC1 with CCT007093 could reverse the function of SIRT1 in regulating the process of microglial activation mediated RGCs loss.Conclusion: Our study reveals a potential novel mechanism of SIRT1-mTORC1 pathway in microglia regulation, and indicates a therapeutic potential for the protection of RGCs in ONI.Keywords: microglia, retinal ganglion cells, optic nerve injury, SIRT1, mTORC1

Retinal ganglion cells (RGCs), the projection neurons of the eye, are irreversibly lost once the optic nerve is injured, which is a critical mechanism of glaucoma. Mobile zinc (Zn2+) levels rapidly increase in retinal interneuron amacrine cells and Zn2+ is then transferred to RGCs via the Zn2+ transporter protein ZnT-3, triggering RGC loss in optic nerve injury. Zn2+ chelation and ZnT-3 deletion promote long-term RGC survival. However, the downstream signaling pathways of Zn2+ in RGCs remains unknown. Here, we show that increased levels of Zn2+ upregulate the expression and activity of mitochondrial zinc metallopeptidase OMA1 in the retina, leading to the cleavage of DELE1 and activation of cytosolic eIF2α kinase PKR, triggering the integrated stress response (ISR) in RGCs. Our study identified OMA1 and ISR as the downstream molecular mechanisms of retinal Zn2+ and potential targets for preventing the progression of Zn2+-associated neuronal damage. [ABSTRACT FROM AUTHOR]

Objective:: Any damage to the optic nerve can potentially lead to degeneration of non-regenerating axons and ultimately death of retinal ganglion cells (RGCs) that in most cases, are not curable by surgery or medication. Neuroprotective functions of different types of stem cells in the nervous system have been evaluated in many studies investigating the effectiveness of these cells in various retinal disease models. Neural progenitor cells (NPCs) secrete an assortment of trophic factors that are vital to the protection of the visual system. We aimed to assess the therapeutic potentials of NPCs in an ONC mouse model. MaterialsAndMethods: : In this experimental study, NPCs were produced using noggin and retinoic acid from human embryonic stem cells (hESCs). Fifty mice were divided into the following three groups: i. Intact, ii. Vehicle [optic nerve crush+Hank’s balanced salt solution (HBSS)], and iii. Treatment (optic nerve crush+NPCs). The visual behavior of the mice was examined using the Visual Cliff test, and in terms of RGC numbers, they were assessed by Brn3a immunostaining and retrograde tracing using DiI injection: Results Intravenous injection of 50,000 NPCs through visual cliff did not produce any visual improvement. However, our data suggest that the RGCs protection was more than two-times in NPCs compared to the vehicle group as examined by Brn3a staining and retrograde tracing. Conclusion: Our study indicated that intravenous injection of NPCs could protect RGCs probably mediated by trophic factors. Due to this ability and good manufacturing practices (GMP) grade production feasibility, NPCs may be used for optic nerve protection. [ABSTRACT FROM AUTHOR]

The ability of light to cause pain is paradoxical. The retina detects light but is devoid of nociceptors while the trigeminal sensory ganglia (TG) contain nociceptors but not photoreceptors. Melanopsin-expressing intrinsically photosensitive retinal ganglion cells (ipRGCs) are thought to mediate light-induced pain but recent evidence raises the possibility of an alternative light responsive pathway independent of the retina and optic nerve. Here, we show that melanopsin is expressed in both human and mouse TG neurons. In mice, they represent 3% of small TG neurons that are preferentially localized in the ophthalmic branch of the trigeminal nerve and are likely nociceptive C fibers and high-threshold mechanoreceptor Aδ fibers based on a strong size-function association. These isolated neurons respond to blue light stimuli with a delayed onset and sustained firing, similar to the melanopsin-dependent intrinsic photosensitivity observed in ipRGCs. Mice with severe bilateral optic nerve crush exhibit no light-induced responses including behavioral light aversion until treated with nitroglycerin, an inducer of migraine in people and migraine-like symptoms in mice. With nitroglycerin, these same mice with optic nerve crush exhibit significant light aversion. Furthermore, this retained light aversion remains dependent on melanopsin-expressing neurons. Our results demonstrate a novel light-responsive neural function independent of the optic nerve that may originate in the peripheral nervous system to provide the first direct mechanism for an alternative light detection pathway that influences motivated behavior.

Background: Because some of its CNS neurons (e.g., retinal ganglion cells after optic nerve crush (ONC)) regenerate axons throughout life, whereas others (e.g., hindbrain neurons after spinal cord injury (SCI)) lose this capacity as tadpoles metamorphose into frogs, the South African claw-toed frog, Xenopus laevis, offers unique opportunities for exploring differences between regenerative and non-regenerative responses to CNS injury within the same organism. An earlier, three-way RNA-seq study (frog ONC eye, tadpole SCI hindbrain, frog SCI hindbrain) identified genes that regulate chromatin accessibility among those that were differentially expressed in regenerative vs non-regenerative CNS [11]. The current study used whole genome bisulfite sequencing (WGBS) of DNA collected from these same animals at the peak period of axon regeneration to study the extent to which DNA methylation could potentially underlie differences in chromatin accessibility between regenerative and non-regenerative CNS. Results: Consistent with the hypothesis that DNA of regenerative CNS is more accessible than that of non-regenerative CNS, DNA from both the regenerative tadpole hindbrain and frog eye was less methylated than that of the non-regenerative frog hindbrain. Also, consistent with observations of CNS injury in mammals, DNA methylation in non-regenerative frog hindbrain decreased after SCI. However, contrary to expectations that the level of DNA methylation would decrease even further with axotomy in regenerative CNS, DNA methylation in these regions instead increased with injury. Injury-induced differences in CpG methylation in regenerative CNS became especially enriched in gene promoter regions, whereas non-CpG methylation differences were more evenly distributed across promoter regions, intergenic, and intragenic regions. In non-regenerative CNS, tissue-related (i.e., regenerative vs. non-regenerative CNS) and injury-induced decreases in promoter region CpG methylation were significantly correlated with increased RNA expression, but the injury-induced, increased CpG methylation seen in regenerative CNS across promoter regions was not, suggesting it was associated with increased rather than decreased chromatin accessibility. This hypothesis received support from observations that in regenerative CNS, many genes exhibiting increased, injury-induced, promoter-associated CpG-methylation also exhibited increased RNA expression and association with histone markers for active promoters and enhancers. DNA immunoprecipitation for 5hmC in optic nerve regeneration found that the promoter-associated increases seen in CpG methylation were distinct from those exhibiting changes in 5hmC. Conclusions: Although seemingly paradoxical, the increased injury-associated DNA methylation seen in regenerative CNS has many parallels in stem cells and cancer. Thus, these axotomy-induced changes in DNA methylation in regenerative CNS provide evidence for a novel epigenetic state favoring successful over unsuccessful CNS axon regeneration. The datasets described in this study should help lay the foundations for future studies of the molecular and cellular mechanisms involved. The insights gained should, in turn, help point the way to novel therapeutic approaches for treating CNS injury in mammals. [ABSTRACT FROM AUTHOR]

Optic nerve damage is a common cause of blindness. Optic nerve injury is often accompanied by fundus vascular disease, retinal ganglion cell apoptosis, and changes in retinal thickness. These changes can cause alterations in protein expression within neurons in the retina. Proteomics analysis offers conclusive evidence to decode a biological system. Furthermore, animal models of optic nerve injury made it possible to gain insight into pathological mechanisms, therapeutic targets, and effective treatment of such injuries. Proteomics takes the proteome as the research object and studies protein changes in cells and tissues. At present, a variety of proteomic analysis methods have been widely used in the research of optic nerve injury diseases. This review summarizes the application of proteomic research in optic nerve injury diseases and animal models of optic nerve injury. Additionally, differentially expressed proteins are summarized and analyzed. Various optic nerve injuries, including those associated with different etiologies, are discussed along with their potential therapeutic targets and future directions.

Retinal ganglion cells (RGCs), the projection neurons of the eye, are irreversibly lost once the optic nerve is injured, which is a critical mechanism of glaucoma. Mobile zinc (Zn2+) levels rapidly increase in retinal interneuron amacrine cells and Zn2+ is then transferred to RGCs via the Zn2+ transporter protein ZnT-3, triggering RGC loss in optic nerve injury. Zn2+ chelation and ZnT-3 deletion promote long-term RGC survival. However, the downstream signaling pathways of Zn2+ in RGCs remains unknown. Here, we show that increased levels of Zn2+ upregulate the expression and activity of mitochondrial zinc metallopeptidase OMA1 in the retina, leading to the cleavage of DELE1 and activation of cytosolic eIF2α kinase PKR, triggering the integrated stress response (ISR) in RGCs. Our study identified OMA1 and ISR as the downstream molecular mechanisms of retinal Zn2+ and potential targets for preventing the progression of Zn2+-associated neuronal damage.

Optic neuropathies are leading causes of irreversible visual impairment and blindness, currently affecting more than 100 million people worldwide. Glaucoma is a group of optic neuropathies attributed to progressive degeneration of retinal ganglion cells (RGCs). We have previously demonstrated an increase in survival of RGCs by the activation of macrophages, whereas the inhibition of macrophages was involved in the alleviation on endotoxin-induced inflammation by antagonist of growth hormone-releasing hormone (GHRH). Herein, we hypothesized that GHRH receptor (GHRH-R) signaling could be involved in the survival of RGCs mediated by inflammation. We found the expression of GHRH-R in RGCs of adult rat retina. After optic nerve crush, subcutaneous application of GHRH agonist MR-409 or antagonist MIA-602 promoted the survival of RGCs. Both the GHRH agonist and antagonist increased the phosphorylation of Akt in the retina, but only agonist MR-409 promoted microglia activation in the retina. The antagonist MIA-602 reduced significantly the expression of inflammation-related genes Il1b, Il6, and Tnf. Moreover, agonist MR-409 further enhanced the promotion of RGC survival by lens injury or zymosan-induced macrophage activation, whereas antagonist MIA-602 attenuated the enhancement in RGC survival. Our findings reveal the protective effect of agonistic analogs of GHRH on RGCs in rats after optic nerve injury and its additive effect to macrophage activation, indicating a therapeutic potential of GHRH agonists for the protection of RGCs against optic neuropathies especially in glaucoma. [ABSTRACT FROM AUTHOR]

Nuclear factor erythroid 2 like (Nfe2l) gene family members 1–3 mediate cellular response to oxidative stress, including in the central nervous system (CNS). However, neuronal functions of Nfe2l3 are unknown. Here, we comparatively evaluated expression of Nfe2l1, Nfe2l2, and Nfe2l3 in singe cell RNA-seq (scRNA-seq)-profiled cortical and retinal ganglion cell (RGC) CNS projection neurons, investigated whether Nfe2l3 regulates neuroprotection and axon regeneration after CNS injury in vivo , and characterized a gene network associated with Nfe2l3 in neurons. We showed that, Nfe2l3 expression transiently peaks in developing immature cortical and RGC projection neurons, but is nearly abolished in adult neurons and is not upregulated after injury. Furthermore, within the retina, Nfe2l3 is enriched in RGCs, primarily neonatally, and not upregulated in injured RGCs, whereas Nfe2l1 and Nfe2l2 are expressed robustly in other retinal cell types as well and are upregulated in injured RGCs. We also found that, expressing Nfe2l3 in injured RGCs through localized intralocular viral vector delivery promotes neuroprotection and long-distance axon regeneration after optic nerve injury in vivo. Moreover, Nfe2l3 provided a similar extent of neuroprotection and axon regeneration as viral vector-targeting of Pten and Klf9, which are prominent regulators of neuroprotection and long-distance axon regeneration. Finally, we bioinformatically characterized a gene network associated with Nfe2l3 in neurons, which predicted the association of Nfe2l3 with established mechanisms of neuroprotection and axon regeneration. Thus, Nfe2l3 is a novel neuroprotection and axon regeneration-promoting factor with a therapeutic potential for treating CNS injury and disease. • Novel role for neuronal Nfe2l3 in promoting neuroprotection and long-distance axonal regeneration after CNS injury in vivo. • AAV2 expression of Nfe2l3 in adult RGCs provides similar neuroprotection and axon regeneration as shRNA KD of Pten or Klf9. • Nfe2l3 is not meaningfully expressed in the adult retinal ganglion cells before or after optic nerve injury. [ABSTRACT FROM AUTHOR]

Optic neuropathies are a major cause of visual impairment due to retinal ganglion cell (RGC) degeneration. Human induced-pluripotent stem cells (iPSCs) represent a powerful tool for studying both human RGC development and RGC-related pathological mechanisms. Because RGC loss can be massive before the diagnosis of visual impairment, cell replacement is one of the most encouraging strategies. The present work describes the generation of functional RGCs from iPSCs based on innovative 3D/2D stepwise differentiation protocol. We demonstrate that targeting the cell surface marker THY1 is an effective strategy to select transplantable RGCs. By generating a fluorescent GFP reporter iPSC line to follow transplanted cells, we provide evidence that THY1-positive RGCs injected into the vitreous of mice with optic neuropathy can survive up to 1 month, intermingled with the host RGC layer. These data support the usefulness of iPSC-derived RGC exploration as a potential future therapeutic strategy for optic nerve regeneration.

Background: The South African claw-toed frog, Xenopus laevis, is uniquely suited for studying differences between regenerative and non-regenerative responses to CNS injury within the same organism, because some CNS neurons (e.g., retinal ganglion cells after optic nerve crush (ONC)) regenerate axons throughout life, whereas others (e.g., hindbrain neurons after spinal cord injury (SCI)) lose this capacity as tadpoles metamorphose into frogs. Tissues from these CNS regions (frog ONC eye, tadpole SCI hindbrain, frog SCI hindbrain) were used in a three-way RNA-seq study of axotomized CNS axons to identify potential core gene expression programs for successful CNS axon regeneration. Results: Despite tissue-specific changes in expression dominating the injury responses of each tissue, injury-induced changes in gene expression were nonetheless shared between the two axon-regenerative CNS regions that were not shared with the non-regenerative region. These included similar temporal patterns of gene expression and over 300 injury-responsive genes. Many of these genes and their associated cellular functions had previously been associated with injury responses of multiple tissues, both neural and non-neural, from different species, thereby demonstrating deep phylogenetically conserved commonalities between successful CNS axon regeneration and tissue regeneration in general. Further analyses implicated the KEGG adipocytokine signaling pathway, which links leptin with metabolic and gene regulatory pathways, and a novel gene regulatory network with genes regulating chromatin accessibility at its core, as important hubs in the larger network of injury response genes involved in successful CNS axon regeneration. Conclusions: This study identifies deep, phylogenetically conserved commonalities between CNS axon regeneration and other examples of successful tissue regeneration and provides new targets for studying the molecular underpinnings of successful CNS axon regeneration, as well as a guide for distinguishing pro-regenerative injury-induced changes in gene expression from detrimental ones in mammals. [ABSTRACT FROM AUTHOR]