ARVO 21 Annual Meeting Abstracts 384 Müller Cells and ... · Guidance cues appear to be present to guide repopulating cells to the correct retinal lamina and to re-establish a mosaic

ARVO 2016 Annual Meeting Abstracts

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384 Müller Cells and Astrocytes In Development and DiseaseTuesday, May 03, 2016 3:45 PM–5:30 PMExhibit/Poster Hall Poster SessionProgram #/Board # Range: 4185–4215/D0204–D0234Organizing Section: Retinal Cell BiologyContributing Section(s): Glaucoma, Lens, Nanotechnology and Regenerative Medicine

Program Number: 4185 Poster Board Number: D0204Presentation Time: 3:45 PM–5:30 PMRepopulation of the retina by myeloid cells following depletion of endogenous retinal microglia in adult miceYikui Zhang, Lian Zhao, Xu Wang, Robert N. Fariss, Wai T. Wong. National Eye Institute, Bethesda, MD.Purpose: Microglia, the resident population of immune cells in the retina, represent a long-lived population of cells that tile the entire retina in an uniform and regular pattern. The factors that maintain microglial homeostasis in terms of their presence and distribution in the retina are not known. We investigate myeloid cell homeostasis in the retina following acute depletion of microglia and characterize the distribution and nature of repopulating myeloid cells.Methods: Microglia were depleted in young, 2-month old CX3CR1CreER; Rosa26-flox-STOP-flox-DTA transgenic mice by inducing a microglial-specific expression of diphtheria toxin subunit alpha by tamoxifen administration. The repopulation of the retina by Iba1+ myeloid cells was monitored by immunohistochemistry at time-points up to 5 months following depletion.Results: Following short-term acute depletion of endogenous retinal microglia (>95%), Iba1+ myeloid cells were observed to repopulate the retina beginning at Day 9 after depletion. These cells emerged predominantly in the peripapillary region and demonstrated rounded or amoeboid morphologies and stained positively for isolectin IB4, F4/80, and CD68. Repopulating cells extended progressively from center to periphery, acquiring an increasingly more ramified morphology with time. Repopulating cells appear targeted initially to the IPL and then the OPL, spacing themselves out in a horizontal mosaic distribution, resembling that observed in endogenous microglia. Repopulating cells can be found extending to all retinal areas by 1 month but full recovery of microglial densities in the IPL and OPL was slow, requiring up to 5 months.Conclusions: Homeostatic mechanisms exist in the retina to induce a repopulation by myeloid cells following microglial depletion. Guidance cues appear to be present to guide repopulating cells to the correct retinal lamina and to re-establish a mosaic distribution of ramified, non-overlapping cells. Current work is focused on understanding the mechanisms for microglial homeostasis and the functional implications of myeloid cell repopulation in the retina.Commercial Relationships: Yikui Zhang, None; Lian Zhao, None; Xu Wang, None; Robert N. Fariss, None; Wai T. Wong, None

Program Number: 4186 Poster Board Number: D0205Presentation Time: 3:45 PM–5:30 PMTumor suppressor p27KIP1 modulates cell-cycle entry and limited neurogenic potential in radial Müller gliaEdward Levine1, 2, Dennis M. Defoe3, Felix Vazquez-Chona2. 1Vanderbilt Eye Institute, Vanderbilt University, Nashville, TN; 2Ophthalmology & Visual Sciences, University of Utah, Salt Lake City, UT; 3Biomedical Sciences, ETSU College of Medicine, Johnson City, TN.Purpose: The proliferative response of Müller glia is a target of regeneration strategies. However, uncontrolled glial activation also contributes to gliosis and tissue remodeling. In an effort to identify key regulators of proliferative reactive gliosis, we address whether

p27KIP1 is a negative of regulator Müller glial cell-cycle entry and neurogenic potential as well as of non-proliferative responses such as migration and GFAP upregulation.Methods: We conditionally targeted the p27 coding region in adult mice harboring the p27LoxP mutation and expressing a tamoxifen-inducible Cre-recombinase under the control of glial promoter GLAST. We also induced transgenic p27 expression in retinal degeneration models using the ROSA26 promoter and Cre-Lox technology. To determine which p27-domain modulates reactive gliosis we induced light damage in mice harboring mutations in either its CDK/cyclin (CK) domain or through its phosphorylation state at serine-10 (S10).Results: Glial-specific p27 inactivation induced enhanced expression cell-cycle entry markers (PCNA and Ki67) with limited expression of mitotic marker phospho-histone H3. p27-deficient Müller glia also displayed re-expression of late progenitor markers such as MCM6 and OTX2 but resulted in additional Müller glia or underwent cell death. Transgenic p27 expression decreased Müller glial proliferation and outer limiting membrane (OLM) breakdowns but was insufficient to block GFAP expression. Mutations in p27 CDK/cyclin domain enhanced nuclear translocation and OLM breakdowns after light-induced retinal degeneration.Conclusions: Our genetic experiments suggest that p27 activity is most closely associated with migratory behavior and cell-cycle entry, whereas GFAP upregulation appears to be a secondary event. Thus, p27 is a prime target to facilitate the first step in regeneration strategies: Müller glial proliferation. Increasing p27 activity may also limit scar formation and OLM breakdowns of during trauma or post-operative eye surgery.Commercial Relationships: Edward Levine, None; Dennis M. Defoe, None; Felix Vazquez-Chona, NoneSupport: EL: RPB, R01-EY013760; P30-EY014800 FV: T32-HD07491, FFS, IRRF, KTF

Program Number: 4187 Poster Board Number: D0206Presentation Time: 3:45 PM–5:30 PMPharmacologic Inhibition of Intermediate Filament Accumulation in Astrocytes and Müller Glia Mediates Retinal NeuroprotectionJeremy M. Sivak1, 2, Darren Chan1, 2, Xiaoxin Guo1, Izzy Livne -Bar3. 1Vision Sciences, Krembil Research Institute, University Health Network, Toronto, ON, Canada; 2Vision Sciences and Ophthalmology, University of Toronto, Toronto, ON, Canada; 3School of Optometry, University of California at Berkeley, Berkeley, CA.Purpose: Astrocyte reactivity in the retina is primarily characterized by accumulations of the type III intermediate filaments (IFs) vimentin and glial fibrillary acidic protein (GFAP). However it has proven challenging to modulate this accumulation under pathological conditions to explore the roles of this process experimentally. We conducted experiments with the small molecule antagonist, withaferin A (WFA), to block type III IF dynamics in vivo in conjunction with metabolic retinal injury, and in combination with a model of induced astrocyte reactivity.Methods: Apoptosis of inner retinal neurons was induced in C57bl/6 mice by excitotoxic injury in combination with vehicle or WFA treatment. This injury model has been established to be dependent on astrocyte and müller glial derived secretion of TNF-α. In addition, this model was combined with induced retinal glial reactivity through transient corneal injury. Glial reactivity and retinal ganglion cell (RGC) apoptosis were imaged and quantified through immunofluorescence microscopy. TNF-α levels were also measured by immunofluorescent and ELISA based methods.



Results: WFA treatment significantly blocked glial reactivity and RGC apoptosis by 80% and 60%, respectively. Induced glial reactivity exacerbated the gliosis by 16 fold, and RGC damage by 7 fold. This increased vulnerability in both parameters was completely rescued by WFA treatment. WFA inhibited p38 mediated TNF-α secretion in cultured retinal astrocytes, and significantly reduced injury induced TNF-α immunoreactivity in the inner retina in vivo.Conclusions: Inhibition of IF dynamics effectively protected the inner retina from excitoxic damage. Our results suggest this mechanism is regulated through release of TNF-α by retinal astrocytes and müller glia.Commercial Relationships: Jeremy M. Sivak, Genentech (F); Darren Chan; Xiaoxin Guo, None; Izzy Livne-Bar, NoneSupport: CIHR (MOP123448), NSERC (RGPIN-2015-06561), GRSC

Program Number: 4188 Poster Board Number: D0207Presentation Time: 3:45 PM–5:30 PMEffect of TNF-α on the expression of glial fibrillary acidic protein (GFAP) and on the photoreceptor differentiation of human Müller glial stem cells in vitro.Erika Aquino, Karen Eastlake, Angshumonik Angbohang, Peng T. Khaw, G. Astrid Limb. NIHR Biomedical Research Centre for Ophthalmology, UCL Institute of Ophthalmology and Moorfields Eye Hospital, London, United Kingdom.Purpose: During retinal gliosis, biochemical and physiological changes occurring in the retina lead to Müller glia proliferation and overexpression of glial fibrillary acidic protein (GFAP), as well as the release of pro-inflammatory factors such as TNF-α. However, the exact role and regulation of GFAP in human Müller glia is not understood, and the role of TNF-α on the neural differentiation of these cells has not been examined. This study investigated the in vitro effect of TNF-α on GFAP expression by the Müller glial stem cell line MIO-M1 as well as on the photoreceptor differentiation of these cells.Methods: MIO-M1 cells were cultured with TNF-α in the presence or absence of FGF-2, taurine, retinoic acid and IGF-1 (FTRI), known to induce photoreceptor differentiation in these cells. RNA was extracted, cDNA produced and GFAP gene expression examined by RT-PCR. Protein expression of GFAP and NR2E3, a marker of photoreceptor differentiation, were examined by western blot analysis. Optical density of bands obtained from PCR and western blot were quantitatively measured and statistically analysed by paired t-test.Results: MIO-M1 cells cultured with increasing concentrations of TNF-α showed that the mRNA and protein expression of GFAP decreased in a dose-dependent manner (p<0.05). Upon culturing cells with FTRI to induce photoreceptor differentiation, there was a significant increase in NR2E3 protein expression (p=0.003). Addition of TNF-α to cells cultured with FTRI did not modify NR2E3 expression as compared to FTRI alone. Moreover, FTRI caused a significant downregulation of GFAP mRNA expression (p<0.0001) in these cells.Conclusions: The present results that a decrease in GFAP expression occurs in MIO-M1 cells cultured with FTRI indicates that the neural differentiation process itself may prevent the development of gliosis. They also suggest that TNF-α may have a protective effect on gliosis in human Müller glia. Further understanding of the mechanisms by which TNF-α may prevent GFAP upregulation may aid in the design of therapeutic approaches to prevent or control retinal gliosis.Commercial Relationships: Erika Aquino, None; Karen Eastlake, None; Angshumonik Angbohang, None; Peng T. Khaw, None; G. Astrid Limb, None

Support: The Special Trustees of Moorfields Eye Hospital and the NIHR Biomedical Research Centre at Moorfields Eye Hospital and UCL Institute of Ophthalmology, London, UK.

Program Number: 4189 Poster Board Number: D0208Presentation Time: 3:45 PM–5:30 PMThe Role of Notch Signaling in the Regenerating Adult Zebrafish RetinaJoshua Hobgood. Biology, University of Notre Dame, South Bend, IN.Purpose: In the damaged zebrafish retina, Müller glia are responsible for regenerating lost cells. We previously showed that Notch signaling is required to maintain Müller glia in a quiescent state, and inhibiting Notch signaling, via intraperitoneal injection of the g-secretase inhibitor RO4929097, is sufficient to induce a regenerative response. Thus, it appears Notch signaling is a negative regulator of retinal regeneration in the zebrafish eye. However, the zebrafish genome encodes four unique Notch receptors; Notch 1a, Notch 1b, Notch 2, and Notch 3. It was unclear if all or only a subset are involved in Müller glia quiescence and if they had any other functions in retinal regeneration. Thus, the purpose of this study was to begin to elucidate the function(s) of the individual Notch receptors in the undamaged and regenerating zebrafish retinas.Methods: Adult albino zebrafish were placed in complete darkness for 14 days, before being subjected to constant light damage. Retinas were isolated for RNA extraction, and qRT-PCR was performed on the four Notch receptor genes using Taqman probes. Dark-adapted albino zebrafish were subjected to morpholino-mediated knockdown of all four Notch receptors, separately, and then subjected to light damage. The retinas were isolated, cryosectioned, and immunolabeled for PCNA, Stat3, and Ascl1a, and analyzed by confocal microscopyResults: The qRT-PCR analysis revealed an increase in expression for notch 1a, 1b, and 2, while notch 3 decreased in expression from at from 0 to 16 hours, and increased subsequently throughout the light timecourse. Morpholino-mediated knockdowns of Notch 1a, 1b, and 2 resulted in fewer proliferating Müller glia and neuronal progenitors at 36 and 72hr of light compared to the controls. In contrast, morpholino-mediated knockdown of Notch 3 resulted in increased numbers of proliferating Müller glia at 36 and 72 hrs.Conclusions: Notch receptors 1a, 1b, and 2, are required for the maximal proliferative response in the light-damaged zebrafish retina. In contrast, downregulation of Notch 3 is necessary for Müller glia to re-enter the cell cycle in response to damage. This suggests that Notch 3 is necessary to maintain Müller glia quiescence and Notch 1a, 1b, and 2 are required for Müller glia and neuronal progenitor proliferation.Commercial Relationships: Joshua Hobgood, None

Program Number: 4190 Poster Board Number: D0209Presentation Time: 3:45 PM–5:30 PMChanges in miRNAs in Müller glia after retinal injury and Dicer deletionStefanie G. Wohl, Thomas A. Reh. Biological Structure, University of Washington, Seattle, WA.Purpose: microRNAs (miRNAs) are negative regulators of gene expression and play roles in retinal development and regeneration (in zebrafish). Less is known about the role of miRNAs in the response to injury in mouse Müller glia (MG). We used NanoString technologies® and quantified miRNAs in 1) mature wild type MG, 2) after light damage (LD), as well as 3) in Dicer conditional knock out (CKO) MG cells.



Methods: We isolated the MG from naïve or light damaged adult wildtype mice (Rlbp-CreER: floxed-stop-tdTomato (TdTom)) mice, as well as from Dicer CKO mice (Rlbp-CreER: floxed-Dicer: floxed-stop-tdTomato), by means of fluorescent activated cell sorting (FACS). To activate the CreER/reporter in the wild type mice, we injected Tamoxifen (Tmx) intraperitoneally 2 days before sorting. The LD mice received Tmx 2 days before the LD (8 h) and the cells were collected 1 week later. To delete Dicer from the MG glia, Tmx was given at postnatal days (P) 11-14 and the cells were collected 4 weeks later. We verified the specificity of the MG labeling using histological analysis and immunofluorescent labeling. Samples of at least 5 sorts (4-6 mice each) were pooled the RNA was purified and analyzed.Results: With multiple Tmx injections the Rlbp1-creER is very efficient: ~ 98% of all MG were tdTom+. For all experiments, the tdTom+ MG were over 90% pure, whereas the reporter negative fractions had no tdTom+ MG. In adult MG, 256 miRNAs were more highly expressed than negative controls. Comparing the TdTom+ cells with the negative fraction allowed us to determine which miRNAs are specifically expressed in MG. After Dicer deletion in the MG, these enriched miRNAs declined between 63-90%. LD caused a number of changes in miRNAs in the MG, with many declining and other increasing in response to injury. Interestingly, there was an increase in the number of tdTom+ MG in the Dicer CKO as assessed by FACS (3.2% versus 1.8%, p = 0.000) or cell counts in sections (18% more MG/ field; p = 0.012). However, the retinas in the Dicer CKO appeared normal and the MG displayed a normal morphology.Conclusions: Our data shows that specific miRNAs are highly expressed in MG, and that LD caused dramatic changes in many of these. The CKO of Dicer caused a decline in most miRNAs and an increase in MG cell number, though the overall retinal structure and MG morphology is not altered.Commercial Relationships: Stefanie G. Wohl; Thomas A. Reh, NoneSupport: National Eye Institute grant NEI R01EY021482, Grant # TA-RM-0614-0650-UWA from the Foundation Fighting Blindness to T.A.R., scholarship Wo 2010/1-1 for S.G.W. from Deutsche Forschungsgemeinschaft (DFG).

Program Number: 4191 Poster Board Number: D0210Presentation Time: 3:45 PM–5:30 PMMüller cell processes occupy the subretinal space in geographic atrophy and Stargardt diseaseMalia M. Edwards1, D. Scott McLeod1, Imran A. Bhutto1, Rhonda Grebe1, Mercedes Villalonga3, Vera L. Bonilha2, Joe G. Hollyfield2, Johanna M. Seddon3, 4, Gerard A. Lutty1. 1Wilmer Eye Institute, Johns Hopkins University School of Medicine, Baltimore, MD; 2Cole Eye Institute, Cleveland Clinic, Cleveland, OH; 3Ophthalmic Epidemiology and Genetics Service, New England Eye Center, Tufts Medical Center, Boston, MA; 4Ophthalmology, Tufts University School of Medicine, Boston, MA.Purpose: Müller cells create the external limiting membrane (ELM) by forming junctions with photoreceptor cells. This study investigated how the focal loss of photoreceptors and retinal pigment epithelium (RPE) in geographic atrophy (GA) and Stargardt disease (STGD) affects Müller cells and the ELM.Methods: Human donor eyes with either no retinal disease (N=4), GA (N=4) or STGD (N=2) were obtained through NDRI, the AMD Registry and Biorepository and the Foundation Fighting Blindness Eye Donor Program. Eyes were imaged after retinal dissection with RPE intact and after RPE removal. Retinas were then stained with GFAP (astrocytes and activated Müller cells), vimentin (Müller cells) and UEA lectin (blood vessels) while the submacular choroids were

stained with UEA lectin. Choroids and retinas (photoreceptors up) were imaged using a Zeiss 710 confocal microscope. After imaging, retinas were cryopreserved and sectioned to better visualize the Müller cells. Additional cross sections were stained with nestin. Three dimensional images were created using Imaris. Eyes were also processed for transmission electron microscopy (TEM).Results: Vimentin staining of the control retina revealed a pan retinal cobblestone-like ELM. This pattern was also observed throughout much of the diseased retinas. Areas with vimentin+ and vimentin+/GFAP+ cells were noted subretinally in GA and STGD eyes. Comparison to the gross photos revealed that these subretinal glial membranes very closely correlated with areas of RPE atrophy. In eyes with GA, smaller glial projections were observed beyond the atrophic region which aligned with drusen in gross photos. The presence of glial elaboration into the subretinal space was confirmed by TEM. Cross sectional analysis of eyes with GA demonstrated that Müller cells anterior to atrophy were disorganized and expressed nestin. Choriocapillaris loss was also evident subjacent to these atrophic areas and was more severe in eyes with STGD.Conclusions: Photoreceptor and RPE cell death appears to stimulate Müller cell remodeling and subretinal membrane formation. These glial membranes likely represent a Müller cell attempt to replace the ELM in regions of degenerated photoreceptors but may interfere with future treatments like photoreceptor or RPE replacement therapy. Subretinal Müller cells may also express VEGF, potentially contributing to the progression from GA to neovascular AMD.Commercial Relationships: Malia M. Edwards, None; D. Scott McLeod, None; Imran A. Bhutto, None; Rhonda Grebe, None; Mercedes Villalonga, None; Vera L. Bonilha, None; Joe G. Hollyfield, None; Johanna M. Seddon, None; Gerard A. Lutty, NoneSupport: NIH/NEI RO1EY016151-09 (GL), EY01765 (Wilmer), R01EY014340 (JH), The Foundation Fighting Blindness (JH), Research to Prevent Blindness Unrestricted grants (Wilmer Eye Institute, Tuft Medical Center, Cole Eye Institute), Wilmer Pooled Professor Fund (ME), and the Macular Degeneration Research Fund Tufts Medical Center, Boston, MA (JMS).

Program Number: 4192 Poster Board Number: D0211Presentation Time: 3:45 PM–5:30 PMGliotic remodeling in long-term organotypic culture of the human retinaArnold Szabo1, Maria Anna Hudak1, Akos Kusnyerik2, Akos Lukats1, Zoltan Zsolt Nagy2, Janos Nemeth2, Agoston Szel1. 1Department of Human Morphology and Developmental Biology, Semmelweis University, Budapest, Hungary; 2Department of Ophthalmology, Semmelweis University, Budapest, Hungary.Purpose: Previously we have shown that in appropriate culture system the human retina can be kept alive with near normal morphology at least for 10 weeks. The overall architecture remains intact in culture and every major cell type survives, however, over time a gliotic reaction develops. In this study we examine this gliotic reaction in detail and describe the temporal changes of Müller cells, astrocytes and microglia.Methods: Adult human eyes with very short (0-4 h) post mortem intervals were used in this study. Approximately 5x5 mm pieces of freshly isolated retina were placed on polycarbonate membranes and were cultured in serum-free medium for up to 10 weeks. The cultures were fixed in different time points and were analyzed by immunohistochemistry using glia-specific markers. The expression levels of glia-specific proteins were determined by Western Blot.Results: The overall morphology was well preserved. All retinal layers were maintained even after ten weeks, but as a sign of retinal



edema the retinal thickness was increased. The Müller cells became hypertrophic with increased expression of vimentin and GFAP. At the outer surface the endfeet of Müller cells developed tiny processes extending beyond the outer limiting membrane. Using antibody against S100 beta we were able to distinguish between astrocytes and Müller cells in gliotic samples. In the central retina S100 beta stained exclusively astrocytes among glial cells, and its expression remained constant during culturing. Reactive astrocytes with swollen cytoplasm were found in the inner retina. In the inner plexiform layer long astrocyte processes formed several horizontal bundles. In non-cultured controls the microglial cells were restricted to the inner retina, while in cultures a fraction of microglial cells invaded the outer retina as well. In some cases close morphological relation between microglia and degenerating photoreceptors was visible.Conclusions: To our knowledge our model provides the first experimental tool which allows the long-term investigation of retinal gliosis in three dimensional human retina with preserved cytoarchitecture. Our results underline differences in the involvement of glial cell subtypes in gliotic reaction and could be a basis of further experiments with clinical significance.Commercial Relationships: Arnold Szabo, None; Maria Anna Hudak, None; Akos Kusnyerik, None; Akos Lukats, None; Zoltan Zsolt Nagy, None; Janos Nemeth, None; Agoston Szel, None

Program Number: 4193 Poster Board Number: D0212Presentation Time: 3:45 PM–5:30 PMInhibition of Muller Cell Circadian Rhythms by siRNA Knockdown of Period GenesLili Xu2, Andrew Liu3, John S. Penn1, Douglas McMahon2. 1Ophthalmology, Vanderbilt University, Nashville, TN; 2Biological Sciences, Vanderbilt University, Nashville, TN; 3Department of Biology, University of Memphis, Memphis, TN.Purpose: The mammalian retina contains a self-sustained circadian clock, but the precise roles of retinal cell types and clock genes in retinal rhythms remain to be defined. We have shown in previously that mouse retinal Muller cells (MMC) of C57BL/6J exhibit self-sustained clock gene rhythms and that Per1, is necessary for overall retinal molecular circadian rhythms. Here we have tested the clock gene rhythms in MMC of C3fh+/+ and MT1-/- and MT1,2-/- and the role of Per1and Bmal1 in MMC circadian rhythms using siRNA knockdown.Methods: Purified C3fh, MT1-/- and MT1,2-/- MMC were seeded into 6 well plates and transfected with Per2Luc or Bmal1Luc vectors. Cell selection was performed with Blasticidin S treatment resulting in stable cell lines expressing Luciferase. C3fh+/+/Per2Luc MMC were serum starved overnight, trypsinized, and then incubated with siRNA (for Per1, Per2, Bmal1, control siRNA) and Lipofectamine for 25min at room temperature. Cells were then seeded onto 35mm dishes containing serum free of DMEM and incubated at 37°C in 5% CO2 and 95% ambient air for 6 hours, switched to 10% FBS DMEM for 6 hours, then changed into recording medium and transferred to a multichannel luminometer for recordingResults: C3fh+/+ and MT1-/- MMC exhibited robust free-running rhythms in PER2::LUC and BMAL1::LUC bioluminescence that were maintained for at least 6 cycles and were partially restores in amplitude following a media change. MT12-/- MMC exhibited the rhythms in BMAL1::LUC bioluminescence and disappeared in PER2::LUC bioluminescence. Rhythms of C3fh+/+/Per2::Luc MMC were suppressed for 14 cycles by treatment with siRNA against Per1, Per2 and Bmal1. Media change increased luminescence, but did not restore rhythmicity. Control siRNA and lipofectamine alone had no effect on MMCrhythms.

Conclusions: Our results show that C3fh+/+ and MT1-/- MMC exhibit molecular circadian rhythms in PER2::LUC and BMAL1::LUC bioluminescence. MT12-/- MMC exhibit the rhythms in BMAL1::LUC bioluminescence. The expression of Per1 and Bmal1 are necessary for rhythmicity in C3fh+/+ MMC populations. These observations could either be the result Per1 and Bmal1 siRNA inhibition of the molecular circadian rhythms in individual C3fh MMC, or due to desynchronization of MMC populations. Future experiments using siRNA transfection in MT1-/- and MT12-/- MMC could differentiate between these possibilities.Commercial Relationships: Lili Xu, None; Andrew Liu; John S. Penn, None; Douglas McMahon, NoneSupport: NIH R01EY015815, P30EY008126

Program Number: 4194 Poster Board Number: D0213Presentation Time: 3:45 PM–5:30 PMChR2 Stimulation in Müller cells Modulates the Retinal Light ResponseCameron Baker, Janette Tang, Mike Berry, Ehud Isacoff, John G. Flannery. Technology, Univeristy California Berkeley, Berkeley, CA.Purpose: Müller cells are the principle glia cells of the retina. Responsible for homeostasis, waste removal, and nutrient delivery, these macroglia cells support their highly specialized neuronal neighbors. Beyond these maintenance roles, Müller glia can potentially augment neuronal communication through their role in neurotransmitter reuptake. Like astrocytes, Müller glia take up excess, and potentially toxic, neurotransmitters (e.g. glutamate) via ion exchange transporters. Müller glia maintain a high membrane potential of -80 mV to support their ion exchange transporters like GLAST and GLT-1. We hypothesize that biasing this membrane potential can inhibit neurotransmitter reuptake systems, modulating the surrounding retinal neuronal light response.Methods: We expressed the canonical ChR2 or a bi-stable point mutant, C128S, in mouse retinal Müller cells via intravitreal injection of ShH10-Y445F, an Müller cell specific AAV capsid previously developed in our group by directed evolution. Mouse retinal light reponses were measured by electroretinogram (ERG) and multielectrode array (MEA).Results: ERG recordings demonstrate a depressed B-wave from retinas expressing ChR2 in Müller cells. We hypothesized that if ChR2 attenuates the membrane potential of Müller cells and inhibits glutamate uptake, we should observe this as a depression of the B-wave amplitude with no change to the A-wave. We indeed found the B-wave for the ChR2-Müller retina to be reduced, and the A-wave remained unaltered.MEA recordings using the bi-stable ChR2(C128S) show a recoverable decreased OFF response after ChR2 stimulation. The bi-stable ChR2(C128S) treated retinas display different channel kinetics than the canonical ChR2. After light stimulation, ChR2(C128S) remains open for ~ 5 minutes. Retinas were exposed to low white light stimulation and then bright activating ChR2 light. Low light stimulation after ChR2(C128S) stimulation showed a decreased OFF response that was able to recover.Conclusions: This electrophysiological data shows neuronal perturbation due to ChR2 stimulation in Müller glia, demonstrating the glial capability to modulate its neuronal neighbors.Commercial Relationships: Cameron Baker, None; Janette Tang, None; Mike Berry, None; Ehud Isacoff, None; John G. Flannery, None



Program Number: 4195 Poster Board Number: D0214Presentation Time: 3:45 PM–5:30 PMThe role of FAK in fibrotic matrix contraction by dedifferentiated Müller cellsRintaro Tsukahara1, Kazuhiko Umazume2, Naoyuki Yamakawa2, Takuya Iwasaki1, Henry J. Kaplan3, Hiroshi Goto2, Shigeo Tamiya3, 4. 1Ophthalmology, Tokyo Med Univ, Ibaraki Med Ctr, Inashikigun, Japan; 2Ophthalmology, Tokyo Med Univ, Shinjuku, Japan; 3Ophthalmology and Visual Sciences, University of Louisville, Louisville, KY; 4Biochemistry and Molecular Biology, University of Louisville, Louisville, KY.Purpose: We have previously reported the inhibitory effect of dasatinib on fibrotic matrix contraction using both in vitro and in vivo models (Umazume K, et al IOVS 2013, Tsukahara R, et al EER 2015). Dasatinib was developed as a dual inhibitor of SFK and ABL tyrosine kinases but has also been reported to inhibit other tyrosine kinases including focal adhesion kinase (FAK). In this study, we examined the effect of FAK inhibition on matrix contraction by dedifferentiated Müller cells, which has been implicated in several ocular fibrotic complications.Methods: Müller cells were isolated from porcine eyes using a papain/DNase kit, and used between passages 3-6. Cells were cultured for 3 days in 25% vitreous fluid supplemented DMEM in the presence or absence of dasatinib or PF573228 (FAK inhibitor). Expression of active (phosphorylated) and total FAK protein expression was examined by Western blot analyses. A type I collagen matrix contraction assays were used to examine matrix contraction.Results: Dedifferentiated Müller cells expressed active FAK. PF573228 treatment significantly reduced active FAK expression and inhibited matrix contraction. When added early (on day 0) prior to cell clustering and pseudo-ERM formation, the inhibitory effect on matrix contraction was comparable to dasatinib. On the other hand, addition of PF573228 after cell clustering (on day 2) resulted in a suppressive effect that was still significant but much diminished in comparison to dasatinib.Conclusions: FAK is involved in matrix contraction by differentiated Müller cells, with the main role early on prior to and/or during cell clustering and ERM formation.Commercial Relationships: Rintaro Tsukahara; Kazuhiko Umazume, None; Naoyuki Yamakawa, None; Takuya Iwasaki, None; Henry J. Kaplan, None; Hiroshi Goto, None; Shigeo Tamiya, NoneSupport: DoD/USAMRAA DMRDP-ARATDA DM090475; Unrestricted institutional grant from Research to Prevent Blindness, NY, NY

Program Number: 4196 Poster Board Number: D0215Presentation Time: 3:45 PM–5:30 PMRole of Dexamethasone preventing Müller glial cells swellingLourdes M. SIQUEIROS1, 2, Hugo Charles-Messance1, Rodrigo Bolanos3, 4, Cecilia Montañez2, José Sahel1, 5, Alvaro Rendon1, Ramin Tadayoni1, 6, Audrey Giocanti7. 1Institut de la Vision/INSERM/UPMC Univ Paris 06/CNRS/CHNO des Quinze-vingts, Paris, France; 2Genetics and Molecular Biology, CINVESTAV, Mexico, Mexico; 3Association to prevent blindness in Mexico, Dr. Luis Sanchez Bulnes Hospital, Mexico, Mexico; 4Ophthalmology, Regional Hospital, Adolfo López Mateos, ISSSTE, Mexico, Mexico; 5Fondation Ophtalmologique Adolphe de Rothschild, Paris, France; 6Ophthalmology, Hôpital Lariboisière, AP-HP, Univ Paris Diderot, Paris, France; 7Ophthalmology, Avicenne hospital, Bobigny, France.Purpose: Retinal edema is due to vascular leakage and/or through cytotoxic events (e.g., glial cell swelling); Müller glial cells (MGC) play a crucial role regulating the volume of the extracellular space

and water and ion homeostasis as well as preserving the blood retinal barrier.Erreur ! Référence de lien hypertexte non valide.We had previously showed that dystrophin Dp71 has a central role in the molecular scaffold responsible for anchoring AQP4 and Kir4.1 in MGC. HSF1 is a transcriptional regulator of Dp71.Dexamethasone (Dex) is a corticosteroid commonly used for the treatment of many retinal diseases (diabetic macular edema, vein occlusions), despite their exact mechanism remaining imperfectly understood.The purpose of this study was to analyze, on retinal explants, the effect of MGC swelling on Dp71 Kir4.1, AQP4, β-DG and ε-SG expression and to determine the role of Dex preventing the swelling and its effects.Methods: According to the ARVO guidelines for animal use for research, 8 weeks WT mice retina were used in a retinal explant model to reproduce the effect of retinal edema on MGC, the retinas were incubated 8h in an hypotonic solution with Barium (Ba).The soma of MGC was measured using the software Image J. We quantified by qPCR Kir4.1, AQP4, β-DG, ε-SG, Dp71 and HSF1 expression.In the same conditions the retinas were treated at baseline with Dex and the same parameters were tested.Results: We observed an increase of MGC soma volume 8h after incubation in hypotonic solution with Ba associated with a decrease of Dp71 (34%), β-DG (23%), AQP4 (56%) and Kir4.1 (50%) mRNA expression comparing with control. ε-SG and HSF1 expression were not modified.When Dex was added to the retinal explants the MGC swelling was prevented and Dp71, β-DG, AQP4 and Kir4.1 expression were comparable to control retina. Moreover, in presence of Dex we observed an increase of HSF1 expression (84%).Conclusions: Edema formation and resolution is a complex mechanism. Using retinal explants, we reproduced MGC swelling and observed an alteration of AQP4, Kir4.1 channels, in this model MGC swelling seems to be related with an inhibition on Dp71 expression. Moreover the addition of Dex to the retinal explants prevents this phenomenon and the results suggest it is related to an increase on HSF1 expression.Commercial Relationships: Lourdes M. SIQUEIROS; Hugo Charles-Messance, None; Rodrigo Bolanos, None; Cecilia Montañez, None; José Sahel, None; Alvaro Rendon, None; Ramin Tadayoni, None; Audrey Giocanti, None

Program Number: 4197 Poster Board Number: D0216Presentation Time: 3:45 PM–5:30 PMCoordinating reactive gliosis and regeneration following damage to the adult zebrafish retinaRyan Thummel2, Jennifer L. Thomas1, Alexandra H. Ranski2, Gregory W. Morgan3. 1Department of Biological Structure, University of Washington, Seattle, WA; 2Department of Anatomy/Cell Biology and Ophthalmology, Wayne State University School of Medicine, Detroit, MI; 3Department of Biochemistry, University of Washington, Seattle, WA.Purpose: In contrast to mammals, zebrafish posses the remarkable ability to regenerate retinal neurons. Damage to the zebrafish retina induces Muller glia to act as stem cells, generating retinal progenitors for regeneration. In contrast, injury in the mammalian retina results in Muller glial reactive gliosis, a characteristic gliotic response that is detrimental to vision. The purpose of this study was threefold. First, we aimed to determine whether zebrafish Muller glia show any signs of reactive gliosis following retinal damage. Next, we tested whether multiple rounds of damage and regeneration resulted in increased gliosis or decreased regeneration. Finally, we tested whether



inhibiting Muller glial cell cycle reentry resulted in an increased gliotic response.Methods: Adult albino zebrafish were exposed to constant intense light to damage photoreceptors. We analyzed the gliosis and regeneration response at multiple timepoints following light onset. In a separate study, we analyzed retinas that underwent six consecutive rounds of damage/regeneration. Finally, we analyzed retinas in which Muller glial proliferation was inhibited by intraocular injections of 5-Fluorouracil.Results: We provide evidence that Muller glia exhibited signs of reactive gliosis immediately following retinal damage, but then downregulated that response as large numbers of progenitors were generated. Next, we show that even after multiple rounds of damage and regeneration, retinas exhibited accelerated photoreceptor degeneration and some signs of increased reactive gliosis. However, photoreceptors were regenerated in normal numbers. Finally, cell cycle inhibition resulted in persistently gliotic Muller glia, which resulted in temporary neuroprotection of the photoreceptors to the cytotoxic lesion, but a significant long-term inhibition of retinal regeneration.Conclusions: Together, these data demonstrate that zebrafish Müller glia possess both gliotic and regenerative potential. Understanding the signaling pathways that determine how Müller glia respond to injury is a critical step toward promoting regeneration in the mammalian retina.Commercial Relationships: Ryan Thummel, None; Jennifer L. Thomas, None; Alexandra H. Ranski, None; Gregory W. Morgan, None

Program Number: 4198 Poster Board Number: D0217Presentation Time: 3:45 PM–5:30 PMImplication of 24-S-hydroxycholesterol in Müller glial cell’s membrane dynamics in the ratArthur Ferrero1, Segolene Gambert-Nicot2, NIyazi Acar2, Alain M. Bron1, 2, Lionel Bretillon2, Catherine P. Garcher1, 2. 1Ophthalmology, University Hospital Dijon, France, Dijon, France; 2CSGA, UMR1324 INRA, 6265 CNRS, Burgundy University, Eye and Nutrition Research Group, Dijon, France.Purpose: The catabolism of cholesterol in neurons leads to a more hydrophilic compound soluble form, the 24-S-hydroxycholesterol by means of an enzyme the CYP46A1. It has been shown that the increase of intraocular pressure in rats leads to glial activation and overexpression of CYP46A1. The aim of this study was to analyse the implication of 24-S-hydroxycholesterol (24-S-OHC) on Müller glial cells (MGC) membrane dynamics in the rat.Methods: MGC were grown in vitro from retinas of 10-day-old Long Evans rats. Cells were treated with 24-S-OHC (treatment) or ethanol (control) for 2 minutes or 6 hours. From twenty millions of MGC in each group, lipid-rafts were obtained after a 1% Lubrol lysis and an ultra centrifugation (180 000g – 20 hours – 4 °C). The following proteins: caveolin, flottilin, connexin 30 and 43, CRALBP, DHAPAT, GFAP and vimentin were analysed using Western blotting on all fractions (lipid-rafts and non-rafts). MGC membrane fluidity was studied in vitro with two different techniques: anisotropy measurements performed with the lipophilic fluorescent probe TMA-DPH and fluorescence recovery after photobleaching (FRAP) observed using confocal microscopy.Results: 24-S-OHC treatment on in vitro MGC increased the expression of GFAP and delocalized GFAP in the lipid-raft fraction; 24-S-OHC treatment induced a delocalization of DHAPAT protein out of the lipid-rafts fraction. Anisotropy was decreased with the 24-S-OHC treatment (difference: 5.1 x 10-3; p < 0.01) revealing an increase of the membrane fluidity. This increase was confirmed by

the FRAP technique, which showed a shorter time of fluorescence recovery for the treated cells.Conclusions: This study showed that 24-S-OHC could be a candidate leading a key role in the activation of MGC, disturbing lipid-raft organization by changing the localization of signalization proteins and increasing membrane’s fluidity.Commercial Relationships: Arthur Ferrero, None; Segolene Gambert-Nicot, None; NIyazi Acar, None; Alain M. Bron, None; Lionel Bretillon, None; Catherine P. Garcher, None

Program Number: 4199 Poster Board Number: D0218Presentation Time: 3:45 PM–5:30 PMThe effect of systemic administration of methylene blue and metformin on photoreceptor degeneration caused by selective Müller cell disruptionWeiyong Shen, Michelle X. Yam, So-Ra Lee, Ying Wang, Sook Chung, Ling Zhu, Mark C. Gillies. Save Sight Institute, The University of Sydney, Sydney, NSW, Australia.Purpose: Derangement of glucose metabolism and energy production is a potential cause of photoreceptor degeneration. Methylene blue (MB) increases shuttling of electrons through the electron transport chain in mitochondria. Metformin (MET) enhances insulin signalling. MB and MET have shown neuroprotective effects in Alzheimer’s and Parkinson’s disease. However, there is little information about their beneficial effects on retinal disease. This study aimed to test the effect of MB and MET on photoreceptor degeneration caused by selective Müller cell disruption in transgenic mice we recently generated (Shen et al. J Neurosci 2012).Methods: Transgenic mice received daily intraperitoneal injection of MB (3mg/kg) and MET (50mg/kg) from 3 days after tamoxifen-induced Müller cell disruption and data analysis was conducted 5 days later. Changes in photoreceptors, microglia and differential expression of heat shock proteins (HSPs), glucose transporter 1 (GLUT1) and key proteins which are vital for energy homeostasis and fatty acid synthesis including acetyl-CoA carboxylase (ACC), AMP-activated protein kinase (AMPK), cytoplasmic acetyl-CoA synthetase (AceCS1) and mammalian long chain acyl-CoA synthetase (ACSL), were examined by immunofluorescent staining and Western blots.Results: We found that systemic administration of MB and MET, either alone or in combination, prevented photoreceptor degeneration and inhibited reactive activation of microglia. The effect on photoreceptor protection was accompanied by reduced expression of HSP60 and an increase in GLUT1 expression. Further analysis showed that MB and MET treatments inhibited phosphorylation of ACC and AMPKα and increased AceCS1 but not ACSL expression.Conclusions: Our data suggest that systemic administration of MB and MET is effective in protecting photoreceptors from degeneration. Regulation of mitochondrial function and insulin signalling may offer a novel approach for the treatment of retinal degenerative diseases.Commercial Relationships: Weiyong Shen; Michelle X. Yam, None; So-Ra Lee, None; Ying Wang, None; Sook Chung, None; Ling Zhu, None; Mark C. Gillies, NoneSupport: Australia National Health and Medical Research Council (NH&MRC, Project Grants 1028393, 1050373).



Program Number: 4200 Poster Board Number: D0219Presentation Time: 3:45 PM–5:30 PMThe influence of X-box Binding Protein 1 and the Unfolded Protein Response on Müller glia differentiation into retinal neuronsTodd McLaughlin1, 2, Jing Yang1, 4, Joshua J. Wang1, 2, Sarah X. Zhang1, 3. 1Departments of Ophthalmology and Ross Eye Institute, University at Buffalo, State University of New York, Buffalo, NY; 2SUNY Eye Institute, State University of New York, Buffalo, NY; 3Department of Biochemistry, University at Buffalo, State University of New York, Buffalo, NY; 4State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangzhou, China.Purpose: Retinal injury or pathologies often result in the loss of retinal cells and subsequent vision impairment. Endogenous repair of the mammalian retina does not occur to any meaningful extent. After retinal insult, Müller glia can de-differentiate into multipotent progenitor cells. We explore factors that influence Müller glia to act as a stem cell-like population able to re-differentiate into neuronal cell types and potentially repair the retina.Methods: We isolate Müller glia from adult wild type mice and inducible, cre-activated conditional knockout mice that lack X-box binding protein-1 (XBP1) in Müller glia. In vitro, we measure endoplasmic reticulum (ER) stress and activation of the Unfolded Protein Response (UPR) as Müller glia de-differentiate to a progenitor-like state and re-differentiate into neuronal subtypes. Protein levels for ER stress and cell-specific markers are determined with Western blotting and immunocytochemical staining. After multiple passages, we induce differentiation of Müller glia into neuron-like cells that express neuronal markers and cell-specific markers. Minor alterations in culture conditions allow us to induce Müller glia into photoreceptor-like cells or retinal ganglion-like cells.Results: We find induction of Müller glia differentiation leads to increased levels of ER stress and activation of all three pathways of the UPR. Conditional deletion of XBP1 in Müller glia amplifies activation of the UPR. Further, we find more rapid and complete differentiation of Müller glia into neuronal cell types in XBP1-negative cells compared to wild type cells. XBP1 deficiency decreases stemness and the proliferative ability of Müller glia while simultaneously increasing the expression of neuronal markers, number of neurites, and neurite length.Conclusions: We conclude that the activation of the UPR is a crucial parameter in promoting Müller glia differentiation. Specifically, XBP1 acts to decrease ER stress during Müller glia differentiation and preserves stemness and proliferative ability. Therefore, manipulating ER stress pathways (e.g. by overexpressing or down-regulating XBP1) at the appropriate time window after injury or disease might allow the Müller glia to revert to a progenitor-like state and proliferate to act as an endogenous repair system in the mammalian retina.Commercial Relationships: Todd McLaughlin, None; Jing Yang; Joshua J. Wang, None; Sarah X. Zhang, NoneSupport: NIH/NEI grants EY019949 and EY025061, ADA research grant #7-11-BS-182, and an Unrestricted Grant to the Department of Ophthalmology, SUNY-Buffalo, from Research to Prevent Blindness.

Program Number: 4201 Poster Board Number: D0220Presentation Time: 3:45 PM–5:30 PMRetinal degeneration triggers the activation of the Hippo pathway effector gene YAP in reactive Müller cellsChristel Masson-Garcia, Annaïg HAMON, Juliette Bitard, Jerome E. Roger, Muriel PERRON. Institute of Neuroscience, CNRS, CERTO, ORSAY, France.

Purpose: Retinitis pigmentosa (RP) is a heterogeneous group of inherited retinal degenerations leading to blindness and currently untreatable. In these diseases, Müller glia cells respond to photoreceptor loss by undergoing reactive gliosis, which has both beneficial and detrimental effects on cell survival. Increasing our knowledge of the complex molecular response of Müller cells to retinal degeneration is thus essential for the development of promising new therapeutic strategies. To identify novel players acting in Müller glia neuroprotective response, we focused our interest on the Hippo pathway, a key regulator of tissue homeostasis.Methods: We used rd10 mice (Pde6Brd10/rd10) as a model of human RP. These mice carry a mutation of the rod-specific phosphodiesterase gene and undergo rod death from P16. We determined mRNA and protein levels of Hippo/Yap pathway components using a combination of quantitative real-time PCR, Western blot, and immunohistochemistryResults: In wild-type postnatal and adult mice, we found that the effectors of the Hippo pathway, YAP1 and TEAD1, are specifically expressed in Müller cells. A comparison of their mRNA and protein levels between wild type and rd10 mice retina revealed an upregulation associated with photoreceptor loss. To determine the transcriptional outcome of this regulation of the Hippo pathway, we examined the expression of YAP1/TEAD1 well known target gene Cyr61 and found a 4-fold increase in rd10 retinas compared to control onesConclusions: This work uncovers a link between the Hippo/YAP pathway and Müller cell-dependent response to photoreceptor loss. Given the Müller cell mediated pro-survival capacities recently reported for Cyr61 (Kucharska et al., Journal of Neurochemistry 2014), our work also suggests that the activation of YAP1/TEAD1 activity in reactive Müller glia may be involved in a mechanism promoting photoreceptor neuroprotection in the degenerating retina.Commercial Relationships: christel Masson-Garcia, None; Annaïg HAMON; Juliette Bitard, None; Jerome E. Roger, None; Muriel PERRON, None

Program Number: 4202 Poster Board Number: D0221Presentation Time: 3:45 PM–5:30 PMMüller cell-derived YAP signaling as a new neuroprotective pathway in the degenerating retinaAnnaïg HAMON2, 1, Jerome E. Roger2, 1, Juliette Bitard2, 1, Christel Masson-Garcia2, 1, Elodie Grellier2, 1, Muriel PERRON2, 1. 1CERTO, Orsay, France; 2Neuro-Psi, Orsay, France.Purpose: Retinal Müller cells are essential for retinal homeostasis and mediate neuroprotection as they release neurotrophic factors during retinal degeneration. The Hippo/YAP signaling pathway has recently emerged as an important regulator of tissue homeostasis through its action on both cell proliferation and survival. Since we recently showed that YAP is specifically expressed in Müller cells, we decided to explore its role in the mouse retina, using a conditional knockout strategy, under both physiological and pathological degenerative conditions.Methods: We crossed Rax-CreERT2; Ai9 mice (Pak et al., Plos One, 2014) with Yapflox/flox mice (Reginensi et al., Plos Genetics, 2013) to generate Yap1 conditional knockout in Müller cells. Deletion of Yap1 was induced by intra-peritoneal injection of hydroxy-tamoxifen (4-OHT, 1mg) at P10. We used N-methyl-N-nitrosourea (MNU, 60mg/kg) intra-peritoneal injections to induce photoreceptor death at P30. Retinal phenotypes were analyzed using quantitative real-time PCR, Western blot, immunohistochemistry and TUNEL essay.Results: Under physiological conditions and up to 1 month, we did not observe any major retinal defects following Yap deletion in Müller cells. However, in retinas with MNU-induced photoreceptor



degeneration, we found that the loss of YAP in Müller cells leads to an increased number of apoptotic photoreceptor cells compared to control mice 16 hrs after injection. In addition, we showed by qPCR that the expression of GFAP, a gliosis marker, is also increased. Finally, following MNU injection in mice lacking Yap expression in Müller cells, we observed a two-fold decrease of Cyr61 expression, a direct target of YAP, compared to control animals.Conclusions: All together, our results reveal that the absence of YAP in Müller cells accelerates the MNU-induced photoreceptor cell death. This suggests that YAP may be involved in the neuroprotective response of reactive Müller glia to retinal injury. Based on our results and the recent pro-survival activity of Cyr61 recently shown in the retina (Kucharska et al., Journal of Neurochemistry, 2014), we propose a model where YAP neuroprotective function in Müller cells would be mediated by Cyr61.Commercial Relationships: Annaïg HAMON, None; Jerome E. Roger, None; Juliette Bitard, None; christel Masson -Garcia, None; Elodie Grellier, None; Muriel PERRON, NoneSupport: Retina France, FRM, ANR, Idex Paris-Saclay

Program Number: 4203 Poster Board Number: D0222Presentation Time: 3:45 PM–5:30 PMLin28a regulates retinal neurogliogenesis and confers ectopic neural potential on Müller gliaXiaohuan Xia, Iqbal Ahmad. Ophthalmology and Visual Science, Univ of Neb Med Center, Omaha, NE.Purpose: During the development of central nervous system (CNS), neurogenesis precedes gliogenesis. However, the mechanism underlying this temporal transition is poorly understood. Although most of studies on CNS showed the effect of Lin28 in regulating the self-renewal of neural stem cells, observations in P19 cells suggested that Lin28 might be involved in regulating the temporal generation of neurons and glia (Balzer et al., 2010, Development. 137:891). Here, we have examined the role of Lin28a in retinal neurogliogenesis to understand the apparent lack of neurogenic potential in mammalian Müller glia (MG).Methods: Lentivirus/retrovirus-mediated gain-of-function (GOF) and loss-of-function (LOF) perturbations were carried out in the late retinal progenitor cells (RPCs) (E18) in neurosphere culture, retinal explant culture, and in vivo (PN1-PN3) to demonstrate the influence of Lin28a on the generation of late born neurons (rod photoreceptors and bipolar cells) and MG. Lin28a LOF and GOF perturbations were carried out in the early RPCs (E14) and enriched MG, respectively, to demonstrate the influence of Lin28a on the generation and neurogenic potential of MG.Results: Lin28a LOF in late RPCs in all models studies significantly promoted differentiation of late born neurons and inhibited the generation of MG. In contrast, when Lin28a was knocked down in late RPCs, more MG and fewer late born neurons were generated, compared to controls. Interestingly, knockdown of Lin28a in early RPCs promoted their premature differentiation along glial lineage. When Lin28 was ectopically expressed in enriched MG, a subset of them acquired neuronal morphology and expressed neuronal markers, while decreasing the expression of glial markers.Conclusions: Lin28a may regulate the timing of neuronal versus glial differentiation in the developing retina and represent a target for facilitating neurogenic properties in MG.Commercial Relationships: Xiaohuan Xia, None; Iqbal Ahmad, NoneSupport: Lincy Foundation, Pearson Foundation, Research for Preventing Blindness

Program Number: 4204 Poster Board Number: D0223Presentation Time: 3:45 PM–5:30 PMImpact of a reduction of plasmalogen levels on Müller cell metabolismCharlotte Pallot1, Julie Mazzocco2, Stephane Gregoire2, Laurent Leclere2, Benedicte Buteau2, Alain M. Bron1, 2, Catherine P. Garcher1, 2, Lionel Bretillon2, Niyazi Acar2. 1Ophthalmology, University hospital, Dijon, France; 2INRA, Eye and Nutrition research group, Dijon, France.Purpose: Retinopathy of Prematurity (ROP) is the leading cause of blindness in premature children resulting from abnormal vascular development. Previous studies have shown that omega-3 polyunsaturated fatty acids (PUFAs) can modulate physiologic and pathologic angiogenesis. Data from our laboratory suggest that plasmalogens, that are phospholipids that concentrate omega-3 PUFAs, are involved in the regulation of post-natal retinal vascular development through the action of a calcium-independent phospholipase A2 (iPLA2) (Saab et al PLoSONE 2014). In the retina, Müller cells are known to play crucial functions in retinal angiogenesis as well as to be rich in plasmalogens and to express phospholipases. The aim of this study is to investigate the roles of plasmalogens and omega-3 PUFAs on Müller cell metabolism.Methods: Primary cultures of rat Müller cells were treated with siRNA against the major enzyme for plasmalogen biosynthesis (DHAP-AT) and/or with a chemical inhibitor of iPLA2, bromoenol lactone (BEL). Lipid profile of cells was determined by gas chromatography, cell proliferation by Ki67 assay and cell migration was evaluated by videomicroscopy. The expression of p38, ERK, JNK in their non-phosphorylated and phosphorylated forms was determined by western blotting.Results: The use of siRNA against DHAP-AT led to a 50%-diminution of plasmalogens levels in cells. Decreasing the cell levels of plasmalogens and/or inhibiting iPLA2 had no impact on cellular viability. A reduction of the ability of cells to migrate was observed in samples treated with siRNA against DHAP-AT at 72 hours. The inhibition of the expression of DHAP-AT led to an increase in the rate of phosphorylation of ERK. No effect of BEL was observed on these parameters.Conclusions: Plasmalogens seem to initiate a MAPK-dependent signalling cascade in retinal Müller cells that might impact cell phenotype. Alteration of Müller cell metabolism seems to be the consequence of a diminution of plasmalogens levels following siRNA treatment rather than that of an inhibition of iPLA2 by BEL.Commercial Relationships: Charlotte Pallot, None; Julie Mazzocco, None; Stephane Gregoire, None; Laurent Leclere, None; Benedicte Buteau, None; Alain M. Bron, None; Catherine P. Garcher, None; Lionel Bretillon, None; Niyazi Acar, NoneSupport: National Institute for Research on Agronomy (INRA); Regional Council of Burgundy; European Regional Development Fund (FEDER)



Program Number: 4205 Poster Board Number: D0224Presentation Time: 3:45 PM–5:30 PMImmunolocalization of Gap Junction Protein Connexin 43 (GJA1) in retina and Glial Müller Cells in WT and Dp-71 null miceRodrigo Bolanos1, 2, Lourdes M. SIQUEIROS3, Yonathan Garfias4, 5, Cecilia Montañez6, José Sahel3, Xavier P. Guillonneau3, Alvaro Rendon3. 1Anterior Segment Department, Association to Prevent Blindness in Mexico, Dr Luis Sánchez Bulnes, Mexico, Mexico; 2Ophthalmology Service, Regional Hospital, Adolfo López Mateos, ISSSTE, Mexico, Mexico; 3Laboratoire de Physiopathologie Cellulaire et Moléculaire de la Rétine, Sorbonne Universités, UPMC Univ Paris 06, INSERM, CNRS, Institut de la Vision, Paris, France; 4Department of Biochemistry, Faculty of Medicine. Universidad Nacional Autónoma de México, UNAM., Mexico, Mexico; 5Research Unit, Institute of Ophthalmology “Conde de Valenciana”, Mexico, Mexico; 6Department of Genetics and Molecular Biology, CINVESTAV, Mexico, Mexico.Purpose: Dp71, the main product of the Duchenne muscular dystrophy gene expressed in retina, is essential for glial cell functions such as retinal water homeostasis and maintenance of the inner blood–retinal barrier (iBRB). Tight junction complexes at the iBRB contain a large number of proteins sucha as connexin 43 (Cx43) among many others. Downregulation of Cx43 expression provokes vascular cell death and increases vascular permeability. We have previously shown that Dp71 is localized in Müller glial cells (MGC) and astrocytes, together with endothelial cells and pericytes surround the retinal vessels to form the iBRB. The purpose of this study is to determine the expression of Cx43 in WT and Dp71-null mice retina in order to explore the role of Dp71 in the maintaining of the iBRB.Methods: According with the ARVO guidelines for animal use for research, 8 weeks WT and Dp71 null mice were used. The expression of Cx43 was determined by qRT-PCR in WT and Dp71 null mice. Sagittal 12-micras cryosections of retinas and isolated retinal cells were used for double-label immunohistochemistry using anti Connexin-43 (Cx43), Glial Fibrillary Acidic Protein (GFAP) and Glutamine synthase (GS) antibodies to determine the expression pattern and the localization of Cx43 in WT and Dp71 null mice retina, astrocytes and MGC.Results: A significant decrease (p=0057) was observed in mRNA expression of Cx43 in Dp71-null mice retina comparing with WT mice. In WT mice retina, Cx43 immunoreactivity was detected on GFAP-positive astrocytes, in the retinal ganglion cell layer and on GS-labeled Müller cells. We observed a impressive decrease of Cx43 expression in MGC and astrocytes of Dp71-null mice retina when compared with WT strain. Moreover, Dp71 deficiency was also associated with an impaired clustering of Cx43. Cx43 immunostaining decreased around blood vessels and in the inner limiting membrane.Conclusions: To our knowledge, this is the first report that explores connexin 43 expression in Dp71-null mice retina. Taken together the results suggest that the absence of Dp71 affects the intercellular tight junctions in mice retina, affecting the expression and localization of Cx43; this impairment could be, at least in part responsible of the abnormalities observed in Dp71 null mice retina, specifically on the increase of iBRB permeability.Commercial Relationships: Rodrigo Bolanos, None; Lourdes M. SIQUEIROS, None; Yonathan Garfias, None; Cecilia Montañez, None; José Sahel, None; Xavier P. Guillonneau, None; Alvaro Rendon, None

Program Number: 4206 Poster Board Number: D0225Presentation Time: 3:45 PM–5:30 PMGlucose effect on Rat and Human Müller Cells Viability and VEGF SecretionSandeep Vellanki1, Ana Ferrigno2, Yuliana Alanis1, Brandi S. Betts-Obregon1, Andrew T. Tsin1. 1Biology, University of Texas at San Antonio, San Antonio, TX; 2Escuela de Medicina, Tecnologico de Monterrey, Monterrey, Mexico.Purpose: Early stage Diabetic Retinopathy is manifested by excessive angiogenesis of which VEGF is a strong promoter. We hypothesize that cultured Rat Müller Cells (RMC) and Human Müller Cells (HMC) respond to high glucose by an increase in cell number and VEGF secretion.Methods: RMC were plated at 23,000 cells/well and HMC were plated 20,000 cells/well in a 24- well plate and treated with 0mM, 5.5mM or 30mM glucose for 24 hours. The number of viable cells were then counted using Trypan Blue Exclusion Method. ELISA was used to determine VEGF levels in cell media. The amount of VEGF secreted per cell was determined by dividing VEGF level in cell media by cell number.Results: A concentration dependent change in cell viability was observed with glucose treatment. After 24 hours, RMC and HMC in 30mM increased in cell number by 23.6% and 46.6% respectively, compared to those in 5.5mM. In contrast, number of cells in 0mM glucose decreased by 50% and 28.3% respectively, and this glucose treatment effect is significant (One-way ANOVA; P≤ 0.05). Compared to 5.5mM the level of VEGF in cell media (pg/ml) increased by 9% in HMC and 19.6% in RMC in 30mM and by 47% in HMC and 9% in RMC in 0mM glucose (One-way ANOVA; P> 0.05). In both RMC and HMC the amount of VEGF secreted per cell decreased by 50% when glucose was increased from 0 to 5.5mM. This decrease is significant in RMC. (One-way ANOVA; P≤ 0.05). However, only a slight decrease (17%) was observed when glucose level increased from 5.5 to 30mM.Conclusions: Our results show that RMC and HMC are highly responsive to change in glucose concentrations. High glucose (30mM compared to 5.5mM) significantly increased HMC cell viability but did not induced a significant change in VEGF secretion (pg/cell). At 5.5mM glucose, HMC secreted a seven-fold higher level of VEGF (pg/cell) than RMC. The mechanism of glucose-induced changes in RMC and HMC cell viability and VEGF secretion remains to be elucidated.Commercial Relationships: Sandeep Vellanki, None; Ana Ferrigno, None; Yuliana Alanis, None; Brandi S. Betts-Obregon, None; Andrew T. Tsin, NoneSupport: National Institute on Minority Health and Health Disparities (G12MD007591) from the National Institutes of Health.

Program Number: 4207 Poster Board Number: D0226Presentation Time: 3:45 PM–5:30 PMPlasmalogens and cell-cell communication between retinal glial cellsJulie Mazzocco1, Charlotte Pallot2, Benedicte Buteau1, Stephane Gregoire1, Laurent Leclere1, Alain M. Bron2, Catherine P. Garcher2, Lionel Bretillon1, Niyazi Acar1. 1Eye & Nutrition Research Group, Dijon, France; 2ophthalmology, university hospital, Dijon, France.Purpose: Plasmalogens (or ether-lipids) are a particular class of glycerophospholipids characterized by the presence of a vinyl-ether bond at the sn-1 position of the glycerol backbone and by the preferential esterification of polyunsaturated fatty acids (PUFAs) at the sn-2 position. Plasmalogens are found at high concentrations in retinal macroglial cells such as Müller cells and astrocytes.



Müller cells and astrocytes are known to communicate by the way of intracellular calcium waves and gap junctions. Connexin 43 is the major protein of these communicating junctions. The aim of this study was to determine the effect of plasmalogens depletion on communication between astrocytes and Müller cells.Methods: Primary retinal Müller cells were isolated from eyes of Wistar rats aged from 9 days whereas astrocytes were obtained from cortex of 21-days old animals. SiRNA against DHAPA-AT -the key enzyme of plasmalogen biosynthesis- were used to decrease the cell content in plasmalogen. After lipid extraction, the concentrations of plasmalogens in cell membranes were monitored by gas chromatography coupled to flame ionization detection. Communication between astrocytes and Müller cells was studied by calcium imaging. Changes in the expression cellular proteins, including connexin 43, were evaluated by proteomics and western-blotting.Results: Lipidomic analyses confirmed that plasmalogens are concentrated in retinal macroglial cells (17% and 13% of total phospholipids for Müller cells and astrocytes, respectively) when compared to the entire retina (7% of total phospholipids). SiRNA against DHAP-AT led to a diminution of plasmalogen content of more than 50%. Decreasing the concentrations of plasmalogens in membranes of glial cells impacted cell-cell communication through decrease in the expression of connexion 43 by more than 50%. Western-blot analyses showed modifications in the expression/phosphorylation of MAPK proteins.Conclusions: Plasmalogens may influence cell-cell communication between retinal Müller cells and astrocytes. As retinal macroglial cells play crucial roles in preserving neuronal integrity and in proper vascular development, modifications of retinal plasmalogen levels are likely to be at the origin of many retinal diseases.Commercial Relationships: Julie Mazzocco, None; Charlotte Pallot, None; Benedicte Buteau, None; Stephane Gregoire, None; Laurent Leclere, None; Alain M. Bron, None; Catherine P. Garcher, None; Lionel Bretillon, None; Niyazi Acar, NoneSupport: French National Institute for Agricultural Research (INRA); National Center for Scientific Research (CNRS); Regional Council of Burgundy; European Regional Development Fund (FEDER); University of Burgundy

Program Number: 4208 Poster Board Number: D0227Presentation Time: 3:45 PM–5:30 PMPrimary muller glial cultures of primate originShweta Singhal1, 2, Venkata Sudheer Babu Pakala2, Milan N. Mehta2, Veluchamy A. Barathi2, Tien Yin Wong1, 2. 1Singapore National Eye Centre, Singapore, Singapore; 2Retina, Singapore Eye Research Institute, Singapore, Singapore.Purpose: Age related retinal degeneration is a significant cause of irreversible blindness worldwide. An alternate approach to cell replacement therapy would be to enhance the ability of the retina to effect its own repair. Muller glia (MG) can mediate complete retinal regeneration after injury in zebrafish by transforming into stem cells. This regenerative capacity is attenuated in mammalian eyes where muller glia respond to injury with a gliotic response instead. Identifying key signals that activate/enhance the regenerative ability of MG could therefore be an elegant therapeutic strategy for irreversible retinal disease. However, differences between rodent and human eyes; and difficulty in sourcing/culturing human MG have hindered research in this area. The aim of this work was to investigate whether primate retinae can be used to establish reliable MG cultures, and ultimately help define critical regulators of the regenerative ability of higher mammalian MG.

Methods: Using previously defined protocols for MG isolation from rodent and human eyes, whole retinae of 6 globes from 4 different adult primates (sp. macaca fascicularis) were isolated after post mortem enucleation, digested using a trypsin based enzyme cocktail and cultured on fibronectin. Cell growth was monitored by phase contrast microscopy.Results: Retinal cell suspensions from all 6 globes demonstrated adhesion of scattered cells after one week in culture. These adherent cells expanded clonally to form colonies by 2 weeks and grew into a confluent monolayer by 6-8 weeks in culture. Once confluent the cells were amenable to passage and expansion in culture using trypsin digestion. They demonstrated a slow doubling time requiring up to 8-10 weeks to reach confluence per passage. We have been able to culture these cells for 9 passages thus far without loss of morphology or self-renewal capability. Their expression of muller glial markers (glutamine synthase, CRALBP), retinal markers (OTX2 and PKC) and stem cell marker LGR5 were characterised using immunocytochemistry and RT PCR.Conclusions: Primary primate muller glial cultures can be established in vitro with a high success rate. While slow growing they are stable in culture and sufficiently expansible to enable molecular characterisation. To our knowledge this is the first report describing the behaviour of primate MG in culture, a potential resource to identify actionable targets for human retinal regenerative therapies.Commercial Relationships: Shweta Singhal, None; Venkata Sudheer Babu Pakala, None; Milan N. Mehta, None; Veluchamy A. Barathi, None; Tien Yin Wong, NoneSupport: NMRC CS-IRG NIG and SNEC HREF

Program Number: 4209 Poster Board Number: D0228Presentation Time: 3:45 PM–5:30 PMTRPV4 and TRPC1 channels regulate strain sensitivity in retinal neurons and gliaAndrew Jo, Sarah Redmon, Anthony Iuso, Tünde Molnar, Tam T. Phuong, David Krizaj. Ophthalmology & Visual Sciences, University of Utah, Salt Lake City, UT.Purpose: Retinal ganglion cells (RGCs) and glia are immersed within a mechanically sensitive environment in which they must constantly cope with and adapt to pressure and osmotic stress. The purpose of this project was to (i) use different stretch paradigms to identify the molecular mechanism that underlies the mechanosensitive properties of mouse RGCs and Muller glial cells (MCs) and (ii) to characterize the relationship between membrane strain, TRP (transient receptor potential) channel activation and [Ca]2+homeostasis in RGC and MCs.Methods: Dissociated or cultured mouse retinal cells were loaded with calcium indicator dyes and stimulated with defined magnitudes and time periods of applied cyclic uniaxial or biaxial substrate strain. RGCs were identified based on immunopanning or by expression of the transgenic Thy1:YFP marker. Calcium concentration [Ca2+]i was assessed with high-resolution optical imaging. Response selectivity was determined with selective TRP channel antagonists, agonists and TRPV4-/- and/or TRPC1-/- cells. Reactive gliosis was determined with GFAP immunolabeling.Results: MCs and a subset of RGCs (57 ± 14 %) responded to cyclical uniaxial or biaxial stretch (0.5 – 15%) with [Ca2+]i elevations. For example, 10% uniaxial stretch elevated [Ca2+]i in Thy1:RGC responders and in Muller glial cells by 89.4 ± 9.7% (n=21) and 72 ± 5.6% fluorescence ratio increase, respectively. Stretch evoked [Ca2+]i responses were reduced in KO cells and following the application of the selective TRPV4 antagonist HC 067047 whereas, capsazepine, an antagonist of TRPV1 channels had no inhibitory effect. The glial response to strain was reduced by



inhibitors of the phospholipase A2 signaling pathway and by genetic elimination of TRPC1 channels. Further consistent with potential mechanosensitive function of glial TRPC1 was the observation of attenuated reactive gliotic response to elevation of intraocular pressure in a TRPC1-/- mouse model of glaucoma.Conclusions: We applied novel mechanobiological paradigms to the retina to demonstrate that stretch- evoked [Ca2+]i signaling in RGCs and Muller cells is mediated by TRPV4 and TRPC1, but not TRPV1, channels. These findings identify possible mechanisms whereby retinal cells sense and transduce intraocular pressure and ECM strain at the optic nerve head, and may thus have implications for the understanding and treatment of blinding diseases such as ischemia and glaucoma.Commercial Relationships: Andrew Jo, None; Sarah Redmon, None; Anthony Iuso, None; Tünde Molnar, None; Tam T. Phuong, None; David Krizaj, NoneSupport: NIH Grants (EY13870, EY022076, P30 EY14800), Department of Defense, and Foundation Fighting Blindness

Program Number: 4210 Poster Board Number: D0229Presentation Time: 3:45 PM–5:30 PMRetinal Ganglion Cells Control Spatial Patterning of Astrocytes During DevelopmentMatthew L. O’Sullivan1, Joseph A. Brzezinski3, Tom M. Glaser4, Jeremy Kay1, 2. 1Neurobiology, Duke University School of Medicine, Durham, NC; 2Ophthalmology, Duke University School of Medicine, Durham, NC; 3Ophthalmology, University of Colorado School of Medicine, Aurora, CO; 4Cell Biology and Human Anatomy, University of California Davis School of Medicine, Davis, CA.Purpose: Retinal astrocytes migrate into the retina from the optic nerve (ON) during late embryonic development and spread centrifugally in the nerve fiber layer (NFL) to cover the inner retina. Astrocytes guide development of the intrinsic retinal vasculature, but factors that pattern the astrocyte network itself remain elusive. We propose that astrocytes use retinal ganglion cell (RGC) axons as directional cues and will not achieve proper spatial distribution without RGCs. We tested this hypothesis by investigating astrocyte development in mice lacking RGCs through genetic ablation via knockout of the transcription factor Math5.Methods: Eyes from Math5 null and littermate control mice were fixed at postnatal day 7 (P7), and retinas removed, immunostained, and imaged by confocal fluorescence microscopy. Antibodies against Pax2, CD31, RBPMS, and neurofilament were used to label astrocytes, blood vessels, RGCs, and RGC axons, respectively, in wholemount retinas. Control and null retinas were analyzed in ImageJ and statistical analyses (t-test, 2-way ANOVA) performed in JMP.Results: Math5 null retinas were severely deficient in RBPMS+ RGCs, lacked axon fascicles in the NFL, and were devoid of intrinsic retinal vasculature. By P7, astrocytes in control retinas have reached the ora serrata and achieved a uniform density across the entire retina. In Math5 mutants, however, the area encompassed by the wavefront of astrocyte migration was significantly reduced (control 98.1 ± 0.5%, KO 72.3 ± 2.6%, p < 0.01). Additionally, the spatial distribution of astrocytes was dysregulated with an abnormally high density of cells near the optic nerve and a steeply declining gradient to the periphery (control 1365 ± 101 cells/mm2 central, 1520 ± 43 middle, 1663 ± 58 peripheral; KO 3188 ± 438 central, 1935 ± 187 middle, 100 ± 28 peripheral; interaction of genotype with eccentricity p<0.001).Conclusions: RGCs are required for normal migration and spatial organization of retinal astrocytes during development, consistent with a model in which the neuroretina guides the maturation of its own astrocytic and vascular networks. Moreover, defects in

astrocytes may underlie the vascular abnormalities in retinas lacking RGCs. Further experiments will describe the trajectory of astrocyte development and investigate cellular and molecular mechanisms underlying RGC-astrocyte-blood vessel interactions.Commercial Relationships: Matthew L. O'Sullivan; Joseph A. Brzezinski, None; Tom M. Glaser, None; Jeremy Kay, NoneSupport: Ruth K Broad Foundation Postdoctoral Fellowship to MO'S, NIH grant EY14259 to TG

Program Number: 4211 Poster Board Number: D0230Presentation Time: 3:45 PM–5:30 PMEndothelin-1 Induced the Reactivation of Primary Rat Ocular AstrocytesShaoqing He1, Hai-Ying Ma1, Yong H. Park1, Junming Wang2, Thomas Yorio1. 1North Texas Eye Research Institute, University of North Texas Hlth Sci Ctr, Fort Worth, TX; 2Depart. of Ophthalmology, TongJi Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China.Purpose: Astrocytes play a crucial role in cell survival and axon function of retinal ganglion cells (RGC) by providing the structural support to neurons, secreting neurotrophic factors to regulate apoptosis and maintenance of the extracellular milieu. Endothelin-1(ET-1) and its receptors are found to be involved in the etiology of glaucoma. However, ET-mediated reactivation of astrocytes affecting RGC survival is still not fully understood. This study aimed to investigating the mechanisms by which ET-1 promotes the reactivation of primary rat ocular astrocytes.Methods: The primary astrocytes were isolated from retinas and optic nerve of rats. Immunostaining of glial fibrillary acid protein (GFAP), RNA binding protein with multiple splicing (RBPMS) and alpha smooth muscle actin (α-SMA) was performed on the cultured primary astrocyte to identify the purity of cells. The cultured primary astrocytes were treated with 100nM endothelin-1 for 24 hours followed the protein detection using Western Blot. ET-1-mediated influx of calcium was monitored in astrocytes using Fura-2 AM calcium imaging.Results: GFAP was uniformly stained on the primary astrocytes, and no staining of RBPMS and α-SMA was identified, whereas the staining of α-SMA was identified in NIH3T3 fibroblast cells. The treatment of ET-1 and ET-3 induced the upregulation of GFAP, neural cell adhesion molecule (NCAM), c-Jun, c-Jun N-terminal kinase (JNK) and Ki67 (a protein marker of cell proliferation). Administration of SP600125, an inhibitor of JNK, attenuated the increased GFAP induced by ET-1 in astrocytes. However, BQ788, an antagonist of ETB receptor, didn’t inhibit ET-1-mediated upregulation of GFAP. In addition, ET-1 triggered augment of intracellular calcium in the primary astrocytes, whereas the application of verapamil, an L-type calcium channel blocker, inhibited the influx of calcium.Conclusions: The hallmark of reactive astrocytes, GFAP, is tightly regulated in astrocytes. An increase in protein levels of GFAP and Ki67 induced by ET-1 reflected the reactivation of astrocytes. Meanwhile, other proteins were also found to be upregulated, such as NCAM, c-Jun and JNK. In addition, intracellular of calcium was also promoted with ET-1 treatment. Taken together, the results suggest that calcium-mediated signaling and JNK/c-Jun pathway are involved in reactivation of astrocytes. This reactivation could lead to dysfunction in the optic nerve and affect RGC survival.Commercial Relationships: Shaoqing He, None; Hai-Ying Ma, None; Yong H. Park, None; Junming Wang, None; Thomas Yorio, NoneSupport: DOD grant: W81XWH-10-2-0003 to Thomas Yorio



Program Number: 4212 Poster Board Number: D0231Presentation Time: 3:45 PM–5:30 PMSigma-1 receptor mediated secretion of BDNF by optic nerve head astrocytesBarbara A. Mysona1, 3, Jing Zhao2, 3, Sylvia B. Smith1, 3, Kathryn E. Bollinger2, 3. 1Department of Cellular Biology and Anatomy, Augusta University, Augusta, GA; 2Department of Ophthalmology, Medical College of Georgia, Augusta, GA; 3James and Jean Culver Vision Discovery Institute, Augusta University, Augusta, GA.Purpose: Sigma-1 receptor (SR1) is a membrane associated molecular chaperone protein expressed in retinal neurons and glia. SR1 agonists are neuroprotective in experimental models of neurodegenerative diseases, including diabetic retinopathy, Alzheimer’s and Parkinson’s disease. The mechanism for neuroprotection within brain and retina is unknown. However, SR1 is known to modulate many cellular processes, including responses to oxidative and endoplasmic reticulum (ER) stress and intracellular Ca2+ signaling. In addition, SR1 mediates secretion of brain derived neurotrophic factor (BDNF) from neuronal and glial cultures. BDNF is a critical factor for neuronal survival and differentiation. Therefore, we chose to investigate whether activation of SR1 induces release of BDNF from retinal tissues and optic nerve head (ONH) astrocytes.Methods: C57BL6 mice were treated by intraperitoneal injection of the SR1 specific agonist (+)-pentazocine (PTZ), 0.5 mg/kg, 3 times a week for 5 weeks. Age-matched C57BL6 controls and SR1 knockout mice were also used. Retina and hippocampus tissues were harvested and analyzed by Western blot and immunohistochemistry for expression of SR1 and BDNF. Primary ONH astrocytes were isolated and cultured from 3-5 day old Sprague Dawley rat pups. ONH astrocytes were treated in presence or absence of the ER stress inducer tunicamycin (5 ng/ml to 5 ug/ml) with or without PTZ (3µM). After 24 hours, media was analyzed by ELISA for secreted BDNF. Cell lysates were analyzed for protein expression of CHOP, SR1, and BDNF.Results: After treatment of C57BL6 mice with PTZ, BDNF levels were increased in hippocampus but not retina while SR1 levels remained unchanged. Retinas from SR1 knockout mice had less BDNF than C57BL6 counterparts. Treatment of ONH astrocytes with tunicamycin (5 ug/ml and 500 ng/ml) blocked secretion of BDNF and increased expression of CHOP while SR1 levels remained unchanged. In contrast, treatment with PTZ increased secretion of BDNF.Conclusions: SR1 mediated release of BDNF may be organ and cell specific. Decreased BDNF observed in SR1 knockout retinas supports a link between SR1 and BDNF secretion. Although treatment with PTZ did not increase BDNF in C57BL6 retinas, PTZ treatment did stimulate BDNF secretion by ONH astrocytes. PTZ-mediated release of BDNF from ONH astrocytes may prove to be neuroprotective in conditions associated with retinal ganglion cell degeneration such as glaucoma.Commercial Relationships: Barbara A. Mysona, None; Jing Zhao, None; Sylvia B. Smith, None; Kathryn E. Bollinger, NoneSupport: NIH grant K08EY021758, American Glaucoma Society young clinician scientist award

Program Number: 4213 Poster Board Number: D0232Presentation Time: 3:45 PM–5:30 PMRetinal astrocytes protect neurons against metabolic stress by inducing the PI3K pathwaySamih Alqawlaq1, 2, Izzy Livne-Bar3, Darren Chan1, 2, Jeremy M. Sivak1, 4. 1Department of vision science, Krembil Research Institute, Toronto, ON, Canada; 2Ophthalmology and Vision Science, University of Toronto, Toronto, ON, Canada; 3School of Optometry, University of California Berkeley, Berkeley, CA; 4Laboratory of Medicine and Pathobiology, University of Toronto, Toronto, ON, Canada.Purpose: Maintenance of retinal function heavily relies on the support roles of astrocytes, which carry out major homeostatic roles to protect neurons from oxidative and metabolic stresses. The purpose of the current research is to identify candidate astrocyte-secreted factors involved in regulating metabolic stress in relevant neuronal cell models.Methods: Astrocyte conditioned media (ACM) was collected from cultures of primary retinal astrocytes, previously described and characterized by our group. ACM significantly reduces metabolic stress induced cell death in the immortalized neural Ht22 cell line, and in primary cortical neurons. Using this model as a screening platform, two directions were pursued: 1) A quantitative antibody (Ab) array to compare enrichment of known neuroprotective factors in ACM, compared to control media. 2) A kinase inhibitor (KI) library screen of over 400 tool compounds, using the Ht22 cell model as a platform for ACM neuroprotection assessment. This screen was followed by pathway validation via western blotting and validation of select KIs in primary cortical neurons.Results: The Ab array showed enrichment of several growth factors in ACM, several of which are known to bind to receptor tyrosine kinases (RTKs), including basic FGF (3-fold enrichment), and NGF (2-fold enrichment). Notably, ACM was not significantly enriched for BDNF or CNTF. In complementary studies the KI screen prominently identified several inhibitors targeting the phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K) pathway. Each inhibitor hit reduced ACM-mediated neuroprotection, without causing any direct neurotoxicity. For example; the PI3K inhibitor ZSTK474, and the AKT inhibitor GSK690693 each eliminated approximately 90% of ACM-mediated effect in both Ht22 cells and primary neurons. Western blotting of Ht22 cells showed that ACM treatment induced AKT phosphorylation by 30 mins, indicating PI3K activation.Conclusions: Neuroprotection of ACM against metabolic stress was established as a screening platform in HT22 cells and primary neurons, allowing high throughput screening of pathway targets. Observed neuroprotection is mediated through the PI3K pathway, and several potential corresponding pathway inducers were identified in ACM. These findings will enable additional testing and validation of candidate pathways and targets to mediate astrocyte secreted neuroprotective signals.Commercial Relationships: Samih Alqawlaq, None; Izzy Livne-Bar, None; Darren Chan, None; Jeremy M. Sivak, NoneSupport: Vision Science Research Program Scholarship

Program Number: 4214 Poster Board Number: D0233Presentation Time: 3:45 PM–5:30 PMA novel method to assess astrocyte calcium in rat retinaHui Li2, Lin Wang1, Grant Cull1. 1Devers Eye Institute, Portland, OR; 2Ophthalmology, Shanghai Tenth People’s Hospital, Shanghai, China.Purpose: To validate the specificity of calcium indicator Fluo-4 AM (F4) binding to the retinal astrocytes and a method for in vivo monitoring of retinal astrocyte calcium (Ca2+) activity with a confocal scanning laser ophthalmoscopy (cLSO).



Methods: (1) Three rat retinas were isolated and loaded with F4 for 90 min in a culture dish. The retinas were imaged with a microscope for F4 positive cells (F4+). Following the imaging, the retinas underwent immunocytochemistry (IHC) staining with glial fibrillary acidic protein (GFAP) antibody, a specific marker for astrocytes. GFAP positive cells (GFAP+) were imaged and overlaid on the images for F4+ cells. The agreement of overlay between the two images was evaluated with Image J.(2) To determine the average time required for F4 fluorescence intensity to reach a stable level after intravitreal (IV) injection, each 5 rat eyes was injected with 5 µl F4 solution (0.1µg/µl). The F4 fluorescence intensity, representing relative astrocytic Ca2+ concentration in the retina, was imaged with the cLSO in fluorescence mode (FM, 488 nm) in vivo. The imaging process began 10 minutes after F4 injection and was repeated every 15 minutes thereafter for the next 2 hours. At each time point, 30-second consecutive images were recorded. The time course change in F4 fluorescence intensity was quantified.Results: (1) Strong fluorescence appeared in cells across the isolated retina particularly along the sides of both arterioles and venules. Overlay of the F4 images with that of GFAP IHC staining in the same retina showed that majority of the F4+ cells were also GFAP+. However, the F4+ cells showed fewer processes compared with GFAP+ cells. (2) cSLO imaging showed fluorescence appeared ten minutes after the IV injection of F4. The F4 fluorescent intensity increased with time and stabilized at ~90 minutes post-injection for at least 30 minutes. However, unlike the F4+ cells under the microscope, no individual cells could be identified due to the limitation in resolution of cSLO. One way RM-ANOVA showed a significant increase in fluorescent intensity over time (P<0.0001). Post-hoc analysis indicated no significant difference in fluorescent intensity from 90 to 120 minutes post-injection.Conclusions: F4 appeared to bind Ca2+ selectively within retinal astrocytes. F4 fluorescence could be imaged in vivo with the cLSO after IV injection and this novel method will allow further studies for the role of astrocytes in the retina.

Commercial Relationships: Hui Li; Lin Wang, None; Grant Cull, NoneSupport: R21 EY024432; Brightfocus Foundation

Program Number: 4215 Poster Board Number: D0234Presentation Time: 3:45 PM–5:30 PMGlial cells of the optic nerve and Persistent Fetal Vasculature (PFV) diseaseStacey L. Hose1, Cheng Zhang2, J S. Zigler1, Jiang Qian1, Debasish Sinha1. 1Ophthalmology, Johns Hopkins Wilmer Eye Inst, Baltimore, MD; 2Montefiore Medical Center, Albert Einstein College of Medicine, Bronx, NY.Purpose: PFV is a human disease that results from failure of the fetal vasculature to regress normally. Failure of all or part of these vessels to regress can lead to serious congenital pathologies. The defects include congenitally small eye, cataract, glaucoma, intravitreal fibrovascular tissue and/or hemorrhage, and retinal detachment. PFV has been reported in other mammalian species, particularly in dogs, and in genetic mouse models. Studies with these animals have helped immensely with our current understanding of the disease. The focus of this study is to determine the role of glial cells of the optic nerve in PFV disease by studying a rat with mutations in both βA3/A1-crystallin (Cryba1) and branched-chain ketoacid dehydrogenase kinase (Bckdk).Methods: Eyes fixed in 2% paraformaldehyde were used for immunohistochemistry for astrocytes (GFAP), oligodendrocytes (APC), blood vessels (lectin), axons (SMI 312) and myelin (myelin basic protein). Animals were perfused with 4% paraformaldehyde and post-fixed in 3% glutaraldehyde prior to electron microscopic analysis of the optic nerve. Human Proteome Microarray v2.0 (CDI, Nextgen Proteomics) was used to study protein-protein interactions.Results: Rats homozygous for mutations in both Cryba1 and Bckdk have severely affected glial populations (astrocytes and oligodendrocytes) of the optic nerve. The PFV phenotype is very severe and the entire fetal vasculature fails to regress. The retina in these double mutant rats is much thinner than normal, showing severe degeneration of the inner retinal layers. Protein-protein interaction studies indicate that βA3/A1-crystallin may regulate abnormal astrocyte migration during hyaloid vessel regression by modulating cortactin and src kinase-associated phosphoprotein 2 (SKAP2).Conclusions: We have developed a novel animal model that mimics clinical signs of severe human PFV, a potentially blinding childhood



disease, for which there are limited treatment options at the present time. Our data suggest that the degree of completeness and severity of the disease depends on both the glial and lens phenotype. The exact prevalence of PFV is unknown; however, a study on childhood blindness and visual loss in the United States showed that PFV accounts for about 5% of all cases of blindness. The clinical benefit in treating PFV is saving the eye from the secondary complications of the disease.Commercial Relationships: Stacey L. Hose, None; Cheng Zhang, None; J S. Zigler, None; Jiang Qian, None; Debasish Sinha, NoneSupport: NIH grant R01EY019037 (to DS) and Research to Prevent Blindness (an unrestricted grant to the Wilmer Eye Institute)

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ARVO 21 Annual Meeting Abstracts 384 Müller Cells and ... · Guidance cues appear to be present to guide repopulating cells to the correct retinal lamina and to re-establish a mosaic