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Andy J Fischer - One of the best experts on this subject based on the ideXlab platform.
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cannabinoid signaling promotes the reprogramming of Muller glia into proliferating progenitor cells
bioRxiv, 2021Co-Authors: Warren A Campbell, Seth Blackshaw, Thanh Hoang, S Blum, A Reske, Andy J FischerAbstract: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 reprogramming of Muller 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 examine how the eCB system impacts glial reactivity and the capacity of MG to become Muller glia-derived progenitor cells (MGPCs). We probed 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 2-Arachidonoylglycerol (2-AG) and Anandamide (AEA) potentiated the formation of MGPCs. Consistent with these findings, CNR1-agonists and MGLL-inhibitor promoted reprogramming, whereas CNR1-antagonist and inhibitors of eCB synthesis suppressed reprogramming. Surprisingly, retinal microglia were largely unaffected by increases or decreases in eCB signaling in both chick and mouse models. However, eCB-signaling suppressed the activation of NFkB-reporter in MG in damaged mouse retinas. We conclude that the eCB system in the retina influences the reactivity of MG and is important for regulating glial reactivity and the reprogramming of MG into proliferating MGPCs, but not for regulating the reactivity of immune cells in the retina.
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midkine is neuroprotective and influences glial reactivity and the formation of Muller glia derived progenitor cells in chick and mouse retinas
Glia, 2021Co-Authors: Warren A Campbell, Seth Blackshaw, Isabella Palazzo, Thanh Hoang, Amanda Fritschkelleher, Andy J FischerAbstract: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 Muller 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 Muller 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 β1, 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 Muller glia during embryonic maturation, following retinal injury, and during reprogramming into MGPCs. MDK mediates glial activity, neuronal survival, and the re-programming of Muller glia into proliferating MGPCs.
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midkine in chick and mouse retinas neuroprotection glial reactivity and the formation of Muller glia derived progenitor cells
bioRxiv, 2020Co-Authors: Warren A Campbell, Seth Blackshaw, Isabella Palazzo, Thanh Hoang, Amanda Fritschkelleher, Andy J FischerAbstract:Recent studies have shown that midkine (MDK), a basic heparin-binding growth factor, is involved in the development and regeneration of the zebrafish retina. However, very little is known about MDK in the retinas of warm-blooded vertebrates. We investigate the expression patterns of MDK and related factors, roles in neuronal survival, and influence upon the formation of Muller glia-derived progenitor cells (MGPCs) in chick and mouse model systems. By using single-cell RNA-sequencing (scRNA-seq), we find that MDK and related factors are dynamically expressed by maturing MG and by MG in retinas damaged by NMDA or treated with insulin and FGF2. Interestingly, MDK is significantly up-regulated by MG in damaged chick retinas, but down-regulated by MG in damaged mouse retinas. In both chick and mouse retinas, exogenous MDK selectively up-regulates cFOS and pS6 (a readout of mTOR-signaling) in Muller glia. In the chick, intraocular injections of MDK before injury decrease numbers of dying cells, decrease microglial reactivity, decrease the accumulation of Non-astrocytic Inner Retinal glial (NIRG) cells, and decrease numbers of proliferating MGPCs. Addition of MDK signaling inhibitor sodium vanadate following retinal injury reverses these effects to increase the number of dying cells, accumulation of NIRG cells, and decrease the number of proliferating MGPCs. Inhibitors of PP2A and Pak1 had specific inhibitory effects on MGPC formation. In mice, MDK administration with NMDA damage drives a small but significant increase in MGPCs. We conclude that MDK expression is dynamically regulated in reactive Muller glia and during reprogramming into MGPCs. MDK acts to coordinate glial activity, neuronal survival, and may act in an autocrine manner to influence the re-programming of Muller glia into proliferating MGPCs.
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NF-κB signaling regulates the formation of proliferating Muller glia-derived progenitor cells in the avian retina.
2019Co-Authors: Isabella Palazzo, Seth Blackshaw, Kyle Deistler, Thanh Hoang, Andy J FischerAbstract:Abstract Neuronal regeneration in the retina is a robust, effective process in some cold-blooded vertebrates, but this process is ineffective in warm-blooded vertebrates. Understanding the mechanisms and cell-signaling pathways that restrict the reprogramming of Muller glia into proliferating neurogenic progenitors is key to harnessing the regenerative potential of the retina. Inflammation and reactive microglia are known to influence the formation of Muller glia-derived progenitor cells (MGPCs), but the mechanisms underlying this response are unknown. Using the chick retina in vivo as a model system, we investigate the role of the Nuclear Factor kappa B (NF-κB) signaling, a critical regulator of inflammation. We find that components of the NF-κB pathway are expressed by Muller glia and are dynamically regulated after neuronal damage or treatment with growth factors. Inhibition of NF-κB enhances, whereas activation suppresses the formation of proliferating MGPCs. Additionally, activation of NF-κB promotes glial differentiation from MGPCs in damaged retinas. With microglia ablated, the effects of NF-κB-agonists/antagonists on MGPC formation are reversed, suggesting that the context and timing of signals provided by reactive microglia influence how NF-κB-signaling impacts the reprogramming of Muller glia. We propose that NF-κB-signaling is an important signaling “hub” that suppresses the reprogramming of Muller glia into proliferating MGPCs and this “hub” coordinates signals provided by reactive microglia.
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retinoic acid signaling regulates the proliferative and neurogenic capacity of Muller glia derived progenitor cells in the avian retina
Stem Cells, 2018Co-Authors: Levi Todd, Lilianna Suarez, Colin Quinn, Andy J FischerAbstract:In the retina, Muller glia have the potential to become progenitor cells with the ability to proliferate and regenerate neurons. However, the ability of Muller 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 Muller 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.
John G Flannery - One of the best experts on this subject based on the ideXlab platform.
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Retinoschisin Gene Therapy in Photoreceptors, Muller glia, or All Retinal Cells in the Mouse Model of X-Linked Retinoschisis
Molecular Therapy, 2014Co-Authors: Leah C Byrne, Bilge E Ozturk, Trevor Lee, Cecile Fortuny, Meike Visel, Deniz Dalkara, David V Schaffer, John G FlanneryAbstract:HHS Public Access Author manuscript Author Manuscript Gene Ther. Author manuscript; available in PMC 2014 December 01. Published in final edited form as: Gene Ther. 2014 June ; 21(6): 585–592. doi:10.1038/gt.2014.31. Retinoschisin gene therapy in photoreceptors, Muller glia, or all retinal cells in the Rs1h−/− mouse Leah C. Byrne, PhD. 1,2 , Bilge E. Ozturk 1 , Trevor Lee 1 , Cecile Fortuny 1 , Meike Visel 1 , Deniz Dalkara, PhD. 1,2,3 , David V. Schaffer, PhD. 2 , and John G. Flannery, PhD. 1,† 1 Department of Molecular and Cellular Biology and The Helen Wills Neuroscience Institute, The University of California, Berkeley, CA 94720 Author Manuscript 2 Department of Chemical and Biomolecular Engineering, Department of Bioengineering, and The Helen Wills Neuroscience Institute, The University of California, Berkeley, CA 94720 Abstract Author Manuscript X-linked retinoschisis, a disease characterized by splitting of the retina, is caused by mutations in the retinoschisin gene, which encodes a secreted cell adhesion protein. Currently, there is no effective treatment for retinoschisis, though viral vector-mediated gene replacement therapies offer promise. We used intravitreal delivery of three different AAV vectors to target delivery of the RS1 gene to Muller glia, photoreceptors, or multiple cell types throughout the retina. Muller glia radially span the entire retina, are accessible from the vitreous, and remain intact throughout progression of the disease. However, photoreceptors, not glia, normally secrete retinoschisin. We compared the efficacy of rescue mediated by retinoschisin secretion from these specific subtypes of retinal cells in the Rs1h−/− mouse model of retinoschisis. Our results indicate that all three vectors deliver the RS1 gene, and that several cell types can secrete retinoschisin, leading to transport of the protein across the retina. The greatest long-term rescue was observed when photoreceptors produce retinoschisin. Similar rescue was observed with photoreceptor-specific or generalized expression, though photoreceptor secretion may contribute to rescue in the latter case. These results collectively point to the importance of cell targeting and appropriate vector choice in the success of retinal gene therapies. Keywords Gene therapy; X-linked retinoschisis; AAV vectors; photoreceptors; Muller glia; cell targeting Author Manuscript Users may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use:http://www.nature.com/authors/editorial_policies/license.html#terms Correspondence should be addressed to J.G.F. Address: University of California at Berkeley,132 Barker Hall, Berkeley, CA 94720-3190, Phone: (510) 642-0209, flannery@berkeley.edu. 3Current address: Institut de la Vision, UMRS 968 UPMC, INSERM, CNRS U7210, F-75012 Paris, France Conflict of Interest We disclose the following conflict of interest: DD, JGF, and DVS are patent holders on ShH10 for gene delivery to the retina. LB, DD, MV, JGF, and DVS are patent holders on 7m8 for delivery of gene products to retinal cells.
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retinoschisin gene therapy in photoreceptors Muller glia or all retinal cells in the rs1h mouse
Gene Therapy, 2014Co-Authors: Leah C Byrne, Bilge E Ozturk, Trevor Lee, Cecile Fortuny, Meike Visel, Deniz Dalkara, David V Schaffer, John G FlanneryAbstract:X-linked retinoschisis, a disease characterized by splitting of the retina, is caused by mutations in the retinoschisin gene, which encodes a putative secreted cell adhesion protein. Currently, there is no effective treatment for retinoschisis, though viral vector-mediated gene replacement therapies offer promise. We used intravitreal delivery of three different AAV vectors to target delivery of the RS1 gene to Muller glia, photoreceptors or multiple cell types throughout the retina. Muller glia radially span the entire retina, are accessible from the vitreous, and remain intact throughout progression of the disease. However, photoreceptors, not glia, normally secrete retinoschisin. We compared the efficacy of rescue mediated by retinoschisin secretion from these specific subtypes of retinal cells in the Rs1h-/- mouse model of retinoschisis. Our results indicate that all three vectors deliver the RS1 gene, and that several cell types can secrete retinoschisin, leading to transport of the protein across the retina. The greatest long-term rescue was observed when photoreceptors produce retinoschisin. Similar rescue was observed with photoreceptor-specific or generalized expression, although photoreceptor secretion may contribute to rescue in the latter case. These results collectively point to the importance of cell targeting and appropriate vector choice in the success of retinal gene therapies.
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aav mediated optogenetic ablation of Muller glia leads to structural and functional changes in the mouse retina
PLOS ONE, 2013Co-Authors: Leah C Byrne, Trevor Lee, Meike Visel, David V Schaffer, Fakhra Khalid, Emilia Zin, Kenneth P Greenberg, John G FlanneryAbstract:Muller glia, the primary glial cell in the retina, provide structural and metabolic support for neurons and are essential for retinal integrity. Muller cells are closely involved in many retinal degenerative diseases, including macular telangiectasia type 2, in which impairment of central vision may be linked to a primary defect in Muller glia. Here, we used an engineered, Muller-specific variant of AAV, called ShH10, to deliver a photo-inducibly toxic protein, KillerRed, to Muller cells in the mouse retina. We characterized the results of specific ablation of these cells on visual function and retinal structure. ShH10-KillerRed expression was obtained following intravitreal injection and eyes were then irradiated with green light to induce toxicity. Induction of KillerRed led to loss of Muller cells and a concomitant decrease of Muller cell markers glutamine synthetase and cellular retinaldehyde-binding protein, reduction of rhodopsin and cone opsin, and upregulation of glial fibrillary acidic protein. Loss of Muller cells also resulted in retinal disorganization, including thinning of the outer nuclear layer and the photoreceptor inner and outer segments. High resolution imaging of thin sections revealed displacement of photoreceptors from the ONL, formation of rosette-like structures and the presence of phagocytic cells. Furthermore, Muller cell ablation resulted in increased area and volume of retinal blood vessels, as well as the formation of tortuous blood vessels and vascular leakage. Electrophysiologic measures demonstrated reduced retinal function, evident in decreased photopic and scotopic electroretinogram amplitudes. These results show that loss of Muller cells can cause progressive retinal degenerative disease, and suggest that AAV delivery of an inducibly toxic protein in Muller cells may be useful to create large animal models of retinal dystrophies.
Thomas A Reh - One of the best experts on this subject based on the ideXlab platform.
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a comparative analysis of reactive Muller glia gene expression after light damage and microrna depleted Muller glia focus on micrornas
Frontiers in Cell and Developmental Biology, 2021Co-Authors: Seoyoung Kang, Thomas A Reh, Daniel Larbi, Monica Andrade, Sara Reardon, Stefanie G. WohlAbstract:Muller glia (MG) are the predominant glia in the neural retina and become reactive after injury or in disease. microRNAs (miRNAs) are translational repressors that regulate a variety of processes during development and are required for MG function. However, no data is available about the MG miRNAs in reactive gliosis. Therefore, in this study, we aimed to profile miRNAs and mRNAs in reactive MG 7 days after light damage. Light damage was performed for 8 h at 10,000 lux; this leads to rapid neuronal loss and strong MG reactivity. miRNAs were profiled using the Nanostring platform, gene expression analysis was conducted via microarray. We compared the light damage dataset with the dataset of Dicer deleted MG in order to find similarities and differences. We found: (1) The vast majority of MG miRNAs declined in reactive MG 7 days after light damage. (2) Only four miRNAs increased after light damage, which included miR-124. (3) The top 10 genes found upregulated in reactive MG after light damage include Gfap, Serpina3n, Ednrb and Cxcl10. (4) The miRNA decrease in reactive MG 7 days after injury resembles the profile of Dicer-depleted MG after one month. (5) The comparison of both mRNA expression datasets (light damage and Dicer-cKO) showed 1,502 genes were expressed under both conditions, with Maff , Egr2, Gadd45b, and Atf3 as top upregulated candidates. (6) The DIANA-TarBase v.8 miRNA:RNA interaction tool showed that three miRNAs were found to be present in all networks, i.e., after light damage, and in the combined data set; these were miR-125b-5p, let-7b and let-7c. Taken together, results show there is an overlap of gene regulatory events that occur in reactive MG after light damage (direct damage of neurons) and miRNA-depleted MG (Dicer-cKO), two very different paradigms. This suggests that MG miRNAs play an important role in a ubiquitous MG stress response and manipulating these miRNAs could be a first step to attenuate gliosis.
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stat pathway activation limits the ascl1 mediated chromatin remodeling required for neural regeneration from Muller glia in adult mouse retina
bioRxiv, 2019Co-Authors: Nikolas L. Jorstad, Matthew S Wilken, Levi Todd, Paul A. Nakamura, Nicholas Radulovich, Marcus J. Hooper, Alex Chitsazan, Brent A. Wilkerson, Fred Rieke, Thomas A RehAbstract:Abstract Muller glia can serve as a source for retinal regeneration in some non-mammalian vertebrates. Recently we found that this process can be induced in mouse Muller glia after injury, by combining transgenic expression of the proneural transcription factor Ascl1 and the HDAC inhibitor TSA. However, new neurons are only generated from a subset of Muller glia in this model, and identifying factors that limit Ascl1-mediated MG reprogramming could potentially make this process more efficient, and potentially useful clinically. One factor that limits neurogenesis in some non-mammalian vertebrates is the STAT pathway activation that occurs in Muller glia in response to injury. In this report, we tested whether injury induced STAT activation hampers the ability of Ascl1 to reprogram Muller glia into retinal neurons. Using a STAT inhibitor, in combination with our previously described reprogramming paradigm, we found a large increase in the ability of Muller glia to generate neurons, similar to those we described previously. Single-cell RNA-seq showed that the progenitor-like cells derived from Ascl1-expressing Muller glia have a higher level of STAT signaling than those that become neurons. Using Ascl1 ChIP-seq and DNase-seq, we found that developmentally inappropriate Ascl1 binding sites (that were unique to the overexpression context) had enrichment for the STAT binding motif. This study provides evidence that STAT pathway activation reduces the efficiency of Ascl1-mediated reprogramming in Muller glia, potentially by directing Ascl1 to inappropriate targets.
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stimulation of functional neuronal regeneration from Muller glia in adult mice
Nature, 2017Co-Authors: Nikolas L. Jorstad, William N Grimes, Rachel O L Wong, Takeshi Yoshimatsu, Matthew S Wilken, Fred Rieke, Leah S. Vandenbosch, Stefanie G. Wohl, Thomas A RehAbstract:Many retinal diseases lead to the loss of retinal neurons and cause visual impairment. The adult mammalian retina has little capacity for regeneration. By contrast, teleost fish functionally regenerate their retina following injury, and Muller glia (MG) are the source of regenerated neurons. The proneural transcription factor Ascl1 is upregulated in MG after retinal damage in zebrafish and is necessary for regeneration. Although Ascl1 is not expressed in mammalian MG after injury, forced expression of Ascl1 in mouse MG induces a neurogenic state in vitro and in vivo after NMDA (N-methyl-d-aspartate) damage in young mice. However, by postnatal day 16, mouse MG lose neurogenic capacity, despite Ascl1 overexpression. Loss of neurogenic capacity in mature MG is accompanied by reduced chromatin accessibility, suggesting that epigenetic factors limit regeneration. Here we show that MG-specific overexpression of Ascl1, together with a histone deacetylase inhibitor, enables adult mice to generate neurons from MG after retinal injury. The MG-derived neurons express markers of inner retinal neurons, synapse with host retinal neurons, and respond to light. Using an assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq), we show that the histone deacetylase inhibitor promotes accessibility at key gene loci in the MG, and allows more effective reprogramming. Our results thus provide a new approach for the treatment of blinding retinal diseases.
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transgenic expression of the proneural transcription factor ascl1 in Muller glia stimulates retinal regeneration in young mice
Proceedings of the National Academy of Sciences of the United States of America, 2015Co-Authors: Yumi Ueki, Masato Nakafuku, Matthew S Wilken, Kristen E Cox, Nikolas L. Jorstad, Laura Chipman, Kristen Sternhagen, Milesa Simic, Kristy Ullom, Thomas A RehAbstract:Muller glial cells are the source of retinal regeneration in fish and birds; although this process is efficient in fish, it is less so in birds and very limited in mammals. It has been proposed that factors necessary for providing neurogenic competence to Muller glia in fish and birds after retinal injury are not expressed in mammals. One such factor, the proneural transcription factor Ascl1, is necessary for retinal regeneration in fish but is not expressed after retinal damage in mice. We previously reported that forced expression of Ascl1 in vitro reprograms Muller glia to a neurogenic state. We now test whether forced expression of Ascl1 in mouse Muller glia in vivo stimulates their capacity for retinal regeneration. We find that transgenic expression of Ascl1 in adult Muller glia in undamaged retina does not overtly affect their phenotype; however, when the retina is damaged, the Ascl1-expressing glia initiate a response that resembles the early stages of retinal regeneration in zebrafish. The reaction to injury is even more pronounced in Muller glia in young mice, where the Ascl1-expressing Muller glia give rise to amacrine and bipolar cells and photoreceptors. DNaseI-seq analysis of the retina and Muller glia shows progressive reduction in accessibility of progenitor gene cis-regulatory regions consistent with the reduction in their reprogramming. These results show that at least one of the differences between mammal and fish Muller glia that bears on their difference in regenerative potential is the proneural transcription factor Ascl1.
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ascl1 reprograms mouse Muller glia into neurogenic retinal progenitors
Development, 2013Co-Authors: Julia Pollak, Matthew S Wilken, Yumi Ueki, Kristen E Cox, Jane M Sullivan, Russell J Taylor, Edward M Levine, Thomas A RehAbstract:Non-mammalian vertebrates have a robust ability to regenerate injured retinal neurons from Muller glia (MG) that activate the gene encoding the proneural factor Achaete-scute homolog 1 (Ascl1; also known as Mash1 in mammals) and de-differentiate into progenitor cells. By contrast, mammalian MG have a limited regenerative response and fail to upregulate Ascl1 after injury. To test whether ASCL1 could restore neurogenic potential to mammalian MG, we overexpressed ASCL1 in dissociated mouse MG cultures and intact retinal explants. ASCL1-infected MG upregulated retinal progenitor-specific genes and downregulated glial genes. Furthermore, ASCL1 remodeled the chromatin at its targets from a repressive to an active configuration. MG-derived progenitors differentiated into cells that exhibited neuronal morphologies, expressed retinal subtype-specific neuronal markers and displayed neuron-like physiological responses. These results indicate that a single transcription factor, ASCL1, can induce a neurogenic state in mature MG.
Daniel Goldman - One of the best experts on this subject based on the ideXlab platform.
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notch signaling via hey1 and id2b regulates Muller glia s regenerative response to retinal injury
bioRxiv, 2021Co-Authors: Daniel Goldman, A Sahu, S Devi, J JuiAbstract:Unlike mammals, zebrafish can regenerate a damaged retina. Key to this regenerative response are Muller glia (MG) that divide and produce progenitors for retinal repair. Although factors regulating MGs decision to divide remain mostly unknown, a certain threshold of neuron death must be exceeded in order for MG to engage in a regenerative response. A role for Notch signaling in this process is indicated since its inhibition expands the zone of injury-responsive MG following a focal injury. Our data show that injury-dependent changes in Dll4 and Dlb control Notch signaling in MG and that Hey1 and Id2b are downstream effectors that regulate proliferation of MG and MG-derived progenitors. Although we find Hey1 and Id2b can inhibit proliferation of MG-derived progenitors, only Hey1 is able to regulate MGs injury response threshold. Remarkably, Hey1 suppression is sufficient to recapitulate the effects of Notch inhibition on MGs injury response threshold.
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zebrafish Muller glia derived progenitors are multipotent exhibit proliferative biases and regenerate excess neurons
Scientific Reports, 2016Co-Authors: Curtis Powell, Jin Wan, Fairouz Elsaeidi, Eli J Cornblath, Daniel GoldmanAbstract:Unlike mammals, zebrafish can regenerate a damaged retina. Key to this regenerative response are Muller glia (MG) that respond to injury by reprogramming and adopting retinal stem cell properties. These reprogrammed MG divide to produce a proliferating population of retinal progenitors that migrate to areas of retinal damage and regenerate lost neurons. Previous studies have suggested that MG-derived progenitors may be biased to produce that are lost with injury. Here we investigated MG multipotency using injury paradigms that target different retinal nuclear layers for cell ablation. Our data indicate that regardless of which nuclear layer was damaged, MG respond by generating multipotent progenitors that migrate to all nuclear layers and differentiate into layer-specific cell types, suggesting that MG-derived progenitors in the injured retina are intrinsically multipotent. However, our analysis of progenitor proliferation reveals a proliferative advantage in nuclear layers where neurons were ablated. This suggests that feedback inhibition from surviving neurons may skew neuronal regeneration towards ablated cell types.
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antiviral drug ganciclovir is a potent inhibitor of the proliferation of Muller glia derived progenitors during zebrafish retinal regeneration
Investigative Ophthalmology & Visual Science, 2016Co-Authors: Shuqiang Zhang, Minmin Zhou, Dong Liu, Xiao Feng Zhao, Daniel GoldmanAbstract:PURPOSE The purpose of this study was to investigate the effect of the antiviral drug ganciclovir (GCV) on Muller glia dedifferentiation and proliferation and the underlying cellular and molecular mechanisms in adult zebrafish. METHODS A Tg(1016tuba1a:GFP) transgenic line was generated to identify injury-induced dedifferentiation of Muller glia. Mechanical retinal damage was induced by a needle-poke injury on the back of the eyes in adult zebrafish. Phosphate-buffered saline or GCV was injected into the vitreous of the eye at the time of injury or through the cornea. The GCV clearance rate from the eye was determined by a reversed-phase HPLC method. Green fluorescent protein (GFP) and bromodeoxyuridine (BrdU) immunofluorescence were used to determine the effect of GCV on retinal regeneration. Cell apoptosis was evaluated by TUNEL staining. Microglia were labeled by vitreous injection of isolectin IB4 conjugates. Quantitative (q)PCR and Western blot analysis were used to determine gene expression in the retina. RESULTS Ganciclovir treatment significantly reduced the number of BrdU+ Muller glia-derived progenitor cells (MGPCs) at 4 days post injury. Further analysis showed that GCV had no impact on Muller glia dedifferentiation and the initial formation of MGPCs. Our data indicate that GCV irreversibly inhibited MGPC proliferation likely through a p53-p21(cip1)-dependent pathway. Interestingly, unlike control cells, GCV-treated Muller glia cells were "locked" in a prolonged dedifferentiated state. CONCLUSIONS Our study uncovered a novel inhibitory effect of GCV on MGPC proliferation and suggests its potential use as a tool to uncover molecular mechanisms underlying retinal regeneration in zebrafish.
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leptin and il 6 family cytokines synergize to stimulate Muller glia reprogramming and retina regeneration
Cell Reports, 2014Co-Authors: Xiao Feng Zhao, Daniel Goldman, Jin Wan, Curtis Powell, Rajesh Ramachandran, Martin G MyersAbstract:Unlike mammals, zebrafish can regenerate a damaged retina. This remarkable regenerative response is mediated by Muller glia (MG) that undergo a reprogramming event that drives their proliferation and the generation of multipotent progenitors for retinal repair. The mechanisms that drive MG reprogramming are poorly understood. Here, we report that Leptin and Gp130-coupled receptors, acting via a Jak/Stat signaling pathway, stimulate MG reprogramming and progenitor formation in the injured retina. Importantly, we find that ascl1a gene expression, which drives MG reprogramming in fish and mammals, is regulated in a Jak/Stat-dependent manner and requires consensus Stat-binding sites for injury-dependent activation. Finally, we identify cytokines that are induced by retinal injury and exhibit a remarkable synergy in their ability to activate Jak/Stat signaling and MG reprogramming in the uninjured retina. Our study not only furthers our understanding of retina regeneration in zebrafish but also suggests new strategies for awakening retina regeneration in mammals.
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insm1a mediated gene repression is essential for the formation and differentiation of Muller glia derived progenitors in the injured retina
Nature Cell Biology, 2012Co-Authors: Rajesh Ramachandran, Xiao Feng Zhao, Daniel GoldmanAbstract:In zebrafish, retinal injury stimulates Muller glia (MG) reprograming, allowing them to generate multipotent progenitors that replace damaged cells and restore vision. Recent studies suggest that transcriptional repression may underlie these events. To identify transcriptional repressors, we compared the transcriptomes of MG and MG-derived progenitors and identified insm1a, a repressor exhibiting a biphasic pattern of expression that is essential for retina regeneration. Insm1a was found to suppress ascl1a and its own expression, and link injury-dependent ascl1a induction with the suppression of the Wnt inhibitor dickkopf (dkk), which is necessary for MG dedifferentiation. We also found that Insm1a was responsible for sculpting the zone of injury-responsive MG by suppressing hb-egf(a) expression. Finally, we provide evidence that Insm1a stimulates progenitor cell-cycle exit by suppressing a genetic program driving progenitor proliferation. Our studies identify Insm1a as a key regulator of retina regeneration and provide a mechanistic understanding of how it contributes to multiple phases of this process.
Abigail S Hackam - One of the best experts on this subject based on the ideXlab platform.
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the effect of extrinsic wnt β catenin signaling in Muller glia on retinal ganglion cell neurite growth
Developmental Neurobiology, 2020Co-Authors: Ganeswara Rao Musada, Galina Dvoriantchikova, Dmitry Ivanov, Ciara Myer, Sanjoy K Bhattacharya, Abigail S HackamAbstract:Muller glia are the predominant glial cell type in the retina, and they structurally and metabolically support retinal neurons. Wnt/β-catenin signaling pathways play essential roles in the central nervous system, including glial and neuronal differentiation, axonal growth, and neuronal regeneration. We previously demonstrated that Wnt signaling activation in retinal ganglion cells (RGC) induces axonal regeneration after injury. However, whether Wnt signaling within the adjacent Muller glia plays an axongenic role is not known. In this study, we characterized the effect of Wnt signaling in Muller glia on RGC neurite growth. Primary Muller glia and RGC cells were grown in transwell co-cultures and adenoviral constructs driving Wnt regulatory genes were used to activate and inhibit Wnt signaling specifically in primary Muller glia. Our results demonstrated that activation of Wnt signaling in Muller glia significantly increased RGC average neurite length and branch site number. In addition, the secretome of Muller glia after induction or inhibition of Wnt signaling was characterized using protein profiling of conditioned media by Q Exactive mass spectrometry. The Muller glia secretome after activation of Wnt signaling had distinct and more numerous proteins involved in regulation of axon extension, axon projection and cell adhesion. Furthermore, we showed highly redundant expression of Wnt signaling ligands in Muller glia and Frizzled receptors in RGCs and Muller glia. Therefore, this study provides new information about potential neurite growth promoting molecules in the Muller glia secretome, and identified Wnt-dependent target proteins that may mediate the axonal growth.
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The effect of extrinsic Wnt/β-catenin signaling in Muller glia on retinal ganglion cell neurite growth.
Developmental neurobiology, 2020Co-Authors: Ganeswara Rao Musada, Galina Dvoriantchikova, Dmitry Ivanov, Ciara Myer, Sanjoy K Bhattacharya, Abigail S HackamAbstract:Muller glia are the predominant glial cell type in the retina, and they structurally and metabolically support retinal neurons. Wnt/β-catenin signaling pathways play essential roles in the central nervous system, including glial and neuronal differentiation, axonal growth, and neuronal regeneration. We previously demonstrated that Wnt signaling activation in retinal ganglion cells (RGC) induces axonal regeneration after injury. However, whether Wnt signaling within the adjacent Muller glia plays an axongenic role is not known. In this study, we characterized the effect of Wnt signaling in Muller glia on RGC neurite growth. Primary Muller glia and RGC cells were grown in transwell co-cultures and adenoviral constructs driving Wnt regulatory genes were used to activate and inhibit Wnt signaling specifically in primary Muller glia. Our results demonstrated that activation of Wnt signaling in Muller glia significantly increased RGC average neurite length and branch site number. In addition, the secretome of Muller glia after induction or inhibition of Wnt signaling was characterized using protein profiling of conditioned media by Q Exactive mass spectrometry. The Muller glia secretome after activation of Wnt signaling had distinct and more numerous proteins involved in regulation of axon extension, axon projection and cell adhesion. Furthermore, we showed highly redundant expression of Wnt signaling ligands in Muller glia and Frizzled receptors in RGCs and Muller glia. Therefore, this study provides new information about potential neurite growth promoting molecules in the Muller glia secretome, and identified Wnt-dependent target proteins that may mediate the axonal growth.
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activation of wnt β catenin signaling in Muller glia protects photoreceptors in a mouse model of inherited retinal degeneration
Neuropharmacology, 2015Co-Authors: Amit K Patel, Krishna Surapaneni, Rei E I Nakamura, Sapir Z Karli, Sarah Syeda, Tinthu Lee, Abigail S HackamAbstract:Abstract The canonical Wnt/β-catenin (“Wnt”) pathway is an essential signaling cascade in the embryonic central nervous system (CNS) that regulates neuronal differentiation and survival. Loss of Wnt signaling in developing and adult tissue has been implicated in numerous CNS diseases, but the precise role of Wnt in regulating neuronal survival, and how its absence could lead to disease, is not understood. In this study, we investigated the effect of Wnt activation on neuronal survival in the adult retina, and identified cellular and molecular mediators. Pan-retinal Wnt signaling activation using Wnt3a induced functional and morphological rescue of photoreceptor neurons in the rd10 mouse model of retinal degeneration. Furthermore, Wnt activation using constitutively active β-catenin specifically targeted to Muller glia increased photoreceptor survival and reduced markers of glial and neuronal remodeling. Wnt-induced photoreceptor protection was associated with elevated levels of the prosurvival protein Stat3, and was reduced by shRNA-mediated knock-down of Stat3, indicating cross-talk between survival pathways. Therefore, these data increase our understanding of the role of Wnt signaling in the retina, and identify radial Muller glia as important cellular mediators of Wnt activity.
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Activation of Wnt/β-catenin signaling in Muller glia protects photoreceptors in a mouse model of inherited retinal degeneration
Neuropharmacology, 2014Co-Authors: Amit K Patel, Krishna Surapaneni, Rei E I Nakamura, Sapir Z Karli, Sarah Syeda, Tinthu Lee, Abigail S HackamAbstract:Abstract The canonical Wnt/β-catenin (“Wnt”) pathway is an essential signaling cascade in the embryonic central nervous system (CNS) that regulates neuronal differentiation and survival. Loss of Wnt signaling in developing and adult tissue has been implicated in numerous CNS diseases, but the precise role of Wnt in regulating neuronal survival, and how its absence could lead to disease, is not understood. In this study, we investigated the effect of Wnt activation on neuronal survival in the adult retina, and identified cellular and molecular mediators. Pan-retinal Wnt signaling activation using Wnt3a induced functional and morphological rescue of photoreceptor neurons in the rd10 mouse model of retinal degeneration. Furthermore, Wnt activation using constitutively active β-catenin specifically targeted to Muller glia increased photoreceptor survival and reduced markers of glial and neuronal remodeling. Wnt-induced photoreceptor protection was associated with elevated levels of the prosurvival protein Stat3, and was reduced by shRNA-mediated knock-down of Stat3, indicating cross-talk between survival pathways. Therefore, these data increase our understanding of the role of Wnt signaling in the retina, and identify radial Muller glia as important cellular mediators of Wnt activity.