Heparin-binding epidermal growth factor and fibroblast growth factor 2 rescue Müller glia-derived progenitor cell formation in microglia- and macrophage-ablated chick retinas.

Recent studies have demonstrated the impact of pro-inflammatory signaling and reactive microglia/macrophages on the formation of Müller glial-derived progenitor cells (MGPCs) in the retina. In chick retina, ablation of microglia/macrophages prevents the formation of MGPCs. Analyses of single-cell RNA-sequencing chick retinal libraries revealed that quiescent and activated microglia/macrophages have a significant impact upon the transcriptomic profile of Müller glia (MG). In damaged monocyte-depleted retinas, MG fail to upregulate genes related to different cell signaling pathways, including those related to Wnt, heparin-binding epidermal growth factor (HBEGF), fibroblast growth factor (FGF) and retinoic acid receptors. Inhibition of GSK3ß, to simulate Wnt signaling, failed to rescue the deficit in MGPC formation, whereas application of HBEGF or FGF2 completely rescued the formation of MGPCs in monocyte-depleted retinas. Inhibition of Smad3 or activation of retinoic acid receptors partially rescued the formation of MGPCs in monocyte-depleted retinas. We conclude that signals produced by reactive microglia/macrophages in damaged retinas stimulate MG to upregulate cell signaling through HBEGF, FGF and retinoic acid, and downregulate signaling through TGFß/Smad3 to promote the reprogramming of MG into proliferating MGPCs.

Fatty acid-binding proteins and fatty acid synthase influence glial reactivity and promote the formation of Müller glia-derived progenitor cells in the chick retina.

A recent comparative transcriptomic study of Müller glia (MG) in vertebrate retinas revealed that fatty acid binding proteins (FABPs) are among the most highly expressed genes in chick ( Hoang et al., 2020). Here, we investigate how FABPs and fatty acid synthase (FASN) influence glial cells in the chick retina. During development, FABP7 is highly expressed by retinal progenitor cells and maturing MG, whereas FABP5 is upregulated in maturing MG. PMP2 (FABP8) is expressed by oligodendrocytes and FABP5 is expressed by non-astrocytic inner retinal glial cells, and both of these FABPs are upregulated by activated MG. In addition to suppressing the formation of Müller glia-derived progenitor cells (MGPCs), we find that FABP-inhibition suppresses the proliferation of microglia. FABP-inhibition induces distinct changes in single cell transcriptomic profiles, indicating transitions of MG from resting to reactive states and suppressed MGPC formation, with upregulation of gene modules for gliogenesis and decreases in neurogenesis. FASN-inhibition increases the proliferation of microglia and suppresses the formation of MGPCs. We conclude that fatty acid metabolism and cell signaling involving fatty acids are important in regulating the reactivity and dedifferentiation of MG, and the proliferation of microglia and MGPCs.

Protein phosphatases regulate the formation of Müller glia-derived progenitor cells in the chick retina.

Different kinase-dependent cell signaling pathways are known to play important roles in glia-mediated neuroprotection and reprogramming of Müller glia (MG) into Müller glia-derived progenitor cells (MGPCs) in the retina. However, very little is known about the phosphatases that regulate kinase-dependent signaling in MG. Using single-cell RNA-sequencing (scRNA-seq) databases, we investigated patterns of expression of Dual Specificity Phosphatases (DUSP1/6) and other protein phosphatases in normal and damaged chick retinas. We found that DUSP1, DUSP6, PPP3CB, PPP3R1 and PPPM1A/B/D/E/G are widely expressed by many types of retinal neurons and are dynamically expressed by MG and MGPCs in retinas during the process of reprogramming. We find that inhibition of DUSP1/6 and PP2C phosphatases enhances the formation of proliferating MGPCs in damaged retinas and in retinas treated with insulin and FGF2 in the absence of damage. By contrast, inhibition of PP2B phosphatases suppressed the formation of proliferating MGPCs, but increased numbers of proliferating MGPCs in undamaged retinas treated with insulin and FGF2. In damaged retinas, inhibition of DUSP1/6 increased levels of pERK1/2 and cFos in MG whereas inhibition of PP2B's decreased levels of pStat3 and pS6 in MG. Analyses of scRNA-seq libraries identified numerous differentially activated gene modules in MG in damaged retinas versus MG in retinas treated with insulin+FGF2 suggesting significant differences in kinase-dependent signaling pathways that converge on the formation of MGPCs. Inhibition of phosphatases had no significant effects upon numbers of dying cells in damaged retinas. We conclude that the activity of different protein phosphatases acting through retinal neurons and MG "fine-tune" the cell signaling responses of MG in damaged retinas and during the reprogramming of MG into MGPCs.

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.

Sex differences in susceptibility to substance use disorder: Role for X chromosome inactivation and escape?

There is a sex-based disparity associated with substance use disorders (SUDs) as demonstrated by clinical and preclinical studies. Females are known to escalate from initial drug use to compulsive drug-taking behavior (telescoping) more rapidly, and experience greater negative withdrawal effects than males. Although these biological differences have largely been attributed to sex hormones, there is evidence for non-hormonal factors, such as the influence of the sex chromosome, which underlie sex disparities in addiction behavior. However, genetic and epigenetic mechanisms underlying sex chromosome influences on substance abuse behavior are not completely understood. In this review, we discuss the role that escape from X-chromosome inactivation (XCI) in females plays in sex-associated differences in addiction behavior. Females have two X chromosomes (XX), and during XCI, one X chromosome is randomly chosen to be transcriptionally silenced. However, some X-linked genes escape XCI and display biallelic gene expression. We generated a mouse model using an X-linked gene specific bicistronic dual reporter mouse as a tool to visualize allelic usage and measure XCI escape in a cell specific manner. Our results revealed a previously undiscovered X-linked gene XCI escaper (CXCR3), which is variable and cell type dependent. This illustrates the highly complex and context dependent nature of XCI escape which is largely understudied in the context of SUD. Novel approaches such as single cell RNA sequencing will provide a global molecular landscape and impact of XCI escape in addiction and facilitate our understanding of the contribution of XCI escape to sex disparities in SUD.

Chromatin access regulates the formation of Müller glia-derived progenitor cells in the retina.

Chromatin access and epigenetic control over gene expression play important roles in regulating developmental processes. However, little is known about how chromatin access and epigenetic gene silencing influence mature glial cells and retinal regeneration. Herein, we investigate the expression and functions of S-adenosylhomocysteine hydrolase (SAHH; AHCY) and histone methyltransferases (HMTs) during the formation of Müller glia (MG)-derived progenitor cells (MGPCs) in the chick and mouse retinas. In chick, AHCY, AHCYL1 and AHCYL2, and many different HMTs are dynamically expressed by MG and MGPCs in damaged retinas. Inhibition of SAHH reduced levels of H3K27me3 and potently blocks the formation of proliferating MGPCs. By using a combination of single cell RNA-seq and single cell ATAC-seq, we find significant changes in gene expression and chromatin access in MG with SAHH inhibition and NMDA-treatment; many of these genes are associated with glial and neuronal differentiation. A strong correlation across gene expression, chromatin access, and transcription factor motif access in MG was observed for transcription factors known to convey glial identity and promote retinal development. By comparison, in the mouse retina, inhibition of SAHH has no influence on the differentiation of neuron-like cells from Ascl1-overexpressing MG. We conclude that in the chick the activity of SAHH and HMTs are required for the reprogramming of MG into MGPCs by regulating chromatin access to transcription factors associated with glial differentiation and retinal development.

NF-κB signaling regulates the formation of proliferating Müller glia-derived progenitor cells in the avian retina.

Retinal regeneration is robust in some cold-blooded vertebrates, but this process is ineffective in warm-blooded vertebrates. Understanding the mechanisms that suppress the reprogramming of Müller glia into neurogenic progenitors is key to harnessing the regenerative potential of the retina. Inflammation and reactive microglia are known to influence the formation of Müller glia-derived progenitor cells (MGPCs), but the mechanisms underlying this interaction are unknown. We used a chick in vivo model to investigate nuclear factor kappa B (NF-κB) signaling, a critical regulator of inflammation, during the reprogramming of Müller glia into proliferating progenitors. We find that components of the NF-κB pathway are dynamically regulated by Müller glia after neuronal damage or treatment with growth factors. Inhibition of NF-κB enhances, whereas activation suppresses, the formation of proliferating MGPCs. Following microglia ablation, the effects of NF-κB-agonists on MGPC-formation are reversed, suggesting that signals provided by reactive microglia influence how NF-κB impacts Müller glia reprogramming. We propose that NF-κB is an important signaling 'hub' that suppresses the reprogramming of Müller glia into proliferating MGPCs and this 'hub' coordinates signals provided by reactive microglia.

Traumatic Brain Injury Causes Chronic Cortical Inflammation and Neuronal Dysfunction Mediated by Microglia.

Traumatic brain injury (TBI) can lead to significant neuropsychiatric problems and neurodegenerative pathologies, which develop and persist years after injury. Neuroinflammatory processes evolve over this same period. Therefore, we aimed to determine the contribution of microglia to neuropathology at acute [1 d postinjury (dpi)], subacute (7 dpi), and chronic (30 dpi) time points. Microglia were depleted with PLX5622, a CSF1R antagonist, before midline fluid percussion injury (FPI) in male mice and cortical neuropathology/inflammation was assessed using a neuropathology mRNA panel. Gene expression associated with inflammation and neuropathology were robustly increased acutely after injury (1 dpi) and the majority of this expression was microglia independent. At 7 and 30 dpi, however, microglial depletion reversed TBI-related expression of genes associated with inflammation, interferon signaling, and neuropathology. Myriad suppressed genes at subacute and chronic endpoints were attributed to neurons. To understand the relationship between microglia, neurons, and other glia, single-cell RNA sequencing was completed 7 dpi, a critical time point in the evolution from acute to chronic pathogenesis. Cortical microglia exhibited distinct TBI-associated clustering with increased type-1 interferon and neurodegenerative/damage-related genes. In cortical neurons, genes associated with dopamine signaling, long-term potentiation, calcium signaling, and synaptogenesis were suppressed. Microglial depletion reversed the majority of these neuronal alterations. Furthermore, there was reduced cortical dendritic complexity 7 dpi, reduced neuronal connectively 30 dpi, and cognitive impairment 30 dpi. All of these TBI-associated functional and behavioral impairments were prevented by microglial depletion. Collectively, these studies indicate that microglia promote persistent neuropathology and long-term functional impairments in neuronal homeostasis after TBI.SIGNIFICANCE STATEMENT Millions of traumatic brain injuries (TBIs) occur in the United States alone each year. Survivors face elevated rates of cognitive and psychiatric complications long after the inciting injury. Recent studies of human brain injury link chronic neuroinflammation to adverse neurologic outcomes, suggesting that evolving inflammatory processes may be an opportunity for intervention. Here, we eliminate microglia to compare the effects of diffuse TBI on neurons in the presence and absence of microglia and microglia-mediated inflammation. In the absence of microglia, neurons do not undergo TBI-induced changes in gene transcription or structure. Microglial elimination prevented TBI-induced cognitive changes 30 d postinjury (dpi). Therefore, microglia have a critical role in disrupting neuronal homeostasis after TBI, particularly at subacute and chronic timepoints.

NFkB-signaling promotes glial reactivity and suppresses Müller glia-mediated neuron regeneration in the mammalian retina.

Müller glia (MG) in mammalian retinas are incapable of regenerating neurons after damage, whereas the MG in lower vertebrates regenerate functional neurons. Identification of cell signaling pathways and gene regulatory networks that regulate MG-mediated regeneration is key to harnessing the regenerative potential of MG. Here, we study how NFkB-signaling influences glial responses to damage and reprogramming of MG into neurons in the rodent retina. We find activation of NFkB and dynamic expression of NFkB-associated genes in MG after damage, however damage-induced NFkB activation is inhibited by microglia ablation. Knockout of NFkB in MG suppressed the accumulation of immune cells after damage. Inhibition of NFkB following NMDA-damage significantly enhanced the reprogramming of Ascl1-overexpressing MG into neuron-like cells. scRNA-seq of retinal glia following inhibition of NFkB reveals coordination with signaling via TGFß2 and suppression of NFI and Id transcription factors. Inhibition of Smad3 signal transducer or Id transcription factors increased numbers of neuron-like cells produced by Ascl1-overexpressing MG. We conclude that NFkB is a key signaling hub that is activated in MG after damage, mediates the accumulation of immune cells, and suppresses the neurogenic potential of MG.

Cannabinoid signaling promotes the de-differentiation and proliferation of Müller glia-derived progenitor cells.

Endocannabinoids (eCB) are lipid-based neurotransmitters that are known to influence synaptic function in the visual system. eCBs are also known to suppress neuroinflammation in different pathological states. However, nothing is known about the roles of the eCB system during the transition of Müller glia (MG) into proliferating progenitor-like cells in the retina. Accordingly, we used the chick and mouse model to characterize expression patterns of eCB-related genes and applied pharmacological agents to investigate how the eCB system impacts glial reactivity and the capacity of MG to become Müller glia-derived progenitor cells (MGPCs). We queried single cell RNA-seq libraries to identify eCB-related genes and identify cells with dynamic patterns of expression in damaged retinas. MG and inner retinal neurons expressed the eCB receptor CNR1, as well as enzymes involved in eCB metabolism. In the chick, intraocular injections of cannabinoids, 2-Arachidonoylglycerol (2-AG) and Anandamide (AEA), stimulated the formation of MGPCs. Cannabinoid Receptor 1 (CNR1)-agonists and Monoglyceride Lipase-inhibitor promoted the formation of MGPCs, whereas CNR1-antagonist and inhibitors of eCB synthesis suppressed this process. In damaged mouse retinas where MG activate NFkB-signaling, activation of CNR1 decreased and inhibition of CNR1 increased NFkB, whereas levels of neuronal cell death were unaffected. Surprisingly, retinal microglia were largely unaffected by increases or decreases in eCB-signaling in both chick and mouse retinas. We conclude that the eCB system in the retina influences the reactivity of MG and the formation of proliferating MGPCs, but does not influence the reactivity of immune cells in the retina.

Midkine is neuroprotective and influences glial reactivity and the formation of Müller glia-derived progenitor cells in chick and mouse retinas.

Recent studies suggest midkine (MDK) is involved in the development and regeneration of the zebrafish retina. We investigate the expression patterns of MDK and related factors, roles in neuronal survival, and influence upon the formation of Müller glia-derived progenitor cells (MGPCs) in chick and mouse model systems. By using single-cell RNA-sequencing, we find that MDK and pleiotrophin (PTN), a MDK-related cytokine, are upregulated by Müller glia (MG) during later stages of development in chick. While PTN is downregulated, MDK is dramatically upregulated in mature MG after retinal damage or FGF2 and insulin treatment. By comparison, MDK and PTN are downregulated by MG in damaged mouse retinas. In both chick and mouse retinas, exogenous MDK induces expression of cFos and pS6 in MG. In the chick, MDK significantly decreases numbers dying neurons, reactive microglia, and proliferating MGPCs, whereas PTN has no effect. Inhibition of MDK-signaling with Na3 VO4 blocks neuroprotective effects with an increase in the number of dying cells and negates the pro-proliferative effects on MGPCs in damaged retinas. Inhibitors of PP2A and Pak1, which are associated with MDK-signaling through integrin ß1, suppressed the formation of MGPCs in damaged chick retinas. In mice, MDK promotes a small but significant increase in proliferating MGPCs in damaged retinas and potently decreases the number of dying cells. We conclude that MDK expression is dynamically regulated in Müller glia during embryonic maturation, following retinal injury, and during reprogramming into MGPCs. MDK mediates glial activity, neuronal survival, and the re-programming of Müller glia into proliferating MGPCs.

mTor signaling is required for the formation of proliferating Müller glia-derived progenitor cells in the chick retina.

We investigate the roles of mTor signaling in the formation of Müller glia-derived progenitor cells (MGPCs) in the chick retina. During embryonic development, pS6 (a readout of active mTor signaling) is present in early-stage retinal progenitors, differentiating amacrine and ganglion cells, and late-stage progenitors or maturing Müller glia. By contrast, pS6 is present at low levels in a few scattered cell types in mature, healthy retina. Following retinal damage, in which MGPCs are known to form, mTor signaling is rapidly activated in Müller glia. Inhibition of mTor in damaged retinas prevented the accumulation of pS6 in Müller glia and reduced numbers of proliferating MGPCs. Inhibition of mTor had no effect on MAPK signaling or on upregulation of the stem cell factor Klf4, whereas Pax6 upregulation was significantly reduced. Inhibition of mTor potently blocked the MGPC-promoting effects of Hedgehog, Wnt and glucocorticoid signaling in damaged retinas. In the absence of retinal damage, insulin, IGF1 and FGF2 induced pS6 in Müller glia, and this was blocked by mTor inhibitor. In FGF2-treated retinas, in which MGPCs are known to form, inhibition of mTor blocked the accumulation of pS6, the upregulation of Pax6 and the formation of proliferating MGPCs. We conclude that mTor signaling is required, but not sufficient, to stimulate Müller glia to give rise to proliferating progenitors, and the network of signaling pathways that drive the formation of MGPCs requires activation of mTor.

Reactive microglia and IL1ß/IL-1R1-signaling mediate neuroprotection in excitotoxin-damaged mouse retina.

BACKGROUND: Microglia and inflammation have context-specific impacts upon neuronal survival in different models of central nervous system (CNS) disease. Herein, we investigate how inflammatory mediators, including microglia, interleukin 1 beta (IL1ß), and signaling through interleukin 1 receptor type 1 (IL-1R1), influence the survival of retinal neurons in response to excitotoxic damage. METHODS: Excitotoxic retinal damage was induced via intraocular injections of NMDA. Microglial phenotype and neuronal survival were assessed by immunohistochemistry. Single-cell RNA sequencing was performed to obtain transcriptomic profiles. Microglia were ablated by using clodronate liposome or PLX5622. Retinas were treated with IL1ß prior to NMDA damage and cell death was assessed in wild type, IL-1R1 null mice, and mice expressing IL-1R1 only in astrocytes. RESULTS: NMDA-induced damage included neuronal cell death, microglial reactivity, upregulation of pro-inflammatory cytokines, and genes associated with IL1ß-signaling in different types of retinal neurons and glia. Expression of the IL1ß receptor, IL-1R1, was evident in astrocytes, endothelial cells, some Müller glia, and OFF bipolar cells. Ablation of microglia with clodronate liposomes or Csf1r antagonist (PLX5622) resulted in elevated cell death and diminished neuronal survival in excitotoxin-damaged retinas. Exogenous IL1ß stimulated the proliferation and reactivity of microglia in the absence of damage, reduced numbers of dying cells in damaged retinas, and increased neuronal survival following an insult. IL1ß failed to provide neuroprotection in the IL-1R1-null retina, but IL1ß-mediated neuroprotection was rescued when expression of IL-1R1 was restored in astrocytes. CONCLUSIONS: We conclude that reactive microglia provide protection to retinal neurons, since the absence of microglia is detrimental to survival. We propose that, at least in part, the survival-influencing effects of microglia may be mediated by IL1ß, IL-1R1, and interactions of microglia and other macroglia.

Retinoic Acid-Signaling Regulates the Proliferative and Neurogenic Capacity of Müller Glia-Derived Progenitor Cells in the Avian Retina.

In the retina, Müller glia have the potential to become progenitor cells with the ability to proliferate and regenerate neurons. However, the ability of Müller glia-derived progenitor cells (MGPCs) to proliferate and produce neurons is limited in higher vertebrates. Using the chick model system, we investigate how retinoic acid (RA)-signaling influences the proliferation and the formation of MGPCs. We observed an upregulation of cellular RA binding proteins (CRABP) in the Müller glia of damaged retinas where the formation of MGPCs is known to occur. Activation of RA-signaling was stimulated, whereas inhibition suppressed the proliferation of MGPCs in damaged retinas and in fibroblast growth factor 2-treated undamaged retinas. Furthermore, inhibition of RA-degradation stimulated the proliferation of MGPCs. Levels of Pax6, Klf4, and cFos were upregulated in MGPCs by RA agonists and downregulated in MGPCs by RA antagonists. Activation of RA-signaling following MGPC proliferation increased the percentage of progeny that differentiated as neurons. Similarly, the combination of RA and insulin-like growth factor 1 (IGF1) significantly increased neurogenesis from retinal progenitors in the circumferential marginal zone (CMZ). In summary, RA-signaling stimulates the formation of proliferating MGPCs and enhances the neurogenic potential of MGPCs and stem cells in the CMZ. Stem Cells 2018;36:392-405.

Sildenafil Administration in Dogs Heterozygous for a Functional Null Mutation in Pde6a: Suppressed Rod-Mediated ERG Responses and Apparent Retinal Outer Nuclear Layer Thinning.

This study was designed to assess risk for retinal toxicity associated with administration of high-dose sildenafil citrate to dogs heterozygous for a functionally null mutation in Pde6a over a 4-month period. Three Pde6a +/- dogs were administered 14.3 mg/kg sildenafil per os and two Pde6a +/- dogs placebo once daily for 16 weeks. Three Pde6a +/+ dogs were administered sildenafil for 7 days. Ophthalmic examination, vision testing, and electroretinography (ERG) were regularly performed. At study termination, dogs were euthanized and globes collected. Retinal layer thickness and photoreceptor nuclei counts were determined from plastic sections. In both Pde6a +/- and Pde6a +/+ sildenafil-treated (ST) dogs, elevation of dark-adapted b-wave threshold and unmasking of the scotopic threshold response (STR) were observed. Sildenafil treated Pde6a +/- dogs had significantly thinner ONL (24.90 +/-1.88 µm, p = 0.004) and lower photoreceptor nuclei counts (273.6 +/- 29.3 cells/100 µm, p = 0.008) compared to measurements (35.90 +/- 1.63 µm) and counts (391.5 +/-27.0 cells/100 µm) from archived untreated Pde6a +/- dogs.

Hedgehog signaling stimulates the formation of proliferating Müller glia-derived progenitor cells in the chick retina.

Müller glia can be stimulated to de-differentiate and become proliferating progenitor cells that regenerate neurons in the retina. The signaling pathways that regulate the formation of proliferating Müller glia-derived progenitor cells (MGPCs) are beginning to be revealed. The purpose of this study was to investigate whether Hedgehog (Hh) signaling influences the formation of MGPCs in the chick retina. We find that Hh signaling is increased in damaged retinas where MGPCs are known to form. Sonic Hedgehog (Shh) is normally present in the axons of ganglion cells, but becomes associated with Müller glia and MGPCs following retinal damage. Activation of Hh signaling with recombinant human SHH (rhShh) or smoothened agonist (SAG) increased levels of Ptch1, Gli1, Gli2, Gli3, Hes1 and Hes5, and stimulated the formation of proliferating MGPCs in damaged retinas. In undamaged retinas, SAG or rhShh had no apparent effect upon the Müller glia. However, SAG combined with FGF2 potentiated the formation of MGPCs, whereas SAG combined with IGF1 stimulated the nuclear migration of Müller glia, but not the formation of MGPCs. Conversely, inhibition of Hh signaling with KAAD-cyclopamine, Gli antagonists or antibody to Shh reduced numbers of proliferating MGPCs in damaged and FGF2-treated retinas. Hh signaling potentiates Pax6, Klf4 and cFos expression in Müller glia during the formation of MGPCs. We find that FGF2/MAPK signaling recruits Hh signaling into the signaling network that drives the formation of proliferating MGPCs. Our findings implicate Hh signaling as a key component of the network of signaling pathways that promote the de-differentiation of Müller glia and proliferation of MGPCs.

A new multichannel method quantitating TUNEL in detached photoreceptor nuclei.

Nuclear co-localization labels are critical to ocular research. Among these, the TUNEL assay has been established as a gold standard of cell death and apoptosis. While several validated computer-based methods exist to quantitate these markers, including ImageJ Retina Analysis (RA) Toolkit and ImagePro, none verify the count with the nuclear counter stain to confirm nuclear co-localization. We established a new ImageJ-based automated multichannel thresholding (MCT) method to quantitate nuclear co-localized labeling. The MCT method was validated by comparing it with the two published TUNEL analysis in TUNEL-positive photoreceptors in an experimental retinal detachment (RD) model. RDs were induced in murine eyes and cross-sectional images of TUNEL and DAPI counter stain were obtained. Images were classified as "typical" or high density "hotspot" TUNEL regions (n = 10/group). Images were analyzed and compared between the MCT method and the published techniques including both "standard" and "high" settings of the RA Toolkit for detecting lower or higher TUNEL densities, respectively. Additional testing of the MCT method with built-in ImageJ thresholding algorithms was performed to produce fully automated measurements. All images were compared with Bland-Altman mean difference plots to assess the difference in counts and linear regression plots to assess correlation. Comparison between the MCT method and the ImagePro method were found to be well correlated (typical: R2 = 0.8972, hotspot: R2 = 0.9000) with minor to non-significant differences. The RA Toolkit settings were found to be mostly well correlated as well (standard/typical: R2 = 0.8036, standard/hotspot: R2 = 0.4309, high/typical: R2 = 0.7895, high/hotspot: R2 = 0.8738) but were often found to have significantly higher counts than the MCT. In conclusion, the MCT method compared favorably with validated computer-based methods of nuclear marker immunofluorescence quantitation and avoids staining artifacts through the incorporation of the nuclear counter stain to confirm positive cells.

BMP- and TGFß-signaling regulate the formation of Müller glia-derived progenitor cells in the avian retina.

Müller glia-derived progenitor cells (MGPCs) have the capability to regenerate neurons in the retinas of different vertebrate orders. The formation of MGPCs is regulated by a network of cell-signaling pathways. The purpose of this study was to investigate how BMP/Smad1/5/8- and TGFß/Smad2/3-signaling are coordinated to influence the formation of MGPCs in the chick model system. We find that pSmad1/5/8 is selectively up-regulated in the nuclei of Müller glia following treatment with BMP4, FGF2, or NMDA-induced damage, and this up-regulation is blocked by a dorsomorphin analogue DMH1. By comparison, Smad2/3 is found in the nuclei of Müller glia in untreated retinas, and becomes localized to the cytoplasm following NMDA- or FGF2-treatment. These findings suggest a decrease in TGFß- and increase in BMP-signaling when MGPCs are known to form. In both NMDA-damaged and FGF2-treated retinas, inhibition of BMP-signaling suppressed the proliferation of MGPCs, whereas inhibition of TGFß-signaling stimulated the proliferation of MGPCs. Consistent with these findings, TGFß2 suppressed the formation of MGPCs in NMDA-damaged retinas. Our findings indicate that BMP/TGFß/Smad-signaling is recruited into the network of signaling pathways that controls the formation of proliferating MGPCs. We conclude that signaling through BMP4/Smad1/5/8 promotes the formation of MGPCs, whereas signaling through TGFß/Smad2/3 suppresses the formation of MGPCs.

Glucocorticoid receptors in the retina, Müller glia and the formation of Müller glia-derived progenitors.

Identification of the signaling pathways that influence the reprogramming of Müller glia into neurogenic retinal progenitors is key to harnessing the potential of these cells to regenerate the retina. Glucocorticoid receptor (GCR) signaling is commonly associated with anti-inflammatory responses and GCR agonists are widely used to treat inflammatory diseases of the eye, even though the cellular targets and mechanisms of action in the retina are not well understood. We find that signaling through GCR has a significant impact upon the ability of Müller glia to become proliferating Müller glia-derived progenitor cells (MGPCs). The primary amino acid sequence and pattern of GCR expression in the retina is highly conserved across vertebrate species, including chickens, mice, guinea pigs, dogs and humans. In all of these species we find GCR expressed by the Müller glia. In the chick retina, we find that GCR is expressed by progenitors in the circumferential marginal zone (CMZ) and is upregulated by Müller glia in acutely damaged retinas. Activation of GCR signaling inhibits the formation of MGPCs and antagonizes FGF2/MAPK signaling in the Müller glia. By contrast, we find that inhibition of GCR signaling stimulates the formation of proliferating MGPCs in damaged retinas, and enhances the neuronal differentiation while diminishing glial differentiation. Given the conserved expression pattern of GCR in different vertebrate retinas, we propose that the functions and mechanisms of GCR signaling are highly conserved and are mediated through the Müller glia. We conclude that GCR signaling directly inhibits the formation of MGPCs, at least in part, by interfering with FGF2/MAPK signaling.

Heparin-binding EGF-like growth factor (HB-EGF) stimulates the proliferation of Müller glia-derived progenitor cells in avian and murine retinas.

Müller glia can be stimulated to de-differentiate, proliferate and form Müller glia-derived progenitor cells (MGPCs) that regenerate retinal neurons. In the zebrafish retina, heparin-binding EGF-like growth factor (HB-EGF) may be one of the key factors that stimulate the formation of proliferating MGPCs. Currently nothing is known about the influence of HB-EGF on the proliferative potential of Müller glia in retinas of birds and rodents. In the chick retina, we found that levels of both hb-egf and egf-receptor are rapidly and transiently up-regulated following NMDA-induced damage. Although intraocular injections of HB-EGF failed to stimulate cell-signaling or proliferation of Müller glia in normal retinas, HB-EGF stimulated proliferation of MGPCs in damaged retinas. By comparison, inhibition of the EGF-receptor (EGFR) decreased the proliferation of MGPCs in damaged retinas. HB-EGF failed to act synergistically with FGF2 to stimulate the formation of MGPCs in the undamaged retina and inhibition of EGF-receptor did not suppress FGF2-mediated formation of MGPCs. In the mouse retina, HB-EGF stimulated the proliferation of Müller glia following NMDA-induced damage. Furthermore, HB-EGF not only stimulated MAPK-signaling in Müller glia/MGPCs, but also activated mTor- and Jak/Stat-signaling. We propose that levels of expression of EGFR are rate-limiting to the responses of Müller glia to HB-EGF and the expression of EGFR can be induced by retinal damage, but not by FGF2-treatment. We conclude that HB-EGF is mitogenic to Müller glia in both chick and mouse retinas, and HB-EGF is an important player in the formation of MGPCs in damaged retinas.