Role of TGF-β Activated Kinase 1(TAK1) in Chick Retinal Development

poster abstractBone morphogenetic proteins (BMPs) play a critical role in vertebrate eye development by regulating cell fate processes in the retina through canonical SMAD and non-canonical MAPK pathways. TGF-β Activated Kinase 1(TAK1) is a MAPKKK that activates the MAPK cascade upon BMP activation. Dysregulation of TAK1 is associated with a variety of diseased states including cancer, but little is known about the role of TAK1 in development. Recent in vitro studies have indicated that TAK1 inhibits the G1-S phase Cyclin D, a process known to be critical to cell cycle exit. Although no studies have focused on the role of TAK1 in retinal development, many studies have indicated that BMPs as well as properly timed cell cycle exit are critical for the differentiation of specific cell types. In studies designed to test the hypothesis that TAK1 is an essential regulator of cell cycle exit in the chick embryonic retina, we have performed immunohistochemistry using an antibody that specifically detects the activated form of TAK1 (pTAK1) and shown the extensive localization of pTAK1 in a subset of differentiated cells and, more prominently, in the mitotic progenitor cells of the retina. Our preliminary studies, aimed at in vivo pharmacological inhibition of TAK1 activity using (5Z)-7-Oxozeaenol in the developing chick eye, show that TAK1 inhibition could lead to a range of developmental defects in the retina. While further studies focusing on molecular changes resulting from TAK1 inhibition and overexpression would shed more light on its functional role in the retina, our results suggest that TAK1 signaling is critical for normal eye development. The heavy localization of pTAK1 in mitotic progenitor cells especially, could be indicative of its role in cell cycle exit. Understanding the functional role of this protein in the context of eye development could aid in understanding the pathophysiology of diseased states associated with TAK1

Role of Interferon Gamma in BMP7 Mediated Gliosis

poster abstractIntroduction: The retina consists of neuronal and non-neuronal cells known as glial cells. The glial cells in the retina include the Müller glia, retinal astrocytes, and microglia. Muller glia and retinal astrocytes are known to undergo gliosis, a protective response, in reaction to diseases or damaging effects such as high blood sugar concentrations or physical injury of the retina. The Belecky-Adams lab has shown that a growth factor, bone morphogenetic protein 7 (BMP7) is capable of triggering reactive gliosis indirectly, by activating secretion of interferon gamma (IFN from microglial cells. The aim of this study is to determine if fludarabine, an inhibitor of IFNγ mediated signaling, is effective in blocking activation by IFNγ in retinal astrocytes. This aim is the first step in testing the hypothesis that a blockage of IFN signaling in BMP7-treated retinas will reduce gliosis. Methods: Retinal astrocytes cells in vitro, were treated with 50 or 100μM of STAT1 inhibitor, fludarabine, for 2 hours, followed by addition of vehicle or 150ng/ml IFNγ. Protein was extracted from cells 24h after addition of vehicle or IFNγ, and quantified using BCA protein assay. Fifty micrograms of protein was loaded onto a denaturing gel and subsequently transferred to PVDF membrane and probed with an antibody against glial fibrillary acidic protein (GFAP) and -tubulin. Bands were quantitated using densitometry and GFAP normalized to the -tubulin loading control. Results: Retinal astrocytes treated with IFN but no inhibitor showed an increase in levels of GFAP, a known marker of gliosis. Cells pre-treated with fludarabine followed by IFN showed a reduction in GFAP expression in comparison to those treated with IFN alone. Conclusions: Fludarabine is effective in blocking the effects of IFNγ in retinal astrocytes. The results will allow further investigation of our hypothesis to determine if blocking IFNin BMP7-treated retinas reduces gliosis. 1Department of Biology, Indiana University-Purdue University Indianapolis, Indianapolis, IN 46202 Mentors: Teri Belecky-Adams, Department of Biology, Indiana University- Purdue University of Indianapolis; Subramanian Dharmarajan, Department of Biology, Indiana University- Purdue University of Indianapolis Funding provided by the Undergraduate Research Opportunity Program (UROP)

MORPHOLOGICAL ANALYSIS OF RETINAL GLIA

poster abstractINTRODUCTION: In addition to neuronal cells, vertebrate retinas also contain non-neuronal cells, referred to as glia. The three types of glial cells found in the retina are the Müller glia, retinal astrocytes, and microglia; together these cells aid in function, support, and maintenance of the retina. Glial cells become reactive upon activation induced by disease or injury to the retina. Activation of the cells is accompanied by morphological, molecular, and functional changes; microglial cells increase production of cytokines and convert to macrophage-like behavior to rid the nervous system of debris following disease, while astrocytes de-differentiate, migrate, proliferate and can form permanent barriers to nervous system regeneration, called glial scars. The aim of this project was characterization of the activation states of these various glial cells based on morphological parameters following treatment of cells with vehicle, known activators, and a suspected activator, the growth factor bone morphogenetic protein 7 (BMP7). Morphological changes, such as cell area, branch points, and branch length, which are indicators of a change in the function of the cells brought about by activation, were measured. METHODS: Cell cultures of mouse retinal astrocytes were treated with vehicle or sodium peroxynitrite (a strong oxidizing agent) while retinal microglia cells in vitro were treated with vehicle, lipopolysaccharide (LPS), or BMP7. Immunocytochemistry (ICC) was performed on the treated samples with cell-specific markers. The astrocytes were labeled with glial fibrillary acidic protein (GFAP) and the microglia labeled with ionized calcium-binding adapter molecule 1 (IBA-1) and submitted to immunofluorescence for imaging. Subsequent determination of morphological changes was done through the software Adobe Photoshop CS6 for preprocessing and then FIJI (ImageJ 2.0) for morphological analysis through the ‘Analyze’ feature and ‘Skeleton’ plugin. RESULTS: ICC analysis demonstrated morphological changes were induced in comparison to vehicle treated controls. The astrocytes treated with sodium peroxynitrite for 32hrs exhibited an increase of 2x in cell area and number of branch points and a decrease of 2x in branch length with increasing branch complexity. The microglia exhibited an increase of 1.2x in cell area, 1.4x in number of branch points and a decrease of 1.2x in branch length at 3hrs and an increase of 1.1x in cell area, 1.6x in number of branch points, and a decrease of 1.3x in branch length at 24hrs with LPS and a 1.5x increase of average cell area and number of branch points and minimal difference in branch length at 24hrs with BMP7. CONCLUSION: Activation of both astrocytes and microglia by known activators and BMP7 brought about increases in cell area, number of branch points and a decrease in branch length. This evidence is consistent with BMP7 triggering activation in both microglia and retinal astrocytes

BMP PATHWAY AND RETINAL ASTROGLIOSIS

poster abstractAstrocytes are the star shaped glial (non-neural) cells present in the cen-tral nervous system. The astrocytes perform a wide range of functions in-cluding: serving as an energy source, aiding neuronal development, main-taining homeostasis and detoxification. Astrocytes are also present in the eye, constituting the majority of the glial cell population and are found in the retina. Any injury to nearby neurons makes the astrocytes reactive, altering their function and morphology. One of the features of reactive astrocytes is to form a physical barrier around the injury site, called the glial scar. One group of molecules thought to play a role is the bone morphogenic proteins (BMPs). Although, the BMPs have been found to increase in CNS following injury, their role in making the retinal astrocytes reactive is not yet known. Our purpose in performing these studies was to clarify the role of BMP7 (a type of the BMP) in retinal astrocytes reactive gliosis. Mouse retinal astrocyte cells were incubated with sodium peroxynitrite (a strong oxidizing agent, which has been previously shown to make astrocytes reactive) or different cocncentrations of BMP7 for different time periods. Cells were lysed and total protein or total RNA was isolated to analyze the protein and gene expression levels of different markers. Treatment with peroxynitrite led to statistically significant increase in levels of expression in including astrocyte specific markers and certain in-hibitory molecules. In the BMP7 treated samples, similar increases in the levels of expression for the astrocyte specific markers and the inhibitory molecules. A comparison between the expression profiles of peroxynitrite- and BMP7-treated cells showed largely (but not completely) overlapping pro-files of expression. The work done here helps ascertain the role of BMP7 in reactive gliosis in vitro. Further, the observation of distinct reactivity profiles under different conditions is indicative of involvement of additional pathways

The Role of Transforming Growth Factorβ (TGF-β)-activated Kinase 1 (TAK1) in Retinal Development

poster abstractPurpose: The formation of the retina is dependent on multiple transcription factors being expressed in the correct time and place. Transforming growth factor-β-activated kinase1 (TAK1), a serine threonine kinase, has been increasingly associated with regulation of proliferation, differentiation and apoptosis of many cell types both within and outside of the central nervous system. However, little is known about its role in development of the retina. Previous results from our lab have indicated that TAK1 is expressed throughout the developing retina; however activated TAK1 is found predominantly in the dividing progenitors of the early developing chick retina. Retinas injected with TAK1 inhibitor appeared to have an increase in progenitor population and a decrease in differentiating retinal ganglion cells. The present study evaluated the potential role of TAK1 in inducing apoptosis in the developing chick retina. Methods: Embryonic day 3(E3) chick retina were injected with vehicle and 1.0 M or 2.0 M concentration (5Z)-7-Oxozeaenol, an irreversible inhibitor of TAK1. 24 hours post inhibition the tissue was harvested. Immunohistochemistry (IHC) was performed to analyze the levels of cleaved caspase 3 expression, a protein activated during apoptosis. Nuclei stained with DAPI were used to quantify the number of cells expressing the caspase3. Lipopolysaccharide (LPS) treated adult, postnatal 30 (P30), mouse retina was used as a positive control for our IHC. Results: No difference in the level of cleaved (activated) caspase 3 immunolabel was found in vehicle-, 1.0 and 2.0 M inhibitor-injected retinas. Conclusion: Lack of cleaved caspase 3 immunolabel in TAK1-inhibited retinas indicates that TAK1 may not be playing any role in inducing cell death through apoptosis in the developing chick retina used in our study. These preliminary results suggest further research should be done to better understand its role in retinal development. Mentors: Teri Belecky-Adams and Sarika Tiwari, Center for Regenerative Biology and Medicine, IUPUI, Department of Biology, IUPU

THE EFFECT OF HDAC INHIBITORS ON RETINAL DEVELOPMENT OF CHICK EMBRYO

poster abstractIntroduction: Gene expression is regulated by the accessibility of regulatory cis-acting DNA elements as well as availability of transcription factors. Histone deacetylase (HDAC) can regulate gene expression by deacetylating histone tails, which leads to a closed conformation of the DNA/histone complex and generally a reduction in expression. HDACs have been proposed to play a key role in cell survival, proliferation and differentiation; however, fewer studies have been focusing on the role of HDACs in the developing vertebrate retina. Methods: Chick retinal explants were treated with vehicle (dimethyl sulphoxide(DMSO)) or 1.0 M Trichstatin A (TSA), a known inhibitor of class 1 and 2 HDACs. Immunohistochemistry (IHC) was performed to analyze the levels of cleaved caspase 3, a protein activated during apoptosis, phospho-histone 3 (pH3) marker for mitotic phase, SOX2 which marks progenitor cells, and Islet-1 which marks differentiated cells. Digital images were analyzed using Image J/FIJI software for numbers of labeled cells. Results: After treatment with control or TSA, numbers of progenitor and differentiating cells were quantified. TSA-treated samples showed a statistically significant increase in SOX2+ (progenitors) and an increase in islet-1+ (differentiating) cells. To assess if any differences in proliferation and/or cell death that might lead to an increase in the number of progenitor and differentiating cells, samples were labeled for pH3 or cleaved caspase 3. Treatment with TSA led to increases in cells positive for pH3 and a statistically significant increase in cells positive for cleaved caspase 3 compared to controls. Conclusions: HDAC inhibitor, TSA, increased the number of progenitor and differentiating cells by increasing proliferation within the developing retina. However, there was also an increase in the number of cells undergoing apoptosis. Ongoing studies will determine which HDACs may be responsible for these results

Standardizing methods and procedures for mouse retinal flat mounts and glial cell counts

poster abstractIntroduction: The mammalian retina contains neuronal cells as well as a number of non-neuronal glial cells. The different types of glial cells include Müller glia, retinal astrocytes, and microglia. Müller glial cells and astrocytes nourish neurons and microglia act as sentinels that respond to injury or disease within the nervous system. The long-term goal of our laboratory has been to study interactions between microglia, Muller glia and astrocytes in healthy and diseased tissue. The focus of the present study was to develop a technique that would allow the laboratory to study changes in cell number in retinal flat mounts and cultures. Methods: Immunohistochemistry (IHC) was performed to fluorescently label mature murine retinal tissue. Retinal flat-mounts were stained with SOX2, a nuclear marker for glial cells or IBA1 for microglial cells, and counter-stained with Hoechst solution to label all nuclei. Pure cultures of mouse microglial cells treated with liposomal clodronate (a drug which specifically targets and ablates microglia) and vehicle were counter stained with Hoechst solution. Cell counts were performed on the images of the fluorescently labeled samples using Image-J software. Results: Convolutions were used to filter images of immunolabeled cultures and retinal flat mounts to make the images clear enough to capture cell number. The cell count assistance protocol yielded acceptable cell count results of the stained cells and determined a detectable difference in the number of clodronate treated cells versus vehicle treated control cells. The images produced of the retinal flat-mounts were analyzed to determine the percentage of SOX2 positive Müller glia in the mature murine retinal tissue. Conclusion: A modified Image J program could be used to determine cellular number in cultures and retinal flat mounts. Mentor: Teri L Belecky-Adams, Department of Biology, Indiana University-Purdue University Indianapoli

Histone deacetylase expression patterns in developing murine optic nerve

BACKGROUND: Histone deacetylases (HDACs) play important roles in glial cell development and in disease states within multiple regions of the central nervous system. However, little is known about HDAC expression or function within the optic nerve. As a first step in understanding the role of HDACs in optic nerve, this study examines the spatio-temporal expression patterns of methylated histone 3 (K9), acetylated histone 3 (K18), and HDACs 1–6 and 8–11 in the developing murine optic nerve head. RESULTS: Using RT-qPCR, western blot and immunofluorescence, three stages were analyzed: embryonic day 16 (E16), when astrocyte precursors are found in the optic stalk, postnatal day 5 (P5), when immature astrocytes and oligodendrocytes are found throughout the optic nerve, and P30, when optic nerve astrocytes and oligodendrocytes are mature. Acetylated and methylated histone H3 immunoreactivity was co-localized in the nuclei of most SOX2 positive glia within the optic nerve head and adjacent optic nerve at all developmental stages. HDACs 1–11 were expressed in the optic nerve glial cells at all three stages of optic nerve development in the mouse, but showed temporal differences in overall levels and subcellular localization. HDACs 1 and 2 were predominantly nuclear throughout optic nerve development and glial cell maturation. HDACs 3, 5, 6, 8, and 11 were predominantly cytoplasmic, but showed nuclear localization in at least one stage of optic nerve development. HDACs 4, 9 and10 were predominantly cytoplasmic, with little to no nuclear expression at any time during the developmental stages examined. CONCLUSIONS: Our results showing that HDACs 1, 2, 3, 5, 6, 8, and 11 were each localized to the nuclei of SOX2 positive glia at some stages of optic nerve development and maturation and extend previous reports of HDAC expression in the aging optic nerve. These HDACs are candidates for further research to understand how chromatin remodeling through acetylation, deacetylation and methylation contributes to glial development as well as their injury response

Class I histone deacetylases in retinal progenitors and differentiating ganglion cells

Background The acetylation state of histones has been used as an indicator of the developmental state of progenitor and differentiating cells. The goal of this study was to determine the nuclear localization patterns of Class I histone deacetylases (HDACs) in retinal progenitor cells (RPCs) and retinal ganglion cells (RGCs), as the first step in understanding their potential importance in cell fate determination within the murine retina. Results The only HDAC to label RPC nuclei at E16 and P5 was HDAC1. In contrast, there was generally increased nuclear localization of all Class I HDACs in differentiating RGCs. Between P5 and P30, SOX2 expression becomes restricted to Müller glial, cholinergic amacrine cells, and retinal astrocytes. Cholinergic amacrine showed a combination of changes in nuclear localization of Class I HDACs. Strikingly, although Müller glia and retinal astrocytes express many of the same genes, P30 Müller glial cells showed nuclear localization only of HDAC1, while retinal astrocytes were positive for HDACs 1, 2, and 3. Conclusion These results indicate there may be a role for one or more of the Class I HDACs in retinal cell type-specific differentiation