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.

Unusual morphology of foveal Müller glia in an adult human born pre-term.

The fovea of the human retina, a specialization for acute and color vision, features a high concentration of cone photoreceptors. A pit on the inner retinal aspect is created by the centrifugal migration of post-receptoral neurons. Foveal cells are specified early in fetal life, but the fovea reaches its final configuration postnatally. Pre-term birth retards migration resulting in a small pit, a small avascular zone, and nearly continuous inner retinal layers. To explore the involvement of Müller glia, we used serial-section electron microscopic reconstructions to examine the morphology and neural contacts of Müller glia contacting a single foveal cone in a 28-year-old male organ donor born at 28 weeks of gestation. A small non-descript foveal avascular zone contained massed glial processes that included a novel class of 'inner' Müller glia. Similar to classic 'outer' Müller glia that span the retina, inner Müller glia have bodies in the inner nuclear layer (INL). These cells are densely packed with intermediate filaments and insert processes between neurons. Unlike 'outer' Müller glia, 'inner' Müller glia do not reach the external limiting membrane but instead terminate at the outer plexiform layer. One completely reconstructed inner cell ensheathed cone pedicles and a cone-driven circuit of midget bipolar and ganglion cells. Inner Müller glia outnumber foveal cones by 1.8-fold in the outer nuclear layer (221,448 vs. 123,026 cells/mm2). Cell bodies of inner Müller glia outnumber those of outer Müller glia by 1.7-fold in the INL (41,872 vs. 24,631 cells/ mm2). Müller glia account for 95 and 80% of the volume of the foveal floor and Henle fiber layer, respectively. Determining whether inner cells are anomalies solely resulting from retarded lateral migration of inner retinal neurons in pre-term birth requires further research.

Repurposing development genes for axonal regeneration following injury: Examining the roles of Wnt signaling.

In this review, we explore the connections between developmental embryology and axonal regeneration. Genes that regulate embryogenesis and central nervous system (CNS) development are discussed for their therapeutic potential to induce axonal and cellular regeneration in adult tissues after neuronal injury. Despite substantial differences in the tissue environment in the developing CNS compared with the injured CNS, recent studies have identified multiple molecular pathways that promote axonal growth in both scenarios. We describe various molecular cues and signaling pathways involved in neural development, with an emphasis on the versatile Wnt signaling pathway. We discuss the capacity of developmental factors to initiate axonal regrowth in adult neural tissue within the challenging environment of the injured CNS. Our discussion explores the roles of Wnt signaling and also examines the potential of other embryonic genes including Pax, BMP, Ephrin, SOX, CNTF, PTEN, mTOR and STAT3 to contribute to axonal regeneration in various CNS injury model systems, including spinal cord and optic crush injuries in mice, Xenopus and zebrafish. Additionally, we describe potential contributions of Müller glia redifferentiation to neuronal regeneration after injury. Therefore, this review provides a comprehensive summary of the state of the field, and highlights promising research directions for the potential therapeutic applications of specific embryologic molecular pathways in axonal regeneration in adults.

Cytokines IL-1ß 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-1ß and IL-10 are essential for Müller glia proliferation in the light-damaged retina. While IL-1ß 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-1ß and IL-10 on Müller glia proliferation following light damage in adult zebrafish.

Dysregulated energy and protein homeostasis and the loss of GABAergic amacrine cells in aging retina.

Aging is a major risk factor for the development or the worsening of retinal degenerative conditions. The intricate network of the neural retina determined that the retinal aging is a complicated process. The aim of this study is to delineate the transcriptomic changes of major retinal neurons during aging in C57BL/6 mice at single-cell level. We analyzed the transcriptional profiles of the photoreceptor, bipolar, amacrine, and Müller glial cells of 1.5-2 and 24-30 months old mice using single-cell RNA sequencing technique. We selectively confirmed the differences in gene expression using immunofluorescence staining and RNA in situ hybridization analysis. We found that each retinal cell type had unique changes upon aging. However, they all showed signs of dysregulated glucose and energy metabolism, and perturbed proteostasis. In particular, old Müller glia exhibited the most profound changes, including the upregulation of cell metabolism, stress-responses, antigen-presentation and immune responses and metal ion homeostasis. The dysregulated gliogenesis and differentiation was confirmed by the presence of Müller glia expressing rod-specific genes in the inner nuclear layer and the outer plexiform layer of the old retina. We further pinpointed the specific loss of GABAergic amacrine cells in old retina. Our study emphasized changes of amacrine and Müller glia during retinal aging, provided resources for further research on the molecular and cellular regulatory mechanisms underlying aging-associated retinal deterioration.

The ketogenic diet and hypoxia promote mitophagy in the context of glaucoma.

Mitochondrial homeostasis includes balancing organelle biogenesis with recycling (mitophagy). The ketogenic diet protects retinal ganglion cells (RGCs) from glaucoma-associated neurodegeneration, with a concomitant increase in mitochondrial biogenesis. This study aimed to determine if the ketogenic diet also promoted mitophagy. MitoQC mice that carry a pH-sensitive mCherry-GFP tag on the outer mitochondrial membrane were placed on a ketogenic diet or standard rodent chow for 5 weeks; ocular hypertension (OHT) was induced via magnetic microbead injection in a subset of control or ketogenic diet animals 1 week after the diet began. As a measure of mitophagy, mitolysosomes were quantified in sectioned retina immunolabeled with RBPMS for RGCs or vimentin for Müller glia. Mitolysosomes were significantly increased as a result of OHT and the ketogenic diet (KD) in RGCs. Interestingly, the ketogenic diet increased mitolysosome number significantly higher than OHT alone. In contrast, OHT and the ketogenic diet both increased mitolysosome number in Müller glia to a similar degree. To understand if hypoxia could be a stimulus for mitophagy, we quantified mitolysosomes after acute OHT, finding significantly greater mitolysosome number in cells positive for pimonidazole, an adduct formed in cells exposed to hypoxia. Retinal protein analysis for BNIP3 and NIX showed no differences across groups, suggesting that these receptors were equivocal for mitophagy in this model of OHT. Our data indicate that OHT and hypoxia stimulate mitophagy and that the ketogenic diet is an additive for mitophagy in RGCs. The different response across RGCs and Müller glia to the ketogenic diet may reflect the different metabolic needs of these cell types.

Astrogliosis in the GFAP-CreERT2:Rosa26iDTR Mouse Model Does Not Exacerbate Retinal Microglia Activation or Müller Cell Gliosis under Hypoxic Conditions.

Diabetic retinopathy (DR) affects over 140 million people globally. The mechanisms that lead to blindness are still enigmatic but there is evidence that sustained inflammation and hypoxia contribute to vascular damage. Despite efforts to understand the role of inflammation and microglia in DR's pathology, the contribution of astrocytes to hypoxic responses is less clear. To investigate the role of astrocytes in hypoxia-induced retinopathy, we utilized a 7-day systemic hypoxia model using the GFAP-CreERT2:Rosa26iDTR transgenic mouse line. This allows for the induction of inflammatory reactive astrogliosis following tamoxifen and diphtheria toxin administration. We hypothesize that DTx-induced astrogliosis is neuroprotective during hypoxia-induced retinopathy. Glial, neuronal, and vascular responses were quantified using immunostaining, with antibodies against GFAP, vimentin, IBA-1, NeuN, fibrinogen, and CD31. Cytokine responses were measured in both the brain and serum. We report that while both DTx and hypoxia induced a phenotype of reduced microglia morphological activation, DTx, but not hypoxia, induced an increase in the Müller glia marker vimentin. We did not observe that the combination of DTx and hypoxic treatments exacerbated the signs of reactive glial cells, nor did we observe a significant change in the expression immunomodulatory mediators IL-1ß, IL2, IL-4, IL-5, IL-6, IL-10, IL-18, CCL17, TGF-ß1, GM-CSF, TNF-α, and IFN-γ. Overall, our results suggest that, in this hypoxia model, reactive astrogliosis does not alter the inflammatory responses or cause vascular damage in the retina.

Intravitreal MPTP drives retinal ganglion cell loss with oral nicotinamide treatment providing robust neuroprotection.

Neurodegenerative diseases have common underlying pathological mechanisms including progressive neuronal dysfunction, axonal and dendritic retraction, and mitochondrial dysfunction resulting in neuronal death. The retina is often affected in common neurodegenerative diseases such as Parkinson's and Alzheimer's disease. Studies have demonstrated that the retina in patients with Parkinson's disease undergoes changes that parallel the dysfunction in the brain. These changes classically include decreased levels of dopamine, accumulation of alpha-synuclein in the brain and retina, and death of dopaminergic nigral neurons and retinal amacrine cells leading to gross neuronal loss. Exploring this disease's retinal phenotype and vision-related symptoms is an important window for elucidating its pathophysiology and progression, and identifying novel ways to diagnose and treat Parkinson's disease. 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) is commonly used to model Parkinson's disease in animal models. MPTP is a neurotoxin converted to its toxic form by astrocytes, transported to neurons through the dopamine transporter, where it causes mitochondrial Complex I inhibition and neuron degeneration. Systemic administration of MPTP induces retinal changes in different animal models. In this study, we assessed the effects of MPTP on the retina directly via intravitreal injection in mice (5 mg/mL and 50 mg/mL to 7, 14 and 21 days post-injection). MPTP treatment induced the reduction of retinal ganglion cells-a sensitive neuron in the retina-at all time points investigated. This occurred without a concomitant loss of dopaminergic amacrine cells or neuroinflammation at any of the time points or concentrations tested. The observed neurodegeneration which initially affected retinal ganglion cells indicated that this method of MPTP administration could yield a fast and straightforward model of retinal ganglion cell neurodegeneration. To assess whether this model could be amenable to neuroprotection, mice were treated orally with nicotinamide (a nicotinamide adenine dinucleotide precursor) which has been demonstrated to be neuroprotective in several retinal ganglion cell injury models. Nicotinamide was strongly protective following intravitreal MPTP administration, further supporting intravitreal MPTP use as a model of retinal ganglion cell injury. As such, this model could be utilized for testing neuroprotective treatments in the context of Parkinson's disease and retinal ganglion cell injury.

Prohibitin 2 deficiency in photoreceptors leads to progressive retinal degeneration and facilitated Müller glia engulfing microglia debris.

Müller glia and microglia are capable of phagocytosing fragments of retinal cells in response to retinal injury or degeneration. However, the direct evidence for their mutual interactions between Müller glia and microglia in the progression of retinal degeneration (RD) remains largely unclear. This study aims to construct a progressive RD mouse model and investigate the activated pattern of Müller glia and the interplay between Müller glia and microglia in the early stage or progression of RD. A Prohibitin 2 (Phb2) photoreceptor-specific knockout (RKO) mouse model was generated by crossing Phb2flox/flox mice with Rhodopsin-Cre mice. Optical Coherence Tomography (OCT), histological staining, and Electroretinography (ERG) assessed retinal structure and function, and RKO mice exhibited progressive RD from six weeks of age. In detail, six-week-old RKO mice showed no significant retinal impairment, but severe vision dysfunction and retina thinning were shown in ten-week-old RKO mice. Furthermore, RKO mice were sensitive to Light Damage (LD) and showed severe RD at an early age after light exposure. Bulk retina RNA-seq analysis from six-week-old control (Ctrl) and RKO mice showed reactive retinal glia in RKO mice. The activated pattern of Müller glia and the interplay between Müller glia and microglia was visualized by immunohistology and 3D reconstruction. In six-week-old RKO mice or light-exposed Ctrl mice, Müller glia were initially activated at the edge of the retina. Moreover, in ten-week-old RKO mice or light-exposed six-week-old RKO mice with severe photoreceptor degeneration, abundant Müller glia were activated across the whole retinas. With the progression of RD, phagocytosis of microglia debris by activated Müller glia were remarkably increased. Altogether, our study establishes a Phb2 photoreceptor-specific knockout mouse model, which is a novel mouse model of RD and can well demonstrate the phenotype of progressive RD. We also report that Müller glia in the peripheral retina is more sensitive to the early damage of photoreceptors. Our study provides more direct evidence for Müller glia engulfing microglia debris in the progression of RD due to photoreceptor Phb2 deficiency.

Dominant role for pigment epithelial CRALBP in supplying visual chromophore to photoreceptors.

Cellular retinaldehyde-binding protein (CRALBP) supports production of 11-cis-retinaldehyde and its delivery to photoreceptors. It is found in the retinal pigment epithelium (RPE) and Müller glia (MG), but the relative functional importance of these two cellular pools is debated. Here, we report RPE- and MG-specific CRALBP knockout (KO) mice and examine their photoreceptor and visual cycle function. Bulk visual chromophore regeneration in RPE-KO mice is 15-fold slower than in controls, accounting for their delayed rod dark adaptation and protection against retinal phototoxicity, whereas MG-KO mice have normal bulk visual chromophore regeneration and retinal light damage susceptibility. Cone pigment regeneration is significantly impaired in RPE-KO mice but mildly affected in MG-KO mice, disclosing an unexpectedly strong reliance of cone photoreceptors on the RPE-based visual cycle. These data reveal a dominant role for RPE-CRALBP in supporting rod and cone function and highlight the importance of RPE cell targeting for CRALBP gene therapies.

Glutamatergic neuronal activity regulates angiogenesis and blood-retinal barrier maturation via Norrin/ß-catenin signaling.

Interactions among neuronal, glial, and vascular components are crucial for retinal angiogenesis and blood-retinal barrier (BRB) maturation. Although synaptic dysfunction precedes vascular abnormalities in many retinal pathologies, how neuronal activity, specifically glutamatergic activity, regulates retinal angiogenesis and BRB maturation remains unclear. Using in vivo genetic studies in mice, single-cell RNA sequencing (scRNA-seq), and functional validation, we show that deep plexus angiogenesis and paracellular BRB maturation are delayed in Vglut1-/- retinas where neurons fail to release glutamate. By contrast, deep plexus angiogenesis and paracellular BRB maturation are accelerated in Gnat1-/- retinas, where constitutively depolarized rods release excessive glutamate. Norrin expression and endothelial Norrin/ß-catenin signaling are downregulated in Vglut1-/- retinas and upregulated in Gnat1-/- retinas. Pharmacological activation of endothelial Norrin/ß-catenin signaling in Vglut1-/- retinas rescues defects in deep plexus angiogenesis and paracellular BRB maturation. Our findings demonstrate that glutamatergic neuronal activity regulates retinal angiogenesis and BRB maturation by modulating endothelial Norrin/ß-catenin signaling.

A novel HDAC8 inhibitor H7E exerts retinoprotective effects against glaucomatous injury via ameliorating aberrant Müller glia activation and oxidative stress.

Glaucoma is considered a neurodegenerative disease characterized by progressive visual field defects that may lead to blindness. Although controlling intraocular pressure (IOP) is the mainstay of glaucoma treatment, some glaucoma patients have unmet needs due to unclear pathogenic mechanisms. Recently, there has been growing evidence that neuroinflammation is a potential target for the development of novel antiglaucoma agents. In this study, we investigated the protective effects and cellular mechanisms of H7E, a novel small molecule inhibits HDAC8, using in vitro and in vivo glaucoma-like models. Importantly, H7E mitigated extracellular MMP-9 activity and MCP-1 levels in glutamate- or S100B-stimulated reactive Müller glia. In addition, H7E inhibited the upregulation of inflammation- and proliferation-related signaling pathways, particularly the ERK and JNK MAPK pathways. Under conditions of oxidative damage, H7E prevents retinal cell death and reduces extracellular glutamate released from stressed Müller glia. In a mouse model of NMDA-induced retinal degeneration, H7E alleviated functional and structural defects within the inner retina as assessed by electroretinography and optical coherence tomography. Our results demonstrated that the newly identified compound H7E protects against glaucoma damage by specifically targeting HDAC8 activity in the retina. This protective effect is attributed to the inhibition of Müller glial activation and the prevention of retinal cell death caused by oxidative stress.

Evolutionary and developmental specialization of foveal cell types in the marmoset.

In primates, high-acuity vision is mediated by the fovea, a small specialized central region of the retina. The fovea, unique to the anthropoid lineage among mammals, undergoes notable neuronal morphological changes during postnatal maturation. However, the extent of cellular similarity across anthropoid foveas and the molecular underpinnings of foveal maturation remain unclear. Here, we used high-throughput single-cell RNA sequencing to profile retinal cells of the common marmoset (Callithrix jacchus), an early divergent in anthropoid evolution from humans, apes, and macaques. We generated atlases of the marmoset fovea and peripheral retina for both neonates and adults. Our comparative analysis revealed that marmosets share almost all their foveal types with both humans and macaques, highlighting a conserved cellular structure among primate foveas. Furthermore, by tracing the developmental trajectory of cell types in the foveal and peripheral retina, we found distinct maturation paths for each. In-depth analysis of gene expression differences demonstrated that cone photoreceptors and Müller glia (MG), among others, show the greatest molecular divergence between these two regions. Utilizing single-cell ATAC-seq and gene-regulatory network inference, we uncovered distinct transcriptional regulations differentiating foveal cones from their peripheral counterparts. Further analysis of predicted ligand-receptor interactions suggested a potential role for MG in supporting the maturation of foveal cones. Together, these results provide valuable insights into foveal development, structure, and evolution.

Cell-cell interaction in the pathogenesis of inherited retinal diseases.

The retina is part of the central nervous system specialized for vision. Inherited retinal diseases (IRD) are a group of clinically and genetically heterogenous disorders that lead to progressive vision impairment or blindness. Although each disorder is rare, IRD accumulatively cause blindness in up to 5.5 million individuals worldwide. Currently, the pathophysiological mechanisms of IRD are not fully understood and there are limited treatment options available. Most IRD are caused by degeneration of light-sensitive photoreceptors. Genetic mutations that abrogate the structure and/or function of photoreceptors lead to visual impairment followed by blindness caused by loss of photoreceptors. In healthy retina, photoreceptors structurally and functionally interact with retinal pigment epithelium (RPE) and Müller glia (MG) to maintain retinal homeostasis. Multiple IRD with photoreceptor degeneration as a major phenotype are caused by mutations of RPE- and/or MG-associated genes. Recent studies also reveal compromised MG and RPE caused by mutations in ubiquitously expressed ciliary genes. Therefore, photoreceptor degeneration could be a direct consequence of gene mutations and/or could be secondary to the dysfunction of their interaction partners in the retina. This review summarizes the mechanisms of photoreceptor-RPE/MG interaction in supporting retinal functions and discusses how the disruption of these processes could lead to photoreceptor degeneration, with an aim to provide a unique perspective of IRD pathogenesis and treatment paradigm. We will first describe the biology of retina and IRD and then discuss the interaction between photoreceptors and MG/RPE as well as their implications in disease pathogenesis. Finally, we will summarize the recent advances in IRD therapeutics targeting MG and/or RPE.

ER-stress response in retinal Müller glia occurs significantly earlier than amyloid pathology in the Alzheimer's mouse brain and retina.

Alzheimer's Disease (AD) pathogenesis is thought to begin up to 20 years before cognitive symptoms appear, suggesting the need for more sensitive diagnostic biomarkers of AD. In this report, we demonstrated pathological changes in retinal Müller glia significantly earlier than amyloid pathology in AD mouse models. By utilizing the knock-in NLGF mouse model, we surprisingly discovered an increase in reticulon 3 (RTN3) protein levels in the NLGF retina as early as postnatal day 30 (P30). Despite RTN3 being a canonically neuronal protein, this increase was noted in the retinal Müller glia, confirmed by immunohistochemical characterization. Further unbiased transcriptomic assays of the P30 NLGF retina revealed that retinal Müller glia were the most sensitive responding cells in this mouse retina, compared with other cell types including photoreceptor cells and ganglion neurons. Pathway analyses of differentially expressed genes in glia cells showed activation of ER stress response via the upregulation of unfolded protein response (UPR) proteins such as ATF4 and CHOP. Early elevation of RTN3 in response to challenges by toxic Aß likely facilitated UPR. Altogether, these findings suggest that Müller glia act as a sentinel for AD pathology in the retina and should aid for both intervention and diagnosis.

Photoreceptor calyceal processes accompany the developing outer segment, adopting a stable length despite a dynamic core.

Vertebrate photoreceptors detect light through a large cilium-based outer segment, which is filled with photopigment-laden membranous discs. Surrounding the base of the outer segment are microvilli-like calyceal processes (CPs). Although CP disruption has been associated with altered outer segment morphology and photoreceptor degeneration, the role of the CPs remains elusive. Here, we used zebrafish as a model to characterize CPs. We quantified CP parameters and report a strong disparity in outer segment coverage between photoreceptor subtypes. CP length is stable across light and dark conditions, yet heat-shock inducible expression of tagged actin revealed rapid turnover of the CP actin core. Detailed imaging of the embryonic retina uncovered substantial remodeling of the developing photoreceptor apical surface, including a transition from dynamic tangential processes to vertically oriented CPs immediately prior to outer segment formation. Remarkably, we also found a direct connection between apical extensions of the Müller glia and retinal pigment epithelium, arranged as bundles around the ultraviolet sensitive cones. In summary, our data characterize the structure, development and surrounding environment of photoreceptor microvilli in the zebrafish retina.

ID factors regulate the ability of Müller glia to become proliferating neurogenic progenitor-like cells.

The purpose of this study was to investigate how ID factors regulate the ability of Müller glia (MG) to reprogram into proliferating MG-derived progenitor cells (MGPCs) in the chick retina. We found that ID1 is transiently expressed by maturing MG (mMG), whereas ID4 is maintained in mMG in embryonic retinas. In mature retinas, ID4 was prominently expressed by resting MG, but following retinal damage ID4 was rapidly upregulated and then downregulated in MGPCs. By contrast, ID1, ID2, and ID3 were low in resting MG and then upregulated in MGPCs. Inhibition of ID factors following retinal damage decreased numbers of proliferating MGPCs. Inhibition of IDs, after MGPC proliferation, significantly increased numbers of progeny that differentiated as neurons. In damaged or undamaged retinas inhibition of IDs increased levels of p21Cip1 in MG. In response to damage or insulin+FGF2 levels of CDKN1A message and p21Cip1 protein were decreased, absent in proliferating MGPCs, and elevated in MG returning to a resting phenotype. Inhibition of notch- or gp130/Jak/Stat-signaling in damaged retinas increased levels of ID4 but not p21Cip1 in MG. Although ID4 is the predominant isoform expressed by MG in the chick retina, id1 and id2a are predominantly expressed by resting MG and downregulated in activated MG and MGPCs in zebrafish retinas. We conclude that ID factors have a significant impact on regulating the responses of MG to retinal damage, controlling the ability of MG to proliferate by regulating levels of p21Cip1, and suppressing the neurogenic potential of MGPCs.

TIMP3/Wnt axis regulates gliosis of Müller glia.

BACKGROUND: Previous studies have confirmed the expression of tissue inhibitor of metalloproteinase-3 (TIMP3) in Müller glia (MG). However, the role of TIMP3 in MG remains unknown. METHODS: A mouse model of laser-induced retinal damage and gliosis was generated using wild-type C57BL/6 mice. TIMP3 and associated proteins were detected using Western blotting and immunofluorescence microscopy. RNA sequencing (GSE132140) of mouse laser-induced gliosis was utilized for pathway analysis. TIMP3 overexpression was induced in human MG. Human vitreous samples were obtained from patients with proliferative diabetic retinopathy (PDR) and healthy controls for protein analysis. RESULTS: TIMP3 levels increased in mouse eyes after laser damage. Morphology and spatial location of TIMP3 indicated its presence in MG. TIMP3-overexpressing MG showed increased cellular proliferation, migration, and cell nuclei size, suggesting TIMP3-induced gliosis for retinal repair. Glial fibrillary acidic protein (GFAP) and vimentin levels were elevated in TIMP3-overexpressing MG and laser-damaged mouse retinas. RNA sequencing and Western blotting suggested a role for ß-catenin in mediating TIMP3 effects on the retina. Human vitreous samples from patients with PDR showed a positive correlation between TIMP3 and GFAP levels, both of which were elevated in patients with PDR. CONCLUSIONS: TIMP3 is associated with MG gliosis to enhance the repair ability of damaged retinas and is mediated by the canonical Wnt/ß-catenin. Changes in TIMP3 could potentially be used to control gliosis in a range of retinal diseases However, given the multifaceted nature of TIMP3, care must be taken when developing treatments that aim solely to boost the function of TIMP3. FUNDING: National Cheng Kung University Hospital, Taiwan (NCKUH-10604009 and NCKUH-11202007); the Ministry of Science and Technology (MOST 110-2314-B-006-086-MY3).

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

BACKGROUND: Unlike 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. METHODS: A 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. RESULTS: Here, 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. CONCLUSION: Our 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.

Regeneration from three cellular sources and ectopic mini-retina formation upon neurotoxic retinal degeneration in Xenopus.

Regenerative abilities are not evenly distributed across the animal kingdom. The underlying modalities are also highly variable. Retinal repair can involve the mobilization of different cellular sources, including ciliary marginal zone (CMZ) stem cells, the retinal pigmented epithelium (RPE), or Müller glia. To investigate whether the magnitude of retinal damage influences the regeneration modality of the Xenopus retina, we developed a model based on cobalt chloride (CoCl2 ) intraocular injection, allowing for a dose-dependent control of cell death extent. Analyses in Xenopus laevis revealed that limited CoCl2 -mediated neurotoxicity only triggers cone loss and results in a few Müller cells reentering the cell cycle. Severe CoCl2 -induced retinal degeneration not only potentializes Müller cell proliferation but also enhances CMZ activity and unexpectedly triggers RPE reprogramming. Surprisingly, reprogrammed RPE self-organizes into an ectopic mini-retina-like structure laid on top of the original retina. It is thus likely that the injury paradigm determines the awakening of different stem-like cell populations. We further show that these cellular sources exhibit distinct neurogenic capacities without any bias towards lost cells. This is particularly striking for Müller glia, which regenerates several types of neurons, but not cones, the most affected cell type. Finally, we found that X. tropicalis also has the ability to recruit Müller cells and reprogram its RPE following CoCl2 -induced damage, whereas only CMZ involvement was reported in previously examined degenerative models. Altogether, these findings highlight the critical role of the injury paradigm and reveal that three cellular sources can be reactivated in the very same degenerative model.