ARVO 2016 Annual Meeting Abstracts
372 Photoreceptor degeneration and phototransduction
Tuesday, May 03, 2016 3:45 PM–5:30 PM
Tahoma 4, TCC Paper Session
Program #/Board # Range: 3841–3847
Organizing Section: Biochemistry/Molecular Biology
Program Number: 3841
Presentation Time: 3:45 PM–4:00 PM
ARL2BP is essential for the function of rod and cone
photoreceptor cells
Abigail R. Hayes1, 2, Ratnesh Singh2, Visvanathan Ramamurthy1, 2.
1
Biochemistry, West Virginia University, Morgantown, WV;
2
Ophthalmology, West Virginia University, Morgantown, WV.
Purpose: Blinding diseases such as retinitis pigmentosa (RP) are
linked to defects in multiple ciliary proteins, including ARL2BP,
a protein thought to be involved in ciliogenesis. Despite the link
between defective ciliogenesis and blindness, little is known
about the mechanisms that control photoreceptor cilia growth and
maintenance. Using an animal model lacking ARL2BP, our goal is
to identify the role of ARL2BP in the development and function of
photoreceptors.
Methods: In this study, we generated a global knockout (KO)
of ARL2BP in mice using the Crispr-Cas9 system. Protein and
mRNA levels were measured by immunoblotting and real-time
PCR. Immunocytochemical staining was used to investigate the
morphology of the retina and protein localization in photoreceptor
cells. Electroretinogram (ERG) recordings were performed to assess
photoreceptor function. Littermates were used in all experiments as
controls (n=4).
Results: At 3 weeks of age, ERGs revealed a significant reduction in
scotopic and photopic responses in animals lacking ARL2BP (↓50%),
while heterozygous littermates were similar in response to wild
type (WT) mice. Immunocytochemical staining of retinal sections
from adult KO animals showed a degeneration of photoreceptor
nuclei compared to WT littermates (↓40%). Despite a reduction
in expression of photoreceptor outer segment proteins, such as
rhodopsin, G-protein transducin, and phosphodiesterase 6, their
localization was unaffected.
Conclusions: Knockout of ARL2BP produced a defect in rod and
cone function phenocopying the patient mutations. Our preliminary
results suggest that ARL2BP is involved in the development of
photoreceptor outer segments, but not in protein trafficking. Our
current efforts are focused on understanding the role of ARL2BP and
its interacting partners to help delineate the mechanism underlying
photoreceptor outer segment biogenesis.
Commercial Relationships: Abigail R. Hayes; Ratnesh Singh,
None; Visvanathan Ramamurthy, None
Support: NH Grant EY017035
Program Number: 3842
Presentation Time: 4:00 PM–4:15 PM
Trafficking of transducin-α by UNC119A/B and ARL3GTP
dependent diffusion
Wolfgang Baehr, Cecilia D. Gerstner, Christin Hanke-Gogokhia,
Guoxin Ying, Jeanne M. Frederick. Ophthalmology, Univ of Utah Sch
of Med, Salt Lake City, UT.
Purpose: UNC119A and UNC119 B are closely related acylbinding proteins expressed in photoreceptors. UNC119A forms an
immunoglobulin-like β-sandwich fold into which the N-terminal acyl
side chain of Tα inserts. Our purpose was to generate UNC119A/B
double knockouts and explore the consequences on transducin
trafficking.
Methods: GST-pull downs, preparation of a monospecific
antibody, western blotting, Unc119 double knockout mouse,
immunohistochemistry.
Results: UNC119A and UNC119B are expressed ubiquitously
among vertebrates. In the mammalian eye, UNC119s are strongly
expressed in photoreceptors. Amino acid sequence comparison of
both UNC119 isoforms reveal perfect conservation of all amino acids
known to interact with the acyl side chain and N-terminal residues
of Tα, and predict nearly identical structures. Both GST-UNC119B
and GST-UNC119/RG4 pull down Tα from retina lysates, suggesting
closely related functions as acyl-binding proteins. We generated
Unc119a/b double knockout mice by breeding doubly heterozygous
parents (Unc119a+/-;Unc119b+/-). Double knockout mice are viable,
fertile and stable. Tα is present in dark-adapted Unc119 dKO rod
outer segments, but mislocalized in the myoid region of the Unc119
dKO inner segment and ONL. These results support the idea that
UNC119 polypeptides traffic Tα by diffusion in the dark-adapted
rod. Trafficking by diffusion is further supported by dependence of
Tα delivery to the outer segments by ARL3GTP, acting as a cargo
dislocation factor. A model of UNC119/ARL3-dependent trafficking
will be presented. Preliminary results show that the Unc119 dKO
a-wave is attenuated at 6 weeks of age. A slowly developing retina
degeneration is complete (single row of ONL nuclei) in the dKO at
11 months of age, while the Unc119a+/-;Unc119b+/- heterozygote
littermate appears normal.
Conclusions: It is unclear how transducin traffics to the distal RIS
and to the ROS. Our results suggest that transducin traffics in darkadapted rods to the OS at least in part by UNC199A/B-dependent
diffusion.
Commercial Relationships: Wolfgang Baehr, None;
Cecilia D. gerstner; Christin Hanke-Gogokhia, None;
Guoxin ying, None; Jeanne M. Frederick, None
Support: NIH grants EY08123; EY019298; EY014800-039003;
RPB Neldon Trust; RPB Senior Investigator; RPB unlimited to the
Ophthalmology, Univesity of Utah.
Program Number: 3843
Presentation Time: 4:15 PM–4:30 PM
The Role of Circadian Rhythms in Regulating the Cone
Phototransduction Cascade Shutoff in Zebrafish Retina
Stephan C. Neuhauss, Jennifer Keim, Jingjing Zang. Institute of
Molecular Life Sciences, University of Zurich, Zurich, Switzerland.
Purpose: A variety of visual behaviors in different species
are regulated by the circadian clock. Here we performed
electrophysiological and behavioral studies to investigate the
molecular mechanism underlying how circadian rhythms influence
photoresponse shutoff in the cone vision This shutoff requires the
phosphorylation of visual pigment by opsin kinase and subsequent
binding of Arrestin, followed by hydrolysis of PDE-Tα-GTPcomplex by GTPase.
Methods: In situ hybridization and qRT-PCR has been used to
(semi-)quantitatively evaluate mRNA expression levelsl throughout
the day on 5dpf larvae and adult fish. Changes in protein level
were assessed by western blotting. Electroretinograms (ERG) and
optokinetic response (OKR) served as functional and behavioral
assessment.
Results: Expression levels of over 10 key phototransduction
regulatory genes (recoverins, arrestins, opsin kinases GRK and
GTPase-accelerating proteins) fluctuate in a circadian rhythm which
is maintained in complete darkness. In extreme cases, e.g. GRK7a,
mRNA decreases 98.4±0.01% (mean ± SED) during the day.
Western blot results show that protein levels also change at 24-hour
cycle with a delayed peak concentration to the mRNA expression
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ARVO 2016 Annual Meeting Abstracts
peak, e.g. the reduction for GRK7a is 82.6±6% (mean ± SED).
ERGs from larvae demonstrate that the photoresponse recovery is
delayed in the evening and accelerated in the morning. When the
interval between two stimuli is 1 second, the response recovery in
the evening is only 62.6% (p<0.01) compared to the morning. This
phenomenon is maintained in continuous darkness (p<0.01), while
constant light exposure disrupts the normal oscillating expression
pattern. GRK7a mRNA declines only 13.8±7% (mean ± SED) over
12 hours. Consistently there is no significant difference (p>0.05) in
photoresponse kinetics between morning and evening under constant
light conditions. An inverted light cycle can successfully reverse the
mRNA fluctuations and a delayed photoresponse recovery and OKR
recorded right before the light is off can still be observed (p<0.05).
Conclusions: The expression level of several important regulators for
cone photoresponse recovery shows robust circadian rhythms, which
may be correlated with the observed photoresponse kinetic and visual
behavioral change throughout the day.
Commercial Relationships: Stephan C. Neuhauss, None;
Jennifer Keim, None; Jingjing Zang, None
Support: Swiss National Science Foundation
Program Number: 3844
Presentation Time: 4:30 PM–4:45 PM
Posttranslational Lipid Modification Controls the Trafficking of
Transducin to the Photoreceptor Cilium
Maxim Sokolov, Marycharmain Belcastro, Joseph Murphy,
Saravanan Kolandaivelu. Ophthalmology, West Virginia Univ Eye
Institute, Morgantown, WV.
Purpose: The signaling functions of various cilia are predicated
based on the trafficking of heterotrimeric G proteins into this
organelle, however, the mechanism underlying these processes
remains poorly understood. This is exemplified in rod photoreceptors,
whose sensory cilia, termed the outer segments, detect photons
using the G protein, transducin. Like all G proteins, the transducin
Gγ1 subunit undergoes a posttranslational lipid modification. Here,
we tested the role of this farnesyl lipid group in the trafficking of
transducin to the rod outer segment.
Methods: Epitope-tagged Gγ1, carrying a Cys71Ser substitution,
which eliminates its farnesylation site (HA-Gγ1-no-farnesyl), was
expressed in the photoreceptors of transgenic mice. An identical
construct without the mutation (HA-Gγ1-wild type) was used as
a control. The protein interactions of HA-Gγ1-no-farnesyl and
HA-Gγ1-wild type were analyzed by pull down assay and Western
blotting, and their subcellular localization was monitored by
immunofluorescence confocal microscopy.
Results: Both HA-Gγ1-no-farnesyl and HA-Gγ1-wild type were clearly
detectable in rods, while their overall levels in the retina were only about
1% that of endogenous Gγ1. No detrimental effects of the transgenes on
photoreceptors were noticed. HA-Gγ1-no-farnesyl and HA-Gγ1-wild
type both formed a complex with the endogenous Gβ1. The subcellular
localization of the resulting Gβ1γ1 complexes was, however, very
different. While HA-Gβ1γ1-no-farnesylwas strictly excluded from the
rod outer segments, HA-Gβ1γ1-wild type exhibited a very pronounced
translocation between the outer segments and the rod inner segments and
synapses that was dependent on the history of light stimulation.
Conclusions: Our data demonstrate that the farnesylation of Gγ1
controls the trafficking of transducin to the photoreceptor cilia, while
being nonessential for Gβ1γ1complex assembly. The mechanism
consistent with this observation will be discussed.
Commercial Relationships: Maxim Sokolov, None;
Marycharmain Belcastro, None; Joseph Murphy, None;
Saravanan Kolandaivelu, None
Support: NIH Grant EY019665
Program Number: 3845
Presentation Time: 4:45 PM–5:00 PM
Ablation of Retbindin Alters Flavin Levels and Leads to Rod and
Cone Photoreceptor Degeneration
Muayyad R. Al-Ubaidi1, Ryan Kelley2, Jianhai Du3, James Hurley4,
Muna I. Naash1. 1Biomedical Engineering, University of Houston,
Houston, TX; 2Cell Biology, University of Oklahoma, Oklahoma
city, OK; 3Ophthalmology, University of Washington, Seattle, WA;
4
Biochemistry, University of Washington, Seattle, WA.
Purpose: Purpose: Retbindin (Retb) is a retina-specific protein
that localizes extracellularly at the outer segment/retinal pigment
epithelium interface and is capable of binding riboflavin in vitro.
Since riboflavin and its derivatives [flavin mononucleotide (FMN)
and flavin adenine dinucleotide (FAD)] are co-factors involved in
regulating metabolic enzymes activities, we hypothesize that Retb is
involved in binding/transport of flavins and its absence will lead to
deleterious metabolic alterations.
Methods: Methods: A knockout mouse (Retb-/-) was generated in which
the coding sequence was replaced with eGFP. Developmental structural,
functional and biochemical analyses were performed. Flavin levels were
measured with HPLC while metabolites were quantified by LC-MS and
data was analyzed using MetaboAnalyst 3.0.
Results: Results: Expression of eGFP in Retb-/- mice was restricted
to rod photoreceptors, consistent with the distribution of Retb in WT
retinas. Retb-/- retinas contained significantly reduced levels of flavins
and exhibited age- and dose-dependent decline, associated with cell
loss, in both rod and cone functions as early as postnatal day 120. We
also observed significant reductions in the binding affinity of Retb/retinas to C14-labeled riboflavin. Finally, a metabolomic analysis
revealed that elimination of Retb led to changes in glycolysis, TCA
cycle and in amino acid metabolism.
Conclusions: Conclusion: Our results are consistent with our
hypothesis and Retb. Further investigation into the role of Retb in the
retina is needed to determine the exact metabolic changes leading to
the degeneration and to determine the role of specific pathways in the
degenerative process in patients with mutations in proteins other than
retbindin. This will enable us to determine the commonality of the
metabolic changes to retinal degenerative diseases.
Commercial Relationships: Muayyad R. Al-Ubaidi, None;
Ryan Kelley, None; Jianhai Du, None; James Hurley, None;
Muna I. Naash, None
Support: NIH R01EY10609, R01EY018137 and the Foundation
Fighting Blindness (C-CMM-07-0405-UOK09 ).
Program Number: 3846
Presentation Time: 5:00 PM–5:15 PM
Design, generation, and initial characterization of gene-edited
mice for the analysis of the peripherin-2/rds (P/rds) cytoplasmic
C-terminal domain
Andrew F. Goldberg1, Breyanna Cavanaugh1, Michelle L. Milstein1,
Victoria A. Kimler1, Kenneth P. Mitton1, Thomas Saunders2. 1Eye
Research Institute, Oakland University, Rochester, MI; 2Transgenic
Animal Model Core, University of Michigan, Ann Arbor, MI.
Purpose: A broad variety of progressive retinal degenerations are
caused by inherited defects in P/rds, a tetraspanin protein essential for
proper formation and stability of the outer segment (OS) organelles
of rod and cone photoreceptors. A subset of pathogenic defects
affect the protein’s C-terminus; however, the normal function of this
domain is not yet known, and hinders a detailed understanding of
disease etiology. The current study investigates the importance of the
P/rds C-terminus in vivo for OS structure/function and photoreceptor
function and viability.
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to access the versions of record.
ARVO 2016 Annual Meeting Abstracts
Methods: P/rds knock-in mice were generated using the CRISPR/
Cas9 system in conjunction with pronuclear microinjection into
single-cell zygotes. Prph2 exon 3 was targeted for cleavage proximal
to Tyr285, and a ssDNA donor template was utilized to introduce a
C>A transversion, mimicking a putatively pathogenic mutation in
human P/rds. Founder mice identified via DNA sequencing were
backcrossed to create C57BL/6J congenic lines. Age-matched
gene-edited mice and littermate controls were analyzed using
histology, immunohistochemistry, Western blotting, transmission
electron microscopy, and electroretinography. Phenotypic effects
of the Tyr285stop mutation were validated using lines derived from
independent founders.
Results: Two independent lines of Tyr285stop P/rds mice have been
generated; each has been transferred to the C57BL6/J background
to eliminate potential off-target effects of gene-editing. Comparison
of the two lines confirmed a consistent phenotype and demonstrates
that a Tyr285stop nonsense mutation associated with progressive
retinal degeneration in humans also causes retinal dystrophy in mice.
This defect disrupts photoreceptor OS morphogenesis in a distinctive
manner, impairs retinal function, and demonstrates an essential role
for the P/rds C-terminal domain in photoreceptor structure/function
and the maintenance of retinal health.
Conclusions: This work presents the first animal model for
Prph2-associated disease to investigate a defect in the protein’s
cytoplasmic C-terminus. It also demonstrates the utility of CRISPR/
Cas9 gene-editing for efficiently producing murine models for
progressive retinal disease. The results to date support the presumed
pathogenicity of a heterozygous Tyr285stop mutation in P/rds, and
allow investigation of protein domain structure/function in vivo.
Commercial Relationships: Andrew F. Goldberg, None;
Breyanna Cavanaugh, None; Michelle L. Milstein, None;
Victoria A. Kimler, None; Kenneth P. Mitton, None;
Thomas Saunders
Support: R01EY013246
analysis revealing increased number of M-opsins in retina of mice
treated with DUNA.
Conclusions: Long term oral treatment with Dunaliella powder rich
in 9-cis-β-carotene preserves retinal function and rescues M-cones
from degeneration in Rpe65rd12 mice. Our findings suggest that
9-cis β-carotene may possibly be an effective treatment for retinal
dystrophies involving the retinoid cycle.
Commercial Relationships: Ifat Sher, None; Victoria Edelshtain,
None; Adi Tzameret, None; Dror Harats, None; Aviv Shaish,
None; Ygal Rotenstreich, None
Support: TEVA NNE2013
Program Number: 3847
Presentation Time: 5:15 PM–5:30 PM
Long-term treatment with 9-cis-β-carotene rich alga Dunaliella
Bardawil inhibits photoreceptor degeneration in a mouse model
of retinoid cycle defect
Ifat Sher1, 2, Victoria Edelshtain1, 2, Adi Tzameret1, 2, Dror Harats3,
Aviv Shaish3, Ygal Rotenstreich1, 2. 1Goldschleger Eye Institute, Sheba
Medical Center, Tel-Hashomer, Israel; 2Sackler Medical School, Tel
Aviv University, Tel Aviv, Israel; 3The Bert W. Strassburger Lipid
Center, Sheba Medical Center, Tel Hashomer, Israel.
Purpose: To examine the therapeutic effect of oral treatment with
9-cis β-carotene rich alga Dunaliella Bardawil on retinal function and
structure in Rpe65rd12 mice, a model of genetic defect in retinoid
cycle.
Methods: Thirteen RPE65rd12 mice at age of 28 days were fed
with Teklad Global 18% Protein Rodent Diet (normal diet), vitamin
A deficiency diet (VAD) or VAD diet supplemented with extracts
of 9-cis β-carotene rich Dunaliella Bardawil algae (DUNA) for 14
months. Retinal function was recorded once a month in both eyes
simultaneously by electroretinograph (ERG). Histological analysis
and immunofluorescence staining with anti- M opsin antibody were
used to determine retinal structure.
Results: At 14 months, mice fed with DUNA presented significantly
higher maximal a-wave coampared with mice fed with VAD or
normal diet (30μV [SE=5] vs. 5 μV [SE=3] p=0.006 and vs. 3 μV
[SE=2] p=0.007, respectivley) and significantly higher maximal
b-wave compared with mice fed normal diet (82μV [SE=8] vs. 30 μV
[SE=10] p=0.002). These results are assosicated with histological
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.