ARVO 2016 Annual Meeting Abstracts
410 RPE-Photoreceptor Interface
Wednesday, May 04, 2016 8:30 AM–10:15 AM
Tahoma 1/2, TCC Paper Session
Program #/Board # Range: 4329–4335
Organizing Section: Retinal Cell Biology
Program Number: 4329
Presentation Time: 8:30 AM–8:45 AM
The Role Of Connexin 43 And PKCδ In The Phagocytosis Of
Photoreceptor Outer Segments
Korina Theodoraki2, 1, Katharina Lueck2, Morgane Gourlaouen1,
Apostolos Georgiannakis2, Stephen E. Moss2. 1KSS Deanary, London,
United Kingdom; 2Ophthalmology and Visual Sciences, Institute of
Ophthalmology, London, United Kingdom.
Purpose: The retinal pigment epithelium (RPE) is responsible for
the daily shedding of photoreceptor outer segments (POS). The RPE
cytoskeleton plays an instrumental role during this process through
activation of signalling cascades leading to its re-organisation and the
uptake of the POS. The role of gap junctions has not been explored.
We performed in vitro studies to test the hypothesis that gap junction
communication participates in the phagocytosis, investigating the role
of Connexin 43 (Cx43) and PKCδ as mediators.
Methods: Freshly enucleated bovine eyes were used to isolate
the RPE cells and the POS. In vitro studies were performed using
primary RPE cells (pRPE), cultured RPE cells (ARPE-19) and
primary POS. Western Blots examined the effect of phagocytosis
on Cx43 and PKCδ phosphorylation at different time points.
PKCδ was suppressed with small interference RNA (siRNA).
Immunofluorescence studies in ARPE-19 cells were performed
to monitor the effect of gap junction inhibition (using 18βglycyrrhetinic acid, 18β-GA) and PKCδ knock-down in POS binding
and internalisation. Experiments were repeated three times. Twotailed Student’s t-test was used for statistical analysis. Statistical
significance was set at P<0.05.
Results: A time-dependent phosphorylation of Cx43 (pS368-Cx43)
and PKCδ (pY311-PKCδ) occurs during phagocytosis in the control
condition and in presence of 18β-GA and PKCδ siRNA respectively.
Immunofluorescence experiments showed that PKCδ knock-down
led to a 41% statistically significant decrease in POS binding and
internalisation, while PKCδ activation by phorbol-12-myristate13-acetate (PMA) led to a non-significant increase in phagocytosis.
PMA was unable to abolish the down-regulatory effect of PKCδ
suppression (PKCδ siRNA+PMA treatment). Use of 18β-GA was
found to have a 1.5 fold increased impact on internalisation and
binding of the POS. The combination of the two treatments (PKCδ
siRNA+18β-GA) recoved phagocytosis.
Conclusions: Our results are consistent with the hypothesis that
gap junction communication particiates in phagocytosis. Cx43 and
PKCδ participate in the same signalling cascade up-regulating POS
binding and internalisation. In response to outer segments, PKCδ
phosphorylates Cx43 leading to gap junction disassembly which
promotes phagocytosis. Src is likely to be the upstream kinase
regulating PKCδ activity, while morphologic experiments will be
needed to understand the structural changes accounting.
Commercial Relationships: korina theodoraki; Katharina Lueck,
None; Morgane Gourlaouen, None; Apostolos Georgiannakis,
None; Stephen E. Moss, None
Program Number: 4330
Presentation Time: 8:45 AM–9:00 AM
Bestrophinopathy is an RPE-photoreceptor interface disease
Karina E. Guziewicz, William A. Beltran, Simone Iwabe,
Artur V. Cideciyan, Samuel G. Jacobson, Anuradha Dhingra,
Kathleen Battaglia, Gustavo D. Aguirre. University of Pennsylvania,
Philadelphia, PA.
Purpose: Bestrophinopathies are caused by mutations in the BEST1
gene expressed in the retinal pigment epithelium (RPE). Both
human and canine bestrophinopathies are characterized by focal or
multifocal separations of the retina from the RPE. The lesions can be
macular or extramacular, and the specific pathomechanism leading to
formation of these lesions remains unclear. We used the spontaneous
dog BEST1 disease model, canine multifocal retinopathy (cmr), to
explore factors contributing to the development of lesions.
Methods: Sixteen cmr dogs (age range 2mo - 6yr) with homozygous
(R25X or P463fs) or compound heterozygous (R25X/P463fs)
mutations in canine BEST1 were monitored clinically and imaged
serially using cSLO/SD-OCT. Retinal structure was examined in
cryosections by H&E, Oil Red O and filipin staining, WGA and
PNA lectins labeling and immunohistochemistry. The sections were
assessed using transmitted light microscopy, epifluorescence and
confocal microscopy. All in vivo and ex vivo analyses were carried
out in comparison to age-matched wild-type control eyes.
Results: Misregulation of lipid metabolism and an indication of
increased levels of oxidative stress within the photoreceptor layer of
cmr-affected retinae were evidenced by Oil Red O, filipin, BODIPY
493/503 and anti-HNE staining. Immunolabeling with anti-EZR,
anti-SLC16A1 and anti-RLBP1 revealed dramatic retraction
of RPE apical cone sheath microvilli in all cmr samples. PNA
staining confirmed compromised interphotoreceptor matrix (IPM)
of cone outer segments. SD-OCT measurements of the distance
between external limiting membrane and RPE showed greater than
normal thickness in cmr implying elongated inner/outer segments.
Histological evaluation revealed areas of subtle dissociation of the
neural retina from RPE with accumulation of subretinal debris.
Conclusions: Compromised IPM and/or retraction of RPE apical
processes that ensheath cones may contribute to the loss of retinal
adhesiveness and lesion formation in bestrophinopathies by retinal
detachment and subretinal deposit formation. These salient alterations
detected at the RPE-photoreceptor interface in dogs, as well as
abnormal electrooculogram light peak detected in many human
patients could be reflecting the underlying pathophysiology resulting
from abnormal volume regulation in mutant RPE cells.
Commercial Relationships: Karina E. Guziewicz,
None; William A. Beltran, None; Simone Iwabe, None;
Artur V. Cideciyan, None; Samuel G. Jacobson, None;
Anuradha Dhingra, None; Kathleen Battaglia, None;
Gustavo D. Aguirre, None
Support: FFB-Center and FFB-TRAP grant, MVRF, NEI/NIH
EY06855, EY17549, EY10420, Van Sloun Fund, Hope for Vision
Program Number: 4331
Presentation Time: 9:00 AM–9:15 AM
Photoreceptor outer segments (POS) induce Retinal pigment
epithelial (RPE) cell multinucleation through modulating the
Protein Kinase C pathway
Dinusha Rajapakse, Mei Chen, Tim Curtis, Heping Xu. Queen’s
university Belfast, Belfast, United Kingdom.
Purpose: Multinuclear RPE cells have been frequently observed
in the aging eye. RPE multinucleation can be induced in vitro by
feeding cells with oxidised photoreceptor outer segments (oxPOS).
We hypothesize that multinucleation is a mechanism of RPE cells to
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ARVO 2016 Annual Meeting Abstracts
repair damage in the aging eye, although the underlying mechanism
remains to be elucidated. The enzymes of Protein kinase C (PKC)
family are critically involved in various cell activation pathways
including cytokinesis. The aim of this study was to understand
whether the PCK pathway is involved in oxPOS-induced RPE
multinucleation.
Methods: The expression of total PKC proteins in RPE cells with/
without oxPOS treatment was determined by Western blotting and
the active PKC levels were determined by ELISA. The expression of
different members of the PCK family in RPE cells was examined by
RT-PCR and immunofluorescence investigations. The contribution
of different PKC members to RPE multinucleation was determined
by using target-specific PKC inhibitors. The involvement of Ca2+
in the activation of PKC was determined using the Fluo-4 calcium
indicators and Ca2+ inhibitors.
Results: RT-PCR study revealed that under normal culture conditions
RPE cells express PKCα, PKCg, PKC ζ, PKCµ, and PKCε. oxPOS
treatment increased the expression of PKCα, PKC ζ, PKCµ, and
PKCε, but decreased the expression of PKCg. The expression of PKC
i was below detectable level in naïve RPE cells and the expression
was significantly upregulated by oxPOS treatment. The expression
levels of total PKC protein was not affected by oxPOS treatment
in Western blot analysis, however the active PKC levels were
significantly increased following oxPOS treatment (p<0.001). In line
with the PKC activation, phosphorylation of p27 kip 1, but not p21
protein was observed in oxPOS treated cells. A 10% reduction in RPE
multinucleation was observed when intracellular Ca2+ was chelated
(p<0.05). Pharmacological inhibition of PKC ζ, PKCα and PKC i
all significantly suppressed oxPOS-induced RPE multinucleation,
although the strongest suppressive effect was observed in PKC ζ
inhibitor treated cells.
Conclusions: oxPOS induces RPE cell multinucleation through
activating the PKC, in particular PKCζ, pathway. Further
understanding the molecular mechanism of RPE multinucleation may
shed light on the pathogenesis of age-related macular degeneration.
Commercial Relationships: Dinusha Rajapakse, None; Mei Chen,
None; Tim Curtis, None; Heping Xu, None
Program Number: 4332
Presentation Time: 9:15 AM–9:30 AM
Dopamine 2 Receptor Signaling Controls the Daily Burst in
Phagocytic Activity by the Retinal Pigment Epithelium
Gianluca Tosini1, Kenkichi Baba1, Virginie Laurent2, David Hicks2.
1
Pharmacology and Toxicology, Morehouse School of Medicine,
Atlanta, GA; 2Institute for Cellular and Integrative Neurosciences,
CNRS UPR 3212, Stassbourg, France.
Purpose: Several lines of evidence suggest that dopamine (DA)
and its receptors are involved in the regulation of rhythmic retinal
pigment epithelium (RPE) functions. For example, inhibition of
DA synthesis during the early part of the light phase induced a
significant reduction of disk shedding and phagocytosis and mice
whose dopaminergic neurons have been destroyed accumulate a
large number of residual bodies in the RPE. Previous work from our
laboratory has shown that the mouse RPE contains a circadian clock
that is synchronized by DA via Dopamine 2 receptor (D2R). In this
study we investigated the effects of D2R removal of the daily rhythm
of RPE phagocytic activity.
Methods: C57/Bl6 and D2R knock-out (KO) in a C57/Bl6 genetic
background were used in this study. Phagocytic activity by the RPE
was measured by counting the number of phagosomes in the RPE
at ZT 23, ZT1 and ZT3 (i.e., before and after light onset) Focal
adhesion kinase (FAK) activation was measured by western blotting.
Photoreceptor viability was assessed by counting the number of
nuclei in the outer nuclear layer (ONL).
Results: The daily burst in phagocytic activity occurring 1-2 hours
after light onset was not longer present in D2R KO mice. FAK
activation is essential for the engulfment of shed outer segments by
the RPE and rhythmic activation of FAK is required for the daily
burst of phagocytic activity. C57/Bl6 mice showed a clear increase
of FAK-P-Y397/FAK 1 hr. after light onset (One way Anova, P <
0.05) whereas D2R KOs mice did not show any significant increase
in the levels of FAK-P-Y397/FAK (One way Anova, P > 0.1).
Morphometric analysis of D2R KO mice also indicated a significant
reduction in the number of nuclei in the ONL of 10 months old D2R
KO (One way Anova P < 0.05). Finally, removal of D2R signaling
induced significant changes in the daily pattern of clock and clock
controlled genes expression.
Conclusions: Our data indicate that D2R signaling is involved in
the timing of the daily burst of phagocytic activity by the RPE. Our
results also indicate that the lack of the daily burst in phagocytic
activity may affect photoreceptor viability.
Commercial Relationships: Gianluca Tosini; Kenkichi Baba,
None; virginie Laurent, None; David Hicks, None
Support: EY022216
Program Number: 4333
Presentation Time: 9:30 AM–9:45 AM
Loss of ATG5 in the RPE slows phagosome degradation
contributing to altered intracellular lipid processing
Kathleen Boesze-Battaglia1, Anuradha Dhingra1, Desiree Alexander1,
Alvina Bragin1, Daniel Lamond1, Vanda S. Lopes2, David Williams2.
1
Biochemistry, University of Pennsylvania, Philadelphia, PA;
2
Ophthalmology and Neurobiology, UCLA, Los Angeles, CA.
Purpose: RPE cells utilize a hybrid degradative pathway which
utilizes components of both autophagy and phagocytosis called LC3
associated phagocytosis (LAP). In the absence of the autophagic
protein, ATG5, LAP is inhibited. In these studies we sought to
determine the role of ATG5-dependent LAP in the breakdown of
ingested outer segments (OS) and to assess the consequences of a
defect in this process.
Methods: Using a cre/lox recombination system we generated
Atg5ΔRPE (BEST1-cre/+;Atg5flox/Atg5flox) and littermate
controls of the following genotypes; BEST1-cre/+;Atg5+/+ and
Atg5flox/Atg5flox. RPE-specific Cre expression was determined
by immunofluorescence, using anti-CRE, with deletion of ATG5
confirmed by double labeling with anti-ATG5 in whole mounts
and eyecups. ATG5 and LC3 levels in retinal and RPE lysates were
compared by Western blot. The phagocytic profile of RPE cells
was determined using ultrathin sections from retinas processed for
conventional TEM from 3 month old Atg5ΔRPE, and control mice
at -1h, 0h, 0.5h, 1h, and 3h relative to light onset. Accumulation of
cholesterol and cholesterol esters was assessed by filipin staining
followed by multi-color confocal microscopy.
Results: In the Atg5ΔRPE mice, cre expression was consistently
found in 85-90% of RPE cells, with only the RPE cells
immunoreactive for CRE. Correspondingly, ATG5 was knocked
down in approximately 90% of the RPE cells. As expected there
was a concomitant decrease in LC3 lipidation. RPE isolated from
Atg5ΔRPE mice 3 hours after light onset showed packets of whorled
disk–like material; such structures were not seen in the littermate
controls. The number of phagosomes in ultrathin sections of RPE in
Atg5ΔRPE mice reached a sharp peak within an hour of lights on.
Phagosome numbers rose similarly in the Atg5ΔRPE mutants but
remained elevated for at least 3 hrs. Lastly, loss of ATG5 resulted in
the accumulation of filipin positive structures in the RPE
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
Conclusions: In the absence of the autophagic protein, ATG5,
degradation of phagosomes containing photoreceptor outer segment
disk membranes is significantly inhibited, consistent with an essential
role of the autophagic pathway in this critical non-autophagic event
in the RPE. The resulting long-term pathology includes an abnormal
accumulation of cholesterol.
Commercial Relationships: Kathleen Boesze-Battaglia,
None; Anuradha Dhingra, None; Desiree Alexander, None;
Alvina Bragin, None; Daniel Lamond, None; Vanda S. Lopes;
David Williams, None
Support: EY-010420-(KBB)
Program Number: 4334
Presentation Time: 9:45 AM–10:00 AM
Retinal Pigment Epithelium (RPE) cells facing cone
photoreceptors display distinct transciptome with implications
for the pathogenesis of Age-related Macular Degeneration (AMD)
Shadi Safuri1, Liat B. Brenner1, Anat Loewenstein2, Ido Perlman1.
1
Ruth & Bruce Rappaport Faculty of Medicine and Rappaport
Institute, Technion-Israel Institute of Technology, Haifa, Israel;
2
Ophthalmology, Tel Aviv Medical Center, Tel Aviv, Israel.
Purpose: RPE Cells interact with photoreceptors in a variety of ways
acting together as a functional unit. As the retinas of most animal models
used in retinal research are dominated by rods, little is known about RPE
cells facing cones - the dominant type of photoreceptors at the human
macula. This study tested whether the transciptome of RPE cells facing
cones differ from that of RPE cells facing rods in a manner that may
explain macular susceptibility and pathogenesis of AMD.
Methods: The retina of NRL knock-out (NRL-ko) mouse is
composed of cone-like photoreceptors, while the retina of wild type
(WT) mouse contains mainly rod photoreceptors. RPE cells were
harvested from eyes of both WT and NRL-ko mice (6 weeks old).
RNA samples were hybridized to Illumina MouseWG-6 v2.0 wholegenome array. After applying filters, including a fold change of 1.7,
and dynamic statistical stringency criteria iteratively modified to
obtain a manageable list size, a list of 108 genes was generated. From
this list, the expression of 25 genes of special interest was validated
using Nanostring’s nCounter technology.
Results: Several genes that were up regulated in RPE facing
cones were previously reported to be associated with macular
degenerations when mutated. Genes coding for proteins serving along
the phagocytosis process, from binding down to degradation, were
up regulated, suggesting a unique phagocytosis process for shed
cone outer segment compared to that of rods. Several genes coding
for proteins serving in the complement system were altered in a
manner consistent with shift in balance toward the classical pathway.
Several other genes were altered, including genes involved in cellular
adhesions, protection from oxidation, visual transduction and retinol
metabolism.
Conclusions: We show that the transciptome of RPE cells can be
modulated by the photoreceptors located adjacent to them. Some of
the genes have relevance to the physiology of RPE-photoreceptors
interactions and others may be of relevance to AMD pathogenesis,
including genes coding for proteins of the complement system. We
suggest that gene expression of RPE cells facing cones can explain, at
least partially, macular susceptibility to age related lesions.
Commercial Relationships: Shadi Safuri, None; Liat B. Brenner,
None; Anat Loewenstein, None; Ido Perlman, None
Support: Israel Science Foundation
Program Number: 4335
Presentation Time: 10:00 AM–10:15 AM
ABCB5 identifies RPE progenitor cells required for normal
retinal development and aging
Bruce Ksander1, 2, Andrew Hertsenberg1, 2, Ruth Y. Lewis1, 2,
Brian Wilson3, Gretchen Berg4, Markus H. Frank3, 5,
Natasha Y. Frank4. 1Harvard Medical School, Boston, MA;
2
Ophthalmology, Mass Eye & Ear, Boston, MA; 3Transplant Research
Program, Boston Children’s Hospital, Boston, MA; 4Division of
Genetics, Brigham & Women’s Hospital, Harvard Medical School,
Boston, MA; 5Harvard Stem Cell Institute, Harvard Medical School,
Boston, MA.
Purpose: RPE might contain subpopulations of progenitor cells with
multipotent differentiation potential; however, no specific molecular
marker for such subpopulations has been described to date. Recently,
we identified ATP binding cassette member B5 (ABCB5) as a marker
for mammalian limbal stem cells, with essential roles in normal
corneal development and repair. In the current study, we describe that
ABCB5 also identifies a novel progenitor subpopulation among RPE
with essential functions in retinal development and regeneration.
Methods: RPE cells were recovered from choroidal flat mounts
isolated from 2 months-old C57BL/6J mice and from human
cadaveric donors. Analysis of ABCB5 expression in mouse and
human RPE was done by dual-color flow cytometry using two
distinct murine and human anti-ABCB5 monoclonal antibodies
(mAb) described previously (Ksander et al., Nature 2014). Abcb5
knock out (KO) mice were generated as previously described
(Ksander et al., Nature 2014) and maintained on a 129S6/SvEvTac/
C57BL/6 mixed genetic background. Gender-matched littermates
were used as controls for experimental analyses. Comparative
histopathology of Abcb5 WT and Abcb5 KO retinas was done by
examination of H&E-stained sections of 9 months old mice.
Results: Flow cytometric analyses of human and murine RPE cell
suspensions revealed that ABCB5 was expressed in 2-8% of RPE
cells. Additional studies demonstrated that a portion of ABCB5positive cells did not express the marker of differentiated RPE cells,
RPE65. Moreover, histological examination of Abcb5 KO mice
revealed significantly reduced RPE cell numbers associated with
abnormal RPE morphology and vacuolization. Additionally, Abcb5
KO animals also exhibited thinning and attenuation of the overlaying
photoreceptor outer and inner segments, and reduced cell numbers in
the outer nuclear layer.
Conclusions: ABCB5 identifies a novel progenitor cell population
in RPE, which lacks expression of the RPE differentiation marker
RPE65, but is essential for retinal development and regeneration.
Commercial Relationships: Bruce Ksander, Rheacell GmbH
& Co. KG (P), Ticeba GmbH (P), Rheacell GmbH & Co. KG (F);
Andrew Hertsenberg, None; Ruth Y. Lewis, None; Brian Wilson,
None; Gretchen Berg, None; Markus H. Frank, Rheacell GmbH
& Co. KG (C), Rheacell GmbH & Co. KG (P), Ticeba GmbH (S),
Ticeba GmbH (P), Rheacell GmbH & Co. KG (F), Rheacell GmbH
& Co. KG (S), Rheacell GmbH & Co. KG (R), Ticeba GmbH (C),
Ticeba GmbH (R); Natasha Y. Frank, Rheacell GmbH & Co. KG
(P), Ticeba GmbH (P)
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.