ARVO 2014 Annual Meeting Abstracts
113 Retina/RPE: Biochemistry and Molecular Biology
Sunday, May 04, 2014 8:30 AM–10:15 AM
Exhibit/Poster Hall SA Poster Session
Program #/Board # Range: 384–429/C0155–C0200
Organizing Section: Biochemistry/Molecular Biology
Contributing Section(s): Retina
Program Number: 384 Poster Board Number: C0155
Presentation Time: 8:30 AM–10:15 AM
Intracellular localisation of human bestrophin in neuronal cells
after transduction with an AAV2.CBA.hBEST1.WPRE vector
Shaun Wood, Michelle McClements, Robert E. MacLaren. Nuffield
Laboratory of Ophthalmology, The University of Oxford, Oxford,
United Kingdom.
Purpose: Mutations in the human BEST1 gene are responsible
for a wide-range of retinal disorders broadly classified as
Bestrophinopathies. The BEST1 protein is known to be expressed
in the retinal pigment epithelium (RPE) where it localises to the
membrane and acts as a Cl- channel. Delivery of hBEST1 to the RPE
cells of the retina is a potential treatment option for patients with
bestrophinopathy. Potential expression of BEST1 in other cell types
of the retina however could be problematic, particularly in neurons
where a channel protein could influence function or even trigger
apoptosis. We therefore sought to identify the membrane localisation
patern of hBEST1 when expressed ectopically from a powerful
expression cassette used in a current clinical trial - AAV2 CAG.
hBEST1.WPRE.pA in HEK293T cells in vitro.
Methods: Human BEST1, driven by the ubiquitous CAG promoter
and with a downstream WPRE sequence, was packaged into
AAV2 capsids and used for in vitro assessment in HEK 293T
cells. Commercially available anti-BEST1 antibodies were tested
for their ability to detect the human BEST1 protein via western
blot. Immunocytochemical (ICC) staining was also performed
to determine localisation of BEST1 within the HEK293T cells.
Histological sections of wild-type mouse eyes were prepared and
immunostained to determine native BEST1 expression throughout the
different layers of the retina using the human-specific antibody.
Results: Western blots using transfected and transduced HEK293T
lysates detected large amounts of hBEST1 protein at the predicted
size of 67 kDa, indicating successful overexpression from the plasmid
and AAV2 vectors respectively. ICC on the HEK293T cells however
showed BEST1 expression in the cytosol with little localisation at the
cell membranes. Staining of the wild type mouse eyes indicated some
expression of BEST1 in the RPE confirming broad species specificity
of the antibodies.
Conclusions: An AAV2 vector carrying CAG.hBEST1 has been
generated and successfully used to express hBEST1 in vitro.
Although initial tests suggest that the localisation of the protein
would render it non-functional in this cell line, further functional tests
will be required to confirm this.
Commercial Relationships: Shaun Wood, None; Michelle
McClements, None; Robert E. MacLaren, None
Program Number: 385 Poster Board Number: C0156
Presentation Time: 8:30 AM–10:15 AM
The effects of SIRT1 on hypoxia induced by cobalt chloride in
human fetal retinal pigment epithelial cells
Huiming Zhang1, 2, Shikun He1, Christine Spee1, David R. Hinton1.
1
Pathology and Ophthalmology, Keck school of medicine at
university southern California, Los Angeles, CA; 2Ophthalmology,
Second Xiangya Hospitol, Central South University, Changsha,
China.
Purpose: SIRT1, a NAD+ dependent histone deacetylase, has been
shown to act as a key regulator of angiogenesis. Hypoxia is a critical
pathological factor in new vessel growth in wet AMD. The current
study was to investigate the role of SIRT1 on a mimic hypoxic
condition in human fetal retinal pigment epithelial (fRPE) cells.
Methods: Human fRPE cells (passage2-4) were incubated in DMED
supplemented with 10% fetal bovine serum at 37°C with 5% CO2.
Addition of cobalt chloride (CoCl2) to DMEM was used to mimic
hypoxic condition. Cell viability was accessed after exposing fRPE
cells to different concentrations of CoCl2 (0, 50, 100, 200, 500,
1000uM) for 24 hours using PrestoBlue™ reagent. The fRPE cells
were transfected using specific SIRT1 siRNA (10nM) following by
CoCl2 (100uM) treatment for 24 hours or pretreating the cells with
resveratrol (SIRT1 activator) (50uM) for 16 hours before CoCl2
treatment. At the end point, the HIF-1a protein accumulation in
the cell lysate was analyzed by western blotting and ELISA were
performed to detect the secreted VEGF protein in the supernatant.
Results: HIF-1a protein expression was increased in a dosedependent manner in CoCl2-treated fRPE cells which reached
a maximum at 100uM CoCl2. Loss of SIRT1 by specific SIRT1
siRNA transfection even further augmented this increase of 1.5-fold
(p<0.05). Treatment of rRPE cells with resveratrol inhibited HIF-1a
accumulation in hypoxia significantly (p<0.01) and this inhibition
effect attenuated by knocking down of SIRT1. Furthermore, VEGF
secreted in the supernatant was up-regulated by 1.5-fold after
incubating fRPE cells with CoCl2 and even further increased with
additionally adding SIRT1 siRNA. While using resveratrol downregulated the VEGF secretion by 2-fold in the supernatant in hypoxia.
This down-regulation effect was also reversed by SIRT1 siRNA.
Conclusions: SIRT 1 and its activator are able to inhibit HIF-1a/
VEGF pathway under a mimic hypoxia condition. The results suggest
that target SIRT1 could be a therapeutic potential for the treatment of
wet AMD.
Commercial Relationships: Huiming Zhang, None; Shikun He,
None; Christine Spee, None; David R. Hinton, None
Support: NIH grants EY01545 and EY03040, RPB, Arnold & Mabel
Beckman Foundation
Program Number: 386 Poster Board Number: C0157
Presentation Time: 8:30 AM–10:15 AM
Inhibition of Rod Outer Segment ectopic FoF1-ATP synthase by
polyphenolic phytochemicals: new Insights on Oxidative Stressrelated Retinopathies
Isabella Panfoli1, Daniela Calzia1, Federico Caicci2, Lucia Manni2,
Paolo Degan3, Silvia Ravera1, Martina Bartolucci1, Paola Ramoino4,
Carlo E. Traverso5. 1DIFAR, University of Genova, Genova,
Italy; 2Biology Department, University of Padova, Padova, Italy;
3
Molecular Mutagenesis and DNA Repair U.O., IRCCS AOU San
Martino – IST, Genova, Italy; 4DISTAV, University of Genova,
Genova, Italy; 5Clinica Oculistica-DINOGMI, University of Genova,
Genova, Italy.
Purpose: The effect of natural polyphenolic phytochemicals,
known antioxidants, was studied on the ectopic ATPase/ATP
synthase activity of the retinal rod Outer Segments (OS). The OS is
a specialized organelle capable of phototransduction. Our previous
studies on purified bovine OS disks reported an extramitochondrial
oxidative phosphorylation (OXPHOS) therein [1]. Notably, OXPHOS
can become a major source of oxidative stress, a common cause of
retinal pathologies.
Methods: The rod OS ATPase/ATP synthase activities were
investigated by luminometry and oxymetry on OS homogenates. The
expression of OXPHOS proteins in the OS was studied by electron
©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 pubs@arvo.org.
ARVO 2014 Annual Meeting Abstracts
transmission and fluorescence confocal microscopy on bovine retinal
sections.
Results: Resveratrol, a stilbene phytoalexin present in grapes and red
wine, and curcumin, a component of turmeric, inhibited ATP synthase
activity by 90% and 20%, respectively. Co-administration of piperine,
a major alkaloid of black pepper, with curcumin enhanced ATP
synthesis inhibition (up to 56%). Piperine alone displayed no effect.
ATPase activity, tested in purified OS in the presence of ouabain,
inhibitor of Na+/K+ATPase, was inhibited by epigallocatechin
gallate, a catechin found in green tea, and quercetin, by 52% and 54%
respectively.
Conclusions: The effect of polyphenols on the OS ATPase/ATP
synthase activity suggests that these natural antioxidant substances
significantly interact with the OS ATP synthase. Multiple lines of
evidence indicate oxidative stress as a common source of retinal
pathologies such as retinitis pigmentosa and age related macular
degeneration, and glaucoma. Therefore, neuroprotective treatments
with these polyphenols can be regarded under new light [2].
references
[1] I.. Panfoli et al. Int J Biochem Cell Biol. 2009; 41, 2555-65.
[2] I. Panfoli . et al Med Hypotheses 2012, 78, 423-7
Commercial Relationships: Isabella Panfoli, None; Daniela
Calzia, None; Federico Caicci, None; Lucia Manni, None; Paolo
Degan, None; Silvia Ravera, None; Martina Bartolucci, None;
Paola Ramoino, None; Carlo E. Traverso, None
Support: MIUR_ Italy
Program Number: 387 Poster Board Number: C0158
Presentation Time: 8:30 AM–10:15 AM
Apoptosis-induced compensatory proliferation in the UVirradiated human conjunctival epithelium cells
Eiji Tomoyori1, 2, Yuko Udaka1, Mayumi Tsuji1, Akiko Sasaki1,
Junichiro Kizaki1, 2, Katsuji Oguchi1. 1Pharmacology, Showa
University School of Medicine, Tokyo, Japan; 2Ophthalmology,
Showa University School of Medicine, Tokyo, Japan.
Purpose: Pterygium is a common eye disease characterized by the
degeneration of fibrous connective tissue of the ocular surface with
epithelial hyperplasia. Pterygum is usually not troublesome unless
it grows large enough to cover cornea to interfere with vision. The
exact cause of pterygium is not known. Possible causes of pterygium
include too much exposure to ultraviolet (UV) light that can lead to
these growths. UV irradiation generates the reactive oxygen species
(ROS) and activates c-jun N- terminal kinase in the cells, both of
which are known to induce apoptosis.
We hypothesized that UV-induced apoptosis may trigger
compensatory proliferation in surrounding cells to maintain tissue
homeostasis. Therefore, we investigated into how activation of
apoptosis is linked to the process of compensatory proliferation in the
UV-irradiated human conjunctival epithelial (HCE) cells.
Methods: In this study, HCE cells were irradiated under UV (312
nm)-emitting lamps (4.94 mW/cm2, 100 mJ/cm2). After irradiation,
HCE cells were analyzed to determine cell viability, apoptotic cells
and cell cycle by Muse™ Cell Analyzer. We used CM-H2DCFDA, a
cell-permeant indicator for ROS for the detection of ROS generated
by UV irradiation. To determine compensatory proliferation,
interleukin-11 (IL-11) was determined using an ELISA kit. To
investigate the distribution of β-catenin in nuclei, β-catenin content
in nuclear fraction was detected by ELISA and immunostaining for
β-catenin was performed.
Results: UV-irradiated cells that underwent apoptosis generated
significantly higher levels of ROS, IL-11 and β-catenin after
irradiation, compared with non-irradiated cells. After UV irradiation,
the percentages of cells occupying the different phases of the cell
cycle changed: cells in S and G2/M phases increased rather than cells
in G0/G1 phase that are expected to result from apoptosis. In UVirradiated cells, nuclear translocation of β-catenin levels increased.
Staining for β-catenin showed negative results in non-irradiated cells,
while high-level nuclear β-catenin staining was observed in UVirradiated HCE cells.
Conclusions: These findings suggested that UV-induced apoptotic
cells may have stimulated the compensatory proliferation of
surrounding healthy cells to maintain homeostasis.
Commercial Relationships: Eiji Tomoyori, None; Yuko Udaka,
None; Mayumi Tsuji, None; Akiko Sasaki, None; Junichiro
Kizaki, None; Katsuji Oguchi, None
Program Number: 388 Poster Board Number: C0159
Presentation Time: 8:30 AM–10:15 AM
3-hydroxykynurenin concentration changes in retina after partial
optic nerve crush and NMDA-induced RGC damage in rat
Tomasz Choragiewicz1, 2, Sebastian Thaler2, Frank Schuettauf2,
Kamila Wertejuk1, Dominika Nowakowska1, Michael J. Koss3, Tomasz
Kocki4, Anselm G. Junemann5, Waldemar A. Turski4, Robert Rejdak1,
6 1
. Department of General Ophthalmology, Medical University of
Lublin, Lublin, Poland; 2Centre for Ophthalmology, University
of Tuebingen, Tuebingen, Germany; 3University Eye Clinic, J W
Goethe-Universitaet FFM, Frankfurt/Main, Germany; 4Deptartment
of Clinical Pharmacology and Toxicology, Medical University of
Lublin, Lublin, Poland; 5Department of Ophthalmology, University
of Erlangen-Nuernberg, Erlangen, Germany; 6Department of
Experimental Pharmacology, PAS Medical Research Centre, Warsaw,
Poland.
Purpose: To investigate concentration of 3-hydroxykynurenin (3OHK) in retinas in a rat model of partial optic nerve crush (PONC) and
NMDA-induced damage of rat retinal ganglion cells (RGCs)
Methods: Female Brown Norway rats (Charles River, Wilmington,
MA), body weight 140-170g were housed under a 12-hour lightdark cycle. The animals were treated in accordance with the ARVO
Statement for the Use of Animals in Vision Research. The rats were
anesthetized with an intraperitoneal injection of chloral hydrate (7%,
6ml/kg body weight), local anaesthesia in form of eye drops were
also applied. One group of animals received intravitreal NMDA
(2ml, 10mM). Control eye received PBS (2ml). In the other group of
animals PONC or sham procedure in control eye were performed.
On day 2 and 7 eyes were enucleated, retinas dissected and
immediately deep frozen. 3OH-K concentration was measured with
HPLC.
Results: Retinal concentration of 3OH-K after 2 days was
significantly higher in retinas 2 days after both NMDA injection and
PONC (27.4% and 40% increases respectively, P<0.05, U MannWhitney). After 7 days 3OH-K concentration was significantly higher
in retinas after NMDA injection (217% increase, P<0.01) but not
after PONC (21% increase, P>0.05).
Conclusions: Retinal concentration of 3OH-K increases in response
both to PONC and NMDA-induced RGC damage in rat, suggesting
its involvement in RGC degenerative processes.
Commercial Relationships: Tomasz Choragiewicz, None;
Sebastian Thaler, None; Frank Schuettauf, None; Kamila
Wertejuk, None; Dominika Nowakowska, None; Michael J.
Koss, None; Tomasz Kocki, None; Anselm G. Junemann, None;
Waldemar A. Turski, None; Robert Rejdak, None
Support: Allergan European Retina Panel
©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 pubs@arvo.org.
ARVO 2014 Annual Meeting Abstracts
Program Number: 389 Poster Board Number: C0160
Presentation Time: 8:30 AM–10:15 AM
Possibility to use human recombinant hyaluronidase as adjuvant
for chemical vitrectomy on porcine vitreous
Koichi Nishitsuka, Mari Narumi, Hidetoshi Yamashita.
Ophthalmology/Vis Sci, Yamagata University Sch of Med, Yamagatashi, Japan.
Purpose: Vitreous plays important roles in the pathogenesis of
vitreo-retinal diseases including proliferative diabetic retinopathy,
proliferative vitreoretinopathy and others. Hyaluronan (HA) is
contained in vitreous and this affects the pathogenesis of those
diseases including fibrovascular membrane formation.To remove
the formed vitreous gel is useful to treat those proliferative diseases
by vitrectomy. Hyaluronidase is one of the vitreolytic substances
to hydrolize HA, and may be applied to the chemical vitrectomy.
To clarify the clinical significance of the degradation of HA, it is
necessary to clarify the correlation between the vitreous status and
HA. In this research project, the degradation process of HA by
hyaluronidase was investigate in the in vitro system.
Methods: Fifty porcine eyes of 2 year old of age were obtained from
a slaughterhouse. In all the experiments, we treated right eyes, and
used left eyes as controls. The vitreous was obtained from eye balls
using the 25-gauge vitrectomy system. Whole vitreous body was
dissected carefully from corneal open sky incision with compression
the eyeball. To investigate the degradation of HA by the human
recombinant hyaluronidase, we measured the HA concentration in
the vitreous specimen (200μl) treated with 1.5 Unit of Hilenex®
(human recombinant hyaluronidase) by ELISA. To observe the effect
of the human recombinant hyaluronidase on the vitreous structure, we
measured the volume of the whole porcine vitreous treated with 15
Unit of Hilenex®.
Results: The mean HA concentration of the vitreous gel after
treatment by the human recombinant hyaluronidase was lower than
control (5.74 vs 142.5 ng/ml, respectively, Mann-Whitney U test
p<0.001). The mean volume of the whole vitreous after treatment
with hyaluronidase was lower the control (1.79 vs 2.45 gram,
respectively, Mann-Whitney U test p<0.001). Human recombinant
hyaluronidase could significantly degradate the HA in the vitreous
specimens and decrease the volume of the whole vitreous.
Conclusions: Human recombinant hyaluronidase could destroy
the vitreous gel structure by the degradation of HA in the porcine
vitreous. Hyaluronidase and/or similar agents can be used as an
adjuvant for the chemical vitrectomy.
Commercial Relationships: Koichi Nishitsuka, None; Mari
Narumi, None; Hidetoshi Yamashita, None
Program Number: 390 Poster Board Number: C0161
Presentation Time: 8:30 AM–10:15 AM
Proteomic Profile of Plasma and Mucosal Samples from Patients
with Stevens-Johnson Syndrome/Toxic Epidermal Necrolysis
Christine M. Mata1, Julia Malalis1, Daneyal Syed2, Daniel Kahn2,
Michael Mosier3, Charles S. Bouchard1, Josephine Cunanan2, Debra
Hoppensteadt2, Jawed Fareed2, Omer Iqbal2. 1Ophthalmology,
Loyola University Medical Center, Maywood, IL; 2Pathology,
Loyola University Medical Center, Maywood, IL; 3Surgery, Loyola
University Medical Center, Maywood, IL.
Purpose: Stevens-Johnson syndrome/toxic epidermal necrolysis
(SJS/TEN) are life-threatening, immune-complex hypersensitivity
adverse drug reactions that affect the skin and mucous membranes,
often resulting in significant ocular inflammation and systemic
morbidity. The aim of this study was to compare the proteomic and
coagulation profiles of sequential mucosal (ocular and oral) and
plasma samples from patients with biopsy-confirmed SJS/TENS
patients (5) and patients with non-SJS/TENS adverse drug reactions
(ADR) (10).
Methods: Multiple plasma samples and swabs from ocular, oral,
and skin lesions were obtained from patients with SJS/TEN (n=5).
Samples from normal healthy controls were also obtained. The
plasma was assayed for thrombin-antithrombin complex (TAT),
prothrombin fragment F1.2, plasminogen activator inhibitor-1
(PAI-2), ZYMUPHEN platelet microparticle activity, HEMOCLOT
protein C, and STACHROME antithrombin, using ELISA kits as per
manufacturer’s instructions. The discharges were isolated following
addition of 0.25 ml of saline to each swab and double centrifugation.
The discharges and plasma samples were analyzed using SELDI-TOF
technique.
Results: Analyses of the SJS/TEN plasma samples revealed a marked
increase in the TAT complexes (6.3±5.9mg/ml), F1.2 (430.4±202.4
pmol/L), platelet microparticles (13.1±9.3nM) and protein C levels
(90.5±63.4 %), with a corresponding decrease in PAI-1 (53.3±18.8ng/
ml) and antithrombin levels (80.7±42.4 %) compared to normal
healthy control plasma, suggesting a procoagulant state. Protein chip
array of the SJS/TEN skin and oral mucosal samples exhibited two
major peaks at 14.2 kDa and 15.6 kDa, in the same molecular weight
range as recombinant human granulysin, a molecule implicated in the
pathophysiology of SJS/TEN. Additional peaks at 11.7, 13.3 and 14.5
kDa were observed to be upregulated in SJS/TEN patients. These
peaks were not present in the normal control group.
Conclusions: Procoagulant factors and unique peaks suggestive of
granulysin may lead to the development of targeted therapy aimed to
attenuate local and systemic inflammatory processes in patients with
SJS/TEN.
Commercial Relationships: Christine M. Mata, None; Julia
Malalis, None; Daneyal Syed, None; Daniel Kahn, None; Michael
Mosier, None; Charles S. Bouchard, None; Josephine Cunanan,
None; Debra Hoppensteadt, None; Jawed Fareed, None; Omer
Iqbal, None
Support: Perritt Foundation, Illinois Society for the Prevention of
Blindness
Program Number: 391 Poster Board Number: C0162
Presentation Time: 8:30 AM–10:15 AM
Secreted Phospholipids of the Organ Cultured Cornea, Iris and
Lens
Yousef A. Alghamdi1, 2, Mitchell Martinez1, 2, Faisal Khattab1, Sanjoy
K. Bhattacharya1, 2, Richard K. Lee1, 2. 1Ophthalmology, Bascom
Palmer Eye Institute, Miami, FL; 2University of Miami Miller School
of Medicine, Miami, FL.
Purpose: To compare different subtypes of phospholipids
(phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine,
phosphatidylinositol,) secreted by organ cultured cornea, iris, and
lens from human donors.
Methods: Human donor eyes were collected from the Lions Eye
Bank, Miami, Florida adhering to the Tenets of the Declaration of
Helsinki. Eyes were carefully dissected to isolate lens, cornea and
iris which were incubated in serum free culture medium for up to 72
hours. Secreted lipids in the culture media was collected every 12
hours and stored at -80C until further use. Total collected media after
incubation was subjected to lipid extraction using the Bligh and Dyer
method. All organ cultures were also subjected to an assessment of
trypan blue exclusion cell viability assay and assessment of pyruvate
kinase activity at the end of incubation period. Cultured organs with
less than 90% viability were excluded from further lipid analyses.
Phospholipids were identified and quantified using established class
specific mass spectrometry settings (ion mode and collision energy)
with appropriate class-specific lipid standards on a TSQ Quantum
©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 pubs@arvo.org.
ARVO 2014 Annual Meeting Abstracts
Access Max mass spectrometer. A Triversa Nanomate was used for
direct infusion with optimized parameters.
Results: Organ cultured secretions of cornea, iris and lens
contain all four classes of phospholipids: phosphatidylcholine,
phosphatidylethanolamine, phosphatidylinositol, phosphatidylserine
albeit to varying quantities. In the secretions of these organ cultures,
most notable was the low presence of phosphatidylcholine as a
percent of total phospholipids. On a relative basis, the amount of
secreted lipids were approximately 10 fold less than that assayed
directly from the tissues or isolated cells from these tissues.
Conclusions: All four classes of phospholipids were found in organ
culture secretions of cornea, iris and lens with phosphatidylcholines
being the lowest by relative amounts to other phospholipid classes.
Commercial Relationships: Yousef A. Alghamdi, None; Mitchell
Martinez, None; Faisal Khattab, None; Sanjoy K. Bhattacharya,
None; Richard K. Lee, None
Support: Supported by an unrestricted grant from RPB to University
of Miami, NIH grants R01EY016112 and P30EYEY014801
Program Number: 392 Poster Board Number: C0163
Presentation Time: 8:30 AM–10:15 AM
Proteomic analysis of the interaction Fusarium solaniStaphylococcus aureus isolated from human keratitis in presence
of antimicrobial agents
Antonio Bautista-Hernández1, Juan Pablo Reyes-Grajeda3, Mariana
Ortiz-Casas1, Aída Verónica Rodríguez-Tovar2, Carolina GaonaJuárez1, Nadia Luz López-Espinosa1, Herlinda Mejia-Lopez1, Victor
M. Bautista1. 1Microbiology and Ocular Proteomics, Institute of
Ophthalmology “Conde de Valenciana”, Mexico, Mexico; 2Medical
Micology, Escuela Nacional de Ciencias Biológicas, Instituto
Politécnico Nacional, Mexico, Mexico; 3Medical Proteomics,
Instituto Nacional de Medicina Genómica, Mexico, Mexico.
Purpose: To study the proteomic profile of the coculture of Fusarium
solani-Staphylococcus aureus in presence of antimicrobials agents
and analyze biofilm formation.
Methods: Staphylococcus aureus and Fusarium solani were
isolated from human keratitis. The microbial were characterized
by microbiological and molecular means. The cocultures were
established in Müller-Hinton Agar with or without Gatifloxacin
and/or Itraconazole during 48h. The cocultures containing 48h
biomass were processed to obtain the cellular extract; the proteins
were quantified, precipitated and 2D electrophoresis performed.
The proteomic profiles were analyzed by Dymension 2.0 software
to determine the differential protein expression in the cocultures
compared to cocultures in presence of antimicrobials. The differential
expressed spots were identified by MALDI-TOF. The biofilms
were established in 96 well with RPMI medium, the concentrations
used for S. aureus was 1X105 and F. solani 1X105 for 24h, the
biofilm formation was determined by crystal violet assay in ELISA
microplate reader at 595 nm. Biofilms were pre-formed in coverslips
and stain with DAPI for fluorescence microscopy.
Results: The coculture fungi grown was modified with antimicrobial
treatment, combinated therapy may not showed the same effect as a
single therapy with Itraconazole. Twenty-two proteins were identified
from proteomics profiles: Enolase, Glyceraldehyde-3-phosphate
dehydrogenase, Fructosa-bisphosphate aldolase, Protein disulfideisomerase, Aconitate Hydratase cytoplasmic of F. solani. The biolfims
microscopy fluorescence showed interactions between S. aureus and
hypha of F. solani.
Conclusions: In the coculture F. solani-S. aureus-Itraconazole, F.
solani showed low growth in presence of Itraconazole compared with
coculture F. solani-S. aureus. Coculture F. solani-S. aureus showed
differential protein expression compared to the cocultures with
Itraconazole and/or Gatifloxacin. The biofilm of F. solani-S. aureus
showed interactions of S. aureus with the hypha of F. solani.
Commercial Relationships: Antonio Bautista-Hernández, None;
Juan Pablo Reyes-Grajeda, None; Mariana Ortiz-Casas, None;
Aída Verónica Rodríguez-Tovar, None; Carolina Gaona-Juárez,
None; Nadia Luz López-Espinosa, None; Herlinda Mejia-Lopez,
None; Victor M. Bautista, None
Support: Conde de Valenciana Foundation and CONACYT-126779
Program Number: 393 Poster Board Number: C0164
Presentation Time: 8:30 AM–10:15 AM
iTRAQ-based quantitative proteomic analysis of tear fluid in
rat penetrating keratoplasty model with acute corneal allograft
rejection
Feifei Huang, JianJiang Xu. ophthalmology, EENT hospital of Fudan
University, Shanghai, China.
Purpose: This study aims to develop further understanding of the
mechanisms of acute corneal allograft rejection by identifying tear
proteins at defined stage and to to discover potential interesting
protein of corneal rejection.
Methods: iTRAQ-2DLC-MS/MS technique was used to uncover
tear proteins that were changed in rat penetrating keratoplasty (PKP)
model at different time points. Bioinformatics technology was
applied in the analysis of significant proteins. The interesting protein
was verified by western blotting.
Results: A total of 269 proteins were quantified, and 118 proteins
were considered as significant proteins by at least 2.0-or 0.5fold. For GO annotation, the top enrichments were neurological
disease, free radical scavenging, cell death and survive and cell
movement. For pathway analysis, the top enrichments were LXR/
RXR activation, acute phase response signaling, clathrin-mediated
endocytosis signaling and coagulation system. Coronin-1A was
considered as an interesting protein for early stage of acute corneal
allograft rejection.
Conclusions: This study first demonstrates that tear proteomics is a
powerful tool for further understanding of the mechanisms of acute
corneal rejection, and coronin-1A in tears might be closely related to
allograft rejection.
©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 pubs@arvo.org.
ARVO 2014 Annual Meeting Abstracts
Identification and relative quantification of coronin-1A using
iTRAQ. A, the expression levels of coronin-1A for 7 iTRAQ
labeled samples; B, the sequences of peptide DAGPLLISLK,
which form coronin-1A; C, another identified peptide sequences
with KSDLFQEDLYPPTAGPDPALTAEEWLSGR leading to the
identification of coronin-1A.
Commercial Relationships: Feifei Huang, None; JianJiang Xu,
None
Support: Shanghai Committee of Science and Technology
Foundation (10411962000).
GO annotation and pathway analysis from IPA database. A, top
enrichments of functions of differential expressed proteins in allograft
group; B, top enrichments of functions of differential expressed
proteins in both allograft and autologous groups. C, top enrichments
of pathways of differential expressed proteins in allograft group; D,
top enrichments of pathways of differential expressed proteins in both
allograft and autologous groups.
Program Number: 394 Poster Board Number: C0165
Presentation Time: 8:30 AM–10:15 AM
Proteomic analysis in pterygium
Sun Woong Kim1, Taiyoun Rhim2. 1Ophthalmology, Hando general
hospital, Ansan, Republic of Korea; 2Bioengineering, Hanyang
University, Seoul, Republic of Korea.
Purpose: To identify differentially expressed proteins in pterygium
compared to healthy conjunctiva using a proteomic analysis.
Methods: Pterygial and healthy conjunctival tissues were obtained
from 24 patients undergoing pterygium excision. Total proteins of
pterygia and healthy conjunctiva were analyzed by one-dimensional
electrophoresis, and protein bands of interest were excised and
subjected to LC-MS/MS analysis using Thermo’s Finnigan
ProteomeX workstation LTQ linear IT MS (Thermo Electron, San
Jose, CA) equipped with an NSI source (San Jose, CA). Using
bioinformatics, differentially expressed proteins were classified, and
three proteins closely involved in the response to oxidative stress
were selected for further validation. Differential expression of these
proteins was confirmed by Western blot and immunohistochemistry.
Results: A web-based gene ontology program, DAVID, was
used to classify 230 proteins that were differentially expressed in
pterygial tissues. Among them, we chose three proteins, aldehyde
dehydrogenase, dimeric NADP-preferring (ALDH3A1), protein
disulfide-isomerase A3 (PDIA3) and peroxiredoxin-2 (PRDX2),
that were significantly overexpressed in pterygium and further
overexpressed in recurrent pterygium. Immunohistochemistry and
Western blot analysis confirmed that these three proteins were
mainly detected in the basal epithelial layer, and their expression was
significantly increased in pterygium compared to normal conjunctiva.
©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
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ARVO 2014 Annual Meeting Abstracts
Conclusions: This study reported increased expressions of
ALDH3A1, PDIA3, and PRDX2 in pterygia using a proteomic
approach. These proteins are presumed to have a protective role
against oxidative stress-induced apoptosis. This result is consistent
with the hypothesis that oxidative stress is a significant factor in the
pathogenesis of pterygium.
Representative images of Western blot analysis, showing increased
expression of Aldehyde dehydrogenase, dimeric NADP-preferring
(ALDH3A1), Protein disulfide-isomerase A3 (PDIA3) and
Peroxiredoxin-2 (PRDX2) in pterygium compared to healthy
conjunctiva.
Commercial Relationships: Sun Woong Kim, None; Taiyoun
Rhim, None
Program Number: 395 Poster Board Number: C0166
Presentation Time: 8:30 AM–10:15 AM
Region-Specific Phosphorylations in Age-related macular
degeneration
O’Donnell Sylvester1, Srinivas R. Sripathi2, Folami Lamoke3,
Manuela Bartoli3, Paul S. Bernstein4, Wan Jin Jahng1. 1Petroleum
Chemistry, American University of Nigeria, Yola, Nigeria;
2
Ophthalmology, Johns Hopkins University, Baltimore, MD;
3
Ophthalmology, Georgia Health Sciences University, Augusta, GA;
4
Ophthalmology and Visual Sciences, University of Utah, Salt Lake
City, UT.
Purpose: The current study explores epigenetic control of
gene expression and oxidative responses through modulating
phosphoproteome in the retina and RPE cells. We examined a novel
role of phosphorylation signaling as an adaptation to extracellular
stress and used phosphoproteome alterations to determine early
molecular mechanism of retina/RPE cell death and survival. Our
hypothesis is that decreased transferrin and increased kinase/
phosphatase activity in AMD could be responsible for regulated
apoptotic phosphorylation signaling during AMD progression.
Methods: Phophoproteomes of peripheral retina and RPE were
compared to age-matching control donor eyes to determine
senescence-associated molecular events during AMD progression.
Phosphoproteins were enriched by charged based spin column
chromatography and resolved by 2D gel electrophoresis. Trypsin
digested modified peptides were enriched using Ga3+/TiO2
immobilized metal ion chromatography. Eluted phosphopeptides
were analyzed using MALDI-TOF and ESI MS/MS. Serine,
threonine and tyrosine phosphorylations were confirmed by phosphowestern blotting analysis.
Results: Our study demonstrated the down-regulation of transferrin
in peripheral retina of AMD donor eyes compared to control.
Phospho-Western blotting analysis revealed the phosphorylation
of intermediate filament vimentin (Ser), Glial fibrillary acidic like
protein and mitochondrial heat shock protein (mtHsp70). Protein
database analysis shows the phosphorylation of transferrin in AMD
donor eyes. Phosphorylation networks in the macular, peripheral
retina, central RPE, and peripheral RPE provide the region-specific
phosphorylation reactions involved in the early progression of AMD.
Conclusions: Our study identifies phosphoprotein changes through
the activation of receptor tyrosine kinases. Our data suggest
that a positive correlation exists between early biomarkers of
phosphoproteome under oxidative stress in vitro and retina/RPE
proteins from AMD patients. The outcome of our work will be the
initial delineation of the underlying physiology of oxidative stressmediated phosphorylation signaling in the retina/RPE apoptosis.
In addition, our study will provide a stimulus for understanding
oxidative stress-induced cytoskeletal changes and the aggregate
formation mechanism by phosphorylations. As a consequence,
an effective therapeutic approach based on the modulation of
phosphorylations is expected to result.
Commercial Relationships: O’Donnell Sylvester, None; Srinivas
R. Sripathi, None; Folami Lamoke, None; Manuela Bartoli, None;
Paul S. Bernstein, None; Wan Jin Jahng, None
Support: Africa Foundation
Immunolocalization of Aldehyde dehydrogenase, dimeric NADPpreferring (ALDH3A1), Protein disulfide-isomerase A3 (PDIA3), and
Peroxiredoxin-2 (PRDX2) in pterygium.
©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
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ARVO 2014 Annual Meeting Abstracts
Program Number: 396 Poster Board Number: C0167
Presentation Time: 8:30 AM–10:15 AM
Mouse embryonic fibroblast derived from Ccp110 knockout
mouse leads to aberrant elongation and defective segregation of
centrioles and failed to produce cilia
Sharda P. Yadav1, Lijin Dong2, Anand Swaroop1. 1N-NRL, National
Eye Institute, National Institutes of Health, Bethesda, MD; 2Genetic
Engineering Core, National Eye Institute, National Institutes of
Health, Bethesda, MD.
Purpose: In most of the mitotic animal cells centrosome duplication
occurs at G2-M phase and defect in it accompanied by extensive
chromosomal abnormalities. Purpose of this study to understand
the mechanism of centrosome biogenesis using mouse embryonic
fibroblast derived from Centrosomal coiled coil protein 110 (Ccp110)
knockout mouse.
Methods: Ccp110 knockout mouse were generated and mouse
embryonic fibroblasts (MEFs) were derived from wild type
heterozygous and Ccp110 knockout E 12.5 embryos. Cilia were
developed by serum starving the cultures cells and looked for
different cilia markers. Centrosomal abnormalities were looked
by immunohistochemistry with specific centriole markers. FACS
analysis of the MEFs was performed for cell cycle analysis after
staining of the DNA with propidium iodide.
Results: Staining of the cilia with acetylated tubulin and ARL13b
suggested that almost 90 % of the Ccp110 knockout MEFs failed to
develop cilia. Ccp110 heterozygous MEFs were able to developed
cilia normally as wild type. Suggesting that one copy of the Ccp110
is sufficient for normal development of cilia in MEFs. Analysis of the
centrioles by staining with γ-tubulin revealed that a subset of Ccp110
mutant MEFs failed to segregate newly formed centrioles and
elongated in size compared to wild type MEFs. Further staining of
the centrioles with Ninein showed a weak staining in Ccp110 mutant
MEFs compared to wild type cells suggesting normal development
of the sub distal appendages. Cell cycle analysis suggested a defect
in the G2-M phase with accumulation of approximately 40% of
polyploidy (8N) cells in Ccp110 mutant MEFs compared to wild
type. Our results suggested a critical role of Ccp110 in regulations of
centriole length and cilia formation in MEFs.
Conclusions: We have successfully generated a Ccp110 knockout
mouse. Mouse embryonic fibroblast derived from Ccp110 knockout
failed to develop cilia, probably due to aberrant segregation of
daughter centrioles, which resulted in the accumulation of G2-M
phase cells with increased polyploidy. Further analysis of the mutant
centrioles with transmission electron microscopy (TEM) is under
progress and will allow us to further understand the ultra structural
defects in it compared to wild type centrioles.
Commercial Relationships: Sharda P. Yadav, None; Lijin Dong,
None; Anand Swaroop, None
Support: NEI Intramural Research Grant
Program Number: 397 Poster Board Number: C0168
Presentation Time: 8:30 AM–10:15 AM
Novel method for quantifying loss of retinal ganglion cells in mice
Micah A. Chrenek, Jana Sellers, Stephanie L. Foster, P M. Iuvone,
Jeffrey H. Boatright. Ophthalmology, Emory University, Atlanta, GA.
Purpose: Retinal ganglion cells (RGC) are damaged in glaucoma
and other optic neuropathies such as traumatic blast injury. Current
methods used to quantify RGC loss are staining RGCs in flatmounts,
imaging and counting the number of surviving cells. This is a
labor intensive and time consuming method. We hypothesized that
a microplate reader assay could quickly quantify the number of
surviving RGCs using either immunostains for RGC specific markers
or mice that express cyan fluorescent protein (CFP) under a Thy1
promoter.
Methods: Thy1-CFP and control (background) C57BL/6J mice
were obtained from Jackson Laboratories. Optic nerve crush was
performed unilaterally in each mouse with the fellow eye serving
as a control. Two weeks after crush, retinas from THY1-CFP mice
were individually homogenized in RIPA buffer with protease
inhibitors and homogenates plated into a black 96-well plate. Average
fluorescence was measured using a fluorescent microplate reader
(Synergy H1 model, BioTek). Retinal flatmounts of C57BL/6J
mice were immunostained for an RGC-specific antigen (Brn3A)
and immunopositive cells counted. After counting, the retinas were
homogenized and fluorescence was measured using the plate reader
to allow direct comparison to counts.
Results: For Thy1-CFP retinas, average fluorescence readings (±
SEM) for uncrushed and crushed retinas were 39095 ± 1507 and
23062 ± 2975 fluorescence units. This corresponds to a 41% decrease
in RGC signal in the eyes that received the optic nerve crush as
compared to control eyes. A similar decrease of 41% was found in
RGC-specific immunopositive cell counts of C57BL/6J flatmounted
retinas. Lysates of the immunostained retinas showed a 44% decrease
in fluorescence readings following crush.
Conclusions: Given these comparable outcomes, we conclude that
the fluorescent plate-reader protocol provides the same quantitative
sensitivity and accuracy as conventional cell counting methods but
with considerably less time and effort, as imaging and cell counting
is avoided. This method could be used as a quick, informative
measurement of the progression of RGC loss following optic nerve
injury.
Commercial Relationships: Micah A. Chrenek, None; Jana
Sellers, None; Stephanie L. Foster, None; P M. Iuvone, None;
Jeffrey H. Boatright, None
Support: USAMRAA DoD W81XWH-12-1-0436, NIH
R01EY14026, NEI P30EY06360, Research to Prevent Blindness
(RPB) and the Abraham and Phyllis Katz Foundation.
Program Number: 398 Poster Board Number: C0169
Presentation Time: 8:30 AM–10:15 AM
Characterization of Autophagy-linked FYVE (Alfy) Protein in
the Retina
Yuhong Wang1, Ammaji Rajala1, Michelle Ranjo-Bishop1, Robert
E. Anderson1, 2, Raju V. Rajala1, 3. 1Ophthalmology, University of
Oklahoma Health Sciences Center, Oklahoma City, OK; 2Cell
Biology, University of Oklahoma Health Sciences Center, Oklahoma
City, OK; 3Cell Biology/Physiology, University of Oklahoma Health
Sciences Center, Oklahoma City, OK.
Purpose: In the central nervous system, autophagy is a normal
cellular process that prevents the formation of protein deposits
from large protein aggregates that are inefficiently degraded by
proteosomes. Autophagy-linked FYVE protein (Alfy) is a recently
discovered, ubiquitously expressed protein that has an FYVE domain
that binds to phosphoinositide 3-kinase (PI3K) -generated PI-3-P with
high affinity. Overexpression of the C-terminal fragment of Alfy has
been shown to increase aggregate clearance and neuroprotection in a
Drosophila eye model of polyglutamine toxicity clearance. There are
no studies available on the expression and role of Alfy in the retina,
so we characterized Alfy in this study.
Methods: Localization of Alfy was examined by
immunohistochemistry performed on wild type and cone-dominant
Nrl-/- retinal sections. The C-terminal fragments of Alfy and PI-3-P
binding mutant were tested both in vitro (mammalian cells) and in
vivo (lipoplex-mediated subretinal injection into mice). The outer
nuclear thickness was used as a function of rod photoreceptor
©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
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ARVO 2014 Annual Meeting Abstracts
degeneration, and cone cell death was assessed by peanut agglutinin
and cone-transducin labeling.
Results: We found that Alfy was expressed in inner segments and
the outer and inner plexiform layers, in both rod- and cone-dominant
retina. The expression of Alfy-WT in HEK-293T cells did not change
the morphology of the cells. However, the PI-3-P-binding mutant
induced autophagosome-like vacuoles and increased cell death. We
also found increased rod and cone cell death in mice injected with
PI-3-P-binding mutant, but not in WT-injected mice.
Conclusions: Our study shows that Alfy is expressed in the retina.
Our research also suggests that the interaction between PI3Kgenerated PI-3-P and the FYVE domain of Alfy is indispensable for
normal functions of autophagy in the retina.
Commercial Relationships: Yuhong Wang, None; Ammaji Rajala,
None; Michelle Ranjo-Bishop, None; Robert E. Anderson, None;
Raju V. Rajala, None
Support: NIH/NEI grant (EY016507, EY00871)
Program Number: 399 Poster Board Number: C0170
Presentation Time: 8:30 AM–10:15 AM
Quantification and comparison of VEGF-B in the vitreous of
patients with diabetic ocular disease and a control group of
patients with non-diabetic ocular disease
Joana Mesquita1, João Paulo Castro Sousa1, 2, Ana S. Rocha1,
Fatima Santos1, Joao Monteiro1, Luís Passarinha1, Cândida Tomaz1.
1
Biochemistry, Centro de Investigação em Ciências da Saúde,
Universidade da Beira Interior, Covilha, Portugal; 2Ophthalmology,
Centro Hospitalar de Leiria-Pombal, Leiria, Portugal.
Purpose: Vascular endothelial growth factor (VEGF) plays a major
role in pathological angiogenesis and in retinal diseases. Evidence
shows that there are increased levels of VEGF-A in the retina and
in the vitreous of patients with retinal diseases, particularly in
diabetic patients. Although many studies have been focused in VEGF
family, VEGF-B remains poorly studied and its function is the most
controversial of the VEGF family. VEGF-B may act as a survival
factor under certain conditions or may act as a growth inhibiting
factor. However there is a gap to know how VEGF-B is expressed in
human eyes and how it is altered in the presence of certain diseases
such as diabetes. The aim of the present study was to quantify and
compare the VEGF-B in the human vitreous of diabetic and nondiabetic patients with ocular disease.
Methods: 33 samples of human vitreous collected at the beginning of
the pars plana vitrectomy from 33 eyes were analyzed and quantified
by ELISA assays. Results obtained of quantified VEGF-B (pg/L)
were analyzed and compared between a group of diabetic patients
with ocular disease (proliferative diabetic retinopathy (PDR)/
epimacular membrane secondary to diabetic maculopathy (ERM))
and a control group of non-diabetic patients with ocular disease
(rhegmatogenous retinal detachment). All patients included in this
study, which adhered to the tenets of the Declaration of Helsinki,
gave their informed consent to surgical treatment.
Results: The mean VEGF–B concentrations in vitreous were
significantly higher in the diabetic patients group (18.82 pg/L
±1.44) vs control group of non-diabetic patients (17.90 pg/L ± 0.32)
(p=0.006).
No statistically significant difference in the mean vitreous
concentrations of VEGF-B was found between the PDR and ERM
group (p=0.449).
Conclusions: The results of the quantification of VEGF-B in
vitreous by the used method showed a statistical significant increase
in diabetic ocular diseases when compared to non-diabetic ocular
diseases. This study shows that VEGF-B may be overexpressed in
vitreous of diabetic patients.
Commercial Relationships: Joana Mesquita, Novartis (E); João
Paulo Castro Sousa, None; Ana S. Rocha, None; Fatima Santos,
None; Joao Monteiro, None; Luís Passarinha, None; Cândida
Tomaz, None
Program Number: 400 Poster Board Number: C0171
Presentation Time: 8:30 AM–10:15 AM
The role of Decorin in ocular ageing and disease
Felicity de Cogan, Jenna ONeill, Richard J. Blanch, Robert A H
Scott, Ann Logan. Clinical and Experimental Medicine, University of
Birmingham, Birmingham, United Kingdom.
Purpose: Pro-fibrotic TGF-β isoforms initiate intraocular scarring
after surgery and also are implied in the aetiology of ocular
hypertension (OHT) and primary open angle glaucoma (POAG),
diseases that are associated with ocular aging. The intravitreal
proteoglycan Decorin has important physiological roles in dampening
cytokine responses in the retina and sequesters TGF-β to regulate its
bioactivity. In this study we investigated the relationship of Decorin
and TGF-β bioactivity within the eye. Our hypothesis is that loss of
Decorin during ageing leads to increased TGF-β bioactivity, which
increases the risk of subsequent OHT/POAG and proliferative
vitreoretinopathy (PVR) after retinal detachment.
Methods: Vitreal samples were collected from patients presenting
with rhegmatogenous retinal detachments, macula holes and ocular
trauma. Patients were excluded if they were under 10 years of age
and had had previous retinal surgery. The vitreous was collected
during surgical vitrectomy and centrifuged, the supernatant frozen
at -80 °C and subsequently analysed by ELISA for the presence of
active TGF-β2, latent TGF-β2 and Decorin.
Results: Intravitreal Decorin significantly decreased with patient age.
Patients under 40 years had a Decorin concentration of 1729±295 pg/
mL, which reduced to 321±204 pg/mL by the age of 50, with levels
remaining low for all patients above this age. TGF-β2 exists in 2
forms, active and latent, in its active form TGF-β2 is highly bioactive
and is linked to fibrosis and ocular degeneration. Although the levels
of total TGF-β2 didn’t change between age groups, the ratio of active
TGF-β2 to total TGF-β2 was significantly enhanced with increasing
patient age. Hence, patients under 40 years had 45.07±2.81 % of
the TGF-β2 in the active form whereas patients above 50 years had
86.68±3.2 % of the TGF-β2 in the active form. The increased ratio of
active TGF-β2 to latent TGF-β2 levels were inversely correlated with
the decrease in Decorin in the vitreous of patients.
©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
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ARVO 2014 Annual Meeting Abstracts
Conclusions: Intravitreal Decorin levels decline with age irrespective
of underlying pathology. This decrease is correlated with an
increase in levels of bioactive TGF-β2. The results imply a causal
link between Decorin decline with age, TGF-β2 activation and the
aetiology of fibrotic pathologies. Targeted supplementation of the
eyes of older patients with Decorin may reduce age related diseases
associated with intraocular fibrosis like OHT/POAG and post-retinal
detachment PVR.
Commercial Relationships: Felicity de Cogan, None; Jenna
ONeill, None; Richard J. Blanch, None; Robert A H Scott, None;
Ann Logan, None
Support: NIHR SRMRC Trauma Centre funding
Program Number: 401 Poster Board Number: C0172
Presentation Time: 8:30 AM–10:15 AM
MERTK Interactions with Src Family Kinases in the Retinal
Pigment Epithelium
Anna Ganios1, Shameka J. Shelby2, Kecia L. Feathers2, Lin Jia2,
Debra A. Thompson2, 1. 1Biological Chemistry, University of
Michigan Medical School, Ann Arbor, MI; 2Ophthalmology and
Visual Sciences, University of Michigan Medical School, Ann Arbor,
MI.
Purpose: Signaling by the receptor tyrosine kinase MERTK is
essential for the phagocytic uptake of shed photoreceptor outer
segments by the retinal pigment epithelium (RPE). We have
previously shown that MERTK activates the intracellular tyrosine
kinase Src, which in turn regulates the Rab effector GDI1. The
purpose of the present study is to further define the potential for
MERTK activation of Src family kinases (SFKs) during RPE
phagocytosis.
Methods: The expression of SFKs in the RPE was assessed using
RT-PCR, immunohistochemical, and western analysis. MERTK
interacting proteins were identified using Ni2+-NTA pulldowns with
the intracellular domain of MERTK (aa 571-999) that was expressed
as a 6xHis fusion protein, purified using Ni2+-NTA agarose, and
phosphorylated by the addition of ATP. 6xHis-rMERTK571-999 was
incubated with cDNA clones encoding the SH2-domains of Src,
Blk, Fgr, Frk, Fyn, Hck, and Yes that were expressed as GST-fusion
proteins and purified using glutathione agarose, or with RPE-J cell
homogenates. MERTK-associated proteins present in Ni2+-NTA
pulldowns were evaluated on coomassie blue stained gels and by
western analysis.
Results: Transcripts encoding multiple SFKs were detected in mouse
RPE, with Src, Hck, Fyn, and Yes present at apparently highest
abundance. Expression of Src, Fyn, and Yes was confirmed by
western analysis of rat RPE/choroid and RPE-J cell homogenates.
Immunohistochemical analysis of mouse RPE/retina cryosections
showed differential expression patterns of SFKs throughout the layers
of the retina. MERTK pulldowns showed interactions with the cloned
SH2-domains of Src, Blk, Fgr, Frk, Fyn, Hck, and Yes, and with
endogenous Src and Fyn.
Conclusions: We find that signaling downstream of MERTK has
the potential to activate multiple SFKs in the RPE. Viewed together
with our earlier findings that MERTK activation of Src regulates
Rab effectors involved in membrane trafficking, and the known role
of Src in regulating cytoskeletal movements, our findings suggest
that MERTK activation of SFKs is a central feature of the molecular
mechanism of RPE phagocytosis.
Commercial Relationships: Anna Ganios, None; Shameka J.
Shelby, None; Kecia L. Feathers, None; Lin Jia, None; Debra A.
Thompson, None
Support: FFB, RPB, NIH Grants EY13934, EY02083, EY07003
Program Number: 402 Poster Board Number: C0173
Presentation Time: 8:30 AM–10:15 AM
Retinal iron accumulation despite an intact blood-retinal barrier
in mice with high serum iron
Liangliang Zhao1, 2, Yafeng Li2, Delu Song2, Ying Song2, Milan
Theurl2, Joshua L. Dunaief2. 1Ophthalmology, The Second Hospital
of Jilin University, Changchun, China; 2Ophthalmology, Scheie Eye
Institute, Philadelphia, PA.
Purpose: The retina is shielded from potentially harmful blood
components by the blood-retinal barrier. Since the retina is
exquisitely sensitive to iron levels, it is important to understand the
roles of systemic versus local influences on retinal iron levels. The
peptide hormone hepcidin (Hepc), which, in turn, is regulated by
Bmp6, is an important regulator of systemic iron metabolism, yet
hepcidin’s role locally in the retina is incompletely understood.
Methods: Retinas of systemic bone morphogenic protein 6 (Bmp6)
knockout (KO) mice were analyzed by Perls’ iron stain and
immunofluorescence to assess the levels of iron and iron-regulated
proteins. Levels of retinal and isolated RPE Hepc mRNA were
measured by qPCR.
Results: H-ferritin and L-ferritin immunoreactivity (which is
directly correlated with iron levels) in the neural retina (NR) and
retinal pigment epithelium (RPE) increased in male Bmp6 KO
mice compared to age-matched WT males. Female Bmp6 KOs had
significantly less retinal ferritin than Bmp6 KO males. Granular
Perls’ iron stain was present in the RPE. Retinal Hepc mRNA levels
increased in parallel with iron levels in Bmp6 KO. Hepc mRNA
levels were not decreased in the NR or RPE at any age in Bmp6 KO
mice.
Conclusions: These findings indicate that elevated serum iron levels
resulting from systemic Bmp6 KO can overwhelm any local retinal
iron regulatory mechanisms. While Hepc may play a local iron
regulatory role in the retina, this is insufficient to prevent retinal iron
overload in the face of markedly elevated serum iron levels. The
retinal regulation of Hepc correlated with retinal iron status rather
than being regulated by a local Bmp6 pathway.
Commercial Relationships: Liangliang Zhao, None; Yafeng Li,
None; Delu Song, None; Ying Song, None; Milan Theurl, None;
Joshua L. Dunaief, None
Support: Research to Prevent Blindness, NEI R01EY015240,
the FM Kirby Foundation, the Paul and Evanina Bell MacKall
Foundation Trust, a gift in honor of Dr. Lee F. Mauger
Program Number: 403 Poster Board Number: C0174
Presentation Time: 8:30 AM–10:15 AM
Soluble Adenylyl cyclase (sAC) in the retinal pigment epithelium
plays a role in generating the light peak of the electrooculogram.
Yong S. Lee1, Minzhong Yu2, Lihua Marmortein1, 3, Neal S. Peachey2,
Alan D. Marmorstein1. 1Ophthalmology, Mayo Clinic, Rochester,
MN; 2Ophthalmic Research, The Cleveland VA and the Cleveland
Clinic, Cleveland, OH; 3The Cole Eye Institute, Cleveland, OH.
Purpose: Niemeyer and Steinberg (Vis. Res. 24:275-28, 1984)
demonstrated that the amplitude of the light peak (LP) of the
electrooculogram (EOG) is diminished by elevated pCO2. In many
cells, sAC an enzyme that catalyzes the hydrolysis of ATP to cAMP
functions as a bicarbonate sensor generating cAMP in response to
HCO3-. In this study we set out to determine if sAC is expressed in
RPE cells and whether it plays a role in generating the LP.
Methods: We used immunofluorescence to localize sAC expression
in the mouse eye. Immunoprecipitation and western blot were used
in conjunction with adenylyl cyclase assays to verify expression of
sAC in RPE lysates. To determine the effect of sAC on the LP, we
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ARVO 2014 Annual Meeting Abstracts
measured the LP using dc-electroretinography in mice treated with
the sAC specific inhibitor KH7 or vehicle alone.
Results: : Immunofluorescence staining suggested that sAC is
broadly expressed in the eye with highest expression apparent
in the ciliary body, corneal endothelium, retina, and RPE.
Expression of sAC in the RPE was confirmed by western blot, and
immunoprecipitation using anti-sAC antibodies. Adenylyl cyclase
assays confirmed that a bicarbonate dependent adenylyl cyclase is
expressed in RPE that is inhibited by KH7. Mice treated with KH7
exhibited a diminished LP in comparison to control mice treated with
vehicle alone.
Conclusions: Our data demonstrate that sAC is expressed by the
RPE and that it contributes to the generation of the LP. Further study
will be necessary to determine how cAMP generated by sAC may be
involved in the LP response as a role for cAMP in generating the LP
has not previously been observed.
Commercial Relationships: Yong S. Lee, None; Minzhong Yu,
None; Lihua Marmortein, None; Neal S. Peachey, None; Alan D.
Marmorstein, None
Support: NEI Grant EY13160, EY21153, EY013847, The
Foundation Fighting Blindness, Research to Prevent Blindness
Program Number: 404 Poster Board Number: C0175
Presentation Time: 8:30 AM–10:15 AM
Genotyping strategy for congenital stationary night blindness
(CSNB): conclusion from a comprehensive study and metaanalysis
Christina Zeitz1, Isabelle Audo1, 2. 1Institut de la Vision, Univ Pierre
et Marie Curie Paris 6, INSERM, UMR_S968, CNRS, UMR_7210,
Paris, France; 2CHNO, INSERM-DHOS CIC 503, Paris, France.
Purpose: CSNB is a genetically and clinically heterogeneous retinal
disease caused by mutations in 17 identified genes. The purpose
of this study was to establish phenotype-genotype correlations,
prevalence, and genotyping strategies for CSNB based on metaanalyses on our cohort and a literature search.
Methods: Detailed phenotypic characterization was performed on
a large CSNB cohort consisting of 350 index patients. To detect
mutations, we used Sanger sequencing, microarray analysis, next
generation targeted (NGS) and whole exome sequencing (WES). An
exhaustive literature search was used to include all described CSNB
causing mutations.
Results: Most of the patients from our CSNB cohort have a
Schubert-Bornschein-type electroretinogram (ERG) with complete
(c) and incomplete (ic) phenotype. We identified novel mutations
in NYX (15), TRPM1 (15), GRM6 (2), and GPR179 (3) in patients
with cCSNB, and in CACNA1F (38) and CABP4 (1) in icCSNB
patients. A few cases in this cohort do not harbour mutations in
known genes. Compiling our data with those from the literature, we
found 67 different mutations associated with gene defects affecting
the phototransduction cascade and leading to CSNB with a Riggstype ERG (RHO = 4, GNAT1 =3, PDE6B = 1, SLC24A1 = 1), or to
CSNB with fundus abnormalities as in Oguchi disease (SAG = 6 and
GRK1 = 9) and Fundus Albipunctatus (RDH5 = 38, RLBP1 = 3 and
RPE65 = 2). Similarly, 293 different mutations have been identified
in gene defects affecting the signalling from photoreceptors to bipolar
cells, of which 132 occurred in cases with icCSNB (CACNA1F =
126, CABP4 = 5 and CACNA2D4 = 1) and 161 in cases with cCSNB
(NYX = 69, GRM6 = 23, TRPM1 = 51, GPR179 = 14 and LRIT3 = 4).
Taking into account founder mutations the following prevalence for
CSNB was established: RHO>GNAT1>SLC24A1≥PDE6B in cases
with a Riggs-type ERG, RDH5>SAG>GRK1>RLBP1>RPE65 in
cases with fundus abnormalities, CACNA1F>CABP4>CACNA2D4
in cases with icCSNB and NYX>TRPM1>GRM6>GPR179>LRIT3 in
cases with cCSNB.
Conclusions: Although CSNB is a heterogeneous disease, precise
clinical examination is a useful first step toward gene-specific
sequencing to identify disease causing mutations. In this context,
Sanger sequencing seems to be the gold standard. WES should
identify the missing gene defects underlying unsolved CSNB cases.
Commercial Relationships: Christina Zeitz, None; Isabelle Audo,
None
Support: ANR-12-BSVS1-0012- 01_GPR179 (CZ), INSERM-ICMR
grant (CZ), Foundation Voir et Entendre (CZ), Dalloz Award (CZ),
FFB:[CD-CL-0808-0466-CHNO] (IA), FFB center grant [C-CMM0907-0428-INSERM04], Ville de Paris and Region Ile de France,
LABEX LIFESENSES [ANR-10-LABX-65] supported by French
state funds managed by the ANR within the Investissements d’Avenir
programme [ANR-11-IDEX-0004-0]
Program Number: 405 Poster Board Number: C0176
Presentation Time: 8:30 AM–10:15 AM
Whole exome sequencing identifies a new ciliary gene in
autosomal recessive rod-cone dystrophy
Isabelle Audo1, 2, Said El Shamieh1, Marion Neuillé1, Angélique
Terray1, Elise Orhan1, Saddek Mohand-Saïd1, 2, Thierry D.
Leveillard1, Olivier Goureau1, Jose A. Sahel1, 2, Christina Zeitz1.
1
Institut de la Vision, Univ Pierre et Marie Curie Paris 6, Inserm
UMR_968, CNRS UMR_7210, Paris, France; 2Inserm -DHOS Centre
d’Investigation Clinique CIC503/CMR « dystrophies rétiniennes
d’origine génétique », Centre Hospitalier National d’Ophtalmologie
des Quinze-Vingts, Paris, France.
Purpose: Autosomal recessive rod-cone dystrophies (arRCD) are
a heterogeneous group of retinal diseases with more than forty
implicated genes. However, comprehensive targeted next generation
sequencing (NGS) still identifies cases with no underlying gene
defect and whole exome sequencing (WES) offers, in these cases,
a method of choice to hunt for new candidates. Our purpose was
to apply WES to identify a novel gene defect in an arRCD case,
to screen a large cohort of 338 patients by direct sequencing, to
establish its prevalence in arRCD and to evaluate the expression and
localization of the respective transcript and protein.
Methods: After exclusion of known gene defects through a
comprehensive method (ARVO 2011, abstract # 6605), WES was
applied to an arRCD case, his unaffected first-cousin-parents, his
unaffected brother and sister. Filtering used available SNP databases,
bioinformatic pathogenic prediction programs, retinal transcriptomic
databases, co-segregation analysis and Sanger sequencing to select
the most likely pathogenic variant. Further Sanger sequencing of the
new candidate was performed on 338 unrelated arRCD. Expression
studies for mRNA (RT-PCR and in situ hybridization) were
performed in retinal sections. In addition, protein immunolocalization
was achieved by co-staining against gamma and acetylated alpha
tubulins human fibroblasts after in vitro induction of monocilia by
serum deprivation.
Results: WES identified a homozygous nonsense change in a novel
gene segregating with the disease. Further Sanger sequencing of a
large arRCD cohort identified two additional cases with nonsense
mutations leading to a prevalence of ~1% of cases in our cohort.
This gene had never been associated with photoreceptor diseases.
Transcriptomic and bioinformatic databases reported the product
of this gene to be associated with the centrosome and expressed
in photoreceptors. In situ hybridization studies revealed mRNA
expression within the outer nuclear layer of mouse retinal sections. In
addition, immunostaining of human monocilia in induced-fibroblasts
confirmed protein localization at the basal body.
©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 pubs@arvo.org.
ARVO 2014 Annual Meeting Abstracts
Conclusions: Whole exome sequencing and further Sanger
sequencing of a large cohort of arRCD patients identify a novel
candidate encoding a ciliary protein critical for photoreceptor
homeostasis. This novel gene defect accounts for 1% of arRCD in the
studied cohort.
Commercial Relationships: Isabelle Audo, None; Said El
Shamieh, None; Marion Neuillé, None; Angélique Terray, None;
Elise Orhan, None; Saddek Mohand-Saïd, None; Thierry D.
Leveillard, None; Olivier Goureau, None; Jose A. Sahel, None;
Christina Zeitz, None
Support: Foundation Voir et Entendre (CZ), Dalloz Award (CZ),
FFB:[CD-CL-0808-0466-CHNO] (IA), FFB center grant [C-CMM0907-0428-INSERM04], Ville de Paris and Region Ile de France,
LABEX LIFESENSES [ANR-10-LABX-65] supported by French
state funds managed by the ANR within the Investissements d’Avenir
programme [ANR-11-IDEX-0004-0], ANR-eRare-RHORCOD.
Program Number: 406 Poster Board Number: C0177
Presentation Time: 8:30 AM–10:15 AM
Absence of GARP2 in mice leads to slowly progressive structural
deficits in rod photoreceptors
Delores A. Davis1, Marci L. Smith1, Youwen Zhang1, Andrew F.
Goldberg2, Steven J. Pittler1, 3. 1Department of Vision Sciences,
University of Alabama at Birmingham, Birmingham, AL; 2Eye
Research Institute, Oakland University, Rochester Hills, MI; 3Vision
Science Research Center, University of Alabama at Birmingham,
BIrmingham, AL.
Purpose: The Cngb1 locus encodes the beta subunit of the rod
photoreceptor CNG channel, and by alternative splicing two
soluble glutamic acid rich proteins, GARP1 and GARP2. We
previously reported that loss of all Cngb1-encoded proteins alters
disk morphogenesis and structural integrity and attenuates the rod
photoresponse, and ultimately leads to photoreceptor death and
blindness (Cngb1-X1; Zhang et al. 2009 J. Cell Sci. 122:1192). Here
we used genome-editing technology to selectively delete expression
of GARP2 to singularly assess its role in the rods.
Methods: Using Sigma’s CompoZr® ZFN technology, exon 12a
that is unique to GARP2 was targeted by injection of ZFN RNA into
the male pronucleus of single cell fertilized mouse embryos that
were allowed to develop using standard mouse transgene techniques.
One allele was identified that contained a complete deletion of exon
12a and upstream acceptor site that represents a true GARP2 null.
Established procedures for OCT, light microscopy, EM and Western
analysis were used to analyze the GARP2 KO retina compared to
WT.
Results: Western analysis of total retina protein demonstrated the
absence of 73 kD murine GARP2 in the knockout retina that is
abundant in WT mouse retina, however beta-subunit and GARP1
expression were unaffected. OCT imaging of 1-6 month old mouse
retina showed no distinct differences in retinal layer thickness but
changes in hyperreflectivity were apparent in the outer segment
region. By light microscopy, as early as 2 months rod outer segments
(ROS) are less uniform and the ROS frequently extend parallel to the
RPE. At this time point in EM micrographs ROS disks appear normal
in all rods examined. By 8.5 months a 20% loss of nuclei in the ONL
was apparent and ROS appear shorter.
Conclusions: GARP2 knockout alone exhibits a more subtle
phenotype than was observed in the Cngb1-X1 total photoreceptor
null mouse. GARP2 does not appear to be required for disk
morphogenesis, however it is necessary to maintain ROS structural
integrity as the animal matures and in aging mice its absence leads
to retinal degeneration. Thus, the GARP2 KO model may represent a
good model for the study of later onset retinal degeneration.
Commercial Relationships: Delores A. Davis, None; Marci L.
Smith, None; Youwen Zhang, None; Andrew F. Goldberg, None;
Steven J. Pittler, None
Support: NIH grant R01 EY018143-05, P30 EY03039-35, UAB
Dept. of Vision Sciences, UAB Vision Science Research Center to
SJP and RR017890 to AFG
Program Number: 407 Poster Board Number: C0178
Presentation Time: 8:30 AM–10:15 AM
Novel technique for sectioning a mouse retinal flat mount
Christopher M. Aderman1, 2, Ye Sun1, Zhuo Shao1, Jean-Sebastien
Joyal1, Lois Smith1. 1Ophthalmology, Children’s Hospital Boston,
Boston, MA; 2Ophthalmology, University of California, San
Francisco, San Francisco, CA.
Purpose: There are currently no reliable, established methods that
allow for isolation of retinal layers in order to enrich for tissue or
cell-specific genes for microarray analysis. Existing techniques,
such as laser capture microdissection, yield very small quantities
of genetic material that must undergo amplification, a step which
invariably induces error and can make microarray studies unreliable.
We have developed a new method to section a flat mounted retina so
that sufficient quantities of tissue specific genes and proteins can be
studied in normal and diseased states.
Methods: We sacrificed neonatal mice at postnatal day 12 and
harvested the eyes. We then dissected the retinas in saline and
made four radial cuts extending from the optic nerve to allow the
retina to lay flat. We performed the dissections on Parafilm-covered
microscope slides, then flipped over the retinas onto pre-frozen and
trimmed blocks of OCT. The exposed portions of the retinas were
embedded in OCT and prepared for sectioning. Optimal sectioning
size was approximately 20 microns, which allowed for a sufficient
tissue quantities while still allowing for resolution of the retinal
layers. High quality RNA was converted to cDNA for real time PCR
analysis.
Results: The left panel is a schematic showing how the flat mounted
retina is sectioned at a thickness of 20 microns, which provides
multiple sections per retinal layer. The right panel compares
Rhodopsin (a photoreceptor marker) mRNA expression in each of
the 12 sections normalized to the housekeeping gene, cyclophillin
A. There is clear enrichment or rhodopsin expression in layers 1
and 2 (54 million and 78 million copies, respectively per 1 million
copies cyclophillin A), which indicates these layers correspond to
photoreceptor layers and represent a 3.8 to 12 fold increase compared
with the other retinal sections.
Conclusions: This novel technique for flat mounting and sectioning
fresh mouse retinas serves as a powerful tool for RNA (or protein)
analysis. The primary advantage of this new method is that we are
able to obtain large amounts of high quality RNA and protein from
specific retinal layers, making large scale, cell-type specific, gene
expression analyses of normal and diseased retinas feasible.
©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 pubs@arvo.org.
ARVO 2014 Annual Meeting Abstracts
research propounds that a phosphorylated BPS region of Grb14
inhibits PTP1B activity, thereby promoting IR activation. We propose
a model in which phosphorylation of the BPS region of Grb14 is
the key element in promoting IR activation, and failure to undergo
phosphorylation on Grb14 leads to both PTP1B and Grb14 exerting
their negative roles in IR. Consistent with this hypothesis, we found
decreased phosphorylation on Grb14 in diabetic type 1 Ins2Akita
mouse retinas, and a decreased retinal IR activation has previously
been reported in this mouse line.
Conclusions: Our results suggest that the phosphorylation status
of the BPS region of Grb14 determines the positive or negative
role it will play in IR signaling. Further, activators of Grb14
phosphorylation may have therapeutic potential to protect the dying
retinal neurons in retinal degenerative diseases.
Commercial Relationships: Raju V. Rajala, None; Ammaji
Rajala, None
Support: NIH/NEI grant (EY016507, EY00871)
Commercial Relationships: Christopher M. Aderman, None; Ye
Sun, None; Zhuo Shao, None; Jean-Sebastien Joyal, None; Lois
Smith, None
Support: EY022275, EY017017, P01 HD18655, RPB Sr
Investigator Award, Lowy Medical Foundation (LEHS)
Program Number: 408 Poster Board Number: C0179
Presentation Time: 8:30 AM–10:15 AM
The Spatial and Temporal Activation of the Retinal Insulin
Receptor: Role of Grb14 and PTP1B
Raju V. Rajala1, 2, Ammaji Rajala1. 1Ophthal/Dean McGee Eye Inst,
Univ of Oklahoma Hlth Sci Ctr, Oklahoma City, OK; 2Physiology,
Univ of Oklahoma Hlth Sci Ctr, Oklahoma City, OK.
Purpose: Retinal cells are post-mitotic tissue. Insulin receptor (IR)
activation is essential for retinal neuron survival. Growth factor
receptor-bound protein 14 (Grb14) is an adapter protein implicated in
receptor tyrosine kinase signaling. Grb14 knockout studies highlight
both the positive and negative roles of Grb14 in receptor tyrosine
kinase signaling, in a tissue specific manner. Retinal cells express
protein tyrosine phosphatase-1B (PTP1B), which dephosphorylates
IR and Grb14, a pseudosubstrate inhibitor of the IR. Thus, the major
question remains: in retinal neurons, how does the IR overcome
inactivation by PTP1B and Grb14?
Methods: Wild type, PTP1B-/-, Grb14-/-, and diabetic type 1
Ins2Akita mice were used in this study. Retinal IR kinase activity,
PTP1B activity and IR phosphorylation were measured.
Results: Our studies suggest that ablation of Grb14 results in
decreased IR activation due to increased PTP1B activity. Our
Program Number: 409 Poster Board Number: C0180
Presentation Time: 8:30 AM–10:15 AM
Role of Complement Components in Retinopathy of Prematurity
Sonika Rathi1, Subhabrata Chakrabarti1, Subhadra Jalali2, 3, Ramesh
Kekunnaya3, Suman Thakur4, Rohit Budhraja4, T. Ramakrishna4, Ch.
Mohan Rao4, Inderjeet Kaur1. 1Prof. Brien Holden Eye Research
Centre, L V Prasad Eye Institute, Hyderabad, India; 2Smt. Kannuri
Santhamma Centre for Vitreo Retinal Diseases, L V Prasad Eye
Institute, Hyderabad, India; 3Jasti V Ramanamma Children’s Eye
Care Centre, L V Prasad Eye Institute, Hyderabad, India; 4Centre for
Cellular and Molecular biology, Hyderabad, India.
Purpose: Retinopathy of Prematurity (ROP) is a proliferative
retinal vascular disorder affecting eye of premature babies with
low gestational age (≤32 weeks) and birth weight (≤1,700g). A
study on mouse model of ischemia induced retinopathy suggested a
protective role for complement C3a and C5a in neovascularisation
while complement is known to mediate angiogenesis in Age-Related
Macular Degeneration (AMD). However, the exact involvement
of complements in ROP pathogenesis is still unclear. The present
study aimed to understand the role of complement components in the
pathogenesis of ROP.
Methods: Vitreous humor (50-100μl) was collected with prior
informed consent from patients (n=40) with stage IV and V of
ROP (classified as per ICROP guidelines) along with infants with
congenital cataract as control subjects (n=40) undergoing vitrectomy.
Prefractionated protein were subjected to trypsin digestion either
in-gel or in solution and the resulting peptides were analysed on a
FT LTQ Orbitrap Velos mass spectrophotometer. The obtained mass
spectra were searched against the International Protein Index (IPI)
database using the Peak studio search engine. Following this strategy,
we examined the complement components in ROP and control
vitreous. Parallely levels of complement component in vitreous
were assessed by multiplex ELISA and further validated by western
blotting.
Results: Proteomic analysis of in-gel trypsin digested proteins
of selected gel pieces ( 160 kDa, 63 kDa and 45 kDa ) detected
371 spectra in ROP while only 95 spectra in controls for C3
fragments,and 323 spectra in ROP while only 102 spectra in control
for C4 fragments. This result showed elevated levels of complement
component C3 and C4 in ROP patients which was also confirmed by
a parallel multiplex ELISA and in-solution digested total proteome
analysis. In gel pieces analysis of 63 kDa and 45 kDa for activated
C3 fragments, 182 spectra were found in ROP while 24 spectra
were found in controls. Increased number of spectra for activated
C3 in ROP vitreous confirmed to the results from western blotting.
©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 pubs@arvo.org.
ARVO 2014 Annual Meeting Abstracts
Cleavage of C5 into C5b was also confirmed by western blotting in
ROP.
Conclusions: The elevated levels of complement factors and its
activation indicated abnormal immune activity in ROP patients.
Activation of complement pathway might be playing an important
role in angiogenesis in ROP patients.
Commercial Relationships: Sonika Rathi, None; Subhabrata
Chakrabarti, None; Subhadra Jalali, None; Ramesh Kekunnaya,
None; Suman Thakur, None; Rohit Budhraja, None; T.
Ramakrishna, None; Ch. Mohan Rao, None; Inderjeet Kaur,
None
Support: Department of Biotechnology, Government of India;Indian
Council of Medical Research
Program Number: 410 Poster Board Number: C0181
Presentation Time: 8:30 AM–10:15 AM
Cysteine proteases expression and secretion by retinal pigmented
epithelium (RPE)
Umar Sharif1, Joe Butler1, Yit C. Yang2, Ian Grierson1, Simon P.
Harding1, Luminita I. Paraoan1. 1Eye and Vision Science, University
of Liverpool, Liverpool, United Kingdom; 2Ophthalmology,
Wolverhampton Med Inst-New Cross, Wolverhampton, United
Kingdom.
Purpose: Control of proteolytic events inside the RPE cell as well
as in its immediate extracellular environment is important in the
maintenance of retinal homeostasis. The purpose of this study was to
therefore investigate the expression and secretion of specific cysteine
proteases in RPE cells.
Methods: Protein expression was analysed by Western blotting
of cell lysates and conditioned media of confluent ARPE-19 and
D407 cell cultures. Immunoreactivity for cathepsin B, L and S
with appropriate intra/extracellular loading controls was assessed.
Quantification was carried out using densitometry analysis and data
was normalised to loading controls/total protein content. In addition,
transcriptional level of cathepsins was quantified by RNA sequencing
in human foetal RPE cells.
Results: Both pro and active forms of cysteine proteases cathepsin B,
L and S were detected in both cell lysates of ARPE-19 and D407 cell
lines. Although expression levels of these cathepsins were slightly
different in the two cell lines, the active form for all cathepsins was
the predominant form in all cases. Results were corroborated by
high expression levels of cathepsin specific transcripts in human
foetal RPE cells. The most abundantly expressed cysteine protease
was cathepsin B followed by L and then S. Active secretion of pro
and active forms of all these cathepsins was evident in all RPE cells
except for cathepsin B which was detected only in its active form.
Conclusions: Cysteine proteases cathepsin B, L and S are
expressed and secreted at high level by RPE cells indicating a likely
extracellular role. These cysteine proteases are expected molecular
targets of one of the most abundantly expressed proteins by the RPE,
the cysteine proteinase inhibitor cystatin C, a variant of which is
associated with AMD pathogenesis. Altered RPE expression of these
cathepsins and/or cystatin C could lead to a proteolytic imbalance
potentially contributing to pathogenic structural and functional
extracellular changes.
Commercial Relationships: Umar Sharif, None; Joe Butler, None;
Yit C. Yang, None; Ian Grierson, None; Simon P. Harding, None;
Luminita I. Paraoan, None
Program Number: 411 Poster Board Number: C0182
Presentation Time: 8:30 AM–10:15 AM
The consequences of deglycosylation of intra-melanosomal
domain of human tyrosinase.
Monika B. Dolinska1, Peter Backlund2, Paul T. Wingfield3, Elena
Kovaleva4, Ewa Grajkowska1, Brian P. Brooks1, Yuri V. Sergeev1.
1
OGVFB, National Eye Institute, Bethesda, MD; 2National Institute
of Child Health and Human Development, Bthesda, MD; 3National
Institute of Arthritis and Musculoskeletal and Skin Diseases,
Bethesda, MD; 4Chesapeake PERL, Savage, MD.
Purpose: Oculocutaneous albinism type 1 is an autosomal recessive
disease associated with decreased melanin pigment production,
decreased best-corrected visual acuity, and mutation in the tyrosinase
(TYR) gene. Tyrosinase is a type 1 trans-membrane and coppercontaining glycoenzyme that catalyzes the initial steps of melanin
pigment production in melanosomes. Human tyrosinase is modified
post-translationally by N-linked glycosylation on asparagine (Asn,
N) residues, which is important for enzymatic activity. Recently we
characterized in vitro 5 N-glycosylation sites in the recombinant
intra-melanosomal domain of human tyrosinase, hTyrCtr (Dolinska
et al,). To explore this study we expressed, purified and characterize
enzymatic activity of 2 N-deglycosylated mutant variants.
Methods: The 5-sites and fully (7-sites) deglycosylated recombinant
mutant variants were prepared using the site-directed mutagenesis
where Asn were replaced with Asp residues to remove the
N-glycosylation sites, respectively. Recombinant human tyrosinase
(residues 19 – 469 of the native protein) and deglycosylated mutants
were produced in larvae (C-PERL, MD) and purified by immobilized
metal affinity and size-exclusion chromatography. N-glycosylation
sites in intra-melanosomal domain were confirmed using mass
spectroscopy. Enzyme activities were measured using L-DOPA color
reaction.
Results: The 5- and 7 residues mutagenesis of the hTyrCtr
glycosylation sites resulted in decreased protein expression and
increased difficulty with protein purification, respectively. Specific
L-DOPA activity measured for the wild type, 5-and 7-deglycosylated
mutants show decrease of enzymes activities with a dramatic change
for the fully deglycosylated protein (50033, 37879, and 95 U/mg,
respectively).
Conclusions: Removal of 5 N-glycosylation sites from the hTyrCtr
causes modest decrease of activity, which is consistent with our
molecular modeling of human tyrosinase which suggests that those
sites are located at the protein surface and do not directly affect the
4-helix bundle structure maintaining the copper-binding sites. The
complete removal of N-glycans affects the protein expression and
diminishes the enzymatic activity, in agreement with previous data in
cell culture.
Commercial Relationships: Monika B. Dolinska, None; Peter
Backlund, None; Paul T. Wingfield, None; Elena Kovaleva, None;
Ewa Grajkowska, None; Brian P. Brooks, None; Yuri V. Sergeev,
None
Program Number: 412 Poster Board Number: C0183
Presentation Time: 8:30 AM–10:15 AM
A fragment of netrin-1 is implicated in the induction of
permeability in diabetic retinopathy
Khalil Miloudi1, Sarah Genest-Brunetta2, Francois Binet4,
Gaelle-Stephanie Mawambo-Tagne2, Agustin Cerani2, Agnieszka
Dejda2, Flavio Rezende3, Timothy Kennedy1, Przemyslaw
Sapieha3. 1Neurology, McGill University, Montreal, QC, Canada;
2
Biochemistry, University of Montreal, Montreal, QC, Canada;
3
Ophtalmology, University of Montreal, Montreal, QC, Canada;
4
Medical biochemistry, Uppsala University, Uppsala, Sweden.
©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 pubs@arvo.org.
ARVO 2014 Annual Meeting Abstracts
Purpose: Although elevated VEGF is frequently associated with
breakdown in barrier function in diabetic retinopathy (DR), we
have recently described roles for neuronal guidance cues in the
pathogenesis of DR. Here we described a new role for a fragment of
netrin-1, the VI-V peptide fragment of netrin-1, in the development of
DR-associated breakdown of barrier function.
Methods: We first investigated retinal levels of full-length and
fragmented netrin-1 by Western Blot in early stages of DR in a
streptozotocin-induced model of diabetes. In a modified version of
the in vivo Miles Assay, we i) subcutaneously and ii) intraviteously
injected recombinant netrin-1 or the shorter VI-V netrin-1 peptide
into wild type mice at 10 weeks of age. A blue dye (Evans blue (EB)),
was later injected intravenously. Permeability was then evaluated
by i) measuring dorsal spots of subcutaneous leaking EB, and ii) by
quantifying EB in retinas after overnight extraction in formamide.
To identify possible signaling pathways involved, human retinal
microvascular endothelial cells were similarly stimulated in vitro.
Results: Our findings reveal a significant increase in the levels of
fragmented netrin-1 at 4 and 8 weeks in diabetic retinas. Intravitreal
injection of the netrin-1 VI-V peptide significantly increased vascular
permeability in vivo both in retina and after subcutaneous injection,
when compared to full-length netrin-1 or vehicle. In agreement, in
vitro data confirmed that the VI-V fragment of netrin-1 provoked the
phosphorylation of permeability-associated pathways such FAK, Src,
and VE-Cadherin.
Conclusions: These results show a new mechanism implicating a
wider spatial effect of netrin-1 via the VI-V peptide fragment. Current
studies aim to elucidate the cellular and molecular mechanism by
which this recently described fragment may be implicated in the
pathogenesis of DR.
Commercial Relationships: Khalil Miloudi, None; Sarah
Genest-Brunetta, None; Francois Binet, None; Gaelle-Stephanie
Mawambo-Tagne, None; Agustin Cerani, None; Agnieszka
Dejda, None; Flavio Rezende, None; Timothy Kennedy, None;
Przemyslaw Sapieha, None
Program Number: 413 Poster Board Number: C0184
Presentation Time: 8:30 AM–10:15 AM
Fetal hemoglobin induction by monomethylfumarate: relevance
to prevention and treatment of sickle cell retinopathy (SR)
Wanwisa Promsote1, Biaoru Li2, Rajalakshmi Veeranan-Karmegam1,
Levi Makala2, Sylvia B. Smith3, 4, Vadivel Ganapathy1, Betty S. Pace2,
Pamela M. Martin1, 4. 1Biochemistry and Molecular Biology, Georgia
Regents University, Augusta, GA; 2Pediatrics, Georgia Regents
University, Augusta, GA; 3Cellular Biology and Anatomy, Georgia
Regents University, Augusta, GA; 4Ophthalmology, Georgia Regents
University, Augusta, GA.
Purpose: SR is a most debilitating and unfortunately common
complication of sickle cell disease (SCD). Abnormal hemoglobin
(HbS) polymerization in red blood cells and consequent vascular
dysfunction is a primary causative factor; clinical and experimental
studies support the critical involvement of other cellular and
molecular factors. RPE cells were reported recently to produce Hb
however this phenomenon was not evaluated in the context of retinal
pathology in SCD. Here, using ARPE-19 and primary RPE cells
established from HbAA (normal)- and HbSS (sickle)-expressing
Townes humanized mice we (a) evaluated globin gene expression
and Hb production in normal and sickle RPE, and (b) validated
monomethylfumarate (MMF) as an effective inducer of γ-globin
expression/fetal Hb (HbF) production in retinal and erythroid cells
and therefore, as a potential therapy for preventing and treating
retinal AND systemic complications of SCD.
Methods: ARPE-19 and HbAA- and HbSS- expressing primary RPE
cells were treated with various concentrations of MMF, followed
by analysis of α-, β- and γ-globin expression and HbF production
by qPCR, western blotting, FACS and immunofluorescence.
Hydroxyurea (HU), at present the only FDA-approved compound
for SCD treatment, was included as a positive control. Parallel
studies were performed using KU812 and primary human erythroid
progenitor cells.
Results: RPE cells express α-, β- and γ-globin mRNA and
consequently synthesize normal (ARPE-19 and HbAA primary RPE)
and sickle (HbSS primary RPE) Hb. MMF induces, in a time- and
dose-dependent manner, γ-globin expression and HbF production
in all cell types evaluated, and in retina following its delivery
intravitreally to live mice. HbF induction by MMF was 2-3 -fold
higher than that by HU.
Conclusions: HbF-inducing therapy is at present the best strategy for
preventing and treating SCD-related complications. However, little is
known regarding the functional relevance of Hb production in retina
by non-erythroid cells or the impact of HbF-inducing therapies in this
tissue. Our present finding that MMF induces γ-globin expression
and HbF production is novel and supports the possible use of this
compound in SCD/SR. The fact that FDA-approved formulations in
which MMF is the main bioactive ingredient are readily available
enhance greatly the translational potential of this study.
Commercial Relationships: Wanwisa Promsote, None; Biaoru Li,
None; Rajalakshmi Veeranan-Karmegam, None; Levi Makala,
None; Sylvia B. Smith, None; Vadivel Ganapathy, None; Betty S.
Pace, None; Pamela M. Martin, None
Program Number: 414 Poster Board Number: C0185
Presentation Time: 8:30 AM–10:15 AM
The Cyclophilin Domain of Ran-binding protein 2 (Ranbp2)
Harbors Discriminating Physiological Activities Towards Distinct
Retinal Substrates
Paulo A. Ferreira1, Kyoung-in Cho1, Hemangi Patil1, Eugene Senda1,
Jessica Wang1, Haiqing Yi1, Sunny Qiu1, Dosuk Yoon1, Minzhong Yu2,
Neal S. Peachey2. 1Ophthalmology, Duke University Medical Center,
Durham, NC; 2Ophthalmic Research, Cleveland Clinic Foundation,
Cleveland, OH.
Purpose: The immunophilins, cyclophilins, catalyze peptidyl cistrans prolyl-isomerization (PPIase), a rate-limiting step in protein
folding and conformational switch in protein function. Cyclophilins
are also chaperones. The C-terminal cyclophilin domain (CY) of
Ranbp2 acts as a selective chaperone towards M-opsin production in
a cell culture system, but its physiological and biological roles in the
retina are ill-defined. This study aims at testing the hypothesis that
the CY of Ranbp2 harbors non-overlapping PPIase and chaperone
activities and discriminating physiological activities towards retinal
substrates.
Methods: We generated transgenic mice lacking endogenous Ranbp2
(Ranbp2-/-) and expressing transgenic bacterial artificial chromosomes
(BAC) of Ranbp2 with impaired C-terminal chaperone and with
(Tg-Ranbp2WT-HA) or without PPIase activities (Tg-Ranbp2R2926A-HA) of
its CY domain followed by examination of the transgenic lines with
molecular, immunocytochemical and physiological approaches.
Results: Compared to wild-type mice, either transgenic line presents
selective deficits in M-opsin biogenesis with its accumulation
and aggregation in M- and M/S-cone photoreceptors, but without
proteostatic impairment of two novel Ranbp2 cyclophilin partners,
the cytokine-responsive effectors, STAT3/STAT5. Stress-induced
STAT3 activation and photoreceptor degeneration are also unaffected
in retinas of Tg-Ranbp2R2926A-HA::Ranbp2-/-. Conversely, proteomic
analyses found the heterogeneous ribonucleic protein A2B1, in which
©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
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ARVO 2014 Annual Meeting Abstracts
mutations cause multisystem proteinopathy and amyotrophic lateral
sclerosis (ALS), is down-regulated post-transcriptionally in inner
retinal neurons of only Tg-Ranbp2R2926A-HA::Ranbp2-/- mice. These
manifestations are accompanied by the age- and tissue-dependent
reductions of diubiquitin and ubiquitylated proteins, increased
deubiquitylation activity, and accumulation of the S1 and S5b
subunits of the 26S proteasome. These effects are also accompanied
by the significant shortening of the implicit times of the scotopic
visual evoked potentials.
Conclusions: These results unveil distinct mechanistic and biological
links between PPIase and chaperone activities of cyclophilin of
Ranbp2 towards proteostasis of selective retinal substrates and with
novel therapeutic potential.
Commercial Relationships: Paulo A. Ferreira, None; Kyoung-in
Cho, None; Hemangi Patil, None; Eugene Senda, None; Jessica
Wang, None; Haiqing Yi, None; Sunny Qiu, None; Dosuk Yoon,
None; Minzhong Yu, None; Neal S. Peachey, None
Support: NIH grants EY019492 & GM083165, Jules & Doris Stein
Research award (RPB), Foundation Fighting Blindness, Dept. of
Veterans Affairs (Cleveland Clinic)
Program Number: 415 Poster Board Number: C0186
Presentation Time: 8:30 AM–10:15 AM
The oral iron chelator deferiprone protects against iron overloadinduced retinal degeneration in Hepcidin knockout mice
Delu Song, Liangliang Zhao, Yafeng Li, Majda Hadziahmetovic,
Ying Song, Joshua L. Dunaief. Ophthalmology, Scheie Eye Institute,
University of Pennsylvania, Philadelphia, PA.
Purpose: To investigate the retinal protective effects of the oral
iron chelator deferiprone (DFP) in Hepcidin (Hepc) knockout mice
with age-dependent retinal iron accumulation and some pathologic
features of AMD.
Methods: Retinas from Hepc knockout mice, with or without
DFP in drinking water, were analyzed by fundus imaging,
electroretinography (ERG), histology, immunofluorescence and
quantitative PCR to investigate the protective effect of DFP on retinal
degeneration.
Results: In Hepc knockout mice, DFP protected against retinal
degeneration indicated by fundus imaging. ERG rod-a, -b and cone-b
wave amplitudes were significantly higher in DFP treated mice.
Plastic sections showed photoreceptor and retinal pigment epithelial
(RPE) cells were preserved in Hepc ko mice treated with DFP. The
mRNA level of oxidative stress-related gene heme oxygenase-1
was significantly decreased in DFP treated Hpec ko retinas.
Consistent with histologic results, the mRNA level of rhodopsin was
significantly higher in retinas treated with DFP. Immunolabeling with
L-ferritin antibody showed significantly decreased signals in RPE
and inner/outer segments. Additionally, autofluorescense in the RPE
layer in cryosections was significantly diminished by DFP which is
consistent with the fundus images.
Conclusions: The oral iron chelator DFP diminished retinal iron
levels and oxidative stress, providing significant protection against
retinal degeneration in Hepc ko mice. This indicates that iron
chelation could be a useful treatment for retinal disease involving
iron overload or oxidative stress.
Commercial Relationships: Delu Song, ApoPharma (F);
Liangliang Zhao, None; Yafeng Li, None; Majda Hadziahmetovic,
None; Ying Song, None; Joshua L. Dunaief, ApoPharma (F)
Support: Research to Prevent Blindness, NEI R01EY015240,
the FM Kirby Foundation, the Paul and Evanina Bell MacKall
Foundation Trust, a gift in honor of Dr. Lee F. Mauger
Program Number: 416 Poster Board Number: C0187
Presentation Time: 8:30 AM–10:15 AM
The target enzyme-interfacing domain in photoreceptor guanylyl
cyclase activating protein 1 (GCAP1)
Igor V. Peshenko1, Elena V. Olshevskaya1, Sunghyuk Lim2, James
Ames2, Alexander M. Dizhoor1. 1Pennsylvania College of Optometry,
Salus University, Elkins Park, PA; 2Department of Chemistry,
University of California, Davis, CA.
Purpose: Retinal guanylyl cyclase (RetGC) activating proteins
(GCAPs) regulate visual photoresponse and trigger various
congenital retinal diseases in humans, but GCAP interaction with
its target enzyme remains obscure. In the present study, we describe
a fine mapping of the residues in GCAP1 using global mutagenesis
of the surface-exposed residues combined with the functional tests
that allow distinguishing between the primary binding to the cyclase
versus its activation.
Methods: Myristoylated GCAP1 mutants expressed in E.coli were
purified and screened for their ability to activate RetGC1 in vitro in
comparison with the wild type GCAP1. Fluorescently tagged GCAP1
mutants were co-expressed in HEK293 cells with fluorescently
tagged RetGC1 and their association was tested in cyto using
confocal microscopy.
Results: In total, 107 residues, surface-exposed based on the
Ca2+GCAP1 crystal structure [1], were altered, mostly using single
point mutations. The side chains strongly affecting GCAP1 ability
to activate RetGC1 localized to a distinct patch on the protein
surface formed by non-metal binding EF hand 1, the loop and the
exiting helix of a metal-binding EF-hand 2 and the entering helix
of a metal binding EF-hand 3. Mutations in that region were able
to completely block activation of the cyclase without affecting Ca2+
binding stoichiometry of GCAP1 or the overall backbone fold of the
molecule. Most of the mutants that failed to activate RetGC1 also
failed to co-localize with RetGC1 in HEK293 membranes. However,
some GCAP1 mutants that completely failed to stimulate RetGC1 not
only retained their ability to bind RetGC1 in cyto, but also markedly
reduced RetGC1 activation by competing with the wild type GCAP1.
These findings indicate that besides the primary binding RetGC1
activation by GCAP1 requires additional secondary interactions.
Conclusions: The residues required for GCAP1 binding to RetGC1
form a distinct “binding patch” on one side of the molecule and also
contains at least two residues that are not essential for the primary
binding but affect secondary interactions required for the RetGC1
activation. References: Stephen et al. (2007) Structure 15, 1392-1402
Commercial Relationships: Igor V. Peshenko, None; Elena V.
Olshevskaya, None; Sunghyuk Lim, None; James Ames, None;
Alexander M. Dizhoor, None
Support: NIH grants EY11522; EY012347; Pennsylvania
Department of Health CURE Formula Grant
Program Number: 417 Poster Board Number: C0188
Presentation Time: 8:30 AM–10:15 AM
The Complex of Proteins Associated with BBS5 in Photoreceptor
Outer Segments
W Clay Smith, Susan N. Bolch, Donald Dugger, Reshmi M. Mathew,
Gina M. D’Ambrosio, J Hugh McDowell. Ophthalmology, University
of Florida, Gainesville, FL.
Purpose: We have previously shown that arrestin1 and arrestin4
associate with BBS5 in a light-dependent manner that is regulated by
the phosphorylation of BBS5 by protein kinase C. The goal of this
current project is to identify other proteins that are associated with
this complex of BBS5 and arrestin in rod outer segments.
Methods: BBS5 was isolated from rod outer segments using
immunoprecipitation with an anti-BBS5 monoclonal antibody
©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 pubs@arvo.org.
ARVO 2014 Annual Meeting Abstracts
cross-linked to Protein G-coated magnetic beads. The isolated
complex of proteins was identified by tandem mass spectrometry.
Non-specific interactions were discarded based on pull downs with
an irrelevant antibody. Potential interaction partners were validated
by immunohistochemistry and direct interaction studies using
reconstitution assays.
Results: The predominant proteins associated with BBS5 in
outer segments were elements of the cytoskeleton, including both
tubulin and actin. Also identified in this complex were arrestin1,
heterotrimeric G-protein subunits, cGMP-phosphodiesterase
holoenzyme, and several chaperone/heat-shock proteins.
Conclusions: Our findings indicate that the BBS5-arrestin
interaction in photoreceptor outer segments occurs as part of a larger
macromolecular complex associated with the cytoskeleton. This
complex includes scaffolding, signaling, and chaperone molecules,
suggestive of a functionally organized signaling complex.
Commercial Relationships: W Clay Smith, None; Susan N. Bolch,
None; Donald Dugger, None; Reshmi M. Mathew, None; Gina M.
D’Ambrosio, None; J Hugh McDowell, None
Support: NIH Grants EY014864 and EY021721, Research to
Prevent Blindness, and C.M. Overstreet Retinal Eye Disease ARMD
Research Fund
Program Number: 418 Poster Board Number: C0189
Presentation Time: 8:30 AM–10:15 AM
Analysis of the complex between transducin-α and UNC119 by
Small Angle X-ray Scattering (SAXS)
Pallavi Cheguru1, Anurima Majumder1, Gopalakrishna Kota1, Lokesh
Gakhar2, Nikolai Artemyev1. 1Molecular Physiology and Biophysics,
University of Iowa, Iowa City, IA; 2Biochemistry, University of Iowa,
Iowa City, IA.
Purpose: UNC119 is a protein partner of transducin-α (Gαt) shown
to be important for transducin trafficking in rods. We have examined
the solution structure of the UNC119/ Gαt complex by SAXS in
order to gain insights into the mechanism of transducin trafficking.
Methods: UNC119 and myristoylated chimeric Gαt were coexpressed in E. coli. A highly pure UNC119/ Gαt complex
with excellent monodispersity was obtained by a three-step
chromatographic procedure. SAXS data on the UNC119/ Gαt
complex were collected using different exposure times of synchrotron
radiation and varying concentrations of the complex. Combined rigidbody/ab initio modeling against the SAXS data was performed using
known atomic structures of UNC119 and Gαt.
Results: Analyses of the scattering curves for the protein complex
indicated a radius of gyration (Rg) of 34.7 Å and a maximum particle
dimension (Dmax) of 117 Å. The model of the UNC119/ Gαt
complex with the best fit to the SAXS data indicated rotation and
bending of the N-terminal α-helix (αN) of Gαt from its position in
the structure of heterotrimeric Gt. This conformation of αN allows a
considerably more compact overall conformation of the complex in
which the two molecules have additional interactions involving the
β3-β4 loop and the α5-helix/C-terminus of Gαt.
Conclusions: The SAXS solution structure of the UNC119/ Gαt
complex suggests a novel interface between UNC119 and Gαt. This
interface is potentially critical to the ability of UNC119 to solubilize
transducin from the membrane and facilitate its trafficking.
Commercial Relationships: Pallavi Cheguru, None; Anurima
Majumder, None; Gopalakrishna Kota, None; Lokesh Gakhar,
None; Nikolai Artemyev, None
Support: NIH RO1 EY-12682
Program Number: 419 Poster Board Number: C0190
Presentation Time: 8:30 AM–10:15 AM
Peripherin-2/rds Function for Outer Segment Structure
Andrew F. Goldberg1, 2, Melanie N. Gary1, Mark E. English1, Linda
Ritter1. 1Eye Research Institute, Oakland University, Rochester, MI;
2
Department of Biological Sciences, Oakland University, Rochester,
MI.
Purpose: Vertebrate vision relies on photon capture by the flattened
membranous disks that comprise rod and cone outer segments (OSs).
The mechanisms that generate and maintain the membrane curvature
required for normal disk morphology are not yet defined, although the
photoreceptor-specific integral membrane protein peripherin/rds (P/
rds) is thought to play a role. The aim of this study was to investigate
the recent proposal that an induced amphipathic helix (AH) within
the cytoplasmic C-terminal domain of P/rds can contribute to the
membrane curvature of OS disks.
Methods: Association of the P/rds C-terminal domain (CTER)
with membranes was assayed in vitro using extruded liposomes
in conjunction with purified recombinant proteins. A gradientbased floatation assay was used to characterize protein-membrane
interaction and derive partition coefficients. The contribution of this
domain to membrane curvature generation was assessed in cellulo,
using WT and mutant variants of P/rds expressed in cultured cells, in
conjunction with immunofluorescence analyses using laser scanning
confocal microscopy and ultrastructural analyses using electron
microscopy.
Results: CTER associated avidly with liposomes generated from
lipids extracted directly from purified OS membranes. It likewise
showed a strong association with liposomes generated from a mixture
of synthetic phospholipids based on OS disk membranes. In contrast,
a significantly weaker association was observed for liposomes
generated from a brain polar lipid extract. Lipid substitution and
protein mutagenesis studies found that avid association required
membrane surface defects (conically-shaped lipids) and was
promoted by (anionic) surface charge. Full-length P/rds and a
deletion mutant lacking only the inducible AH, were each released
from the cellular secretory pathway when expressed in cultured
cells, and accumulated within networks of high-curvature membrane
tubules.
Conclusions: We found lipid composition to be a key regulator
of P/rds cytoplasmic C-terminus interaction with membranes.
In particular, the results indicate an essential role for conicallyshaped phospholipids (such as DHA-containing species), which
introduce surface packing defects into membranes. These findings
are consistent with a hydrophobic insertion (wedging) model for
curvature generation, and support a model in which the C-terminal
AH contributes to disk rim formation/stability in parallel with other
protein domains.
Commercial Relationships: Andrew F. Goldberg, None; Melanie
N. Gary, None; Mark E. English, None; Linda Ritter, None
Program Number: 420 Poster Board Number: C0191
Presentation Time: 8:30 AM–10:15 AM
Retinitis Pigmentosa 2 regulates transport of isoprenylated
proteins to photoreceptor outer segments
Houbin Zhang1, 2, Li Jiang2, Christin Hanke3, 2, Wolfgang Baehr2.
1
Sichuan Academy of Medical Sciences &Sichuan Provincial
People’s Hospital, Chengdu, China; 2Ophthalmology, University of
Utah, Salt Lake City, UT; 3Biochemistry and Biology, University of
Potsdam, Potsdam-Golm, Germany.
Purpose: X-linked retinitis pigmentosa (XLRP) is a devastating form
of retinal degeneration, manifesting early in life with symptoms of
night blindness, visual field defects, and decreased visual function.
©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 pubs@arvo.org.
ARVO 2014 Annual Meeting Abstracts
In-vitro, RP2 functions as a GAP for the small GTPase ARL3, a GDI
displacement factor (GDF). Mutations in the Rp2 gene account for
approximately one quarter of all XLRPs. The purpose of this study
was to investigate the consequences of RP2 deletion and identify
mechanisms causative of XLRP.
Methods: Intracellular localization of RP2 in photoreceptors was
determined by neonatal electroporation of an RP2-EGFP expression
vector. An Rp2 knockout mouse was generated using a EUCOMM
ES cell line containing a gene trap in intron 1. The knockout mice
were characterized by Western blot, immunocytochemistry, and
electroretinography (ERG).
Results: RP2-eGFP was localized to the plasma membrane of inner
segments, axons and synaptic termini in photoreceptors, but not
in outer segments. The Rp2 gene knockout mice were viable and
developed normally. Ablation of Rp2 gene expression led to slowly
progressing degeneration of cone and rod photoreceptors as indicated
by ERG recordings. Scotopic a-wave and photopic b wave amplitudes
were reduced as early as one month of age in the knockout mice. The
Rp2Y/- ERG amplitudes were further reduced at 6 months of age.
Trafficking of transmembrane phototransduction proteins, including
cone opsins, to Rp2Y/- photoreceptors outer segments was normal
up to 14 months of age. While targeting of transducin α and βγ to the
Rp2Y/- outer segments was not affected in the knockout, transport of
rod and cone PDE6 as well as GRK1 to outer segments was impeded.
Conclusions: RP2 is distributed to plasma membrane of inner
segments and synaptic termini in photoreceptors. RP2 is not essential
for trafficking cone opsins and transducin to photoreceptor outer
segments, but regulates transport of isoprenylated proteins to
photoreceptor outer segments. Our results suggest that RP2/ARL3
may allosterically release prenylated proteins from their soluble
complex with PDE6D and unload them to donor membranes (e.g.,
TGN vesicles). ). In the Rp2 knockout, this process is impeded.
Commercial Relationships: Houbin Zhang, None; Li Jiang, None;
Christin Hanke, None; Wolfgang Baehr, None
Support: NIH EY008123
Program Number: 421 Poster Board Number: C0192
Presentation Time: 8:30 AM–10:15 AM
Arl3 rod-specific knockout displays RP-like rod photoreceptor
degeneration
Christin Hanke1, 2, Houbin Zhang3, 1, Cecilia Gerstner1, Jeanne
Frederick1, Wolfgang Baehr1. 1Ophthalmology, University of Utah,
Salt Lake City, UT; 2Physical Biochemistry, University of Potsdam,
Potsdam-Golm, Germany; 3Biology and Genetics, Sichuan Academy
of Medical Sciences & Sichuan Provincial People’s Hospital,
Chengdu, China.
Purpose: Arf-like protein 3 (Arl3) localizes predominantly in the
photoreceptor inner segment. Germline Arl3 knockout mice do not
survive beyond PN 21 and display multiple organ ciliary defects as
well as retinal regeneration (Schrick et al., (2006). Am. J. Pathol. 168,
1288-1298). We therefore generated rod-specific Arl3 knockouts to
elucidate the role of Arl3 in transport of photoreceptor membraneassociated proteins.
Methods: Knockouts containing a gene trap in intron 1 of the Arl3
gene were generated using a EUCOMM cell line. Breeding with Flp
mice, followed by mating with iCre75+ mice, generated rod-specific
knockouts. Photoreceptor function and retina morphology of wildtype (WT) and mutant mice were analyzed by confocal microscopy,
ERG and immunohistochemistry. An Arl3-specific polyclonal
antibody (Ab) was generated using a full-length recombinant Arl3
polypeptide expressed in bacteria.
Results: Western blot of WT retina with anti-Arl3-Ab identified
a 20 kDa protein, which was significantly reduced in two month-
old mutant (Arl3flox/flox;iCre75+) retina. Immunohistochemistry
revealed Arl3 localization predominantly in the inner segments
of WT photoreceptor cells. Arl3 immunoreactivity was absent
in homozygous rod knockouts, but still present in cones and the
inner retina. Scotopic and photopic ERGs of rod knockout and
WT mice at PN15 had comparable amplitudes suggesting normal
phototransduction. Retina histology of PN15 knockout mice was
comparable to WT. One month-old Arl3flox/flox;iCre75+ mice
showed reduced (80-90%) scotopic, but normal photopic ERG
responses. In retinas of two month-old knockout mice, scotopic ERGs
were extinguished, whereas cone ERGs were highly attenuated.
Retinas of one month-old homozygous knockout mice had 4-5
rows of nuclei in the ONL, and only one row in two month-old
mice. Immunohistochemistry of PN 15 and one month-old retina
sections revealed that rhodopsin transport, as shown by rho1D4
labeling of ROS, is normal. Rhodopsin was undetectable in two
month-old conditional knockout mice due to complete photoreceptor
degeneration.
Conclusions: Rod-specific knockout of Arl3 revealed rapidly
progressing photoreceptor degeneration, with knockout mice being
completely blind at two months of age. Outer segment development
appeared to be unimpaired by Arl3 deletion and rod photoreceptor
function was normal at P14.
Commercial Relationships: Christin Hanke, None; Houbin
Zhang, None; Cecilia Gerstner, None; Jeanne Frederick, None;
Wolfgang Baehr, None
Support: NIH Grants EY08123, EY014800-01A2, EY019298
Program Number: 422 Poster Board Number: C0193
Presentation Time: 8:30 AM–10:15 AM
Modulation of severity of RPGR-associated retinal degeneration
in mice due to mutations in RPGR-interacting proteins
Linjing Li1, Nageswara Rao Kollu1, Cecinio Ronquillo2, Hemant
Khanna1, Wolfgang Baehr2. 1Ophthalmology, UMASS Medical
School, Worcester, MA; 2Department of Ophthalmology and Visual
Sciences, John A. Moran Eye Center, Salt Lake City, UT.
Purpose: In humans, over 80% of X-linked retinitis pigementosa
(XLRP) is caused by mutations in RPGR. RPGR associated disease
is clinically heterogeneous, indicating involvement of genes that
can influence the associated phenotype. RPGR is known to interact
with selected ciliary proteins including CEP290, RPGRIP1, NPHP1,
NPHP4 and NPHP5. The purpose of this study is to assess the
contribution of these RPGR-interacting proteins on the severity of
RPGR-associated retinal degeneration in RpgrKO mice.
Methods: RpgrKO female mice were bred with male Cep290rd16/
rd16, Nphp1-/-, Nphp4nmf192/nmf192, Nphp5-/-, Rpgrip1-/-.
Males from F1 generation with genotype RpgrKO/Cep290+/rd16,
RpgrKO/Nphp1+/-, RpgrKO/Nphp4+/nmf192, RpgrKO/Nphp5+/-,
RpgrKO/Rpgrip1+/- were selected for further analysis. Structural
and function studies were performed using histology, transmission
electron microscopy (TEM), immunofluorescence staining, and
Electroretinopgraphy (ERG).
Results: The RpgrKO mice exhibit degeneration of retina and
relatively mild decrease in cone and rod function by 6 months of age.
Our analysis of double mutant mice (10-12 months) revealed that
RpgrKO/Cep290+/rd16 exhibit accelerated retinal degeneration as
compared to age-matched RpgrKO mice. TEM analysis of RpgrKO/
Cep290+/rd16 retina showed vesicle accumulation at the base of the
outer segments of photoreceptors, which were not detected in the
RpgrKO mice. We also detected decreased cone-specific staining of
M-opsin. No significant effect on the retina was observed in RpgrKO/
Nphp1+/-, RpgrKO/Nphp4+/nmf192, RpgrKO/Nphp5+/-, RpgrKO/
©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 pubs@arvo.org.
ARVO 2014 Annual Meeting Abstracts
Rpgrip1+/- (n=3) mice up to 6 months of age, as compared to
RpgrKO.
Conclusions: Our studies suggest that mutations in CEP290
can potentially influence the severity of retinal phenotype due to
mutations in RPGR. Further studies are in progress to assess the
influence of additional RPGR-interacting proteins on RPGR-disease.
Commercial Relationships: Linjing Li, None; Nageswara Rao
Kollu, None; Cecinio Ronquillo, None; Hemant Khanna, None;
Wolfgang Baehr, None
Support: NEI, FFB
Program Number: 423 Poster Board Number: C0194
Presentation Time: 8:30 AM–10:15 AM
Cysteine targets multiple phototransduction components
Tomoki Isayama1, Junchao Wu1, Vanessa Lee1, Anita L. Zimmerman2,
Clint L. Makino1. 1Ophthalmology, Mass Eye & Ear Infirmary,
Boston, MA; 2Molecular Pharmacology, Physiology and
Biotechnology, Brown University, Providence, RI.
Purpose: Sulfhydryl-binding and reducing agents affect the
photoresponse of vertebrate rods in a manner that suggests action at
multiple points in the phototransduction cascade. Thus, compounds
containing thiol groups such as the amino acid, cysteine, could alter
photoreceptor physiology by forming disulfide bonds. We probed for
such effects on salamander photoreceptors using electrophysiological
methods.
Methods: Photoresponses were recorded from the inner segment
of salamander rods and cones using the suction electrode method,
while perfusing the outer segment (OS) with different cysteine
analogs. D-cysteine, L-cysteine and D-cysteine with a methyl ester
moiety were used to test for stereo-specificity and to ensure access
to the interior of the OS, respectively. Dark-adapted and partially
bleached cells were recorded to test for effects on visual pigment
and apo-opsin. Patch clamp recordings of membranes excised from
salamander rod OSs were carried out to clarify the effects on the
cyclic nucleotide-gated (CNG) channel.
Results: Both D- and L-cysteine increased circulating current and
flash response size of dark-adapted rods, consistent with a higher
fraction of CNG channels in the open state. Partially bleached
cones exhibited a two-fold recovery of sensitivity, while recovery
of sensitivity was less pronounced in partially bleached rods. The
greater effect in cones suggested that cysteine might not easily
enter the rod OS. Indeed, when D-cysteine methyl ester was given
to partially bleached rods, there was a rapid increase in circulating
current, followed by a slower rise in current and recovery of absolute
sensitivity. The latter effect indicated relief of bleaching adaptation.
Unexpectedly, flash response kinetics sped up for cells given the
cysteine methyl ester. Patch clamp recording of rod OS membrane
showed increased currents in the presence of D-cysteine, confirming
a direct effect on the channels.
Conclusions: These results indicate that cysteine can affect at least
two components of the phototransduction cascade. First, cysteine
opens more CNG channels, resulting in larger responses. Second,
in partially bleached photoreceptors, cysteine appears to partially
quench ongoing cascade activation, as shown by the recovery
of sensitivity, possibly by suppressing the activity of opsin. The
acceleration of response kinetics in partially bleached cells by
cysteine methyl ester suggests that an additional cascade component
may be affected.
Commercial Relationships: Tomoki Isayama, None; Junchao Wu,
None; Vanessa Lee, None; Anita L. Zimmerman, None; Clint L.
Makino, None
Support: NIH EY011358, Lions of Massachusetts, Howe Laboratory
Endowment of the Massachusetts Eye & Ear Infirmary, Research to
Prevent Blindness, Inc.
Program Number: 424 Poster Board Number: C0195
Presentation Time: 8:30 AM–10:15 AM
Role of cAMP in the Recovery of the Cone Photoresponse in
Zebrafish Larvae
Jared D. Chrispell, Shoji Osawa, Ellen R. Weiss. Cell Biology and
Physiology, University of North Carolina at Chapel Hill, Chapel Hill,
NC.
Purpose: In the vertebrate retina, cAMP is known to play a role in
circadian rhythm and dysregulation of cAMP has been associated
with retinal degeneration. Light-dependent changes in cAMP levels
have been observed in rod inner segments in mice and cone inner
segments in goldfish, where they can influence the phosphorylation
state of proteins involved in phototransduction. The retina-specific
G protein-coupled receptor kinases Grk1 and Grk7 are substrates of
Protein kinase A (PKA), the major downstream effector of cAMP.
Elevated cAMP levels leads to increased phosphorylation of Grk1
in mice and Grk 7 in frogs – an indication of a link between cAMP
and proteins involved in phototransduction in cones. cAMP has
been studied most extensively in rods, where levels are found to
be high in the dark and low in the light. In zebrafish, cones mature
prior to rods and electroretinogram (ERG) analyses detect cone
photoresponses as early as 4 days post fertilization (dpf). In contrast,
ERG responses in rods appear between 15 and 21 dpf. Therefore, the
zebrafish retina at 4-7 dpf serves functionally as an all-cone model.
In order to better understand how changes in cAMP influence the
cone photoresponse in vertebrates, we employed the use of drugs that
increase intracellular cAMP levels and measured their effects on cone
recovery by ERG analysis of zebrafish larvae.
Methods: Zebrafish larvae (5 dpf) were incubated for 30 min in
forskolin (50 μM) or IBMX (1 mM). This was followed by coincubation for 5 min with L-AP4 (500 μM) to block inner retina
signaling and allow recording of the cone mass receptor potential.
Recovery was measured using a dual flash paradigm with increasing
interstimulus intervals.
Results: Zebrafish larvae exposed to forskolin, an activator of
adenylyl cyclase, display a decreased rate of recovery when
compared to untreated larvae. A similar result is obtained when larvae
are exposed to IBMX, a phosphodiesterase inhibitor.
Conclusions: Increased levels of intracellular cAMP in the zebrafish
all-cone retina result in decreased visual function, in the form a lower
recovery rate in response to bright flashes of light. This observation
is in agreement with our hypothesis that intracellular levels of cAMP
affect proteins critical for efficient recovery of the photoresponse
in cones. Further studies will be carried out to identify the specific
proteins involved and the mechanism by which they are affected.
Commercial Relationships: Jared D. Chrispell, None; Shoji
Osawa, None; Ellen R. Weiss, None
Support: NEI Grant EY022279, NEI Grant EY012224
Program Number: 425 Poster Board Number: C0196
Presentation Time: 8:30 AM–10:15 AM
Circadian regulated genes underlying retinal susceptibility and
resistance to light-induced damage
Alison C. Ziesel1, Daniel T. Organisciak2, Micah A. Chrenek1,
Paul Wong1. 1Ophthalmology, Emory University, Atlanta, GA;
2
Biochemistry and Molecular Biology, Wright State University,
Dayton, OH.
Purpose: Light-induced retinal damage has long served as a model
of retinal dysfunction and visual cell loss due to inherited disease or
©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 pubs@arvo.org.
ARVO 2014 Annual Meeting Abstracts
caused by oxidative stress. Numerous extrinsic factors are known
to influence the extent of visual cell loss. Visual cell damage is
also affected by intrinsic factors, including circadian rhythms. The
purpose of the current study is to screen for circadian related factors
that affect visual cell loss.
Methods: Gene profiles were generated for both dark-reared and
cyclic light-reared animals using standard methodologies. Retinal
tissues taken at 1 am, 9 am, and 5 pm were analyzed. For darkreared animals 1 am and 9 am define light-induced damage (LID)
susceptible periods, while 5 pm defines a LID resistant period. In the
case of cyclic light-reared animals 1 am defines a LID susceptible
period, while 9 am and 5 pm define LID resistant periods. We took
a comparative analysis to screen for changes at the level of gene
expression to see if there were any conserved events between LID
resistance and susceptibility. These results in turn were compared
from one profiling study to the other to examine for consistencies in
fold changes, FC.
Results: 13 genes were consistently repressed during the LID
susceptible state as compared to the resistant state. 17 genes were
consistently elevated during the LID susceptible state as compared to
the resistant state. We regard these 30 genes as those that putatively
define retinal susceptibility/resistance to light-induced damage and
an ontological interrogation of these genes was undertaken. Gene
products for 15 of the 30 LID susceptibility/resistance genes fall into
a single functional protein network. Included in this network is DRD4
the functional product of the dopamine receptor D4 gene (Drd4).
Conclusions: With respect to retinal susceptibility/resistance to
light damage we have found consistent changes in 30 genes. The
identification of Drd4 as one of these genes provides some validation
to the authenticity of these LID retinal susceptibility/resistance genes.
In mice with a disruption of the Drd4 gene, cyclic AMP levels in the
dark-adapted retina are significantly lower compared to wild-type
retina, unresponsive to light, and resistant to light-induced damage
(Dr. M. Iuvone, Emory University, personal communication, 2013).
Commercial Relationships: Alison C. Ziesel, None; Daniel T.
Organisciak, None; Micah A. Chrenek, None; Paul Wong, None
Support: This work was supported in part with funding from
NSERC, RPB, NIH (NEI) P30 EY006360, FFB, the Parker
Foundation and the Knights Templar of Georgia (PW) and NIH
(NEI) grant EY01959, the International Retina Research Foundation,
Ohio Lions Eye Research Foundation, and the Petticrew Research
Laboratory (DTO).
Program Number: 426 Poster Board Number: C0197
Presentation Time: 8:30 AM–10:15 AM
Metabolic Differences Between Light- and Dark-Adapted Mouse
Retinas
Ellen R. Weiss1, Shoji Osawa1, Suraj Dhungana2, Susan McRitchie2,
Susan Sumner2. 1Cell Biology & Physiology, The University of
North Carolina at Chapel Hill, Chapel Hill, NC; 2RTI International,
Research Triangle Park, NC.
Purpose: In normal retinas, production of large amounts of ATP, as
well as exposure to UV light and high levels of oxygen all give rise
to reactive oxygen species (ROS). Excess production of ROS can
damage proteins, lipids and DNA in photoreceptors. Additionally,
these cells have high levels of photosensitive retinoid derivatives
that are easily oxidized, becoming toxic to the cell. The goal of the
present work is to define metabolic changes that occur in retinas
from light- and dark-adapted mice. These results will be compared
with animal models of retinal degeneration where light is often an
exacerbating factor.
Methods: C57BL/6J mice raised under a normal light/dark cycle
were exposed to 250 lux for 3 h or adapted overnight in complete
darkness. Eyes from these animals were enucleated and the retinas
dissected in PBS, followed by rapid freezing in liquid nitrogen. Four
retinas (~15-20 mg) were treated as a single sa¬mple. Each sample
was homogenized in buffer containing 50:50 acetonitrile:water.
The homogenate was centrifuged, the supernatant was dried and
re-suspended in 95:5 H2O:methanol for UPLC-TOF-MS analysis.
Samples were chromatographed on a BEH HSST3 column
followed by analysis on a G-2 SYNAPT-QTOF mass spectrometer
equipped with the Acquity UPLC system. Data were analyzed using
TransOmics software to determine the group differentiating markers
of dark- and light-adapted retinas.
Results: Approximately 2,000 compound ions were detected.
Differences in the metabolic profiles of retinas from dark- and lightadapted animals were observed using Orthogonal Projections to
Latent Structure-Discriminant Analysis (OPLS-DA). Analysis of the
group differentiating markers using the Human Metabolome Data
Base revealed members of the retinol metabolism pathway as well as
several classes of lipids, amino acid derivatives and nucleotides that
were distinguished between the dark- and light-adapted retinas.
Conclusions: We evaluated the metabolic profile of wild type mice to
determine the normal metabolic changes that occur in the retina under
dark- and light-adapted conditions. Since light is an exacerbating
factor in a number of retinal degenerative diseases, our results serve
as a foundation for evaluating changes in metabolism that occur
in animal models for retinal degeneration. We anticipate that these
studies will reveal novel pathways leading to improved therapeutic
strategies.
Commercial Relationships: Ellen R. Weiss, None; Shoji Osawa,
None; Suraj Dhungana, None; Susan McRitchie, None; Susan
Sumner, None
Support: A grant supplement to 5 R01 EY012224 from the NIH
Common Fund (ERW) and 1U24DK097193-01 from the NIH
Common Fund administered through the NIDDK for the Regional
Comprehensive Metabolomics Resource Core at RTI International
(RTI RCMRC; SS).
Program Number: 427 Poster Board Number: C0198
Presentation Time: 8:30 AM–10:15 AM
The response of the retinal pigmented epithelium to a novel,
nanosecond laser, in vivo: comparison with a conventional
continuous wave laser
Marzieh Tahmasebi1, 2, Robert J. Casson1, 2, Glyn Chidlow1, 2, John P.
Wood1, 2, Malcolm J. Plunkett2. 1University of Adelaide, Adelaide, SA,
Australia; 2South Australian Institute of Ophthalmology, Adelaide,
SA, Australia.
Purpose: We have recently characterised the effects of a novel
nanosecond pulse laser, the Retinal Regeneration Therapy (2RT) laser
on the rat retina. It is believed, however, that the closely apposed
retinal pigmented epithelium (RPE), which is selectively targeted
by the 2RT laser, also plays an important role in the response of the
retina to laser treatment. We therefore sought to disseminate the
effects of the 2RT laser on the RPE cell layer, in situ.
Methods: Deeply anaesthetised pigmented Dark Agouti rats
were treated in one or both eyes with either the 2RT laser, or a
conventional, continuous wave (CW), non-RPE-selective laser, for
comparative purposes. Animals were euthanized immediately, or
after 6 hours, 24 hours, 3 days or 7 days. RPE whole-mounts were
prepared and the physical status of cells and expression of factors
of interest were delineated by immunohistochemistry and scanning
electron microscopy (SEM).
Results: Both lasers caused immediate ablation of RPE cells in
irradiated regions. In both cases, repair of the RPE cell-layer over the
subsequent 7 days occurred. Each laser also induced up-regulations
©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 pubs@arvo.org.
ARVO 2014 Annual Meeting Abstracts
in the expression of αβ-crystallin, heat shock 27kD protein
(HSP27), nestin, basic fibroblast growth factor (FGF-2) and ciliary
neurotrophic factor (CNTF) in RPE cells bordering irradiated regions.
In all cases expression of these factors peaked at 1-3 days after
laser-treatment. Interestingly, peak expression of such factors was
concurrent with the expression of the cellular proliferation marker,
proliferating cell nuclear antigen (PCNA). There were no significant
differences between the induced expression changes seen in any of
the factors of interest with either laser.
Conclusions: These findings demonstrate that either selective (2RT)
or non-selective (CW) laser treatment of the RPE can promote similar
gene expression changes within this cell layer in situ. Furthermore,
the spatiotemporal expression pattern of such factors is demonstrated.
Future research will explore the possibility of using the 2RT laser,
in particular, to induce expression of potentially protective factors
which may combat retinal disease, in situ.
Commercial Relationships: Marzieh Tahmasebi, None; Robert
J. Casson, None; Glyn Chidlow, None; John P. Wood, None;
Malcolm J. Plunkett, Ellex Medical Lasers (E)
Program Number: 428 Poster Board Number: C0199
Presentation Time: 8:30 AM–10:15 AM
Signaling Pathways Elicited By Light In Photoreceptor Nuclei
From Bovine Retina
Paola M. Natalini1, 2, Melina Mateos1, 2, Norma Giusto1, 2, Mónica
Ilincheta de Boschero1, 2. 1Instituto de Investigaciones Biquímicas
de Bahía Blanca (INIBIBB), Bahía Blanca, Argentina; 2Universidad
Nacional del Sur, Bahia Blanca, Argentina.
Purpose: Phototransduction occurs in the outer segments of
photoreceptor cells, and the signaling mechanisms elicited by light
stimulation, such as Insulin receptor (IR) activation, have been well
characterized. However, much less is known about the possible
consequences of these mechanisms of signal transduction at the
nuclear level in photoreceptor cells. The aim of this study was to
evaluate whether ex-vivo light exposure of bovine retinas modulates
different signaling pathways in nuclei from photoreceptor cells. The
activity and localization of diacylglycerol kinases (DAGK) and the
phosphorylation of extracellular signal-regulated kinase (ERK),
protein kinase C (PKC) and Akt as well as the presence of the IR in
the photoreceptor nuclei were studied.
Methods: Bovine eyes were adapted to darkness (2 h, 0° C), and
then eye cups were exposed to light (280 cd, 30 min) or to darkness.
To obtain photoreceptor nuclei, isolated retinas were homogenized
in 0.25 M sucrose, filtrated and purified by centrifugation onto a
discontinuous sucrose gradient. To evaluate the purification protocol,
immunofluorescence (IF), electronic microscopy (EM) and western
blot (WB) techniques were employed. Light-induced damage was
evaluated by IF (DNA damage) and by measuring the generation of
thiobarbituric acid reactive substances (TBARS). DAGKs (α, β, γ, ε,
and ζ isoforms), PKCα, Akt and ERK1/2 were detected by WB. IR
presence was detected by WB and IF. DAGKs activity was measured
using radioactive substrates.
Results: WB and IF revealed the presence of IR in photoreceptor
nuclei. Light exposure neither affected DNA fragmentation nor
increased TBARS levels in retinal homogenates. However, light
induced a differential localization and activation of DAGK isoforms
in photoreceptor nuclei with respect to the darkness condition. In
addition, an increase in the contents of phospho-PKCα (2.5 fold) and
phospho-Akt (3 fold) without changes in total PKCα and Akt was
seen in nuclei from light exposed retinas. A significant increase in
response to light in the content of total and phosphorylated forms of
ERK1/2 (2 fold) was also observed.
Conclusions: Our results demonstrate that light activates ERK
and signaling pathways linked to G-protein coupled receptors and
tyrosine kinase receptors in photoreceptor nuclei. Light-dependent
activation of IR, which is present in these nuclei, might be mediating
these effects.
Commercial Relationships: Paola M. Natalini, None; Melina
Mateos, None; Norma Giusto, None; Mónica Ilincheta de
Boschero, None
Support: IBRO International Travel Grants
Program Number: 429 Poster Board Number: C0200
Presentation Time: 8:30 AM–10:15 AM
Brief-Period Light Preconditioning: Effects of Intensity
Preston Girardot, Micah A. Chrenek, Priscila Cunha, Jana Sellers, J
M. Nickerson, Jeffrey H. Boatright. Ophthalmology, Emory School of
Medicine, Atlanta, GA.
Purpose: Preconditioning refers to exposure to stressful but nontoxic
stimuli that protects against subsequent toxic insult. We previously
reported that exposing mice to bright light for a brief period protects
against light-induced retinal degeneration. Here we investigated
whether there is a dose-response relationship between the intensity of
light used in preconditioning and the amount of resulting protection.
Methods: 129S2/SvPasCrl mice were obtained from Charles River
and kept in a 12:12 hour light: dark cycle. Mice were dark adapted
one day prior to light preconditioning. The day of preconditioning,
mice were treated with 1% atropine eye drops with a 30 min
dilation period, followed by a 4 h exposure to no preconditioning
(approximately 50 lux), 1000, 3000, and 5000 lux light. Twenty four
hours later, mice either exposed to dim light (50 lux) or bright toxic
light (50,000 lux) for 4 h. At least one week later, retinal function was
assessed by scotopic electroretinography (ERG) and visual acuity
was assessed by optokinetic tracking system (OKT).
Results: In mice that had no preconditioning, toxic light exposure
diminished mean ERG amplitudes to 27±3.7% (a-wave) and 23±3.2%
(b-wave) of dim controls (p<0.0001). Preconditioning using 3000
and 5000 lux light preserved ERG a-wave amplitudes (p<0.0005) and
preconditioning at all intensities preserved ERG b-wave amplitudes
(p<0.0001). The amount of preservation increased with increasing
light intensity (r2=0.96 and 0.97 for a- and b-wave amplitudes,
respectively). Preconditioning using 3000 and 5000 lux light did not
significantly alter ERG responses in mice not exposed to toxic light.
Exposure to toxic light significantly decreased visual acuity from
0.4981±0.0044 to 0.2675±0.037 cycles/degree (n=8, p<0.0001).
Visual acuity of mice exposed to toxic light was significantly greater
if with preconditioning (p<0.003, no preconditioning versus any
intensity) and was statistically indistinguishable in mice not exposed
to toxic light.
Conclusions: These data suggest that protective light preconditioning
can be as brief as a single 4 hour exposure and can be optimized
by altering intensity, all without diminishing retinal function.
This dosing approach provides an alternative to long-established
preconditioning approaches that require several days of cyclic
light exposure and may be confounded by inherent damage to
photoreceptors.
Commercial Relationships: Preston Girardot, None; Micah A.
Chrenek, None; Priscila Cunha, None; Jana Sellers, None; J M.
Nickerson, None; Jeffrey H. Boatright, None
Support: NEI R01 EY014026, NEI P30 EY06360, Research to
Prevent Blindness (RPB), The Abraham & Phyllis Katz Foundation
©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 pubs@arvo.org.