Retinal Regeneration Research Articles

Current Challenges of Retinal Ganglion Cell Regeneration

Current Challenges of Retinal Ganglion Cell Regeneration

MTOR activity is essential for retinal pigment epithelium regeneration in zebrafish.

The retinal pigment epithelium (RPE) plays numerous critical roles in maintaining vision and this is underscored by the prevalence of degenerative blinding diseases like age-related macular degeneration (AMD), in which visual impairment is caused by progressive loss of RPE cells. In contrast to mammals, zebrafish possess the ability to intrinsically regenerate a functional RPE layer after severe injury. The molecular underpinnings of this regenerative process remain largely unknown yet hold tremendous potential for developing treatment strategies to stimulate endogenous regeneration in the human eye. In this study, we demonstrate that the mTOR pathway is activated in RPE cells post-genetic ablation. Pharmacological and genetic inhibition of mTOR activity impaired RPE regeneration, while mTOR activation enhanced RPE recovery post-injury, demonstrating that mTOR activity is essential for RPE regeneration in zebrafish. RNA-seq of RPE isolated from mTOR-inhibited larvae identified a number of genes and pathways dependent on mTOR activity at early and late stages of regeneration; amongst these were components of the immune system, which is emerging as a key regulator of regenerative responses across various tissue and model systems. Our results identify crosstalk between macrophages/microglia and the RPE, wherein mTOR activity is required for recruitment of macrophages/microglia to the RPE injury site. Macrophages/microglia then reinforce mTOR activity in regenerating RPE cells. Interestingly, the function of macrophages/microglia in maintaining mTOR activity in the RPE appeared to be inflammation-independent. Taken together, these data identify mTOR activity as a key regulator of RPE regeneration and link the mTOR pathway to immune responses in facilitating RPE regeneration.

Intravitreal Administration of rhNGF Enhances Regenerative Processes in a Zebrafish Model of Retinal Degeneration.

Nerve growth factor (NGF) is the best characterized neurotrophin, and it is known to play an important role in ocular homeostasis. Here, we demonstrated the expression of NGF receptors in adult zebrafish retina and optimized a light-induced retina degeneration (LID) zebrafish model that mimics human cone-rod disorders, demonstrating that intravitreal (IV) administration of rhNGF can boost zebrafish retinal regeneration in this model. Adult zebrafish retinae exposed to 60 h of light irradiation (60 h LID) displayed evident reduction of outer nuclear layer (ONL) thickness and cell number with presence of apoptotic cells. Retinal histologic evaluation at different time points showed that IV therapeutic injection of rhNGF resulted in an increase of ONL thickness and cell number at late time points after damage (14 and 21 days post injury), ultimately accelerating retinal tissue recovery by driving retinal cell proliferation. At a molecular level, rhNGF activated the ERK1/2 pathway and enhanced the regenerative potential of Müller glia gfap- and vim-expressing cells by stimulating at early time points the expression of the photoreceptor regeneration factor Drgal1-L2. Our results demonstrate the highly conserved nature of NGF canonical pathway in zebrafish and thus support the use of zebrafish models for testing new compounds with potential retinal regenerative properties. Moreover, the pro-regenerative effects of IV-injected NGF that we observed pave the way to further studies aimed at evaluating its effects also in mammals, in order to expedite the development of novel rhNGF-based therapeutic approaches for ophthalmological disorders.

Pigment Epithelia of the Eye: Cell-Type Conversion in Regeneration and Disease.

Pigment epithelial cells (PECs) of the retina (RPE), ciliary body, and iris (IPE) are capable of altering their phenotype. The main pathway of phenotypic switching of eye PECs in vertebrates and humans in vivo and/or in vitro is neural/retinal. Besides, cells of amphibian IPE give rise to the lens and its derivatives, while mammalian and human RPE can be converted along the mesenchymal pathway. The PECs’ capability of conversion in vivo underlies the lens and retinal regeneration in lower vertebrates and retinal diseases such as proliferative vitreoretinopathy and fibrosis in mammals and humans. The present review considers these processes studied in vitro and in vivo in animal models and in humans. The molecular basis of conversion strategies in PECs is elucidated. Being predetermined onto- and phylogenetically, it includes a species-specific molecular context, differential expression of transcription factors, signaling pathways, and epigenomic changes. The accumulated knowledge regarding the mechanisms of PECs phenotypic switching allows the development of approaches to specified conversion for many purposes: obtaining cells for transplantation, creating conditions to stimulate natural regeneration of the retina and the lens, blocking undesirable conversions associated with eye pathology, and finding molecular markers of pathology to be targets of therapy.

Disruption of miR-18a Alters Proliferation, Photoreceptor Replacement Kinetics, Inflammatory Signaling, and Microglia/Macrophage Numbers During Retinal Regeneration in Zebrafish.

In mammals, photoreceptor loss causes permanent blindness, but in zebrafish (Danio rerio), photoreceptor loss reprograms Müller glia to function as stem cells, producing progenitors that regenerate photoreceptors. MicroRNAs (miRNAs) regulate CNS neurogenesis, but the roles of miRNAs in injury-induced neuronal regeneration are largely unknown. In the embryonic zebrafish retina, miR-18a regulates photoreceptor differentiation. The purpose of the current study was to determine, in zebrafish, the function of miR-18a during injury-induced photoreceptor regeneration. RT-qPCR, in situ hybridization, and immunohistochemistry showed that miR-18a expression increases throughout the retina between 1 and 5days post-injury (dpi). To test miR-18a function during photoreceptor regeneration, we used homozygous miR-18a mutants (miR-18ami5012), and knocked down miR-18a with morpholino oligonucleotides. During photoreceptor regeneration, miR-18ami5012 retinas have fewer mature photoreceptors than WT at 7 and 10 dpi, but there is no difference at 14 dpi, indicating that photoreceptor regeneration is delayed. Labeling dividing cells with 5-bromo-2'-deoxyuridine (BrdU) showed that at 7 and 10 dpi, there are excess dividing progenitors in both mutants and morphants, indicating that miR-18a negatively regulates injury-induced proliferation. Tracing 5-ethynyl-2'-deoxyuridine (EdU) and BrdU-labeled cells showed that in miR-18ami5012 retinas excess progenitors migrate to other retinal layers in addition to the photoreceptor layer. Inflammation is critical for photoreceptor regeneration, and RT-qPCR showed that in miR-18ami5012 retinas, inflammatory gene expression and microglia activation are prolonged. Suppressing inflammation with dexamethasone rescues the miR-18ami5012 phenotype. Together, these data show that in the injured zebrafish retina, disruption of miR-18a alters proliferation, inflammation, the microglia/macrophage response, and the timing of photoreceptor regeneration.

Neuroinflammation, Microglia and Implications for Retinal Ganglion Cell Survival and Axon Regeneration in Traumatic Optic Neuropathy.

Traumatic optic neuropathy (TON) refers to a pathological condition caused by a direct or indirect insult to the optic nerves, which often leads to a partial or permanent vision deficit due to the massive loss of retinal ganglion cells (RGCs) and their axonal fibers. Retinal microglia are immune-competent cells residing in the retina. In rodent models of optic nerve crush (ONC) injury, resident retinal microglia gradually become activated, form end-to-end alignments in the vicinity of degenerating RGC axons, and actively internalized them. Some activated microglia adopt an amoeboid morphology that engulf dying RGCs after ONC. In the injured optic nerve, the activated microglia contribute to the myelin debris clearance at the lesion site. However, phagocytic capacity of resident retinal microglia is extremely poor and therefore the clearance of cellular and myelin debris is largely ineffective. The presence of growth-inhibitory myelin debris and glial scar formed by reactive astrocytes inhibit the regeneration of RGC axons, which accounts for the poor visual function recovery in patients with TON. In this Review, we summarize the current understanding of resident retinal microglia in RGC survival and axon regeneration after ONC. Resident retinal microglia play a key role in facilitating Wallerian degeneration and the subsequent axon regeneration after ONC. However, they are also responsible for producing pro-inflammatory cytokines, chemokines, and reactive oxygen species that possess neurotoxic effects on RGCs. Intraocular inflammation triggers a massive influx of blood-borne myeloid cells which produce oncomodulin to promote RGC survival and axon regeneration. However, intraocular inflammation induces chronic neuroinflammation which exacerbates secondary tissue damages and limits visual function recovery after ONC. Activated retinal microglia is required for the proliferation of oligodendrocyte precursor cells (OPCs); however, sustained activation of retinal microglia suppress the differentiation of OPCs into mature oligodendrocytes for remyelination after injury. Collectively, controlled activation of retinal microglia and infiltrating myeloid cells facilitate axon regeneration and nerve repair. Recent advance in single-cell RNA-sequencing and identification of microglia-specific markers could improve our understanding on microglial biology and to facilitate the development of novel therapeutic strategies aiming to switch resident retinal microglia’s phenotype to foster neuroprotection.

Rod genesis driven by mafba in an nrl knockout zebrafish model with altered photoreceptor composition and progressive retinal degeneration.

Neural retina leucine zipper (NRL) is an essential gene for the fate determination and differentiation of the precursor cells into rod photoreceptors in mammals. Mutations in NRL are associated with the autosomal recessive enhanced S-cone syndrome and autosomal dominant retinitis pigmentosa. However, the exact role of Nrl in regulating the development and maintenance of photoreceptors in the zebrafish (Danio rerio), a popular animal model used for retinal degeneration and regeneration studies, has not been fully determined. In this study, we generated an nrl knockout zebrafish model via the CRISPR-Cas9 technology and observed a surprising phenotype characterized by a reduced number, but not the total loss, of rods and over-growth of green cones. We discovered two waves of rod genesis, nrl-dependent and -independent at the embryonic and post-embryonic stages, respectively, in zebrafish by monitoring the rod development. Through bulk and single-cell RNA sequencing, we characterized the gene expression profiles of the whole retina and each retinal cell type from the wild type and nrl knockout zebrafish. The over-growth of green cones and mis-expression of green-cone-specific genes in rods in nrl mutants suggested that there are rod/green-cone bipotent precursors, whose fate choice between rod versus green-cone is controlled by nrl. Besides, we identified the mafba gene as a novel regulator of the nrl-independent rod development, based on the cell-type-specific expression patterns and the retinal phenotype of nrl/mafba double-knockout zebrafish. Gene collinearity analysis revealed the evolutionary origin of mafba and suggested that the function of mafba in rod development is specific to modern fishes. Furthermore, the altered photoreceptor composition and abnormal gene expression in nrl mutants caused progressive retinal degeneration and subsequent regeneration. Accordingly, this study revealed a novel function of the mafba gene in rod development and established a working model for the developmental and regulatory mechanisms regarding the rod and green-cone photoreceptors in zebrafish.

Rescue of photoreceptor with human mesenchyme stem cell and human mesenchyme stem cell expressing erythropoietin in total degeneration of retina animal model.

Purpose:This study aimed to investigate the efficacy of human-derived umbilical cord mesenchymal stem cells (HDUMSC) and human-derived umbilical cord mesenchymal stem cells expressing erythropoietin (HDUMSC-EPO) to rescue total degenerated retina in a rat model.Methods:The study included four treatment groups, namely negative control using normal saline (HBSS) injection, positive control using sodium iodide 60 mg/kg (SI), SI treated with HDUMSC, and SI treated with HDUMSC-EPO given via subretinal and intravenous routes, to test the efficacy of retinal regeneration following SI-induced retinal degeneration. Retinal function in both phases was tested via electroretinography (ERG) and histological staining examining the outer nuclear layer (ONL).Results:There was a statistically significant result (P < 0.05) in the SI treated with HDUMSC-EPO only when comparing day 11 (mean = 23.6 μv), day 18 (mean = 25.2 μv), day 26 (mean = 26.3 μv), and day 32 (mean = 28.2 μv) to the b-wave ERG on day 4 rescue injection day (mean = 12.5 μv). The SI treated with HDUMSC-EPO showed significant improvement in b-wave ERG readings in the Sprague–Dawley (SD) rat but did not restore baseline readings prior to degeneration (day 0). Both treated groups’ ONL thicknesses did not show significant changes compared to the negative control group (HBSS) following rescue therapy.Conclusion:Total retinal degeneration following intravenous SI injection was observed at 60 mg/kg. SI treated with HDUMSC and HDUMSC-EPO showed no regenerative potential compared to baseline in SI-induced total retina degeneration on ERG or histology, whereas SI treated with HDUMSC-EPO group showed a substantial increase in b-wave ERG amplitude over time.

Mdka Expression Is Associated with Quiescent Neural Stem Cells during Constitutive and Reactive Neurogenesis in the Adult Zebrafish Telencephalon.

In contrast to mammals, adult zebrafish display an extraordinary capacity to heal injuries and repair damage in the central nervous system. Pivotal for the regenerative capacity of the zebrafish brain at adult stages is the precise control of neural stem cell (NSC) behavior and the maintenance of the stem cell pool. The gene mdka, a member of a small family of heparin binding growth factors, was previously shown to be involved in regeneration in the zebrafish retina, heart, and fin. Here, we investigated the expression pattern of the gene mdka and its paralogue mdkb in the zebrafish adult telencephalon under constitutive and regenerative conditions. Our findings show that only mdka expression is specifically restricted to the telencephalic ventricle, a stem cell niche of the zebrafish telencephalon. In this brain region, mdka is particularly expressed in the quiescent stem cells. Interestingly, after brain injury, mdka expression remains restricted to the resting stem cell, which might suggest a role of mdka in regulating stem cell quiescence.

Visual Function is Gradually Restored During Retina Regeneration in Adult Zebrafish.

In comparison to mammals, zebrafish are able to regenerate many organs and tissues, including the central nervous system (CNS). Within the CNS-derived neural retina, light lesions result in a loss of photoreceptors and the subsequent activation of Müller glia, the retinal stem cells. Müller glia-derived progenitors differentiate and eventually restore the anatomical tissue architecture within 4 weeks. However, little is known about how light lesions impair vision functionally, as well as how and to what extent visual function is restored during the course of regeneration, in particular in adult animals. Here, we applied quantitative behavioral assays to assess restoration of visual function during homeostasis and regeneration in adult zebrafish. We developed a novel vision-dependent social preference test, and show that vision is massively impaired early after lesion, but is restored to pre-lesion levels within 7 days after lesion. Furthermore, we employed a quantitative optokinetic response assay with different degrees of difficulty, similar to vision tests in humans. We found that vision for easy conditions with high contrast and low level of detail, as well as color vision, was restored around 7–10 days post lesion. Vision under more demanding conditions, with low contrast and high level of detail, was regained only later from 14 days post lesion onwards. Taken together, we conclude that vision based on contrast sensitivity, spatial resolution and the perception of colors is restored after light lesion in adult zebrafish in a gradual manner.

Iron contributes to photoreceptor degeneration and Müller glia proliferation in the zebrafish light-treated retina

Zebrafish possess the ability to completely regenerate the retina following injury, however little is understood about the damage signals that contribute to inducing Müller glia reprogramming and proliferation to regenerate lost neurons. Multiple studies demonstrated that iron contributes to various retinal injuries, however no link has been shown between iron and zebrafish retinal regeneration. Here we demonstrate that Müller glia exhibit transcriptional changes following injury to regulate iron levels within the retina, allowing for increased iron uptake and decreased export. The response of the zebrafish retina to intravitreal iron injection was then characterized, showing that ferrous, and not ferric, iron induces retinal cell death. Additionally, iron chelation resulted in decreased numbers of TUNEL-positive photoreceptors and fewer proliferating Müller glia. Despite the contribution of iron to retinal cell death, inhibition of ferroptosis did not significantly reduce cell death following light treatment. Finally, we demonstrate that both the anti-ferroptotic protein Glutathione peroxidase 4b and the Transferrin receptor 1b are required for Müller glia proliferation following light damage. Together these findings show that iron contributes to cell death in the light-damaged retina and is essential for inducing the Müller glia regeneration response.

Opportunities for agent based modeling of retinal stem cell transplantation.

Opportunities for agent based modeling of retinal stem cell transplantation.

Effect of Sei and Fin whale Müller glia in the survival and neurite growth of RGCs in vitro

AbstractPurposeMüller cells are crucial in retinal homeostasis. In zebrafish and lower vertebrates, spontaneous retinal regeneration was observed. In the mammalian retina, there is no such evidence. However, recent studies have been demonstrated that Müller cells promote the survival and the neurite outgrowth of RGCs in vitro. Here, the effect of Müller cells from two of the largest mammals in the world, Fin and Sei whales, was analyzed on RGCs survival and neurite outgrowth.MethodsThe retinas of beached Balaenoptera physalus and Balaenoptera borealis whales, 24 hr post‐mortem, were studied. Cultures of Müller cells were grown and, once whale Müller cells reached confluence, adult rat RGCs were seeded on Müller cells. The cells were cultured during six days. The number and length of the neurites of the RGCs were quantified. The conditioned media (CM) from whale Müller cells was analysed by mass spectrometry to identify and quantify proteins. Results were compared to the CM from pig Müller cells. NELL2, SEMA3F, CD56, NRCAM, OGN and PEDF were selected as candidate factors to promote neurite outgrowth and tested in pure rat RGCs cultures.ResultsRGCs survival increased 60% when cultured with whale Müller cells. The length of the neurites (&gt;200 μm) increased 90% and the length of the longest neurites (&gt;1000 μm) increased more than 500%. Proteomic analysis from whale Müller cells CM showed that the function of the 10% of the most represented proteins detected was related to neurite outgrowth, compared to only 2% of the proteins in pig CM. The selected proteins induced an increase in the percentage of RGCs with long neurites (&gt;200 μm) and an increase of 200% in RGCs with very long neurites (&gt;1000 μm).ConclusionsWhale Müller cells increase RGCs neurite growth, suggesting that whale Müller cells secrete a combination of neurotrophic factors promoting regeneration of RGCs. Besides, the selected proteins from the CM increase neurite outgrowth, confirming the capacity of the factors secreted by whale Müller cells to regenerate RGCs.Supported by ELKARTEK (KK‐2019/00086), MINECO‐Retos (PID2019‐111139RB‐I00) Grupos UPV/EHU (GIU2018/50).

Implantable patches assembled with mesenchymal stem cells and gelatin/silk fibroin composite microspheres for the treatment of traumatic optic neuropathy

The therapeutic benefits of transplanting mesenchymal stem cells (MSCs) to the injury site of traumatic optic neuropathy (TON) represents a crucial, yet unresolved question, the answer to which may facilitate development of innovative therapies for TON, a frustrating disease with no effective treatment. Herein, we address this issue by showing that MSC transplantation to the injury site is able to promote optic nerve regeneration in terms of retinal ganglion cell survival and axon regeneration using MSC@MS, implantable patches assembled with MSCs and gelatin/silk fibroin composite microspheres, which integrate 3D culture and trypsin-free harvesting, and can increase in vivo retention of transplanted MSCs. In contrast, conventional bolus injection of MSC suspension is not potent enough to produce significant benefits. We also show MSCs in MSC@MS demonstrate superior gene expression profiles over monolayer culture after IGF-1 stimulation. In future study, microspheres in MSC@MS can further be harnessed to deliver therapeutic cargoes that boost the potency of MSCs or treat TON synergistically with MSCs, making MSC@MS a versatile platform to develop tailored MSC-based therapies for TON.

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, multiomics 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.

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

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

A Comparative Analysis of Gene and Protein Expression Throughout a Full 28-Day Retinal Regeneration Time-Course in Adult Zebrafish

Following photoreceptors ablation by intense light exposure, adult zebrafish are capable of complete regeneration due to the ability of their Müller glia (MG) to re-enter the cell cycle, creating progenitors that differentiate into new photoreceptors. The majority of previous reports on retinal regeneration focused on the first few days of the regenerative response, which include MG cell-cycle re-entry and progenitor cell proliferation. With this study, we analyzed the full 28-day time-course of regeneration by pairing a detailed morphological/immunological analysis with RNA-seq transcriptional profiling at 8 key time points during retinal regeneration. We observed several novel findings. First, we provide evidence for two separate peaks of MG gliosis, with the secondary gliotic peak occurring after MG cell-cycle re-entry. Second, we highlight a distinct transcriptional shift between 5- and 10-days post lesion that highlights the transition from progenitor proliferation to differentiation into new photoreceptors. Third, we show distinctly different patterns of transcriptional recovery of the photoreceptor opsins at 28 days post lesion. Finally, using differential gene expression analysis, we revealed that the established functional recovery of the retina at 28 days post lesion does not, in fact, return to an undamaged transcriptional state, potentially redefining what the field considers complete regeneration. Together, to our knowledge, this work represents the first histological and transcriptomic map of a 28-day time-course of retinal regeneration in adult zebrafish.

Retinal Lineage Therapeutic Specific Effect of Human Orbital and Abdominal Adipose-Derived Mesenchymal Stem Cells.

Retinal degenerative diseases are one of the main causes of complete blindness in aged population. In this study, we compared the therapeutic potential for retinal degeneration of human mesenchymal stem cells derived from abdominal subcutaneous fat (ABASCs) or from orbital fat (OASCs) due to their accessibility and mutual embryonic origin with retinal tissue, respectively. OASCs were found to protect RPE cells from cell death and were demonstrated to increase early RPE precursor markers, while ABASCs showed a raise in retinal precursor marker expression. Subretinal transplantation of OASCs in a mouse model of retinal degeneration led to restoration of the RPE layer while transplantation of ABASCs resulted in a significant restoration of the photoreceptor layer. Taken together, we demonstrated a lineage-specific therapeutic effect for either OASCs or ABASCs in retinal regeneration.

Evidence of regional specializations in regenerated zebrafish retina

Adult zebrafish are capable of functional retinal regeneration following damage. A goal of vision science is to stimulate or permit a similar process in mammals to treat human retinal disease and trauma. Ideally such a process would reconstitute the stereotyped, two-dimensional topographic patterns and regional specializations of specific cell types, functionally important for representation of the visual field. An example in humans is the cone-rich fovea, essential for high-acuity color vision. Stereotyped, global topographic patterns of specific retinal cell types are also found in zebrafish, particularly for cone types expressing the tandemly-replicated lws (long wavelength-sensitive) and rh2 (middle wavelength-sensitive) opsins. Here we examine whether regionally specialized patterns of LWS1 and LWS2 cones are restored in regenerated retinas in zebrafish. Adult transgenic zebrafish carrying fluorescent reporters for lws1 and lws2 were subjected to retinal lesions that destroy all neurons but spare glia, via intraocular injection of the neurotoxin ouabain. Regenerated and contralateral control retinas were mounted whole or sectioned, and imaged. Overall spatial patterns of lws1 vs. lws2 opsin-expressing cones in regenerated retinas were remarkably similar to those of control retinas, with LWS1 cones in ventral/peripheral regions, and LWS2 cones in dorsal/central regions. However, LWS2 cones occupied a smaller fraction of regenerated retina, and several cones co-expressed the lws1 and lws2 reporters in regenerated retinas. Local patterns of regenerated LWS1 cones showed modest reductions in regularity. These results suggest that some of the regional patterning information, or the source of such signals, for LWS cone subtypes may be retained by undamaged cell types (Müller glia or RPE) and re-deployed during regeneration.

Targeting senescent retinal pigment epithelial cells facilitates retinal regeneration in mouse models of age-related macular degeneration

Although age-related macular degeneration (AMD) is a multifactorial disorder with angiogenic, immune, and inflammatory components, the most common clinical treatment strategies are antiangiogenic therapies. However, these strategies are only applicable to neovascular AMD, which accounts for less than 20% of all AMD cases, and there are no FDA-approved drugs for the treatment of dry AMD, which accounts for ~ 80% of AMD cases. Here, we report that the elimination of senescent cells is a potential novel therapeutic approach for the treatment of all types of AMD. We identified senescent retinal pigment epithelium (RPE) cells in animal models of AMD and determined their contributions to retinal degeneration. We further confirmed that the clearance of senescent RPE cells with the MDM2-p53 inhibitor Nutlin-3a ameliorated retinal degeneration. These findings provide new insights into the use of senescent cells as a therapeutic target for the treatment of AMD.