ARVO 2016 Annual Meeting Abstracts
517 RPE Pathology
Thursday, May 05, 2016 8:00 AM–9:45 AM
Exhibit/Poster Hall Poster Session
Program #/Board # Range: 6027–6055/D0246–D0274
Organizing Section: Retinal Cell Biology
Program Number: 6027 Poster Board Number: D0246
Presentation Time: 8:00 AM–9:45 AM
Nuclear factor-erythroid 2-related factor-2 (Nrf2) and
peroxisome proliferator-activated receptor γ coactivator-1α
(PGC-1α) deficient mice: Dissecting the antioxidant regulation
pathways affects retinal autophagy
Jussi J. Paterno1, 2, Adrian Smedowski2, 3, Maria Hytti2, 4,
Marialaura Amandio5, Anna-Kaisa Ruotsalainen6,
Henri O. Leinonen6, Heikki Tanila6, Anna-Liisa Levonen6,
Pasi Tavi6, Anu Kauppinen1, 4, Kai Kaarniranta1, 2. 1Department
of Ophthalmology, Kuopio University Hospital, Kuopio, Finland;
2
Department of Ophthalmology, University of Eastern Finland,
Kuopio, Finland; 3Department of Physiology, Medical University
of Silesia, Katowice, Poland; 4School of Pharmacy, University of
Eastern Finland, Kuopio, Finland; 5Department of Drug Sciences,
University of Pavia, Pavia, Italy; 6A.I. Virtanen Institute of Molecular
Sciences, University of Eastern Finland, Kuopio, Finland.
Purpose: Autophagic clearance participates in the regulation
of proteostasis in the retina. Here, we describe a mouse model,
deficient in two major transcriptional factors regulating
antioxidant defense response, that mimic autophagy decline in
retinal pigment epithelium (RPE).
Methods: Nrf2–/– PGC-1α–/– double knock-out (DKO) mice and
age-matched wild-type controls on a C57BL/6J background were
housed in groups on a 12-hour light/dark cycle and given food
and water ad libitum. All procedures were in compliance with the
ARVO statement for the Use of Animals in Ophthalmic and Vision
Research and approved by the Finnish National Animal Experiment
Board. Optical coherence tomography (OCT) of retina and darkadapted electroretinogram (ERG) were recorded in young adult (3.5
to 4.5 months old) and aged (12 to 13.5 months old) DKO mice and
controls. Retinal tissue sections were immunostained with primary
antibodies against 4-hydroxynonenal (4-HNE), ubiquitin, human
antigen R (HuR, embryonic lethal abnormal vision (ELAV)-like
protein 1), sequestosome 1 (SQSTM1, nucleoporin p62), beclin-1,
and microtubule-associated proteins 1A/1B light chain 3B (LC3).
Results: Aged Nrf2/PGC1α–/– mice developed RPE degeneration,
associated with upregulation of oxidative stress (4-HNE), protein
aggregation (ubiquitin), selective autophagy of aggregate-prone
proteins (HuR, p62) and overall autophagy (beclin-1, LC3).
These changes were accompanied by impaired visual function as
assessed by ERG. Young adult DKO (n = 6) and control (n = 6)
mice did not differ in their a- and b-wave amplitudes (p > 0.05),
but among the aged mice both the a- and b-wave amplitudes were
smaller in DKO (n = 7) compared to control mice (n = 7, a-wave:
p < 0.05; b-wave: p < 0.01).
Conclusions: Our findings suggest that combined Nrf2 and PGC-1α
deficiency increases oxidative stress and protein aggregation, and
affects autophagy. This coincides with the retinal degeneration and
decreased visual function observed in this promising animal model of
age-related retinal degeneration.
Commercial Relationships: Jussi J. Paterno; Adrian Smedowski,
None; Maria Hytti, None; Marialaura Amandio, None;
Anna-Kaisa Ruotsalainen, None; Henri O. Leinonen, None;
Heikki Tanila, None; Anna-Liisa Levonen, None; Pasi Tavi, None;
Anu Kauppinen, None; Kai Kaarniranta, None
Support: Finnish Eye Foundation Grant
Program Number: 6028 Poster Board Number: D0247
Presentation Time: 8:00 AM–9:45 AM
Exogenous claudin-3 and claudin-19 rescue ARPE19 from
dedifferentiation
SHAO-BIN WANG1, Shaomin Peng1, 2, Deepti Singh1,
Ron A. Adelman1, Bo Chen1, Lawrence J. Rizzolo1.
1
Surgery&Ophthalmology, YALE UNIVERSITY, NEW HAVEN, CT;
2
Aier School of Ophthalmology, Central South University, Changsha,
China.
Purpose: The ARPE19 cell line gradually loses RPE properties with
cell passage. One potential cause is that ARPE19 fails to uniformly
express the major claudins found in native RPE tight junctions. As
tight junctions regulate cell shape, morphology and gene expression,
we explored whether exogenous expression of claudin-3 or
claudin-19 might restore native RPE properties.
Methods: ARPE19 were transduced with adenoviral vectors
that expressed claudin-3, claudin-19, or as a control, green
fluorescent protein (GFP). We used the transepithelial electrical
resistance (TER) to assess barrier properties; RT-PCR arrays of
RPE markers or junctional proteins to assess gene expression;
and immunoblotting and immunofluorescence to assess protein
expression. Comparisons were made with human fetal RPE
(hfRPE). A scratch assay and inhibitors of EGF-stimulated
pathways were used to evaluate wound-healing.
Results: PCR arrays revealed substantial quantitative differences
between hfRPE and ARPE19 with respect to RPE signature/
maturation genes. Exogenous claudin-3 and claudin-19 increased
the TER and the expression of ~50% of the genes that were underexpressed in ARPE19. The claudins had little effect on the expression
of other tight, adherens, and gap junction proteins. Expression of
the EGF receptor was decreased, which prompted us to examine
wound-healing. In ARPE19, EGF stimulated cell proliferation and
wound-healing using the AKT arm of the EGF-signaling pathway. In
contrast, wound-healing was retarded in claudin-expressing ARPE19
and was insensitive to EGF. Instead, AKT was constitutively
activated. Wound-healing was more epithelial-like in that wounds
were slowly closed by a combination of cell spreading and cell
proliferation.
Conclusions: Directly or indirectly, claudin-3 and -19 effected
changes in gene expression that partially restored a native RPE
phenotype. The results suggest claudins and AKT signaling might be
therapeutic targets for treating proliferative vitreoretinopathy.
Commercial Relationships: SHAO-BIN WANG, None;
Shaomin Peng, None; Deepti Singh, None; Ron A. Adelman, None;
Bo Chen, None; Lawrence J. Rizzolo, None
Support: Dept. of Defense MR130036; CT Regen Med Rsch Fund
14-SCB-Yale-18; Leir Family Fund; Alonzo Family Fund
Program Number: 6029 Poster Board Number: D0248
Presentation Time: 8:00 AM–9:45 AM
The role of IP-10 in RPE degeneration during AMD progression
Jacob Roney, Kabhilan Mohan, Kyung Jung, Dingyuan Lou,
Subhash C. Prajapati, Jennifer Brown, Mark E. Kleinman.
Ophthalmology & Visual Sciences, University of Kentucky,
Lexington, KY.
Purpose: Interferon gamma-induced protein 10 (IP-10/CXCL10)
is a pro-inflammatory cytokine that is upregulated in the retinal
pigment epithelium (RPE) during progression of age-related
macular degeneration (AMD). Previous data suggest significantly
enhanced expression of IP-10 after RPE treatment with doublestranded RNA (dsRNA) which is also increased in eyes with
AMD. In this study, we set out to determine whether this cytokine
is critical to dsRNA mediated RPE degeneration in a mouse model
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ARVO 2016 Annual Meeting Abstracts
of Ip10 deficiency as well as phenotypic features and cytokine
expression in the aged mouse.
Methods: Ip10 deficient mice (Jackson Labs, C57BL/6J background,
young (4-8 weeks) and aged (12-18 months) were compared to
age-matched Wild type C57BL/6J mice (N=6-8). Intravitreous
injections (PBS, poly I:C or Alu RNA; both 1μg) were performed in
Wild-type and Ip10-/- mice (N=4-6, 4-6 weeks) and analyzed at day
7. Fundus photography and autofluorescence were obtained using
Topcon TRC-50IX; electroretinograms (ERG; Espion) were obtained
under scotopic conditions. RPE/choroid flatmounts were prepared
and immuno-stained for the tight junction protein, zona occludens-1
(ZO-1). RPE levels of inflammatory cytokines in the aging model
were analyzed via multiplex bead arrays (Luminex, N=3). The Ip10-/mouse strain was sequenced for spontaneous rd8 mutations (N=3).
Results: Ip10-/- mice are not protected from dsRNA induced RPE
degeneration. Funduscopy, autofluorescent imaging, and posterior
segment OCT revealed widespread moderate retinal degeneration
in the aged Ip10-/- mice compared to age-matched controls. Protein
studies revealed significantly decreased levels of multiple proinflammatory cytokines in the RPE of aged Ip10-/- mice. Sequencing
for rd8 mutations was positive.
Conclusions: A mouse model of Ip10 deficiency was not protected
from dsRNA induced RPE degeneration suggesting that this
cytokine is not critical during AMD progression. Aged Ip10-/mice developed spontaneous retinal degeneration compared to age
matched Wild-type BLC57/6J mice. We discovered that this strain
harbors a previously unreported rd8 mutation which may account
for this abnormal retinal phenotype.
Program Number: 6030 Poster Board Number: D0249
Presentation Time: 8:00 AM–9:45 AM
Retinal pigment epithelium atrophy in the Cln3Δex7/8 knock-in
mouse model of juvenile neuronal ceroid lipofuscinosis
Kabhilan Mohan1, Yu Zhong2, Jinpeng Liu3, Ahmad Al-Attar4,
Huijuan Liu2, Kyung Jung1, Morgan Dow4, Jinze Liu3, Teresa Fan4,
Qing Jun Wang2, Mark E. Kleinman1. 1Ophthalmol & Visual
Sciences, University of Kentucky, Lexington, KY; 2Molecular
and Cellular Biochemistry, University of Kentucky, Lexington,
KY; 3Computer Science, University of Kentucky, Lexington, KY;
4
University of Kentucky, Lexington, KY.
Purpose: Juvenile neuronal ceroid lipofuscinosis (JNCL or Batten’s
disease) is a blinding pigmented retinal dystrophy with homozygous
deletion of the exons 7 and 8 of the CLN3 gene in a large population
of JNCL patients. Although JNCL is primarily a lysosomal storage
disease, the retinal pigment epithelium (RPE) atrophy and the role of
autophagy in mediating such atrophy remains unclear. In this study,
we aim to characterize RPE pathology and the status of various
autophagic markers in the Cln3Δex7/8 knock-in mouse model of JNCL.
Methods: Cln3Δex7/8 mutant mice (Jackson Labs) were analyzed
for spontaneous rd1/rd8 mutation; 8-month (n=5) and 18-monthold (n=5) mutant mice were compared to age-matched Wild type
C57BL/6J mice (8 month, n=4; 18 month; n=3). Fundus photography
and autofluorescence were obtained using Topcon TRC-50IX;
electroretinograms (ERG; Espion) were obtained under scotopic
conditions. RPE/choroid flatmounts were prepared and immunostained for the tight junction protein, zona occludens-1 (ZO-1).
RNA interference of the CLN3 gene was performed in the RPE-1
cell line (ATCC). Autophagic flux was studied with Western blot for
the known markers p62 and LC3II. Autophagy and lysosome gene
expression profiles were captured by RNA-Seq and qPCR.
Results: No rd1/rd8 mutations were found in the Cln3Δex7/8
strain. Funduscopic analyses revealed a mottled appearance with
extensive hypopigmentation in aged Cln3Δex7/8 mutant mice.
Hyper-autofluorescent aggregates were observed in Cln3Δex7/8 mice
at both ages. Significant losses in both a- and b-waves of aged
mutant mice were observed compared to age-matched controls.
ZO-1 immunofluorescence revealed widespread disruption of RPE
tight junctions exclusively in the aged mutant mice. CLN3 siRNA
treatment of RPE-1 cells led to greatly enhanced autophagic flux as
well as autophagy and lysosome gene expression alterations that are
consistent with increased autophagic and lysosomal degradation.
Conclusions: We have observed extensive RPE atrophy and
gradual loss of retinal function loss in the Cln3Δex7/8 mouse model;
interestingly, autofluorescent aggregates occur even in young
mutant mice. Cell culture studies show enhanced autophagic activity
resulting from CLN3 deficiency. Our data suggest a role for Cln3
function in autophagy which may contribute to RPE degeneration in
JNCL and serve as a novel therapeutic in this blinding disease.
Commercial Relationships: Jacob Roney, None;
Kabhilan Mohan; Kyung Jung, None; Dingyuan Lou,
None; Subhash C. Prajapati, None; Jennifer Brown, None;
Mark E. Kleinman, 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
Human umbilical tissue-derived cell (hUTC) is an allogeneic cellbased medicinal product for the treatment of geographic atrophy,
an advanced form of dry AMD. We previously demonstrated that
subretinal injection of hUTC preserves visual function and retinal
architecture in the RCS rat with reduced apoptosis in the retina,
suggesting that hUTC may have a protective effect on retina. The
retina is particularly susceptible to oxidative stress and this plays an
important role in the pathogenesis of AMD and contributes to RPE
damage. Here, we aimed to determine whether hUTC could improve
the health of RPE cells exposed to oxidative stress.
Methods: The ARPE-19 cell line was obtained from American
Type Culture Collection. Primary human RPE (hRPE) cultures were
prepared from postmortem human eyes obtained from donors and
the purity was confirmed by cytokeratin staining. The MTT and
crystal violet assays were performed to evaluate the cell viability of
ARPE-19 and hRPE exposed to a lethal dose (1500 µM) of hydrogen
peroxide (H2O2) after co-culturing with hUTC for 72 hours in
transwell inserts. Western blots were preformed to examine the level
of protein carbonylation, a key marker of oxidative stress induced
damage, in ARPE-19 cells co-cultured with hUTC and then subject
to sublethal doses of H2O2 (200 and 400 µM) for 3 and 6 hours,
respectively.
Results: ARPE-19 co-cultured with hUTC for 72 hours showed
significantly improved viability following treatment with 1500 µM
H2O2 compared to untreated control cells, as assessed by both MTT
and crystal violet assays. Similar results were obtained in hRPE.
Moreover, a significantly lower level of protein carbonylation was
observed in hUTC pretreated ARPE-19 cells exposed to 400 µM
H2O2 for 6 hours as determined by Western blot analysis.
Conclusions: These findings suggest that hUTC may secrete factors
that can potentially protect RPE from oxidative damage. Investigation
is currently underway to examine the mechanisms of action.
Commercial Relationships: Sayak K. Mitter, Janssen R&D (F);
Michael E. Boulton, Janssen R&D (F); Ian Harris; Jing Cao,
Janssen R&D
Support: The work described was performed under a sponsored
research agreement between Indiana University and Janssen R&D
and a Research to Prevent Blindness Unrestricted Grant.
Commercial Relationships: Kabhilan Mohan, None; Yu Zhong,
None; Jinpeng Liu, None; Ahmad Al-Attar, None; Huijuan Liu,
None; Kyung Jung, None; Morgan Dow, None; Jinze Liu, None;
Teresa Fan, None; Qing Jun Wang, None; Mark E. Kleinman,
None
Support: Mark Kleinman - K08EY021757; Foundation Fighting
Blindness; Research to Prevent Blindness. Qingjun Wang - Ellison
Medical Foundation
Program Number: 6031 Poster Board Number: D0250
Presentation Time: 8:00 AM–9:45 AM
Human umbilical tissue-derived cells protect retinal pigment
epithelial cells from oxidative damage
Sayak K. Mitter1, Michael E. Boulton1, Ian Harris2, Jing Cao2.
1
Ophthalmology, Indiana University School of Medicine,
Indianapolis, IN; 2Janssen R&D, Spring House, PA.
Purpose: Retinal pigment epithelium (RPE) cells perform many
functions crucial for retinal homeostasis and vision. RPE cell
dysfunction results in various retinal degenerative diseases including
age-related macular degeneration (AMD) for which there are
currently no effective treatment. Cell therapies targeting RPE cells are
being developed in the clinic for the treatment of retinal degeneration.
Program Number: 6032 Poster Board Number: D0251
Presentation Time: 8:00 AM–9:45 AM
Neuroprotectin D1 (NPD1) modulates amyloid precursor protein
(APP) processing in human retinal pigment epithelial cells (RPE)
Khanh Do, Jorgelina M. Calandria, Nicolas G. Bazan. Neuroscience
Center of Excellence, Louisiana State University Health, NEW
ORLEANS, LA.
Purpose: Amyloid beta is a major component of drusen in agerelated macular degeneration (AMD) (Ratnayaka et al; Eye, 2015). In
neuronal cells in culture from human brain expressing APP mutations
that cause familial forms of Alzheimer’s disease, NPD1 shifts APP
processing from the tandem beta and gamma secretase to alpha
secretase (Zhao et al; PLOS One, 2011). We hypothesize that NPD1
deters formation of amyloid-β towards the prosurvival sAPPa, as it
occurs in human brain cells. Here we have studied the bioactivity of
NPD1 on APP processing in RPE cells.
Methods: Plasmid construct containing Swedish double mutant APP
protein (APPsw), which dominantly produce sAPPβ (a precursor
of amyloid beta), was transfected into human ARPE-19 cells. After
24h, NPD1 was added at 50, 100 and 500 nM concentrations in the
oxidative stress-inducing condition (10 ng/mL of tumor necrosis
factor alpha (TNFα) plus 600 nM H2O2). The synthesized nonamyloidogenic sAPPα and amyloidogenic sAPPβ were analyzed
by western blot after 48h of NPD1 treatment. Non-transfected and
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ARVO 2016 Annual Meeting Abstracts
non-oxidative stress conditions were used as negative controls.
Furthermore, we also investigated the anti-oxidant activity of
2 nM sAPPα protein in preventing oxidative stress-induced
apoptosis in ARPE-19 cells. The cell survival ratio was analyzed
by Hoechst staining.
Results: At 48h, higher sAPPα production was observed in the
increase of NPD1 concentration. The sAPPα expression peaked
1.5 times higher than the non-treated control at 500 nM of NPD1.
In contrast, reverse production of sAPPβ was observed, where the
highest amount of sAPPβ was detected in the absence of NPD1. An
equal amount of holo-APP, regarded as total of all sAPP forms, was
detected in all studied condition. Also, we saw that NPD1 decreased
the level of amyloid-β secretion to the control level. For anti-oxidant
action, our results showed that the presence of 2 nM sAPPα could
prevent oxidative stress-induced apoptosis in RPE cells.
Conclusions: We found an NPD1 concentration-dependent increase
of non-amyloidogenic sAPPα and decrease of amyloidogenic sAPPβ
in RPE cells. These results suggest that NPD1 shifts production of
amyloidogenic to the non-amyloidogenic pathway in human RPE
cells. Additionally, the protective activity of sAPPα modulated by
NPD1 represents a new RPE cell survival route.
Commercial Relationships: Khanh Do, None;
Jorgelina M. Calandria, None; Nicolas G. Bazan, None
Support: NIGMS grant P30 GM103340, NEI grant R01 EY005121
(NGB) and Research to Prevent Blindness
Program Number: 6033 Poster Board Number: D0252
Presentation Time: 8:00 AM–9:45 AM
Bruch’s Membrane is Leaky and RPE lacks Apoptosis,
Autophagy and Lysosomes in AMD
Mones S. Abu-Asab, Christopher P. Ardeljan, Maria M. Campos.
Histology Section, National Eye Institute, Bethesda, MD.
Purpose: This study aims to interpret the ultrastructural aberrations
in RPE and Bruch’s membrane of AMD in order to identify
contributing factors to its etiology. Very few published ultrastructural
reports on AMD have identified pathological abnormalities in RPE
and Bruch’s membrane. We have observed that some aberrations
could cause the accumulation of drusen and cellular debris, as well
as other abnormalities. We are also investigating whether changes
within the macula differ from those in the peripheral fundus.
Methods: Ultrastructural examination of Bruch’s membrane and
RPE, from the macula and peripheral fundus, was performed on
the eyes of 7 AMD donors, all of whom were over 80 years old.
Eyes were fixed in formalin and samples from the macula and
temporal fundus were dissected out. Preparation for TEM was
done as follows: tissues were embedded in epoxy resin, sectioned,
double-stained with uranyl acetate and lead citrate, and viewed
with a JOEL JM-1010 TEM.
Results: Ultrastructural aberrations were seen in all of the examined
specimens. Normal and necrotic RPE cells appeared without
lysosomes or autophagosomes, and none of the specimens had
apoptotic RPE cells. In some specimens, the RPE layer had doubled
by producing a new healthy layer of cells on top of the older
degenerate one. Drusen and debris of necrotic cells were present
below the RPE layer. Bruch’s membrane was leaky, allowing drusen
contents and cellular debris of necrotic RPE cells to pass from the
RPE side onto the choroidal side; these encompassed lipid droplets,
lipofuscin, chromatin vesicles, degenerate melanosomes, and
aqueous vesicles. Additionally, Bruch’s membrane showed collagen
degeneration and loss of elastin.
Conclusions: Our survey of AMD specimens revealed a number of
ultrastructural aberrations, some of which have not been previously
described in AMD. These RPE aberrations indicate dysfunctional
cellular clearance mechanisms such as apoptosis, lysosomes, and
autophagy. Lack of apoptosis seems to be forcing RPE cells into
necrosis leading to an accumulation of cellular debris on top of and
within Bruch’s membrane. Debris is also being passed onto the
choroidal side of Bruch’s membrane. Necrosis and not apoptosis
appears to be the prominent type of cell death in AMD. Both the
macular and peripheral regions shared the same abnormalities and
there was no significant differences between them.
Commercial Relationships: Mones S. Abu-Asab;
Christopher P. Ardeljan, None; Maria M. Campos, None
Program Number: 6034 Poster Board Number: D0253
Presentation Time: 8:00 AM–9:45 AM
Altered Transepithelial Resistance of Induced Pluripotent
Stem Cell-derived Retinal Pigment Epithelium Obtained
from Age-related Macular Degeneration (AMD) Patients
Ernesto F. Moreira, Jie Gong, Sibylle Rosendahl, Amanda Barrett,
Mark A. Fields, Zsolt Ablonczy, Baerbel Rohrer, Lucian V. Del Priore.
Ophthalmology, Medical University of South Carolina, Charleston, SC.
Purpose: We have previously reported the generation of induced
pluripotent stem cells (iPSC) derived from fibroblasts of patients
with advanced macular degeneration, as well as the differentiation
of iPSCs into retinal pigment epithelium (RPE). Here, we report the
transepithelial resistance (TER) analysis of iPSC-derived RPE cells
from AMD patients and unaffected controls.
Methods: Fibroblasts were grown and expanded from punch
biopsies obtained from patients with advanced (dry or wet) AMD
and unaffected controls. Sendai virus technology (ThermoFisher
Scientific) was used to deliver Yamanaka factors to reprogram
fibroblasts into iPSCs. Subsequently, iPSCs were differentiated into
RPE using our recently published protocol (Gong et al, 2015). Pure
iPSC-RPE cells, grown in transwell plates for 1-2 months, were
characterized morphologically (honeycomb monolayer appearance
and pigmentation) and by immunocytochemistry for RPE specific
molecules (i.e., OTX2, MITF, Bestrophin). RPE trans-epithelial
resistance (TER) was assessed with a Voltohmmeter averaging three
measurements in each (duplicate) transwell.
Results: iPSC-RPE cell lines derived from AMD patients (N=4)
and controls (N=2) formed characteristic monolayers showing
typical honeycomb organization and pigmentation. These cells
also expressed specific RPE cell markers important for their
differentiation and function, including MITF, RPE65. Baseline
TER analysis showed that dry AMD samples had a significantly
reduced TER (29 +/-10 (#1) and 37 +/-16 (#2) and 117 +/-21 (#3))
as compared to the wet AMD and controls (411 +/-16 (wet AMD);
629 +/-26 (control #1); 330 +/- 48 (control #2)).
Conclusions: These studies suggest that intrinsic differences in RPE
cells between AMD patients and unaffected controls may exist in the
ability to make tight junctions necessary for RPE barrier function.
In addition, these results further indicate the potential use of these
cells to investigate features of AMD based on the genotype and/
or phenotype of the patients. Ongoing experiments are aimed at
analyzing complement activation and VEGF levels in the supernatant
of iPSC-RPE before and after challenging them with specific AMDrelevant stressors, in addition to determining expression of tightjunction markers for these cells.
Commercial Relationships: Ernesto F. Moreira, None; Jie Gong,
None; Sibylle Rosendahl, None; Amanda Barrett, None;
Mark A. Fields, None; Zsolt Ablonczy, None; Baerbel Rohrer;
Lucian V. Del Priore, Mesoblast (C), Angioblast (C), Advanced Cell
Technology (C), Ocata (C)
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
Support: This work is supported in part by a Brightfocus Foundation
grant and a faculty funding program from the Dean’s office of
the Medical University of South Carolina (EFM); NIH grants
EY019320 (BR), EY019065 (ZA), and an unrestricted grant to the
Ophthalmology Department, MUSC, from the Research to Prevent
Blindness. BR is also sponsored in part by a Department of Veterans
Affairs merit award RX000444, and the Feldberg Endowment.
LVDP is supported in part by a grant from the Foundation Fighting
Blindness.
Program Number: 6035 Poster Board Number: D0254
Presentation Time: 8:00 AM–9:45 AM
Compromised phagosome maturation underlies defective RPE
clearance in an in vitro model of Smith-Lemli-Opitz Syndrome
Sriganesh Ramachandra Rao1, 3, Nestor Mas Gomez2,
Bruce A. Pfeffer1, 3, Aryn Rowsam1, 3, Claire H. Mitchell2,
Steven J. Fliesler1, 3. 1Ophthalmology and Biochemistry,
SUNY-Buffalo Sch Med & Biomed Sci and SUNY Eye Institute,
Buffalo, NY; 2Anatomy & Cell Biology, University of Pennsylvania
Sch Dental Med, Philadelphia, PA; 3Research Service, VAWNYHSBuffalo VAMC, Buffalo, NY.
Purpose: The retinal pigmented epithelium (RPE) in the AY9944 rat
model of Smith-Lemli-Opitz syndrome (SLOS), a genetic disorder of
cholesterol biosynthesis, exhibits accumulation of phagosomes and
other inclusions, compared to controls [Fliesler et al., 2004, Arch.
Ophthalmol.]. We examined SLOS patient iPSC-derived (SLOS
RPE), vs. normal human embryonic stem cell-derived (nhRPE), cells
in vitro to determine the underlying mechanism of this defect.
Methods: SLOS RPE (harboring both T93M and IVS8 G-C DHCR7
mutations) and nhRPE were treated with bovine rod outer segments
(ROS) for 48 h; rhodopsin levels were quantified by Western blotting/
densitometry (WB/D; probed with 1D4 MAb) to assess phagosome
clearance. Lysosomal protease (mature Cathepsin-D), and markers
of autophagic flux (p62 and LC3-I/II) were assessed by WB/D,
normalized to GAPDH levels, with corresponding antibodies. SLOS
RPE and nhRPE lysosomal pH was measured using LysoSensor
Yellow/Blue DND-160, and lysosomal Cathepsin-D activity was
estimated using a BODIPY-Pepstatin assay kit (Molecular Probes).
Statistical significance of mean/S.E. values was determined by paired
Student’s t-test (criterion, p ≤ 0.05).
Results: Autophagic marker levels in SLOS RPE (expressed as
% change, vs. nhRPE) were as follows (p<0.05): Beclin-1, +62%;
p62, +18%; LC3-II, -50%. Mature Cathepsin-D levels were
unaltered. Lysosomal pH in SLOS hRPE was statistically more
acidic (4.50 ± 0.02, p<0.05) compared to nhRPE (4.56 ± 0.01),
but Cathepsin-D activity was not significantly altered. However,
heterophagic clearance of exogenous ROS was defective in SLOS
RPE, as evidenced by persistence of 1D4+ material (rod opsin
C-terminus), compared to nhRPE cells.
Conclusions: Decreased LC3-II levels, elevated p62 levels, and
persistence of 1D4+ material indicate compromised phagosome
maturation in SLOS RPE compared to nhRPE. Elevated Beclin1
levels obviate defective initiation of autophagy/heterophagy; also,
compromised lysosomal physiology was not involved. However,
increased 7-dehydrocholesterol (7DHC) levels (a SLOS hallmark)
and/or oxysterols derived therefrom may underlie the SLOSassociated RPE defect.
Commercial Relationships: Sriganesh Ramachandra Rao, None;
Nestor Mas Gomez, None; Bruce A. Pfeffer, None; Aryn Rowsam,
None; Claire H. Mitchell, None; Steven J. Fliesler, None
Support: NIH RO1 EY007361 (SJF); RPB Unrestricted Grant (SJF);
VAWNYHS facilities and resources (SRR, BAP, AMR, SJF). NIH
R01 EY013434 (CHM)
Program Number: 6036 Poster Board Number: D0255
Presentation Time: 8:00 AM–9:45 AM
Semi-automated analysis of lipofuscine and melanolipofuscine
granules in the RPE using structured illumination microscopy
Nil Celik1, Gerrit Best2, Florian Schock2, Alena Bakulina3,
Gerd U. Auffarth1, Christoph Cremer2, Juergen Hesser3,
Stefan Dithmar4. 1Department of Ophthalmology, University
Hospital Heidelberg, Heidleberg, Germany; 2Institute of Molecular
Biology, Mainz, Germany; 3University Medical Center Mannheim,
Experimental Radiation Oncology, Mannheim, Germany;
4
Department of Ophthalmology, Dr. Horst Schmidt Kliniken,
Wiesbaden, Germany.
Purpose: We already introduced a new analyzing tool for lipofuscine
granules (LG) in retinal pigment epithelium (RPE) cells in highly
resolved 3D-Structured illumination microscopy (SIM) images
automatically. In a further analysis we additionally investigated
melanolipofuscine granules (MLG).
Methods: 3D-SIM images were examined for LG and MLG using
three different excitation wavelengths (488, 568 and 647 nm). Our
automated software identifies and quantifies (number, size and
position) single granules in RPE flatmounts. The LG and MLG of an
88-year-old donor were examined for the macular region.
Results: From 541 granules, 411 (76.0%) were identified as LG
and 130 (24.0%) as MLG. LG and MLG were distributed towards
the cell edge.
Conclusions: Highly resolved 3D-images of LG as well as MLG
are obtainable with SIM. LG and MLG can be differentiated and
characterized regarding their number and position with a semiautomated analysis of the established SIM data. Quantitative
characteristics of autofluorescent granules help to understand cellular
processes within the RPE.
Commercial Relationships: Nil Celik; Gerrit Best, None;
Florian Schock, None; Alena Bakulina, None; Gerd U. Auffarth,
Abbott Medical Optics, Alcon Laboratories, Inc., Alimera Sciences,
Inc., Bausch & Lomb Surgical, Carl Zeiss Meditec, Contamac,
Glaukos Corporation, Heidelberg Engineering, HumanOptics,
Novartis Pharmaceuticals Corporation, Oculentis, Physiol, Power
Vision, Rayner Intraocular Lenses Ltd, Technolas (F), Abbott
Medical Optics, Alcon Laboratories, Inc., Allergan, Bausch & Lomb
Surgical, Bayer Healthcare Pharmaceuticals, Carl Zeiss Meditec,
Hoya, HumanOptics, Kowa, Oculentis, OPHTEC, Physiol, Rayner
Intraocular Lenses Ltd, Technolas (R), Abbott Medical Optics, Alcon
Laboratories, Inc., Allergan, Bausch & Lomb Surgical, Novartis
Pharmaceuticals Corporation, Rayner Intraocular Lenses Ltd,
Technolas (C); Christoph Cremer, None; Juergen Hesser, None;
Stefan Dithmar, None
Support: Supported by Gertrud Kusen Foundation
Program Number: 6037 Poster Board Number: D0256
Presentation Time: 8:00 AM–9:45 AM
Mutations in the Mitf gene affect autophagy in mouse primary
RPE cells
Andrea García-Llorca1, Margret Helga Ogmunsdottir2,
Eirikur Steingrimsson2, Thor Eysteinsson1. 1Physiology, University of
Iceland, Reykjavík, Iceland; 2Biochemistry and Molecular Biology,
University of Iceland, Reykjavík, Iceland.
Purpose: Mutations at the mouse microphthalmia locus (Mitf) affect
the development of different cell types including melanocytes, retinal
pigment epithelium and osteoclasts. The MITF protein is a member
of the MYC supergene family of basic-helix-loop-helix-leucinezipper (bHLHZip) transcription factors and is known to regulate
the expression of cell-specific target genes by binding DNA as
homodimer or as heterodimer with related proteins. The purpose of
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ARVO 2016 Annual Meeting Abstracts
this study was to examine the role of Mitf in regulating autophagy in
mouse primary RPE cells by studying mouse Mitf mutations.
Methods: Primary RPE cells from wild type and MITF mutant mice
(Mitfmi-enu122(398), MitfMi-Wh/+ and MitfMi-Wh/Mitfmi-mi) were isolated
by enzymatic dissociation. The characteristic morphology and the
expression of RPE65 of these RPE cells were observed by antibody
staining and microscopy. Expression of LC3 and MITF was analyzed
with western blot analysis and confocal microscopy in primary RPE
cells from C57BL/6J mice, untreated or treated with the mTOR
inhibitor Torin1 for 3 hours or after incubation in starvation media for
1 hour. Torin1 and starvation are known activators of autophagy. The
levels of LC3 and MITF were measured and compared by western
blot in RPE cells from wild type and the Mitf mutant mice.
Results: Wild type RPE cells express MITF and also basal levels
of LC3. The treatment with starvation media and Torin1 treatment
increased the levels of LC3 in RPE cells. Furthermore, both
starvation and Torin1treatment resulted in reduced MITF protein
levels. Wild type treated RPE cells with starvation media showed
phagosomes around the nuclei. Only the LC3II protein was detected
in RPE cells from MITF mutant whereas wild type RPE cells showed
both LC3I and II, suggesting that the degradation pathway of LC3 is
stalled in the RPE from Mitf mutant mice.
Conclusions: This study suggests that autophagy is affected in Mitf
mutant mice. This is consistent with in vitro data showing that MITF
regulates expression of genes involved in autophagy.
Commercial Relationships: Andrea García-Llorca, None;
Margret Helga Ogmunsdottir, None; Eirikur Steingrimsson,
None; Thor Eysteinsson, None
Support: Icelandic Research Council Grants, Helga Jonsdottir and
Sigurlidi Kristjansson Memorial Fund
Program Number: 6038 Poster Board Number: D0257
Presentation Time: 8:00 AM–9:45 AM
Age-related degeneration of the retinal pigment epithelium (RPE)
of DJ-1 mice
Vera L. Bonilha1, 2, Brent A. Bell1, Mary E. Rayborn1,
Joe G. Hollyfield1, 2, Ivy S. Samuels1, 3, Chengsong Xie4, Huaibin Cai4.
1
Ophthalmic Research, Cleveland Clinic, Cleveland, OH;
2
Ophthalmology, Cole Eye Institute/CCLCM, Cleveland, OH;
3
Research Service, Louis Stokes Cleveland Veterans Affairs Medical
Center, Cleveland, OH; 4Lab. of Neurogenetics, National Institute of
Aging/ NIH, Bethesda, MD.
Purpose: DJ-1 is ubiquitously expressed in many tissues including
the brain where it functions as an antioxidant, redox-sensitive
molecular chaperone and transcription regulator robustly protecting
cells from oxidative stress. We previously reported mild structural
and physiological changes including RPE thinning, central decrease
in red/green cone opsin staining, decreased labeling of ezrin, broader
distribution of ribeye labeling, decreased tyrosine hydroxylase in
dopaminergic neurons, and increased DNA oxidation in the retinas
of 3 and 6 month old DJ-1 knockout (KO) in comparison to control
mice. The severity of these changes increased with age; therefore, in
the present study we extended our analysis to older (18 month-old)
mice and compared the results to young (3 month-old) DJ-1 KO and
control mice.
Methods: The effects of DJ-1 deletion were examined in KO
mice through non-invasive, fundus imaging in vivo using SLO and
OCT. A profile analysis of the outer retina was performed from the
four retinal quadrants to assess OCT signal reflectivity and lamina
morphology. Photoreceptor and RPE function were assessed by ERG.
Histological and immunohistological evaluation of the retinas of DJ-1
KO and control mice were also performed in cryosections as well as
whole-mounted retina and RPE/choroid. Retina/RPE lysates were
assayed for oxidation using antibodies.
Results: SLO showed no differences between the fundus of DJ-1
KO and control mice. OCT imaging demonstrated all retinal layers in
both young and older DJ-1 KO retinas. The profile analysis revealed
significant hyper-reflectivity of photoreceptor outer segments and
outer retina thinning in DJ-1 KO in comparison to control mice for all
retinal quadrants examined. ERG studies of older DJ-1 KO mice noted
significant decreased values under dark- and light-adapted conditions.
Histologically, changes in the RPE of the DJ-1 KO mice progressed
from increased presence of vacuoles at 3 months of age to the presence
of vacuoles and lesions at 18 months of age. The RPE lesions were also
visible by OCT and in whole mounted RPE/choroid samples labeled
with phalloidin. Photoreceptors were degenerated when opposing the
RPE lesions. Increased protein carbonyl derivatives were detected in
retina/RPE lysates from older DJ-1 KO mice.
Conclusions: DJ-1 KO mice display progressive signs of retinal/
RPE degeneration in association with increased levels of oxidative
stress markers.
Commercial Relationships: Vera L. Bonilha, None; Brent A. Bell,
None; Mary E. Rayborn, None; Joe G. Hollyfield, None;
Ivy S. Samuels, None; Chengsong Xie; Huaibin Cai, None
Support: Research to Prevent Blindness, Wolf Family
Foundation,National Eye Institute (EY014240-08) and a VA Merit
Award i01-BX002754 from Biomedical Laboratory Research and
Development (ISS).
Program Number: 6039 Poster Board Number: D0258
Presentation Time: 8:00 AM–9:45 AM
TFEB-mediated clearance of the lipofuscin fluorophore A2E
Ivana Trapani1, Elisabetta Toriello1, Renato Minopoli1,
Alberto Auricchio1, 2. 1Telethon Institute of Genetics and Medicine
(TIGEM), Pozzuoli, Italy; 2Medical Genetics, Department of
Translational Medicine, Federico II University, Naples, Italy.
Purpose: Abnormal accumulation of various by-products of the
visual cycle, including the diretinoid-pyridinium-ethanolamine
(A2E), in retinal pigment epithelium (RPE) is an hallmark of both
Stargardt disease (STGD) and age-related macular degeneration
(AMD). This is responsible for RPE and, consequently, photoreceptor
(PR) cell death. A2E storage in RPE lysosomes has been shown to
reduce both the capacity of RPE to degrade phagocytized PR outer
segments and autophagosome biogenesis, trafficking and autophagic
flux. The transcription factor EB (TFEB) is a master regulator of
cellular clearance. Here we tested if TFEB overexpression induces
A2E clearance from the RPE cells.
Methods: We have generated a plasmid encoding for a double-serine
mutant (S142A, S211A) form of TFEB, which is constitutively
active. We have transfected this plasmid in the human RPE-derived
ARPE19 cells to evaluate TFEB activation of its transcriptional
targets and ability to clear A2E after loading.
Results: We found that TFEB overexpression in ARPE19 cells results
in the induction of TFEB transcriptional targets involved in cellular
clearance. Importantly, TFEB overexpression in ARPE19 cells was
associated with reduction of intracellular A2E accumulation.
Conclusions: Taken together these results suggest that TFEB
overexpression is an effective strategy to promote A2E clearance
in vitro. Further studies will clarify if this holds true in vivo. These
results may have implications for the therapy of both STGD and the
more common AMD.
Commercial Relationships: Ivana Trapani, None;
Elisabetta Toriello, None; Renato Minopoli, None;
Alberto Auricchio, None
Support: Italian Telethon Foundation grant TGM11MT1
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: 6040 Poster Board Number: D0259
Presentation Time: 8:00 AM–9:45 AM
Chloroquine toxicity of the retinal pigment epithelium and retina
in vitro and a mouse model of retinal toxicity
Dhanesh Amarnani1, 2, Leo A. Kim2, Patricia A. D’Amore2,
Lindsay Wong2. 1Ophthalmology, Harvard Medical School, Boston,
MA; 2Retina, Schepens Eye Research Institute/ Massachusetts Eye
and Ear Infirmary, Boston, MA.
Purpose: To evaluate the cell death mechanisms of chloroquineinduced toxicity of human cultured epithelial cells(RPE). To
develop and characterize a murine model of chloroquine-induced
retinal toxicity
Methods: ARPE-19 cells were cultured until confluence, treated
with 500uM chloroquine for 24 hours and cell death was measured
by lactate dehydrogenase(LDH) release. Cytoskeletal structure
and lysosomal permeability were assessed using phalloidin and
lysosomal-associated membrane protein(LAMP-1) staining,
respectively. The roles of lysosomal enzymes cathepsin B and L were
examined by blocking their activity using specific cell permeable
inhibitors. The contribution of different cell death mediators was
evaluated by specific inhibition of caspase-1, 3, 8, 9 and receptorinteracting protein(RIP) kinases. To develop an animal model of
retina chloroquine toxicity, subretinal chloroquine (0.2 mg/kg)
injections were performed. Phenotypic changes were evaluated using
optical coherence tomography(OCT) and functional assessment was
performed by electroretinography(ERG). Histological changes to the
RPE were assessed.
Results: 500uM chloroquine induced 30-40% of cell death at
24 hours. Lysosomal permeabilization and cytoskeletal structure
disruption were observed at 3 and 6 hours. Blockage of cathepsin B/L
activity resulted in a significant decrease in cell death, indicating that
lysosomal destabilization and resulting cathepsin release are upstream
of these cell death pathways. Partial rescue of cell death was
observed with caspases and RIP kinase inhibitors. Preliminary results
from OCT demonstrate loss of the RPE layer and thinning of the
outer retina within two weeks of injection. ERG revealed a decreased
scotopic response in both the A and B-waves of the ERG waveform.
H&E staining of retinal cross sections in chloroquine-treated mice
revealed loss of RPE and disruption of outer segments. ZO-1 staining
of RPE flat mounts demonstrated RPE cells death, enlarged cells and
loss of cell junctions.
Conclusions: Chloroquine-induced cell death occurs through
multiple cell death mechanisms. Inhibition of upstream activators
such as cathepisn B/L may be a feasible approach to block multiple
cell death pathways. Some of the phenotypic and functional changes
observed in our mouse model are similar to those observed in human
subjects with chloroquine toxicity, and may be model of RPE and
retinal degeneration.
Commercial Relationships: Dhanesh Amarnani, None;
Leo A. Kim, None; Patricia A. D'Amore, None; Lindsay Wong
Program Number: 6041 Poster Board Number: D0260
Presentation Time: 8:00 AM–9:45 AM
Prominin 1 Plays a Central Role in Regulating Stress-induced
Autophagy in the Retinal Pigment Epithelium
Edward Chaum, Christina S. Winborn, Sujoy Bhattacharya,
Jinggang Yin. Ophthalmology, Univ of Tennessee Health Sci Ctr,
Memphis, TN.
Purpose: Prominin1 (Prom1) is a transmembrane glycoprotein
that localizes to plasma membrane protrusions and is a known
cancer stem cell marker. It also plays a role in photoreceptor disk
morphogenesis; mutations in the Prom1 gene cause Type 4 Stargardt
disease. Our previous studies have shown that Prom1 is also a novel
regulator of autophagy in the RPE. The purpose of this study was to
examine the cellular mechanisms of Prom1-mediated autophagy. We
show that loss of Prom1 expression impairs stress-induced autophagy
in the human RPE.
Methods: We used the CRISPR/CAS9 nuclease system to knockout
(KO) Prom1 in ARPE19 cells. FlowSight imaging cytometry and
confocal immunofluorescence assays were used to quantify LC3
puncta accumulation in the presence and absence of chemical
modulators of autophagy in control APRE19 cells and in Prom1
KO cells. We also monitored autophagosome formation in response
to autophagic regulators using a tandem RFP-GFP-LC3 construct
containing a GFP marker that is quenched when the autophagosome
fuses with the lysosome.
Results: We show that knocking out Prom1 expression significantly
reduces starvation-induced LC3-puncta accumulation in the human
RPE. Flow cytometry and confocal microscopy demonstrated that
Prom1 KO; 1) decreases LC3 puncta accumulation in response to
starvation or rapamycin, 2) reduces autophagy flux and increases
chloroquine-induced LC3-puncta accumulation, and 3) delays
autophagosome maturation.
Conclusions: Prom1 expression plays in important role in the
activation of stress-induced autophagy in the human RPE.
Commercial Relationships: Edward Chaum, None;
Christina S. Winborn, None; Sujoy Bhattacharya, None;
Jinggang Yin, None
Support: The Shulsky Foundation, New York, NY, an unrestricted
UTHSC departmental grant from Research to Prevent Blindness,
New York, NY, The Lions of Arkansas Foundation Inc., and the
Plough Foundation, Memphis, TN. UTHSC Hamilton Eye Institute
Core Grant for Vision Research, supported by the National Eye
Institute (P30 EY013080).
Program Number: 6042 Poster Board Number: D0261
Presentation Time: 8:00 AM–9:45 AM
Molecular Mechanisms Regulating Prom1-dependent Autophagy
in Human Retinal Pigment Epithelial Cells
Sujoy Bhattacharya, Qiuhua Zhang, Jinggang Yin,
Christina S. Winborn, Edward Chaum. Ophthalmology, University of
Tennessee Health Science Center Hamilton Eye Institute, Memphis, TN.
Purpose: Beyond its role in photoreceptor disk morphogenesis,
our previous studies have demonstrated that Prom1 (CD133) is a
novel regulator of autophagy in the RPE. However, the cellular and
molecular mechanisms governing Prom1-mediated RPE autophagy
have not previously been determined. We show that Prom1 modulates
autophagy in the RPE through inhibition of Akt/mTOR activities
and through its association with the selective autophagy receptor,
sequestosome (SQSTM1)/p62.
Methods: The CRISPR/Cas 9 nuclease system was used to
knockout Prom1 in the RPE and Prom1 lentiviral constructs
were also used to overexpress Prom1 in ARPE19 cells. Western
blotting and flow cytometry assays were used to quantify LC3-II
processing and punctae formation in the presence and absence
of lysosomal inhibitors as cellular readouts for autophagic flux.
Immunoprecipitation was used to detect Prom1 interacting proteins
and Western blotting was performed to analyze the mTOR and Akt
pathway-associated proteins: p-ULK1 (Ser555), p-AMPKα (Thr172),
p-mTOR (Ser2448), pAkt (Ser473), p4EBP1(Thr37/46), and LC3-II/
LC3-I after stimulation of autophagy.
Results: Overexpression of Prom1, significantly reduced
phosphorylation of S6 ribosomal protein at Ser235/236 and Akt at
Ser473, surrogate markers for mTORC1 and mTORC2 activities,
respectively, and decreased p62 levels. Prom1 overexpression
increased Atg5, and Atg7 expression, LC3I/LC3-II turnover, EBSS-
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ARVO 2016 Annual Meeting Abstracts
induced autophagy flux, and decreased Bafilomycin-induced p62
accumulation. Immunoprecipitation experiments revealed that Prom1
interacted with p62 and HDAC6, both critical mediators of autophagy
flux and autophagosome maturation. Conversely, the Prom1 KO
increased mTOR activity, delayed autophagosome trafficking to the
lysosome, increased p62 accumulation, and impaired stress-induced
autophagy.
Conclusions: We demonstrate a novel regulation of Prom1dependent autophagy via upstream inhibition of the mTOR/Akt
pathway, and through the protein acting as a molecular scaffold for
p62/HDAC6 association in the autophagosomal-lysosomal fusion
pathway.
Commercial Relationships: Sujoy Bhattacharya, None;
Qiuhua Zhang, None; Jinggang Yin, None; Christina S. Winborn,
None; Edward Chaum, None
Support: The Shulsky Foundation, New York, NY; an unrestrcited
UTHSC departmental grant from Research to Prevent Blindness,
New York, NY; The Lions of Arkansas Foundation Inc., and the
Plough Foundation, Memphis, TN; UTHSC Hamilton Eye Institute
Core Grant for Vision Research; supported by the National Eye
Institute (P30 EY013080)
Program Number: 6043 Poster Board Number: D0262
Presentation Time: 8:00 AM–9:45 AM
Loss of MREG dependent LC3 associated phagocytosis (LAP) by
the RPE leads to altered intracellular lipid processing
Desiree Alexander1, Anuradha Dhingra1, William C. Gordon2,
Bokkyoo Jun2, Nicolas G. Bazan2, Kathleen Boesze-Battaglia1,
Alvina Bragin1. 1Biochemistry, University of Pennsylvania,
Philadelphia, PA; 2Neuroscience Center of Excellence School of
Medicine, Louisiana State University Health, New Orleans, LA.
Purpose: RPE cells utilize a hybrid process that incorporates
aspects of phagocytosis and autophagy to efficiently degrade
ingested photoreceptor outer segments. Our previous studies have
established that this LC3 dependent process is mediated by a cargo
sorting protein, melanoregulin (MREG). The purpose of this study
was to determine if defective phagosome maturation as observed in
the absence of MREG and LC3B contributes to alterations in lipid
deposition in the RPE.
Methods: In all studies we utilized RPE isolated from Mreg -/-,
LC3B -/- and C57Bl6/J mice. Accumulation of cholesterol and
cholesterol esters was assessed by filipin staining followed by
multi-color confocal microscopy. Levels of cholesterol and 7-ketocholesterol were determined using LC/MS and docosahexaenoic acid
(DHA) levels by LC MS/MS. iPLA2-VIA activity was determined
using a BODIPY® FL-based fluorometric assay.
Results: Cholesterol accumulated in the Mreg -/- RPE as indicated
by the prevalence of filipin positive structures. Biochemical analyses
also showed elevated cholesterol levels in Mreg -/- RPE, however
the most significant increases were in 7-ketocholesterol, in which
case loss of MREG was associated with 5- fold increase in this
immuno-modulatory lipid. Moreover, an increase in cholesterol
accumulation (or filipin positive structures) was also seen in LC3B/- RPE. OS lipids provide a massive daily supply of DHA esterified
to phospholipids for cleavage by the iPLA2-VIA phospholipase
A2 isoform. Defective phagosome maturation in Mreg -/- RPE also
contributed to a decrease in free DHA, with a majority of the DHA
found to be esterified to phospholipids. This decrease in phospholipid
hydrolysis was not due to alterations in iPLA2-VIA activity but likely
due to defective phagocyte processing.
Conclusions: Our results suggest that defective LC3 associated
phagocytosis, specifically regulated by MREG, leads to accumulation
of cholesterol over the long term. Furthermore, loss of MREG leads
to a decrease in the intracellular DHA pool, likely compromising the
generation of protective docosanoids.
Commercial Relationships: Desiree Alexander,
None; Anuradha Dhingra, None; William C. Gordon,
None; Bokkyoo Jun, None; Nicolas G. Bazan, None;
Kathleen Boesze-Battaglia, None; Alvina Bragin, None
Support: NEI grant(s) EY-10420 (KBB) and EY-005121 (NGB).
Program Number: 6044 Poster Board Number: D0263
Presentation Time: 8:00 AM–9:45 AM
Aging and antioxidants differentially modulate the effect of
phagocytized lipofuscin granules on expression of antioxidant
enzymes in ARPE-19 cells
Anna K. Pilat1, Michal Sarna1, Dawid Wnuk2, Magdalena Olchawa1,
Tadeusz J. Sarna1. 1Biophysics, Faculty of Biochemistry, Biophysics
and Biotechnology, Jagiellonian University, Krakow, Poland; 2Cell
Biology, Faculty of Biochemistry, Biophysics and Biotechnology,
Jagiellonian University, Krakow, Poland.
Purpose: Aged RPE cells are at elevated risk of oxidative stress
due accumulation of lipofuscin (LF). It was postulated that chronic
oxidative stress, mediated in the human RPE by LF, could contribute
to the development of age-related macular degeneration (AMD).
We have previously shown that LF under sub-lethal photic stress
conditions photooxidizes cellular proteins in ARPE-19 cells. Here we
ask whether LF-mediated oxidative stress modulates expression of
antioxidant enzyme proteins such as heme oxygenase-1 (HO-1) and
catalase in ARPE-19 cells.
Methods: Lipofuscin granules (LF), isolated from human RPEs from
two age groups, were enriched with a combination of zeaxathin and
alpha tocopherol (An). Control or antioxidant enriched lipofuscin
granules (An-LF) were introduced to APRE-19 cells by phagocytosis.
Control cells, An-treated cells or cells with LF and An-LF, irradiated
with blue light for selected time intervals, were analyzed by Western
blot for selected antioxidant enzymes. To monitor sub-lethal
oxidative stress in ARPE-19 cells mediated by phagocytized human
RPE lipofuscin atomic force microscopy (AFM) and laser–scanning
confocal fluorescence microscopy were employed.
Results: Irradiation of LF containing cells with blue light induced
HO-1 protein with the effect being light dose dependent. The extent
of HO-1 expression by lipofuscin was higher for lipofuscin isolated
from older donors (age: 50-59) compared to younger donors (age:
18-29). Enrichment of lipofuscin granules isolated from both age
groups with zeaxathin and alpha tocopherol reduced HO-1protein
level. AFM analysis revealed that after blue light-treatment, cells with
lipofuscin granules, were significantly softer. Fluorescence analysis
showed that lipofuscin-mediated photic stress to RPE cells induced
significant reduction in the formation of actin stress fibers.
Conclusions: HO-1 upregulation may help to protect RPE cells
form LF-mediated oxidative stress. Phototoxicity of lipofuscin
granules in RPE can be modulated by combination of antioxidants.
AFM and fluorescence image analysis could be employed as
sensitive methods for detecting early sub-lethal photic changes in
RPE cells mediated by LF.
Commercial Relationships: Anna K. Pilat, None; Michal Sarna,
None; Dawid Wnuk, None; Magdalena Olchawa, None;
Tadeusz J. Sarna, None
Support: Poland National Science Center (grant Maestro 2013/08/A/
NZ1/00194). Faculty of Biochemistry, Biophysics and Biotechnology
of Jagiellonian University is a partner of the Leading National
Research Center (KNOW) supported by the Ministry of Science and
Higher Education.
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: 6045 Poster Board Number: D0264
Presentation Time: 8:00 AM–9:45 AM
Proteomic changes in ARPE-19 cells stimulated with complement
serum and UV-irradiated photoreceptor outer segments
(UV-POS)
Martin Busch1, Susanne Wasmuth1, Albrecht Lommatzsch2,
Solon Thanos3, Daniel Pauleikhoff2. 1Ophtha-Lab, Department of
Ophthalmology at St. Franziskus Hospital, Muenster, Germany;
2
Department of Ophthalmology at St. Franziskus Hospital, Muenster,
Germany; 3Institute of Experimental Ophthalmology, University of
Muenster, Muenster, Germany.
Purpose: ARPE-19 cells as a model for retinal pigment epithelial
(RPE) cells were shown to undergo proinflammatory and
proangiogenic changes upon complement stimulation and UV-POS
treatment suggesting a role in AMD pathology. The aim of our study
was to reveal potential intracellular signaling pathways mediating
these functional changes of RPE cells using proteomics and
stimulation with complement serum and UV-POS.
Methods: For complement stimulation, ARPE-19 cells were
incubated in DMEM/F12 medium supplemented with 5 %
human complement serum (HCS) for 24 hours. Medium alone or
supplemented with either 5 % heat-inactivated HCS or C7-deficient
serum were used as controls. Further groups of cells were pre-treated
every other day with 10 µg/ml UV-POS for one week to a total of
three pre-treatments. Afterwards the cells were washed and incubated
in medium containing 5 % HCS or medium alone. Proteins were
separated by 2D-gel electrophoresis and differentially occurring
protein spots were peptide mapped with MALDI-TOF mass
spectrometry.
Results: Some protein spots were differentially expressed in the
various treatment groups and differed between two independent
experimental sets. From the two experimental sets, a total of 27
protein spots were selected and processed for MALDI-TOF analysis
that identified a total of 56 different proteins, which could be grouped
into 7 functional entities including metabolic (12), structural (16),
globular (3), cell-cell and cell-matrix interaction-associated proteins
(2), gene expression (4), proteins related to inflammatory processes
and angiogenesis (7), and proteins involved in signal transduction
(10). 60 percent of the identified proteins participating in signal
transduction are associated with cellular and oxidative stress
response. Out of two cases, expression of these proteins was observed
in response to HCS, UV-POS, or their combination.
Conclusions: The results show that complement stimulation and
UV-POS uptake regulate the cell proteome. Further experiments are
required to clarify how far the identified differentially expressed
proteins associated with signal transduction are involved in the
mediation of the functional changes of RPE cells in response to
complement stimulation and UV-POS.
Commercial Relationships: Martin Busch, None;
Susanne Wasmuth, None; Albrecht Lommatzsch, None;
Solon Thanos, None; Daniel Pauleikhoff, None
Support: Dr. Werner Jackstädt Foundation and Voltmann Foundation
Program Number: 6046 Poster Board Number: D0265
Presentation Time: 8:00 AM–9:45 AM
Mutations in CFH perturb RPE homeostasis in vitro
Roni Hazim, David S. Williams. Jules Stein Eye Institute, UCLA, Los
Angeles, CA.
Purpose: Single nucleotide polymorphisms (SNPs) in the
complement factor H gene (CFH) are closely linked with increased
risk of age-related macular degeneration (AMD). In particular,
SNPs in exons 2 and 9, responsible for I62V and Y402H amino
acid substitutions, respectively, constitute a genetic haplotype that
increases the risk of developing AMD in late adulthood by 7-fold.
Individually, both of these SNPs have been shown to compromise
the function of CFH. Although most abundant in the circulating
plasma, CFH is locally expressed and secreted by the retinal pigment
epithelium (RPE), a primary site of insult in AMD pathogenesis.
Numerous models have been made to elucidate the role of CFH,
yet the full extent of CFH function and the underlying cellular
mechanisms responsible for perturbing RPE homeostasis remain
elusive. The purpose of this study is to investigate the role of CFH,
in relation to RPE biology and pathology, using an in vitro model of
human RPE cells.
Methods: We used the genome-editing technology of the CRISPR/
Cas9 system to specifically target and modify the CFH locus in an
immortalized human RPE cell line (ARPE-19). Two stable cell lines
were generated, one of which exhibited a knockdown of CFH while
the other expressed the mutated form of the protein (VV62, HH402).
Brightfield microscopy was used to assess the morphology of the
stable cell lines, and immunocytochemistry and western blotting
were used to analyze cytoskeletal organization and to test for signs of
inflammation and cellular stress.
Results: Sanger sequencing verified the genetic modifications to the
CFH locus, and western blotting confirmed the reduction in CFH
protein levels in the CFH-knockdown line. Brightfield microscopy
revealed a striking morphological phenotype in the CFH-knockdown
line, which exhibited an elongated, fusiform shape. Immunolabeling
of α-tubulin confirmed an abnormal arrangement of the microtubule
cytoskeleton in these cells. Additionally, both CFH-mutant cell
lines were found to possess an aberrant expression of ZO-1, a
tight junction protein known to associate with actin filaments.
Characteristics of AMD, including inflammation and cellular stress,
were also more prominent in the CFH-knockdown line, as indicated
by increased expression of C5b-9 and 3-NT, respectively.
Conclusions: Our results indicate that compromised CFH function
has a significant effect on RPE morphology, as well as increasing
inflammation and cellular stress.
Commercial Relationships: Roni Hazim, None;
David S. Williams, None
Support: NIH grant EY07042
Program Number: 6047 Poster Board Number: D0266
Presentation Time: 8:00 AM–9:45 AM
Growth differentiation factor 6 (GDF6) is a novel inducer of
the epithelial-to-mesenchymal transition in retinal pigmented
epithelial cells
Amanda M. Hurley1, Monte J. Radeke2, Pete J. Coffey2. 1Molecular,
Cellular, and Developmental Biology, University of California,
Santa Barbara, Santa Barbara, CA; 2Neuroscience Research Institute,
University of California, Santa Barbara, Santa Barbara, CA.
Purpose: As retinal pigmented epithelium (RPE) cells are passaged,
they undergo an irreversible epithelial-to-mesenchymal transition
(EMT). We have shown previously, that GDF6, a member of the
transforming growth factor-beta (TGFβ) family, is highly upregulated
in RPE cells that have undergone EMT. We hypothesize that GDF6
plays an integral role in the irreversible transition of an RPE cell from
an epithelial state to a mesenchymal state.
Methods: Passage 0 fetal RPE cells were transduced with a lentiviral
construct encoding GDF6 or an empty vector control. The GDF6
positive cells were collected, re-plated at a high density, and allowed
to differentiate for 32 days. At this point images were obtained
for pigmentation analysis and cells were harvested for RNA.
Pigmentation was analyzed using Matlab software. The images were
binarized and the number of back pixels was divided by the total
number of pixels. qRT-PCR was performed using a variety of EMT
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ARVO 2016 Annual Meeting Abstracts
and RPE markers. Student’s T tests were used to determine statistical
significance
Results: RPE transduced with GDF6 produce a significantly lower
level of pigmentation (21%) than cells infected with an empty
vector control (68%, p=0.0001). This reduction in pigmentation is
accompanied with a change in cell morphology; where the control
cells maintain a cuboidal morphology and the GDF6 secreting cells
have a spindle-like morphology. qRT-PCR analysis reveals that RPE
transduced with GDF6 significantly upregulate known EMT markers
like ACTA2, CTGF, and COL1A1 and downregulate classical RPE
markers such as LRAT, APOE, and PMEL compared to control cells.
Conclusions: GDF6 is a novel inducer of EMT in RPE. Cells that
overexpress GDF6 will prematurely undergo EMT, simultaneously
downregulating traditional RPE markers while upregulating EMT
and wound response markers. Inhibition of GDF6 may be integral in
prolonging the integrity of RPE cultures.
Commercial Relationships: Amanda M. Hurley, None;
Monte J. Radeke; Pete J. Coffey, None
Support: CIRM LA1-02086, Garland Initiative for Vision Research,
Arnold & Mable Beckman Initiative for Macular Research
Program Number: 6048 Poster Board Number: D0267
Presentation Time: 8:00 AM–9:45 AM
Cellular effects of lysosomal dysfunction in retinal pigment
epithelial cells
Mallika Valapala2, Abdulla Alamri2, Rhonda Grebe1, J S. Zigler1,
James T. Handa1, Debasish Sinha1. 1UVEA, Johns Hopkins Univ
Sch of Med, Baltimore, MD; 2Optometry, Indiana University,
Bloomington, IN.
Purpose: The retinal pigment epithelial (RPE) is not only one of
the most active phagocytic cell types in the body but also has a high
rate of autophagy. Lysosomes are involved in the terminal stages of
phagocytosis and autophagy, hence dysfunctional lysosomes lead to
impaired cellular clearance in the RPE. The objective of this study
was to analyze if the transcriptional program regulating lysosomal
biogenesis and function can be induced in the RPE following
lysosomal disruption by treatment with ammonium chloride. We also
examined the cellular effects of lysosomal dysfunction in the RPE
using Cryba1 (gene encoding βA3/A1-crystallin) knockout mice.
Methods: Confluent monolayers of ARPE-19 cells were treated with
1 mM ammonium chloride (to block endolysosomal acidification)
for 18 hrs. Ammonium chloride was later removed and the cells
were subjected to a 6 hr recovery period. Quantitative real time
PCR (qPCR) analysis was performed to study the expression
of the following genes involved in lysosomal biogenesis and
function: Lysosome associated membrane protein 1(LAMP-1),
Cathepsin D (CTSD), Cathepsin A (CTSA), Cathepsin F (CTSF),
hexosaminidase A, and ATPase, H+ transporting, lysosomal V0
subunit a1 (ATP6V0A1) encoding for a subunit of V-ATPase.
Immunofluorescence and immunoblotting was used to determine the
expression of the lysosomal marker, LAMP-1. Cellular ultrastructure
of the Cryba1 KO RPE was studied by transmission electron
microscopy (TEM).
Results: qPCR analysis of ARPE-19 cells treated with
ammonium chloride and subjected to a 6 hr recovery period
revealed significant induction of genes regulating both lysosomal
biogenesis (LAMP-1,HexA) and function (CTSD, CTSA,CTSF
and ATP6V0A1) compared to untreated cells. Immunoblotting and
immunofluorescence studies showed increased expression of LAMP1 in cells recovered from ammonium chloride treatment compared
to untreated cells. TEM of 25 month old Cryba1 KO mice revealed
many vacuole-like structures containing undigested cellular material
and photoreceptor outer segments.
Conclusions: Our studies suggest that perturbation of lysosomal
function by pre-treatment with ammonium chloride followed by a
recovery period results in a rapid induction of the transcriptional
program that induces lysosomal biogenesis and function in ARPE-19
cells. Impaired lysosomal function seen in the Cryba1 KO mice leads
to RPE cellular abnormalities and eventual degeneration
Commercial Relationships: Mallika Valapala, None;
Abdulla Alamri, None; Rhonda Grebe, None; J S. Zigler, None;
James T. Handa, None; Debasish Sinha, None
Program Number: 6049 Poster Board Number: D0268
Presentation Time: 8:00 AM–9:45 AM
Nrf2 and Hif1a have opposite responses to oxidative stress in
ARPE-19 cells
Hong Wei, James T. Handa. OPHTHALMOLOGY, JOHNS
HOPKINS UNIVERSITY, BALTIMORE, MD.
Purpose: Smoking causes oxidative stress and damage to the retina,
reduces blood flow in eye tissue, and promotes ischemia, hypoxia,
and micro-infarctions. It also induces cell death to retinal pigment
epithelial (RPE) cells. This study explores how the RPE cell responds
to stress induced by cigarette smoking and hypoxia, and how the
transcription factor NF-E2-related factor 2 (Nrf2) and Hypoxia
Inducible Factor 1 alpha (Hif1a), which has been reported to be
induced by oxidative stress in addition to hypoxia, can influence the
anti-oxidant response through JNK and p38 signaling.
Methods: ARPE-19 cells were grown to confluence, serum starved
for 24h, and then treated with cigarette smoke extract (CSE) and 1%
O2 for 24h. The transcription factors Nrf2 and Hif1a proteins and
MAPK pathway signaling were analyzed by Western blotting.
Results: CSE induced a significant dose dependent increase in NRF2
protein (p<0.01) and a remarkable reduction in Hif1a (p<0.001) in
ARPE-19 cells, while treatment with 1% O2 resulted in a significant
increase in Hif1a (p<0.01) with no change in Nrf2. Since oxidative
stress and hypoxia can either induce or impair JNK or p38 signaling,
we next looked at phosphorylated JNK and p38 after CSE treatment.
CSE decreased phospho-JNK (p<0.01) and increased phosphor-p38
(p<0.01) compared to control treated cells while 1% O2 increased
phospho-JNK (p<0.01) and decreased phospho-p38 (p<0.05).
Conclusions: NRF2 and Hif1a respond differently to oxidative stress
in RPE cells, with differential signaling though Phospho-JNK and
Phospho-p38, which can mediate inflammation, neovascularization,
and apoptosis.
Commercial Relationships: Hong Wei; James T. Handa, None
Support: : EY019044, EY14005, RPB Physician Scientist Award,
Unrestricted grant from RPB. Dr. Handa is the Robert Bond Welch
Professor.
Program Number: 6050 Poster Board Number: D0269
Presentation Time: 8:00 AM–9:45 AM
Impaired lysosomal calcium signaling in RPE cells with an in
vitro model of chloroquine retinopathy
Nestor Mas Gomez2, Wennan Lu2, Jason Lim2, Alan Laties1,
Kirill Kiselyov3, Claire H. Mitchell2, 4. 1Ophthalmology, University
of Pennsylvania, Philadelphia, PA; 2Department of anatomy and
cell biology, School of dental medicine, University of Pennsylvania,
Philadelphia, PA; 3Department of Biological Sciences, University of
Pittsburgh, Pittsburgh, PA; 4Physiology, University of Pennsylvania,
Philadelphia, PA.
Purpose: The incidence of chloroquine retinopathy is increasing,
with patients experiencing a loss of central vision and photoreceptors.
While the initial step in the pathology is widely acknowledged
to involve lysosomal alkalinization by chloroquine, the links
between alkalinization and photoreceptor death remain unknown.
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ARVO 2016 Annual Meeting Abstracts
The lysosome is a major source of intracellular Ca2+, and release
of lysosomal Ca2+ through the TRPML1 channel is necessary for
signaling and lysosomal exocytosis. In this study we examined the
effect of an in vitro model of chloroquine retinopathy on the release
of Ca2+ through TRPML1 in RPE cells and asked whether lysosomal
exocytosis contains protective components
Methods: For Ca2+ measurements, RPE cells from C57Bl6J mice
and ARPE19 cells were grown on coverslips, loaded with fura-2 and
the 340/380 nm signal was recorded using microscope or fluorimeter
based readings. Exocytosis was recorded from mouse cells grown
on transwell filters. IL-6 was measured with an Elisa kit and acid
phosphatase (AP) with a colorimetric assay. TRPML1 mRNA levels
were determined with qPCR. The in vitro model of chloroquine
retinopathy consisted of ARPE-19 cells treated with 10 µM CHQ for
7 days and 1 µM U18666A for 1 day
Results: The TRPML1 channel agonist ML-SA1 led to reversible and
repeatable elevations in cytoplasmic Ca2+ in mouse RPE and ARPE19 cells. The response to ML-SA1 was significantly reduced in the
chloroquine model. Treatment increased TRPML1 mRNA levels,
while lysosomal Ca2+ levels released by glycyl-L-phenylalanine-betanaphthylamide were not altered; together this suggests the reduced
Ca2+ efflux in the chloroquine model reflected a reduction in channel
function. In polarized mouse RPE cells, ML-SA1 triggered a release
of lysosomal enzyme AP across the apical membrane. Importantly,
the cytokine IL-6 was also released in the same samples, suggesting
lysosomal release of IL-6 following activation of TRPML1
Conclusions: The ability of ML-SA1 to trigger lysosomal Ca2+
release and concurrent exocytosis of acid phosphatase and IL-6
implies that lysosomal Ca2+ contributes to exocytosis in RPE
cells. The reduced response to ML-SA1 in the in vitro chloroquine
retinopathy model predicts that IL-6 release will also be reduced. As
IL-6 is reported to protect photoreceptors, this reduced signal may be
detrimental, although this awaits direct confirmation
Commercial Relationships: Nestor Mas Gomez, None;
Wennan Lu, None; Jason Lim, None; Alan Laties, None;
Kirill Kiselyov, None; Claire H. Mitchell, None
Support: EY-013434
Program Number: 6051 Poster Board Number: D0270
Presentation Time: 8:00 AM–9:45 AM
The tyrosine phosphatase receptor PTPRZ1 inhibits β-catenindependent gene expression in RPE cells
Jorgelina M. Calandria, Khanh Do, Swornim Shrestha,
Nicolas G. Bazan. Neuroscience Center of Excellence, LSU Health
Sciences Center, New Orleans, LA.
Purpose: PTPRZ1 is a tyrosine phosphatase receptor that stabilizes
the cadherin/catenin complex at cell-cell junctions. The retinal
pigment epithelium (RPE) relies on these cell-cell interactions to
maintain the integrity and polarization of the monolayer in the retina.
β-catenin may be released from the complex and translocated into
the nucleus. Neuroprotectin D1 (NPD1), a bioactive derivative of
docosahexaenoic acid (DHA), modulates gene expression upon stress
conditions (Calandria et al, 2015. Cell Death Differ). We propose
that the inhibition of PTPRZ1 by HMGB1 promotes the release of
β-catenin from the cell-cell junction complex and, along with NPD1,
favors its nuclear translocation to modulate gene expression and
promote survival of RPE.
Methods: Primary RPE cells were exposed to 600 µM H2O2
and 10ng/ml TNFα to induce oxidative stress. β-catenin release
and translocation was assessed by immunocytochemistry. Gene
expression was measured by real-time PCR. Silencing and
overexpression of PTPRZ1 and HMGB1 was carried out by
transfecting RPE cells with siRNA and vectors carrying the open
reading frame. TCF/LEF-β-catenin activity was measured using a
TOP Flash system driving the expression of luciferase.
Results: The addition of HMGB1 in the medium induced an increase
in the mobilization of β-catenin from the membrane to the cytoplasm.
The silencing of PTPRZ1 induced the same effect, suggesting that
HMGB1 acts through the inhibition of the tyrosine phosphatase
receptor. NPD1 and HMGB1 separately increased the nuclear
translocation of β-catenin. Intriguingly, HMGB1, and not NPD1,
induced a rise in TCF/LEF activity, suggesting that both effects
proceed through different pathways. The wnt/β-catenin canonical
genes CCND1, CLDN1 and MMP2 were modulated by HMGB1
but not affected by NPD1. On the other hand, FOXO3a-driven
genes BIM, CDKN1A and CDKN1B expression was influenced by
HMGB1 and NPD1 in an additive manner.
Conclusions: Altogether, these results suggest that NPD1 and
HMGB1 act cooperatively, but through different pathways, to
modulate β-catenin activity. Suppression of PTPRZ1 plus addition of
NPD1 lead to an increased mobilization and nuclear translocation of
β-catenin, affecting its activity in a differently. The modulation of the
β-catenin activity may be used as a therapeutic instrument to promote
retinal cell remodeling and survival.
Commercial Relationships: Jorgelina M. Calandria, None;
Khanh Do, None; Swornim Shrestha, None; Nicolas G. Bazan,
None
Support: Supported by NIGMS grant P30 GM103340 and NEI grant
R01 EY005121 (NGB) and Research to Prevent Blindness
Program Number: 6052 Poster Board Number: D0271
Presentation Time: 8:00 AM–9:45 AM
Effects of Anticancer Drug Cisplatin on Cybrids Containing A, B,
D or L Haplogroup mtDNA
Gregory Yung, Kunal Thaker, Marilyn Chwa, Sina Abedi, Tej Patel,
Theresa Thai, Cristina M. Kenney. Gavin Herbert Eye Institute-UCI,
Irvine, CA.
Purpose: Mitochondria (mt) DNA can be categorized into
haplogroups according to the accumulation of single nucleic
polymorphisms (SNPs). While the influence of mitochondria in
metabolism has been established, the extent to which mtDNA
haplogroups influence intercellular metabolic activities is not
well understood. It is recognized that different racial/ethnic
populations have different susceptibilities to diseases and responses
to medication. The present study characterizes the response of
transmitochondrial cybrids containing either A (non-white Hispanic,
B (non-white Hispanic), D (East Asian) and L (African origin)
haplogroups to Cisplatin, a commonly used cancer drug.
Methods: Cybrids were created by fusing platelets from donors with
different ethnic/racial backgrounds with Rho0 ARPE-19 cells (lacking
mtDNA). Cybrids containing either A (n=2), B (n=2), D (n=3), or
L(n=5) haplogroups were treated with 40 µM Cisplatin for 48 hours
and then cell viabilities were measured with the MTT assay. Data
were calculated based upon 6-12 wells per cell line. The untreated
cybrid values were normalized to 100% to serve as the baseline. An
unpaired t-test was used to analyze the data (GraphPad Prism 5).
Results: The L cybrids showed significantly reduced viability
between Untreated cybrids (100% ± 5.42) and Cisplatin-treated
groups (69.75 ± 5.81%, p = 0.0003). D cybrids had decreased cell
viability after Cisplatin treatment (77.39 ± 2.13%, p < 0.0001)
compared to Untreated D cybrids (100% ± 1.58). The A cybrids also
showed lower cell viability (80.11 ± 3.75%) compared to Untreated
A cybrids (100% ± 3.83, p = 0.0007). In contrast, Cisplatin-treated B
cybrids had similar levels of cell viability compared to Untreated B
cybrids (95.27 ± 4.96% versus 100% ± 8.09, p = 0.62).
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
Conclusions: Our results show the degree of cytotoxic effects of
Cisplatin can vary depending upon the mtDNA haplogroup contained
within the cells; L (African origin) cybrids > D (Asian origin) cybrids
> A (non-white Hispanic) cybrids > B (non-white Hispanic) cybrids.
These data suggest an individual’s mtDNA background may play a
greater role than previously thought in responses and side effects to
Cisplatin and may redefine the appropriate anticancer treatment and
chemotherapy for different racial groups.
Commercial Relationships: Gregory Yung, None; Kunal Thaker,
None; Marilyn Chwa, None; Sina Abedi, None; Tej Patel, None;
Theresa Thai, None; Cristina M. Kenney, None
Support: Discovery Eye Foundation, Guenther Foundation,
Beckman Initiative for Macular Research, Polly and Michael Smith
Foundation, Max Factor Family Foundation, Iris and B. Gerald
Cantor Foundation
Program Number: 6053 Poster Board Number: D0272
Presentation Time: 8:00 AM–9:45 AM
Varying Mitochondrial Haplogroups Show Differential
Expression Levels of Histone Proteins
Jon L. Norman, Shari R. Atilano, Marilyn Chwa, Kunal Thaker,
Cristina M. Kenney. Gavin Herbert Eye Institute - UCI, Irvine, CA.
Purpose: Gene expression can be regulated by how tightly DNA
associates with the nuclear proteins called histones, which are
responsible for nucleosome structure integrity and gene regulation
in eukaryotes. Recent studies show that cybrids (cytoplasmic
hybrids), with identical nuclei but different mitochondrial (mt)
DNA, differentially express methylation and acetylation genes.
Our hypothesis is that different mtDNA haplogroups, representing
populations from different geographic origins, have altered
expression levels of histone proteins genes. The present study was
conducted to determine if cybrids with mtDNA haplogroups H
(European origin), L (African origin), and K (European origin – most
common in Ashkenazi Jews) show differences in expression levels
for histone genes.
Methods: Cybrids were created by fusing Rho0 ARPE-19 cells
(devoid of mtDNA) with platelets isolated from Haplogroup H (n=8),
L (n=6), or K (n=7) individuals. Haplogroups were determined
by DNA analyses with PCR/restriction enzyme digestion and
sequencing. Gene expression was analyzed by RT-qPCR for four
histone genes using ALASv1 as a reference gene.
Results: L cybrids had lower expression levels for the histone genes
HIST1H3F (2-fold, p=0.0003), HIST1H3H (0.44-fold, p=0.008), and
HIST1H4H (0.63-fold, p=0.003) when compared to haplogroup H
cybrids. K cybrids had lower expression levels for the histone genes
HIST1H3A (0.3-fold, p<0.0001), HIST1H3H (0.28-fold, p<0.0001),
and HIST1H4H (0.5-fold, p<0.0001) when compared to haplogroup
H cybrids. The H cybrids values were assigned a value of 1.
Conclusions: Cybrids, which have identical nuclei but either
H, L, or K haplogroup mtDNA, have differing RNA expression
levels for histone genes. Previous studies have demonstrated that
different mtDNA haplogroups mediate different expression levels
of genes related to complement, inflammation, and apoptosis, but
the mechanism(s) of retrograde signaling are not understood. Our
results show that H, L and K cybrids also have unique expression
levels of HIST1H3A, HIST1H3F, HIST1H3H and HIST1H4H, which
would lead to variability in the nucleosome core and may influence
acetylation/methylation status of cells and their retrograde signaling
abilities. These factors may play a key role in gene regulation and
in determining why cells from unique ethnic/racial origins are more
susceptible to certain eye diseases.
Commercial Relationships: Jon L. Norman, None;
Shari R. Atilano, None; Marilyn Chwa, None; Kunal Thaker,
None; Cristina M. Kenney, None
Support: Discovery Eye Foundation, Guenther Foundation,
Beckman Initiative for Macular Research, Polly and Michael Smith
Foundation, Max Factor Family Foundation, Iris and B. Gerald
Cantor Foundation.
Program Number: 6054 Poster Board Number: D0273
Presentation Time: 8:00 AM–9:45 AM
Comparison of Lipid and Cholesterol-Biosynthesis Gene
Products in H- and K-mt-DNA haplogroup cybrids
Christine Garabetian1, Kunal Thaker1, Javier Cáceres-del-Carpio1,
Shari R. Atilano1, Marilyn Chwa1, Anthony B. Nesburn1, 2,
Cristina M. Kenney1, 3. 1Gavin Herbert Eye Institute, University of
California Irvine, Irvine, CA; 2Cedars-Sinai Medical Center, Los
Angeles, CA; 3Department of Pathology and Laboratory Medicine,
University of California Irvine, Irvine, CA.
Purpose: Cholesterol has been associated in the pathogenesis of
neurodegenerative and systemic pathologies, and certain populations
have higher incidence of cholesterol-linked familial diseases. The
purpose of this experiment was to determine if mitochondrial DNA
(mt-DNA) haplogroup H and K transmitochondrial cybrids regulate
the cholesterol and lipid biosynthesis pathways differently. Using
transmitochondrial cybrids, cell lines with identical nuclei but
either H or K mtDNA, we looked at the relationship between the
gene expression levels of cholesterol-related genes and neutral lipid
deposits in H and K cybrids.
Methods: Transmitochondrial cybrids were created by fusing
mitochondria (mt) isolated from human platelets and cells lacking
mtDNA; thus these cybrids have identical nuclei but either H or K
mtDNA. RNA from H and K cybrids were analyzed using GeneChip
arrays (n=3). Specific cholesterol-related genes FDFT1, SQLE, LSS,
CYP51A1, EBP, and DHCR24 were analyzed using Q-PCR (H, n=7;
K, n=9). Corresponding reference genes were used as housekeepers
for each of the six genes studied. H and K cybrids (n=10) were
seeded at 104/well and stained with HCS LipidTox Green Neutral
Lipid Stain. Statistical analyses were performed using student’s t-test.
Results: Q-PCR showed insignificant differences of the cholesterol
biosynthesis genes in H vs. K; however, one K cybrid (CY#13-57)
showed elevated expression of 4 out of 6 cholesterol biosynthesis
genes compared to H cybrids; (17.4-fold increase in FDFT1
expression, a 31-fold increase in SQLE expression, a 10.8-fold
increase in CYP51A1 expression, and a 5.4-fold increase in EBP).
LipidTox staining showed cytoplasmic distribution of the lipids
with significantly elevated intensity in the CY#13-57 compared to H
cybrids or other K cybrids (p<0.001).
Conclusions: Due to all cybrids differing only in their mtDNA
content, our findings suggest that H- and K-haplogroup mt-DNA do
not necessarily affects genes involved in cholesterol biosynthesis.
However, the increased expression of cholesterol-related genes and
larger and more vibrant lipid deposits seen in CY#13-57 suggest the
mt-DNA in that specific K cybrid is different than other K cybrids
and is mediating cholesterol production and lipid transport through
some mechanism.
Commercial Relationships: Christine Garabetian,
None; Kunal Thaker, None; Javier Cáceres-del-Carpio,
None; Shari R. Atilano, None; Marilyn Chwa, None;
Anthony B. Nesburn, None; Cristina M. Kenney, 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
Support: Discovery Eye Foundation, Guenther Foundation,
Beckman Initiative for Macular Research, Polly and Michael Smith
Foundation, Max Factor Family Foundation, Iris and B. Gerald
Cantor Foundation, J.C.d.C. was the David & Julianna Pyott PanAmerican-Retina Research Fellowship
Program Number: 6055 Poster Board Number: D0274
Presentation Time: 8:00 AM–9:45 AM
Pax6 role in the regulation of retinal pigmented epithelium
maturation
Yamit Cohen1, Pablo Blinder2, Maria Idelson3, Benjamin Reubinoff3,
Shalev Itzkovitz4, Ruth Ashery-Padan1. 1Department of Molecular
Genetics and Biochemistry, Tel-Aviv University, Tel-Aviv, Israel;
2
Department of Neurobiology, Tel Aviv University, Tel Aviv, Israel;
3
Department of Gynecology, Hadassah-Hebrew University Medical
Center, Jerusalem, Israel; 4Department of Molecular Cell Biology,
Weizmann Institute of Science, Rehovot, Israel.
Purpose: Haploinsufficiency of Pax6 was long shown to cause
aniridia and was reported to involve neovascularization of the
cornea. However, Pax6 involvement in vascularization remained
elusive and lack of imaging techniques for the choroid vasculature
limited further inquiry regarding the effect on this tissue. This
research aims to determine the molecular mechanism in which Pax6
regulates retinal pigmented epithelium (RPE) maturation and choroid
development using somatic mutagenesis in mouse models and RPE
cells derived from human embryonic stem cells (hES-RPE).
Methods: To this purpose, conditional mutations are induced in
mice and gain of function analysis is conducted using sub-retinal
injections followed by electroporation. Quantitative expression
levels are measured using single molecule FISH and the choroidal
phenotype is analyzed using a novel perfusion technique. Further
investigation of the molecular mechanism involved in this regulation
is performed using hES-RPE cells and includes chip-seq, RNA-seq
and knockdown using viral infections.
Results: Pax6 deletion in the specified RPE resulted in a phenotype
of aniridia and among the changes in gene expression we observed
an up-regulation in Sox9 expression level, a key transcription
factor in organogenesis which is related to late stages of RPE
differentiation. Pax6 and Sox9 expression patterns were determined
in course of RPE differentiation in wild type mice and mice with
Pax6 specific ablation from the RPE. Quantitative expression
analysis illustrated opposite correlation and gain of function analysis
confirmed this observation since miss-expression of Pax6 resulted in
inhibition of Sox9 expression.
Conclusions: This study is the first to document Pax6 role in timing
RPE differentiation through its regulatory relations with Sox9.
Future efforts are aimed to elucidate the molecular mechanism of this
regulation and examine a possible effect on the choroid vasculature.
Commercial Relationships: Yamit Cohen, None; Pablo Blinder,
None; Maria Idelson, None; Benjamin reubinoff, None;
shalev Itzkovitz, None; Ruth Ashery-Padan, None
Support: ISVER travel grant award for best presentation
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.