Retinal regeneration requires dynamic Notch signaling

Retinal damage in the adult zebrafish induces Müller glia reprogramming to produce neuronal progenitor cells that proliferate and differentiate into retinal neurons.Notch signaling,which is a fundamental mechanism known to drive cell-cell communication,is required to maintain Müller glia in a quiescent state in the undamaged retina,and repression of Notch signaling is necessary for Müller glia to reenter the cell cycle.The dynamic regulation of Notch signaling following retinal damage also directs proliferation and neurogenesis of the Müller glia-derived progenitor cells in a robust regeneration response.In contrast,mammalian Müller glia respond to retinal damage by entering a prolonged gliotic state that leads to additional neuronal death and permanent vision loss.Understanding the dynamic regulation of Notch signaling in the zebrafish retina may aid efforts to stimulate Müller glia reprogramming for regeneration of the diseased human retina.Recent findings identified DeltaB and Notch3 as the ligand-receptor pair that serves as the principal regulators of zebrafish Müller glia quiescence.In addition,multi-omics datasets and functional studies indicate that additional Notch receptors,ligands,and target genes regulate cell proliferation and neurogenesis during the regeneration time course.Still,our understanding of Notch signaling during retinal regeneration is limited.To fully appreciate the complex regulation of Notch signaling that is required for successful retinal regeneration,investigation of additional aspects of the pathway,such as post-translational modification of the receptors,ligand endocytosis,and interactions with other fundamental pathways is needed.Here we review various modes of Notch signaling regulation in the context of the vertebrate retina to put recent research in perspective and to identify open areas of inquiry.

A novel mode of retinal regeneration:the merit of a new Xenopus model

Retinal regeneration： The retina is a part of the central nervous system （CNS） and has long attracted neurobiologists as an excellent model organ for the study of CNS regeneration. In classical studies using urodele amphibians like the salamander newt, it has been shown that the retina regenerates after the removal of the whole tissue even in the adulthood. This type of regeneration is considered as an example of ＂transdifferentiation＇, since the source of the regenerating retina is the retinal pigmented epithelial cells （RPE cells） （Okada, 1991;

Live-cell imaging:new avenues to investigate retinal regeneration

Sensing and responding to our environment requires functional neurons that act in concert. Neuronal cell loss resulting from degenerative diseases cannot be replaced in humans, causing a functional impairment to integrate and/or respond to sensory cues. In contrast, zebrafish（Danio rerio） possess an endogenous capacity to regenerate lost neurons. Here, we will focus on the processes that lead to neuronal regeneration in the zebrafish retina. Dying retinal neurons release a damage signal, tumor necrosis factor α, which induces the resident radial glia, the Müller glia, to reprogram and re-enter the cell cycle. The Müller glia divide asymmetrically to produce a Müller glia that exits the cell cycle and a neuronal progenitor cell. The arising neuronal progenitor cells undergo several rounds of cell divisions before they migrate to the site of damage to differentiate into the neuronal cell types that were lost. Molecular and immunohistochemical studies have predominantly provided insight into the mechanisms that regulate retinal regeneration. However, many processes during retinal regeneration are dynamic and require live-cell imaging to fully discern the underlying mechanisms. Recently, a multiphoton imaging approach of adult zebrafish retinal cultures was developed. We will discuss the use of live-cell imaging, the currently available tools and those that need to be developed to advance our knowledge on major open questions in the field of retinal regeneration.

Induction of Differentiation of Mesenchymal Stem Cells into Retinal Pigment Epithelial Cells for Retinal Regeneration by Using Ciliary Neurotrophic Factor in Diabetic Rats

Diabetic retinopathy(DR)is a common cause of blindness all over the world.Bone marrow mesenchymal stem cells(BMSCs)have been considered as a promising strategy for retinal regeneration in the treatment of DR.However,the poor viability and low levels of BMSCs engraftment limit the therapeutic potential of BMSCs.The present study aimed to examine the direct induction of BMSCs differentiation into the cell types related to retinal regeneration by using soluble cytokine ciliary neurotrophic factor(CNTF).We observed remarkably increased expression of cellular retinaldehyde-binding protein(CRALBP)and retinoid isomerohydrolase(RPE65)in BMSCs treated with CNTF in vitro,indicating the directional differentiation of BMSCs into the retinal pigment epithelium(RPE)cells,which are crucial for retinal healing.In vivo,the diabetic rat model was established by use of streptozotocin(STZ),and animals treated with BMSCs+CNTF exhibited better viability and higher delivery efficiency of the transplanted cells than those treated with BMSCs injection alone.Similar to the in-vitro result,treatment with BMSCs and CNTF combined led to the differentiation of BMSCs into beneficial cells(RPE cells),and accelerated retinal healing characterized by the activation of rod photoreceptor cells and phagocytosis function of RPE cells.In conclusion,CNTF contributes to the differentiation of BMSCs into RPE cells,which may help overcome the current stem cell therapy limitations in the field of retinal regeneration.

Paving the way toward retinal regeneration with mesencephalic astrocyte-derived neurotrophic factor

Neurodegenerative diseases are a leading cause of disability worldwide,and despite significant resources put toward the discovery of potential therapeutic targets,there are currently no effective treatments.The rise of methods to derive and propagate stem cells in vitro offered

Gene therapy and the regeneration of retinal ganglion cell axons

Because the adult mammalian central nervous system （CNS） has only limited intrinsic capacity to regenerate connections after injury, due to factors both intrinsic and extrinsic to the mature neuron （Shen et al., 1999; Berry et al., 2008; Lingor et al., 2008; Sun and He, 2010; Moore et al., 2011 ）, therapies are required to support the survival of injured neu-rons and to promote the long-distance regrowth of axons back to their original target structures. The retina and optic nerve （ON） are part of the CNS and this system is much used in experiments designed to test new ways of promoting regeneration after injury （Harvey et al., 2006; Benowitz and Yin, 2008; Berry et al., 2008; Fischer and Leibinger, 2012）. Testing of therapies designed to improve retinal ganglion cell （RGC） viability also has direct clinical relevance because there is loss of these centrally projecting neurons in many ophthalmic diseases.

Mesenchymal stem cell therapy for retinal ganglion cell neuroprotection and axon regeneration

Retinal ganglion cells （RGCs） are responsible for propagat- ing signals derived from visual stimuli in the eye to the brain, along their axons within the optic nerve to the superior colliculus, lateral geniculate nucleus and visu- al cortex of the brain. Damage to the optic nerve either through trauma, such as head injury, or degenerative dis- ease, such as glaucoma causes irreversible loss of function through degeneration of non-regenerating RGC axons and death of irreplaceable RGCs, ultimately leading to blindness （Berry et al., 2008）. The degeneration of RGCs and their axons is due to the loss of the necessary source of retrogradely transported neurotrophic factors （NTFs） being hindered by axonal injury. NTFs are survival factors for neurons and play a pivotal part in axon regeneration. Stem cells particularly mesenchymal stem cells （MSCs） have been shown to possess a natural intrinsic capacity for paracrine support, releasing multiple signalling mol- ecules including NTFs. By transplanting MSCs into the vitreous, they are positioned adjacent to the injured reti- na to provide paracrine-mediated therapy for the retinal neuronal cells （Johnson et al., 2010a; Mead et al., 2013）. Additionally, MSCs may be pre-differentiated into sup- portive glial-like cells, such as Schwann cells, which could further increase their potential for paracrine support of injured neurons （Martens et al., 2013）. Thus, MSCs have received considerable attention as a new cellular therapy for both traumatic and degenerative eye disease, acting as an alternative source of NTFs, protecting injured RGCs and promoting regeneration of their axons （Figure 1）.

Editor's Choice——Retinal ganglion cell damage and regeneration

Retinal ganglion cell apoptosis is considered to be the main cause of loss of vision in glaucoma patients. Microglia cells are phagocytic cells present in the retina. In the retina of glaucoma rat models, microglia cells become activated, which suggests a role for microglia in the pathogenesis of optic nerve injury in glaucoma patients. The retinal ganglion cell is the only cell that can produce action potential in the retina,

The role of Toll-like receptors in retinal ischemic diseases

Toll-like receptors（TLRs） are commonly referred to a series of evolutionary conserved receptors which recognize and respond to various microbes and endogenous ligands.Growing evidence has demonstrated that the expression of TLRs in the retina is regulated during retinal ischemic diseases,including ischemia-reperfusion injury,glaucoma,diabetic retinopathy（DR） and retinopathy of prematurity（ROP）.TLRs can be expressed in multiple cells in the retina,such as glial cells,retinal pigment epithelium（RPE）,as well as photoreceptor cells and endothelium cells.Activation of TLRs in retina could initiate a complex signal transduction cascade,induce the production of inflammatory cytokines and regulate the level of costimulatory molecules,which play prominent roles in the pathogenesis of retinal ischemic diseases.In this review,we summarized current studies about the relationship between TLRs and ischemic retinopathy.A greater understanding of the effect of TLRs on ischemic injuries may contribute to the development of specific TLR targeted therapeutic strategies in these conditions.

Correlation between photoreceptor injury-regeneration and behavior in a zebrafish model

Direct exposure to intensive visible light can lead to solar retinopathy, including macular injury. The signs and symptoms include central scotoma, metamorphopsia, and decreased vision. However, there have been few studies examining retinal injury due to intensive light stimulation at the cellular level. Neural network arrangements and gene expression patterns in zebrafish photoreceptors are similar to those observed in humans, and photoreceptor injury in zebrafish can induce stem cell-based cellular regeneration. Therefore, the zebrafish retina is considered a useful model for studying photoreceptor injury in humans. In the current study, the central retinal photoreceptors of zebrafish were selectively ablated by stimulation with high-intensity light. Retinal injury, cell proliferation and regeneration of cones and rods were assessed at 1, 3 and 7 days post lesion with immunohistochemistry and in situ hybridization. Additionally, a light/dark box test was used to assess zebrafish behavior. The results revealed that photoreceptors were regenerated by 7 days after the light-induced injury. However, the regenerated cells showed a disrupted arrangement at the lesion site. During the injury-regeneration process, the zebrafish exhibited reduced locomotor capacity, weakened phototaxis and increased movement angular velocity. These behaviors matched the morphological changes of retinal injury and regeneration in a number of ways. This study demonstrates that the zebrafish retina has a robust capacity for regeneration. Visual impairment and stress responses following high-intensity light stimulation appear to contribute to the alteration of behaviors.

Absence of ephrin-A2/A3 increases retinal regenerative potential for Müller cells in Rhodopsin knockout mice

Müller cells(MC) are considered dormant retinal progenitor cells in mammals.Previous studies demonstrated ephrin-As act as negative regulators of neural progenitor cells in the retina and brain.It remains unclear whether the lack of ephrin-A2/A3 is sufficient to promote the neurogenic potential of MC.Here we investigated whether the MC is the primary retinal cell type expressing ephrin-A2/A3 and their role on the neurogenic potential of Müller cells.In this study, we showed that ephrin-A2/A3 and their receptor EphA4 were expressed in retina and especially enriched in MC.The level of ephrin As/EphA4 expression increased as the retina matured that is correlated with the reduced proliferative and progenitor cell potential of MC.Next, we investigated the proliferation in primary MC cultures isolated from wild-type and A2~(–/–) A3~(–/–) mice by 5-ethynyl-2′-deoxyuridine(EdU) incorporation.We detected a significant increase of EdU~+ cells in MC derived from A2~(–/–) A3~(–/–) mice.Next, we investigated the role of ephrin-A2/A3 in mice undergoing photoreceptor degeneration such as Rhodopsin knockout(Rho~(–/–)) mice.To further evaluate the role of ephrin-A2/A3 in MC proliferation in vivo, EdU was injected intraperitoneally to adult wild-type, A2~(–/–) A3~(–/–), Rho~(–/–) and Rho~(–/–) A2~(–/–) A3~(–/–) mice and the numbers of EdU~+ cells distributed among different layers of the retina.Ephrin As/EphA4 expression was upregulated in the retina of Rho~(–/–) mice compared to the wild-type mice.In addition, cultured MC derived from ephrin-A2~(–/–) A3~(–/–) mice also expressed higher levels of progenitor cell markers and exhibited higher proliferation potential than those from wild-type mice.Interestingly, we detected a significant increase of EdU~+ cells in the retinas of adult ephrin-A2~(–/–) A3~(–/–) mice mainly in the inner nuclear layer;and these EdU~+ cells were co-localized with MC marker, cellular retinaldehyde-binding protein, suggesting some proliferating cells are from MC.In Rhodopsin knockout mice(Rho~(–/–) A2~(–/–) A3~(–/–) mice), a significantly greater amount of EdU~+ cells were located in the ciliary body, retina and RPE than that of Rho~(–/–) mice.Comparing between 6 and 12 weeks old Rho~(–/–) A2~(–/–) A3~(–/–) mice, we recorded more EdU~+ cells in the outer nuclear layer in the 12-week-old mice undergoing severe retinal degeneration.Taken together, Ephrin-A2/A3 are negative regulators of the proliferative and neurogenic potentials of MC.Absence of ephrin-A2/A3 promotes the migration of proliferating cells into the outer nuclear layer and may lead to retinal cell regeneration.All experimental procedures were approved by the Animal Care and Use Committee at Schepens Eye Research Institute, USA(approval No.S-353-0715) on October 24, 2012.

Imipramine protects retinal ganglion cells from oxidative stress through the tyrosine kinase receptor B signaling pathway

Retinal ganglion cell（RGC） degeneration is irreversible in glaucoma and tyrosine kinase receptor B（Trk B）-associated signaling pathways have been implicated in the process.In this study,we attempted to examine whether imipramine,a tricyclic antidepressant,may protect hydrogen peroxide（H_2O_2）-induced RGC degeneration through the activation of the Trk B pathway in RGC-5 cell lines.RGC-5 cell lines were pre-treated with imipramine 30 minutes before exposure to H_2O_2.Western blot assay showed that in H_2O_2-damaged RGC-5 cells,imipramine activated Trk B pathways through extracellular signal-regulated protein kinase/Trk B phosphorylation.TUNEL staining assay also demonstrated that imipramine ameliorated H_2O_2-induced apoptosis in RGC-5 cells.Finally,Trk B-Ig G intervention was able to reverse the protective effect of imipramine on H_2O_2-induced RGC-5 apoptosis.Imipramine therefore protects RGCs from oxidative stress-induced apoptosis through the Trk B signaling pathway.

Construction of a plasmid for human brain-derived neurotrophic factor and its effect on retinal pigment epithelial cell viability

Several studies have investigated the protective functions of brain-derived neurotrophic factor（BDNF） in retinitis pigmentosa. However, a BDNF-based therapy for retinitis pigmentosa is not yet available. To develop an efficient treatment for fundus disease, an eukaryotic expression plasmid was generated and used to transfect human 293 T cells to assess the expression and bioactivity of BDNF on acute retinal pigment epithelial-19（ARPE-19） cells, a human retinal epithelial cell line. After 96 hours of co-culture in a Transwell chamber, ARPE-19 cells exposed to BDNF secreted by 293 T cells were more viable than ARPE-19 cells not exposed to secreted BDNF. Western blot assay showed that Bax levels were downregulated and that Bcl-2 levels were upregulated in human ARPE-19 cells exposed to BDNF. Furthermore, 293 T cells transfected with the BDNF gene steadily secreted the protein. The powerful anti-apoptotic function of this BDNF may be useful for the treatment of retinitis pigmentosa and other retinal degenerative diseases.

Retinal ganglion cells regenerate long-distance axons through neural activity stimulation and find their way back to the brain

Human central nerve system(CNS)is an extremely complex and delicate structure.While regeneration is possible in some reptiles and fish CNS,the regeneration capacity seems completely lost in adult mammals.Therefore,the classic concept is that once neurons in mammal

A cascade model of information processing and encoding for retinal prosthesis

Retinal prosthesis offers a potential treatment for individuals suffering from photoreceptor degeneration diseases.Establishing biological retinal models and simulating how the biological retina convert incoming light signal into spike trains that can be properly decoded by the brain is a key issue.Some retinal models have been presented,ranking from structural models inspired by the layered architecture to functional models originated from a set of specific physiological phenomena.However,Most of these focus on stimulus image compression,edge detection and reconstruction,but do not generate spike trains corresponding to visual image.In this study,based on stateof-the-art retinal physiological mechanism,including effective visual information extraction,static nonlinear rectification of biological systems and neurons Poisson coding,a cascade model of the retina including the out plexiform layer for information processing and the inner plexiform layer for information encoding was brought forward,which integrates both anatomic connections and functional computations of retina.Using MATLAB software,spike trains corresponding to stimulus image were numerically computed by four steps：linear spatiotemporal filtering,static nonlinear rectification,radial sampling and then Poisson spike generation.The simulated results suggested that such a cascade model could recreate visual information processing and encoding functionalities of the retina,which is helpful in developing artificial retina for the retinally blind.

Glaucomatous optic neuropathy treatment options:the promise of novel therapeutics,techniques and tools to help preserve vision

Peripheral vision loss followed by ＂tunnel vision＂ and eventual irreversible blindness is the fate of patients afflicted by various forms of glaucoma including primary open-angle glaucoma（POAG） and normotensive glaucoma（NTG）.These complex and heterogeneous diseases are characterized by extensive death of retinal ganglion cells（RGCs） accompanied by retraction and severance of their axonal connections to the brain and thus damage to and thinning of the optic nerve.Since patients suffering from this glaucomatous optic neuropathy（GON） first notice visual impairment when they have lost 〉 40% of their RGCs,early diagnosis is the key to retard the progression of glaucoma.Elevated intraocular pressure（IOP）,low cerebrospinal and/or low intracranial fluid pressure,advancing age,and ethnicity are major risk factors associated with POAG.However,retinal vascular abnormalities and a high sensitivity of RGCs and optic nerve head components to neurotoxic,inflammatory,oxidative and mechanical insults also contribute to vision loss in POAG/GON.Current treatment modalities for POAG and NTG involve lowering IOP using topical ocular drugs,combination drug products,and surgical interventions.Two recently approved multi-pharmacophoric drugs（e.g.,rho kinase inhibitor,Netarsudil;a drug conjugate,Latanoprostene Bunod） and novel aqueous humor drainage devices（i Stent and Cy Pass） are also gaining acceptance for treating POAG/NTG.Neuroprotective and regenerative agents,coupled with electroceutical,mechanical support systems,stem cell transplantation and gene therapy are emerging therapeutics on the horizon to help combat GON.The latter techniques and approaches hope to rejuvenate RGCs and repair the optic nerve structures,thereby providing a gain of function of the visual system for the glaucoma patients.

Macular thickness as a predictor of loss of visual sensitivity in ethambutol-induced optic neuropathy

Ethambutol is a common cause of drug-related optic neuropathy.Prediction of the onset of ethambutol-induced optic neuropathy and consequent drug withdrawal may be an effective method to stop visual loss.Previous studies have shown that structural injury to the optic nerve occurred earlier than the damage to visual function.Therefore,we decided to detect structural biomarkers marking visual field loss in early stage ethambutol-induced optic neuropathy.The thickness of peripapillary retinal nerve fiber layer,macular thickness and visual sensitivity loss would be observed in 11 ethambutol-induced optic neuropathy patients（22 eyes） using optical coherence tomography.Twenty-four healthy age-and sex-matched participants（48 eyes） were used as controls.Results demonstrated that the temporal peripapillary retinal nerve fiber layer thickness and average macular thickness were thinner in patients with ethambutol-induced optic neuropathy compared with healthy controls.The average macular thickness was strongly positively correlated with central visual sensitivity loss（r2=0.878,P=0.000）.These findings suggest that optical coherence tomography can be used to efficiently screen patients.Macular thickness loss could be a potential factor for predicting the onset of ethambutol-induced optic neuropathy.

The promise of stem cell-based therapeutics in ophthalmology

The promising role of cellular therapies in the preservation and restoration of visual function has prompted intensive efforts to characterize embryonic, adult, and induced pluripotent stem cells for regenerative purposes. Three main approaches to the use of stem cells have been described： sustained drug delivery, immunomodulation, and differentiation into various ocular structures. Studies of the differentiation capacity of all three types of stem cells into epithelial, neural, glial and vascular phenotypes have reached proof-of-concept in culture, but the correction of vision is still in the early developmental stages, and the requirements for effective in vivo implementation are still unclear. We present an overview of some of the preclinical findings on stem-cell rescue and regeneration of the cornea and retina in acute injury and degenerative disorders.

Genetic and epigenetic regulators of retinal Müller glial cell reprogramming

Background:Retinal diseases characterized with irreversible loss of retinal nerve cells,such as optic atrophy and retinal degeneration,are the main causes of blindness.Current treatments for these diseases are very limited.An emerging treatment strategy is to induce the reprogramming of Müller glial cells to generate new retinal nerve cells,which could potentially restore vision.Main text:Müller glial cells are the predominant glial cells in retinae and play multiple roles to maintain retinal homeostasis.In lower vertebrates,such as in zebrafish,Müller glial cells can undergo cell reprogramming to regenerate new retinal neurons in response to various damage factors,while in mammals,this ability is limited.Interestingly,with proper treatments,Müller glial cells can display the potential for regeneration of retinal neurons in mammalian retinae.Recent studies have revealed that dozens of genetic and epigenetic regulators play a vital role in inducing the reprogramming of Müller glial cells in vivo.This review summarizes these critical regulators for Müller glial cell reprogramming and highlights their differences between zebrafish and mammals.Conclusions:A number of factors have been identified as the important regulators in Müller glial cell reprogramming.The early response of Müller glial cells upon acute retinal injury,such as the regulation in the exit from quiescent state,the initiation of reactive gliosis,and the re-entry of cell cycle of Müller glial cells,displays significant difference between mouse and zebrafish,which may be mediated by the diverse regulation of Notch and TGFβ(transforming growth factor-β)isoforms and different chromatin accessibility.

Advances in optic nerve regeneration and neuroprotection strategies

The most common irreversible blindness diseases are age-related macular degeneration, glaucoma, anddiabetic retinopathy which involve the optic nerve or retina. These diseases share a common condition of causing blindness - progressive neural cells loss of retina （photoreceptor ceils, retinal ganglion cells （RGCs））. Although many advances in the treatment for these diseases have been achieved in recent years, the visual function often cannot be reversed. To improve the visual outcomes, the retinal neuron cells must be rescued. Optic nerve diseases including glaucoma were mostly studied for the effort to rescue the injured neurons and regenerate the neuron axons.