ARVO 2016 Annual Meeting Abstracts
225 Retina/RPE: Biochemistr and molecular biology
Monday, May 02, 2016 8:30 AM–10:15 AM
Exhibit/Poster Hall Poster Session
Program #/Board # Range: 1722–1765/D0118–D0161
Organizing Section: Biochemistry/Molecular Biology
Contributing Section(s): Anatomy/Pathology
Program Number: 1722 Poster Board Number: D0118
Presentation Time: 8:30 AM–10:15 AM
The Role of Free Radicals in Thermal, Light-driven and Enzymecatalyzed Isomerization of Retinoids
Tongzhou Xu, Quan Yuan, Joanna J. Kaylor, Avian Tsan,
Gabriel H. Travis. Stein Eye Institute, University of California, Los
Angeles School of Medicine, Los Angeles, CA.
Purpose: The visual chromophore for most opsin pigments is
11-cis-retinaldehyde (11-cis-RAL), which is isomerized to all-transretinaldehyde (all-trans-RAL) upon absorption of a photon. After
briefly stimulating visual transduction, vertebrate opsins dissociate
to yield free all-trans-RAL. Restoration of light sensitivity follows
re-isomerization of all-trans-RAL to 11-cis-RAL by enzymes of
the visual cycle, which include the isomerases, Rpe65 and DES1.
Retinoid isomerization also occurs in the absence of proteins upon
exposure to light or molecular iodine. In this study, we tested the
effects of spin trap and spin probe reagents on several modes of
retinoid isomerization, attempting to understand their mechanisms.
Retinoids contain a system of conjugated double bonds that stabilize
carbocation or radical-cation intermediates through electron
delocalization. If retinoid isomerization involves a free-radical
mechanism, the intermediate should be stabilized by spin trap and
spin probe reagents, which would inhibit the isomerization.
Methods: The effects of 4-hydroxy-TEMPO, DMPO, DMPIO,
PBN and PTMIO reagents on DES1 and Rpe65 retinoid isomerase
activities were tested in homogenates of HEK-293T cells that stably
express these proteins. We also tested tissue homogenates of chicken
retinas (for DES1) and RPE cells (for Rpe65). Inhibition of alltrans-retinol or all-trans-RAL isomerization catalyzed by iodine was
tested in n-heptane. Inhibition of all-trans-RAL photoisomerization
was tested in methanol using 380-nm monochromatic light, in the
presence of the same spin traps and probes.
Results: DES1-catalyzed retinol isomerization was strongly
inhibited by 4-hydroxy-TEMPO and PTMIO, while Rpe65-catalyzed
isomerization was inhibited by DMPIO, PBN and PTMIO. Iodinecatalyzed isomerization of retinol and retinaldehyde were inhibited
by 4-hydroxy-TEMPO and PTMIO. In contrast, photoisomerization
of retinaldehyde was not significantly inhibited by any reagents
tested.
Conclusions: Rpe65-, DES1-, and iodine-catalyzed isomerization
of retinol and retinaldehyde appear to involve free-radical
intermediate(s). Photoisomerization of retinaldehyde appears not to
involve a free-radical intermediate.
Commercial Relationships: Tongzhou Xu, None; Quan Yuan,
None; Joanna J. Kaylor, None; Avian Tsan, None;
Gabriel H. Travis, None
Program Number: 1723 Poster Board Number: D0119
Presentation Time: 8:30 AM–10:15 AM
Purification and biochemical characterization of full-length
human tyrosinase
Nicole J. Kus, Monika B. Dolinska, Yuri V. Sergeev. National Eye
Institute, National Institutes of Health, Bethesda, MD.
Purpose: The human tyrosinase (hTyr) is a Type I membrane bound
glycoenzyme located in the melanosome. Tyrosinase is responsible
for the production of melanin, catalyzing the initial and rate-limiting
steps in melanogenesis, namely the hydroxylation of L-tyrosine to
L-dihydroxyphenylalanine (L-dopa) and subsequent oxidation of
o-diphenol to L-dopaquinone. About ~300 mutations in the hTyr gene
are associated with oculocutaneous albinism (OCA-1), an autosomal
recessive disorder. Previously, the intra-melanosomal domain
of human tyrosinase (hTyrCtr) has been expressed, purified and
characterized in our group. Here, we purified and characterized the
hTyr and compared this protein with properties of the hTyrCtr.
Methods: Both hTyr and hTyrCtr were produced in T. ni larvae. The
membrane-bound hTyr was solubilized in the presence of 1.0% Triton
X-100. HisTrap affinity and size exclusion chromatography were
used in the purification process, where all buffers contained 0.1%
Triton X-100. The obtained fractions were treated with Proteinase
K (100 mg/mL) followed by Superose 12 10/300 gel filtration.
Production of the hTyr obtained a yield of >0.1 mg per 10 g of larval
biomass. hTyrCtr was purified as a regular soluble protein. Molecular
masses were determined from SEC profiles using Bio-Rad protein
standards. Michaelis-Menten kinetics measured using a Spectramax
i3 plate reader.
Results: Western Blot and SDS-Page showed the hTyr polypeptide
molecular weight from 67 to 98 kDa. The polydispersion caused
a range of molecular mass attributed to the variability of protein
glycosylation. The molecular mass of hTyr was obtained from a
single peak in the gel filtration chromatogram, approximately 74,131
Da, in comparison to hTyrCtr, which had a molecular mass of 56,742
Da. Activity of the protein was measured in the presence of detergent.
An active, approximately 10% glycosylated, hTyr protein was
obtained which had a Km of 0.769±0.267 mM, similar to hTyrCtr
(0.77±0.335 mM).
Conclusions: Full-length human tyrosinase was successfully over
expressed in T. ni larvae, solubilized, and purified in the presence of
Triton X-100. Similarity of the Michaelis constant suggests similar
ligand binding affinities for hTyr and hTyrCtr. Purified tyrosinase
is critical for the search of suitable activators of mutant variants in
treatment of genetic disorders, such as oculocutaneous albinism
(OCA-1), and dermatological disorders, like hyperpigmentation.
Commercial Relationships: Nicole J. Kus, None;
Monika B. Dolinska, None; Yuri V. Sergeev, None
Program Number: 1724 Poster Board Number: D0120
Presentation Time: 8:30 AM–10:15 AM
Mitochondrial Trafficking by Kinesin-Myosin-Cadherin Complex
in the RPE
Wan Jin Jahng1, Thagriki Dluya2, Weilue He3, O’Donnell Sylvester1,
Musa Neksumi4, Ji-Yeon Um5, Srinivas R. Sripathi6. 1Retina
Proteomics Lab, American University of Nigeria, Yola, Nigeria;
2
Biochemistry, MAUTECH, Yola, Nigeria; 3Biomedical Engineering,
Michigan Tech, Houghton, MI; 4Chemistry, MAUTECH, Yola,
Nigeria; 5Optometry, SNUST, Seoul, Korea (the Republic of);
6
Ophthalmology, The Johns Hopkins University, Baltimore, MD.
Purpose: How mitochondria communicate with the nucleus in
apoptosis remain an intriguing question. The current study tested
the hypothesis that mitochondrial trafficking could be determined
by the retrograde signaling complex under stress conditions. To test
our hypothesis, normal and aberrant mitochondrial networks were
examined quantitatively based on mitochondrial size, shape, position,
composition, and dynamics.
Methods: The mitochondrial trafficking complex was isolated by
immunoprecipitation using a prohibitin antibody. To understand
mitochondrial structure and function, twelve categories, including
(1) mitochondrial area, (2) cellular area, (3) mitochondrial content,
(4) perimeter, (5) circularity, (6) average perimeter, (7) average
mitochondrial area, (8) average circularity, (9) area/perimeter, (10)
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ARVO 2016 Annual Meeting Abstracts
area/perimeter normalized to minor axis, (11) minor axis, (12) area/
perimeter normalized to circularity, were calculated using ARPE19 cells under oxidative stress. Mitochondrial localization and
morphology were examined by immunocytochemistry.
Results: The mitochondrial trafficking complex- kinesin, unknown
protein at 110 kDa (myosin head motor domain, SH3 domain, ATP
binding domain), unknown protein at 88 kDa (cadherin repeat,
Ca2+ binding)- regulates mitochondrial size, morphology, and
compositions. Kinesin-myosin-prohibitin interaction is involved in
anterograde mitochondrial trafficking, whereas PKU beta S/T kinasemyosin-PI3K-lamin B2 bindings regulate an energy demanding
retrograde transport of mitochondria. Prohibitin binding with a
trafficking protein complex may regulate the bidirectional transport
of mitochondria along actin microfilaments and microtubules.
The mitocondrial trafficking complex may imply that a specific
mechanism of communication may exist in the ATP and Ca+2
demanding regions. Total area of mitochondria decreased in 4050% and both perimeter/circular mitochondria were downregulated
up to 60-70%. Area/perimeter normalized to circularity ratio of
mitochondria was decreased to 63% under oxidative stress.
Conclusions: Mitochondrial dysfunction, altered dynamics, impaired
transport, and turnover perturbation are associated with AMD.
Impaired mitochondrial transport decreases the release of healthy
mitochondria to distal processes, and disrupted eliminations of
injured mitochondria may cause energy reduction and alteration of
Ca2+ concentration.
Commercial Relationships: Wan Jin Jahng; Thagriki Dluya,
None; Weilue He, None; O'Donnell Sylvester, None;
Musa Neksumi, None; Ji-Yeon Um, None; Srinivas R. Sripathi,
None
Program Number: 1725 Poster Board Number: D0121
Presentation Time: 8:30 AM–10:15 AM
Retinoid x receptor (RXR) expression in the rodent retina and
effects of its modulation in neuronal cells
Yogita Dheer1, Nitin Chitranshi1, Roshana Vanderwall1,
Stuart L. Graham1, 2, Vivek Gupta1. 1FMHS, Macquarie university,
Sydney, NSW, Australia; 2University of Sydney, Save Sight Institute,
Sydney, NSW, Australia.
Purpose: Retinoid x receptors (RXR) belong to the family of nuclear
receptors comprising RXR α, β and γ isoforms which are well
expressed in the brain, however minimal data exists regarding their
expression and distribution in the retina. The exact physiological role
of RXRs has yet to been fully elucidated but they are believed to play
important role in regulating transcriptional signaling and apoptosis.
The purpose of this study was to investigate the presence and
distribution of RXRs in the rodent retina. In addition, RXR activation
was evaluated by treating neuronal cells with an RXR agonist.
Methods: Immunofluorescence (IF) staining using specific antibodies
and DAPI was used to determine the expression of each of the RXR
α, β and γ isoforms in various layers of the C57BL/6 mice retinas
(n=3). A combination of western blotting (WB) and IF techniques
was also used to establish expression of various RXRs in the SHSY5Y neuronal cell line. The biological effects of activation of RXRs
on cell viability was determined by treating cells with RXR agonist
bexarotene (12 hrs) in a concentration range of 0.001 µM to 10 µM.
Results: Differential immuno-reactivity against RXR α, β and γ
isoforms was observed in the normal mouse retina. RXRα was
observed to localise mainly to the ganglion cell layer (GCL) and the
inner nuclear layer (INL) while γ immuno-reactivity was primarily
detected in the GCL. We observed only a weak expression of RXR
β isoform in the retina. Expression of each of the various RXRs
was established in the SH-SY5Y neuronal cells using WB and
IF, mainly perinuclear in location. Densitimetric quantification
using WB revealed that bexarotene treatment (0.1-1.00 µM range)
resulted in increased expression of the α, β, γ receptors compared
to controls (p<0.05) (5 replications). Treatment with higher agonist
concentrations (5-10 µM range) resulted in significantly reduced
neuronal cell viability (p<0.05).
Conclusions: Our initial findings reveal the expression of RXRs
within SH-SY5Y cells and in the laminar structure of mouse retina.
Bexarotene treatment suggests that RXRs can be targeted and
upregulated by using this specific agonist. Experiments are underway
to further localize these receptors to specific cell types within the
retina and determine their role and potential use in neuroprotective
strategies.
Commercial Relationships: Yogita Dheer, None; Nitin Chitranshi,
None; Roshana Vanderwall, None; Stuart L. Graham, None;
Vivek Gupta, None
Program Number: 1726 Poster Board Number: D0122
Presentation Time: 8:30 AM–10:15 AM
Modulation of cathepsin L expression and NF-kB signalling
pathway effectors by advanced glycation end-products in retinal
pigment epithelial cells
Nur Musfirah Mahmud1, Umar Sharif1, Paul Kay1,
Tengku Ain Fathlun Tengku Kamalden2, Yit C. Yang3,
Simon P. Harding1, Luminita Paraoan1. 1Eye and Vision Science,
University of Liverpool, Liverpool, United Kingdom; 2University of
Malaya, Kuala Lumpur, Malaysia; 3Department of Ophthalmology,
Wolverhampton Med Inst-New Cross, Wolverhampton, United
Kingdom.
Purpose: We have previously demonstrated that an accumulation of
advanced glycation end-products (AGEs) in the Bruch’s membrane
(BrM) can alter retinal pigment epithelium (RPE) lysosomal activity
by changing expression of key effectors and inhibitors. Altered
activity of lysosomal cysteine proteases, the cathepsins, can in turn
impact signalling via the NF-kB pathway. The aim of this study
therefore was to analyse the effects of AGEs on specific lysosomal
cathepsins, and the endogenous levels of effectors of the NF-kB
signalling pathway in RPE.
Methods: ARPE-19 cells were cultured on AGE-containing BrM
mimics in vitro for 7-14 days. Intracellular processing of the
cysteine proteases cathepsins B, L and S were assessed by qPCR
and immunoblotting, while their intracellular activity was assessed
using fluorescence-based cleavage assays. Expression of NF-kB
(p65) and its main regulatory protein, IκBα, was assessed by qPCR
and immunoblotting. Statistical analysis was performed using the
independent T-test.
Results: Levels of the active form of cathepsin L were significantly
decreased following AGE exposure (33%, p=<0.027). This decrease
was also manifested as a decrease (36%, p=0.02) in its intracellular
activity. Cathepsin S active form decreased by 74% (p=0.004), yet
this change had no effect on its activity. Transcript expression of
cathepsins L and S was unaltered, suggesting effects on protein
processing rather than at the transcriptional level. Expression,
processing and activity of cathepsin B were unaltered. Under the
same conditions, NF-κB p65 and Ser536P p65 protein levels were
reduced by 37% (p=0.0003) and 52% (p=0.03) respectively. In
addition, IκBα expression was significantly down regulated (31%,
p=0.02).
Conclusions: AGE accumulation in the BrM, a common
consequence of the ageing process, affects the expression and activity
of the key lysosomal effector, cathepsin L. Following exposure to
AGEs, the overall endogenous levels of key effectors of the NFkB signalling pathway are also decreased. These results, together
These abstracts are licensed under a Creative Commons Attribution-NonCommercial-No Derivatives 4.0 International License. Go to http://iovs.arvojournals.org/
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ARVO 2016 Annual Meeting Abstracts
with data from previous studies, support the hypothesis that these
processes are related. Altered NF-kB-driven transcription affects
expression and secretion of pro- and anti- inflammatory cytokines
by the RPE, potentially contributing to drusen biogenesis and AMD
pathogenesis.
Commercial Relationships: Nur Musfirah Mahmud, None;
Umar Sharif, None; Paul Kay, None; Tengku Ain Fathlun Tengku
Kamalden, None; Yit C. Yang, None; Simon P. Harding, None;
Luminita Paraoan, None
Support: University of Malaya Scholarship
Program Number: 1727 Poster Board Number: D0123
Presentation Time: 8:30 AM–10:15 AM
Robust automated processing and analysis of images of RPE cells
Priyanka Priyadarshani1, Shuman Guo2, Kevin Donaldson1,
Haitao Huang2, Micah A. Chrenek1, Hans E. Grossniklaus1, Yi Jiang2,
J M. Nickerson1. 1Opthalmology, Emory University, Athens, GA;
2
Mathematics and Statistics, Georgia State Uinversity, Atlanta, GA.
Purpose: This study aimed to develop an automated method
for analyzing large series of retinal pigment epithelium (RPE)
cells in flat mounts. Bulk analysis of such data sets helps us to
measure individual cell morphometry, and to determine texture
and spatial point properties of RPE sheets that reflect cell-cell and
cell-environment interactions and cell organization in normal and
diseased eyes.
Methods: RPE flat mounts were compared from two groups of mice,
rd10 and C57BL/6J at different ages (P30, P45, P60 and P100). The
RPE sheet was prepared and imaged as before (Boatright et al. Mol
Vis 2015; 21:40-60). Images were processed using MATLAB scripts
and an automated “Cutbox selector”. CellProfiler software was used
to perform morphometric analysis of cell shape, area, number of
neighboring cells, and 20 other metrics.
Results: A major bottleneck in processing images is to select parts
of the RPE sheet that lacked dissection, processing, and staining
artifacts. Manual selection was time-consuming. We implemented
an automated method based on statistical analysis using k-means
clustering of the tissue patterns to automatically generate and select
artifact-free boxes from the RPE sheet. Further, the scripts numbered
the boxes, and collected x,y coordinates automatically, and organized
them for input into CellProfiler version 2.1.1, which then performed
downstream analysis automatically. While the manual selection
method took 1.5-2.5 hours for each eye, our automated counterpart
reduced completion time to a few seconds. With this method,
coverage of the whole RPE sheet was increased from about 20% to
50%. This method allows almost completely automated processing
once images were collected, ensuring collection of more quantitative
and unbiased data.
Conclusions: Manual analysis of image files, such as images of
retinal pigment epithelium (RPE) sheet, is difficult and in many
cases, quantitative manual analysis of cell shapes is impossible. The
risk of human errors, heavy time commitment, and sampling bias
are just some of the drawbacks of manual implementations. This
automated method proved useful for the analysis of RPE cells in flat
mounts with greater accuracy, more efficient sampling of data points,
and less human bias and errors. Our automated tool has become an
integral part of our RPE sheet and cell analysis system. This approach
would work well with many other histology or EM image sets.
Commercial Relationships: Priyanka Priyadarshani;
Shuman Guo, None; Kevin Donaldson, None; Haitao Huang,
None; Micah A. Chrenek, None; Hans E. Grossniklaus, None;
Yi Jiang, None; J M. Nickerson, None
Support: NIH R01EY016470, R01EY021592, P30EY06360,
an internal pilot grant from the Neuroscience Initiative at Emory
University, and an unrestricted grant to the Emory Eye Center from
Research to Prevent Blindness, Inc
Program Number: 1728 Poster Board Number: D0124
Presentation Time: 8:30 AM–10:15 AM
Expression of an N-terminal GARP region truncation of CNG
channel β-subunit on a KO background partially rescues
structure/function and alters calcium feedback
Steven J. Pittler1, Youwen Zhang2, Alex S. McKeown1,
Timothy W. Kraft1, Boris Reidel3, Vadim Y. Arshavsky3,
Marie E. Burns4, Marci L. DeRamus1. 1Department of Vision
Sciences, Vision Science Research Center, University of Alabama at
Birmingham, School of Optometry, Birmingham, AL; 2Department of
Ophthalmology, Mayo Clinic, Rochester, MN; 3Albert Eye Research
Institute, Duke Eye Center, Duke University, Durham, NC; 4Center
for Neuroscience, UC Davis, Davis, CA.
Purpose: The rod cGMP-gated cation channel β-subunit and two
associated GARP proteins encoded by the Cngb1 locus are required
for normal phototransduction, disk morphogenesis, and rod structural
integrity. Deletion of the promoter region and first coding exon in
mice (X1 KO) eliminates expression of these proteins and leads to
structural/ functional photoreceptor deficits and retinal degeneration.
The aim of this study is to determine if a truncated β-subunit (Tβ) can
substitute for full length β in the X1 KO mouse retina.
Methods: The murine β subunit is composed of 1326 aa, of which
550 correspond to GARP1 and 326 to GARP2. We introduced a 285
aa N-terminal truncation product (Tβ) into WT mice (WT Tβ) and
crossed the transgene allele onto the X1 KO background (X1 Tβ).
Mice were assessed by OCT, histology, EM Western, ERG, and rod
single cell recordings.
Results: At 1 month, OCT and light histology in X1 Tβ mice was
normal, but by four months EM revealed overgrowth of rod outer
segments (OS). By 8 months, OS were widely spaced and discs not
as tightly packed. In WT Tβ mice the transgene localized exclusively
in the rod photoreceptor in both inner and outer segments, with levels
3-fold above WT levels. At 1 month, functional recovery assessed
by ERG was normal. Rod single cell suction electrode recordings
also showed that the X1 Tβ rods were functioning similarly to WT,
with similar dark currents and collecting areas. However, X1 Tβ rods
displayed greater dark calcium levels; indicated by a 2-fold larger
Na+/Ca++, K+ exchange current that led to larger than normal single
photon responses, smaller Io, and slower kinetics.
Conclusions: Tβ expression partially restores structure/function in
the X1 KO mouse. This partial recovery does not persist for the life
of the animal, indicating that the N-terminus of the β-subunit, and
possibly GARPs are required for sustained normal structure/function.
The truncated β-subunit is sufficient to at least partially establish
a plasma/disk membrane connection and significantly improve the
photoresponse compared to X1 KO mice. Lastly, higher than normal
calcium levels may be indicative of abnormal calcium feedback in the
X1 Tβ mouse that may normally be mediated by the low affinity, high
capacity Ca++-binding GARP region.
Commercial Relationships: Steven J. Pittler, None;
Youwen Zhang, None; Alex S. McKeown, None;
Timothy W. Kraft, None; Boris Reidel, None;
Vadim Y. Arshavsky, None; Marie E. Burns, None;
Marci L. DeRamus, None
Support: UAB School of Optometry, UAB Vision Science Research
Center, P30 EY003039 and R01 EY018143 (SJP), R01 EY012859
and R01 EY005722 (VYA), and R01 EY014047 (MEB).
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ARVO 2016 Annual Meeting Abstracts
Program Number: 1729 Poster Board Number: D0125
Presentation Time: 8:30 AM–10:15 AM
An Usher Syndrome Type IIA knockin model leads to hair cell
abnormalities, light dependent retinal dysfunction, and late-onset
retinitis pigmentosa
Muna I. Naash1, Michael A. Gratton2, Maggie Mwoyosvi3, 1.
1
Biomedical Engineering, University of Houston, Houston, TX;
2
Otolaryngology, University of Saint Louis, Saint Louis, MO; 3Cell
Biology, University of Oklahoma Health Sciences Center, Oklahoma
City, OK.
Purpose: Usher syndrome (USH) is a devastating disease that leads
to combined deafness and blindness; the disease mechanisms of this
sensory loss remain elusive. The c.2299delG mutation in usherin is
the most common cause for USH type 2A in patients. To study the
role of usherin in audiovisual impairment we have generated and
characterized a knockin (KI) mouse model expressing the human
c.2299delG mutation.
Methods: The c.2299delG mutation causes a frame shift
and premature stop codon. Message and protein levels were
evaluated by RT-PCR and immunoblotting. Transmission electron
microscopy (TEM), scanning electron microscopy (SEM) and
immunohistochemistry (IHC) were used to evaluate retinal and
cochlear structure. Electroretinography (ERG) was used to assess
retinal function in animals reared in both 30 lux and 1200 lux 12hr
light/12hr dark cyclic lighting conditions.
Results: The KI mutation is present in the endogenous locus,
truncated message is stable, and the mutant protein is expressed
in photoreceptors and hair cells. SEM evaluation of the cochlea
showed outer hair cell abnormalities consistent with hearing loss at
postnatal day 180 (P180). Retinal evaluations via IHC and ERG up
to P180 appear normal; however there is a delay in transducin and
arrestin translocation upon light exposure as early as P30. KI mice at
P360 have a significant decline in scotopic amplitudes and delay in
scotopic ERG recovery as early as P30. Furthermore, KI mice reared
in a higher cyclic lighting condition (1200 lux) show a rescue of the
delay in scotopic recovery and arrestin translocation at P30. This
light dependent rescue seen in KI animals is only evident at P30, not
in older animals, and is dependent on mice being born in the higher
lighting conditions (1200 lux).
Conclusions: This mouse is the first USH2A model with a human
mutation. This model also shows hair cell abnormalities and retinal
degeneration. Data are consistent with USH patients having a later
onset visual impairment compared to hearing loss. Interestingly, this
is the first USH model to show a light dependent rescue of retinal
dysfunction at early ages. Full characterization of this model will lead
to a better understanding of the human disease and will be a valuable
resource to develop targeted therapies for treatment.
Commercial Relationships: Muna I. Naash, None;
Michael A. Gratton, None; Maggie Mwoyosvi, None
Support: Foundation Fighting blindness, Fight for Sight Summer
Fellowship, and NEI (EY10609, EY18656, EY022778).
Program Number: 1730 Poster Board Number: D0126
Presentation Time: 8:30 AM–10:15 AM
Chicken Embryonic Retina as a Model for Studying the Influence
of Diet on VLC-PUFAs During Development
Aruna Gorusupudi, Binxing Li, Yumna Subhani, Rajalekshmy Shyam,
Paul S. Bernstein. Department of Opthamology and Visual sciences,
Moran Eye Center, Salt lake city, UT.
Purpose: Recently, it has been shown that a new class of nondietary polyunsaturated fatty acids (C>26), the very long-chain
polyunsaturated fatty acids (VLC-PUFAs), are specifically present
in vertebrate retina. VLC-PUFA deficiency may be a key factor in
dominant Stargardt disease 3 (STGD3), which leads to vision loss in
children. We hypothesize that changes in diet influence the LC-PUFA
levels in chicken egg yolk, which in turn could affect the VLC-PUFA
levels in developing chicken retinas. We selected this model because
of its relative procedural simplicity, larger embryonic eye size
compared to rodents, and because it provides an optimal setting to
study developmental changes.
Methods: Eggs were incubated, and the embryos were dissected
from embryonic development days 15 (E15) through 21 (E21)
to collect developing eyes. Eyes were dissected under a light
microscope to separate retina and RPE/choroid. Retina and RPE/
choroid were separately extracted using a standardized method to
isolate their fatty acid methyl esters, which were then analyzed by
GC-MS (electron ionization mode). Analytical Method A was used to
analyze the LC-PUFAs, and Method B was used to analyze C24-C36
VLC-PUFAs.
Results: The VLC-PUFA (C28-C34) levels (%) in developing
chicken retinas increase significantly from E15 (0.0108 ± 0.0007) to
E21 (0.0131 ± 0.0002), while RPE/choroid is devoid of VLC-PUFAs.
The retinal n-3/n-6 VLC-PUFA ratios also increased significantly
from E15 (0.22 ± 0.02) to E21 (0.31±0.006). In addition, our results
showed an 18% increase in retinal DHA levels and a 12% increase in
n-3/n-6 LC-PUFA ratios from E15 to E21, while n-3/n-6 VLC-PUFA
precursor ratios decreased by 27%.
Conclusions: The results of this study suggest that chicken embryos
could serve as a promising new model for studying VLC-PUFA
synthesis in retina. In the present study, we have observed an increase
in the VLC-PUFA (C28-C34) levels in the embryonic chicken retinas
starting from E15 through E21. Further experiments with n-3 fatty
acid enriched eggs will help us to better understand how maternal
diet plays a role in altering n-3/n-6 VLC-PUFA ratios and levels in
developing retina and may provide further support for the addition of
omega-3 polyunsaturated fatty acids in prenatal vitamins to enhance
infant ocular health.
Commercial Relationships: Aruna Gorusupudi, None;
Binxing Li; Yumna Subhani, None; Rajalekshmy Shyam, None;
Paul S. Bernstein, None
Support: Research to Prevent Blindness. NIH Core Grant EY14800.
Program Number: 1731 Poster Board Number: D0127
Presentation Time: 8:30 AM–10:15 AM
VAMP7 as a regulator of rhodopsin transport carrier fusion in
rod photoreceptors
Vasundhara Kandachar1, Beatrice M. Tam2, Orson L. Moritz2,
Dusanka Deretic1. 1Department of Surgery/Division of
Ophthalmology, University of New Mexico School of Medicine,
Albuquerque, NM; 2Department of Ophthalmology and Visual
Sciences, University of British Columbia, Vancouver, BC, Canada.
Purpose: The identity of the R-SNARE involved in the fusion of
rhodopsin transport carriers (RTC) that pairs with Qabc-SNAREs
Syntaxin 3 and SNAP-25 on the rod inner segment plasma membrane
to deliver rhodopsin to the outer segment of rod photoreceptors is
unknown. We have tested vesicle-associated membrane protein 7
(VAMP7) as a candidate R-SNARE. VAMP7 consists of a regulatory
longin domain (LD), a SNARE motif and a transmembrane domain.
LD is phosphorylated at Tyr-45 by c-Src kinase, which activates both
SNARE binding and exocytosis.
Methods: Immunoprecipitation (IP) using post-nuclear supernatant
from frog retinas was performed. Multiple constructs of VAMP7-GFP
fusion proteins were expressed in Xenopus laevis photoreceptors
under the control of Xenopus rhodopsin promoter. A combination
of proximity ligation assay (PLA), immunostaining and in vitro
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ARVO 2016 Annual Meeting Abstracts
biochemical interaction studies were used to identify the localization
of VAMP7 and its potential interacting partners.
Results: We have examined VAMP7 regulation during formation,
trafficking and targeting of RTCs to the fusion site. Based on the
IP data, VAMP7 forms a complex with Syntaxin 3 and SNAP-25.
Immunostaining and PLA show that VAMP7 co-localizes with
Syntaxin 3 at the RTC fusion site. Based on immunostaining, VAMP7
also co-localizes with rhodopsin, both in the Golgi/TGN and on
RTCs. The localization of the phosphomimetic GFP-VAMP7 (Y45E)
and GFP-VAMP7-ΔLD in transgenic Xenopus are similar to that
of endogenous VAMP7, whereas the non-phosphorylatable GFPVAMP7 (Y45F) mutant is retained in the Golgi. The GFP-VAMP7
(R150E) with a mutation in the SNARE motif shows aberrant
localization in or around the Golgi while the double mutant (Y45E/
R150E) localizes predominantly to the plasma membrane. Based on
protein interaction and PLA assays, VAMP7 interacts with Rab11,
Rabin8 and Rab8, which form the RTC targeting complex.
Conclusions: The ability of VAMP7 to form a complex with
Syntaxin 3 and SNAP-25, along with its localization on RTCs at the
fusion site, strongly suggest that VAMP7 is the R-SNARE involved
RTC fusion. The Tyr-45 phosphorylation plays a critical role in
the subcellular localization of VAMP7. The R150E mutation in the
SNARE motif appears to preclude its interaction with the components
of the Rabin8-Rab11-Rab8 targeting complex.
Commercial Relationships: Vasundhara Kandachar, None;
Beatrice M. Tam, None; Orson L. Moritz, None; Dusanka Deretic,
None
Support: EY12421, CIHR-MOP64400, NSERC, Foundation
Fighting Blindness-Canada
freezing medium and frozen in liquid nitrogen. Sagittal cross-sections
were cut at 10 μm. We performed indirect immunofluorescence
(IF) for beta-, gamma-, delta-, and epsilon-sarcoglycans. Also,
hematoxylin-eosin (H&E) staining on the tissues was performed.
Results: We compared WT against SGCD-null mice in regards
of retinal architecture by means of H&E staining. In figure 1, we
observe that all layers appear frailer. Also, there is a notably decrease
on the thickness in the inner plexiform layer (IPL). After treatment,
SGCD null mice show a discrete increase in thickness in the IPL
and the cross-sections appear more firm. Overall, SGC IF staining
exhibits an up-regulation after (-)-epicatechin treatment in both WT
and null.
Conclusions: Taken together all the results we suggest that
SGCD null mice is a model of RD. Furthermore, we propose the
(-)-epicatechin regimen as a possible treatment for RD as it can reestablish in part the retinal architecture in this model. Also, there are
several changes in distribution and expression of the SGC proteins in
the retina of SGCD (-/-) mice.
Program Number: 1732 Poster Board Number: D0128
Presentation Time: 8:30 AM–10:15 AM
Changes in the sarcoglycan complex and effects of (-)-epicatechin
in SGCD-null mice as a potential animal model for retinal
degeneration
Andric C. Perez-Ortiz1, Gabriela Solano-García2,
Alexandra Luna-Angulo2, Ramon M. Coral-Vazquez3,
Yonathan Garfias6, Viridiana Garcia-Perez4,
Sergio De los Santos-Enriquez4, Alvaro Rendon5,
Israel Ramirez-Sanchez1, Francisco J. Estrada-Mena1. 1Laboratory of
Molecular Biology, Universidad Panamericana School of Medicine,
Benito Juarez, Mexico; 2Department of Neuroscience, Instituto
Nacional de Rehabilitación, Distrito Federal, Mexico; 3Escuela
Superior de Medicina, Instituto Politécnico Nacional, Distrito
Federal, Mexico; 4Centro médico nacional 20 de noviembre, ISSSTE,
Distrito Federal, Mexico; 5Institut de la Vision, Paris, France;
6
Instituto de Oftalmologia F.A.P. Conde de Valenciana, I.A.P., Distrito
Federal, Mexico.
Purpose: Molecular underpinnings of retinal degeneration (RD)
are poorly understood, there is an increasing need of therapeutic
molecules to revert or prevent further damage in patients. The
aim of this work is to investigate the changes in expression of the
sarcoglycan complex (SGC) in a delta-sarcoglycan (SGCD) null
mice as a potential model for RD. We also tested the effects of
(-)-epicatechin as a treatment option.
Methods: Sixteen C57BL/6J sgcd-/- mice and 12 C57BL/6J wild
type (WT) mice were equally divided into two groups. All animals
were 3 months old and were handled according to the ARVO
Statement for the use of animals in Ophthalmic and Visual Research.
Intervention group received 1mg/kg/day of (-)-epicatechin q12h PO
diluted in DMSO 2% for 14 days. After enucleation, mice eyes were
fixed for 1hr in 4% paraformaldehyde and dehydrated in a sucrose
gradient (10-30%). Eyes were cryoprotected before embedding in
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ARVO 2016 Annual Meeting Abstracts
mass spectrometric studies indicated that Cys112 is the required
palmitoylation site for membrane association. Controversially,
another study, also using mass spectrometry methods, denied RPE65
palmitoylation and concluded that membrane association occurred
via electrostatic interactions with acidic phospholipid headgroups
as earlier suggested. To resolve this contradiction, we used an
independent method to determine the existence and relevance of
palmitoylation in RPE65 structure and function.
Methods: To assay RPE65 palmitoylation, we used the acyl-biotinyl
exchange (ABE) and acyl-RAC (resin assisted capture) methods. In
both methods, free thiols are blocked by thiol reagent followed by
cleavage of cysteine-palmitoyl linkages by hydroxylamine (HA). In
ABE, the newly free thiols are labeled with biotin-HPDP followed
by avidin-agarose affinity-purification; while for acyl-RAC, the
HA-treated protein with free thiols is directly applied to thiopropyl
sepharose 6B resin for pull-down. Eluates from both approaches were
analyzed by western blotting and mass spectrometry.
Results: We found that both the ABE and acyl-RAC methods
were sensitive enough to detect palmitoyl modification of RPE65.
However, we found that RPE65 was incompletely palmitoylated,
showing only 3-5% palmitoylation by either method, and depended
on whether RPE65 was co-expressed with LRAT or not. We
confirmed that the structurally palmitoylated rhodopsin was
fully palmitoylated, while CRALBP, a negative control, was not
palmitoylated. Mass spectrometric analysis of ABE-labeled RPE65
peptides confirms that Cys231 is not the site of palmitoylation; other
Cys-peptides are being sought. We also found that RPE65 binds to
palmitoyl-CoA-agarose and is eluted by 10 mM palmitoyl-CoA,
but not by 10 mM CoA, further suggesting RPE65’s affinity for the
palmitoyl moiety.
Conclusions: We conclude that RPE65 is dynamically palmitoylated,
with significant turnover in acylation. It is evident, as previously
suggested, that RPE65 is not palmitoylated at all times. The
functional importance of this putative dynamic palmitoylation is
being studied. It is possible it may play a heretofore unappreciated
role in the visual cycle.
Commercial Relationships: Tingting Liu, None;
Eugenia Poliakov, None; Susan Gentleman, None; T.
Michael Redmond
Support: Intramural Research Program of the National Eye Institute,
National Institutes of Health
Figure 1. H&E staining. Comparison between wild type (A), SGCD
null treated with DMSO (B), and SGCD null mice treated with
(-)-epicatechin. Arrows show areas of retinal fragility.
Commercial Relationships: Andric C. Perez-Ortiz, None;
Gabriela Solano-García, None; Alexandra Luna-Angulo,
None; Ramon M. Coral-Vazquez, None; Yonathan Garfias,
None; Viridiana Garcia-Perez; Sergio De los Santos-Enriquez,
None; Alvaro Rendon, None; Israel Ramirez-Sanchez, None;
Francisco J. Estrada-Mena, None
Program Number: 1733 Poster Board Number: D0129
Presentation Time: 8:30 AM–10:15 AM
Probing RPE65 palmitoylation by acyl-exchange labeling
Tingting Liu, Eugenia Poliakov, Susan Gentleman,
T. Michael Redmond. National Eye Institute, National Institute of
Health, Bethesda, MD.
Purpose: Early studies showed that RPE65 is a membrane-associated
protein. This membrane association was later partly attributed to
S-palmitoylated cysteine residues. However, the existence, site
and role of palmitoylation(s) in RPE65 are controversial. Recent
Program Number: 1734 Poster Board Number: D0130
Presentation Time: 8:30 AM–10:15 AM
Very long chain polyunsaturated fatty acids (VLC-PUFAs) in
cone- and rod-rich regions of the human retina
William C. Gordon1, 2, Bokkyoo Jun2, Nicolas G. Bazan2.
1
Ophthalmology, LSU Health Sciences Center, New Orleans, LA;
2
Neuroscience, LSU Health Sciences Center, New Orleans, LA.
Purpose: Docosahexaenoic acid (22:6) is concentrated in
photoreceptors, located at the sn-2 position on PC VLC-PUFAs,
necessary for vision, and is released from phospholipids during stress
to form neuroprotective pro-homeostatic mediators that include
neuroprotection D1. In AdipoR1 knockout mice 22:6 uptake/retention
is inhibited, PC-containing VLC-PUFAs are greatly reduced, and
photoreceptors degenerate, displaying a flecked retina (Rice et al,
Nature Com, 2015). Since the onset of retinal degenerations are
observed at specific retinal locations (cone-rich macula vs rod-rich
periphery), we asked if the VLC-PUFA profile changed in different
retinal areas.
Methods: Normal human eyes were obtained from NDRI and
punches taken of the macula and periphery. VLC-PUFAs were
characterized by LC/MS/MS and the data normalized by internal
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ARVO 2016 Annual Meeting Abstracts
standard (deuterated arachidonic acid) or by percent of total species.
Phosphatidylcholine (PC) species were also standardized against
PC(44:0) and (28:0). PC-containing VLC-PUFAs have 22:6 esterified
at the sn-2 position. MALDI lipid profiles of cone- and rod-rich
regions were obtained and compared to determine relative abundance
of lipid species.
Results: PC sn-1 fatty acids (FAs) from 22 carbons (C) to 32C
were in low abundance, 32 and 34C were abundant, 36C were less
abundant, and 38C very scarce. PC(40:8; 20:4/20/4) was uniformly
distributed among cone- and rod-rich regions, but PC(44:12;
22:6/22:6) was more prevalent in peripheral retina. Total VLC-PUFAcontaining PCs were also more abundant peripherally, however, 58C
PCs were evenly distributed. Comparisons of macular and peripheral
spectra by MALDI imaging revealed significant differences in PC
distribution. For example, 34:6, 36:9, and 46:10 were more prevalent
in cone-rich retina; 36:4, 38:4, 56:10 were more abundant in the rodrich region.
Conclusions: Synthesis of 32 and 34C VLC-PUFAs results in few
24-32C FAs, indicating shorter chain FAs are steps in long chain
synthesis; 32C and 34C VLC-PUFA abundance, with some 36C and
38C, thus indicate end points. PC spectra from rod-rich areas are
distinctly different from the cone-rich region, implying a difference
in molecular makeup of each photoreceptor type, and suggesting
that differences in photoreceptor lipid profiles may contribute to
susceptibility of disease-inducing cellular and molecular events.
Commercial Relationships: William C. Gordon, None;
Bokkyoo Jun, None; Nicolas G. Bazan, None
Support: NEI EY005121, NIGMS GM103340, and Research to
Prevent Blindness
Program Number: 1735 Poster Board Number: D0131
Presentation Time: 8:30 AM–10:15 AM
Lipid profiling in a diurnal frog retina shows no VLC-PUFA
Bokkyoo Jun1, Robert F. Rosencrans1, Hamilton E. Farris1,
Corinne Richards-Zawacki2, William C. Gordon1, Nicolas G. Bazan1.
1
Neuroscience Center of Excellence, LSU Health Sciences Center,
New Orleans, LA; 2University Of Pittsburgh, Pittsburgh, PA.
Purpose: The goal of this project is to understand the fundamental
roles of Docosahexaenoic acid (DHA) and Very-long-chain-polyunsaturated-fatty-acids (VLC-PUFAs) in retinal function. DHA and
VLC-PUFAs are relatively abundant in mouse, rat and human retina,
but vary in other non-mammalian taxa with different visual ecologies.
From a clinical point of view, VLC-PUFAs are important because
they are synthesized by ELOVL4 enzyme, and mutated ELOVL4
results in photoreceptor degeneration. Evolutionarily, humans are
diurnal primates. In order to understand the evolutionary conservation
of this fundamental aspect of retinal biochemistry, we asked whether
or not a diurnal frog exhibits similar DHA and VLC-PUFA enriched
retinal cellular membranes.
Methods: Lipids were extracted from superior and inferior regions
of diurnal frog retinas and loaded onto a liquid chromatography-mass
spectrometer (LC-MS/MS) for analysis. We analyzed VLC-PUFA in
phosphatidylcholine and phosphatidylethanolamine molecular species
(with PC(28:0) as our internal standard) after naturally occurring
isotopes were corrected using self-made programs. Triacylglycerols
(TAG) are identified and analyzed as well.
Results: PC spectra for both superior and inferior regions show
no significant contribution from VLC-PUFAs. For both regions,
DHA containing PCs are predominantly paired with fatty acids 16:0
and 18:0. Across the entire retina, a preponderance of PE species
contain DHA, whereas relatively fewer PCs contain DHA. We also
observe up to PE48:12 (22:6/26:6) species. With respect to regional
differences, the superior has more DHA containing PCs than in the
inferior. Additionally, greater diversity of TAG species is observed in
the inferior retina as compared to the superior retina. Finally, higher
concentrations of total esterified DHA are observed in the inferior
retina, as compared to the superior retina.
Conclusions: Differing from human retina and murine retinas,
VLC-PUFAs ranging from 32 carbons to 38 carbons are absent from
the frog retina, especially in the PC species. Furthermore, diurnal
amphibian retina appears to store DHA primarily in PEs, as opposed
the typical mammalian DHA contained within PCs (including
PC44:12). In conclusion, our results suggest the role of VLC-PUFAs
differs across mammalia and amphibia.
Commercial Relationships: Bokkyoo Jun, None;
Robert F. Rosencrans, None; Hamilton E. Farris, None;
Corinne Richards-Zawacki, None; William C. Gordon, None;
Nicolas G. Bazan, None
Support: NEI 005121 and NIGMS GM103340 (NGB) and Research
to Prevent Blindness
Program Number: 1736 Poster Board Number: D0132
Presentation Time: 8:30 AM–10:15 AM
Peropsin Effects Light-dependent Modulation of Retinyl Esters
in the RPE
Jeremy D. Cook, Eunice Ng, Marcia Lloyd, Shannan Eddington,
Dean Bok, Hui Sun, Roxana A. Radu, Gabriel H. Travis. Stein Eye
Institute, UCLA, Los Angeles, CA.
Purpose: Peropsin (Rrh) is a non-visual opsin of unknown function
expressed solely in the apical microvilli of the retinal pigment
epithelium (RPE). The aim of the current study is to understand the
function of peropsin. Its unique expression pattern suggests that
peropsin conveys the illumination status to RPE cells, possibly to
affect light-dependent regulation of visual-retinoid metabolism.
Methods: In vivo studies were performed on dark-adapted mice
homozygous for a null mutation in the Rrh gene (Rrh-/-) compared
to age-matched 129/Sv wild-type (WT) mice. Retinoid dynamics
experiments were performed on overnight dark-adapted mice.
Subsets were subjected to photobleach followed by incremental
amounts of dark recovery. Retinoids were analyzed from RPE and
retina separately. Retinoid uptake was measured in whole eyecups
incubated with IRBP-bound [3H]-retinol in dark or in light conditions.
LRAT and retinyl-ester hydrolase (REH) activity were measured
using RPE homogenates and all-trans-retinol or all-trans-retinyl
acetate as substrates, respectively. Retina sections were studied by
light and fluorescence microscopy.
Results: Retinas from Rrh-/- mice had normal morphology, with no
evidence of degeneration. All-trans-retinyl ester levels were severalfold lower in RPE from Rrh-/- versus WT mice under dark-adapted
and immediate post-bleach conditions. These retinoids returned
to WT levels after five minutes of recovery in the dark. Possible
explanations for this biochemical phenotype include (i) impaired
uptake of retinol into RPE cells in the dark; (ii) impaired synthesis
of retinyl esters in the RPE; or (iii) accelerated hydrolysis of retinyl
esters in the dark. To test these possibilities, we measured uptake of
[3H] retinol into dark- and light-exposed eyecups. We observed no
difference between WT and Rrh-/- in either [3H] retinol uptake or
[3H] retinyl-ester synthesis. In vitro ester synthesis was decreased
in homogenate prepared from Rrh-/- mice. Finally, we assayed
for differences in REH activity in eye cup homogenates, and we
observed a moderate increase in REH activity in Rrh-/- samples.
Conclusions: Loss of peropsin in dark-adapted Rrh-/- mice causes
accelerated hydrolysis of retinyl esters by the RPE. Therefore,
peropsin appears to inhibit the hydrolysis of retinyl esters in the dark.
Peropsin may act to sequester substrate for Rpe65-isomerase and
hence the RPE visual cycle under dim light conditions.
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to access the versions of record.
ARVO 2016 Annual Meeting Abstracts
Commercial Relationships: Jeremy D. Cook, None; Eunice Ng,
None; Marcia Lloyd, None; Shannan Eddington, None; Dean Bok,
None; Hui Sun, None; Roxana A. Radu, None; Gabriel H. Travis,
None
Support: NEI Grant EY024379, NEI Grant EY00031
Program Number: 1737 Poster Board Number: D0133
Presentation Time: 8:30 AM–10:15 AM
A2E adducts in the human retinal pigment epithelium
Masahiro Kono1, Zsolt Ablonczy1, Patrice W. Goletz1,
Joe G. Hollyfield2, Rosalie K. Crouch1. 1Ophthalmology, Medical
Univ of South Carolina, Charleston, SC; 2Cole Eye Institute,
Cleveland Clinic Lerner College of Medicine, Cleveland, OH.
Purpose: To identify new fluorophores in human retinal pigment
epithelial (RPE) cells.
Methods: Human RPE was brushed from donor eyes, homogenized,
and extracted with a series of organic solvents. Phase separated
fractions were collected and run on thin layer chromatography
(TLC) plates. Prominent fluorescent bands were isolated and eluted
off the silica. These samples were then subjected directly to mass
spectrometry (MS), and the major peaks were further fragmented
(MS-MS) to gain structural information.
Results: Two TLC bands fluoresced orange. One correlated to
the bisretinoid A2E. The other migrated farther on the TLC plate,
indicative of compounds more hydrophobic than A2E. The MS of
this orange band included peaks with mass/charge (m/z) of 970
and 999. Fragmentation of both compounds gave a primary peak
corresponding to A2E (m/z = 592). Closer examination of the MSMS spectra indicated fragments below 592 mass units are identical
to A2E fragmentation, and those above 592 are consistent with A2E
containing a phosphoglycerol adduct with monoacyl chains.
Conclusions: Additional A2E adducts are being isolated and
identified from the RPE of human donor eyes upon a re-examination
of extractable, fluorescent components. Our long-term goal is to
identify and map the spatial distribution of additional fluorescent
compounds within the human RPE and to understand the relative
toxicity of the fluorescent compounds that accumulate in human
lipofuscin granules of the RPE during aging.
Commercial Relationships: Masahiro Kono, None;
Zsolt Ablonczy, None; Patrice W. Goletz, None; Joe G. Hollyfield,
None; Rosalie K. Crouch, None
Support: Human donor eyes were collected through the Foundation
Fighting Blindness Eye Donor Program. Supported by The
Foundation Fighting Blindness, Research to Prevent Blindness,
and the National Eye Institute (NIH) gratns: R01EY014240 (JGH),
R01EY019065 (ZA), and R01EY019515 (MK)
Program Number: 1738 Poster Board Number: D0134
Presentation Time: 8:30 AM–10:15 AM
Protease Nexin-1 (PN-1): A Novel Survival Factor for Retina
Cells
Preeti Subramanian1, Jeanee Bullock1, 2, Paige Winokur1,
Veronique Arocas3, S. Patricia Becerra1. 1National Eye Institute,
Bethesda, MD; 2Biochemistry and Molecular & Cellular Biology,
Georgetown University medical Center, Washington, DC; 3U1148
Inserm, Batiment Inserm, Hopital Bichat, Secteur Claude Bernard,
Paris, France.
Purpose: Protease Nexin-1 (PN-1) is a member of the serine protease
inhibitor (serpin) superfamily. It has demonstrable neurotrophic
effects in the brain. Its gene SERPINE2 is expressed in the retina.
PN-1 inhibits serine protease thrombin. Recently it was shown that
PN-1 inhibits angiogenesis in the retina. The neurotrophic capacities
of PN-1 in the retina have not been evaluated. The purpose of this
study was to investigate the efficacy of the PN-1 in protecting the
retina.
Methods: Rat retinal progenitor R28 cells and human ARPE-19
cells were cultured. PN-1 was analyzed by western blots and probed
vs anti-PN-1 antibody. Bacterially-derived human PN-1 and RS10
and PN-1-derived 17mer peptide and mammalian-derived pigment
epithelium-derived factor (PEDF) were used. TUNEL assays
were performed to quantitate cell-death. Binding assays to ligand
binding domain of PEDF-receptor was done by peptide affinity
chromatography. RT-PCR was performed for Bcl-2.
Results: A PN-1 immuno-reactive band was detected in total lysates
from ARPE-19 cells but was undetectable in media or washes of
cells with high ionic strength buffers. We tested PN-1 and RS10, a
modified PN-1 that lacks the ability to inhibit serine proteases, for
survival of retina cells. Both versions of PN-1 (at 2.5 nM and 10
nM) decreased the percentage of TUNEL positive cells in R28 cells
relative to untreated cells. PN-1 is similar to PEDF, a neurotrophic
factor for the retina. PN-1-derived 17mer peptide was designed from
the alignment with the neurotrophic region of PEDF. PN1-17mer
also decreased the percentage of TUNEL positive cells relative
to untreated cells, like full-length PN-1 and PEDF. Percentage
of binding to PEDF-receptor of fluoresceinated PN-1-17mer and
fluoresceinated PEDF-17 mer peptides was 11% and 45% of the total
ligand input, respectively. Expression of the anti-apoptotic Bcl-2 gene
increased upon treatment with PN-1 for 6 h relative to untreated and
was similar to treatments with fetal bovine serum (FBS).
Conclusions: PN-1 is a survival factor for retina cells in culture
and its mechanism of action is independent of inhibition of serine
protease inhibition. A small region towards the amino terminus
confers the survival activity to the PN-1 polypeptide.
Commercial Relationships: Preeti Subramanian, None;
Jeanee Bullock, None; Paige Winokur, None; Veronique Arocas,
None; S. Patricia Becerra, None
Program Number: 1739 Poster Board Number: D0135
Presentation Time: 8:30 AM–10:15 AM
Investigating the Cell Death Mechanisms of ARPE-19 Cells using
Modified ARPE-Derived ECM to Model Aging and Disease
Elizabeth R. Gaillard1, 2, Jennifer C. Tournear1. 1Chemistry and
Biochemistry, Northern Illinois University, DeKalb, IL; 2Biological
Sciences, Northern Illinois University, DeKalb, IL.
Purpose: RPE cell death is a symptom of age related macular
degeneration (AMD), but it is unclear what mechanisms of cell death
are involved in this process. This study aims to modify ARPE-derived
extra cellular matrix (ECM) in order to investigate the ARPE-19 cell
death mechanism associated with various stresses modeling both agerelated modifications and inflammation.
Methods: A series of modifications were performed on ECM derived
from ARPE-19 cells to model aging and inflammation. These
modifications included non-enzymatic glycation, A2E, blue light
irradiated A2E and non-enzymatic nitration. These modifications
were done on ECM coating 24-well plates. Post modification, healthy
ARPE-19 cells were seeded onto the ECM and allowed to attach for
30 min. Unattached cells are removed and fixed for further study.
Attached cells were allowed to grow prior to viability analysis.
After 24 hours of growth, both unattached and attached cells were
stained with Annexin-V and PI then subjected to analysis using flow
cytometry to investigate apoptosis versus necrosis as mechanisms of
cell death.
Results: Cell viability is observed to decrease under all stresses when
compared to unmodified ECM. Cells grown on blue light irradiated
A2E modified ECM showed not only a loss in viability, but also a
change in proliferation and cell morphology. There was evidence
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ARVO 2016 Annual Meeting Abstracts
of both apoptosis and necrosis as mechanisms of cell death. When
analyzing both the unattached and attached simultaneously, a further
decrease in cell viability was observed suggesting that attachment to
modified ECM is impaired.
Conclusions: These data suggest that more than one mechanism
of cell death is involved under aging and disease conditions. Both
necrotic and apoptotic cells were observed supporting the idea that
cells have difficulty in both cell attachment and proliferation. This
study provides insight into the mechanisms of RPE cell death in
AMD and can help to further understand the pathogenesis of the
disease.
Commercial Relationships: Elizabeth R. Gaillard, None;
Jennifer C. Tournear, None
Program Number: 1740 Poster Board Number: D0136
Presentation Time: 8:30 AM–10:15 AM
Identifying Novel Fluorophores in Human RPE Melanolipofuscin
Michael Vega1, Elizabeth R. Gaillard1, 2. 1Department of Chemistry
and Biochemistry, Northern Illinois University, Genoa, IL;
2
Department of Biological Sciences, Northern Illinois University,
DeKalb, IL.
Purpose: To identify novel fluorophores in human RPE
melanolipofuscin. Identifying novel fluorophores in melanolipofuscin
extracts may lead to the development of new diagnostic techniques
for the early detection of age related macular degeneration.
Methods: Human RPE melanolipofuscin is extracted from human
donor eyes as previously described by Feeney-Burns. Folch
extraction is performed to obtain the organic soluble portion. The
organic soluble melanolipofuscin is collected, dried under argon, and
reconstituted in HPLC grade methanol for use in high performance
liquid chromatography tandem mass spectrometry (LC/MS/MS)
coupled to a fluorescent detector (Surveyor LC with PDA, Thermo
Finnigan LCQ Advantage MS, Surveyor FL). Fluorescence detection
and tandem mass spectrometry data are analyzed for the identification
and structure elucidation of the fluorescent components of human
RPE melanolipofuscin.
Results: Melanolipofuscin extracts from human donor tissue have
been subjected to LC/MS/MS. The fluorophore, A2E, has been
observed as a component of human RPE melanolipofuscin. The
presence of additional fluorophores has been confirmed. Tandem
mass spectrometry analysis of these unidentified fluorophores has
provided structural information for these vitamin A derivatives.
Conclusions: Human RPE melanolipofuscin is observed to
accumulate in the RPE with age and this accumulation has been
suggested to closely correlate with the onset of AMD. Fluorescent
components of melanolipofuscin have been identified in human
melanolipofuscin extracts. These fluorophores may lead to the
development of a new fluorescence based diagnostic technique for
the early detection of AMD.
Commercial Relationships: Michael Vega, None;
Elizabeth R. Gaillard, None
Program Number: 1741 Poster Board Number: D0137
Presentation Time: 8:30 AM–10:15 AM
Evaluating the Influence of Melanin in Cytokine Secretion in
Photo-stressed Retinal Pigment Epithelial Cells
Sally Yacout1, Elizabeth R. Gaillard1, 2. 1Chemistry and Biochemistry,
Northern Illinois University, DeKalb, IL; 2Biological Sciences,
Northern Illinois University, Dekalb, IL.
Purpose: It is well known that retinal pigment epithelial cells
are able to express and secrete cytokines and present antigens,
however the role of melanin in RPE cell immune response has not
been established. This study evaluates immune response in photo-
stressed pigmented and unpigmented ARPE-19 cells by monitoring
interleukin-6 (IL-6) expression and secretion.
Methods: ARPE-19 cells were pigmented with bovine melanin and
subjected to UVC irradiation or dark treatment. Unpigmented ARPE19 cells were used as a control. IL-6 secretion was measured using an
enzyme-linked immunosorbent assay (ELISA) and gene expression
was detected using PCR. Relative IL-6 secretion was evaluated in
comparison to dark-treated unpigmented ARPE-19 cells. Statistical
analysis was performed using a two-tailed Student’s t-test.
Results: IL-6 secretion was significantly increased in UVC stressed
unpigmented (p = 0.004) and pigmented (p < 0.0001) ARPE-19
cells. Elevated IL-6 secretion was observed in pigmented cells under
dark (111±9%) and photo-stress (164±8%) conditions compared
to unpigmented dark control cells. Additionally, photo-stressed
pigmented cells showed a 113±6% increase compared to irradiated
unpigmented cells.
Conclusions: The presence of melanin in ARPE-19 cells results in
an increase in IL-6 secretion under dark and photo-stress conditions
compared to unpigmented cells. This finding suggests that RPE cell
pigmentation may be related to the immune response of these cells.
Monitoring expression and secretion of related pro-inflammatory
cytokines and signaling factors is needed to further investigate
melanin-dependent immune response.
Commercial Relationships: Sally Yacout, None;
Elizabeth R. Gaillard, None
Program Number: 1742 Poster Board Number: D0138
Presentation Time: 8:30 AM–10:15 AM
Label-free proteomic analysis of human retinal pigment
epithelium (RPE) reveals loss of RPE abilities after RPE
depolarization
Yao-Tseng Wen, Rong-Kung Tsai. Buddhist Tzu Chi General Hospital,
Hualien, Taiwan.
Purpose: The retinal pigment epithelium (RPE) a monolayer of
polarized cells that plays many essential roles in the maintenance
and homeostasis of photoreceptor cells. The apical ends of RPE
abut and engulf photoreceptor OS through phagocytosis whereas the
basolateral sides lie on Bruch’s membrane and transport nutrients,
digested metabolic wastes, ions and water between the retina and
the choroidal vasculature in the back of the eye. RPE provides the
recycle of retinoids for the phototransduction pathway as well as
the blood-retinal barriers. De-polarization of RPE may influence the
functions of RPE in maintaining the homeostasis of the retina and
choroid. Therefore, the purpose of this study is to investigate the RPE
proteome changes under the de-polarized condition.
Methods: The label-free LC-MS/MS analysis was used to the
proteome samples from the polarized-RPE and the non-polarized
RPE. The real-time RT-PCR analysis was used to confirm the depolarization-influenced proteins. Phagocytotic ability, tight junction,
and microvilli formation were evaluated in the polarized-RPE and the
non-polarized RPE.
Results: The label-free proteomic analysis identified 216 proteins,
which are influenced by de-polarization condition. Six RPE specific
proteins and 3 tight junction-related proteins were downregulated by
depolarization condition and were confirmed the RNA expression in
the polarized-RPE and the non-polarized RPE. The de-polarized RPE
reduced 2.3-fold of phagocytosis ability and lose the tight junction. In
addition, the microvilli were less in the de-polarized RPE than in the
polarized PRE.
Conclusions: RPE de-polarization can dramatically change the RPE
gene expression and RPE biological functions. Our findings provided
crucial information for investigating the role of RPE polarity in
maintaining the homeostasis of the retina and choroid.
These abstracts are licensed under a Creative Commons Attribution-NonCommercial-No Derivatives 4.0 International License. Go to http://iovs.arvojournals.org/
to access the versions of record.
ARVO 2016 Annual Meeting Abstracts
Commercial Relationships: Yao-Tseng Wen, None; RongKung Tsai, None
Support: MOST 103-2314-B-303-007-MY3
Program Number: 1743 Poster Board Number: D0139
Presentation Time: 8:30 AM–10:15 AM
Delineation of the Farnesyl-Binding Site on AIPL1 by NMR
Ravi Prakash Yadav1, Liping Yu2, Nikolai Artemyev1. 1Molecular
Physiology and Biophysics, University of Iowa, Iowa City, IA;
2
Biochemistry and NMR Core Facility, University of Iowa, Iowa
City, IA.
Purpose: AIPL1 is a specialized chaperone of phosphodiesterase-6
(PDE6) in rods and cones. Mutations in AIPL1 lead to destabilization
of PDE6 and cause Leber congenital amaurosis type 4 (LCA4),
a severe form of childhood blindness. Binding of the prenylated
C-termini of PDE6 to the AIPL1 FKBP domain is critical to AIPL1
chaperone function. We have investigated the farnesyl-binding site of
AIPL1 by NMR.
Methods: The uniformly 15N- and selectively 13C-methyl-labeled
AIPL1 FKBP domain was obtained by growing E. coli with 15NH4Cl,
13
C-α-ketobutyrate, and 13C-α-ketoisovalerate. The 15N/1H and 13C/1H
HSQC spectra were collected for the purified AIPL1 FKBP and select
mutants in the absence and presence of S-farnesyl-L-cysteine methyl
ester (FC). The FC-binding site was determined and the FC ligand
was docked to the homology model of the AIPL1 FKBP using the
restraint data obtained from the NMR experiments. Binding of FC to
the AIPL1 FKBP and its mutants was also assessed using FRET.
Results: Binding of FC caused selective changes in the 15N/1H HSQC
spectra as well as in the 13C/1H HSQC spectra of the Ile CδH3 and
Leu/Val methyl regions of AIPL1 FKBP. The Ile and Leu methyl
peaks that shifted upon FC binding were assigned based on the NMR
analysis of select Ile→Leu and Leu→Ile mutants of the AIPL1 FKBP.
Conclusions: The NMR analysis delineated a novel prenyl-binding
site at the interface of the unique insert region and the core FKBP
fold of AIPL1. A model of the complex of AIPL1 FKBP with FC
generated from this study facilitates identification of LCA4 mutations
perturbing the ligand-binding to AIPL1.
Commercial Relationships: Ravi Prakash Yadav, None;
Liping Yu, None; Nikolai Artemyev, None
Support: NIH Grant EY010843
Program Number: 1744 Poster Board Number: D0140
Presentation Time: 8:30 AM–10:15 AM
Ocular parameters changes in the IRBP knockout mouse eye
Shanu Markand1, Sara A. Wetzstein1, Natecia Williams1,
Ranjay Chakraborty1, 2, Priyanka Priyadarshani1, Kevin Donaldson1,
Jeffrey H. Boatright1, 2, Machelle T. Pardue2, 3, J M. Nickerson1.
1
Opthalmology, Emory University, Decatur, GA; 2Rehab Center of
Excellence, Atlanta VA Medical Center, Atlanta, GA; 3Biomedical
Engineering, Georgia Institute of Technology, Atlanta, GA.
Purpose: Despite the high prevalence of myopia in the worldwide
population, underlying mechanisms are unclear. Interphotoreceptor
retinoid-binding protein (IRBP) is major protein of the subretinal
space. It plays a crucial role in the visual cycle. Our laboratory
previously reported eye size defects at postnatal day 8 (P8), profound
myopia and retinal degeneration at P30 in the IRBP knockout (KO)
mice, indicating a role for IRBP in eye development. The purpose of
this study was to determine which ocular parameters affecting optical
power are altered in IRBP KO mice.
Methods: Both male and female C57BL/6J (WT) and congenic
IRBP KO mice at P30 (WT, n=8; KO, n=6) and P55 (WT, n=3;
KO, n=5) were subjected to whole-eye biometrical imaging using a
Bioptigen R4310 deep imaging spectral domain optical coherence
tomography system (SD-OCT). Parameters included central corneal
thickness (CCT), anterior chamber depth (ACD), lens thickness (LT),
vitreous depth (VD), and retinal thickness (RT). Mean thicknesses
(+/- standard deviation; SD) were recorded. An unpaired t-test with
Welch’s correction assuming unequal variances was used to assess
statistical significance between groups.
Results: SD-OCT analysis revealed that at both P30 and P55, VD
was significantly deeper in IRBP KO (P30: 1018 ± 51.26 μm in
KO, 638 ± 37.59 in WT, p<0.0001; P55: 899.6± 15.12 in KO, 564±
8.72 in WT, p<0.0001). The VD increase in IRBP KO mice was
accompanied by an increase in total axial length (P30: 3354 ± 35.44
in KO, 3068 ± 56.52 in WT, p<0.0001; P55: 3396 ± 36.34 μm in KO,
3130 ± 9.91 in WT, p<0.0001). Conversely, ACD was significantly
decreased in IRBP KO (P30: 259.4 ± 14.14 μm in KO, 289.6
±12.49 in WT, p<0.01; P55: 294.7 ± 10.92 μm in KO, 314.5 ± 4.48
in WT, p<0.05). At P30, RT was significantly reduced in KO mice
(172.5 ± 16.39 in KO, 210.5 ± 24.94 in WT, p<0.01). No significant
differences in CCT or lens thickness were observed in IRBP KO
versus WT mice at P30 or P55.
Conclusions: Our data support selective axial length growth as the
primary contributor to profound myopic shift in IRBP KO mice,
similar to clinical myopia. It may be that an increase in VD is the
primary contributor to profound myopic shift in IRBP KO mice and
that this increase also resulting results in compensatory compression
of other compartments of the eye. Our data indicate an importance of
IRBP in normal eye development and emmetropization.
Commercial Relationships: Shanu Markand; Sara A. Wetzstein,
None; Natecia Williams, None; Ranjay Chakraborty, None;
priyanka priyadarshani, None; Kevin Donaldson, None;
Jeffrey H. Boatright, None; Machelle T. Pardue, None;
J M. Nickerson, None
Support: NIH R01EY016470, R01EY021592, P30EY006360,
R01EY016435, R01EY014026, Research to Prevent Blindness, Katz
Foundation.
Program Number: 1745 Poster Board Number: D0141
Presentation Time: 8:30 AM–10:15 AM
The Effects of Platelet Gel on the Cultured Human Retinal
Pigment Epithelial Cells
Mozhgan Rezaeikanavi1, Sahar Balagholi1, 2, Abouzar Bagheri1, 3.
1
Ocular Tissue Engineering Research Center, Shahid Beheshti
University of Medical Sciences, Tehran, Iran (the Islamic Republic
of); 2Department of Hematology, Faculty of Allied Medicine, Tehran
University of Medical Sciences, Tehran, Iran (the Islamic Republic
of); 3University of Social Welfare and Rehabilitation Sciences,
Tehran, Iran (the Islamic Republic of).
Purpose: To investigate the cellular and molecular changes of
cultured human retinal pigment epithelial (hRPE) when treated with
different concentrations of platelet gel.
Methods: Confluent cultured hRPE cells in 3rd, 5th, and 7th were
treated with 10%, 20%, and 30% platelet gels. Cultivated hRPE cells
in 20% fetal bovine serum were considered as control. Cell viability
was determined by MTT assay and by light microscopy. Total RNA
was isolated and the gene expression profile was determined using
real time RT-PCR. The real-time RT–PCR analysis was performed in
duplicate as three independent experiments. Differences between the
control and experimental groups were analyzed using the Student t
test. P value less than 0.05 was considered statically significant.
Results: Viability of cultivated hRPE cells treated with different
concentrations of platelet gel was higher than the controls on day
7 (p value=0.044); however, it was comparable with control on
day 3 (p value=0.108) and showed a borderline increase on day 1
(p value=0.074). The viability of hRPE cells was not significantly
These abstracts are licensed under a Creative Commons Attribution-NonCommercial-No Derivatives 4.0 International License. Go to http://iovs.arvojournals.org/
to access the versions of record.
ARVO 2016 Annual Meeting Abstracts
different between different concentrations of platelet gel on days 1,
3 and 7; however, hRPE cells demonstrated a significant increase of
viability with 30% as compared to 10% platelet gel on day 7. hRPE
cells treated with different concentrations of platelet gel did not
show a significant change in the expression of Ki67, α-SMA, and
PAX6; however, expressions of MMP-9, MMP2 and KDR1 were not
significantly changed with 10% platelet gel and a high expression of
RPE65 was observed following 30% platelet gel treatment.
Conclusions: It seems that platelet gel as a 3D substrate has a
positive effect on viability of cultivated hRPE cells; however, 10%
platelet gel seems to be the proper concentration in termshttps://
arvo2016.abstractcentral.com/submission of low risk of
inflammation, angiogenesis, and induction of cell proliferation.
Commercial Relationships: Mozhgan Rezaeikanavi, None;
Sahar Balagholi, None; Abouzar Bagheri, None
Program Number: 1746 Poster Board Number: D0142
Presentation Time: 8:30 AM–10:15 AM
Lysophosphatidic acid (LPA) elicits pro-inflammatory responses
in ARPE-19 cells via activation of LPA2 receptors
Christoph Ullmer, Elisabeth A. Zirwes, Anja Osterwald. Roche
Pharma Research & Early Development, F. Hoffmann-La Roche AG,
Basel, Switzerland.
Purpose: The consequences of increased levels of the bioactive
phospholipid LPA in the vitreous fluid from patients with proliferative
diabetic retinopathy (PDR) compared to non-diabetic patients remain
unknown. Due to the high inflammatory pathophysiology of PDR, we
hypothesized that LPA acts on retinal pigment epithelial (RPE) cells
to increase the production of inflammatory mediators by activating
LPA receptors and respective signaling pathways.
Methods: Expression of LPA receptors was examined in ARPE19 cells by real-time PCR. The induction of cytokine levels was
determined by ELISA and real-time PCR. Cells were transfected with
a NF-kappa B (NFkB) responsive reporter gene using a lentivirus
and LPA mediated NFkB activation was measured as luciferase
activity. LPA receptor activity was investigated by transfection of
LPA receptor siRNA, and by pre-incubation with LPA receptor
antagonists, pertussis toxin or inhibitors of signal transduction
proteins.
Results: ARPE-19 cells predominantly express LPA2 receptor
mRNA; LPA3, LPA4, LPA5 and LPA6 receptors were below level
of detection. Exogenously applied LPA (2.5 μM for 24h) induces
cells to secrete interleukin-6 (IL-6), and interleukin-8 (IL-8). LPAmediated interleukin secretion and mRNA induction could be fully
inhibited by co-incubation with a LPA2 receptor antagonist (cpd
35) but not or partially by a LPA1 receptor antagonist (AM095).
Furthermore, LPA activated the NFkB reporter gene by 17-fold,
which was fully prevented by transfection of a LPA2 selective
siRNA. The EC50 of LPA to activate NFkB (304 nM) matched the
induction of IL-6 (275 nM) and IL-8 (294 nM) mRNA. The LPA
response was fully antagonized by cpd 35, but not by AM095. LPA
failed to modulate cAMP or intracellular Ca2+ levels. Full inhibition
of the pro-inflammatory LPA2 pathway was achieved by inhibition of
JAK3, PI3K, AKT, and IKK.
Conclusions: This study demonstrates that LPA promotes ARPE19 cells to secrete inflammatory mediators such as IL-6 and IL-8
via activation of LPA2 receptors. These data suggest that activation
of LPA2 receptors may potentially contribute to the inflammatory
pathophysiology in PDR that displays elevated vitreous LPA as well
as IL-6 and IL-8 levels. Activation of JAK3, PI3K, AKT, and NFkB
are important signaling components in LPA2 receptor-mediated
cytokine secretion, which is unprecedented and will be further
analyzed.
Commercial Relationships: Christoph Ullmer, F. HoffmannLa Roche Ltd; Elisabeth A. Zirwes, F. Hoffmann-La Roche Ltd;
Anja Osterwald, F. Hoffmann-La Roche Ltd
Program Number: 1747 Poster Board Number: D0143
Presentation Time: 8:30 AM–10:15 AM
Acetazolamide and inner retinal fluid homeostasis: gliotic
changes in cultured porcine retina
Jens Nääv Ottosson, Linnea Taylor, Karin Arner, Fredrik K. Ghosh.
Department of Ophthalmology, Institute of clinical sciences, Lund
University, Lund, Sweden.
Purpose: To determine the effects of acetazolamide (AZ), a carbonic
anhydrase inhibitor used to treat glaucoma, on protein expression
related to retinal fluid homeostasis, gliosis and apoptosis in adult
porcine retinal explants.
Methods: Retinal explants were cultured using a standardized
protocol, with and without AZ added to the medium, for two and
five days. Specimens were fixed, cryosectioned and stained with
hematoxylin and eosin as well as immunohistochemical markers
for Müller cells and photoreceptors. Apoptosis was assessed using
terminal deoxynucleotidyl transferase dUTP nick end labeling
(TUNEL). Fluorescence intensity was analyzed using ImageJ, and
acquired data was processed using an ANOVA with a Tukey post hoc
test. Eyes fixed immediately after enucleation were used as in vivo
controls.
Results: Hallmarks of retinal gliosis and neuronal degeneration were
seen in all cultured explants, especially in the 5 DIV specimens,
including: an upregulation of glial fibrillary acidic protein (GFAP)
and a decrease in the number of ganglion cells. A reduced number
of ganglion cells was seen in 5DIV specimens treated with AZ
compared to 5DIV controls, although this difference was not
statistically significant. Apoptosis was significantly increased in AZ
5DIV compared to 5DIV controls (P < 0,05). All cultured specimens
displayed an increased expression of Kir4.1, and there was a reduced
expression of Kir4.1 in the 5 DIV specimens treated with AZ
compared to their untreated counterparts (P < 0,01). Significant loss
of polarization in cultured retinas was observed in Kir4.1 as well as
aquaporin-4 (AQP4) labeling, although the total expression of AQP4
remained unaltered. In contrast with carbonic anhydrase II (CAII),
which revealed no differences in expression, carbonic anhydrase XIV
(CAXIV) was upregulated in all cultured specimens.
Conclusions: All cultured explants displayed clear signs of gliosis, as
well as an upregulation of Kir4.1 and CAXIV and a loss of polarity
in the expression of Kir4.1 and AQP4. In the 5DIV specimens treated
with AZ, there was a downregulation of Kir4.1 and an increase in the
number of apoptotic cells compared with untreated counterparts, as
well as a trend towards increased ganglion cell death. Considering the
frequent use of AZ in the treatment of glaucoma, these results merit
further investigation.
Commercial Relationships: Jens Nääv Ottosson, None;
Linnea Taylor, None; Karin Arner, None; Fredrik K. Ghosh,
None
Program Number: 1748 Poster Board Number: D0144
Presentation Time: 8:30 AM–10:15 AM
Intravitreal injection vs. topical eye drop application of
nanoparticles using a mouse model
Sara Wetzstein1, Jana T. Sellers1, Kevin Donaldson1,
Gretchen Unger2, Shanu Markand1, Priyanka Priyadarshani1,
Jeffrey H. Boatright1, 3, J M. Nickerson1. 1Ophthalmology, Emory,
Decatur, GA; 2Genesegues, Inc., Chaska, MN; 33Center for Visual
and Neurocognitive Rehabilitation, Atlanta VA Medical Center,
Decatur, GA.
These abstracts are licensed under a Creative Commons Attribution-NonCommercial-No Derivatives 4.0 International License. Go to http://iovs.arvojournals.org/
to access the versions of record.
ARVO 2016 Annual Meeting Abstracts
Purpose: The purpose of this study was to test the hypothesis that
intravitreal injection of nanoparticles (NPs) coated with hyaluronic
acid (HA) impacts retinal morphology more than topical application
in mouse eyes.
Methods: Male 129S2 mice at postnatal day 185 were subjected to
either topical eye drop application (n=8) or intravitreal injection using
a transcorneal route (n=8) of nanoparticles. Two hours post injection,
mice were euthanized and eyes were enucleated with neutral buffered
formalin with zinc fixation, sectioning, H&E staining, and imaging.
A score system was employed using 3 separate blind examiners for
evaluation of representative eye sections at 10X. Each section was
graded for cracks, peeling, and histological damage. Evaluation
followed a 4-point grading system for cornea, retina, lens, and an
overall score, with 0=intact morphology, 1=light damage, 2=mild
damage, and 3=heavy damage. A Mann-Whitney test was used to
assess statistical significance between treatment groups.
Results: Histological analysis revealed that intravitreal injection
significantly damaged overall eye morphology (Topical: 1.055;
Intravitreal: 2.055, p<0.0002) with a lower score indicating more
intact morphology. Intravitreal injected eyes were significantly
damaged for the cornea (Topical: 0.670; Intravitreal: 1.915, p
<0.0061), lens (Topical: 1.165; Intravitreal: 2.250, p <0.0025), and
retina (Topical: 1.250; Intravitreal: 2.000, p <0.0146) when analyzed
individually. More intravitreal eyes hemorrhaged (50%) compared
with topical eyes (12.5%).
Conclusions: Our data support the hypothesis that intravitreal
nanoparticle injection significantly impacts eye morphology. Topical
drops had fewer lens fractures, better-attached retinas, straight
irises, and undamaged corneal epithelium and endothelium in H&E
sections. Thus, the adverse impact of intravitreal injections should
be counterbalanced with the potential benefit of direct nanoparticle
delivery.
Commercial Relationships: Sara Wetzstein; Jana T. Sellers,
None; Kevin Donaldson, None; Gretchen Unger, Genesegues;
Shanu Markand, None; priyanka priyadarshani, None;
Jeffrey H. Boatright, None; J M. Nickerson, None
Support: NIH R01EY016470, R01EY021592, P30EY006360,
R01EY014026, Research to Prevent Blindness, Katz Foundation.
Program Number: 1749 Poster Board Number: D0145
Presentation Time: 8:30 AM–10:15 AM
Fenofibrate is a competitive inhibitor of the RPE65 isomerase
Gennadiy P. Moiseyev1, Younghwa Shin1, Yusuke Takahashi3, 2,
Jian-Xing (Jay) Ma1, 2. 1Department of Physiology, Univ of Oklahoma
Hlth Sci Ctr, Oklahoma City, OK; 2Harold Hamm Diabetes Center,
Oklahoma City, OK; 3Department of Medicine, Univ of Oklahoma
Hlth Sci Ctr, Oklahoma City, OK.
Purpose: Fenofibrate, a drug originally developed to treat high
cholesterol and triglycerides, has recently attracted a major attention
as a novel medical treatment for diabetic retinopathy (DR). The
mechanism for the benefits of fenofibrate in DR is not known.
Recently, it has been shown that retinyl amine, retinoid visual cycle
inhibitor, delayed the development of early DR lesions in diabetic
mice. We hypothesized that fenofibrate may inhibit the visual cycle.
To this end, we studied the effect of fenofibrate on the RPE65
isomerase activity.
Methods: All-trans-[3H]-retinol was used as a substrate to measure
the activities of lecithin:retinol acyltransferase (LRAT) and
isomerase in bovine RPE microsomes. Fenofibrate dissolved in
dimethylformamide was added in a small volume (less than 2%)
from the stock solution into the reaction mixture. The generated
retinoids were extracted and analyzed by HPLC with a Radiomatic
flow scintillation analyzer. To determine the inhibition mode of the
fenofibrate, an adenoviral expression vector was used to express
chicken RPE65 in 293A cells, and inhibition of RPE65 was measured
in a liposome-based isomerase assay.
Results: Fenofibrate showed a concentration-dependent inhibition
of the isomerase activity as measured in bovine microsomes, with
an IC50 of 90 µM. In the same reaction system, LRAT activity
was not affected by fenofibrate at a concentration as high as 500
µM. To analyze the inhibition type, all-trans retinyl palmitate was
incorporated in liposomes and used as a substrate for recombinant
RPE65. The concentration dependence of the RPE65 reaction
was measured in the absence and presence of fenofibrate. The
Lineweaver-Burk plot demonstrated two lines crossing Y-axis at the
same point suggesting that fenofibrate inhibits the retinoid isomerase
reaction in a competitive manner. Analysis of these results yielded Ki
= 42 ± 4 µM.
Conclusions: Fenofibrate potently and selectively inhibited
conversion of all-trans-retinyl ester to 11-cis-retinol catalyzed by
RPE65 isomerase. The competitive mode of the fenofibrate inhibition
suggests that it is likely bound to the RPE65 active site. This
inhibition may be used to slow down the visual cycle and to decrease
the accumulation of A2E in the RPE indicating its therapeutic
potential in Stargardt’s disease and age-related macular degeneration.
Commercial Relationships: Gennadiy P. Moiseyev, None;
Younghwa Shin, None; Yusuke Takahashi, None; Jian-Xing
(Jay) Ma, None
Support: NIH grants (EY018659, EY012231, EY019309,
P20GM104934), a JDRF grant (2-SRA-2014-147-Q-R) and an
OCAST grants (HR13-076)
Program Number: 1750 Poster Board Number: D0146
Presentation Time: 8:30 AM–10:15 AM
Rod bipolar cell degeneration in Pik3c3/Vps34 conditional
knockout mice
Feng He1, Melina A. Agosto1, Ralph Nichols2, Theodore G. Wensel1, 2.
1
Biochemistry, Baylor College of Medicine, Houston, TX;
2
Ophthalmology, Baylor College of Medicine, Houston, TX.
Purpose: The type III phosphoinositide 3-kinase (Pik3c3/Vps34)
participates in various cellular functions, including intracellular
trafficking and cell survival. Our previous studies showed aggressive
rod degeneration in rod-specific Vps34 conditional knockout mice
due to impairment of autophagy and endosomal pathways. In this
study, we investigated Vps34 function in bipolar cells, which are the
downstream neurons of the visual phototransduction pathway, using
bipolar-specific Vps34 conditional knockout mice.
Methods: Electroporation of plasmid DNA directing expression
of DsRed fused to a phosphoinositide binding domain (DsRed2xHrs) was used to localize PI(3)P in bipolar cells. A mouse line
with a conditional functional deletion of Vps34 in bipolar cells was
generated by crossing Vps34 floxed mice with a transgenic mouse
line that expresses Cre recombinase in bipolar cells using the Purkinje
cell protein-2 (PCP2) promoter. Structural changes in the retina were
determined by immunofluorecence and electron microscopy.
Results: PI(3P) was localized to discrete puncta of various sizes
in bipolar cells. Loss of Vps34 function in bipolar cells caused
significant degeneration of the inner retina. The number of rod
ON-bipolar cells, determined by immunostaining with PCP2 and
PKCa antibodies, was significantly reduced in Vps34 knockout
mice at 3 months, while there were no significant changes in cone
ON- or OFF-bipolar cells, horizontal cells, or amacrine cells.
The thickness of the inner nuclear and inner plexiform layers was
remarkably reduced. No significant degeneration was observed in
the photoreceptor cell or ganglion cell layers. Transmission electron
microscopy showed an accumulation of vesicles in bipolar cell bodies
These abstracts are licensed under a Creative Commons Attribution-NonCommercial-No Derivatives 4.0 International License. Go to http://iovs.arvojournals.org/
to access the versions of record.
ARVO 2016 Annual Meeting Abstracts
and a lack of invaginating rod bipolar dendrites in synaptic triads
of the outer plexiform layer. Autophagy markers LC3 and p62, as
well as ubiquitinated proteins, accumulated and co-localized in rod
ON-bipolar cells in Vps34 KO mice. LAMP-1 also accumulated, but
did not co-localize with the p62/LC3 puncta, indicating abnormal
autophagy in bipolar cells in the absence of Vps34.
Conclusions: Vps34 is essential for rod ON bipolar cell survival. The
mechanisms of degeneration in the absence of Vps34 may involve
disruption of autophagy and/or related pathways.
Commercial Relationships: Feng He, None; Melina A. Agosto,
None; Ralph Nichols, None; Theodore G. Wensel, None
Support: NIH grant R01-EY07981 and P30-EY002520. Knights
Templar Eye Foundation grant.
Program Number: 1751 Poster Board Number: D0147
Presentation Time: 8:30 AM–10:15 AM
Nutrient utilization and metabolite transport in retinal pigment
epithelium
Jianhai Du, Kaitlen Knight, Christina Lieu, Connor Jankowski,
Van Tran, James Hurley, Jennifer R. Chao. University of Washington,
Seattle, WA.
Purpose: The retinal pigment epithelium (RPE) is essential to
maintaining metabolic homeostasis in the outer retina. How RPE
utilizes nutrients and transports metabolites is not well understood.
We have used 13C tracers to study nutrient consumption and
metabolite transport in human fetal RPE cells.
Methods: Human fetal RPE cells were grown for 4-6 weeks on
transwell filters and then switched into culture medium with 13C
tracers including glucose, glutamine and proline on either the apical
or basal side. Media from the apical and basal sides were collected at
8h, 24h and 48h for analysis of labeled and unlabeled metabolites by
GC-MS and LC-MS.
Results: RPE cells utilize 13C glucose, 13C glutamine and 13C proline
from both apical and basal sides. 13C label from glucose appears
in intermediates from glycolysis and the mitochondrial TCA cycle
including lactate, pyruvate, citrate, oxoglutarate and malate. 13C
also appears in amino acids including glutamate, glutamine, serine,
glycine, and alanine on both sides. Remarkably, these 13C-glucosederived metabolites appear predominantly on the apical side at 8h
(8-35 times more than basal side). Most of these metabolites are
transported gradually to the basal side at 24 h and 48 h (3-9 times
and 1.5-5 times more than the basal side, respectively). Both 13C
glutamine and 13C proline can provide carbons for synthesis of
glutamate and mitochondrial intermediates that are released primarily
to the apical side. 13C from 13C-glutamine also labels pyruvate
and lactate that accumulate quickly at the apical side and before
being transported to basal side. RPE also exports a high level of
3-hydroxybutyrate (3-HB) to the apical side. The 3-HB does not
incorporate labeled carbons from glucose, glutamine or proline.
Conclusions: In addition to nutrient transport, RPE also actively
utilizes nutrients to synthesize and export metabolic intermediates
and amino acids to the apical side to support the outer retina.
Commercial Relationships: Jianhai Du, None; Kaitlen Knight,
None; Christina Lieu, None; Connor Jankowski, None; Van Tran,
None; James Hurley, None; Jennifer R. Chao, None
Support: NIH Grant EY06641
Program Number: 1752 Poster Board Number: D0148
Presentation Time: 8:30 AM–10:15 AM
Selective ablation of dehydrodolichyl diphosphate synthase
(Dhdds) expression in RPE alters retinal structure and function
Stephanie J. Davis1, 2, Marci L. DeRamus1, Bruce A. Pfeffer3,
Sriganesh Ramachandra Rao3, Delores A. Davis1, Steven J. Fliesler3,
Steven J. Pittler1, 4. 1Department of Vision Sciences, School of
Optometry, University of Alabama at Birmingham, Birmingham,
AL; 2Department of Vision Sciences, Post-baccalaureate
Research Education Program, Birmingham, AL; 3Department of
Ophthalmology, Biochemistry/Research Service, SUNY-Buffalo/VA
Med Ctr-Buffalo, Buffalo, NY; 4Department of Vision Sciences, UAB
Vision Science Research Center, Birmingham, AL.
Purpose: Mutations in the human gene encoding dehydrodolichyl
diphosphate synthase (DHDDS), which is required for N-linked
protein glycosylation, cause retinitis pigmentosa (RP). We generated
a conditional Dhdds knockout mouse model to study the effects of
cell type-specific ocular Dhdds deficiency.
Methods: A Dhdds conditional knockout construct was obtained
from KOMP (UC Davis), then introduced into murine ES cells,
confirmed by PCR and then FRT was used to excise a LacZ cassette.
Mouse lines were established and bred to homozygosity. Mice were
crossed to RPE-Cre mice (Yun Le, OUHSC), and were assessed by
OCT, ERG, histology, immunofluorescence, and TEM at 1, 2, and 3
mo. postnatal.
Results: OCT analysis of RPE-Dhdds-/- mice showed a significant
reduction (vs. WT) in ONL and total retinal thickness at all ages
analyzed. No OCT changes were observed in RPE-Dhdds+/- mice.
Histologic analysis showed panretinal degeneration affecting the
RPE and photoreceptor layers. TEM of 3-mo old homozygous mice
revealed ectopic RPE migration and displacement of the ELM. Credependent GFP reporter expression indicated that >90% of RPE cells
expressed Cre by 3 mo of age; however, at 1 mo, expression was
barely detectable. In 1-mo old RPE-Dhdds+/- mice, no differences
from WT were observed in the scotopic ERG. However, RPEDhdds-/- mice exhibited significant reduction (42%; p <0.01) in
b-wave amplitudes compared to WT, without altered a-wave. By 2
mo of age, hets (RPE-Dhdds+/-) showed a significant reduction in both
the a-wave (17%; p <0.05) and b-wave (44%; p <0.001) amplitudes.
These deficits were even more pronounced in RPE-Dhdds-/- mice,
with significant reductions in both the a-wave (42%; p <0.01) and
b-wave (52%; p <0.001) amplitudes.
Conclusions: RPE-specific Dhdds heterozygous and homozygous
knockout mice have been generated, using Cre-lox technology.
Homozygous knockout mice exhibit progressive retinal degeneration,
with structural and functional deficits, suggesting that RPE protein
N-glycosylation is required for retinal function and viability.
Functional deficits in heterozygous mice predict that human RP
carriers of DHDDS mutations may exhibit a haploinsufficiency
phenotype.
Commercial Relationships: Stephanie J. Davis,
None; Marci L. DeRamus; Bruce A. Pfeffer, None;
Sriganesh Ramachandra Rao, None; Delores A. Davis, None;
Steven J. Fliesler, None; Steven J. Pittler, None
Support: UAB School of Optometry, UAB Vision Science
Research Center, P30 EY003039 and R01 EY018143 (SJP); and by
R01 EY007361, an Unrestricted Grant from Research to Prevent
Blindness, and facilities and resources provided by the Dept. of
Veterans Affairs/VAWNYHS (SJF), R25 GM086256 (UAB PREP)
These abstracts are licensed under a Creative Commons Attribution-NonCommercial-No Derivatives 4.0 International License. Go to http://iovs.arvojournals.org/
to access the versions of record.
ARVO 2016 Annual Meeting Abstracts
Program Number: 1753 Poster Board Number: D0149
Presentation Time: 8:30 AM–10:15 AM
Retinal Proteomics: Protein Changes in Porcine Retinas
Following Experimental BRVO and Ranibizumab Intervention
Lasse J. Cehofski1, 2, Anders Kruse1, Sigridur Olga Magnusdottir3,
Allan Stensballe4, Bent Honoré5, Henrik Vorum1, 2. 1Department of
Ophthalmology, Aalborg University Hospital, Aalborg, Denmark;
2
Department of Clinical Medicine, Aalborg University, Aalborg,
Denmark; 3Biomedical Research Laboratory, Aalborg University
Hospital, Aalborg, Denmark; 4Department of Health Science and
Technology, Aalborg University, Aalborg, Denmark; 5Department of
Biomedicine, Aarhus University, Aarhus, Denmark.
Purpose: Retinal proteins involved in focal adhesion and
extracellular matrix remodeling have recently been shown to be
upregulated in experimental branch retinal vein occlusion (BRVO).
We hypothesized that the expression of a subset of these proteins
would be blocked by inhibition of VEGF-A after experimental
BRVO.
Methods: In five Danish Landrace pigs experimental BRVO was
induced in both eyes with an argon laser by applying laser burns
onto a branch vein in the inferior retina (Danish Animal Experiments
Inspectorate permission 2013-15-2934-00775). After 24 hours an
intravitreal injection of 0.05 ml Ranibizumab was administered
in the right eyes of the animals while left eyes were given an
intravitreal injection of 0.05 ml 0.9% sodium chloride water. Three
days after BRVO the retinas were excised and prepared for liquid
chromatography tandem mass spectrometry by a filter-aided sample
preparation method. In MaxQuant mass spectrometry data were
searched against a pig protein database with a peptide and protein
false discovery rate of 1%. In Perseus the proteins were filtered
requiring at least 2 unique peptides per protein. A two-tailed paired
t-test was used for statistic analysis.
Results: In retinas treated with Ranibizumab the analysis identified
29 significantly upregulated proteins (p < 0.05, fold change > 1.25)
and 86 significantly downregulated proteins (p < 0.05, fold change <
0.80). Bioinformatic analysis showed a downregulation of proteins
involved in cell adhesion including integrin β-1, nectin-2, nidogen-2,
protocadherin 7, contactin associated protein 1 and transmembrane
glycoprotein NMB. Significantly downregulated proteins also
included lipocalin-7 and platelet derived growth factor receptor-β.
KEGG pathway analysis identified a downregulation of pathways
involved in protein processing in endoplasmic reticulum, cell
adhesion and adherens junction.
Conclusions: Integrin-β1, nidogen-2 and lipocalin-7 were
downregulated in retinas treated with Ranibizumab. In an earlier
study these proteins were found to be upregulated when porcine
retinas with experimental BRVO were compared to controls.
Therefore, we propose the hypothesis that experimental BRVO
is associated with an upregulation of integrin-β1, nidogen-2 and
lipocalin-7 that is reversed through intervention with Ranibizumab.
Commercial Relationships: Lasse J. Cehofski, None;
Anders Kruse; Sigridur Olga Magnusdottir, None;
Allan stensballe, None; Bent honoré, None; Henrik Vorum, None
Support: Svend Andersen Foundation, Bagger-Sørensen Foundation,
Obel Family Foundation, Herta Christensen Foundation, North
Denmark Region
Program Number: 1754 Poster Board Number: D0150
Presentation Time: 8:30 AM–10:15 AM
Mapping protein-protein interactions of Bestrophin1 - a potential
insight into the development of Bestrophinopathies
Elena Segal1, 2, Ronit Heinrich1, Shadi Safuri1, Ami Aronheim3,
Naim Shehadeh2, Ido Perlman1. 1Physiology and neuroscience,
The Ruth & Bruce Rappaport Faculty of Medicine, Technion-Israel
Institute of Technology, Haifa, Israel; 2Pediatrics A, Meyer Children’s
Hospital, Rambam Health Care Campus, Haifa, Israel; 3Molecular
Genetics, The Rappaport Family Institute for Research in the Medical
Sciences, Technion – Israel Institute of Technology, Haifa, Israel.
Purpose: Specific mutations in hBest1 gene result in different
phenotypes, depending upon the mutation, including juvenile-onset
Best Vitelliform Macular Degeneration, adult-onset Vitelliform
Macular Dystrophy, Autosomal Dominant Vitreoretinochoroidopathy
and Autosomal Recessive Bestrophinopathy. The expression of Best1
is confined to the retinal pigment epithelium (RPE), responsible
for phagocytosis of photoreceptors outer segments (POS). We have
recently found that mutated Best1, expressed in ARPE-19 cells, alter
POS phagocytosis compared to the wild type Best1 in a manner
depending upon the mutation. We hypothesize that Best1 modulates
POS phagocytosis via protein-protein interactions with regulatory
proteins
Methods: The Ras recruitment system (Broder et al., 1998) is based
on the ability of Ras mutant to be localized to the plasma membrane
through the interaction between two proteins. Best1 is fused to Ras
whereas human RPE cDNA library is fused to v-Src membrane
localization signal. Ras membrane localization via protein-protein
interaction results in the complementation of a yeast temperature
sensitive mutant strain in the Ras guanyl nucleotide exchange factor,
Cdc25-2 (Aronheim, 2001a and b). The screening is performed as
follows: Best1 is prepared on Met425 plasmid whereas the library is
constructed on galactose inducible plasmid. The Met425 promoter
is repressed in the presence of methionine, thus Best1 expression
is induced when cells are grown on a medium lacking methionine.
The library expression is induced in medium containing galactose
and repressed in the presence of glucose. In order to induce the
expression of Best1 and library, cells are grown on galactose medium
lacking methionine. The candidate clones are further analyzed by
DNA extraction and sequencing and screened with 4 different Best1
mutations
Results: Mapping protein-protein interactions of wild type Best1
and RPE library resulted in 13 candidates, from which two DNA
sequences analyzed by BLAST were consistent with 2 different
proteins that may potentially interact with Best1. Those 2 proteins
failed to interact with mutated Best1; Arg47His and Arg200X
mutations
Conclusions: Our findings suggest that Best1 may interact with 2
different proteins, previously reported to regulate cell signaling, and
that those interactions may be altered by mutations, thus contributing
to disease manifestation
Commercial Relationships: Elena Segal, None; Ronit Heinrich,
None; Shadi Safuri, None; Ami Aronheim, None; Naim Shehadeh,
None; Ido Perlman, None
Program Number: 1755 Poster Board Number: D0151
Presentation Time: 8:30 AM–10:15 AM
Retinal pigment epithelial cells oxidize fatty acid from ingested
photoreceptor outer segments to produce ketone bodies
Juan Reyes-Reveles1, Kathleen Boesze-Battaglia1,
Desiree Alexander1, Anuradha Dhingra1, Alvina Bragin1,
Nancy J. Philp2. 1Biochemistry, University of Pennsylvania,
Philadelphia, PA; 2Thomas Jefferson University, Philadelphia, PA.
These abstracts are licensed under a Creative Commons Attribution-NonCommercial-No Derivatives 4.0 International License. Go to http://iovs.arvojournals.org/
to access the versions of record.
ARVO 2016 Annual Meeting Abstracts
Purpose: To determine if RPE utilizes phospholipids from
photoreceptor outer segments (OSs) for fatty acid oxidation and
ketogenesis.
Methods: Polarized Human fetal RPE (hfRPE) and ARPE19 cells
were fed, palmitate, docosahexaenoic acid (DHA) or outer segments
for various periods of time and β-Hydroxybutyrate (β-HB) levels
were measured using a commercially available kit (LiquiColor,
Stanbio). The ketogenic enzymes, 3-hydroxy-3-methylglutarylCoA synthase 2 (HMGCS2) and β-hydroxybutyrate dehydrogenase
(BDH1) were accessed by immunoblotting and subcellular
localization determined by immunostaining and confocal microscopy.
Results: HMGCS2, the enzyme catalyzing the committed step in
ketogenesis was detected in lysates prepared from ARPE-19 and
hfRPE. Consistent with previous studies showing that hfRPE cells
utilize the 16 carbon fatty acid, when cells were incubated with the
22:6 substrate, DHA, β-HB was released. Twice as much β-HB was
secreted by the apical RPE when the cells were fed DHA as compared
to the 16 carbon palmitate. To determine if the endogenous substrate
for RPE-ketogenesis is derived from ingested OS, polarized hfRPE
or ARPE19 were fed OS in the apical chamber and β-HB levels
measured. Ingestion of OSs in the presence of glucose stimulated
ketogenesis resulting in the preferential release of β-HB into the
apical medium in both hfRPE and to a lesser extent in ARPE19, with
no detectable β-HB in the basal media. The extent of β-HB released
was does dependent, with 0.3+/- 0.028 nmoles of β-HB released
with 25uM OS and 1.65 +/-0.148 nmoles released when cells were
fed 250uM OS. The addition of OS also resulted in an increase in
the free fatty acid content of the RPE cells, to 20+/-1.87 uM with OS
addition. No β-HB release was observed when the RPE cells were
challenged with beads and decreased levels of β-HB were released
upon challenge with oxidized OS, from 3.16+/- 0.29 nmoles with
OS to 0.42 +/- 0.54 nmoles (oxOS). In both human and mouse RPE,
HMGCS2 co-localized with the mitochondrial marker COX-4.
Conclusions: Human RPE cells can utilize both 16:0 and 22:6 fatty
acid substrates and OS to generate β-HB, which is preferentially
exported across the apical membrane and serves as a metabolic
substrate or neuroprotectant for photoreceptor cells.
Commercial Relationships: Juan Reyes-Reveles, None;
Kathleen Boesze-Battaglia, None; Desiree Alexander, None;
Anuradha Dhingra, None; Alvina Bragin, None; Nancy J. Philp,
None
Support: NEI grant(s) EY-10420 (KBB) and EY-012042 (NJP).
Program Number: 1756 Poster Board Number: D0152
Presentation Time: 8:30 AM–10:15 AM
Differential Activity of Systemic and Retinal 12/15-Lipoxygenases
in a Mouse Model of Diabetes
Ahmed S. Ibrahim1, 2, Heba M. Saleh1, 6, Khaled Hussein1, 5,
Babak Baban1, 3, Nader Sheibani4, Mohamed A. Al-Shabrawey1, 6.
1
Department of Oral Biology, College of Dental Medicine, Augusta
University, Augusta, GA; 2Department of Biochemistry, Faculty
of Pharmacy, Mansoura University, Mansoura, Egypt; 3Section of
Plastic Surgery, Department of Surgery, Augusta University, Augusta,
GA; 4Department of Ophthalmology and Visual Sciences, University
of Wisconsin School of Medicine and Public Health, Madison, WI;
5
Oral and Dental Research Division, Department of Surgery and
Medicine, National Research Center, Cairo, Egypt; 6Culver Vision
Discovery Institute and Ophthalmology, Medical College of Georgia,
Augusta, GA.
Purpose: The 12/15-lipoxygenase (12/15-LOX) has proinflammatory
effects such as enhanced chemotaxis and vascular adhesion of
leukocytes, which has been implicated in the pathogenesis of diabetic
retinopathy (DR). Our previous studies have shown increased
retinal 12/15-LOX expression and activity in the vitreous of diabetic
patients with retinopathy, diabetic mouse retinas, and human retinal
endothelial cells treated with high glucose. In the current study we
aimed to evaluate the change in circulating 12/15-LOX activity in a
type 1 diabetic mouse model and to characterize the contribution of
endothelial 12/15-LOX versus circulating 12/15-LOX to leukocyte
adhesion.
Methods: A lipidomic approach using liquid chromatography
coupled with mass-spectrometry (LC-MS) was used to investigate the
activity of circulating 12/15-LOX on linoleic acid (LA), arachidonic
acid (AA), docosahexaenoic acid (DHA), and eicosapentaenoic
acid (EPA) in the plasma of streptozotocin-induced diabetic mice
(6-months of diabetes). Mouse retinal endothelial cells (mREC) as
well as leukocytes isolated from 12/15-LOX knockout (KO) or wild
type (WT) mouse were used to study leukocyte adhesion using the
myeloperoxidase assay.
Results: Out of 29 bioactive lipids screened, only 3 metabolites
showed significant changes in diabetic mouse plasma compared to
normal controls. The increased metabolites included two products
derived from the effect of 12/15-LOX on DHA (Resolvin D2 and
14-HDoHE) and one product derived from the lipoxygenation of
LA (13-OxoODE). In contrast, there was no significant change in
the plasma level of metabolites derived from 12/15-lipoxygenation
of AA or EPA including 12- and 15-HETE or 12- and 15-HEPE,
respectively. These data presumably reflect a differential role of
circulating 12/15-LOX versus endothelial 12/15-LOX in mediating
leukocyte adhesion. To this end, we performed in vitro leukocyte
adhesion assay on LPS-activated mREC using leukocytes isolated
from 12/15-LOX KO versus WT mice. Activated mREC significantly
augmented the number of adherent leukocytes isolated from 12/15LOX KO relatively to the same extent as those derived from WT
mice.
Conclusions: Our current and previously studies suggest a
differential role of endothelial 12/15-LOX versus the one in
circulating blood cells in mediating the inflammatory responses
during DR. This may facilitate the development of more precisely
targeted treatment strategies.
Commercial Relationships: Ahmed S. Ibrahim, None;
Heba M. Saleh, None; Khaled Hussein, None; Babak Baban,
None; Nader Sheibani, None; Mohamed A. Al-Shabrawey, None
Support: National Eye Institute (5R01EY023315-02)
Program Number: 1757 Poster Board Number: D0153
Presentation Time: 8:30 AM–10:15 AM
Characterization of Src-homology phosphotyrosyl phosphatase 2
in the Retina
Raju V. Rajala1, 2, Yuhong Wang1, Michelle Ranjo-Bishop1,
Ammaji Rajala1. 1Ophthalmology, Univ of Oklahoma Hlth Sci Ctr,
Oklahoma City, OK; 2Physiology and Cell Biology, University of
Oklahoma Health Sciences Center, Oklahoma City, OK.
Purpose: Reactive oxygen species (ROS) play a multitude of
signaling roles in different organisms from bacteria to mammalian
cells. They were initially thought to be toxic byproducts of aerobic
metabolism, but have now been acknowledged as central players
in the complex signaling network of cells. Mild concentrations of
ROS have been shown to inhibit the activity of protein tyrosine
phosphatase, PTP1B through oxidative inactivation of catalytic
cysteine, which is in the active site of PTP1B. Studies from
our laboratory show that pharmacological inhibition or genetic
deletion of PTP1B in photoreceptors resulted in the activation of
a neuroprotective insulin receptor survival signaling in both rods
and cones. Our laboratory is working towards identification of
endogenous proteins that can stimulate ROS production to inhibit
These abstracts are licensed under a Creative Commons Attribution-NonCommercial-No Derivatives 4.0 International License. Go to http://iovs.arvojournals.org/
to access the versions of record.
ARVO 2016 Annual Meeting Abstracts
PTP1B activity. Src-homology phosphotyrosyl phosphatase 2
(Shp2) has recently been shown to stimulate ROS production in
macrophages. In this study, we characterized Shp2 in the retina and
studied the effect of Shp2 on ROS generation in cultured R28 retinal
neurons.
Methods: Immunohistochemistry was used to characterize Shp2
expression in the retina, using cryo-, paraffin-, prefer-, and methanolfixed tissues from bovine, wild-type mouse, and cone-dominant Nrl-/mouse retinas. Mouse retinas were isolated from dark- and lightadapted (300 lux) conditions. Retinas were subjected to immunoblot
analysis to study the state of phosphorylation on Shp2 as a function
of its activation. Shp2 activity was also measured. Cultured R28
retinal neurons were transfected with Shp2 wild-type and inactive
mutant constructs, and the production of ROS was examined.
Results: Our results show that Shp2 is expressed in both rod and
cone photoreceptor cells, in addition to the inner retinal layers. We
also found significantly higher levels of tyrosine phosphorylation
and Shp2 phosphatase activity in light-adapted retinas than in darkadapted retinas. We found that catalytically inactive Shp2 (cysteine
to serine substitution) fails to generate ROS, whereas wild-type Shp2
promotes ROS production in R28 retinal neurons.
Conclusions: Our data show the expression of Shp2 in various layers
of the retina. Our neuronal cell culture data show that Shp2 promotes
ROS production in vitro.
Commercial Relationships: Raju V. Rajala, None; Yuhong Wang;
Michelle Ranjo-Bishop, None; Ammaji Rajala, None
Support: NIH/NEI grant (EY016507, EY00871, EY021725)
Program Number: 1758 Poster Board Number: D0154
Presentation Time: 8:30 AM–10:15 AM
Correlation between concentration of vitreous angiogenic
cytokines (VEGF-B and PIGF) with central retinal thickness and
macular volume in diabetic retinopathy patients
Joana Mesquita1, João Paulo Castro Sousa2, 1, Sara Vaz-Pereira3, 4,
Arminda Neves2, Paulo Tavares-Ratado1, Luís Passarinha1,
Cândida Tomaz1. 1CICS-UBI – Health Sciences Research Centre,
University of Beira Interior, Covilhã, Portugal; 2Ophthalmology,
Centro Hospitalar de Leiria-Pombal, Leiria, Portugal;
3
Ophthalmology, Hospital de Santa Maria, Lisbon, Portugal; 4Faculty
of Medicine, University of Lisbon, Lisbon, Portugal.
Purpose: Placental growth factor (PlGF) and vascular endothelial
growth factor B (VEGF-B) are members of the vascular endothelial
growth factor family, binding to VEGF-R1. PIGF inhibition
suppresses pathological angiogenesis in inflammatory disorders
and diabetic retinas. PIGF levels are overexpressed in the vitreous
of diabetic retinopathy (DR) patients and levels may increase
with the severity of the disease. VEGF-B is a vascular survival
factor, safeguarding the balance between blood vessel growth and
degeneration. Under degenerative conditions VEGF-B is a survival
factor to protect cells from apoptosis; during certain pathologies can
act as antiangiogenic factor to prevent overgrowth of blood vessels.
Angiogenic VEGF-B activity during ocular neovascularization may
be due to its survival effect, rescuing neovessels from apoptosis.
Vitreous VEGF-B levels are significantly increased in DR patients.
The purpose of this study was to correlate vitreous angiogenic
cytokines (VEGF-B and PIGF) levels in DR measured by ELISA
and demonstrate its correlation with quantitative measurements of
central retinal thickness (CRT) and macular volume (MV) by optical
coherence tomography (OCT).
Methods: Vitreous samples were obtained from 42 DR patients
undergoing vitrectomy. ELISA was used to quantify vitreous
VEGF-B (pg/ml) (n=24) and vitreous PIGF (pg/ml) (n=18). OCT
scans were evaluated to measured CRT (μm) and MV (mm3). Results
obtained of VEGF-B (pg/ml) and PIGF (pg/ml) were correlated
with CRT (μm) and MV (mm3). All patients included in this study,
which adhered to the tenets of the Declaration of Helsinki, gave their
informed consent to surgical treatment.
Results: Correlation between vitreous VEGF-B and CRT was
statistically significant, positive and moderate: 0.441 (p ≤ 0,05).
Correlation between vitreous VEGF-B and MV was statistically
significant, positive and robust: 0.716 (p ≤ 0,01). Correlation
between vitreous PIGF and CRT or vitreous PIGF and MV, was not
statistically significant.
Conclusions: These results suggest that CRT and MV observed in
OCT increase with increased levels of VEGF-B. This study shows
that overexpression of VEGF-B may have an impact on CRT and
MV of DR patients, thus targeting VEGF-B inhibition may have
therapeutic beneficial implications.
Commercial Relationships: Joana Mesquita, Alimera Sciences;
João Paulo Castro Sousa, None; Sara Vaz-Pereira, None;
Arminda Neves; Paulo Tavares-Ratado, None; Luís Passarinha,
None; Cândida Tomaz, None
These abstracts are licensed under a Creative Commons Attribution-NonCommercial-No Derivatives 4.0 International License. Go to http://iovs.arvojournals.org/
to access the versions of record.
ARVO 2016 Annual Meeting Abstracts
Program Number: 1759 Poster Board Number: D0155
Presentation Time: 8:30 AM–10:15 AM
The Effect of Electrical Stimulation on Cell viability and
Proliferation in 661W cells
Ji Yang, Qing-Feng Wang, Men Lin, Zi-Bing Jin. Laboratory for Stem
Cell & Retinal Regeneration (Jin Lab), The Eye Hospital of Wenzhou
Medical University, WenZhou, China.
Purpose: Recent studies suggested Electrical stimulation had the
ability to rescue retina function from retinal disease. However, the
effect of electrical stimulation on photoreceptor cells is still poorly
understood. The purpose of this study was to detect the effect of
electrical stimulation on cell viability and proliferation in 661W cells.
In addition, we tried to preliminarily explore mechanism of the effect
on 661W cells after electrical stimulation.
Methods: We designed an experimental instrument, in vitro cell
electrical stimulation instrument (ESI). The ESI is composed of
following parts: a two-channel power supply (STG-4002), the
connecting circuits and 6-well plate’s lid equipped with four parallel
U-shaped electrodes. 661W cells were plated into the 6-well plate
of ESI and cultivated for 24h. 661W cells were supplemented with
serum-free medium. And we divided 661W cells into two groups.
One was stimulated by 600mv while the other one was stimulated by
900mv for 24h. We detected cell viability and proliferation by Cell
Counting Kit-8 and EdU. The changes of mRNA and protein level
were determined by Quantitative real-time RT-PCR and western blot.
Results: After electrical stimulation for 24h, the cell viability
increased in the electrical stimulation group compared to the control
group (P<0.001). The rate of proliferating cells increased significantly
in the electrical stimulation group compared to the control group
(P<0.001). And the brain derived neurotrophic factor (Bdnf) mRNA
(P<0.01) and protein (P<0.001) level were found increased after
electrical stimulation compared to the control group.
Conclusions: Our results suggest that electrical stimulation has the
ability to promote 661W cells’ viability and proliferation. In addition,
we found the effect of electrical stimulation on 661W cells may
depend on increasing Bdnf expression.
Commercial Relationships: Ji Yang; Qing-Feng Wang, None;
Men Lin, None; Zi-Bing Jin, None
Support: National Key Basic Program of China (2013CB967502 to
Z.BJ), National Natural Science Foundation of China (81371059 to
Z.BJ)
Program Number: 1760 Poster Board Number: D0156
Presentation Time: 8:30 AM–10:15 AM
Confocal imaging reveals glucose uptake by photoreceptors in
vivo
Michelle Giarmarco, Mark A. Kanow, Ken J. Lindsay, Jianhai Du,
James Hurley. University of Washington, Seattle, WA.
Purpose: Identifying the metabolic fuels used by neurons and glia
in the retina is of fundamental importance for understanding retinal
function and viability. In a previous study1 we showed that pyruvate
kinase, the enzyme that catalyzes the final step in glycolysis, is
abundant in photoreceptors (PRs) and absent from Müller glia. We
hypothesize that the primary entry site for glucose in the outer retina
is PRs, not Müller glia, and that PRs provide fuel to Müller glia in the
form of lactate2. To test this hypothesis, we used an in vivo approach
to decipher which retinal cell types take up glucose in live zebrafish
larvae. We also tested the hypothesis by using immunohistochemistry
(IHC) to assess the distribution of glucose transporters (GLUTs) in
adult mouse retinas.
(References: 1. Lindsay, K. J. et al. (2014). PNAS, 111(43), 15579–
15584. 2. Hurley, J. B., Lindsay, K. J., & Du, J. (2015). J Neurosci
Res, 93(7), 1079–1092.)
Methods: For in vivo glucose uptake imaging experiments, a
fluorescent glucose analog, 2-NBDG, was injected into yolks of
transgenic larval zebrafish expressing a red fluorescent protein
marker (tdTomato) in cone PRs. Larvae were then embedded in
agarose and imaged live via confocal microscopy, and colocalization
between 2-NBDG and tdTomato was determined. For IHC, adult
mouse eyecups were fixed in 4% paraformaldehyde and cut into
20-µm cryosections. Sections were incubated overnight with primary
antibodies against CRALBP, arrestin and GLUT1, followed by
incubation with fluorescent-tagged secondary antibodies. Sections
were imaged via confocal microscopy.
Results: In vivo imaging of larval zebrafish retinas revealed robust
2-NBDG uptake in cone PRs and the inner plexiform layer within
minutes of injection. IHC showed that GLUT1 in mouse retina is
abundant in PRs above the Müller glia apical processes, and appears
to be absent from Müller glia. Because the apical processes of Müller
glia partially overlap with the most inner portion of the PR cell body
layer, we are developing methods to resolve definitively whether or
not GLUT1 is present on the apical processes of Müller glia.
Conclusions: Rapid in vivo uptake of glucose into zebrafish cone
photoreceptors and the presence of glucose transporters on mouse rod
PR inner segments is consistent with our hypothesis that PRs and not
Müller glia are the initial site of glucose entry at the outer retina.
Commercial Relationships: Michelle Giarmarco;
Mark A. Kanow, None; Ken J. Lindsay, None; Jianhai Du, None;
James Hurley, None
Support: NSF GRF #2013158531, NIH NEI #EY06641, NIH NEI
#EY017863
Program Number: 1761 Poster Board Number: D0157
Presentation Time: 8:30 AM–10:15 AM
Changes in the Retinal Cholinergic System (RCS) in the Tg-SwDI
Alzheimer’s Disease Mouse Model
Fred Oliveira-Souza1, Mark Bolding2, Marci L. DeRamus1,
Thomas vanGroen3, Christianne E. Strang4. 1Vision Sciences,
University of Alabama at Birmingham, Birmingham, AL; 2Radiology,
University of Alabama at Birmingham, Birmingham, AL; 3Dept. Cell,
Developmental and Integrative Biology, University of Alabama at
Birmingham, Birmingham, AL; 4Psychology, University of Alabama
at Birmingham, Birmingham, AL.
Purpose: Alzheimer’s disease (AD), the most common type of
dementia, is characterized by severe cognitive deficits, which may
arise as a result of tauopathy, senile plaques, substantial neuronal
loss and abnormalities of several neurotransmitter systems (e.g.
cholinergic). Impairment in motion perception, contrast sensitivity,
acuity and color are also present in AD. We believe that these visual
deficits may stem from disturbances in the RCS. To assess this
hypothesis, we compared acetylcholine receptor (AChR) transcript
expression in the retinas of Tg-SwDI mice in three age groups: 6-8.9,
9-11.9 and 12-15 months old (mo). Tg-SwDI mice express the human
amyloid precursor protein with the Swedish KM670/671NL, Dutch
E693Q, and Iowa D694N mutations.
Methods: We conducted quantitative real-time polymerase chain
reaction (qPCR) with validated and optimized primers using wholeretina RNA.
Results: We observed statistically significant (≤0.05) differences
in all age groups, in nicotinic (nAChR) and muscarinic (mAChR)
mRNA transcripts in the retinas of AD mice as compared to agematched wild type (WT) mice; fold-change is shown in parentheses.
At 6-8.9 mo, there was upregulation (UR), in α2 (3.7), α4 (2.7), α5
(2.4), α6 (1.9), α7 (2.5), β3 (2.4) and β4 (3.0) nAChR subunits; and
downregulation (DR) in α9 (3.2) and α10 (7.1) nAChR subunits,
m4 (2.6) and m5 (6.1) mAChRs. At 9-11.9 mo, there was UR in m1
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ARVO 2016 Annual Meeting Abstracts
mAChR (13.3) and in α2 (14.5), α3 (19.3), α4 (8.7), α5 (6.5), α6
(19.3), α9 (4.7), β3 (3.8) and β4 (5.2) nAChR subunits. They also
exhibited DR in α10 nAChR subunit (4.9) and m5 mAChR (8.5). At
12-15 mo, there was DR in m4 (3.3) and m5 (5.8) mAChRs, α4 (5.1),
α7 (3.8), α9 (6.3) and α10 (7.9) nAChR subunits.
Conclusions: The early UR of AChR transcripts might compensate,
in part, for the loss of cholinergic cells. As the cell loss becomes
extensive, this compensatory mechanism can no longer mitigate the
cholinergic deficits. Knowing the causes of the visual deficits may
be crucial in early AD diagnosis, as they may occur before cognitive
decline is observed. The retina provides an enormous opportunity
to develop non-invasive biomarkers for AD and early diagnosis
through visual assessment. This work is an important step in the quest
towards attaining a better understanding of AD and facilitating its
early detection and treatment.
Commercial Relationships: Fred Oliveira-Souza, None;
Mark Bolding, None; Marci L. DeRamus; Thomas vanGroen,
None; Christianne E. Strang, None
Program Number: 1762 Poster Board Number: D0158
Presentation Time: 8:30 AM–10:15 AM
Distinct bipolar cell subtypes carry parallel streams of temporal
information under scotopic conditions
Christopher Fortenbach3, 1, Marie E. Burns3, 2. 1School of Medicine,
University of California, Davis, Davis, CA; 2Ophthalmology &
Vision Science and Cell Biology and Human Anatomy, University of
California, Davis, Davis, CA; 3Center for Neuroscience, University of
California, Davis, Davis, CA.
Purpose: Visual signal processing begins at the bipolar cell within
the retina. Rods, which are responsible for vision in scotopic
conditions, form direct chemical synapses with rod bipolar cells and
signal indirectly to a dozen subtypes of cone bipolar cells. Cone
bipolar cells display distinct temporal properties when stimulated
directly by cones. Given that the photoresponses of rods are
significantly slower than cones and that this limits the ability of
rods to convey high frequency visual information to second-order
retinal neurons, it remains unknown whether rod-driven visual signal
separates into parallel channels carrying distinct temporal information
at the level of the bipolar cell.
Methods: Whole-cell recordings of rods, rod bipolar cells, cone
bipolar cells, and AII amacrine cells were performed in darkadapted mouse retina slices. Voltage-responses were elicited by
calibrated flashes and sinusoidal flickering stimuli. Light-evoked
flicker responses were then subjected to Fourier analysis to extract
both the magnitude and phase of the cellular response at the
stimulus frequency. Following recording, slices were subject to
immunohistochemical analysis to determine bipolar cell subtype.
Results: At the scotopic intensities investigated, each class of cell
displayed low-pass frequency tuning with different bipolar cell
subtypes displaying different characteristic cutoff frequencies and
phase shifts. Some bipolar classes showed improved temporal
performance as background intensities increased. The temporal
performance of individual bipolar cell subtypes was further impacted
by the addition of strychnine, which blocks glycinergic signaling
in the retina. In the presence of DNQX, which blocks synaptic
output from rod bipolar cells, rod bipolar cells displayed smaller
flicker magnitudes and a reduced ability to convey high-frequency
information.
Conclusions: Rod-driven responses within the retina separate into
parallel channels with distinct temporal properties at the level of
the bipolar cell. Visual information conveyed by these channels is
influenced by feedback within the inner retina in a subtype-dependent
manner, which can further shape their temporal performance.
Commercial Relationships: Christopher Fortenbach, None;
Marie E. Burns
Support: National Eye Institute R01-EY14047, UC Davis NEI
Vision Training Grant (T32-EY015387), the UC Davis NEI Core
Grant (P30- EY012576), and the UC Davis Physician Scientist
Training Program.
Program Number: 1763 Poster Board Number: D0159
Presentation Time: 8:30 AM–10:15 AM
Metabolic activity of single isolated mouse rod photoreceptors
Chunhe Chen, Leopold Adler, Yiannis Koutalos. Department of
Ophthalmology, Medical University of South Carolina, Charleston, SC.
Purpose: To determine the effectiveness of different metabolites
as metabolic substrates of mouse rod photoreceptors. In mouse rod
photoreceptor outer segments, the all-trans retinal released from
photoactivated rhodopsin is reduced to all-trans retinol via a reaction
that utilizes metabolic input in the form of NADPH. We have used
the conversion of all-trans retinal to all-trans retinol to measure the
metabolic activity of single rod photoreceptors isolated from mouse
retinas.
Methods: Experiments were carried out with dark-adapted living rod
photoreceptors isolated from 2-3 month old 129/sv wild type mice.
NADPH generation in a single cell was measured from the ratio
Fex-340/Fex-380 of the fluorescence intensities excited by 340 and
380 nm light with emission collected >420 nm. For an experiment,
all-trans retinal was generated by exposing a single dark-adapted cell
to >530 nm light for 1 min, and the extent of conversion to all-trans
retinol was measured at 30 min after light exposure. The value of the
Fex-340/Fex-380 ratio was used to calculate the fraction of all-trans
retinal converted to all-trans retinol and the corresponding fraction
of NADPH (Adler et al. 2014, J Biol Chem 289:1519). Amino acids
were used as metabolic substrates at a concentration of 0.5 mM.
Results: In the presence of 5 mM glucose as metabolic substrate,
single isolated mouse rods converted ~70-80% of all-trans retinal
to retinol, corresponding to an NADPH fraction of ~10-20%. From
among the 20 amino acids, glutamine (0.5 mM) supported a similar
level of conversion of all-trans retinal to retinol as 5 mM glucose,
corresponding to similar fraction of NADPH. The rest of the amino
acids (at 0.5 mM concentration) supported the conversion of all-trans
retinal to retinol to a much lesser extent, indicating NADPH fractions
of 1-2% at the most. With either glucose or glutamine as metabolic
substrate, the presence of formic acid (5 mM) resulted in significantly
lower conversion of all-trans retinal to retinol, indicating lower
NADPH fractions.
Conclusions: Glucose and glutamine are the preferred metabolic
substrates of mouse rod photoreceptors. Other amino acids can
support metabolic activity only to a limited extent, perhaps due to the
absence of efficient transporters or lack of the required intracellular
metabolic machinery. Formic acid, the toxic metabolite of methanol,
can significantly suppress rod photoreceptor metabolic activity.
Commercial Relationships: Chunhe Chen, None; Leopold Adler,
None; Yiannis Koutalos, None
Support: Supported by NEI grant EY014850 and by an unrestricted
award to the Department of Ophthalmology at Medical University of
South Carolina from Research to Prevent Blindness, Inc.
Program Number: 1764 Poster Board Number: D0160
Presentation Time: 8:30 AM–10:15 AM
Evaluation of the membrane affinity of peripheral membrane
proteins in live rod photoreceptors
Nycole A. Maza1, 2, Peter D. Calvert1, 2. 1Neuroscience and
Ophthalmology, SUNY Upstate Medical University, Syracuse, NY;
2
SUNY Eye Institute, Syracuse, NY.
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ARVO 2016 Annual Meeting Abstracts
Purpose: Transducin is a peripheral membrane protein that
undergoes light-dependent translocation between the rod outer
segment (ROS) and cell body. The mechanisms underlying this
translocation have been the subject of intense investigation, but
remain unclear. We tested the hypothesis that hydrophobic and
electrostatic membrane interactions, due to lipid moieties and surface
charges on proteins and disc membranes, play a role in governing
peripheral membrane protein distribution by setting membrane
affinity.
Methods: A myristoyl (MYR) and/or farnesyl (FAR) transferase
motif was added to EGFP with a 15aa polybasic (+), acidic (-) or
neutral (0) charge domain in between. Each probe was placed under
the control of the XOP promoter and expressed in Xenopus laevis
rod photoreceptors via REMI transgenics. Retinas were harvested
and analyzed via live cell confocal microscopy and multiphoton
fluorescence relaxation after photoconversion (mpFRAP). Diffusion
coefficients were calculated from mpFRAP data. Two-tailed student
T-test was used for statistical analysis.
Results: The mobilities of the probes varied as follows: MYR(0)
EGFP(0)FAR << MYR(+)EGFP ≤ MYR(0)EGFP ≤ EGFP(0)FAR
<< MYR(-)EGFP ≤ EGFP(-)FAR <<<EGFP. Mutating the lipid
transferase motif resulted in probes with mobilities comparable
to EGFP alone. Myristoylated proteins were enriched in the ROS,
despite differences in charge and mobility. The distribution and
mobility of farnesylated proteins varied based on charge character.
The dually lipidated probe was distributed throughout the rod.
Conclusions: The mobility of peripheral membrane proteins in the
ROS depends on adjacent charge character. The data suggest that
positive charge and lipidation work together to increase membrane
affinity, while negative charge reduces the membrane affinity
provided by the lipid. Dually lipidated proteins had the lowest
mobility, indicating an additional lipid moiety conveys greater
membrane affinity than adjacent positive charge. Ultimately, our
results support the hypothesis that hydrophobic and electrostatic
interactions play a role in setting the membrane affinity, however the
distribution differs based on lipid identity.
Commercial Relationships: Nycole A. Maza; Peter D. Calvert,
None
Support: NIH Grant EY018421
saline solution at 4 °C. Metabolically active rod photoreceptors
were isolated from the peripheral region of human retinas.
Interphotoreceptor retinoid binding protein (IRBP) was extracted
from bovine retinas and purified to homogeneity by combination of
Con-A affinity, ion exchange, and size-exclusion chromatography. Its
concentration was determined by amino acid analysis and absorption
spectrophotometry. Rod photoreceptors were isolated from the
bleached retinas and incubated in the presence of either IRBP and 11cis retinal to regenerate rhodopsin, or IRBP alone. After incubation,
cells were washed with a physiological saline solution and placed
on an epifluorescence microscope stage at 37 °C. Cells were then
exposed to different concentrations of 11-cis retinal with different
concentrations of BSA as carrier. The levels of rod outer segment
lipofuscin precursors were determined from their fluorescence (ex,
490 nm; em >520 nm).
Results: Exposure to 11-cis retinal resulted in a significant increase
in lipofuscin precursor levels, which was higher for cells that
contained rhodopsin. Higher extracellular concentrations of BSA
resulted in lower levels of lipofuscin precursors formed by the
exogenous 11-cis retinal.
Conclusions: 11-cis retinal can generate lipofuscin precursors in
the outer segment of human rod photoreceptors. The extracellular
presence of a retinal carrier limits the formation of lipofuscin
precursors, likely by buffering 11-cis retinal.
Commercial Relationships: Leopold Adler, None; Chunhe Chen,
None; Federico Gonzalez-Fernandez, None; Yiannis Koutalos,
None
Support: Supported by: NEI Grant EY014850 (YK); an unrestricted
award to the Department of Ophthalmology at Medical University of
South Carolina from Research to Prevent Blindness; Start-up Award,
Research! Mississippi; Activation award, VA Office of Research and
Development (ORD); Merit Review Award I01BX007080, ORD
(F.G.-F.); NEI grant EY09412 (F.G.-F.)
Program Number: 1765 Poster Board Number: D0161
Presentation Time: 8:30 AM–10:15 AM
Extracellular Presence of a Non-Specific Retinal Carrier Prevents
the Formation of Lipofuscin Precursors from 11-Cis Retinal in
Human Rod Photoreceptor Outer Segments
Leopold Adler1, Chunhe Chen1, Federico Gonzalez-Fernandez2, 3,
Yiannis Koutalos1. 1Ophthalmology, Medical University of South
Carolina, Charleston, SC; 2Departments of Ophthalmology &
Pathology, University of Mississippi School of Medicine, Jackson,
MS; 3R&D Division, Veterans Affairs Medical Center, Jackson, MS.
Purpose: The 11-cis retinal chromophore of rhodopsin, the rod
visual pigment, is supplied to rod photoreceptor outer segments
extracellularly via the interphotoreceptor matrix. 11-cis retinal
can react with outer segments to generate precursors of lipofuscin
that eventually accumulate in lysosomes of the retinal pigment
epithelium. We tested whether extracellular bovine serum albumin
(BSA), a non-specific retinal carrier, can prevent the formation
of lipofuscin precursors from 11-cis retinal in isolated human rod
photoreceptors.
Methods: Fresh human donor eyes were procured through the
National Disease Resource Interchange. Eyes were dissected and
the retinas isolated within 48 hrs after death. Isolated retinas were
bleached to destroy residual rhodopsin, and stored in a physiological
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