Eye / Vision
Permanent URI for this collectionhttps://hdl.handle.net/20.500.12503/21711
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Browsing Eye / Vision by Author "Clark, Abbot F."
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Item Phosphoproteomic changes in the retina following optic nerve crush(2016-03-23) Clark, Abbot F.; Pang, Iok-Hou; Liu, YangPurpose: Phosphorylation is a major type of protein post-translational modification. In this study, we evaluated the phosphoproteomic changes in the retina induced by optic nerve crush (ONC) in the mouse, an acute model of central nervous system (CNS) axonal injury. The functional role of an identified major phosphoprotein was further studied. Methods: Intraorbital ONC was performed in adult C57BL/6J mice. Retinas were collected at 0, 6, and 12 h following optic nerve injury. Retinal proteins labeled with CyDye-C2 were subjected to 2D-PAGE. 2D gel phosphoprotein staining was performed, followed by in-gel and cross-gel image analysis. The ratio change of protein differential phosphorylation following ONC was obtained. Proteins with significant changes in phosphorylation (ratios ≥ 1.5) in retinas of the injured eyes compared to the control eyes were spot-picked, tryptic digested, and peptide fragments were analyzed by MALDI-TOF (MS) and TOF/TOF (tandem MS/MS). Proteins identity was based on 10 or more peptides. Identified proteins were validated by western blotting and immunofluorescence staining in separate experiments (n ≥ 3). Cell migration assay and flow cytometry-based phagocytosis assay were performed using primary cultured mouse optic nerve astrocytes. Results: Intraorbital ONC increased phosphorylation of many retinal proteins. Among them, 53 significantly phosphorylated proteins were identified. Significantly phosphorylated proteins in optic nerve crushed retinas include protein kinase C alpha, glycogen phosphorylase, tubulin-folding cofactor B, among others. One of the identified phosphoproteins, PEA-15, was confirmed by western blot analysis; ONC increased phosphorylation of this protein without affecting its basal protein expression level. Immunofluorescence staining using phospho-PEA-15-specific antibody demonstrated that increased phosphorylated PEA-15 co-localized with GFAP, a marker for Müller cells and astroglia in the retina and optic nerve. PEA-15 knockdown significantly promoted optic nerve astrocyte migration and suppressed phagocytosis. Conclusions: Our novel approach identified specific proteins whose phosphorylation was increased by ONC. One of these proteins, PEA-15, mediates major optic nerve astrocytic functions, which likely affect retinal neuronal survival and regeneration after injury. These new insights will lead to novel therapeutic targets for retinal and CNS neurodegeneration.Item RGC death in a mouse model of congenital glaucoma(2016-03-23) Anderson, Michael; McDowell, Colleen; Clark, Abbot F.; Daniel, SteffiPurpose:Mutation in the podosomal adaptor protein SH3PXD2B (nee) causes anterior segment dysgenesis, elevated intraocular pressure (IOP), and congenital glaucoma, as previously described in B10.A-H2h4/(4R)SgDvEg mice. We investigated the effect of the nee mutation in C57BL/6J mice with respect to IOP, total retinal ganglion cell (RGC) death, and RGC subtype specific death in nee mice containing the Trhr-GFP transgene (selectively expresses GFP in ON-OFF direction selective RGCs). Methods:IOP and RGC death were measured in B6.Sh3pxd2bnee mutant (MUT) and wild type (WT) mice at post-natal days 30, 60, 75, and 90. C57BL/6J mice containing the Trhr-GFP transgene were crossed with B6.Sh3pxd2bnee to obtain nee mutant mice expressing GFP in ON-OFF direction selective RGCs. IOP was measured using a TonoLab tonometer. RGC damage was assessed by immunofluorescence of labeled retinal flat mounts using the GFP biomarker and NeuN. Results:Significant IOP elevation was observed in MUT mice at days 30, 60, 75, and 90 compared to WT mice (p2 value of 0.742. Significant differences in the percent cell survival of GFP positive RGCs was observed in MUT mice containing the Trhr-GFPtransgene at 30 days (55.1±15%; n=4-6; p=0.0017), 60 days (16.5±4.6%; n=4-6; p Conclusions:These studies characterized the nee glaucoma phenotype in C57BL/6J mice and demonstrate the specific susceptibility of ON-OFF direction selective RGCs. Future studies will identify susceptibility to additional subtypes of RGCs using this model system. These data are important to determine timing and onset of disease as well as identifying novel therapeutic targets.Item The role of canonical Wnt signaling and K cadherin in the regulation of intraocular pressure(2016-03-23) Bermudez, Jaclyn; Millar, J. Cameron; Clark, Abbot F.; Mao, Weiming; Webber, HannahPurpose: Primary open angle glaucoma (POAG) is the most prevalent form of glaucoma and has been associated with pathological changes in the trabecular meshwork (TM), the primary site of aqueous humor outflow in the eye. We have found that inhibition of canonical Wnt signaling in the TM raises intraocular pressure (IOP), and restoration of Wnt signaling normalizes IOP, though the mechanisms by which Wnt signaling maintains TM homeostasis are unknown. We hypothesize that the canonical Wnt signaling pathway in the TM regulates IOP via cadherins junctions. Materials and methods: We studied five cadherin isoforms abundant in the TM as shown by exome sequencing of normal and glaucomatous human TM (NTM and GTM, respectively) tissues. For in vitro studies, NTM cells (gift from Novartis) were treated with or without recombinant 100ng/ml Wnt3a or 1ug/ml sFRP-1 or both for 4-48 hours. Membrane protein fractions were isolated for western immunoblotting (WB) and probed for the cadherin isoforms. TM cells were also immunostained for cadherin isoforms or β-catenin. RNA was extracted from TM cells for cDNA synthesis and qPCR analysis of cadherins. Ad5.CMV recombinant adenoviruses encoding E cadherin, K cadherin, and/or sFRP-1 were injected unilaterally into the eyes of 4-6 month old female BALB/cJ mice (n=6). Conscious IOP of both eyes was then non-invasively measured for up to 35 days. Results: WB showed that Wnt3a TM cell membrane associated K-cadherin, which was inhibited with the addition of the Wnt antagonist sFRP-1. Immunostaining showed that -catenin accumulated on TM cell membrane upon Wnt3a treatment, and filopodia-like connections formed between TM cells. qPCR showed that Wnt3a also significantly increased K cadherin expression (n=3, p Conclusion: Our results suggested that cadherins play a role in the regulation of TM homeostasis and IOP via the Wnt signaling pathway.Item Title: BMP4 induced ID proteins inhibit the fibrotic effects of TGFβ2 in primary human TM cells.(2016-03-23) Wordinger, Robert J.; Clark, Abbot F.; Mody, AvaniPurpose: Elevated intraocular pressure (IOP) is a major risk factor associated with primary open angle glaucoma (POAG). Increase expression of transforming growth factor β2 (TGFβ2) in POAG aqueous humor (AH) and trabecular meshwork(TM) causes extracellular matrix (ECM) protein deposition in TM, leading to increase in outflow resistance and elevating IOP. However underlying mechanism of BMP4 pathway that regulates the inhibition of TGFβ2 induced fibrosis remains undetermined. BMP4 regulates variety of cellular processes by induction of inhibitor of DNA binding protein (ID1, ID3), which bind and suppress specific transcription factors complex and in turn regulates the gene function. This study will determine whether ID1/ID3 are downstream target of BMP4 that attenuates TGFβ2 induction of ECM proteins. Methods: Primary human TM cells were treated with BMP4 (10ng/ml) for 0-48hr and ID1 and ID3 mRNA and protein expression was determined by Q-PCR and western immunoblotting. Primary TM cells were treated with a BMPR inhibitor to confirm that BMP4 signaling is necessary for induction of ID1 and ID3 protein expression. Further GTM3 cells were transfected with ID1 or ID3 expression vectors to study inhibitory effects on TGFβ2 induced fibronectin and plasminogen activator inhibitor-I (PAI-1) protein expression. Additionally GTM3 cells were transfected with ID1 and ID3 siRNA to determine whether these ID proteins are responsible for the BMP4 suppression of TGFβ2 induced fibronectin expression. Results: BMP4 (10ng/ml) induced early expression of ID1 and ID3 in primary TM cells. Overexpression of ID1 and ID3 significantly inhibited TGFβ2 induced expression of fibronectin and PAI-1 in TM cells. Further, knockdown of ID1 and ID3 suppressed the inhibitory effects of BMP4 on the TGFβ2 induction of fibronectin. Conclusion: BMP4 induced ID1 and ID3 expression suppresses TGFβ2 pro-fibrotic activity in human TM cells. In the future, targeting specific regulators may control the TGFβ2 pro-fibrotic effects on the TM, leading to disease modifying IOP lowering therapies.Item Validate Grx2 gene knockout mice as a new model for age-related retinal degeneration(2016-03-23) Xavier, Christy; Liu, Yang; Chavala, Sai; Clark, Abbot F.; Pang, Iok-Hou; Wu, Hongli; Liu, XiaobinPurpose: Age-related macular degeneration (AMD) is a leading cause of blindness worldwide. The poorly understood pathogenesis has greatly hindered our progress in therapeutic development. To address this shortcoming, this project was designed to examine how retinal redox dysregulation leads to AMD and characterize glutaredoxin 2 (Grx2), a mitochondrial thiol redox regulating enzyme, knockout mice as a new animal model for AMD. Methods: The retinal pigment epithelium (RPE) layers were isolated from healthy and AMD donor eyes. Grx2 protein levels were measured by Western blot analysis. Primary RPE cells were isolated from wild-type (WT) and Grx2 knockout (KO) mice for the in vitro study. The visual function of WT and Grx2 KO mice were examined by fundus photography and scotopic electroretinography (ERG). H&E staining was used for histological exams. RPE structural changes were assessed by immunostaining of tight junction protein ZO-1. Lipofuscin autofluorescence was examined on cryostatsections. The level of protein glutathionylation (PSSG) was measured by immunoblotting using anti-PSSG antibody. Results: Grx2 protein level and enzyme activities were decreased by approximately 30% in AMD donor eyes. Primary RPE cells isolated from Grx2 KO mice were more sensitive to H2O2-induced oxidative damage than WT RPE cells. Grx2 KO mice developed age-dependent retinal degenerative pathology. By 12 months of age, Grx2 null mice showed ~50% decrease in a-wave and ~30% decline in b-wave amplitudes (n=8, P Conclusions: Grx2 plays a critical role in maintaining the mitochondrial redox homeostasis in the aging retina. Grx2 deficiency causes PSSG accumulation and sensitizes RPE cells to age-related oxidative damage, leading to RPE degeneration and photoreceptor damage. As a new animal model for AMD, Grx2 KO mice will provide new insights into the pathogenesis and therapeutics of AMD.