Eye / Vision

Permanent URI for this collectionhttps://hdl.handle.net/20.500.12503/21711

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Effect of BAMBI expression on intraocular pressure and aqueous humor outflow facility in mice
(2016-03-23) Millar, Cameron; Clark, Abbot; McDowell, Colleen; Hernandez, Humberto
Purpose: Elevated intraocular pressure (IOP) is an important risk factor in the development of glaucoma. TGFβ2 is well known to be involved in regulating both the extracellular matrix in the trabecular meshwork (TM) as well as ocular hypertension. BAMBI (BMP and activin membrane-bound inhibitor), a TGF-β pseudoreceptor, has been shown to be a negative regulator of TGF-β2. However, the role of BAMBI in regulating IOP is unknown. We investigated whether knockdown of BAMBI results in ocular hypertension in mice due to uninhibited TGFβ2 signaling. Methods: B6;129S1-Bambitm1Jian/J mice were injected intravitreally with 2.5x107 pfu of either Ad5.TGFβ2 (n=10), Ad5.Cre (n=9), or Ad5.TGFβ2 + Ad5.Cre (n=10), in one eye of each animal. The contralateral uninjected eyes were used as negative controls. IOP was measured using a TonoLab rebound tonometer. Aqueous humor outflow facility was assessed using a constant flow infusion method. Student’s t-test was used to compare between vector-treated and control uninjected eyes. Results: Injection with either Ad5.Cre, Ad5.TGFβ2, or Ad5.TGFβ2 + Ad5.Cre each induced ocular hypertension starting at day 7 post-injection and maintained significant IOP elevation throughout the 56 day time course compared to uninjected control eyes (p Conclusions: Here we show for the first time that conditional knockdown of BAMBI in the TM with Ad5.Cre induces ocular hypertension by reducing aqueous humor outflow facility. These data further explain the mechanisms involved in the development of glaucomatous TM damage and provide potential new targets to lower IOP.
ETA receptor upregulation may be associated with ERK signaling in a rat model of glaucoma.
(2016-03-23) Krishnamoorthy, Raghu; McGrady, Nolan
Purpose: The endothelin system has been shown to play a causative role in the neurodegenerative effects seen in animal models of glaucoma. However, the mechanisms leading to neurodegeneration need to be examined further. The goal of this study was to investigate the endothelin signaling pathway to determine the contribution of extracellular signal-regulated kinases 1 and 2 (ERK1/2) to endothelin-mediated cell death. Methods: Male retired breeder Brown Norway rats were subjected to IOP elevation by the Morrison’s method and maintained for 2 and 4 weeks. Retinal sections obtained from the rats were subjected to immunohistochemical analysis of ETA receptor expression. In a separate set of experiments, western blots were performed on transformed 661W cells transiently transfected with either the ETA receptor or ETB receptor cDNA expression vector. Another set of experiments was performed with stable clones overexpressing the ETA receptor. The cells were grown on 100 mm dishes and treated for 24 hr with 100nM endothelin-1 (ET-1) or endothelin-3 (ET-3). Immunoblot analysis of levels of endothelin receptor and ERK1/2 phosphorylation was carried out. Results: An increase in immunostaining for ETA receptors was observed mainly in the inner plexiform layer and a modest increase was also observed in the RGC layer which was significant at 4 weeks of IOP elevation. Cell culture experiments showed an appreciable upregulation of ETB receptors following overexpression of ETA receptors and a reciprocal upregulation of ETA receptors following overexpression of ETB receptors. A 1.8-fold increase in ERK1/2 phosphorylation was observed in stable clones overexpressing ETA receptors, which was further elevated 2-3 fold after treating cells with either endothelin-1 or endothelin-3, compared to empty vector transfected cells. Conclusions: While the two endothelin receptors may have distinct functions, there is a significant overlap of the ETA and ETB receptor mediated signal transduction pathways and there appears to be some level of cross-talk between the two receptors. While there is a substantial body of evidence for the pro-survival role of ERKs, prolonged activation of ERK1/2 has been shown to be associated with cell death. While the mechanisms are not completely clear, the current study points to an association of ERK1/2 with cell death following overexpression of ETA and ETB receptors.
Overexpression of Endothelin A and B Receptors Enhances Calcium Mobilization in Ocular Astrocytes and Ciliary Epithelial Cells
(2016-03-23) He, Shaoqing; Ma, Hai-Ying; Park, Yong; Yorio, Thomas; Broyles, Heather V.
Purpose: Endothelin-1 (ET-1), a vasoactive peptide, binds ETA receptor and ETB receptor to exert its role in multiple cellular processes. A growing body of evidence suggests that elevated levels of ET-1 and activation of its receptors contributes to neurodegeneration in glaucoma, where reactive astrocytes are found to induce damage of retinal ganglion cells. Overexpression of c-Jun, a transcription factor, has been shown to increase levels of ETB receptor, suggesting that the expression of ETB receptor is regulated by c-Jun. This study attempts to determine if overexpression of ET-1 receptors affect calcium influx in response to ET-1 treatment. Methods: Primary astrocytes were isolated from retina and optic nerve of rat pups postnatal 4-7 days. Calcium imaging using Fura-2-AM fluorescent dye was used to determine calcium influx following treatment of ET-1, in the presence and absence of BQ610 (ETA selective antagonist), or BQ788 (ETB selective antagonist) or no treatment (control). ETA, ETB and c-Jun were also overexpressed in Human Non-Pigmented Epithelial (HNPE) cells using DNA transfection and calcium mobilization was measured. Results: Overexpression of ETA or ETB in HNPE cells significantly increased [Ca2+]i levels compared to control following ET-1 treatment at p2+]i in primary astrocytes. Treatment with either BQ610 or BQ788 in primary astrocytes significantly (p2+]i levels compared to control following ET-1 treatment. Conclusion: This study demonstrated that ETA and ETB can mediate calcium influx in HPNE cells and primary astrocytes. ETA receptor stimulation produced a similar calcium influx in HPNE cells as ETB receptor activation, suggesting that both receptors may be involved in [Ca2+]i signaling. The increase in calcium can result in activation of cell death pathways that may explain the ET-1 neurodegenerative actions.
RGC death in a mouse model of congenital glaucoma
(2016-03-23) Anderson, Michael; McDowell, Colleen; Clark, Abbot F.; Daniel, Steffi
Purpose: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.
Risk-based Exposure Evaluation Process: Protecting Worker Exposure to Active Pharmaceutical Ingredients
(2016-03-23) Gruner, Janet; Rich, Alisa PhD, MPH; Fields, Alexandra
The purpose of this project is to support Alcon’s Risk-based Exposure Evaluation Process (REEP). The objective of REEP is to transfer Novartis’s decision-making process from a hazard-based approach to a risk-based, data driven approach in order to protect their pharmaceutical workers’ exposure to active pharmaceutical ingredients (APIs) during the manufacturing process. Personal breathing zone air sampling was conducted for the high risk APIs to determine personal exposure during different unit operations. Sampling results were compared to Novartis Internal Occupational Exposure Limits (NIOEL). Bayesian statistical analysis was performed to determine if a change in control is necessary and to interpret findings. While REEP is a continuous process, these preliminary results revealed the first set of risk-based API exposure data for Alcon’s six pharmaceutical sites. Based on the results, improvements to multiple levels of control such as engineering, administrative, and respiratory protection equipment (RPE) are currently in the process of implementation.
Phosphoproteomic changes in the retina following optic nerve crush
(2016-03-23) Clark, Abbot F.; Pang, Iok-Hou; Liu, Yang
Purpose: 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.
Perfusion-cultured bovine anterior segments as a model for studying TGFB2-induced ocular hypertension and glaucoma
(2016-03-23) Bermudez, Jaclyn; Clark, Abbot; Mao, Weiming; Wileman, Justin
Purpose: Primary open angle glaucoma (POA) is a leading cause of blindness worldwide. The most important risk factor is elevated intraocular pressure (IOP), which is due to glaucomatous damage to the trabecular meshwork (TM). Damaged TM does not function properly and creates an obstruction to aqueous humor outflow, and therefore elevates IOP. Transforming growth factor beta 2 (TGFβ2) is elevated in about 50% POAG patients, and is known to increase IOP in several experimental models. The purpose of this study was to determine if TGFβ2 induces IOP elevation in perfusion cultured bovine eyes, which are an important glaucoma research model. Methods: Fresh bovine eyes were obtained, transferred to the lab and carefully dissected. Vitreous humor, uvea, retina, retinal pigment epithelium, and lens were removed. The remaining anterior segment tissue, which contained the sclera, cornea, and TM, was mounted and sealed on a custom-made plexiglass dish with an O-ring using four screws. Perfusion medium was infused by a syringe pump at a constant infusion rate of 5 ul/min. After baseline IOP was established, bovine eyes were perfused with or without 10ng/ml TGFB2 for up to 7 days. IOP was recorded by a pressure transducer and a computerized system. Changes in IOP were calculated by subtracting baseline IOP from IOP post treatment. Conditioned medium was collected for Western immunoblotting (WB). Results: TGFβ2 increased IOP in treated bovine eyes by about 4.5 mmHg while the fellow control eye did not show significant changes in IOP. WB showed that fibronectin, a TGFβ2-inducible protein, was increased in perfusate collected from the TGFβ2 treated eyes. Conclusion: Our study showed that TGFβ2 is able to induce ocular hypertension in perfusion cultured bovine eyes. This will provide researchers a useful model to study POAG.
Epigenetic regulation of TGFβ2 in the pathogenesis of glaucoma
(2016-03-23) Webber, Hannah; Liu, Xiangyang; Cheng, Yi-Qiang; Clark, Abbot; Mao, Weiming; Bermudez, Jaclyn Y.
Primary open angle glaucoma (POAG) is a leading cause of blindness worldwide. The primary risk factor for the development and progression of this optic neuropathy is increased intraocular pressure (IOP) caused by glaucomatous damage to the trabecular meshwork (TM). The glaucoma-associated factor, transforming growth factor beta 2 (TGFβ2) is increased in the TM of POAG patients. TGFβ2 elevates IOP in perfusion cultured human eyes and in rodents. We hypothesize that histone acetylation plays a role in dysregulated TGFβ2 expression. To test our hypothesis, we treated primary non-glaucomatous human TM (NTM) cells as well as perfusion cultured bovine eyes with 10 nM thailandepsin-A (TDP-A), a potent histone deacetylase inhibitor. We found that TDP-A increased protein acetylation in the TM using Western immunoblotting. Chromatin immunoprecipitation showed that TDP-A induced histone hyperacetylation associated with the TGFβ2 promoter. This change of acetylation significantly increased TGFβ2 expression in NTM cells as shown by quantitative PCR (n=6, p
Glutaredoxin 2 (Grx2) Protects Retinal Pigment Epithelial Cells from Oxidative Damage by Regulating Autophagy
(2016-03-23) Liu, Xiaobin; Nguyen, Yen; Tran, Tyler; Wu, Hongli; Xavier, Christy
Purpose: Glutaredoxin 2 (Grx2) is an oxidoreductase present in the mitochondria where it protects the organelle from oxidative damage and maintains its redox homeostasis.The purpose of this study is to evaluate the cytoprotective effects of Grx2 in human retinal pigment epithelial (RPE) cells and characterize its potential function in regulating autophagy.Methods: Primary RPE cells were isolated from Grx2 knockout (KO) mice and treated with or without 400 µM H2O2 for 4 h. Human retinal pigment epithelial (ARPE-19) cells were transfected with either a human Grx2 cDNA-containing plasmid (pCR3.1-hGrx2) or an empty vector pCR3.1. Cells were treated with or without 200 µM H2O2 for 16 h. Grx2 protein expression was detected by western blot analysis. Cell viability was measured by a colorimetric assay with WST8. The morphology of nuclear chromatin was assessed by staining with Hoechst 33342. Apoptosis was quantitatively analyzed by flow cytometry. The level of protein glutathionylation (PSSG) and autophagy pathway proteins were measured by immunoblotting.Results: Primary RPE cells that lack Grx2 were more sensitive to oxidative damage. On the other hand, Grx2 overexpression protected RPE cells from H2O2-induced cell viability loss. Assessment of apoptosis indicated that cells transfected with Grx2 were more resistant to H2O2 with fewer cells undergoing apoptosis as compared to vector control cells. PSSG accumulation was also attenuated by Grx2 overexpression with acute H2O2 challenge. Furthermore, protein levels of LC3II and Beclin-1, which are key molecules to initiate autophagy, were inhibited in Grx2 overexpressed cells with H2O2 treatment. Conversely, primary Grx2 KO RPE cells showed higher levels of LC3II and Beclin-1 under oxidative stress.Conclusion: Grx2 rescues RPE cells from lethal oxidative damage, possibly through alleviation of ROS-triggered autophagy and prevention of PSSG accumulation.
Endothelin-1 Induced the Reactivation of Primary Rat Ocular Astrocytes
(2016-03-23) Ma, Hai-Ying; Park, Yong; Wang, Junming; Yorio, Thomas; He, Shaoqing
Purposes: Astrocytes play a crucial role in cell survival and axon function of retinal ganglion cells (RGCs) 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 astrocytes 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.
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, Avani
Purpose: 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.
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, Hannah
Purpose: 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.
Over Expression of Sigma-1 Receptor Protects Against Retinal Ganglion Cell loss in an Optic Nerve Crush Model.
(2016-03-23) Li, Linya; Liu, Yang; Yorio, Thomas; Ellis, Dorette
Purpose: Recent studies in sigma-1 receptors (σ-1r)- deficient mice demonstrated increased losses of retinal ganglion cells (RGCs) in oxidative stress and optic nerve crush mice models (ONC) . This study determines the neuroprotective role of the σ-1r in σ-1r- deficient mice ONC model. Methods: ONC was performed in σ-1r k/o mice and wild type mice. Briefly, the left optic nerve (ON) were exposed intraorbitally through a small window made between the surrounding muscles. The ON was crushed approximately 1mm behind the globe with self-closing forceps for 4 seconds under visualization. The contralateral eye was used as controls for ONC. Two weeks prior to ONC, mice were intravitreally injected with AAV2-CAG-σ-1r-GFP vector (1.2 x 1010) and expression of σ-1r was assessed; AAV2 empty vector (1.2 x 1010) served as controls. Termination experiments include counting RGCs using RNA-binding protein with multiple splicing antibodies (RBPMS antibodies) in experimental animals to determine neuroprotective activities of the σ-1r. Results: RGC death was accelerated in σ-1r k/o ONC animals when compared with wild-type mice. σ-1r k/o ONC mice injected with AAV2-CAG-σ-1r-GFP vector demonstrated significant increases in RGCs numbers and activity when compared with σ-1r k/o ONC mice injected with empty vector or non-injected σ-1r k/o ONC animals. Conclusion: Our studies which involved the expression of σ-1r in a system devoid of σ-1rs, provided direct evidence of neuroprotection of RGCs due to the expression of σ-1rs. These findings support a protective role of σ-1r in RGCs when they are challenged by neurodegenerative insults. Financial Disclosure: Supported by a grant from DOD (W81XWH-10-2-0003)
GRα and GRβ expression levels in trabecular meshwork determines steroid responsiveness upon glucocorticoid treatment
(2016-03-23) Zode, Gulab; Mao, Weiming; Clark, Abbot; Patel, Gaurang
Purpose Glucocorticoid (GC) induced ocular hypertension (OHT) is a serious side effect of prolonged GC therapy with patients showing elevated intraocular pressure (IOP). Two major isoforms of glucocorticoid receptor (GRα and GRβ) regulate GCs sensitivity and specificity in various tissues. GRβ acts as a dominant negative regulator of GC activities and has been shown to regulate GC responsiveness in trabecular meshwork (TM). We evaluated GRα and GRβ expression levels in two mouse strains and studied how expression levels regulate GC and GC-induced OHT. Methods TM cells from C57BL/6J and BALB/cJ mice strains were isolated and characterized. RNA was isolated from TM cells and evaluated for GRα and GRβ expression levels using quantitative (Q)-PCR. To study how both TM cell lines respond to Dexamethasone (DEX) they were treated with DEX (100nM) and myocilin (MYOC) expression in TM cells was determined by Q-PCR analysis. Three month old C57BL/6J and BALB/cJ mice strains were used to evaluate changes in IOP upon DEX treatment. Mice were peri-ocularly injected with DEX-Acetate (100ug/eye) in both eyes. Conscious IOP measurements were taken using a TonoLab tonometer. Two-tailed Student’s t-test and One-way ANOVA were used for statistical analysis. Results MTM cells from both strains (C57BL/6J and BALB/cJ) expressed TM markers, including collagen IV, laminin and α-smooth muscle actin. GRα expression levels between both strains were similar. TM cells from BALB/cJ mice expressed significantly higher levels of GRβ compared to TM cells from C57BL/6J. When TM cells were treated with 100nM DEX, TM cells from C57BL/6J showed induction of myocilin expression compared to untreated controls whereas; TM cells from BALB/cJ did not show myocilin induction. IOP measurements upon DEX-Acetate treatment showed significant IOP elevation in C57BL/6J mice (ΔIOP of 3.5mmHg, p Conclusions In mouse, GRα and GRβ expression levels determines GC responsiveness. Higher GRβ expression levels leads to GC resistance. The current findings provide an important foundation for comparisons of GRα and GRβ expression levels in the TM among different strains. Also, manipulating GRα to GRβ expression levels holds a promise for desensitizing cells and tissues to GCs effects.
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, Xiaobin
Purpose: 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.
Tissue transglutaminase causes intraocular pressure elevation in mice
(2016-03-23) Millar, Cameron; McDowell, Colleen; Clark, Abbot; Raychaudhuri, Urmimala
Purpose: The profibrotic cytokine TGF-β2 increases expression of the crosslinking enzyme tissue transglutaminase (TGM2). In the trabecular meshwork (TM), excessive crosslinking of ECM proteins mediated by TGM2 could increase extracellular matrix (ECM) protein deposition, thereby decreasing the aqueous humor outflow facility. We hypothesize that increased expression of TGM2 increased ECM crosslinking in TM cells, and increases aqueous humor outflow resistance leading to elevated intraocular pressure (IOP) in mice. Methods: MTM cells were grown to confluency and transduced with Ad5.TGM2 (MOI of 75). On Day 5, MTM cells were fixed with 4% PFA for immunocytochemistry (ICC). Ad5.TGM2 (1.28 - 106 pfu in 2ml) was injected intravitreally into the left eye of female BALBc/J retired breeder mice (n = 18). The uninjected (right) eye served as a control. Daytime conscious IOP measurements were taken twice a week using a TonoLab rebound tonometer for approximately 3 weeks. Aqueous humor outflow facilities (C) was studied on day 23 (n = 6) using our published constant flow infusion method. Results: In cultured MTM cells, treatment with Ad5.TGM2 increased immunostaining of ε-(γ-glutamyl)lysine (GGEL) bonds, demonstrating increased TGM2 crosslinking activity after treatment with Ad5.TGM2. In BALBc/J mice, injection of Ad5.TGM2 significantly increased IOP from day 14 to 22, with the maximum difference elevation at Day 19, (15.86 +/- 1.06 mmHg (injected) versus 10.7 +/- 0.48 mmHg (control) (p Conclusion: Increased expression of TGM2 in mouse TM cells increases the ECM cross-linking activity of TGM2. Increased expression of TGM2 in the TM of the living mouse increases aqueous outflow resistance and elevates IOP. In the future, we will study whether TGM2 is responsible for TGF-β2 induced ocular hypertension.
Genetic and pharmacological inhibition of ER stress-induced ATF4/CHOP pro-death pathway prevents myocilin misfolding and rescues mouse models of glaucoma
(2016-03-23) Phan, Tien; Zode, Gulab; Kasetti, Ramesh
Purpose: We recently demonstrated that ER stress-induced pro-death markers including ATF4 and CHOP are significantly increased in human post-mortem glaucomatous trabecular meshwork (TM) tissues. The purpose of this study was to further explore the role of ATF4/CHOP in myocilin misfolding in TM and IOP elevation. Methods: ATF4 and CHOP levels were analyzed in the TM of Tg-MYOCY437H and dexamethasone (Dex)-treated mice by Western blotting (n=3). Conscious IOP was monitored by rebound tonometer in C57BL/6 mice injected intravitreally with Ad5 Null or ATF4 virus. IOP was measured in Chop-/-Tg-MYOCY437H mice and compared to Chop+/+Tg-MYOCY437H and WT mice. The effects of CRISPR-Cas9 mediated knockdown of ATF4/CHOP on myocilin accumulation was studied in TM-5 cells expressing mutant myocilin. Myocilin accumulation and ER stress was examined in TM cells treated with pharmacological inhibition of the ATF4/CHOP pathway with 100 and 200nM of ISRIB (an inhibitor of the integrated stress response) for 48 hours. Furthermore, ocular hypertensive Tg-MYOCY437H mice were treated with an intravitreal injection of ISRIB (2ul, 2mM) and IOP was measured 4 days post-injections. Results: Western blot analysis demonstrated increased ATF4 and CHOP levels in the iridocorneal angle tissues of Tg-MYOCY437H and Dex-treated mice. Forced expression of Ad5.ATF4 led to significant IOP elevation in WT mice (16mmHg in null vs 23mmHg in Ad5.ATF4-injected mice; p+/+Tg-MYOCY437H mice (22mmHg, n=16) had significantly elevated IOP compared to WT Chop+/+ littermates (16.5mmHg, n=4), while Chop-/-Tg-MYOCY437H mice (19mmHg, n=8) did not elevate IOP compared to Chop-/- littermates (18mmHg, n=6) indicating that deletion of Chop rescues ocular hypertension in Tg-MYOCY437H mice. In addition, Cas9 targeting of ATF4 or CHOP dramatically reduced ATF-4 and CHOP levels and also reduced myocilin accumulation and ER stress in TM-5 cells. Treatment of TM cells with ISRIB (100nM) dramatically reduced myocilin accumulation and also prevented ER stress. Pharmacological inhibition of the ATF4/CHOP pathway with ISRIB (2mM) significantly reduced elevated IOP in Tg-MYOCY437H mice (14.5mmHg in vehicle vs 10mmHg in ISRIB treated Tg-MYOCY437Hmice, p Conclusion: These studies indicate that genetic or pharmacological inhibition of the ATF4/CHOP pathway may provide a novel approach to glaucoma treatment.

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