Regeneration Of Retinal Ganglion Cells Research Articles

Large-scale CRISPR screen reveals context-specific genetic regulation of retinal ganglion cell regeneration.

Many genes are known to regulate retinal regeneration following widespread tissue damage. Conversely, genes controlling regeneration following limited cell loss, per degenerative diseases, are undefined. As stem/progenitor cell responses scale to injury levels, understanding how the extent and specificity of cell loss impact regenerative processes is important. Here, transgenic zebrafish enabling selective retinal ganglion cell (RGC) ablation were used to identify genes that regulate RGC regeneration. A single cell multiomics-informed screen of 101 genes identified seven knockouts that inhibited and eleven that promoted RGC regeneration. Surprisingly, 35 of 36 genes known/implicated as being required for regeneration following widespread retinal damage were not required for RGC regeneration, and seven even enhanced regeneration kinetics, including proneural factors neurog1, olig2, and ascl1a. Mechanistic analyses revealed ascl1a disruption increased the propensity of progenitor cells to produce RGCs; i.e., increased "fate bias". These data demonstrate plasticity in how Müller glia can convert to a stem-like state and context-specificity in how genes function during regeneration. Increased understanding of how the regeneration of disease-relevant cell types is specifically controlled will support the development of disease-tailored regenerative therapeutics.

Neuritin 1 Drives Therapeutic Preservation of Retinal Ganglion Cells in an Ex Vivo Human Glaucoma Model.

Purpose: Glaucoma is a leading cause of irreversible blindness. Glaucomatous intraocular pressure (IOP) triggers deleterious effects, including gliosis, optic nerve (ON) axonal retraction, neurotrophic factor deprivation, inflammation, and other pathological events, leading to retinal ganglion cell (RGC) loss. Trophic factor impairment enhances RGC apoptosis susceptibility. Neuritin 1 (NRN1), a neurotrophic protein downstream of various neurotrophins, exhibited RGC protection and regeneration in axotomy models. We evaluated human recombinant NRN1's impact on human RGCs cultured in pressurized conditions within the ex vivo translaminar autonomous system to simulate glaucoma pathogenesis. Methods: Human glaucomatous and non-glaucomatous donor eyes were obtained from eye banks according to the Declaration of Helsinki. Initially, we evaluated NRN1and RGC marker expression in glaucoma and non-glaucomatous retina to determine the NRN1 level and its association with RGC loss. Further, we evaluated NRN1's therapeutic potential by treating pressurized human eyes at normal and high IOP for seven days. Retina, ON, and conditioned medium were analyzed for RGC survival (THY1, RBPMS), gliosis (GFAP), apoptosis (CASP3, CASP7), and extracellular matrix deposition (COLIV, FN) by qRT-PCR and western blotting. Paraphenylenediamine staining assessed ON axonal degeneration, whereas ex vivo electroretinogram assessed retinal activity. Results: Glaucomatous retinas exhibited significant reductions in both NRN1 (*p = 0.007, n = 5) and RGC marker expression (*p = 0.04, n = 5). NRN1 treatment reduced gliosis, extracellular matrix deposition, ON degeneration, and increased retinal activity in pressure-perfused eyes. Conclusions: Our study confirms that NRN1 enhances human RGC survival and improves retinal function in degenerative conditions, substantiating it as a promising candidate for rescuing human RGCs from degeneration.

CX3CL1-CX3CR1 axis protects retinal ganglion cells by inhibiting microglia activation in a distal optic nerve trauma model

BackgroundThe chemokine CX3CL1 has been reported to play an important role in optic nerve protection, but the underlying mechanism is still unclear. CX3CR1, the only receptor of CX3CL1, is specifically expressed on retinal microglia, whose activation plays a role in the pathological process of optic nerve injury. This study aimed to evaluate whether CX3CL1 exerts optic neuroprotection by affecting the activation of microglia by combining with CX3CR1.MethodsA mouse model of distal optic nerve trauma (ONT) was used to evaluate the effects of the CX3CL1-CX3CR1 axis on the activation of microglia and survival or axonal regeneration of retinal ganglion cells (RGCs). The activation of microglia, loss of RGCs, and damage to visual function were detected weekly till 4 weeks after modeling. CX3CL1 was injected intravitreally immediately or delayed after injury and the status of microglia and RGCs were examined.ResultsIncreases in microglia activation and optic nerve damage were accompanied by a reduced production of the CX3CL1-CX3CR1 axis after the distal ONT modeling. Both immediate and delayed intravitreal injection of CX3CL1 inhibited microglia activation, promoted survival of RGCs, and improved axonal regenerative capacity. Injection with CX3CL1 was no longer effective after 48 h post ONT. The CX3CL1-CX3CR1 axis promotes survival and axonal regeneration, as indicated by GAP43 protein and gene expression, of RGCs by inhibiting the microglial activation after ONT.ConclusionsThe CX3CL1-CX3CR1 axis could promote survival and axonal regeneration of RGCs by inhibiting the microglial activation after optic nerve injury. The CX3CL1-CX3CR1 axis may become a potential target for the treatment of optic nerve injury. Forty-eight hours is the longest time window for effective treatment after injury. The study is expected to provide new ideas for the development of targeted drugs for the repair of optic nerve.

Shootin1 Regulates Retinal Ganglion Cell Neurite Development: Insights From an RGC Direct Somatic Cell Reprogramming Model.

Retinal ganglion cells (RGCs) connect the retina to the brain. Proper development of the axons and dendrites of RGCs is the basis for these cells to function as projection neurons to deliver visual information to the brain. The purpose of this study was to investigate the function of Shtn1 (which encodes shootin1) in RGC neurite development. Immunofluorescence (IF) was used to characterize the expression pattern of marker genes. An in vitro direct somatic cell reprogramming system was used to generate RGC-like neurons (iRGCs), which was subsequently used to study the function of Shtn1. Short-hairpin RNAs (shRNAs) were used to knock down Shtn1, and the coding sequence (CDS) of Shtn1 was used to overexpress the gene. Lentiviruses were used to deliver shRNAs or CDSs into iRGCs. The patch clamp technique was used to measure the electrophysiological properties of the iRGCs. RNA sequencing (RNA-seq) was used to examine transcriptome expression. Using IF, we demonstrated that shootin1 is distinctively expressed in RGCs during the period in which RGCs actively develop and adjust the connections of their neurites with upstream and downstream neurons. Using the iRGC system, we demonstrated that Shtn1 promotes the growth and complexity of neurites and thus the electrophysiological maturation, of iRGCs. RNA-seq analyses showed that Shtn1 may also regulate gene expression and neurogenesis in iRGCs. Shtn1 promotes RGC neurite development. These findings improve our understanding of the molecular machinery governing RGC neurite development and may help to optimize future RGC regeneration methods.

Interleukin-4 protects retinal ganglion cells and promotes axon regeneration

BackgroundThe preservation of retinal ganglion cells (RGCs) and the facilitation of axon regeneration are crucial considerations in the management of various vision-threatening disorders. Therefore, we investigate the efficacy of interleukin-4 (IL-4), a potential therapeutic agent, in promoting neuroprotection and axon regeneration of retinal ganglion cells (RGCs) as identified through whole transcriptome sequencing in an in vitro axon growth model.MethodsA low concentration of staurosporine (STS) was employed to induce in vitro axon growth. Whole transcriptome sequencing was utilized to identify key target factors involved in the molecular mechanism underlying axon growth. The efficacy of recombinant IL-4 protein on promoting RGC axon growth was validated through in vitro experiments. The protective effect of recombinant IL-4 protein on somas of RGCs was assessed using RBPMS-specific immunofluorescent staining in mouse models with optic nerve crush (ONC) and N-methyl-D-aspartic acid (NMDA) injury. The protective effect on RGC axons was evaluated by anterograde labeling of cholera toxin subunit B (CTB), while the promotion of RGC axon regeneration was assessed through both anterograde labeling of CTB and immunofluorescent staining for growth associated protein-43 (GAP43).ResultsWhole-transcriptome sequencing of staurosporine-treated 661 W cells revealed a significant upregulation in intracellular IL-4 transcription levels during the process of axon regeneration. In vitro experiments demonstrated that recombinant IL-4 protein effectively stimulated axon outgrowth. Subsequent immunostaining with RBPMS revealed a significantly higher survival rate of RGCs in the rIL-4 group compared to the vehicle group in both NMDA and ONC injury models. Axonal tracing with CTB confirmed that recombinant IL-4 protein preserved long-distance projection of RGC axons, and there was a notably higher number of surviving axons in the rIL-4 group compared to the vehicle group following NMDA-induced injury. Moreover, intravitreal delivery of recombinant IL-4 protein substantially facilitated RGC axon regeneration after ONC injury.ConclusionThe recombinant IL-4 protein exhibits the potential to enhance the survival rate of RGCs, protect RGC axons against NMDA-induced injury, and facilitate axon regeneration following ONC. This study provides an experimental foundation for further investigation and development of therapeutic agents aimed at protecting the optic nerve and promoting axon regeneration.

NFATc4 Knockout Promotes Neuroprotection and Retinal Ganglion Cell Regeneration After Optic Nerve Injury.

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.

Neutrophil-inflicted vasculature damage suppresses immune-mediated optic nerve regeneration

In adult mammals, injured retinal ganglion cells (RGCs) fail to spontaneously regrow severed axons, resulting in permanent visual deficits. Robust axon growth, however, is observed after intra-ocular injection of particulate β-glucan isolated from yeast. Blood-borne myeloid cells rapidly respond to β-glucan, releasing numerous pro-regenerative factors. Unfortunately, the pro-regenerative effects are undermined by retinal damage inflicted by an overactive immune system. Here, we demonstrate that protection of the inflamed vasculature promotes immune-mediated RGC regeneration. In the absence of microglia, leakiness of the blood-retina barrier increases, pro-inflammatory neutrophils are elevated, and RGC regeneration is reduced. Functional ablation of the complement receptor 3 (CD11b/integrin-αM), but not the complement components C1q-/- or C3-/-, reduces ocular inflammation, protects the blood-retina barrier, and enhances RGC regeneration. Selective targeting of neutrophils with anti-Ly6G does not increase axogenic neutrophils but protects the blood-retina barrier and enhances RGC regeneration. Together, these findings reveal that protection of the inflamed vasculature promotes neuronal regeneration.

Ciliary neurotrophic factor mediated growth of retinal ganglion cell axons on PGS/PCL scaffolds

Ciliary neurotrophic factor (CNTF) promotes survival and/or differentiation of a variety of neuronal cells including retinal ganglion cells (RGCs). Delivery of CNTF requires a suitable medium capable of mediating diffusion and premature release of CNTF within the target tissue. Polymeric tissue-engineered scaffolds have been readily used as substrates for cell transplantation, expansion, and differentiation and, as carriers of cell growth factors. Their functions to CNTF release for RGC proliferation have remained so far unexplored, especially to CNTF affinity to the scaffold and subsequent RGC fate. Electrospun poly(glycerol sebacate)/poly(ϵ-caprolactone) (PGS/PCL) biopolymer scaffolds have recently shown promising results in terms of supporting regeneration of RGC neurites. This work explores covalent immobilization of CNTF on PGS/PCL scaffold and the way immobilised CNTF mediates growth of RGC axons on the scaffold. An ex-vivo three-dimensional model of rodent optic nerve on PGS/PCL revealed that RGC explants cultured in CNTF mediated environment increased their neurite extensions after 20 d of cell culture employing neurite outgrowth measurements. The CNTF secretion on PGS/PCL scaffold was found bio-mimicking natural extracellular matrix of the cell target tissue and, consequently, has shown a potential to improve the overall efficacy of the RGC regeneration process.

Combined treatment of human mesenchymal stem cells and green tea extract on retinal ganglion cell regeneration in rats after optic nerve injury

Retinal ganglion cell (RGC) death and axonal loss cause irreversible vision loss upon optic nerve (ON) injury. We have independently demonstrated that mesenchymal stem cells (MSCs) and green tea extract (GTE) promote RGC survival and axonal regeneration in rats with ON injury. Here we aimed to evaluate the combined treatment effect of human bone marrow-derived MSCs (hBM-MSCs) and GTE on RGC survival and axonal regeneration after ON injury. Combined treatment of hBM-MSCs and GTE promoted RGC survival and neurite outgrowth/axonal regeneration in ex vivo retinal explant culture and in rats after ON injury. GTE increased Stat3 activation in the retina after combined treatment, and enhanced brain-derived neurotrophic factor secretion from hBM-MSCs. Treatment of 10 μg/mL GTE would not induce hBM-MSC apoptosis, but inhibited their proliferation, migration, and adipogenic and osteogenic differentiation in vitro with reducing matrix metalloproteinase secretions. In summary, this study revealed that GTE can enhance RGC protective effect of hBM-MSCs, suggesting that stem cell priming could be a prospective strategy enhancing the properties of stem cells for ON injury treatment.

Retinal circuit disassembly, plasticity, and repair in glaucoma

Precise connections between synaptic partners are shaped during development to ensure proper neural circuit function, but how these connections are disassembled and rearranged after injury is less well understood. In glaucoma, a neurodegenerative disease of the optic nerve, the ganglion cell is injured but very little is known about how the presynaptic circuitry is perturbed. Furthermore, the ability to diagnose and treat glaucoma at an optimal stage before irreversible retinal ganglion cell loss occurs requires a comprehensive understanding of inner retina circuit disassembly and plasticity. This lecture will provide insights into retinal circuit disassembly and plasticity, the role of microglia in synapse disassembly and the opportunities for retinal repair of inner retinal circuitry in glaucoma. These findings expand knowledge of how adult neural circuits react and rearrange in the face of injury and permit.The design of novel psychophysics testing paradigms and retinal ganglion cell regeneration or neuroprotection strategies.

Retinal Ganglion Cell Fate Induction by Ngn-Family Transcription Factors.

Retinal ganglion cells (RGCs) are the projection neurons of the retina. Loss of RGCs is the cellular basis for vision loss in patients with glaucoma. Finding ways to regenerate RGCs will aid in the development of regenerative therapies for patients with glaucoma. The aim of this study was to examine the ability of Ngn-family transcription factors (TFs) to induce RGC regeneration through reprogramming in vitro and in vivo. In vitro, lentiviruses were used to deliver Ngn-TFs into mouse embryonic fibroblasts (MEFs). In vivo, mouse pup retina electroporation was used to deliver Ngn-TFs into late-stage retinal progenitor cells (RPCs). Immunofluorescence staining and RNA sequencing were used to examine cell fate reprogramming; patch-clamp recording was used to examine neuronal electrophysiologic functions. In vitro, all three Ngn-TFs, Ngn1, Ngn2, and Ngn3, were able to work alone to reprogram MEFs into RGC-like neurons that resembled RGCs at the transcriptome level, exhibited typical neuronal membrane electrophysiologic properties, and formed functional synaptic communications with retinal neurons. In vivo, Ngn-TFs reprogrammed the differentiation-competent state of late-stage RPCs to generate RGCs. Ngn-TFs are effective in inducing an RGC-like fate both in vitro and in vivo and might be explored further in the future for glaucoma translational applications.

Modifying treatment of degenerative retinal diseases. Part 2. Conditioning therapy techniques and the problem of maximizing retinal plasticity

In the first part of the review [ROJ, 2023; 16 (2): 160–2], we discussed the common and specific features of adaptive and non-adaptive retinal plasticity characteristic of glaucoma, age-related macular degeneration, retinitis pigmentosa, diabetic retinopathy, and retinopathy of prematurity. The presented part of the review discusses the issues of axon regeneration of retinal ganglion cells and analyzes therapeutic approaches aimed at maximizing the plasticity and stimulating the reparative potential of the retina. The protective effects of "conditioning" stimuli in the modifying treatment of retinal diseases are considered. Some of the present-day visual rehabilitation strategies based on visual perception training and visual fixation training using biofeedback systems are reported.

The Retinal Ganglion Cell Repopulation, Stem Cell Transplantation, and Optic Nerve Regeneration Consortium

The Retinal Ganglion Cell (RGC) Repopulation, Stem Cell Transplantation, and Optic Nerve Regeneration (RReSTORe) consortium was founded in 2021 to help address the numerous scientific and clinical obstacles that impede development of vision-restorative treatments for patients with optic neuropathies. The goals of the RReSTORe consortium are: (1) to define and prioritize the most critical challenges and questions related to RGC regeneration; (2) to brainstorm innovative tools and experimental approaches to meet these challenges; and (3) to foster opportunities for collaborative scientific research among diverse investigators. The RReSTORe consortium currently includes > 220 members spanning all career stages worldwide and is directed by an organizing committee comprised of 15 leading scientists and physician-scientists of diverse backgrounds. Herein, we describe the structure and organization of the RReSTORe consortium, its activities to date, and the perceived impact that the consortium has had on the field based on a survey of participants. In addition to helping propel the field of regenerative medicine as applied to optic neuropathies, the RReSTORe consortium serves as a framework for developing large collaborative groups aimed at tackling audacious goals that may be expanded beyond ophthalmology and vision science. The development of innovative interventions capable of restoring vision for patients suffering from optic neuropathy would be transformative for the ophthalmology field, and may set the stage for functional restoration in other central nervous system disorders. By coordinating large-scale, international collaborations among scientists with diverse and complementary expertise, we are confident that the RReSTORe consortium will help to accelerate the field toward clinical translation. Proprietary or commercial disclosure may be foundin the Footnotes and Disclosures at the end of this article.

Promoting axon regeneration by inhibiting RNA N6-methyladenosine demethylase ALKBH5.

A key limiting factor of successful axon regeneration is the intrinsic regenerative ability in both the peripheral nervous system (PNS) and central nervous system (CNS). Previous studies have identified intrinsic regenerative ability regulators that act on gene expression in injured neurons. However, it is less known whether RNA modifications play a role in this process. Here, we systematically screened the functions of all common m6A modification-related enzymes in axon regeneration and report ALKBH5, an evolutionarily conserved RNA m6A demethylase, as a regulator of axonal regeneration in rodents. In PNS, knockdown of ALKBH5 enhanced sensory axonal regeneration, whereas overexpressing ALKBH5 impaired axonal regeneration in an m6A-dependent manner. Mechanistically, ALKBH5 increased the stability of Lpin2 mRNA and thus limited regenerative growth associated lipid metabolism in dorsal root ganglion neurons. Moreover, in CNS, knockdown of ALKBH5 enhanced the survival and axonal regeneration of retinal ganglion cells after optic nerve injury. Together, our results suggest a novel mechanism regulating axon regeneration and point ALKBH5 as a potential target for promoting axon regeneration in both PNS and CNS.

Novel nano-micro-macro multiple-nested hydrogel with gradient ciliary neurotrophic factor distribution induces directional axon regeneration of retinal ganglion cells

Ciliary neurotrophic factor (CNTF) shows potential for stimulating axon regeneration of retinal ganglion cells (RGCs), whereas unordered regeneration of axons does not have any biological significance. Given the chemotaxis characteristics of RGCs, materials with gradient CNTF distribution can chemically stimulate axon extension based on the CNTF concentration gradient. Therefore, a novel nano-micro-macro multiple-nested hydrogel with gradient CNTF distribution was developed in this study to induce directional axonal outgrowth of RGCs. The CNTF and magnetic nanoparticles (MNPs) were co-loaded into gelatin microspheres (CNTF&PEG-MNP@GMs), and subsequently suspended in a gelatin methacryloyl (GelMA) solution (viscous). Using a magnet, the CNTF&PEG-MNP@GMs were moved from the far-magnet side toward the near-magnet side forming gradient CNTF&PEG-MNP@GMs distribution inside the GelMA solution. The GelMA was then cross-linked via ultraviolet (365 nm) to form a hydrogel with gradient CNTF distribution. This hydrogel was further applied as the matrix for retinal explant culturing. The results showed that the number of axons in the segments of 200–300 µm and > 300 µm was 10.21 times and 15.02 times of those in the Gradient than those in the Without Gradient. The axons area distribution was 3.28 times and 13.41 times, respectively. 86.1% of the axons area in the Gradient were distributed in the segments > 200 µm, whereas only 56.27% in the Without Gradient. The results suggested that the hydrogel-matrix with gradient CNTF distribution could intensely induce directional axonal extension of RGCs in the retinal explant. The present study provides a new method for CNTF distribution, which could induce directional axonal regeneration of RGCs, and in turn have potential application in optic nerve injury repair to some extent.

Promotion of axon regeneration and protection on injured retinal ganglion cells by rCXCL2

BackgroundIn addition to rescuing injured retinal ganglion cells (RGCs) by stimulating the intrinsic growth ability of damaged RGCs in various retinal/optic neuropathies, increasing evidence has shown that the external microenvironmental factors also play a crucial role in restoring the survival of RGCs by promoting the regrowth of RGC axons, especially inflammatory factors. In this study, we aimed to screen out the underlying inflammatory factor involved in the signaling of staurosporine (STS)-induced axon regeneration and verify its role in the protection of RGCs and the promotion of axon regrowth.MethodsWe performed transcriptome RNA sequencing for STS induction models in vitro and analyzed the differentially expressed genes. After targeting the key gene, we verified the role of the candidate factor in RGC protection and promotion of axon regeneration in vivo with two RGC-injured animal models (optic nerve crush, ONC; retinal N-methyl-D-aspartate, NMDA damage) by using cholera toxin subunit B anterograde axon tracing and specific immunostaining of RGCs.ResultsWe found that a series of inflammatory genes expressed upregulated in the signaling of STS-induced axon regrowth and we targeted the candidate CXCL2 gene since the level of the chemokine CXCL2 gene elevated significantly among the top upregulated genes. We further demonstrated that intravitreal injection of rCXCL2 robustly promoted axon regeneration and significantly improved RGC survival in ONC-injured mice in vivo. However, different from its role in ONC model, the intravitreal injection of rCXCL2 was able to simply protect RGCs against NMDA-induced excitotoxicity in mouse retina and maintain the long-distance projection of RGC axons, yet failed to promote significant axon regeneration.ConclusionsWe provide the first in vivo evidence that CXCL2, as an inflammatory factor, is a key regulator in the axon regeneration and neuroprotection of RGCs. Our comparative study may facilitate deciphering the exact molecular mechanisms of RGC axon regeneration and developing high-potency targeted drugs.

Phosphorylated S6K1 and 4E-BP1 play different roles in constitutively active Rheb-mediated retinal ganglion cell survival and axon regeneration after optic nerve injury.

Ras homolog enriched in brain (Rheb) is a small GTPase that activates mammalian target of rapamycin complex 1 (mTORC1). Previous studies have shown that constitutively active Rheb can enhance the regeneration of sensory axons after spinal cord injury by activating downstream effectors of mTOR. S6K1 and 4E-BP1 are important downstream effectors of mTORC1. In this study, we investigated the role of Rheb/mTOR and its downstream effectors S6K1 and 4E-BP1 in the protection of retinal ganglion cells. We transfected an optic nerve crush mouse model with adeno-associated viral 2-mediated constitutively active Rheb and observed the effects on retinal ganglion cell survival and axon regeneration. We found that overexpression of constitutively active Rheb promoted survival of retinal ganglion cells in the acute (14 days) and chronic (21 and 42 days) stages of injury. We also found that either co-expression of the dominant-negative S6K1 mutant or the constitutively active 4E-BP1 mutant together with constitutively active Rheb markedly inhibited axon regeneration of retinal ganglion cells. This suggests that mTORC1-mediated S6K1 activation and 4E-BP1 inhibition were necessary components for constitutively active Rheb-induced axon regeneration. However, only S6K1 activation, but not 4E-BP1 knockdown, induced axon regeneration when applied alone. Furthermore, S6K1 activation promoted the survival of retinal ganglion cells at 14 days post-injury, whereas 4E-BP1 knockdown unexpectedly slightly decreased the survival of retinal ganglion cells at 14 days post-injury. Overexpression of constitutively active 4E-BP1 increased the survival of retinal ganglion cells at 14 days post-injury. Likewise, co-expressing constitutively active Rheb and constitutively active 4E-BP1 markedly increased the survival of retinal ganglion cells compared with overexpression of constitutively active Rheb alone at 14 days post-injury. These findings indicate that functional 4E-BP1 and S6K1 are neuroprotective and that 4E-BP1 may exert protective effects through a pathway at least partially independent of Rheb/mTOR. Together, our results show that constitutively active Rheb promotes the survival of retinal ganglion cells and axon regeneration through modulating S6K1 and 4E-BP1 activity. Phosphorylated S6K1 and 4E-BP1 promote axon regeneration but play an antagonistic role in the survival of retinal ganglion cells.

The Optic Nerve Crush Injury Paradigm in African Turquoise Killifish to Study Axonal Regeneration in an Aged Environment.

In our graying world population, we are increasingly facing brain injuries and age-associated neurodegenerative diseases, which are often characterized by axonal pathology. Here, we propose the killifish visual/retinotectal system as a model for investigating central nervous system repair, more specifically axonal regeneration, in an aging context. We first describe an optic nerve crush (ONC) injury paradigm in killifish to induce and study both de- and regeneration of retinal ganglion cells (RGCs) and their axons. Subsequently, we summarize several methods for mapping different steps of the regenerative process-namely, axonal regrowth and synapse reformation-using retro- and anterograde tracing methods, (immuno)histochemistry, and morphometrical analyses.

Purified regenerating retinal neurons reveal regulatory role of DNA methylation-mediated Na+/K+-ATPase in murine axon regeneration

While embryonic mammalian central nervous system (CNS) axons readily grow and differentiate, only a minority of fully differentiated mature CNS neurons are able to regenerate injured axons, leading to stunted functional recovery after injury and disease. To delineate DNA methylation changes specifically associated with axon regeneration, we used a Fluorescent-Activated Cell Sorting (FACS)-based methodology in a rat optic nerve transection model to segregate the injured retinal ganglion cells (RGCs) into regenerating and non-regenerating cell populations. Whole-genome DNA methylation profiling of these purified neurons revealed genes and pathways linked to mammalian RGC regeneration. Moreover, whole-methylome sequencing of purified uninjured adult and embryonic RGCs identified embryonic molecular profiles reactivated after injury in mature neurons, and others that correlate specifically with embryonic or adult axon growth, but not both. The results highlight the contribution to both embryonic growth and adult axon regeneration of subunits encoding the Na+/K+-ATPase. In turn, both biochemical and genetic inhibition of the Na+/K+-ATPase pump significantly reduced RGC axon regeneration. These data provide critical molecular insights into mammalian CNS axon regeneration, pinpoint the Na+/K+-ATPase as a key regulator of regeneration of injured mature CNS axons, and suggest that successful regeneration requires, in part, reactivation of embryonic signals.

Assaying Optic Nerve Regeneration in Larval Zebrafish.

Zebrafish have a remarkable capacity for spontaneously regenerating their central nervous system. Larval zebrafish are optically transparent and therefore are widely used to dynamically visualize cellular processes in vivo, such as nerve regeneration. Regeneration of retinal ganglion cell (RGC) axons within the optic nerve has been previously studied in adult zebrafish. In contrast, assays of optic nerve regeneration have previously not been established in larval zebrafish. In order to take advantage of the imaging capabilities in the larval zebrafish model, we recently developed an assay to physically transect RGC axons and monitor optic nerve regeneration in larval zebrafish. We found that RGC axons rapidly and robustly regrow to the optic tectum. Here, we describe the methods for performing the optic nerve transections, as well as methods for visualizing RGC regeneration in larval zebrafish.