Adult Retina Research Articles

Monocyte invasion into the retina restricts the regeneration of neurons from Müller glia.

Endogenous reprogramming of glia into neurogenic progenitors holds great promise for neuron restoration therapies. Using lessons from regenerative species, we have developed strategies to stimulate mammalian Müller glia to regenerate neurons in vivo in the adult retina. We have demonstrated that the transcription factor Ascl1 can stimulate Müller glia neurogenesis. However, Ascl1 is only able to reprogram a subset of Müller glia into neurons. We have reported that neuroinflammation from microglia inhibits neurogenesis from Müller glia. Here we find that the peripheral immune response is a barrier to CNS regeneration. We show that monocytes from the peripheral immune system infiltrate the injured retina and negatively influence neurogenesis from Müller glia. Using CCR2-knockout mice of both sexes we find that preventing monocyte infiltration improves the neurogenic and proliferative capacity of Müller glia stimulated by Ascl1. Using scRNA-seq analysis we identified a signaling axis wherein Osteopontin, a cytokine highly expressed by infiltrating immune cells is sufficient to suppress mammalian neurogenesis. This work implicates the response of the peripheral immune system as a barrier to regenerative strategies of the retina.Significance Statement Regeneration of neurons in the central nervous system is extremely limited in mammals. Transgenic overexpression of the proneural transcription factor Ascl1 enables mammalian retinal glia to regenerate some neurons lost to injury. We found that during this regenerative response to injury, monocytes from the periphery invade the neural retina and these inflammatory cells negatively regulate the ability of Müller glia to reprogram into neurogenic progenitors. When monocytes are inhibited from infiltrating the retina, regeneration of neurons from Müller glia is significantly enhanced. This work implicates peripheral immunomodulation as a tool to enhance endogenous neuronal replacement strategies.

REGULATION OF RETINOGENESIS IN PRENATAL ONTOGENESIS AND ITS DISRUPTION IN AGE-RELATED MACULAR DEGENERATION

One of the main causes of blindness in the elderly in developed countries is age-related macular degeneration (AMD) [1, 2, 3]. Active research is being conducted to identify putative molecular targets for the development of an effective strategy for conservative treatment of both atrophic and neovascular forms of this disease [4, 5, 6], but at the present stage, pharmacotherapy is effective only for some patients, and is insufficient to stop the progression of the disease or eliminate the damage that has already occurred. The most promising and promising potential treatment options are considered to be the introduction of cellular technologies with retinal cell replacement and targeted pharmacotherapy. The aim of this study was to investigate the role of the retinal development inducer, SIRT1 [7, 8, 9], in retinogenesis to justify pharmacological inhibition of this protein as a possible treatment option for neovascular AMD. Using immunohistochemistry and immunocytochemistry, we assessed the expression and subcellular localization of SIRT1 and its innate inhibitor DBC1 in the retina of fetuses, adults, and mice. We also studied SIRT1 in retinal progenitor cells derived from mice and humans, the former having been used in small interfering RNA studies. SIRT1 is widely expressed in the developing and adult retina and is a regulator of key genes in retinal development, namely PAX6, Nestin, and CRX [10, 11, 12]. Moreover, we found that photoreceptor progenitor cells were among the smallest cells in a heterogeneous mouse retinal progenitor cell population [13, 14, 15]. Collectively, these results provide a basis for manipulating SIRT1 expression in small retinal progenitors as a means to increase the yield of photoreceptors for transplantation in retinal degeneration models [16, 17]. Furthermore, SIRT1 was found to be highly expressed in human-derived choroidal neovascular membranes, allowing its activity to be pharmacologically inhibited by the drug nicotinamide as a potent regulator of angiogenic and hypoxic signaling in a human retinal pigment epithelial cell line at both the protein level using angiogenesis arrays and at the RNA level using whole genome microarrays [18, 19]. These results point to the SIRT1 inhibitor, Nicotinamide, as a possible agent for treatment of neovascular AMD. Further studies of Nicotinamide are warranted in animal models of AMD. To the best of our knowledge, this is the first time that a detailed analysis of SIRT1 as a regulator of both retinal development and choroidal neovascularization has been reported.

Restoration of cone-circuit functionality in the regenerating adult zebrafish retina

Unlike humans, teleosts like zebrafish exhibit robust retinal regeneration after injury from endogenous stem cells. However, it is unclear if regenerating cone photoreceptors regain physiological function and integrate correctly into post-synaptic circuits. We used two-photon calcium imaging of living adult retina to examine photoreceptor responses before and after light-induced lesions. To assess functional recovery of cones and downstream outer retinal circuits, we exploited color opponency; UV cones exhibit intrinsic Off-response to blue light, but On-response to green light, which depends on feedback signals from outer retinal circuits. Accordingly, we assessed the presence and quality of Off- vs. On-responses and found that regenerated UV cones regain both Off-responses to short-wavelength and On-responses to long-wavelength light within 3months after lesion. Therefore, physiological circuit functionality is restored in regenerated cone photoreceptors, suggesting that inducing endogenous regeneration is a promising strategy for human retinal repair.

Dopaminergic amacrine cells express HCN channels in the developing and adult mouse retina.

To determine the molecular and functional expression of hyperpolarization-activated cyclic nucleotide-gated (HCN) channels in developing and mature dopaminergic amacrine cells (DACs), the sole source of ocular dopamine that plays a vital role in visual function and eye development. HCN channels are encoded by isoforms 1-4. HCN1, HCN2, and HCN4 were immunostained in retinal slices obtained from mice at postnatal day 4 (P4), P8, and P12 as well as in adults. Each HCN channel isoform was also immunostained with tyrosine hydroxylase, a marker for DACs, at P12 and adult retinas. Genetically-marked DACs were recorded in flat-mount retina preparation using a whole-cell current-clamp technique. HCN1 was expressed in rods/cones, amacrine cells, and retinal ganglion cells (RGCs) at P4, along with bipolar cells by P12. Different from HCN1, HCN2 and HCN4 were each expressed in amacrine cells and RGCs at P4, along with bipolar cells by P8, and in rods/cones by P12. Double immunostaining shows that each of the three isoforms was expressed in approximately half of DACs at P12 but in almost all DACs in adults. Electrophysiology results demonstrate that HCN channel isoforms form functional HCN channels, and the pharmacological blockade of HCN channels reduced the spontaneous firing frequency in most DACs. Each class of retinal neurons may use different isoforms of HCN channels to function during development. HCN1, HCN2, and HCN4 form functional HCN channels in DACs, which appears to modulate their spontaneous firing activity.

Robust reprogramming of glia into neurons by inhibition of Notch signaling and nuclear factor I (NFI) factors in adult mammalian retina.

Generation of neurons through direct reprogramming has emerged as a promising therapeutic approach for treating neurodegenerative diseases. In this study, we present an efficient method for reprogramming retinal glial cells into neurons. By suppressing Notch signaling by disrupting either Rbpj or Notch1/2, we induced mature Müller glial cells to reprogram into bipolar- and amacrine-like neurons. We demonstrate that Rbpj directly activates both Notch effector genes and genes specific to mature Müller glia while indirectly repressing expression of neurogenic basic helix-loop-helix (bHLH) factors. Combined loss of function of Rbpj and Nfia/b/x resulted in conversion of nearly all Müller glia to neurons. Last, inducing Müller glial proliferation by overexpression of dominant-active Yap promotes neurogenesis in both Rbpj- and Nfia/b/x/Rbpj-deficient Müller glia. These findings demonstrate that Notch signaling and NFI factors act in parallel to inhibit neurogenic competence in mammalian Müller glia and help clarify potential strategies for regenerative therapies aimed at treating retinal dystrophies.

Cytokines IL-1β and IL-10 are required for Müller glia proliferation following light damage in the adult zebrafish retina

Zebrafish possess the ability to regenerate dying neurons in response to retinal injury, with both Müller glia and microglia playing integral roles in this response. Resident Müller glia respond to damage by reprogramming and undergoing an asymmetric cell division to generate a neuronal progenitor cell, which continues to proliferate and differentiate into the lost neurons. In contrast, microglia become reactive, phagocytose dying cells, and release inflammatory signals into the surrounding tissue following damage. In recent years, there has been increased attention on elucidating the role that microglia play in regulating retinal regeneration. Here we demonstrate that inflammatory cytokines are differentially expressed during retinal regeneration, with the expression of a subset of pro-inflammatory cytokine genes upregulated shortly after light damage and the expression of a different subset of cytokine genes subsequently increasing. We demonstrate that both cytokine IL-1β and IL-10 are essential for Müller glia proliferation in the light-damaged retina. While IL-1β is sufficient to induce Müller glia proliferation in an undamaged retina, expression of IL-10 in undamaged retinas only induces Müller glia to express gliotic markers. Together, these findings demonstrate the essential role of inflammatory cytokines IL-1β and IL-10 on Müller glia proliferation following light damage in adult zebrafish.

Towards Stem/Progenitor Cell-Based Therapies for Retinal Degeneration.

Retinal degeneration (RD) is a leading cause of blindness worldwide and includes conditions such as retinitis pigmentosa (RP), age-related macular degeneration (AMD), and Stargardt's disease (STGD). These diseases result in the permanent loss of vision due to the progressive and irreversible degeneration of retinal cells, including photoreceptors (PR) and the retinal pigment epithelium (RPE). The adult human retina has limited abilities to regenerate and repair itself, making it challenging to achieve complete self-replenishment and functional repair of retinal cells. Currently, there is no effective clinical treatment for RD. Stem cell therapy, which involves transplanting exogenous stem cells such as retinal progenitor cells (RPCs), embryonic stem cells (ESCs), induced pluripotent stem cells (iPSCs), and mesenchymal stem cells (MSCs), or activating endogenous stem cells like Müller Glia (MG) cells, holds great promise for regenerating and repairing retinal cells in the treatment of RD. Several preclinical and clinical studies have shown the potential of stem cell-based therapies for RD. However, the clinical translation of these therapies for the reconstruction of substantial vision still faces significant challenges. This review provides a comprehensive overview of stem/progenitor cell-based therapy strategies for RD, summarizes recent advances in preclinical studies and clinical trials, and highlights the major challenges in using stem/progenitor cell-based therapies for RD.

Retinoid Synthesis Regulation by Retinal Cells in Health and Disease.

Vision starts in retinal photoreceptors when specialized proteins (opsins) sense photons via their covalently bonded vitamin A derivative 11cis retinaldehyde (11cis-RAL). The reaction of non-enzymatic aldehydes with amino groups lacks specificity, and the reaction products may trigger cell damage. However, the reduced synthesis of 11cis-RAL results in photoreceptor demise and suggests the need for careful control over 11cis-RAL handling by retinal cells. This perspective focuses on retinoid(s) synthesis, their control in the adult retina, and their role during retina development. It also explores the potential importance of 9cis vitamin A derivatives in regulating retinoid synthesis and their impact on photoreceptor development and survival. Additionally, recent advancements suggesting the pivotal nature of retinoid synthesis regulation for cone cell viability are discussed.

Unique spatially and temporary-regulated/sex-specific expression of a long ncRNA, Nb-1, suggesting its pleiotropic functions associated with honey bee lifecycle

Honey bees are social insects, and each colony member has unique morphological and physiological traits associated with their social tasks. Previously, we identified a long non-coding RNA from honey bees, termed Nb-1, whose expression in the brain decreases associated with the age-polyethism of workers and is detected in some neurosecretory cells and octopaminergic neurons, suggesting its role in the regulation of worker labor transition. Herein, we investigated its spatially and temporary-regulated/sex-specific expression. Nb-1 was expressed as an abundant maternal RNA during oogenesis and embryogenesis in both sexes. In addition, Nb-1 was expressed preferentially in the proliferating neuroblasts of the mushroom bodies (a higher-order center of the insect brain) in the pupal brains, suggesting its role in embryogenesis and mushroom body development. On the contrary, Nb-1 was expressed in a drone-specific manner in the pupal and adult retina, suggesting its role in the drone visual development and/or sense. Subcellular localization of Nb-1 in the brain during development differed depending on the cell type. Considering that Nb-1 is conserved only in Apidae, our findings suggest that Nb-1 potentially has pleiotropic functions in the expression of multiple developmental, behavioral, and physiological traits, which are closely associated with the honey bee lifecycle.

Identifying Hmga2 preserving visual function by promoting a shift of Müller glia cell fate in mice with acute retinal injury

BackgroundUnlike in lower vertebrates, Müller glia (MG) in adult mammalian retinas lack the ability to reprogram into neurons after retinal injury or degeneration and exhibit reactive gliosis instead. Whether a transition in MG cell fate from gliosis to reprogramming would help preserve photoreceptors is still under exploration.MethodsA mouse model of retinitis pigmentosa (RP) was established using MG cell lineage tracing mice by intraperitoneal injection of sodium iodate (SI). The critical time point for the fate determination of MG gliosis was determined through immunohistochemical staining methods. Then, bulk-RNA and single-cell RNA seq techniques were used to elucidate the changes in RNA transcription of the retina and MG at that time point, and new genes that may determine the fate transition of MG were screened. Finally, the selected gene was specifically overexpressed in MG cells through adeno-associated viruses (AAV) in the mouse RP model. Bulk-RNA seq technique, immunohistochemical staining methods, and visual function testing were used to elucidate and validate the mechanism of new genes function on MG cell fate transition and retinal function.ResultsHere, we found the critical time point for MG gliosis fate determination was 3 days post SI injection. Hmga2 was screened out as a candidate regulator for the cell fate transition of MG. After retinal injury caused by SI, the Hmga2 protein is temporarily and lowly expressed in MG cells. Overexpression of Hmga2 in MG down-regulated glial cell related genes and up-regulated photoreceptor related genes. Besides, overexpressing Hmga2 exclusively to MG reduced MG gliosis, made MG obtain cone’s marker, and retained visual function in mice with acute retinal injury.ConclusionOur results suggested the unique reprogramming properties of Hmga2 in regulating the fate transition of MG and neuroprotective effects on the retina with acute injury. This work uncovers the reprogramming ability of epigenetic factors in MG.

The impact of timing and injury mode on induced neurogenesis in the adult mammalian retina

Regeneration of neurons has important implications for human health, and the retina provides an accessible system to study the potential of replacing neurons following injury. In previous work, we generated transgenic mice in which neurogenic transcription factors were expressed in Müller glia (MG) and showed that they stimulated neurogenesis following inner retinal damage. It was unknown, however, whether the timing or mode of injury mattered in this process. Here, we explored these parameters on induced neurogenesis from MG and show that MG expressing Ascl1 will generate new bipolar neurons with similar efficiency irrespective of injury mode or timing. However, MG that express Ascl1-Atoh1 produce a new type of retinal ganglion-like cell after outer retinal damage, which is absent with inner retinal damage. Our data suggest that although cell fate is primarily dictated by neurogenic transcription factors, the inflammatory state of MG relative to injury can influence the outcome of induced neurogenesis.

Non-haematopoietic Sca-1+ Cells in the Retina of Adult Mice Express Functional TLR2.

The mammal retina does not have the capacity to regenerate throughout life, although some stem and progenitor cells persist in the adult retina and might retain multipotentiality, as previously described in many tissues. In this work we demonstrate the presence of a small lineage- Sca-1+ cell population in the adult mouse retina which expresses functional TLR2 receptors as in vitro challenge with the pure TLR2 agonist Pam3CSK4 increases cell number and upregulates TLR2. Therefore, this population could be of interest in neuroregeneration studies to elucidate its role in these processes.

Inflammation is a critical factor for successful regeneration of the adult zebrafish retina in response to diffuse light lesion.

Inflammation can lead to persistent and irreversible loss of retinal neurons and photoreceptors in mammalian vertebrates. In contrast, in the adult zebrafish brain, acute neural inflammation is both necessary and sufficient to stimulate regeneration of neurons. Here, we report on the critical, positive role of the immune system to support retina regeneration in adult zebrafish. After sterile ablation of photoreceptors by phototoxicity, we find rapid response of immune cells, especially monocytes/microglia and neutrophils, which returns to homeostatic levels within 14days post lesion. Pharmacological or genetic impairment of the immune system results in a reduced Müller glia stem cell response, seen as decreased reactive proliferation, and a strikingly reduced number of regenerated cells from them, including photoreceptors. Conversely, injection of the immune stimulators flagellin, zymosan, or M-CSF into the vitreous of the eye, leads to a robust proliferation response and the upregulation of regeneration-associated marker genes in Müller glia. Our results suggest that neuroinflammation is a necessary and sufficient driver for retinal regeneration in the adult zebrafish retina.

Quantification of Proliferating and Mitotically Active Retinal Cells in Mice by Flow Cytometry.

Adult mammals lack the ability to regenerate retinal neurons after injury. However, in previous studies from this lab, topical application of the selective alpha7 nicotinic acetylcholine receptor (nAChR) agonist, PNU-282987, has been associated with an increase in the number of retinal neurons in adult murine models both in the presence and absence of injury to the retina. Additionally, studies assaying mitotic markers have shown a substantial increase in the amount of mitotically active and proliferating cells with the topical application of the alpha7 nAChR agonist. However, these previous studies were performed using fluorescent immunolabeling and subsequent confocal microscopy, thus limiting the number of antibodies that can be multiplexed. As a result, we have developed a flow cytometry method that allows for the multiplexing and analysis of multiple external and internal markers in dissociated retinal cells. In this paper, a step-by-step protocol is described for the labeling of multiple retinal cell types such as retinal ganglion cells, rod photoreceptors, and Müller glia, concurrently with Müller glia-derived progenitor cells that arise after treatment with PNU-282987. Key features • Neurogenesis in the adult mammalian retina. • Flow cytometry of retinal cells. • PNU-282987-induced mitotic activity in the retina. • Dissociation of the retina for flow cytometry analysis. Graphical overview Schematic demonstrating the protocol for preparation of retinal cells for flow cytometry analysis. (A) Adult mice (3-6 months) are subjected to topical PBS eyedrop treatment containing DMSO (control groups) or PNU-282987 (experimental groups). Both eyedrop treatments contain 1 mg/mL of BrdU to label proliferating cells. After treatment, mice are euthanized, and retinae are harvested for dissociation using papain. (B) Dissociated retina cells are fixed and permeabilized before aliquots are taken for cell counts on a hemocytometer. After determining the number of cells present, conjugated antibodies and unconjugated primary antibodies are added at the appropriate dilutions. Fluorescent secondary antibodies are added for markers that are unconjugated. Cells are then subjected to flow cytometric analysis using a BD LSRFortessa.

Characterization of the whole Advillin expressing retinal ganglion cell population in the rodent retina

Aims/Purpose: We have quantified and examined the topography of the total population of retinal ganglion cells (RGC) expressing Advillin (Advillin+RGCs), an actin‐binding protein expressed in somatic sensory neurons, in the retinas, as well as their projections to the brain, in control adult Advillin‐Cretg/+ mice (Zurborg et al. Mol Pain 2011, 7:66).Methods: Retinas from adult Advillin‐Cretg/+ Ai14fl/fl C57BL/6J mice (Frutos‐Rincón et al., Int J Mol Sci 2022, 23:2997) expressing tdTomato were prepared as whole‐mounts, labelled with antibodies recognizing Brn3a and melanopsin. The entire Advillin+RGC population was quantified and its retinal distribution analysed by nearest neighbour maps. The brains were serial coronal crysectioned and the axonal Advillin+RGC projections in the brain were anatomically analysed.Results: Advillin protein was mainly located in the somas and axons of a subset of RGCs. Total number of Advillin+RGCs was 1947 ± 157 (mean ± SD; n = 7), which constitutes 5% of the total RGC population. Approximately 17.5% (340 ± 76) and 0% (3 ± 1) of the Advillin+RGC population co‐expressed Brn3a or melanopsin proteins, respectively. The Advillin+RGC population was distributed across the entire retina, with a higher concentration in the temporal region. The axons of this RGC subpopulation project to the main retinal recipient regions in the brain, such as the lateral geniculate nucleus (LGN) and superior colliculus (SC). However, none of these axons project to the suprachiasmatic nucleus. These data indicates that Advillin+RGCs is not a subpopulation of ipRGCs.Conclusions: The Advillin protein, which is characteristic of sensory neurons, is also expressed in a specific RGC subpopulation of the adult mouse retina. A small percentage of Advillin+RGCs expressed the Brn3a transcription factor, while none expressed melanopsin. The axons of Advillin+RGCs project to the typical retino‐recipient areas in the brain, excluding the suprachiasmatic nucleus.

Consequences of early life stress on the structure and function of the adult mouse retina.

Consequences of early life stress on the structure and function of the adult mouse retina.

Molecular genetic background for the development of early neurodegenerative processes in retina

All neurodegenerative retinal diseases are characterized by decreased metabolic and regenerative processes, impaired microcirculation, and structural abnormalities of the retina. Age is a significant risk factor for age-related macular degeneration (AMD), which is the primary cause of irreversible vision loss in individuals aged over 60 years. Since its pathogenesis is not completely understood, there is currently no effective treatment for AMD. The pathogenesis of age-related retinal changes is still uncertain, despite being grounded on alterations in characteristic features of the retina due to aging. The molecular events preceding and accompanying clinical disease manifestations pose a challenge for human study. The retina shares a uniform basic structure throughout all vertebrate species, enabling the use of animals in exploring the mechanisms responsible for maintaining a healthy physiological structure of the retina and for the pathogenesis of numerous diseases. With the knowledge acquired, novel remedies for these ailments can be developed for humans [1]. The study analyzed the OXYS rat line, known for its premature aging and retinopathy which mimics the dry form of AMD in humans. The aim was to examine how postnatal retinal neurogenesis changes contribute to the development of AMD-like retinopathy in these rats. By approximately 3–4 months of age, all structural components of the retina in OXYS rats demonstrate pathological changes, including vessels (both choroidal and intraretinal), Bruch’s membrane, photoreceptors, ganglion neurons, interneurons, and RPE. This is supported by our research findings [2, 3]. By approximately 3–4 months of age, all structural components of the retina in OXYS rats demonstrate pathological changes, including vessels (both choroidal and intraretinal), Bruch’s membrane, photoreceptors, ganglion neurons, interneurons, and RPE. One hundred percent of OXYS rats exhibit clinical symptoms of retinopathy. As they age, pathological changes intensify and coincide with photoreceptor death, hindered autophagy, and active gliosis [3–5]. Due to its limited capacity for neurogenesis, the adult mammalian retina’s structural and functional properties during its development can exert lasting impacts on ontogeny. We discovered that OXYS rats exhibited a notable reduction in the population of amacrine neurons during birth, along with an increment in the populations of ganglion and horizontal neurons in the retina as a form of compensation. The postnatal development of the rat retina is finished by the 20th day of life. In OXYS rats, this development is distinct in that it causes a shift in the timing of differentiation of bipolar cells and photoreceptors, leading to the later formation of the outer retinal layer. This layer is composed of synapses between photoreceptors, bipolar cells, and horizontal cells. The delayed onset of synaptogenesis in the OXYS rat retina results in elevated apoptosis levels and a heightened reduction of neurons. Consequently, the processes of photoreceptor differentiation and synaptogenesis remain incomplete by the time of eye opening in OXYS rats. This incomplete development can significantly impact the retina’s structure and functions. These findings indicate that a delay in retinal formation could serve as a predictor of the development of AMD in OXYS rats, and potentially this disease in humans.

Pressure-Related Effects on Homeostatic Müller Cell Proteins in the Adult Porcine in Vitro Retina

PurposeTo explore early pressure-related effects on Müller cell homeostatic proteins in the in vitro adult porcine retina.MethodsRetinal explants were subjected to 0-, 10-, 30-, or 60-mmHg of pressure for 24 or 48 h in culture. Retinal explants fixed immediately after enucleation were used as controls. Müller cell proteins were evaluated by GFAP, GS, CRALBP, and bFGF immunohistochemistry.ResultsGFAP-labeling revealed no differences in fluorescence intensity after 24 or 48 h in any of the pressure groups compared with control retinas. However, a higher intensity was found in the 30- and 60-mmHg groups compared with 0-mmHg counterparts after 24 and 48 h. A higher intensity in GS-labeled sections was found in the 10-and 60-mmHg groups compared with controls and remaining pressure groups after 48 h. Compared with control retinas, CRALBP labeling revealed a higher intensity in the 60-mmHg group after 24 h and in the 10-, 30-, and 60-mmHg groups after 48 h. After 24 and 48 h, a lower intensity was found in bFGF-labeled cells in the 0-, 10-, and 30-mmHg groups compared with controls, while no difference was seen for the 60-mmHg group.ConclusionsMüller cells in the cultured porcine adult retina respond early to pressure by altering the expression of GFAP as well as the homeostatic proteins GS, CRALBP, and bFGF.

Invited Session II: Retinal remodeling and regeneration: Stimulation of functional neural regeneration in adult mouse retina.

Neurons are not regenerated in the adult mammalian retina. However, in non-mammalian vertebrates, specialized glial cells (Muller glia, MG) spontaneously generate neural progenitors, which go on to replace neurons after injury. We have recently developed strategies to stimulate regeneration of functional neurons in the adult mouse retina by over-expressing developmental transcription factors, including Ascl1, Atoh1, Islet1 and Pou4f2. We have found that over-expressing Ascl1 in MG in vivo, followed by injury and TSA, induces regeneration of functional retinal neurons in adult mice. Adding additional TFs, such as Atoh1, substantially increases the efficiency of neurogenesis from the MG, while combining other TFs with Ascl1 can alter the types of neurons that are regenerated after injury. Together our data show that we can considerably recapitulate in mice much of the regeneration that occurs naturally in fish.

ASCL1 induces neurogenesis in human Müller glia

In mammals, loss of retinal cells due to disease or trauma is an irreversible process that can lead to blindness. Interestingly, regeneration of retinal neurons is a well established process in some non-mammalian vertebrates and is driven by the Müller glia (MG), which are able to re-enter the cell cycle and reprogram into neurogenic progenitors upon retinal injury or disease. Progress has been made to restore this mechanism in mammals to promote retinal regeneration: MG can be stimulated to generate new neurons invivo in the adult mouse retina after the over-expression of the pro-neural transcription factor Ascl1. In this study, we applied the same strategy to reprogram human MG derived from fetal retina and retinal organoids into neurons. Combining single cell RNA sequencing, single cell ATAC sequencing, immunofluorescence, and electrophysiology we demonstrate that human MG can be reprogrammed into neurogenic cells invitro.