ARVO 2014 Annual Meeting Abstracts 125 Lens cell biology and ...

ARVO 2014 Annual Meeting Abstracts 125 Lens cell biology and structure Sunday, May 04, 2014 1:30 PM–3:15 PM Exhibit/Poster Hall SA Poster Session Program #/Board # Range: 724–750/C0267–C0293 Organizing Section: Lens Program Number: 724 Poster Board Number: C0267 Presentation Time: 1:30 PM–3:15 PM Transfection of ARE-GFP Construct in Mouse Lens Shambhu D. Varma, Krish Chandrasekaran. Ophthal & Visual Sci & Biochem, Univ of Maryland Sch of Med, Baltimore, MD. Purpose: Oxidative stress plays a significant role in the pathogenesis of cataracts. While administration of nutritional antioxidants and metabolic agonists attenuates the process by scavenging the reactive oxygen species, the effect is short lived and highly variable due to constraints that limit their availability. Also, oxidative stress enhances transcription of several microRNAs that interfere with the biogenesis of transcription factors (Nrf2 and others) that regulate the transcription of antioxidant enzymes by binding with the AREs. Oxidative stress can affect this process through several routes. It was thus considered desirable to examine the feasibility of transfecting the lens with ingredients that influence the course of cataract development. Here, we examined the feasibility of using liposome incorporated ARE –GFP construct for such transfection studies. Methods: Lenses from 6-week-old C57BL6J mice were incubated with the above construct incorporated in a liposome using Mirius 3D Transfection System. Briefly, the TransiT3D reagent was mixed with the construct in OptiMEM medium and incubated at room temperature for 15 min. It was then added to 1 ml of medium-199 containing the lens. The lens with the construct was then cultured for 24 hrs at 37 ° in an incubator gassed with 95% air and 5% CO2. Medium contained either 5mM glucose or 25mM galactose. The expression of GFP was monitored by fluorescence microscopy. Results: That the liposome penetrated the lens with its construct was highly apparent by the expression of ARE-GFP fusion protein, noticed by fluorescence in the lenses incubated with 5mM glucose as well as 25 mM galactose. It was also notable that fluorescence was significantly greater in the lenses incubated with 25mM galactose. Conclusions: The results demonstrate that the liposome incorporated ARE- GFP DNA constructs can penetrate the intact lens. The construct also remained active, indicated by GFP florescence. The florescence was prominent in the equatorial region, the region of active cell differentiation. Also, it was noticeably greater in the galactosemic lenses, indicating a stress response. In summary, the results clearly indicate that the method can be used successfully to assess the functionality of the antioxidant system. Hence further studies are in progress to assess the transcription of ARE-GFP construct in the lens exposed to oxidative stress, as could be affected by purported anticataractogenic agents.

Commercial Relationships: Shambhu D. Varma, None; Krish Chandrasekaran, None Support: NIH Grant EY1292 Program Number: 725 Poster Board Number: C0268 Presentation Time: 1:30 PM–3:15 PM EGR1 deletion partially rescues the abnormalities of β1-integrin null lenses Yichen Wang, Anne Terrell, Melinda K. Duncan. Biological Sciences, University of Delaware, Newark, DE. Purpose: β1-integrin is the major β-integrin subunit expressed in both lens epithelial and fiber cells. Previous research has shown that β1-integrin is essential for the maintenance of lens epithelial integrity and survival in late embryonic lens development. Lack of β1-integrin in the lens will lead to severe micropthalmia and lack of lens in adult mice. RNAseq revealed that early growth response 1 (EGR1), a major regulator of fibrosis and apoptosis, was upregulated upon the conditional deletion of β1-integrin in the lens. This work investigates the possible role of EGR1 in the abnormalities found in the β1-MLR10 lens. Methods: Mice homozygous for the β1-integrin floxed allele and carrying the MLR10 Cre gene (which can express Cre recombinase in all lens cells from lens vesicle stage, β1-MLR10) were bred to EGR1 null mice (EGR1 -/-). Mice heterozygous for the β1-integrin floxed allele, MLR10 Cre, and the EGR1 null allele (β1 F/+ MLR10 EGR1+/-) were further crossed to generate mice homozygous for the β1-integrin floxed allele and EGR1 null allele, carrying MLR10 Cre (β1-MLR10 EGR1 -/-). PCR and immunostaining were performed to confirm the loss of both β1-integrin and EGR1. General morphology was assessed by darkfield microscopy. Expression of molecular markers in the lens was studied by immunofluorescence staining. Results: Conditional deletion of β1-integrin from the lens led to a more than 10 fold upregulation of EGR1 mRNA at 15.5 dpc. Mice that lack both the β1-integrin and EGR1 genes from the lenses were created and loss of β1-integrin was confirmed via immunofluorescence staining. As previously reported, β1-MLR10 mice were extremely microphthalmic and lacked lenses as adults. In contrast, β1-MLR10 EGR1 -/- mice showed larger eyes as adults, with identifiable lens tissue, although it was irregular-shaped and cloudy. Analysis of β1-MLR10 EGR1 -/- lenses at 16.5 dpc revealed differences in the expression pattern of Pax6, cMaf and αSMA, compared to that of β1-MLR10 lenses. These data suggested that EGR1 deletion modified the β1-MLR10 lens phenotype. Conclusions: Mice lacking both the β1-integrin and EGR1 genes from the lens exhibited a different phenotype than β1-MLR10 mice which lack β1-integrin but upregulate EGR1 expression. These data indicated that β1-integrin cross talks with the EGR1 pathway to regulate lens epithelial phenotype during development. Commercial Relationships: Yichen Wang, None; Anne Terrell, None; Melinda K. Duncan, None Support: NEI Grant EY015279 Program Number: 726 Poster Board Number: C0269 Presentation Time: 1:30 PM–3:15 PM Expression of CHIP and Ubc5 in lens epithelial cells enhances ubiquitin-dependent protein degradation Shuhong Jiang1, 2, Lei Lyu1, Zhenzhen Liu1, Allen Taylor1, Fu Shang1. 1USDA Human Nutrition Research Center on Aging, Tufts University, Boston, MA; 2Department of Ophthalmology, The People’s Hospital of Baoan Shenzhen, Southern Medical University, Shenzhen, China. Purpose: Accumulation and precipitation of abnormal proteins in the lens is causally related to cataract formation. The ubiquitin

©2014, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission to reproduce any abstract, contact the ARVO Office at [email protected].

ARVO 2014 Annual Meeting Abstracts proteasome system (UPS) is an important mechanism for identification and selective removal of abnormal proteins. Age-or stress-related compromise to the UPS contributes to accumulation of abnormal proteins in the lens. The purpose of this study is to test whether overexpression of CHIP, a bifunctional molecule that participates in ubiquitin conjugation and chaperone function, or Ubc5, a ubiquitin conjugating enzyme with broad substrate specificity, or CHIP and Ubc5 together increase ubiquitination and UPS-dependent degradation of abnormal proteins from lens cells. Methods: Overexpression of CHIP and Ubc5 in cultured human lens epithelial cells (HLEC) was achieved using adenovirus-mediated transfection. Levels of CHIP, Ubc5 and ubiquitin conjugates were determined by Western blotting. The capacity to catalyze de novo ubiquitination was determined using 125I-labeled ubiquitin and endogenous substrates or using 125I-labeled luciferase as a specific substrate, with supplemental ubiquitin. UPS-dependent degradation capacity was determined using 125I-labeled luciferase. Results: Adenovirus-mediated overexpression increased the levels of CHIP and Ubc5 by 2-5 fold relative to control cells. When both CHIP and Ubc5 were overexpressed in the cells, a majority of the overexpressed CHIP was in mono-ubiquitinated form. Overexpression of CHIP alone did not effect ubiquitination or ubiquitin-dependent degradation. Overexpression of Ubc5 increased ubiquitination and UPS-dependent degradation by ~20%. However, when both CHIP and Ubc5 were overexpressed, ubiquitination and UPS-dependent degradation increased by >50%. Conclusions: Levels of CHIP and Ubc5 HLEC are limited in lens cells. Overexpression of CHIP and Ubc5 together increases the ubiquitination and UPS-dependent degradation capacity. These data suggest that ubiquitination is the rate-limiting step of UPS-mediated degradation and that enhancement of UPS capacity can accelerate the degradation of abnormal proteins. These studies open up new avenues for delaying cataract. Commercial Relationships: Shuhong Jiang, None; Lei Lyu, None; Zhenzhen Liu, None; Allen Taylor, None; Fu Shang, None Support: NIH Grant EY11712, EY13250, EY021212; USDA 1950510000-060-01A Program Number: 727 Poster Board Number: C0270 Presentation Time: 1:30 PM–3:15 PM Increase of Apoptosis in the Lenses of Foxe3 Mutant Mice Louis Zhang, Lixing W. Reneker. Ophthalmology, University of Missouri, Columbia, MO. Purpose: Mutations in human FOXE3 forkhead transcription factor are linked to congenital eye disease called Peters’ anomaly, which is characterized by central corneal opacity and lens-corneal adhesions. Programmed cell death (PCD or apoptosis) plays an essential role in separation of nascent lens vesicle from the overlaying surface ectoderm. In this study, we investigated whether loss of Foxe3 function had an effect on apoptosis during lens development. Methods: Homozygous dysgenetic lens (dyl) mice, which are lossof-function mutant of Foxe3, were used in the study. Apoptosis was detected by TUNEL assay. The expression of phosphorylated ERK (pERK), pAKT and survivin in wild type (WT) and dyl lenses was analyzed by immunostaining. Results: In E11.5 dyl lenses, apoptotic cells were detected in both anterior and posterior part of the lens vesicle, and the level was increased when compared to the WT lenses at the same age. Apoptosis was also found in E18.5 dyl but not WT lenses. These results suggest that Foxe3 is essential for lens cell survival in addition to its function in other cellular processes such as cell proliferation during lens development. It is known that expression of PDGF receptor alpha (PDGFRalpha) requires Foxe3 and the level of

PDGFRalpha is significantly reduced in dyl lenses. To access whether increase of apoptosis in dyl lenses was due to reduction in growth factor receptor signaling activity, we examined the levels of pERK, pAKT and surviving in E18.5 lenses by immunostaining. We found no significant differences between the WT and dyl lenses. Conclusions: In addition to its function in cell proliferation and differentiation, Foxe3 is also required for cell survival, particularly at the stage of lens vesicle development. Apoptosis in E18.5 dyl lenses is not due to the reduction of pERK, pAKT and surviving levels. Commercial Relationships: Louis Zhang, None; Lixing W. Reneker, None Support: EY13146 and RPB Program Number: 728 Poster Board Number: C0271 Presentation Time: 1:30 PM–3:15 PM PARP-1 in the human lens: a story of life and death Andrew J. Smith1, Richard P. Bowater1, John R. Reddan2, Michael Wormstone1. 1School of Biological Sciences, University of East Anglia, Norwich, United Kingdom; 2Oakland University, Rochester, MI. Purpose: Poly(ADP-ribose) polymerase-1 (PARP-1) is a protein best known for its involvement in DNA repair processes. PARP-1 activity is also linked to the oxidative stress response, which involves pathways associated with cell fate. In the current study, we assessed the functional roles of PARP-1 within lens cells in response to oxidative stress. Methods: The human lens epithelial cell line FHL124, which has a 99.5% similarity in gene expression to native human lens tissue and human lenses obtained from donor eyes were used as the experimental models. Hydrogen peroxide (H2O2) was employed to induce oxidative stress and cell death assessed by LDH release. PARP-1 activity was suppressed by targeted siRNA and chemical inhibition. Immunocytochemistry and western blotting were used to assess PARP-1 expression. To determine DNA single and double strand break damage the alkaline comet assay was performed and quantified using Comet IV lite analysis software (Perceptive Instruments). Results: PARP-1 expression was demonstrated by immunocytochemistry in both the FHL124 cell line and whole human lenses; in both instances expression was predominantly nuclear. Chemical inhibition of PARP-1 rendered FHL124 cells more susceptible to H2O2 (30 μM)-induced DNA strand breaks. PARP-1 expression was also depleted with targeted siRNA resulting in a knockdown of greater than 80%; these cells were similarly more susceptible to H2O2-induced DNA strand breaks than control cells, treated with scrambled control siRNA. Interestingly, chemical inhibition of PARP-1 significantly inhibited H2O2 (100 μM)-induced cell death relative to control cells. Chemical inhibition of PARP-1 in whole human lenses resulted in a reduced level of opacity and cell death following exposure to 1 mM H2O2 relative to match pair controls without PARP-1 inhibition. Conclusions: PARP-1 is expressed in both FHL124 cells and whole human lenses. Inhibition of PARP-1 protects FHL124 cells against H2O2-induced cell death whilst rendering FHL124 cells more susceptible to DNA strand breaks. PARP-1 inhibition in whole human lenses protects them from H2O2-induced opacity and related cell death. PARP-1 can play a number of important roles in the fate of lens cells and further studies are required to elucidate the regulatory processes that give rise to these effects. Commercial Relationships: Andrew J. Smith, None; Richard P. Bowater, None; John R. Reddan, None; Michael Wormstone, None Support: The Humane Research Trust


ARVO 2014 Annual Meeting Abstracts Program Number: 729 Poster Board Number: C0272 Presentation Time: 1:30 PM–3:15 PM Chaperone-independent mitochondrial translocation, oxidative stress protection and prevention of apoptosis by αB-crystallin Bettina Teng1, Rebecca S. McGreal2, Daniel Chauss1, Lisa A. Brennan1, Marc Kantorow1. 1Biomedical Sciences, Florida Atlantic University, Boca Raton, FL; 2Department of Ophthalmology & Visual Sciences, Albert Einstein College of Medicine, New York, NY. Purpose: We have previously demonstrated that αB-crystallin protects lens epithelial cell and retinal pigmented epithelial cell (RPE) mitochondrial function through its ability to directly protect cytochrome c against oxidative inactivation and to preserve the ability of methionine sulfoxide reductase A (MsrA) to repair cytochrome c through reduction of met 80 sulfoxide. We hypothesized that αB-crystallin might protect mitochondria through its chaperone function, here we evaluated the relationship between αB-crystallin chaperone function, mitochondrial membrane potential, cytochrome c release and apoptosis in multiple ocular cell types including lens and RPE cells. Methods: Three forms of αB-crystallin exhibiting different chaperone activity levels including wild type, R120G (decreased chaperone activity) and M68A (increased chaperone activity) were evaluated for their abilities to translocate to the mitochondria, maintain mitochondrial membrane potential, prevent cytochrome c release and maintain viability of ARPE19 cells, SRA04/01 human lens epithelial cells and primary chicken lens epithelial cells. Results: As expected R120G αB-crystallin exhibited decreased chaperone activity relative to wild type αB-crystallin while M68A exhibited increased chaperone activity relative to wild type αBcrystallin. αB-crystallin protected both lens and RPE cells against oxidative stress damage. Interestingly these forms of αB-crystallin exhibited comparable levels of mitochondrial protection against oxidative stress in RPE cells. Conclusions: These results confirm that αB-crystallin plays an important role in protecting the mitochondria of multiple cells against oxidative stress damage and therefore plays an important role in the maintenance and protection of multiple ocular cell-types against oxidative stress damage. Surprisingly, the protection of the mitochondria by αB-crystallin appears to be independent of its chaperone activity suggesting dual cellular functions for this important ocular chaperone. Commercial Relationships: Bettina Teng, None; Rebecca S. McGreal, None; Daniel Chauss, None; Lisa A. Brennan, None; Marc Kantorow, None Support: EY13022 MK Program Number: 730 Poster Board Number: C0273 Presentation Time: 1:30 PM–3:15 PM Characterizing the diffusion of molecules in the anterior lens capsule using fluorescence recovery after photobleaching (FRAP) Vivian M. Sueiras1, Vincent T. Moy2, Noel M. Ziebarth1. 1Biomedical Engineering, College of Engineering, University of Miami, Coral Gables, FL; 2Physiology and Biophysics, Miller School of Medicine, University of Miami, Miami, FL. Purpose: To determine the size-dependent transport of molecular probes through the lens capsule by means of fluorescence recovery after photobleaching (FRAP). Methods: Experiments were conducted on 4 porcine lens capsules. The eyes were obtained from an abattoir and shipped in saline to the laboratory overnight. A capsulorhexis was performed to separate a portion of the anterior capsule from the lens. Each excised capsule was submerged in a prepared solution of anionic, fluorescein-labeled dextran in PBS: 2 samples in 10kD MW (d-F10) and 2 samples in

40kD MW (d-F40). The samples were soaked overnight, allowing the molecules to diffuse into the capsule and to reach chemical and diffusional equilibrium. The capsules were then removed from the bath, washed 3 times with PBS, plated on a glass bottom dish and hydrated with PBS. A Nikon A1R confocal microscope was used to conduct FRAP experiments. Prior to bleaching, a single image was acquired to establish the baseline fluorescent intensities. A 20mmx20mm square in the center of the field of view (FOV) at a plane within the capsule was bleached for 2 seconds using an argon laser (488nm) set to full power. After bleaching, time-lapse images of the full FOV were continuously acquired each second for 1 minute. From these images, 2 different regions were identified. Changes in fluorescence intensity stemming from the diffusion of the fluorescent tracer were monitored in a 7mmx7mm region within the bleached area. Since the amount of fluorescence decreases during time-lapse imaging, a reference area located at the periphery of the FOV was used for bleaching correction. These experiments were repeated in at least 4 different areas within 10mm of the central depth along the z-axis of the capsule. The Nikon NIS-Elements software was used to determine the time to half maximum recovery for each measurement. Results: The time to half maximum recovery was 2.87±0.73s and 2.75±0.38s for the d-F10 and 2.92±0.19s and 3.53±0.52s for the d-F40. The time to half maximum recovery was significantly slower (p=0.049) for the larger anionic, fluorescein-labeled dextran. Conclusions: Our results indicate that diffusion of anionic particles in the lens capsule is size-dependent. Additional experiments with molecules of varied size/charge are needed to further our understanding of diffusion in the capsule and its role in maintaining lens optical clarity. Commercial Relationships: Vivian M. Sueiras, None; Vincent T. Moy, None; Noel M. Ziebarth, None Support: NSF MRI 0722372; James & Esther King Biomedical Research Program Shared Instrument Grant 24157 Program Number: 731 Poster Board Number: C0274 Presentation Time: 1:30 PM–3:15 PM Expression of a mutant Cx46 leads to changes in lens fiber ultrastructure Sarah M. El-Khazendar1, Rebecca K. Zoltoski1, Viviana M. Berthoud2, Peter J. Minogue2, Eric C. Beyer2. 1Illinois College of Optometry, Chicago, IL; 2University of Chicago, Chicago, IL. Purpose: Inherited human cataracts have been linked to mutations in connexin46 (Cx46) including Cx46fs380. Mice expressing Cx46fs380 were generated by a knock-in strategy. Although lenses of young mutant mice appear transparent, anterior cataracts were evident in homozygous Cx46fs380 mice at 2 months and in some heterozygotes at 4 months. The experiments were designed to characterize the structural changes in lens fiber cell membranes that occur in these mice with age. Methods: Levels of connexins in lens homogenates were determined by immunoblotting. Lenses were examined by scanning electron microscopy, and the number of furrows, interdigitations, and ball and socket joints were quantified in different regions of the lenses from wild type and homozygous mice. Data are presented as mean ± SEM. Nonparametric t-tests were used to assess the significance of differences between genotypes and ages (p 50 unique regulators and >400 targets, representing ~1300 regulatory relationships in 40 stages from distinct cell types. Based on these integrative approaches, we identified several active networks in different stages. Not surprisingly, Pax6 was identified as a major regulator in all stages, while Fgfr1-3 were critical for regulating gene expression in differentiation. Furthermore, overlay of cataractassociated genes from Cat-map onto these GRNs identified regulatory relationships for 44 genes, with some clustering in distinct pathways. Interestingly, the GRNs revealed that expression of many cataractassociated genes was initiated early in lens development. Conclusions: We have constructed lens-GRNs based on hundreds of molecular interactions in the lens. The principle advantage of the curated lens GRN resource is that every edge (relationship) between individual nodes (regulators and targets) is supported by at least one piece of published experimental evidence. In addition to effective visualization of regulatory relationships in lens development, these GRNs will assist in predicting and interpreting how specific perturbations in genetic pathways cause cataract. Commercial Relationships: Deepti Anand, None; Djordje Djordjevic, None; Sylvie Smith, None; Joshua W K. Ho, None; Salil A. Lachke, None Support: NIH/NEI R01 EY021505 Program Number: 736 Poster Board Number: C0279 Presentation Time: 1:30 PM–3:15 PM Investigation of Caprin2 (RNG140 RNA granule protein 140) function in mouse lens Soma Dash1, Christine Dang1, David C. Beebe3, Salil A. Lachke1, 2 1 . Department of Biological Sciences, University of Delaware, Newark, DE; 2Center for Bioinformatics & Computational Biology, University of Delaware, Newark, DE; 3Department of Ophthalmology and Visual Sciences, Washington University, St. Louis, MO. Purpose: We recently identified two post-transcriptional regulatory proteins (Tdrd7 and Celf1) that are essential for vertebrate lens development as demonstrated by the severe lens defects caused by their deficiency in multiple animal models. Here, we explore the function of a third conserved lens-expressed protein Caprin2 (RNG140, Eeg1, C1qdc1) that has multiple conserved domains, including the coiled-coil and RGG domains (for RNA binding) as well as the C1q domain, found in TNF super-family of proteins, that mediates protein-protein interactions. Methods: iSyTE predicted Caprin2 as a candidate with high lensenriched expression and potential function in lens development and/ or homeostasis. In situ hybridization, qRT-PCR, western blotting and immunofluorescence were used to detect Caprin2 RNA and protein expression in the lens. To test Caprin2 function in the


ARVO 2014 Annual Meeting Abstracts lens, we obtained mice carrying Caprin2 conditional mutant allele from EUCOMM (Caprin2tm2a(EUCOMM)Wtsi) and crossed them with an established lens deleter mouse line Pax6GFPCre to generate lensspecific Caprin2 null mutants (hereafter referred to as Caprin2 cKO). Results: Caprin2 expression has been previously shown to be responsive to induction by FGF8 in chicken lens fiber cells, indicating a potential role in the lens (Loren et al. 2009 Differentiation. 77:386-94). These findings, together with its high rank in iSyTE expression datasets and its association with RNA granules in neurons, encouraged us to investigate Caprin2 function in the lens. We confirmed previous findings that Caprin2 exhibits enriched expression in mouse lens from E10.5 onwards and is expressed in postnatal lens. We recently generated Caprin2 cKO mutants and have initiated the phenotypic characterization of lens defects in these animals. Although there is no overt opacity in young adult Caprin2 cKO mutant lenses, initial microscopic analyses suggest a mild but consistent abnormality in the central part of the lens. Compound mouse mutants carrying combinations of Caprin2, Tdrd7 or Celf1 null alleles are being characterized for further insight into its function in the lens. Conclusions: We have investigated the function of Caprin2 in the lens, and demonstrate that its nullizygosity in mouse mutants causes mild lens defects. In sum, Caprin2 represents a new regulatory protein that, along with Tdrd7 and Celf1, may function in a conserved post-transcriptional regulatory network in the lens. Commercial Relationships: Soma Dash, None; Christine Dang, None; David C. Beebe, None; Salil A. Lachke, None Support: NIH/NEI R01 EY021505 Program Number: 737 Poster Board Number: C0280 Presentation Time: 1:30 PM–3:15 PM The mechanism of lens regeneration Weiju Wu1, Noemi Lois2, Shane Richards1, Christopher Saunter3, Roy Quinlan1. 1Biological and Biomedical Sciences, Durham University, Durham, United Kingdom; 2Queen’s University of Belfast, Belfast, United Kingdom; 3Biophysical Sciences Institute, Durham University, Durham, United Kingdom. Purpose: To investigate lens regeneration in terms of cell growth, cell differentiation and cell polarity in a rodent model. Methods: An extracapsular crystalline lens extraction was undertaken to preserve the lens capsular bag and anterior lens epithelium on the right eye of 60 Sprague-Dawley rats. Contralateral, unoperated eyes served as controls. Cell proliferation was tracked using EdU, which was injected intra-peritoneally before sacrifice on 0, 1, 2, 3, 7, 10, 15, 30 and 50 days postoperatively. EdU labelled cells were detected by Click-iT labelling. The expression of beta-catenin, E-/N-cadherin, AQP0, ZO-1 and gama-tubulin was monitored by confocal immunofluorescence light microscopy using a Leica SP5 system. Results: Newly regenerated lenses were optically clear and the capsular bag filled steadily with cellular material post-extracapsular cataract surgery. A focal opacity was usually observed at the capsulotomy site. A monolayer of cells lined both the anterior and posterior capsule at the early stage of lens regeneration. A burst of cell proliferation occurred in the lens cells on day 1 after surgery, but then decreased rapidly. There was extensive cell reorganisation within the capsular bag. By day 7, epithelial cells at the lens periphery were starting to differentiate, which was followed by changes in the cells only on the posterior capsule. In contrast, epithelial cells on the anterior capsule surface continue to proliferate. The reorganisation of the cell layers during these early stages of lens regeneration adheres to a programme that results in the re-establishment of

central, germinative (GZ) and transitional zones (TZ) by day 30 postoperatively. Conclusions: Lens regeneration involves both temporal and spatial cues to re-establish the cell compartments that typify the unoperated mammalian lens. Commercial Relationships: Weiju Wu, None; Noemi Lois, None; Shane Richards, None; Christopher Saunter, None; Roy Quinlan, None Support: Fight for Sight Program Number: 738 Poster Board Number: C0281 Presentation Time: 1:30 PM–3:15 PM Aging and Oxidative Stress-Induced Dysregulation of LEDGF Gene in Lens Epithelial Cells and Lenses Released by Sulforaphane Through Epigenetic Reprograming Bhavana Chhunchha1, Eri Kubo2, Dhirendra P. Singh1. 1 Ophthalmology & Visual Sciences, Univ of Nebraska Medical Center, Omaha, NE; 2Ophthalmology, Kanazawa Medical University, Kanazawa, Japan. Purpose: We examined the functional connection between dysregulation of activity of the stress-inducible Lens Epithelial Derived Growth Factor (LEDGF) and aberrant epigenetic signaling, and restoration of LEDGF expression and activity by Sulforaphane (SFN), a naturally occurring isothiocyanate. Methods: Prdx6-deficient (Prdx6-/-) and Prdx6+/+ lens epithelial cells (LECs) or lenses from mice of different ages and human LECs were treated with SFN and exposed to different doses of UVB/H2O2 for different periods. Expression of LEDGF, DNA methyltransferases (DNMTs), histone deacetylases (HDACs), acetyl H3 and H2B, SUV39H1, trimethyl-H3K9 and heat shock proteins (hsps) mRNA and protein were assessed by q PCR and Western blot. Levels of reactive oxygen species (ROS) were quantified with H2DCFHDA dye assay. Methylation status of LEDGF gene was analyzed by methylation specific PCR (MSP) bisulfite sequencing. ChIP analysis was used with acetyl-H3, acetyl-H3K9, acetyl 2B, Sp1, a transactivator of LEDGF and HDAC1, SUV39H1, di- and trimethylH3K9, 5-methyl cytosine antibodies to examine status of active and inactive chromatin and Sp1 on CpG island of LEDGF gene promoter (-170/-10). Effect of depletion of Sp1 (SiRNA) on LECs was monitored by MTS and TUNEL assays. Results: SFN treatment restored activity of LEDGF and its target genes hsp27/αB-crystallin, and cells had reduced ROS levels. SFN treatment significantly promoted cell viability and attenuated expression levels of DNMT1, DNMT3b and reduced site-specific CpG methylation. SFN also reduced expression levels of HDAC1, SUV39H1 and dimethyl-H3 and increased levels of active chromatin markers, acetyl H3/acetyl-H3K9 and acetyl-H2B. ChIP analysis of LEDGF promoter revealed that SFN increased the active chromatin markers acetyl-H3K9, acetyl-2B and Sp1 access at CpG island. In contrast the dimethyl-H3K9, SUV39H1 inactive chromatin markers were decreased at the site in dose-dependent fashion. SFN induced acetylation and reduced methylation, facilitating binding of LEDGF activator Sp1 to its site in LEDGF. Knockdown of Sp1 reduced SFNinduced upregulation of LEDGF mRNA and facilitated cell death. Conclusions: Our studies provide insights into SFN-mediated epigenetic upregulation of LEDGF and its target genes, hsps, and may open avenues for SFN/antioxidant-mediated prevention or delay of cataractogenesis. Commercial Relationships: Bhavana Chhunchha, None; Eri Kubo, None; Dhirendra P. Singh, None Support: EY013394


ARVO 2014 Annual Meeting Abstracts Program Number: 739 Poster Board Number: C0282 Presentation Time: 1:30 PM–3:15 PM Reprogramming Lens Mesenchymal Cells to Induced Pluripotent Stem Cells Katie L. Bales, Roy Joseph, Om P. Srivastava. University of Alabama Birmingham, Birmingham, AL. Purpose: To determine a specific timeline for lens epithelial to mesenchemymal cell transition (EMT) and reprogramming of the transitioned mesenchymal cells to induced pluripotent stem cells (iPSCs) using viral vectors. Methods: Lens epithelial cells were isolated from lenses of onemonth old C57BL/6 mice and were cultured in 6-well plates in M199 medium with 10% fetal bovine serum (FBS) and 1% antibiotics. Cells were maintained for five days for mesenchymal transition. Mesenchymal cells were directly reprogramed, delivering reprogramming factors in a single virus using 2A “selfcleaving” peptides, using a single polycistronic lentiviral vector co-expressing four transcriptional factors (Oct4, Sox2, Klf4 and C-Myc) to yield iPSCs. After twenty days, clones were removed for immunohistochemical cell analysis and RNA isolation. Cells were fixed in 4% paraformaldehyde at room temperature and examined for epithelial markers (Connexin-43, E-cadherin), mesenchymal markers (Alpha smooth muscle actin), lens-specific markers (CryAB) and stem cell markers (Sox1, Oct4, SSEA4). Results: Based on results from immunohistochemical cell analysis and Quantitative PCR, at day zero, there was an increased expression of epithelial markers and by day two, there was an up-regulation of mesenchymal markers. Stem cell markers were not present at both the time-points. Mesenchymal cells were then reprogramed directly to iPSCs after twenty days, obtaining four to six embryonic stem celllike colonies per 105 cells. Immunohistochemical analysis showed clones were positive for all three stem cell markers. Conclusions: Transitioning of lens epithelial cells to mesenchymal cells resulted in the loss of epithelial markers and up-regulation of mesenchymal cell markers, suggesting EMT. Our data show that using a single polycistronic lentiviral vector co-expressing four transcriptional factors can successfully reprogram mesenchymal cells generated from lens epithelial cells into iPSCs. Commercial Relationships: Katie L. Bales, None; Roy Joseph, None; Om P. Srivastava, None Support: NIH Grant EY-06400 Program Number: 740 Poster Board Number: C0283 Presentation Time: 1:30 PM–3:15 PM DNA methylation activity of Dnmt3a and Dnmt3b is not essential for normal mouse lens development Thanh Hoang, Evan K. Horowitz, Blake R. Chaffee, Devin G. Bruney, Savana E. Rosalez, Brad D. Wagner, Michael L. Robinson. Department of Biology, Miami University, Oxford, OH. Purpose: Lens fiber cell differentiation from epithelial cells involves a coordinated change in gene expression. Despite the wealth of knowledge of transcription factors involved in lens development, very little is known about the epigenetic regulation of lens fiber cell differentiation. In mammals, Dnmt3a and Dnmt3b enzymes are responsible for creating de novo DNA methylation changes during development. Our study sought to determine the role of DNA methylation mediated by Dnmt3a and Dnmt3b activities during lens development in mice. Methods: To generate Dnmt3 single knockout (KO) mice, mice with a conditional “loxP-flanked” flanked mutation in Dnmt3a (Dnmt3LL) and mice with a conditional “loxP-flanked” flanked mutation in Dnmt3b (Dnmt3bLL) were bred independently with three different Cre-expressing mouse strains: Sox2-Cre, Le-Cre and MLR10-

Cre mice. Sox2-Cre mice express Cre recombinase at 2 cell-stage embryos. Le-Cre mice express Cre in the lens placode by embryonic day 9.0 (E9.0). Cre expression in MLR10-Cre mice initiates at lens vesicle stage (E10.5). Dnmt3ab double KO mice were generated by crossing Dnmt3a KO mice with Dnmt3b KO mice. The levels of Dnmt3a and Dnmt3b mRNA transcripts in the lens were quantified by RT-qPCR. Lens morphology and integrity were examined by hematoxylin and eosin staining. Results: Dnmt3a/Sox-2 KO mice died within 4 weeks after birth with no obvious eye defect. Dnmt3b/Sox-2 KO mice and Dnmt3ab/ Sox-2 double KO mice died early during embryonic development, thus effect on eye phenotype was not examined. Conditional Dnmt3a, Dnmt3b, and Dnmt3ab double KO mice with LeCre and MLR10 Cre did not show any obvious lens defects. RT-qPCR confirmed more than 90% loss of Dnmt3a and Dnmt3b mRNAs in the lens. Conclusions: Mouse lenses lacking DNA methylation activity of Dnmt3a and Dnmt3b appeared morphologically normal well into adulthood. Whether de novo DNA methylation really has any role in lens development and whether de novo DNA methylation in Dnmt3ab double KO mice were compensated by other Dnmt enzymes needs further studies. Current analyses are focused on determining if loss of Dnmt3a or Dnmt3b or both results in reduced levels of DNA methylation in the lens. Commercial Relationships: Thanh Hoang, None; Evan K. Horowitz, None; Blake R. Chaffee, None; Devin G. Bruney, None; Savana E. Rosalez, None; Brad D. Wagner, None; Michael L. Robinson, None Support: NH grant NEI 402110 Program Number: 741 Poster Board Number: C0284 Presentation Time: 1:30 PM–3:15 PM Formation of a second lens in the zebrafish occhiolino/ collagen4a5 mutant Owen Lawrence1, Masamoto Aose3, Tor Linbo1, Rachel Tittle4, Pawat Seritrakul4, David W. Raible1, Jeff M. Gross4, John I. Clark1, 2. 1Biological Structure, University of Washington, Seattle, WA; 2Ophthalmology, University of Washington, Seattle, WA; 3 Ophthalmology, Dokkyo Medical University, Tochigi, Japan; 4 Molecular Biosciences, Institute for Cellular and Molecular Biology, University of Texas at Austin, Austin, TX. Purpose: Characterization of the mechanism for the visual defects in the zebrafish occhiolino (occ) mutant. Methods: In an N-ethyl-N-nitrosourea (ENU) screen for eye phenotypes occ was identified as having a small pupil and lens. Because of visual deficits, occ mutants behave as blind fish and have difficulty feeding beyond 5 days post fertilization (dpf). Tissue sections of occ and wild-type (WT) embryos were compared using H&E staining. Live occ and WT embryos were mounted in soft agar and lens development was compared by multiphoton imaging over the first 5dpf. Impairment of visual function was evaluated using the optokinetic response (OKR). Retinal function was evaluated using electroretinograms (ERG). Whole genome sequencing and SNPmapping was utilized to identify the causative mutation. Results: In H&E stained sections, disruption of the original lens and formation of a second lens in occ mutants was observed by 5dpf. Live embryo imaging with multiphoton microscopy starting at 1dpf determined that occ mutants and WT embryos developed similarly to approximately 3dpf. After 3dpf, a second lens cell mass separated from the surface ectoderm and formed anterior to the original lens in occ. Retinal structure was similar in occ mutants and WT embryos. OKR screening confirmed visual defects in occ embryos. The number of saccades per 20 sec decreased in occ to less than one-half of WT embryos. ERG wave amplitudes and timing were nearly identical


ARVO 2014 Annual Meeting Abstracts in occ and WT embryos confirming normal photoreceptor function. The formation of the second lens leads to defective optics without an effect on retinal structure or function. Whole genome sequencing and SNP-mapping identified a T>G mutation in the intron 41 splice-donor site of the collagen4a5 (col4a5) gene. This mutation resulted in the inclusion of intron 41 and a premature stop codon. Complementation testing with two other col4a5 mutant alleles (col4a5uta8 and col4a5sa1609) resulted in embryos with lens defects, supporting the identification of col4a5 as the causative mutation in occ. Conclusions: A mutation in the splice-donor site of the collagen4a5 gene leads to development of a second lens mass at 3 dpf in the zebrafish mutant occhiolino, leading to altered optical function with normal retinal ERGs. Marked thinning of the human lens capsule is associated with collagen4a5 mutation. Zebrafish occ is a model for the importance of lens capsule in the development of visual function. Commercial Relationships: Owen Lawrence, None; Masamoto Aose, None; Tor Linbo, None; Rachel Tittle, None; Pawat Seritrakul, None; David W. Raible, None; Jeff M. Gross, None; John I. Clark, None Support: EY04542, P30-EY01730 and R21-EY022770 Program Number: 742 Poster Board Number: C0285 Presentation Time: 1:30 PM–3:15 PM Protein Serine/Threonine Phosphatases Dephosphorylate p53 at Multiple Sites and Negatively Regulates Its Ability on Lens Differentiation Xiangcheng Tang1, 2, Fangyuan Liu1, Zhongwen Luo1, Weike Ji2, Wenfeng Hu2, Xiaohui Hu2, Yizhi Liu1, David W. Li2, 1. 1Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangzhou, China; 2 Ophthalmology & VIsual Sciences, University of Nebraska Medical Center, Omaha, NE. Purpose: p53 is a transcription factor and play essential role in the ocular lens. Previous studies have shown that inactivation of p53 by viral gene expression or knockout leads to cataractogenesis. Our recent results provide first evidence that p53 regulates lens differentiation through control of both transcription factors, C-Maf and Prox-1, and also lens crystallin genes encoding alpha- and betacrystallins. In the present study, we present evidence to show that PP-1 and PP-2A can dephosphorylate p53 at multiple sites (Ser-6, Ser-9 and Ser-20) to control its ability in regulating C-Maf, Prox-1, alphaA- and betaA3/A1-crystallin genes. Methods: Human lens epithelial cells and alphaTN4-1 mouse lens epithelial cells were used as testing systems. Co-immunoprecipitation assays, in vitro dephosphorylation assay, inhibition of phosphatase activity, overexpression and knockdown of various subunits from PP-1 and PP-2A were used to determine the specific dephosphorylation of p53 by PP-1 and PP-2A. Reverse transcription polymerase chain reaction, Western-blot analysis and reporter gene activity assays were used to study gene expression involved in lens differentiation. Results: p53 was differentially phosphorylated at different sites during mouse lens development. PP-1 and PP-2A can dephosphorylate p53 at Ser-6, Ser-9 and Ser-20. Changes in the phosphorylation status of p53 at these sites alters its transcriptional activity and negatively regulates the expression of C-Maf, Prox-1, alphaA- and betaA3/A1-crystallin genes. Conclusions: PP-1 and –2A directly dephosphorylate p53 to regulate lens differentiation via control of expression of the downstream genes. Commercial Relationships: Xiangcheng Tang, None; Fangyuan Liu, None; Zhongwen Luo, None; Weike Ji, None; Wenfeng Hu, None; Xiaohui Hu, None; Yizhi Liu, None; David W. Li, None Support: EY018380

Program Number: 743 Poster Board Number: C0286 Presentation Time: 1:30 PM–3:15 PM Molecular characterization of mouse lens epithelial cell lines and their applicability to study lens RNA granules Anne Terrell1, Stephanie Waters1, Christine Dang1, David C. Beebe3, Salil A. Lachke1, 2. 1Department of Biological Sciences, University of Delaware, Newark, DE; 2Center for Bioinformatics & Computational Biology, University of Delaware, Newark, DE; 3Department of Ophthalmology and Visual Sciences, Washington University, St. Louis, MO. Purpose: The discovery of RNA granule proteins associated with mammalian cataract has initiated an interest in investigating posttranscriptional control mechanisms in the lens. However, deleting the core components (e.g. Dcp1a, Ddx6, etc.) of RNA granules like Processing Bodies (PBs) and Stress Granules (SGs) is expected to cause early embryonic lethality in mice, and conditional null mutant alleles for these genes are as yet unavailable. Mouse lens epithelial cell lines offer an alternate reagent, but have not been characterized for their suitability in these studies. Therefore, we investigated several mouse lens epithelial cell lines for their expression of lens marker genes as well as their potential to form distinct RNA granules. Methods: Mouse lens epithelial cell lines (LECs) 21EM15, 17EM15 and αTN4 were characterized in this study. Mouse fibroblast cell line NIH3T3 was analyzed as a non-lens derived reference. Comparative gene expression analysis between 21EM15 cell line microarrays and iSyTE expression datasets were used to determine lens-enriched genes that are potentially expressed in these cell lines. Expression of candidate genes was validated using regular and quantitative RT-PCR. For testing their potential to form PBs and SGs, cells were stressed with sodium arsenite and were examined by confocal microscopy. Results: We find that LECs 21EM15, 17EM15 and αTN4 exhibit significantly higher expression of the following key lens genes when compared to NIH3T3 cells: Pax6, Foxe3, Prox1, Jag1, Aldh1a1 and Trpm3. Additionally, these LECs also expressed 35 other lens marker genes. When tested for RNA granules, all three LECs were found to have significantly higher (1.5 to 6-fold) levels of PBs when compared to NIH3T3 cells in unstressed conditions. In stress conditions, all cell lines exhibited elevated levels of both classes of RNA granules, but the extent of elevation was higher for LECs when compared to the reference cell line. Conclusions: In sum, our data demonstrate that lens epithelial cell lines 21EM15, 17EM15 and αTN4 retain the expression of several important lens markers genes. Significantly, these LECs exhibit elevated levels of P-bodies and Stress granules when compared to a non-lens cell line in both normal and stress conditions, indicating their suitability for investigating the function of these cytosolic ribonucleoprotein complexes in lens cells. Commercial Relationships: Anne Terrell, None; Stephanie Waters, None; Christine Dang, None; David C. Beebe, None; Salil A. Lachke, None Support: NIH/NEI R01 EY021505 Program Number: 744 Poster Board Number: C0287 Presentation Time: 1:30 PM–3:15 PM Substructure of the germinative zone of the mouse lens Steven Bassnett, Yanrong Shi. Ophthal & Vis Science, Washington Univ Sch of Med, Saint Louis, MO. Purpose: The germinative zone (GZ) of the lens epithelium contains the mitotically-active cell population and serves as the growth engine of the lens. Despite its key role, the GZ is geographically ill-defined. As part of ongoing efforts to model lens growth, we analyzed cell proliferation patterns within the GZ across the mouse life span.


ARVO 2014 Annual Meeting Abstracts Methods: Mice (2 weeks to 46 months of age) were given an IP injection of EdU to label S-phase cells. 1 hr after EdU administration mice were euthanized by CO2 inhalation and whole lenses were processed for confocal microscopy. The polar and equatorial regions of the lens were imaged in three dimensions. Maximum intensity projections were generated from stacks of optical sections and analyzed by computer-assisted morphometry. Age-dependent changes in epithelial cell population and age/latitude-dependent variations in cell size and mitotic index were computed. Results: Over the age range studied here, equatorial and polar diameters of the mouse lens increased by >40%. Despite a concomitant increase in lens surface area, the number of epithelial cells decreased from approximately 50,000 at 2 weeks to 40,000 at six months. This decline was paralleled by an increase in the surface area of individual cells that was first evident in the central epithelium and subsequently spread towards the equator. Analysis of mitotic index within the GZ revealed a decline in peak index from 7% at 2 weeks to