PBN (phenyl-N-tert-butylnitrone)-derivatives are effective in slowing the visual cycle and rhodopsin regeneration and in protecting the retina from light-induced damage

Article abstract of DOI:10.1371/journal.pone.0145305

A2E and related toxic molecules are part of lipofuscin found in the retinal pigment epithelial (RPE) cells in eyes affected by Stargardt's disease, age-related macular degeneration (AMD), and other retinal degenerations. A novel therapeutic approach for t

Full text of DOI:10.1371/journal.pone.0145305

RESEARCH ARTICLE
PBN (Phenyl-N-Tert-Butylnitrone)-Derivatives
Are Effective in Slowing the Visual Cycle and
Rhodopsin Regeneration and in Protecting
the Retina from Light-Induced Damage
Megan Stiles1,7, Gennadiy P. Moiseyev2, Madeline L. Budda3, Annette Linens1,7, Richard
S. Brush1,7, Hui Qi1,7, Gary L. White4, Roman F. Wolf4, Jian-xing Ma2,5, Robert Floyd8,
Robert E. Anderson1,4,7, Nawajes A. Mandal1,3,5,6,7
*
1 Department of Ophthalmology, OUHSC, Oklahoma City, Oklahoma, United States of America,
2 Department of Physiology, OUHSC, Oklahoma City, Oklahoma, United States of America, 3 Department
of Cell Biology, OUHSC, Oklahoma City, Oklahoma, United States of America, 4 Department of Pathology,
OUHSC, Oklahoma City, Oklahoma, United States of America, 5 Department of Endocrinology and
Diabetes, OUHSC, Oklahoma City, Oklahoma, United States of America, 6 Oklahoma Center for
Neuroscience, OUHSC, Oklahoma City, Oklahoma, United States of America, 7 Dean McGee Eye Institute,
Oklahoma City, Oklahoma, United States of America, 8 Experimental Therapeutics, Oklahoma Medical
Research Foundation, Oklahoma City, Oklahoma, United States of America
* mmandal@ouhsc.edu
OPEN ACCESS
Citation: Stiles M, Moiseyev GP, Budda ML, Linens
A, Brush RS, Qi H, et al. (2015) PBN (Phenyl-N-Tert-
Butylnitrone)-Derivatives Are Effective in Slowing the
Visual Cycle and Rhodopsin Regeneration and in
Protecting the Retina from Light-Induced Damage.
PLoS ONE 10(12): e0145305. doi:10.1371/journal.
pone.0145305
Abstract
A2E and related toxic molecules are part of lipofuscin found in the retinal pigment epithelial
(RPE) cells in eyes affected by Stargardt’s disease, age-related macular degeneration
(AMD), and other retinal degenerations. A novel therapeutic approach for treating such
degenerations involves slowing down the visual cycle, which could reduce the amount of
A2E in the RPE. This can be accomplished by inhibiting RPE65, which produces 11-cis-reti-
nol from all-trans-retinyl esters. We recently showed that phenyl-N-tert-butylnitrone (PBN)
inhibits RPE65 enzyme activity in RPE cells. In this study we show that like PBN, certain
PBN-derivatives (PBNDs) such as 4-F-PBN, 4-CF3-PBN, 3,4-di-F-PBN, and 4-CH3-PBN
can inhibit RPE65 and synthesis of 11-cis-retinol in in vitro assays using bovine RPE micro-
somes. We further demonstrate that systemic (intraperitoneal, IP) administration of these
PBNDs protect the rat retina from light damage. Electroretinography (ERG) and histological
analysis showed that rats treated with PBNDs retained ~90% of their photoreceptor cells
compared to a complete loss of function and 90% loss of photoreceptors in the central retina
in rats treated with vehicle/control injections. Topically applied PBN and PBNDs also signifi-
cantly slowed the rate of the visual cycle in mouse and baboon eyes. One hour dark adapta-
tion resulted in 75–80% recovery of bleachable rhodopsin in control/vehicle treated mice.
Eye drops of 5% 4-CH3-PBN were most effective, inhibiting the regeneration of bleachable
rhodopsin significantly (60% compared to vehicle control). In addition, a 10% concentration
of PBN and 5% concentration of 4-CH3-PBN in baboon eyes inhibited the visual cycle by
60% and by 30%, respectively. We have identified a group of PBN related nitrones that can
reach the target tissue (RPE) by systemic and topical application and slow the rate of
Editor: Zsolt Ablonczy, Medical University of South
Carolina, UNITED STATES
Received: April 17, 2015
Accepted: December 2, 2015
Published: December 22, 2015
Copyright: © 2015 Stiles et al. This is an open
access article distributed under the terms of the
Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any
medium, provided the original author and source are
credited.
Data Availability Statement: All relevant data are
within the paper and its Supporting Information files.
Funding: The research is conducted by the fundings
from The Foundation Fighting Blindness (United
States), Beckman Initiative for Macular Research,
RPB and National Institutes of Health fundings. The
funders had no role in study design, data collection
and analysis, decision to publish, or preparation of
the manuscript.
PLOS ONE | DOI:10.1371/journal.pone.0145305 December 22, 2015
1 / 17

Nitrones for Retinal Degeneration
Competing Interests: The authors have declared
that no competing interests exist.
rhodopsin regeneration and therefore the visual cycle in mouse and baboon eyes. PBNDs
can also protect the rat retina from light damage. There is potential in developing these com-
pounds as preventative therapeutics for the treatment of human retinal degenerations in
which the accumulation of lipofuscin may be pathogenic.
Introduction
At present, approximately 1.75 million Americans have age-related macular degeneration
(AMD) [1]. Diseases like AMD and Stargardt’s Disease (STGD), a juvenile form of macular
degeneration, are particularly devastating because they destroy central vision and inhibit nor-
mal daily function. The atrophic, non-exudative, or drusenoid macular degeneration, collec-
tively called 'dry AMD,' accounts for about 90% of all AMD cases. Dry AMD does not usually
cause complete loss of vision, but significantly impairs central vision required for reading, driv-
ing, and other visually detailed tasks. A substantial proportion of advanced dry AMD trans-
forms to 'wet' or neovascular AMD, which is vision threatening. Anti-angiogenic therapies
have been developed for wet AMD, but there is no proven therapy for dry AMD [2].
One of the hallmarks of AMD and several other retinal degenerative diseases is the presence
of lipofuscin in the retinal pigmented epithelium (RPE). One of the components of lipofuscin
is A2E, a bisretinoid that is a product resulting from the condensation of all-trans-retinal with
the membrane lipid phosphatidylethanolamine which has been studied in great detail for their
role in RPE pathology [3–5]. All-trans-retinal is generated when the visual pigment rhodopsin
absorbs a photon of light. All-trans-retinal is reduced by retinal dehydrogenases to all-trans-
retinol and transported to the RPE where it is converted to all-trans-retinyl ester by lecithin ret-
inol acyl transferase (LRAT) localized in the RPE cells. This is followed by conversion to 11-
cis-retinol by RPE65, which is further oxidized to 11-cis retinal and then returned to the retina
and incorporated into rhodopsin, thus completing the ‘visual cycle’. The excess of all-trans-ret-
inal after rhodopsin bleaching can induce toxicity in photoreceptors at certain unfavorable
conditions such as high intensity light or genetic mutations [6–8] in addition to the toxic effects
of A2E and other bisretinoids.
With age, and in some inherited diseases, there is an accumulation of lipofuscin (and A2E)
in the RPE. Previous studies demonstrated that A2E is toxic to RPE and other cells, and its
accumulation in the RPE is thought to be one of the events that ultimately lead to the death of
RPE cells [9–12].
One strategy currently being studied to reduce the accumulation of A2E in the RPE is to
slow down the ‘visual cycle’, which will reduce the amount of 11-cis-retinal available for forma-
tion of rhodopsin, and thus reduce the amount of all-trans-retinal that is produced by light.
The key step of the visual cycle is the cleavage-isomerization of all-trans-retinyl ester to 11-cis-
retinol. Previous studies have established that the enzyme that catalyzes the isomerization step
is RPE65 (retinal pigment epithelium-specific protein of 65 kDa) isomerase, which is expressed
in the RPE [13–15]. Intact RPE65 function is essential for vision, as mutations in the RPE65
gene cause several forms of inherited retinal dystrophies [16–20].
Over 10 years ago, we discovered that phenyl-N-tert-butylnitrone (PBN), a spin-trap agent
used for measuring the production of oxygen free radicals, protected the retina from light-
induced retinal degeneration (LIRD) [21]. In in vitro enzyme assay, Poliakov et al., 2011,
showed inhibition of RPE65 by PBN [22]. We performed a series of in vivo studies and wit-
nessed PBN inhibition of RPE65 in rats [23]. We also demonstrated that PBN does not affect
PLOS ONE | DOI:10.1371/journal.pone.0145305 December 22, 2015
2 / 17

Nitrones for Retinal Degeneration
or inhibit the function of retinal dehydrogenases (RDHs) present in the photoreceptor outer
segments and LRAT localized in the RPE cells [23]. PBN injected intraperitoneally in rats sig-
nificantly affected the rate of regeneration of rhodopsin and recovery of the maximal a-wave
response of the electroretinogram, consistent with a slowing of the visual cycle [23]. Under
these conditions, there was no effect on the photoresponse of cones, indicating that the slowing
down of the rod visual cycle in these animals did not affect the ability of their cones to respond
to light.
Here we report the development and testing of certain PBN-derivatives (PBNDs) for their
effect on the visual cycle and light-induced retinal degeneration. We also report the efficacy of
PBN and PBNDs in slowing down the rate of rhodopsin regeneration and the visual cycle
when applied topically to mouse and baboon eyes.
Materials and Methods
Animal care
All procedures were performed according to the Association for Research in Vision and Oph-
thalmology Statement for the Use of Animals in Ophthalmic and Vision Research. Albino
Sprague-Dawley rats and BALB/c mice were born and raised in the Dean A. McGee Eye Insti-
tute vivarium and maintained from birth under dim cyclic light (5 lux, 12 h on/off, 7 a.m. to 7
p.m. CST). All the procedures, tissue harvest and the methods of euthanasia for mice and rats
were reviewed and approved by the OUHSC Institutional Animal Care and Use Committee
(OUHSC IACUC #12–063). Rats and mice were euthanized by carbon dioxide asphyxiation
before harvesting the eye or retinal tissues.
Adult male and female baboons (Papio anubis) were obtained from the OUHSC Baboon
Research Resource breeding colony and housed in an AAALAC-accredited vivarium at
OUHSC. During the study, baboons were housed either individually or in pairs under regular
indoor cyclic light (approximately 325 lux, 12 h on/off, 7 a.m. to 7 p.m. CST). They were fed a
commercial diet of monkey chow along with fresh fruit and vegetables. Baboons were housed
individually in Group 6 enclosures for nonhuman primates (84 inches in height, with 25.1
square-feet of floor space), or they were housed in pairs in two joined Group 6 enclosures (84
inches in height, with 50.2 square-feet of floor space) [cit: Code of Federal Regulations 2013 9
CFR §3.80]. Each baboon received social contact and structural, manipulable, and food enrich-
ment in accordance with the OUHSC Baboon Research Resource Environmental Enrichment
program. All animal procedures, including experiments and interventions, as well as euthana-
sia, were reviewed and approved by the OUHSC Institutional Animal Care and Use Committee
(OUHSC IACUC #12–063).
Materials
PBN was purchased from Sigma-Aldrich (St. Louis, MO) and the other derivatives were cus-
tom synthesized. Briefly, to synthesize 4-Fluorophenyl t-butyl nitrone (4-F-PBN), 4-fluoroben-
zaldehyde (8 mmol) and N-tertbutylhydroxylamine (120 mmol) were mixed in chloroform
with molecular sieves (50 g, 4 A) and silica gel (10 g). The mixture was sealed under argon gas
and stirred for 70 h at room temperature. The mixture was then filtered and the solid washed
with ethyl acetate and the combined solution was rotary evaporated to give the crude product.
The compound 4-trifluoromethylphenyl t-butylnitrone (4-CF3-PBN) was synthesized in a sim-
ilar process of 4-F-PBN from 4-trifluoromethylbenzaldehyde and t-butylhydroxylamine. Simi-
larly, 4-Methylphenyl t-butyl nitrone (4-CH3-PBN) was prepared by the reaction of
4-methylbenzaldehyde with t-butylhydroxylamine; 4-methoxyphenyl t-butylnitrone
(4-CH3O-PBN was synthesized from 4-methoxybenzaldehyde and t-butylhydroxylamine;
PLOS ONE | DOI:10.1371/journal.pone.0145305 December 22, 2015
3 / 17

Nitrones for Retinal Degeneration
4-Ethoxyphenylethoxyphenyl t-butylnitrone (4-EtO-PBN) from 4-ethoxybenzaldehyde and t-
butylhydroxylamine; 2-fluorophenyl t-butylnitrone (2-F-PBN) from 2-fluorobenzaldehyde
and t-butylhydroxylamine; 3-fluorophenyl t-butylnitrone (3-F-PBN) from 3-fluorobenzalde-
hyde with t-butylhydroxylamine; 3,5-difluorophenyl t-butylnitrone (3,5-diF-PBN) from
3,5-difluorobenzaldehyde and t-butylhydroxylamine; and 3,4-difluorophenyl t-butylnitrone
(3,4-diF-PBN) from a reaction of 3,4-difluorobenzaldehyde with t-butylhydroxylamine. All of
the compounds were purified by HPLC to >98% purity.
In vitro assay for RPE65
The effect of several PBN analogs on RPE65 retinoid isomerase activity was investigated in an
in vitro assay detailed in Mandal et al., 2011 [23]. Bovine RPE microsomes were used as a
source of the RPE65 protein, and all-trans-[3H] retinol was a substrate for the retinoid isomer-
ase assay. Although all-trans-retinyl ester was shown to be a direct substrate of the retinoid
isomerase [24], very poor water solubility of retinyl ester limits its use in the in vitro retinoid
isomerase assay. Therefore, all-trans-[3H]-retinol was used to generate retinyl esters in the
microsomes of the bovine RPE, which is catalyzed by LRAT localized in RPE microsomes.
Therefore, our assay tested activities of both LRAT and RPE65. The retinyl esters in RPE
microsomes were then isomerized/hydrolyzed to 11-cis-retinol by the bovine RPE65. We previ-
ously showed that PBN specifically inhibits RPE65, not the RDHs and LRAT [23]. The pro-
duced 11-cis-[3H]-retinol was quantified by normal phase HPLC with flow scintillation
analyzer.
Intraperitoneal dosing of PBN derivatives in Sprague Dawley rats for
rhodopsin measurement
PBNDs 4-F-PBN, 4-CF3-PBN and 4-CH3-PBN were dissolved in saline with saline alone used
as vehicle. PBNDs and vehicle were injected intraperitoneally into light-adapted (200–400 lux
room light) rats. The light source for this bench top light adaptation was cool white light bulbs
(13w, F13T5/CW from Sylvania s088). The rats were kept in ambient light for 2h and then
moved into total darkness for 2.5h, after which these dark-adapted rats were euthanized and
retinas were immediately harvested under dim red light. Dark adapted-controls were rats that
experienced dark adaptation for 12h and received no treatment and no light exposure before
euthanasia, as described above, followed by retinal harvest. Light adapted-controls were rats
that experienced ambient light (200–400 lux) for 2h and received no treatment before euthana-
sia followed by retinal harvest in light. Rhodopsin measurement was conducted following the
classical method of David Papermaster [25] with slight modification that we published earlier
[21,23,26,27]. Briefly, under red light, each retina was homogenized in 450 μl of buffer contain-
ing 10 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1 mM EDTA, 2% (w/v) octyl glucoside, and 50
mM hydroxylamine for 1 minute. Hydroxlyamine is used to rapidly form a stable oxime with
all-trans-retinal that is produced by the bleaching of rhodopsin. This complex absorbs around
360 nm, which is further into the UV spectrum than the bleaching intermediates of rhodopsin
and its more stable product meta-II (380 nm). This oxime guarantees that the difference spec-
trum (native vs. bleached rhodopsin) at 500 nm will not be contaminated with the bleaching
intermediates. Homogenates were centrifuged at 16,000 × g, and soluble lysates were scanned
from 270 to 800 nm in a spectrophotometer (Ultrospec 3000 UV-visible spectrophotometer,
GE Healthcare). Samples were then bleached under room light (200–400 lux) for 10–20 min
and scanned again. The difference in spectra at 500 nm between pre- and post-bleached sam-
ples was used to determine rhodopsin content using a molar extinction coefficient of 42,000
M
−1 [28]. The rhodopsin content was calculated as picomoles/retina.
PLOS ONE | DOI:10.1371/journal.pone.0145305 December 22, 2015
4 / 17

Nitrones for Retinal Degeneration
Light-damage of the retina following dosing of PBNDs
PBNDs were dosed following the same dosing scheme as rhodopsin measurement. After dos-
ing, SD rats were placed in dim-light conditions for 30 min before exposure to 2,700 lux white
reflected light from white electric bulb (32w, F32T8/TL84, Philips Long Life Plus) fitted on the
top of a specially fabricated light box. All the sides and the bottom of the box are painted white.
Rats are housed single in wire-top transparent plastic cages with the water bottle from the side
not to block light for 6h of exposure. There is approximately 3 feet distance between the light
source and the rats and there is ample air circulation in the box to prevent heating up. The lux
level is measured inside the cage at the level of the rats’ eyes. After exposure, the rats were
returned to a dim-light cyclic room (5–10 lux) to recover for 7 days. Retinal damage/protection
was assessed by functional analysis using ERG and structural analysis of histological sections
following the procedure published previously [23,26,29,30].
Topical administration of PBNDs in BalbC mice for rhodopsin
measurement
For topical administration of PBN, a 10% concentration was prepared in 1x phosphate-buff-
ered saline, (pH 7.4, PBS) with 30% 1-Methyl-2-pyrrolidinone (MPA) used as a solubilizer.
PBNDs were made to a 5% concentration in PBS using 15% MPA. BalbC mice raised in dim
light conditions (5–10 lux) were dark-adapted overnight. The following morning the mice
were moved to ambient room light (200–400 lux) and given 1 drop of the PBN solutions in
each eye once every 3 hours over a 6 hour period resulting in 3 total doses before returning to
the dark for 1 hour. The mice were euthanized by CO2 asphyxiation in the dark followed by
whole eye harvest for rhodopsin measurement. 30% MPA in PBS was used as PBN vehicle con-
trol. For the PBNDs 15% MPA in PBS was used as vehicle control. Dark adapted no treatment
controls were used as a baseline for 100% rhodopsin. Light adapted no treatment controls were
used to verify 100% rhodopsin photo bleaching. Rhodopsin was measured as described above
and the rhodopsin content calculated as picomoles/eye.
Topical administration of PBN and 4-CH3-PBN in baboons to measure
rhodopsin regeneration
A total of 12 baboons used for this study (4 for PBN and 8 for 4-CH3-PBN). All baboons used
for this study were scheduled for euthanasia as determined by the OUHSC Attending Veteri-
narian for reasons unrelated to this study. No baboon died without euthanasia. The baboons
were evaluated at least twice daily by clinical veterinarians and a staff environmental enrich-
ment coordinator to ensure that humane endpoints were observed and animals did not experi-
ence pain or distress. Once the decision was made to euthanize an animal, the veterinary staff
promptly notified the investigator and the acute procedure was performed under the approval
of the Attending Veterinarian. Reasons for scheduled euthanasia included chronic illness that
was nonresponsive to therapy (e.g., idiopathic epilepsy, irritable bowel syndrome, septic
shock), or social incompatibility with demonstrated aggression despite multiple efforts to
habilitate animals to a colony environment. Other factors including participation in previous
experimental procedures were also taken into account for euthanasia criteria. The baboons
were evaluated at least twice daily by clinical veterinarians and a staff environmental enrich-
ment coordinator to ensure that humane endpoints were observed and animals did not experi-
ence pain or distress until euthanasia occurred. Baboons were fasted 12 hours prior to the
procedure. On the day of the procedure, each animal was chemically restrained using Ketamine
(10mg/kg IM). Once immobile, Propofol (4mg/kg IV) was administered to induce anesthesia.
PLOS ONE | DOI:10.1371/journal.pone.0145305 December 22, 2015
5 / 17

Nitrones for Retinal Degeneration
Anesthesia was maintained using isoflurane (1–2%). Blood pressure, pulse oximetry, heart rate,
respiratory rate, body temperature, and anesthetic depth were monitored throughout the dura-
tion of the procedure. Baboons were dosed in the left eye with 1 drop of either 10% PBN or 5%
4-CH3-PBN every 15 minutes over a 2h period (total of 8 doses). Vehicle was applied to the
right eyes using the same schedule. Two hours after the first drops, Euthasol (1mg/5kg IV) was
administered for euthanasia. The eyes were thoroughly flushed with vehicle, enucleated, and
rinsed again with vehicle. Eyes were wrapped in aluminum foil and placed on ice for 2 hours,
after which each eye was hemisected under dim red light and the anterior segment discarded.
The retina was dissected from the retinal pigment epithelium and suspended in a petri dish
containing PBS. Four pieces of tissue from each retina were removed (still under dim red light)
and placed into individual plastic tubes for rhodopsin measurement as described above. Pro-
tein values were determined by absorbance at 280 nm (for 1 cm path length cuvette, we consid-
ered 1 absorbance = 1 mg/ml protein) and the photobleachable rhodopsin content calculated
as picomoles/mg protein. The percent regeneration in PBN-treated eyes was determined rela-
tive to values for the vehicle treated eyes, which were set at 100%.
Statistical analyses
Statistical analyses were performed by using GraphPad Prism 5.0 software (GraphPad Software
Inc., La Jolla, CA). The quantitative data are expressed as mean standard error of the mean
(SEM) for each group. An unpaired Student t test was performed for the means of two
unmatched groups; for three or more groups, one-way ANOVA was used to compare each pair
of the test groups.
Results
Synthesis of new PBN Derivatives and their inhibition of RPE 65
Working with a custom synthesis company, we synthesized nine derivatives of PBN that
includes various fluorine-derivatives (4-F-PBN, 4-CF3-PBN, 2-F-PBN, 3-F-PBN, 3,4-di-
F-PBN, and 3,5-di-F-PBN), methyl derivatives (4-CH3-PBN, 4-O-CH3-PBN) and an ethyl
derivative (4-O-C2H5-PBN). We have tested three of these derivatives (4-F-PBN, 4-CF3-PBN,
and 4-CH3-PBN) along with PBN in various in vivo assays to determine their efficacy in rho-
dopsin regeneration and protection of retina from light-induced damage (Fig 1A–1D). This
selection was based on their efficacy in inhibiting RPE65 activity in vitro in a bovine microsome
assay as described in the Materials and Methods section. We calculated IC50 values for all the
derivatives that inhibit 50% of the enzymatic activity. The inhibition of retinoid isomerase
activity was dependent on the concentration of the PBNDs (Fig 1E–1G; the enzyme inhibition
graph is not shown for the other derivatives). Although all-trans-retinyl ester is the endogenous
substrate for the RPE65 retinoid isomerase, the insolubility of retinyl ester in water prevented
its direct use in the reaction. Therefore, all-trans-[3H] retinol was used to produce retinyl esters
by LRAT in bovine RPE microsomes; these esters were then converted directly to 11-cis-[3H]
retinol by RPE65. Incubation of the bovine RPE microsomes with all-trans-[3H] retinol
resulted in formation of all-trans-retinyl esters and significant amounts of 11-cis-[3H] retinol,
as shown by the HPLC elution profile (Fig 1H, representative for 4-CF3-PBN). The addition of
250 μM of 4-CF3-PBN to the retinoid isomerase assay reaction resulted in a significant inhibi-
tion of 11-cis-retinol production (peak 4), as shown by HPLC (Fig 1I), whereas the generation
of retinyl ester (peak 1) was not decreased, suggesting that LRAT was not inhibited by 4-CF3-
PBN. Some amounts of all-trans-retinal (peak 3) and 13-cis-retinal (peak 2) were observed due
to the oxidation and thermal isomerization of all-trans-retinol (Fig 1H and 1I). Fig 1J repre-
sents a table of all the derivatives we tested for RPE65 activity assay. The fluorinated analogs
PLOS ONE | DOI:10.1371/journal.pone.0145305 December 22, 2015
6 / 17

Nitrones for Retinal Degeneration
Fig 1. PBND tested for RPE-65 enzyme inhibition efficiency. The structure of PBN and the derivatives of PBN that are used in various in vitro and in vivo
assays are shown in Fig 1A–1D. Concentration-dependent inhibition of RPE65 and generation of 11-cis-retinol in in vitro microsome assays were performed
with representative images shown in Fig 1E, 4-F-PBN; Fig 1F, 4-CF3-PBN; and Fig 1G, 4-CH3-PBN. A table of the IC50 values of the compounds tested for
their inhibition efficiency for RPE 65, (Fig 1J), show various inhibition rates for which we determined which PBN-derivatives looked the most promising to
continue our analysis.
doi:10.1371/journal.pone.0145305.g001
PLOS ONE | DOI:10.1371/journal.pone.0145305 December 22, 2015
7 / 17

Nitrones for Retinal Degeneration
4-F-PBN, 4-CF3-PBN, and 3,4-di-F-PBN were the most efficient inhibitors. 4-CH3-PBN
proved as effective as PBN.
A representative HPLC tracing of inhibition of the retinoid isomerase activity by 4-CF3-
PBN is shown in Fig 1H and 1I. Bovine RPE microsomes (20 μg) were incubated with 0.2 μM
of all-trans-[3H] retinol in the presence or absence of 4-CF3-PBN for 2 h at 37°C. The retinoids
generated were analyzed by HPLC and flow scintillation counter. Fig 1H, HPLC elution profile
without inhibitor; and Fig 1I, with 250 μM of 4-CF3-PBN. Fig 1H and 1I: Peak 1, retinyl esters;
2, 13-cis-retinal; 3, all-trans-retinal; 4, 11-cis-retinol; 5, all-trans-retinol.
Effect of systemic administration of PBNDs on rhodopsin regeneration in
rats
To determine if PBNDs delayed dark adaptation, we gave IP injections of our PBNDs (50 mg/
kg) to light-adapted rats and kept them in the light for two additional hours, after which they
were placed in total darkness for 2.5 h. Eyes were harvested under dim red light and rhodopsin
content determined spectrophotometrically. As shown in Fig 2, rhodopsin regeneration was
significantly reduced by administration of all PBNDs, compared to controls, which had 75%
recovery of fully dark-adapted values. These results demonstrate profound inhibition of rho-
dopsin recovery by systemic administration of PBN and PBNDs.
PBNDs protect retina from light stress
In previous studies, we have shown that 50 mg/kg systemic PBN administered intraperitoneally
0.5 h before light stress (2,700 lux for 6 h) provided significant (80–90%) protection of retinal
structure and function in rats [23]. Here, using similar parameters, we tested PBNDs in rats
and compared their effects with PBN and saline (vehicle). ERGs were done 7 days after light
exposure, after which eyes were harvested for histological analyses. ERG responses were signifi-
cantly reduced in vehicle-treated rats after light stress (saline LD), whereas PBNDs significantly
Fig 2. PBN and certain PBNDs inhibited rhodopsin regeneration. Rats were treated with either PBN or
certain PBNDs before 2h of light adaptation followed by 2.5h of dark adaptation after which the animals were
euthanized and retinas harvested for rhodopsin assay. Saline treated retina recovered ~ 75% rhodopsin in
2.5h dark. PBN and its derivatives, however, blocked rhodopsin regeneration significantly. LA, light adapted;
DA, dark adapted. [* = p<0.01; ** = p<0.001; n = 4–6]
doi:10.1371/journal.pone.0145305.g002
PLOS ONE | DOI:10.1371/journal.pone.0145305 December 22, 2015
8 / 17

Nitrones for Retinal Degeneration
reduced the loss of ERG responses. All PBNDs were comparable to or better than PBN in pre-
venting ERG losses (Fig 3A and 3B).
The findings obtained by functional ERG analysis were confirmed by quantitative morphom-
etry of the thickness of the ONL of the retina. By 7 days, the dead photoreceptors were removed
and the remaining nuclei in the ONL represent the viable photoreceptors. The spidergram in
Fig 4A shows a thickness map of the retina in cross section through the vertical meridian. The
light stress paradigm caused significant loss of photoreceptor cells, which was more pronounced
in superior central retina. Pre-treatment with systemic PBN or PBNDs resulted in retention of
most of the photoreceptor cells (Fig 4A). The average ONL thickness in the superior and inferior
retina is shown in Fig 4B. PBN and all PBNDs significantly protected rod photoreceptors from
light stress-induced degeneration. The protective effect of 4-F-PBN and 4-CF3-PBN were com-
parable to PBN, whereas the effect of 4-Me-PBN was slightly lower than that found for PBN
(Fig 4A and 4B). Representative histological images of the retina are shown in (S1 Fig).
Topical administration of PBNDs inhibits rhodopsin regeneration
To determine if any of the PBNDs can reach the retina following topical application (specifi-
cally to the RPE-choroid) and inhibit rhodopsin regeneration, we designed eye-drop formula-
tions with six PBN derivatives as presented in Fig 1. Based on their IC50 values, we predicted
that compounds with lower IC50 values would inhibit RPE65 enzyme activity to a greater
extent, which would be evidenced by lower levels of rhodopsin regeneration after photobleach-
ing. We applied 3 topical drops over a period of 6h in completely light-adapted mice with
bleached rhodopsin (0% expected level). After the last eye-drop the mice were moved to dark
for 1h to allow rhodopsin regeneration. One hour dark adaptation allowed 75–82% recovery of
bleachable rhodopsin in untreated and vehicle-treated mice. A 10% PBN eye-drop inhibited
rhodopsin regeneration significantly, whereas 1% PBN showed little inhibition (Fig 5). Pres-
ence of PBN in the RPE-choroid tissue was determined by mass spectrometry (S2 Fig). In addi-
tion, three other derivatives, 4-CF3-PBN, 3,4-di-F-PBN, and 4-CH3-PBN at 5% concentration
also showed an inhibiting effect on rhodopsin regeneration with the greatest inhibition by
4-CH3-PBN (Fig 5).
Fig 3. Systemically administered PBN and PBND protect rat retina from light damage. Electroretinographic (ERG) responses were recorded for a
series of flash stimuli intensities at 0.04, 4, 200, 400, and 2000 cd.sec/m2. ERG responses presented here are from the intensities of 4 and 400 cd.sec/m2.
The dim flash (4 cd.sec/m2) stimulates the rod photoreceptor cells and the bright flash (400 cd.sec/m2) stimulates both rod and cone photoreceptors.
Therefore, the blue bars represent only rod responses and the red bars represent mixed response from both rod and cone photoreceptors. Fig 3A shows A-
wave responses, and Fig 3B shows B-wave responses. NLD represents non-light-damaged group and LD represents light-damaged groups. [## = p<0.001:
NLD vs. LD saline-treated; * = p<0.01 and ** = p<0.001: LD saline vs. PBNDs; n = 4–6]
doi:10.1371/journal.pone.0145305.g003
PLOS ONE | DOI:10.1371/journal.pone.0145305 December 22, 2015
9 / 17

Nitrones for Retinal Degeneration
Fig 4. Histological analysis of rat retina confirms protection from light damage. Vehicle or Saline treated (Vehicle LD) rats lost most of their
photoreceptors from the central retina. Effect is more pronounced in the superior retina. NLD is non-light-damage control. Fig 4A shows the ONL thickness
across the retina in the vertical meridian. PBN treatment (PBN LD) retinas retained ~90% of photoreceptor cells. 4-CF3-PBN is comparable to PBN in
protection, whereas 4-F-PBN appears better than PBN. 4-CH3-PBN also protects the retina significantly, although its effect is slightly less than PBN. Fig 4B
shows central retinal outer retinal layer (ONL) thickness in both inferior and superior retina. [** = p<0.001; n = 8–10]
doi:10.1371/journal.pone.0145305.g004
PLOS ONE | DOI:10.1371/journal.pone.0145305 December 22, 2015
10 / 17

Nitrones for Retinal Degeneration
Fig 5. Mice treated topically with PBN or PBND’s showed reduction in rhodopsin regeneration. Dark adapted no treatment (DA-NT), no treatment light-
adapted followed by dark adapted (NT LA-DA), and vehicle controls were used. Both 1% and 10% PBN were tested against 5% concentrations of PBNDs.
Topical administration of assigned drug consisted of 1 drop in each eye once every 3 hours over a 6 hour period resulting in 3 total applications in light
adapted mice eyes. Immediately following the 3rd and final dose mice were placed in the dark for 1h before harvest. LA, light adapted; DA, dark adapted. [* =
p<0.01; ** = p<0.001; n = 6]
doi:10.1371/journal.pone.0145305.g005
Topical administration of 10% PBN vs 5% 4-CH3-PBN in baboon eyes
With 4-CH3-PBN showing the greatest inhibition of rhodopsin in our mouse models, it was
selected for study in baboon eyes alongside 10% PBN. Using the same formulation used in
mice, 10% PBN and 5% 4-CH3-PBN were used for dosing anesthetized baboon eyes over a 2
hour period before harvest for rhodopsin measurement. PBN determinations in baboon eyes
clearly show that the compound reached the RPE/choroid tissue (S1 Table). Rhodopsin regen-
eration was significantly lower in the eyes treated with 10% PBN compared to controls (Fig 6).
These results support our finding in mouse eyes treated with PBN, namely that the drug reaches
the RPE and inhibits the enzymatic activity of RPE65. 5% 4-CH3-PBN exhibited a similar effect
to PBN in inhibiting rhodopsin regeneration when administered as an eye drop (Fig 6).
Discussion
The goal for developing any kind of therapy for the treatment of macular degeneration should
target the underlying cause of the disease and halt or slow the loss of vision. The accumulation
of fluorescent materials such as lipofuscin in the RPE cells of the aging retina, which is most
pronounced in the macula, induces RPE cell dysfunction and death in AMD and Stargardt’s
patients. One strategy for protecting RPE cells is to develop drugs that slow down the normal
visual cycle. The rationale for this approach is that the visual cycle produces all-trans-retinal in
PLOS ONE | DOI:10.1371/journal.pone.0145305 December 22, 2015
11 / 17

Nitrones for Retinal Degeneration
Fig 6. PBN and 4-CH3-PBN topically administered in baboon eyes slows rate of rhodopsin
regeneration. Baboon eyes were treated topically with drug or vehicle every 15 minutes over a 2h period
before eyes were harvested and dark adapted 2h before retinal harvest. 10% PBN showed a roughly 50%
higher inhibition rate compared to 5% CH3-PBN suggesting both drugs to be effective. [** = p<0.001; n = 6–8
eyes]
doi:10.1371/journal.pone.0145305.g006
the photoreceptors, which serves as one of the precursors for the biosynthesis of A2E (a pyridi-
nium bis-retinoid that is derived from two molecules of all-trans-retinal and one molecule of
ethanolamine), a key component of lipofuscin [3–5]. These toxic retinoids can generate free
radicals upon light exposure that can damage RPE cell membranes [10], inhibit RPE lysosomes
(which leads to drusen formation) [9], and activate complement factors [12], a major genetic
risk factor for AMD [31–34]. Therefore, inhibiting or slowing down the visual cycle should
reduce the accumulation of A2E in the RPE cells.
We recently showed that PBN, when delivered systemically, can substantially slow down the
visual cycle by inhibiting RPE65 [23]. In this study, we investigated the effect of several PBN
analogs in both in vitro and in vivo assays to determine their effectiveness. Since PBN is a
widely studied nitrone, we decided to explore the potential that modifications of the chemical
structure may reveal increased understanding of its interaction with the active site and perhaps
find a derivative that may have enhanced activity. Since specific PBN derivatives have been
shown to have potent activities in other pre-clinical models [35], we first chose to test: A) the
4-hydroxyl-phenyl derivative of PBN (4-OH-PBN), which has been shown to be active in pre-
venting noise-induced hearing loss [36], and B) the 2,4-disulfonyl-phenyl derivative of PBN
(DS-PBN) which is seen to be active in pre-clinical models of acute ischemic stroke [35,37] and
in models of glioma [38,39]. We found that 4-OH-PBN as well as DS-PBN had very little if any
activity in slowing down the visual cycle in our previous studies [23]. Since the 4-hydroxyl
group as well as the di-sulfonyl groups attached to the phenyl moiety of PBN represents major
perturbations to the phenyl chemical structure and to its electronic nature, we rationalized that
the intact phenyl group was very important and that other more minor chemical and electronic
density modifications to its chemical structure may turn out to be informative. Working with a
custom synthesis company we had them synthesize various fluorine-derivatives and methyl
PLOS ONE | DOI:10.1371/journal.pone.0145305 December 22, 2015
12 / 17

Nitrones for Retinal Degeneration
derivatives of the phenyl group of PBN and tested their activity in both in vitro and in vivo
assays to determine their effectiveness in slowing the visual cycle by inhibiting RPE65 retinoid
isomerase activity (Fig 1).
In vitro assays evaluated the inhibitory effect of PBN analogs on RPE65 retinoid isomerase
in microsomal preparation of bovine RPE cells, whereas in vivo assays were carried out in rats,
mice and baboons. The rat studies involved delivery of the compounds intraperitoneally,
whereas the mouse and baboon in vivo studies were carried out by topical application of some
selected compounds in the form of eye drops. In all in vivo studies, the activity of RPE65 was
measured indirectly by rhodopsin regeneration. Inhibition of RPE65 will inhibit the formation
of 11-cis-retinal in the RPE cells, which will lower its availability for regeneration of rhodopsin
in rod photoreceptors. After treatment with PBN and PBNDs in the light (with fully bleached
rhodopsin), the mice or rats were moved to the dark room for a specified time period to recover
bleachable rhodopsin. Baboon eyes, on the other hand, were harvested in the light and wrapped
in aluminum foil and stored at 4°C for 2 hours to allow recovery of rhodopsin, similar to what
is done with porcine or bovine eyes obtained at an abattoir. In all animals tested in this study,
PBN and PBNDs reduced the recovery of photobleachable rhodopsin when applied either by
IP injection or topically.
The chemical structures of the PBN analogs that inhibited RPE65 activity similar to or more
than PBN are shown in Fig 1. The fluorinated analogs 4-F-PBN, 4-CF3-PBN, and 3,4-di-
F-PBN were the most efficient inhibitors. These three analogs were more effective at inhibiting
RPE65 activity than the native PBN molecule. The IC50 values of 4-CH3-PBN were similar to
PBN (Fig 1). Therefore, in search of more effective nitrones to inhibit RPE65, we found a few
fluorinated derivatives that were superior to PBN. In vivo studies involving these derivatives,
however, required tests concerning their delivery to determine bioavailability in the target tis-
sue and inhibition of RPE65 in the eye.
In our previous studies, we tested PBN by systemic administration intraperitoneally [23].
Systemic PBN can inhibit rhodopsin regeneration and protect retina from light-induced dam-
age. We therefore tested the new derivatives systemically for their effect on these parameters.
Interestingly, the PBN analogs that were effective in inhibiting RPE65 in vitro were also effec-
tive in vivo (Figs 1, 2, 5, and 6). IP injections of these compounds with similar doses of PBN
also prevented intense light-induced damage of the retina (Figs 3 and 4). We thus identified
and verified three new compounds that can inhibit RPE65 in vivo, slow-down rhodopsin
regeneration and, if administered prior to intense light exposure, can prevent retina from
photo-damage. This could prove significant in terms of drug discovery; these compounds can
be formulated for human use in cases where slowing rhodopsin regeneration would have thera-
peutic benefits.
Regeneration of rhodopsin during light exposure is essential for intense light-mediated
damage of photoreceptor cells [40–42]. Regeneration of rhodopsin depends on the availability
of 11-cis-retinal. Accordingly, vitamin A-deficient rats, rhodopsin knock-out (KO) mice that
lack the opsin apoprotein, and RPE-65 KO mice that have opsin apoprotein but lack the ability
to generate 11-cis-retinal are all protected against light damage [40,41,43–45]. RPE65 is one of
the key enzymes of the visual cycle. It is the only retinoid isomerase in RPE cells and it catalyzes
the rate-limiting step of the visual cycle converting all-trans-retinyl esters to 11-cis-retinol.
Inhibition of RPE65 with a competitive inhibitor such as 13-cis-retinoic acid or retinylamine
provides efficient protection of the retina from light induced damage [46–48]. RPE65 is there-
fore a potential target for slowing down the visual cycle. One of the strategies for treating blind-
ing eye diseases that accumulate excessive undigested materials, including visual cycle
byproducts in the RPE cells, is to slow the rod visual cycle, which is not required for vision in
ambient light and can potentially reduce the accumulation of the byproducts such as the retinal
PLOS ONE | DOI:10.1371/journal.pone.0145305 December 22, 2015
13 / 17

Nitrones for Retinal Degeneration
fluorophore A2E, a component of lipofuscin [49]. We previously showed that the amount and
dosing schedule of PBN we used in rats affected only rod function [23]. Our therapeutic goal is
to slow the visual cycle during the daylight hours when rod vision is not needed. Because of the
short half-life (~2 h) of PBN in the rat retina [50], we anticipate that dosing only during the
day would allow sufficient time for PBN to be cleared before rod function would be needed,
thus avoiding the possibility of chronic night blindness. Also, we do not need to inhibit ‘rod
visual cycle’ completely; a dose of day time nitrones that inhibits 30–40% RPE65 could be
proved effective in the long term in reducing accumulation of A2E. Therefore, the PBN deriva-
tives we tested herein could potentially be developed as therapeutics for those retinal degenera-
tions in which accumulation of A2E leads to pathogenesis.
Our ultimate goal is to develop a human drug for Stargardt’s Disease and dry-AMD thera-
pies. The method of delivery is thus quite salient. A topical application will have significant
advantages over systemic (oral) or intravitreal delivery due to a reduced systemic effect and/or
ease of application for a prolonged period of time. We therefore tested topical application of
the PBN derivatives in mice to determine their bioavailability and effect on RPE65, which was
measured by rhodopsin regeneration. We found that 4-CH3-PBN was the most effective
through topical application followed by 3,4-di-F-PBN. PBN and 4-CH3-PBN were further
tested in larger mammal (baboon) eyes and were found to reach the RPE and effectively reduce
rhodopsin regeneration.
In summary, we have identified novel nitrones that can inhibit RPE65 enzyme activity in
vitro and in vivo. Some of these nitrones can be delivered topically in mice and baboons, which
suggests a greater opportunity for developing therapeutic drugs for human retinal degenera-
tions based on molecular nitrones.
Supporting Information
S1 Fig. Representative images of H&E sections of rat eyes treated with PBNDs and light
damaged. After ERG recordings, eyes we enucleated for histology, marked for orientation, and
fixed. Five-μm sections were cut along the vertical meridian through the optic nerve and
stained with H&E. Representative sections from each treatment were imaged from the supe-
rior-central retina, which is specifically affected by exposure to damaging light. A, no light
damage control (NLD); B, no treatment light damage control (LD); C, PBN treated before light
damage; D, 4-F-PBN treated before light damage; E, 4-CF3-PBN treated before light damage; F,
4-CH3-PBN treated before light damage. Two headed arrows showing the thickness of the reti-
nal outer nuclear layer (ONL) that represents the photoreceptor cells. RPE, retinal pigment epi-
thelium; PR, photoreceptors; OPL, outer plexiform layer; INL, inner nuclear layer; IPL, inner
plexiform layer; and GCL, ganglion cell layer. The scale bar = 50 μm.
(PDF)
S2 Fig. MS-MS analysis using nanospray direct infusion (Advion TriVersa Nanomate) with
a Thermo Scientific TSQ Ultra triple quadrupole mass spectrometer.
(PDF)
S1 Table. Determination of PBN present in the RPE-choroids of baboon. Along with the
retinal tissues for rhodopsin assay as described in the methods section, pieces of RPE-Choroid
tissue were collected for determination of PBN by mass spectrometry. The amount of PBN was
determined by MS-MS analysis as described in S2 Fig. Concentrations were calculated using
XCalibur software (Thermo Scientific). PBN determinations in two baboon eyes clearly show
that the compound reached the RPE/choroid complex. These results support our finding in
mouse eyes treated with PBN, namely that the drug reaches the RPE/choroid complex and
PLOS ONE | DOI:10.1371/journal.pone.0145305 December 22, 2015
14 / 17

Nitrones for Retinal Degeneration
inhibits the enzymatic activity of RPE65.
(PDF)
Acknowledgments
We are thankful to Dr. Michael Elliott (University of Oklahoma Health Sciences Center, Okla-
homa City, OK) for thoughtful discussion; Mark Dittmar and his team (Dean A. McGee Eye
Institute, Oklahoma City, OK) for assistance with animal breeding and feeding; and Linda S.
Boone (Dean A. McGee Eye Institute, Oklahoma City, OK) for assistance in histology.
Author Contributions
Conceived and designed the experiments: NAM REA GPM. Performed the experiments: MS
AL MLB RSB GPM HQ. Analyzed the data: NAM GPM. Contributed reagents/materials/anal-
ysis tools: RF REA JM GLW RFW. Wrote the paper: MS MLB NAM REA RSB.
References
1. Friedman DS, O'Colmain BJ, Munoz B, Tomany SC, McCarty C, de Jong PT, et al. Prevalence of age-
related macular degeneration in the United States. Arch Ophthalmol. 2004; 122: 564–572. PMID:
15078675
2. Nguyen DH, Luo J, Zhang K, Zhang M. Current therapeutic approaches in neovascular age-related
macular degeneration. Discov Med 2013; 15: 343–348. PMID: 23819948
3. Ben-Shabat S, Parish CA, Vollmer HR, Itagaki Y, Fishkin N, Nakanishi K, et al. Biosynthetic studies of
A2E, a major fluorophore of retinal pigment epithelial lipofuscin. J Biol Chem 2002; 277: 7183–7190.
PMID: 11756445
4. Liu J, Itagaki Y, Ben-Shabat S, Nakanishi K, Sparrow JR. The biosynthesis of A2E, a fluorophore of
aging retina, involves the formation of the precursor, A2-PE, in the photoreceptor outer segment mem-
brane. J Biol Chem 2000; 275: 29354–29360. PMID: 10887199
5. Sparrow JR, Nakanishi K, Parish CA. The lipofuscin fluorophore A2E mediates blue light-induced dam-
age to retinal pigmented epithelial cells. Invest Ophthalmol Vis Sci 2000; 41: 1981–1989. PMID:
10845625
6. Maeda A, Maeda T, Golczak M, Palczewski K. Retinopathy in mice induced by disrupted all-trans-reti-
nal clearance. J Biol Chem 2008; 283: 26684–26693. doi: 10.1074/jbc.M804505200 PMID: 18658157
7. Maeda A, Maeda T, Golczak M, Chou S, Desai A, Hoppel CL, et al. Involvement of all-trans-retinal in
acute light-induced retinopathy of mice. J Biol Chem 2009; 284: 15173–15183. doi: 10.1074/jbc.
M900322200 PMID: 19304658
8. Palczewski K. Retinoids for treatment of retinal diseases. Trends Pharmacol Sci 2010; 31: 284–295.
doi: 10.1016/j.tips.2010.03.001 PMID: 20435355
9. Sparrow JR, Boulton M. RPE lipofuscin and its role in retinal pathobiology. Exp Eye Res 2005; 80:
595–606. PMID: 15862166
10. Suter M, Reme C, Grimm C, Wenzel A, Jaattela M, Esser P, et al. Age-related macular degeneration.
The lipofusion component N-retinyl-N-retinylidene ethanolamine detaches proapoptotic proteins from
mitochondria and induces apoptosis in mammalian retinal pigment epithelial cells. J Biol Chem 2000;
275: 39625–39630. PMID: 11006290
11. Wu Y, Yanase E, Feng X, Siegel MM, Sparrow JR. Structural characterization of bisretinoid A2E photo-
cleavage products and implications for age-related macular degeneration. Proc Natl Acad Sci U S A
2010; 107: 7275–7280. doi: 10.1073/pnas.0913112107 PMID: 20368460
12. Zhou J, Jang YP, Kim SR, Sparrow JR. Complement activation by photooxidation products of A2E, a
lipofuscin constituent of the retinal pigment epithelium. Proc Natl Acad Sci U S A 2006; 103: 16182–
16187. PMID: 17060630
13. Jin M, Li S, Moghrabi WN, Sun H, Travis GH. Rpe65 is the retinoid isomerase in bovine retinal pigment
epithelium. Cell 2005; 122: 449–459. PMID: 16096063
14. Moiseyev G, Chen Y, Takahashi Y, Wu BX, Ma JX. RPE65 is the isomerohydrolase in the retinoid
visual cycle. Proc Natl Acad Sci U S A 2005; 102: 12413–12418. PMID: 16116091
PLOS ONE | DOI:10.1371/journal.pone.0145305 December 22, 2015
15 / 17

Nitrones for Retinal Degeneration
15. Redmond TM, Poliakov E, Yu S, Tsai JY, Lu Z, Gentleman S. Mutation of key residues of RPE65 abol-
ishes its enzymatic role as isomerohydrolase in the visual cycle. Proc Natl Acad Sci U S A 2005; 102:
13658–13663. PMID: 16150724
16. Gu SM, Thompson DA, Srikumari CR, Lorenz B, Finckh U, Nicoletti S, et al. Mutations in RPE65 cause
autosomal recessive childhood-onset severe retinal dystrophy. Nat Genet 1997; 17: 194–197. PMID:
9326941
17. Lorenz B, Gyurus P, Preising M, Bremser D, Gu S, Andrassi M, et al. Early-onset severe rod-cone dys-
trophy in young children with RPE65 mutations. Invest Ophthalmol Vis Sci 2000; 41: 2735–2742.
PMID: 10937591
18. Marlhens F, Bareil C, Griffoin JM, Zrenner E, Amalric P, Eliaou C, et al. Mutations in RPE65 cause
Leber's congenital amaurosis. Nat Genet 1997; 17: 139–141. PMID: 9326927
19. Morimura H, Fishman GA, Grover SA, Fulton AB, Berson EL, Dryja TP. Mutations in the RPE65 gene in
patients with autosomal recessive retinitis pigmentosa or leber congenital amaurosis. Proc Natl Acad
Sci U S A 1998; 95: 3088–3093. PMID: 9501220
20. Thompson DA, Gyurus P, Fleischer LL, Bingham EL, McHenry CL, Apfelstedt-Sylla E, et al. Genetics
and phenotypes of RPE65 mutations in inherited retinal degeneration. Invest Ophthalmol Vis Sci 2000;
41: 4293–4299. PMID: 11095629
21. Ranchon I, LaVail MM, Kotake Y, Anderson RE. Free radical trap phenyl-N-tert-butylnitrone protects
against light damage but does not rescue P23H and S334ter rhodopsin transgenic rats from inherited
retinal degeneration. J Neurosci 2003; 23: 6050–6057. PMID: 12853423
22. Poliakov E, Parikh T, Ayele M, Kuo S, Chander P, Gentleman S, et al. Aromatic lipophilic spin traps
effectively inhibit RPE65 retinol isomerohydrolase activity. Biochemistry 2011; 50: 6739–6741. doi: 10.
1021/bi200532m PMID: 21736383
23. Mandal MN, Moiseyev GP, Elliott MH, Kasus-Jacobi A, Li X, Chen H, et al. Alpha-phenyl-N-tert-butylni-
trone (PBN) prevents light-induced degeneration of the retina by inhibiting RPE65 protein isomerohy-
drolase activity. J Biol Chem 2011; 286: 32491–32501. doi: 10.1074/jbc.M111.255877 PMID:
21785167
24. Moiseyev G, Crouch RK, Goletz P, Oatis J Jr., Redmond TM, Ma JX. Retinyl esters are the substrate
for isomerohydrolase. Biochemistry 2003; 42: 2229–2238. PMID: 12590612
25. Papermaster DS, Dreyer WJ. Rhodopsin content in the outer segment membranes of bovine and frog
retinal rods. Biochemistry 1974; 13: 2438–2444. PMID: 4545509
26. Chen H, Tran JT, Eckerd A, Huynh TP, Elliott MH, Brush RS, et al. Inhibition of de novo ceramide bio-
synthesis by FTY720 protects rat retina from light-induced degeneration. J Lipid Res. 2013; 54: 1616–
1629. doi: 10.1194/jlr.M035048 PMID: 23468130
27. Tanito M, Brush RS, Elliott MH, Wicker LD, Henry KR, Anderson RE. High levels of retinal membrane
docosahexaenoic acid increase susceptibility to stress-induced degeneration. J Lipid Res. 2009; 50:
807–819. doi: 10.1194/jlr.M800170-JLR200 PMID: 19023138
28. Irreverre F, Stone AL, Shichi H, Lewis MS. Biochemistry of visual pigments. I. Purification and proper-
ties of bovine rhodopsin. J Biol Chem. 1969; 244: 529–536. PMID: 5768853
29. Chen H, Tran JT, Anderson RE, Mandal MN. Caffeic acid phenethyl ester protects 661W cells from
H2O2-mediated cell death and enhances electroretinography response in dim-reared albino rats. Mol
Vis. 2012; 18: 1325–1338. PMID: 22690111
30. Mandal MN, Patlolla JM, Zheng L, Agbaga MP, Tran JT, Wicker L, et al. Curcumin protects retinal cells
from light-and oxidant stress-induced cell death. Free Radic Biol Med. 2009; 46: 672–679. doi: 10.
1016/j.freeradbiomed.2008.12.006 PMID: 19121385
31. Edwards AO, Ritter R 3rd, Abel KJ, Manning A, Panhuysen C, Farrer LA. Complement factor H poly-
morphism and age-related macular degeneration. Science 2005: 308: 421–424. PMID: 15761121
32. Hageman GS, Anderson DH, Johnson LV, Hancox LS, Taiber AJ, Hardisty LI, et al. (2005) A common
haplotype in the complement regulatory gene factor H (HF1/CFH) predisposes individuals to age-
related macular degeneration. Proc Natl Acad Sci U S A 2005; 102: 7227–7232.
33. Haines JL, Hauser MA, Schmidt S, Scott WK, Olson LM, Haynes C, et al. Complement factor H variant
increases the risk of age-related macular degeneration. Science 2005; 308: 419–421. PMID:
15761120
34. Klein RJ, Zeiss C, Chew EY, Tsai JY, Sackler RS, Haynes C, et al. Complement factor H polymorphism
in age-related macular degeneration. Science 2005; 308: 385–389. PMID: 15761122
35. Floyd RA, Kopke RD, Choi CH, Foster SB, Doblas S, Towner RA. Nitrones as therapeutics. Free Radic
Biol Med. 2008; 45: 1361–1374. doi: 10.1016/j.freeradbiomed.2008.08.017 PMID: 18793715
36. Choi CH, Chen K, Vasquez-Weldon A, Jackson RL, Floyd RA, Kopke RD. Effectiveness of 4-hydroxy
phenyl N-tert-butylnitrone (4-OHPBN) alone and in combination with other antioxidant drugs in the
PLOS ONE | DOI:10.1371/journal.pone.0145305 December 22, 2015
16 / 17

Nitrones for Retinal Degeneration
treatment of acute acoustic trauma in chinchilla. Free Radic Biol Med. 2008; 44: 1772–1784. doi: 10.
1016/j.freeradbiomed.2008.02.005 PMID: 18328271
37. Maples KR, Green AR, Floyd RA. Nitrone-related therapeutics: potential of NXY-059 for the treatment
of acute ischaemic stroke. CNS Drugs 2004; 18: 1071–1084. PMID: 15581379
38. He T, Doblas S, Saunders D, Casteel R, Lerner M, Ritchey JW, et al. Effects of PBN and OKN007 in
rodent glioma models assessed by 1H MR spectroscopy. Free Radic Biol Med. 2011; 51: 490–502.
doi: 10.1016/j.freeradbiomed.2011.04.037 PMID: 21600283
39. Towner RA, Gillespie DL, Schwager A, Saunders DG, Smith N, Njoku CE, et al. Regression of glioma
tumor growth in F98 and U87 rat glioma models by the Nitrone OKN-007. Neuro Oncol. 2013; 15: 330–
340. doi: 10.1093/neuonc/nos337 PMID: 23328810
40. Grimm C, Reme CE, Rol PO, Williams TP. Blue light's effects on rhodopsin: photoreversal of bleaching
in living rat eyes. Invest Ophthalmol Vis Sci. 2000; 41: 3984–3990. PMID: 11053303
41. Humphries MM, Rancourt D, Farrar GJ, Kenna P, Hazel M, Bush RA, et al. Retinopathy induced in
mice by targeted disruption of the rhodopsin gene. Nat Genet. 1997; 15: 216–219. PMID: 9020854
42. Noell WK, Walker VS, Kang BS, Berman S. Retinal damage by light in rats. Invest Ophthalmol. 1966;
5: 450–473. PMID: 5929286
43. Noell WK, Albrecht R. Irreversible effects on visible light on the retina: role of vitamin A. Science 1971;
172: 76–79. PMID: 5546288
44. Noell WK, Delmelle MC, Albrecht R.Vitamin A deficiency effect on retina: dependence on light. Science
1971; 172: 72–75. PMID: 5546287
45. Redmond TM, Yu S, Lee E, Bok D, Hamasaki D, Chen N, et al. Rpe65 is necessary for production of
11-cis-vitamin A in the retinal visual cycle. Nat Genet. 1998; 20: 344–351. PMID: 9843205
46. Gollapalli DR, Rando RR. The specific binding of retinoic acid to RPE65 and approaches to the treat-
ment of macular degeneration. Proc Natl Acad Sci U S A 2004; 101: 10030–10035. PMID: 15218101
47. Maeda A, Maeda T, Golczak M, Imanishi Y, Leahy P, Kubota R, et al. Effects of potent inhibitors of the
retinoid cycle on visual function and photoreceptor protection from light damage in mice. Mol Pharma-
col. 2006: 70: 1220–1229. PMID: 16837623
48. Sieving PA, Chaudhry P, Kondo M, Provenzano M, Wu D, Carlson TJ, et al. Inhibition of the visual
cycle in vivo by 13-cis retinoic acid protects from light damage and provides a mechanism for night
blindness in isotretinoin therapy. Proc Natl Acad Sci U S A 2001; 98: 1835–1840. PMID: 11172037
49. Maiti P, Kong J, Kim SR, Sparrow JR, Allikmets R, Rando RR. Small molecule RPE65 antagonists limit
the visual cycle and prevent lipofuscin formation. Biochemistry 2006; 45: 852–860. PMID: 16411761
50. Ranchon I, Chen S, Alvarez K, Anderson RE. Systemic administration of phenyl-N-tert-butylnitrone pro-
tects the retina from light damage. Invest Ophthalmol Vis Sci. 2001; 42: 1375–1379. PMID: 11328754
PLOS ONE | DOI:10.1371/journal.pone.0145305 December 22, 2015
17 / 17