Aberrant buildup of all-Trans-retinal dimer, a nonpyridinium bisretinoid lipofuscin fluorophore, contributes to the degeneration of the retinal pigment epithelium

Article abstract of DOI:10.1167/iovs.16-20734

PURPOSE. Nondegradable fluorophores that accumulate as deleterious lipofuscin of RPE are involved in pathological mechanisms leading to the degeneration of RPE in AMD. A2E, a major component of RPE lipofuscin, could cause damage to RPE cells. Nevertheless

Full text of DOI:10.1167/iovs.16-20734

Biochemistry and Molecular Biology
Aberrant Buildup of All-Trans-Retinal Dimer, a
Nonpyridinium Bisretinoid Lipofuscin Fluorophore,
Contributes to the Degeneration of the Retinal Pigment
Epithelium
Junli Zhao,1,2 Yi Liao,1 Jingmeng Chen,1 Xinran Dong,3 Zhan Gao,1 Houjian Zhang,1 Xiaodan
Wu,4 Zuguo Liu,1 and Yalin Wu1
1Fujian Provincial Key Laboratory of Ophthalmology and Visual Science, Eye Institute of Xiamen University, College of Medicine,
Xiamen University, Xiamen, China
2College of Pharmaceutical Sciences, Zhejiang University, Hangzhou, China
3Department of Ophthalmology, Guangdong Academy of Medical Science, Guangdong General Hospital, Guangzhou, China
4Center of Analysis and Measurement, Zhejiang University, Hangzhou, China
Correspondence: Yalin Wu, Fujian
Provincial Key Laboratory of Oph-
thalmology and Visual Science, Eye
Institute of Xiamen University, Col-
lege of Medicine, Xiamen University,
Xiang’an South Road, Xiamen
361102, China;
PURPOSE. Nondegradable fluorophores that accumulate as deleterious lipofuscin of RPE are
involved in pathological mechanisms leading to the degeneration of RPE in AMD. A2E, a major
component of RPE lipofuscin, could cause damage to RPE cells. Nevertheless, all-trans-retinal
dimer (atRAL dimer) was found to be much more abundant than that of A2E in eyes of
Abca4ꢀ/ꢀRdh8ꢀ/ꢀ double-knockout (DKO) mice, a rodent model showing the typical
characteristics of retinopathies in AMD patients. Our aim was to elucidate the effect and
mechanism of atRAL dimer–induced RPE degeneration.
yalinw@xmu.edu.cn.
METHODS. Eyes harvested from C57BL/6J wild-type (WT) and Abca4ꢀ/ꢀRdh8ꢀ/ꢀ DKO mice
were examined by HPLC. Cellular uptake, subcellular localization, 5-bromo-2-deoxyuridine
(BrdU), Cdc25C, DNA strand breaks, mitochondrial membrane potential (DWm), and
cytochrome c were analyzed by fluorescence microscopy. Cellular toxicity was assayed by
lactate dehydrogenase (LDH) assay and dead cell staining. Apoptosis and cell-cycle stages
were detected by flow cytometry. Furthermore, in vitro and in vivo expression of proteins
associated with cell cycle and apoptosis was measured by immunoblot assays.
Submitted: September 12, 2016
Accepted: January 11, 2017
Citation: Zhao J, Liao Y, Chen J, et al.
Aberrant buildup of all-trans-retinal
dimer, a nonpyridinium bisretinoid
lipofuscin fluorophore, contributes to
the degeneration of the retinal pig-
ment epithelium. Invest Ophthalmol
Vis Sci. 2017;58:1063–1075. DOI:
10.1167/iovs.16-20734
RESULTS. All-trans-retinal dimer clearly could damage RPE cell membrane and inhibit the
proliferation of RPE cells as well as induce DNA damage and cell-cycle arrest at the G2/M
phase via activating the ATM/ATR-Chk2-p53 signaling pathway. Moreover, this di-retinal
adduct triggered mitochondrion-associated apoptosis in RPE cells. Evidence from the cell-
based experiments was also corroborated by a remarkable abnormality in expression of
proteins associated with cell cycle (Cyclin B1 and Cdc2) and apoptosis (p53, Bcl-2 and Bax) in
the RPE of Abca4ꢀ/ꢀRdh8ꢀ/ꢀ DKO mice.
CONCLUSIONS. These findings suggest that atRAL dimer that accumulates beyond a critical level,
facilitates age-dependent RPE degeneration.
Keywords: all-trans-retinal dimer, bisretinoid lipofuscin, retinal pigment epithelium, cell
cycle, proliferation inhibition, mitochondrial dysfunction
ge-related macular degeneration (AMD) is a retinal degen-
Aerative disease that is a major cause of irreversible vision
loss in individuals older than 50 years in many developed
countries, especially in the United States.1–3 In the latest
estimates undertaken by the World Health Organization, AMD
affects more than 30 million people, and incurs legal blindness
in more than 500,000 patients with AMD annually. The etiology
of AMD is implicated with RPE and photoreceptor degenera-
tion.4 Gradual accumulation of autofluorescent lipofuscin in
the lysosomal storage bodies of the RPE is a hallmark of aging in
vertebrates.5 High levels of RPE lipofuscin have been consid-
ered to be associated with degeneration of the RPE6,7 and
concomitant disturbance in the daily phagocytosis of shed
photoreceptor outer segments by RPE cells, a critical process
for the maintenance of the retina.8 Although excess deposition
of lipofuscin pigment granules in the RPE contributes to
increased morbidity of AMD, the mechanism underlying RPE
lipofuscin-induced retinal degeneration is not fully understood
as yet. An inadvertent consequence of retinal metabolism
necessary for regeneration of visual chromophore 11-cis-retinal
is the formation of retinal-derived compounds that undergo
synthesis in photoreceptor outer segments of neural retina and
are secondarily deposited in the RPE as predominant hydro-
phobic components of toxic lipofuscin.9,10 Several lipofuscin
fluorophores have been structurally identified in vertebrate
RPE, including A2E (Fig. 1A),11 isoA2E,12 iisoA2E,13 all-trans-
retinal dimer (atRAL dimer) (Fig. 1B),14 A2-DHP-PE,15 pdA2E,16
and isopdA2E.17 In all of these pigments, the most intense and
Copyright 2017 The Authors
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FIGURE 1. Measurement of atRAL dimer and A2E in Abca4ꢀ/ꢀRdh8ꢀ/ꢀ DKO and WT mice. (A, B) A2E and atRAL dimer: structures, UV-visible
absorbance maxima (kmax), mass-to-charge ratio (m/z), and electronic transition assignments (ꢀ). (C) A representative reverse-phase HPLC
chromatogram of a hydrophobic extract of six eyes harvested from 6-month-old Abca4ꢀ/ꢀRdh8ꢀ/ꢀ DKO mice was overlaid with that of synthesized
A2E (red) and atRAL dimer (blue) as well as endogenous atRAL dimer (dotted pink) in 10 eyes from Abca4ꢀ/ꢀRdh8ꢀ/ꢀ DKO mice at 6 to 10 months
of age. Top insets: UV-visible absorbance spectra of A2E and atRAL dimer peak fractions isolated from eyes of 6-month-old Abca4ꢀ/ꢀRdh8ꢀ/ꢀ DKO
mice. Inset on the right: ESI-MS spectrum of atRAL dimer from 10 eyes of Abca4ꢀ/ꢀRdh8ꢀ/ꢀ DKO mice at 6 to 10 months of age. All-trans-retinal
dimer has a mass of 550.86. (D) All-trans-retinal dimer in C57BL/6J WT and Abca4ꢀ/ꢀRdh8ꢀ/ꢀ DKO mice at ages 2, 4, 6, and 8 months was measured
using HPLC. Integrated peak areas (mVꢁs) were calculated using Empower version 3 software, and presented as mean 6 SEM of three to four
samples. (E) All-trans-retinal dimer and A2E in mutant (Abca4ꢀ/ꢀRdh8ꢀ/ꢀ) mice at 2, 4, 6, 8, and 10 months of age were quantified by reverse-phase
HPLC. Integrated peak areas (mVꢁs) were converted into picomolar quantities by using standard calibration curves (Supplementary Fig. S1). Data
were expressed as mean 6 SEM of three samples.
extensive studies are directed to A2E due to its abundance and
typical age-dependent accumulation in humans,18 and C57BL/
6J and BALB/cByJ wild-type (WT) mice.15
of Nanjing University (Nanjing, China). Genotyping of mice and
sequencing of mutations were carried out according to a
previous report.22 Abca4ꢀ/ꢀRdh8ꢀ/ꢀ DKO mice were kindly
donated by the Krzysztof Palczewski Laboratory (Case Western
Reserve University, Cleveland, OH, USA). All these mice were
raised under 12 hour on/off cyclic lighting with an in-cage
illuminance of approximately 180 to 230 lux in the Laboratory
Animal Center of Xiamen University. Experiments with animals
were performed in accordance with protocols approved by the
Institutional Animal Care and Use Committee of Xiamen
University Medical College, and adherent to the ARVO Statement
for the Use of Animals in Ophthalmic and Vision Research.
Previous studies have indicated that Abca4ꢀ/ꢀRdh8ꢀ/ꢀ
double-knockout (DKO) mice reproduce the main features of
human AMD in a rodent model.19–21 Palczewski and coworkers
carried out HPLC quantification of atRAL dimer in eyes of
Abca4ꢀ/ꢀRdh8ꢀ/ꢀ DKO mice versus WT mice at 3 and 6
months of age, and demonstrated that the amount of atRAL
dimer in Abca4ꢀ/ꢀRdh8ꢀ/ꢀ DKO mice increased significantly
and age-dependently.19 Additionally, histological evidence
revealed that the dramatic retinal degeneration in Abca4ꢀ/ꢀ
Rdh8ꢀ/ꢀ DKO mice initiated at the age of 1.0 to 1.5 months,
and rapidly worsened with age ranging from 3.0 to 6.0
months.19 Based on these phenomena, this age-dependent
phenotype in the retina of Abca4ꢀ/ꢀRdh8ꢀ/ꢀ DKO mice is
possibly associated with the exacerbated accumulation of
atRAL dimer in the RPE. Accordingly, the objective of this study
was to elucidate the potential role of atRAL dimer in the
occurrence and development of RPE degeneration.
Eye Extracts and HPLC Analysis
Murine eyes (4–13 eyes/sample) were homogenized in a vol/
vol solution of PBS/deuterium-depleted water and 50%
methanolic chloroform, as previously reported.13 Each extract
was examined by reverse-phase HPLC using an Alliance System
(Waters Corp., Milford, MA, USA) equipped with a 2695
separation module, a 2998 photodiode array detector, and a
2475 multichannel (k) fluorescence detector. For compound
elution, an analytical scale Atlantis (Milford, MA, USA) dC18 (3
lm, 4.6 3 150 mm) column was used with a gradient mobile
phase consisting of acetonitrile and water in the presence of
0.1% trifluoroacetic acid: 75% to 90% acetonitrile (0–30
minutes), 90% to 100% acetonitrile (30–40 minutes), and
100% acetonitrile (40–100 minutes), with a flow rate of 0.5
mL/min. Photodiode array detection was monitored at 430 nm.
MATERIALS AND METHODS
Animals
C57BL/6J WT mice were purchased from Shanghai SLAC
Laboratory Animal Co. Ltd. (Shanghai, China), and C57BL/6N
mice were purchased from the Model Animal Research Center
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Peak area (lVꢁs; 1 mVꢁs ¼ 1000 lVꢁs) was integrated using
Empower version 3 software (Waters Corp.). Molar quantity
per eye was calculated using calibration curves constructed
from known concentrations and integrated HPLC peak areas of
synthesized standards (Supplementary Fig. S1). Compounds in
the extract of 10 eyes from Abca4ꢀ/ꢀRdh8ꢀ/ꢀ DKO mice at 6 to
10 months of age were ionized by electrospray ionization (ESI)
operated in a positive ion mode and analyzed by using an API
3200 Q-Trap LC/MS/MS instrument supplied by Applied
Biosystems (Foster City, CA, USA). The same dC18 column
and mobile phase was used as described above for HPLC.
103 cells/well) were incubated with atRAL dimer (0–80 lM).
Lactate dehydrogenase assay was performed by using an LDH
Cytotoxicity Assay Kit (Beyotime, Haimen, China). Cytoplasmic
LDH was measured in culture medium after 2- and 5-day
incubations of atRAL dimer with cells in fresh serum-free
medium by using the microplate reader at 490 nm absorbance.
After 3-day treatment, the nuclei of membrane-permeabilized
cells were also labeled with 5 nM Dead Red (Molecular Probes,
Invitrogen), and nuclei of all cells were stained by DAPI
(1:1000 dilution; Beyotime).
Apoptosis Detection by Flow Cytometry
Synthesis
Differentiation between apoptotic versus necrotic types of cell
death was studied by Annexin V and propidium iodide (PI)
staining using the apoptosis detection kit (MultiSciences,
Hangzhou, China). Briefly, after ARPE-19 cells were treated
with atRAL dimer (0, 20, 40, and 80 lM) for 3 days, cells were
harvested, washed in cold PBS, and then pelleted. Cells were
resuspended in 500 lL binding buffer, and stained with 5 lL
Annexin V-FITC (fluorescein isothiocyanate) and 10 lL PI in
the dark for 5 minutes at room temperature, followed by flow
cytometry analysis using a Cytomics FC500 flow cytometer
(Beckman Coulter, Brea, CA, USA).
All-trans-retinal dimer was synthesized in vitro as described
previously,14 with minor modifications. Briefly, a chemical
reaction mixture of atRAL (50 mg, 0.176 mM) and NaH (4.23
mg, 0.176 mM) in 3 mL anhydrous tetrahydrofuran (THF) was
stirred for 3 hours at room temperature in the dark, and
terminated by slow addition of a saturated ammonium chloride
(NH4Cl) solution. The resulting mixture was extracted with
diethyl ether (Et2O), and dried using saturated sodium chloride
and anhydrous sodium sulphate. After the solvents were
concentrated to dry with a rotary evaporator, the silica gel
column chromatography with an eluent of hexane/methylene
chloride (4:1, vol/vol) afforded approximately 12 mg of atRAL
dimer in pure form. The A2E was synthesized as published.12
The identity and purity of synthesized A2E and atRAL dimer
were corroborated by proton nuclear magnetic resonance
spectroscopy, UV-visible absorbance, and reverse-phase HPLC
with monitoring at 430 nm (Supplementary Figs. S2–S4).
Single-Cell Gel Electrophoresis Assay
DNA strand breaks were probed using alkaline single-cell gel
electrophoresis (comet assay). To prevent additional DNA
damage, all processes were conducted under dim red light.
Three days after exposure to atRAL dimer (0, 20, 40, and 80
lM), ARPE-19 cells were harvested, embedded in 0.5% agarose
gel at a density of 1 3 106 cells/mL, and spread onto the fully
frosted slides. Slides were immersed in ice-cold lysis solution at
pH 10 and kept at 48C for 3 hours; the lysis solution consisted
of 2.5 M NaCl, 100 mM Na2 EDTA, 10 mM Trizma base, 1%
Triton X-100, and 10% DMSO. Then cellular DNA was allowed
to unwind in the electrophoresis buffer, including 300 mM
NaOH and 1 mM Na2EDTA (pH > 13) for 20 minutes, and
electrophoresed for 20 minutes at a constant voltage of 25 V
and 300 mA at 48C. Next, the slides were neutralized with 0.4
M Tris-HCl (pH 7.5), dehydrated in ice-cold ethanol, and left to
air dry. Samples were stained with 20 lg/mL ethidium bromide
(EB) and then examined under a fluorescence inverted
microscope (Axio Observer A1; Zeiss, Heidelberg, Germany).
Fifty to 100 randomly selected cells per treatment were scored
from three independent experiments and measured for the
length of DNA comet tails.
Cell Culture and Treatments
A human RPE cell line (ARPE-19), purchased from FuDan IBS
Cell Center (Shanghai, China), was grown in Dulbecco’s
modified Eagle medium (DMEM) (HyClone, Logan, UT, USA)
supplemented with 10% heat-inactivated fetal bovine serum
(HyClone), penicillin (100 units/mL), and streptomycin (100
lg/mL) in a humidified incubator with 5% CO2 at 378C. A stock
solution of atRAL dimer (40 mM) was prepared with dimethyl
sulfoxide (DMSO), and ARPE-19 cells were incubated with
serial dilutions of atRAL dimer (0, 2.5, 5, 10, 20, 40, and 80 lM)
for 1 to 3 days.
Cellular Uptake and Subcellular Localization
ARPE-19 cells were incubated with atRAL dimer at indicated
concentrations for 3 days in 24-well cell culture plates. Uptake
of atRAL dimer by cells was detected and imaged using a laser
scanning confocal microscope (LSM510 Meta; Carl Zeiss,
Heidelberg, Germany) with an argon-krypton laser (atRAL
dimer, Ex 488 nm and Em 530 nm; 40,6-diamidino-2-phenyl-
indole [DAPI], Ex 405 nm and Em 426–489 nm). To determine
where atRAL dimer localized within RPE cells, cells grown in
24-well cell culture plates were simultaneously incubated with
atRAL dimer (20 lM) and LysoTracker Red DND-99 (50 nM;
Molecular Probes, Invitrogen, Waltham, MA, USA) in culture
medium for 45 minutes. Cells were washed in fresh and
prewarmed PBS, and fixed with 4% paraformaldehyde for 15
minutes at 48C in the dark. After rinsing the cells with PBS,
photographs were taken by the confocal microscope.
Measurement of Mitochondrial Membrane
Potential
Determination of the mitochondrial inner transmembrane
potential (DWm) was carried out by a mitochondrial mem-
brane potential assay kit with JC-1 (Beyotime). Briefly, after
treating ARPE-19 cells with atRAL dimer (0, 20, 40, and 80 lM)
for 3 days, 105 cells were harvested, resuspended in 500 lL
DMEM medium, and stained with 500 lL JC-1 at 378C for 20
minutes. After being washed using ice-cold staining buffer
twice, cells were finally suspended in 500 lL staining buffer,
and analyzed by the flow cytometer. Changes in MMP also were
evaluated in situ by an inverted fluorescence microscope
(BX61W1-FV1000; Olympus, Tokyo, Japan). For imaging of JC-1
monomers, the wavelengths were set at 490 nm for excitation
and 530 nm for emission, and for JC-1 aggregates, the
wavelengths were set at 525 nm and 590 nm for excitation
and emission, respectively.
Cellular Toxicity Tests
Cellular toxicity was assessed by lactate dehydrogenase (LDH)
assay and dead cell staining. Cells seeded in 96-well plates (6 3
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FIGURE 2. Detection of internalization and subcellular localization of atRAL dimer by confocal laser scanning fluorescence microscopy. (A) Retinal
pigment epithelial cells were untreated or incubated with atRAL dimer (5, 10, 20, 40, and 80 lM) for 3 days. Then cells were fixed, and nuclei were
stained with DAPI (blue). Internalized atRAL dimer granules exhibit green autofluorescence at excitation/emission of 488/505 to 550 nm, and
perinuclear distribution of this adduct is evident. All-trans-retinal dimer granules that fluoresce green are augmented with increasing of atRAL dimer
concentrations. Scale bars: 20 lm. (B) Colocalization of atRAL dimer with LysoTracker Red (Molecular Probes, Invitrogen). Microscopic
autofluorescence signals at the left and in the middle are superimposed on the right; particles that colocalize in the left and middle appear
yellowish orange on the right when green is combined with red.
(1:100 dilution; Jackson ImmunoResearch, West Grove, PA,
USA) for 2 hours at room temperature. After extensive washes
with 20 mM Tris-HCl (pH ¼ 7.5), 137 mM NaCl, 0.1% Tween-20
(TTBS) and mounting by glycerol, these slides were examined
by the confocal microscope.
Detection of Cell-Cycle Stages
Human RPE cells (ARPE-19) were treated with atRAL dimer (0,
20, 40, and 80 lM) for 3 days before testing the distribution of
cell-cycle stages. Control and treated cells were collected into
polypropylene tubes and pelleted at 2000 rpm for 5 minutes.
Cells were then washed with precooling PBS twice and fixed
with chilled 70% ethanol overnight at 48C. Fixed cells were
washed with PBS, and incubated in 0.5 mL PBS containing 50
lg RNase A, 25 lg PI, and 0.2% Triton X-100 at 48C for 30
minutes in the dark. The DNA content was determined by the
flow cytometer, and the percentage of cells at G0/G1, S, and
G2/M phases was calculated using the MultiCycle software
(Beckman Coulter).
Immunofluorescence Staining of Cdc25C
Human RPE cells (ARPE-19) seeded in 96-well culture plates
were incubated with 0, 20, 40, and 80 lM atRAL dimer for 2
days. Cells were fixed with 4% paraformaldehyde for 20
minutes, and permeabilized with 0.5% Triton X-100 for 20
minutes. Human RPE cells (ARPE-19) that underwent pretreat-
ment were incubated with immunol staining blocking buffer to
block nonspecific staining for 30 minutes, followed by
incubation with anti-Cdc25C antibody (1:200; Cell Signaling
Technology) at 48C overnight. After three washes with PBS, the
resulting cells were incubated with Alexa Fluor 594-conjugated
AffiniPure Donkey Anti-Rabbit IgG (HþL) for 2 hours at 378C,
counterstained with DAPI, and then analyzed with the
fluorescence microscope.
Immunofluorescence Staining of Cytochrome c
Human RPE cells (ARPE-19) grown on glass cover slips were
treated with atRAL dimer (0, 20, 40, and 80 lM) for 3 days, and
subsequently incubated with 1 lM MitoTracker Red CM-
H2Xros at 378C for 45 minutes. Cells were fixed with 4%
paraformaldehyde at 48C for 15 minutes, permeabilized with
0.5% Triton X-100 for 20 minutes, and then blocked with
immunol staining blocking buffer (Beyotime) for 40 minutes.
Afterward, cells were exposed to a monoclonal anti–Cyto-
chrome c antibody (1:200 dilution; Cell Signaling Technology,
Danvers, MA, USA) overnight at 48C, followed by incubation
with FITC-labeled Donkey anti-Rabbit secondary antibody
Staining With 5-Bromo-2-Deoxyuridine
For 5-bromo-2-deoxyuridine (BrdU) (Sigma-Aldrich Corp., St.
Louis, MO, USA) staining, 30 lM BrdU was added to the culture
for the final 24 hours before fixation of ARPE-19 cells. Fixed
cells were permeabilized with 1% Triton X-100, and denatured
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FIGURE 3. Cytotoxicity and proliferation inhibition induced by atRAL dimer in RPE cells. (A, B) Lactate dehydrogenase release from cultured RPE
cells receiving atRAL dimer. Cells were incubated with atRAL dimer (0, 5, 10, 20, 40, and 80 lM) for 2 (A) and 5 (B) days in fresh serum-free
medium. ***P < 0.001, significantly different compared with control. (C) Fluorescence labeling of the nuclei of membrane-compromised cells.
Cultured RPE cells were untreated or exposed to 20, 40, and 80 lM atRAL dimer for 3 days. Microscopic visualization of nonviable cells was
accomplished by labeling the nuclei of nonviable cells with Dead Red (Molecular Probes, Invitrogen) and nuclei of all cells with DAPI (blue). (D)
Typical fluorescence photomicrographs of RPE cells stained with BrdU: BrdU, 24 hours after treatment of RPE cells with atRAL dimer (0, 20, 40, and
80 lM), was added to the culture, and the incubation was continued for another 24 hours. Proliferating cells were visualized by BrdU
immunofluorescence and total cells were stained with DAPI. (E) A statistical chart exhibited the percentage of BrdU (þ) cells in DAPI (þ) cells.
Values were based on counts performed in triplicate wells in each of two independent experiments, with approximately three to five fields counted
per well, and were presented as mean 6 SEM. ***P < 0.001.
by 2 N HCl for 30 minutes at 378C. Then the samples were
neutralized by 0.1 M borate buffer for 10 minutes and fixed
once again. Following incubation with immunol staining
blocking buffer to block nonspecific staining for 30 minutes,
cells were treated with anti-BrdU antibody (1:100; Santa Cruz
Biotechnology Co., Ltd., Shanghai, China) at 48C overnight.
After three washes with PBS, cells were incubated with Alexa
Fluor 594-conjugated AffiniPure Donkey Anti-Mouse IgG (HþL)
(1:100 dilution; Jackson ImmunoResearch Laboratories) for 2
hours at 378C. Finally, cells were counterstained with DAPI and
analyzed with the Leica DMI3000B fluorescence microscope
(Leica, Wetzlar, Hessen, Germany).
cells were lysed in ice-cold lysis buffer (RIPA Lysis Buffer;
Beyotime) for 5 minutes and centrifuged at 13,000g for 5
minutes at 48C. Protein concentrations in the supernatants
were measured using the BCA protein assay kit (Beyotime).
Fifty micrograms of proteins in each sample were subjected to
SDS-PAGE, and the gels were transferred to 0.2 lm nitrocellu-
lose membranes by electroblotting. Nitrocellulose membranes
were exposed to 5 mg/mL nonfat milk for 70 minutes at room
temperature with an aim of blocking nonspecific binding, and
incubated overnight at 48C with primary antibodies (Cell
Signaling Technology) against Bcl-2 (1:1000 dilution), Bax
(1:1000 dilution), Caspase-3 (1:1000 dilution), Tubulin (1:1000
dilution), p-wee1 (1:1000 dilution), Cdc25C (1:1000 dilution),
Cyclin B1 (1:1000 dilution), Cdc2 (1:1000 dilution), Chk2
(1:1000 dilution), p53 (1:1000 dilution), p21 (1:1000 dilution),
and glyceraldehyde 3-phosphate dehydrogenase (GAPDH)
(1:5000 dilution). After being washed with TTBS three times,
Immunoblot Assays
Human RPE cells (ARPE-19) that underwent treatment with
atRAL dimer (0, 20, 40, and 80 lM) for 3 days were harvested
from six-well cell culture plates and washed with PBS. Then
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FIGURE 4. All-trans-retinal dimer caused G2/M cell cycle arrest and activated the ATM/ATR-Chk2-p53 signaling pathway. (A) Flow cytometry
analysis of cell-cycle phase distribution in RPE cells treated with 0, 20, 40, and 80 lM atRAL dimer for 3 days. Cell number shown in the y axle
indicates peak value of the cell cycle phase. (B) Histograms displayed the percentage of cell-cycle distribution. Data were expressed as mean 6 SEM
from three independent experiments. **P < 0.01 and ***P < 0.001 versus control cells not exposed to atRAL dimer. (C–E) Expression levels of cell-
cycle–associated proteins Chk2, p53, p21, p-wee1, Cdc25C, Cyclin B1, and Cdc2 were measured by Western blot after treating RPE cells with atRAL
dimer for 3 days. The full-length blots are shown in Supplementary Figure S5. Each protein intensity was expressed as mean 6 SEM of three
independent experiments. *P < 0.05, **P < 0.01, and ***P < 0.001. (F) Colocalization of Cdc25C protein and the nuclei of RPE cells displayed the
atRAL dimer–induced nuclear exclusion of Cdc25C after 2 days; Cdc25C was visualized via immunofluorescence with a specific Cdc25C antibody
followed by Alexa Fluor 594-conjugated AffiniPure Donkey Anti-Rabbit IgG (HþL). Nuclear staining was performed with DAPI and is shown in blue.
(G) The area percentage of Cdc25C protein in DAPI was quantified by Image J software (http://imagej.nih.gov/ij/; provided in the public domain by
the National Institutes of Health, Bethesda, MD, USA) from three independent experiments. Data were presented as mean 6 SEM. ***P < 0.001 as
compared with control cells.
the membranes were incubated with secondary antibodies
(IRDye 800/700CW Donkey anti-Rabbit/Mouse; LI-COR, Lin-
coln, NE, USA) for 2 hours at room temperature. Specific
antibody binding was detected by an Odyssey Fc Dual-Mode
Imaging System (LI-COR), and band intensity was quantified
using the software of Image Studio Lite Version 4.0 (LI-COR).
With regard to in vivo expression of apoptosis/cell-cycle–
associated proteins, 4-month-old C57BL/6J WT and Abca4ꢀ/ꢀ
Rdh8ꢀ/ꢀ DKO mouse eyecups without cornea, lens, and
vitreous were homogenized in complete EDTA-free protease
inhibitor mixture (MAIBIO, Shanghai, China) and centrifuged
at 13,000g for 5 minutes at 48C. Protein concentrations in the
homogenates were determined by the BCA protein assay kit.
Protein extracts from retinal tissues of mouse eyes were tested
for protein expression of Bcl-2, Bax, Caspase-3, Cyclin B1,
Cdc2, p53, and GAPDH as described above.
Statistical Analysis
All experiments were independently repeated at least three
times. Data were analyzed using 1-way ANOVA and Newman-
Keuls test for multiple comparisons (Prism 5.0; GraphPad
Software, La Jolla, CA, USA), and expressed as mean 6 SEM. A
P value less than 0.05 was considered to be statistically
significant.
RESULTS
Quantification, Internalization, Subcellular
Location, Cytotoxicity, and Cellular Proliferation
Inhibition
The constituents of RPE lipofuscin in eyes of C57BL/6J WT and
Abca4ꢀ/ꢀRdh8ꢀ/ꢀ DKO mice were reexamined. Reverse-phase
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amassed in RPE cells to perturb membrane integrity; mem-
brane damage was examined by assaying the release of
cytoplasmic LDH into culture medium. As shown in Figure
3A, cultured RPE cells exposed to atRAL dimer did not exhibit
elevated LDH levels at 5 and 10 lM after 2 days of incubation in
fresh serum-free medium, but did display a remarkable increase
in LDH levels at concentrations ranging from 20 to 80 lM.
When atRAL dimer treatment stretched over 5 days, a
significant increase of LDH levels was readily observed with
atRAL dimer concentrations starting from 10 lM (Fig. 3B). All-
trans-retinal dimer–induced cell death was visualized by
labeling all cells with DAPI and compromised cells with Dead
Red, a membrane-impermeant fluorescent dye. Compared with
the control in which cell damage/death did not occur, the
percentage of nonviable/dead cells in total cells increased with
increasing atRAL dimer exposure concentrations (Fig. 3C).
Based on the phenomenon that atRAL dimer substantially
decreased count and density of RPE cells (Fig. 3C), we inferred
that atRAL dimer likely inhibited natural growth of RPE cells.
Accordingly, the proliferating cells were examined by labeling
with BrdU immunofluorescence after incubating atRAL dimer
with RPE cells. Indeed, nearly 80% of control cells were
proliferating, but atRAL dimer–laden RPE cells were signifi-
cantly inhibited to reproduce in a concentration-dependent
manner (Fig. 3D). By counting cell number in triplicate wells in
each of two experiments with three to five fields per well, the
rates of BrdU (þ) cells/DAPI (þ) cells clearly decreased with
increasing atRAL dimer exposure concentrations (Fig. 3E),
thereby confirming the occurrence of RPE cell proliferation
suppression.
FIGURE 5. Induction of DNA damage by atRAL dimer in RPE cells. (A)
After 3 days following exposure to 0, 20, 40, and 80 lM atRAL dimer,
cells were subjected to the alkaline comet assay for detecting DNA
strand breaks. Typical fluorescence photomicrographs of atRAL dimer
induced DNA comets stained with EB. (B) Quantification of DNA strand
breakage at 2 or 3 days after treatment of RPE cells with atRAL dimer at
indicated concentrations. Tail moment is indicative of the extent of
DNA damage. Data are expressed as mean 6 SEM of results in two
independent experiments; approximately 50 to 100 nuclei per
treatment were randomly selected to measure the comet tail length
from the trailing edge of the nucleus to the leading edge of the tail. ***P
< 0.001 as compared with control cells.
All-Trans-Retinal Dimer Induces DNA Strand
Breaks, and Triggers Cell-Cycle Arrest at the G2/M
Phase by Activating the ATM/ATR-Chk2-p53
Signaling Pathway
To further examine the mechanism by which atRAL dimer may
inhibit proliferation of RPE cells, we investigated the effects of
atRAL dimer on cell cycle. Retinal pigment epithelium cells
were incubated with serial concentrations of atRAL dimer for 3
days, stained with PI, and finally evaluated by flow cytometry
(Fig. 4A). As expected, starting at 40 lM, atRAL dimer induced
cell-cycle arrest in the G2/M phase (Fig. 4B).
HPLC chromatogram showed a significant peak corresponding
to atRAL dimer in a hydrophobic extract derived from six eyes
of 6-month-old Abca4ꢀ/ꢀRdh8ꢀ/ꢀ DKO mice while monitoring
eluents at 430 nm (Fig. 1C). We quantified atRAL dimer and
A2E in eyes of WT and Abca4ꢀ/ꢀRdh8ꢀ/ꢀ DKO mice at 2, 4, 6,
8, or 10 months of age. The data indicated that atRAL dimer
was remarkably upregulated in Abca4ꢀ/ꢀRdh8ꢀ/ꢀ DKO mice
versus age-matched WT mice (Fig. 1D), as previously report-
ed.19 Even more strikingly, the amount of atRAL dimer was
observed to be much greater than that of A2E in Abca4ꢀ/ꢀ
Rdh8ꢀ/ꢀ DKO mice (Fig. 1E).
Confocal imaging in the horizontal plane confirmed that
exogenously delivered atRAL dimer was internalized by RPE
cells in culture (Fig. 2A). Intensity of green autofluorescence
from internalized atRAL dimer granules was augmented at
concentrations from 5 to 80 lM. Because internalized atRAL
dimer was found to be distributed in a punctate perinuclear
pattern that is characteristic of lysosomes, we sought to define
the intracellular compartmentation of atRAL dimer. According-
ly, cultured RPE cells were simultaneously incubated with
atRAL dimer and LysoTracker Red DND, a membrane-diffusible
acidophilic fluorophore. On examination by confocal laser
scanning microscopy, loads of intracellular granules containing
atRAL dimer were observed to colocalize with the LysoTracker
probe (Fig. 2B).
To obtain further insight into the mechanism of atRAL
dimer–induced RPE cell-cycle arrest, we studied the expression
of several cell-cycle–associated regulators by a Western blotting
assay as well as an immunofluorescence staining of Cdc25C, a
tyrosine phosphatase that plays a key role in the regulation of
cell division.23 Once Cdc25C is phosphorylated at the site of
Ser216 under the action of Chk2, phosphorylated Cdc25C will
bind to 14-3-3 and then block mitotic entry under normal
conditions and after DNA damage.24,25 In the current study,
immunofluorescence analysis with anti-Cdc25C antibody
revealed that atRAL dimer clearly triggered the nuclear
exclusion of Cdc25C in RPE cells at concentrations starting
from 20 lM (Figs. 4F, 4G). With immunoblot assays, RPE cells
treated with atRAL dimer significantly exhibited an increase in
protein levels of Chk2, p53, and p21, but the expression of
Cdc25C, Cyclin B1, Cdc2, and p-wee1 was clearly decreased in
a concentration-dependent manner (Figs. 4C–E). It has been
previously reported that Chk2 and p53 are activated via the
ataxia telangiectasia–mutated (ATM)/ATM and Rad3-related
(ATR) signaling pathway.26 p21, a p53 downstream effector,
serves as an inhibitor of Cyclin B1-Cdc2 complexes that control
the G2/M transition.27 As depicted in Figure 4C, the
overexpression of p53 and p21 in atRAL dimer–laden RPE
cells impeded the activation of the Cyclin B1-Cdc2 complexes,
To understand the level at which atRAL dimer is tolerated
by RPE cells in vitro, we tested the ability of atRAL dimer
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FIGURE 6. Evidence that atRAL dimer induced apoptosis in RPE cells. (A) Flow cytometry analysis via Annexin V/PI staining was used to identify
apoptosis after incubations of RPE cells with atRAL dimer (0, 20, 40, and 80 lM) for 3 days. (B) A bar graph manifested the percentage of cells at
different stages of the apoptotic program; mean 6 SEM from three independent experiments. (C) Typical dot plots of MMP changes in RPE cells
treated with atRAL dimer (0, 20, 40, and 80 lM) for 3 days were generated by JC-1 staining flow cytometry. (D) A bar chart displayed the percentage
of cells with low MMP (DWm); mean 6 SEM of three independent experiments. (E) Representative fluorescence photomicrographs of in situ JC-1
staining output by the inverted fluorescence microscopy. Scale bars: 100 lm. (F) Green/red ratio was quantified from three dependent experiments
by Image Pro Plus software (Media Cybernetics, Rockville, MD, USA). Data were presented as mean 6 SEM. ***P < 0.001 as compared with control
cells.
which was associated with the G2/M cell-cycle arrest. Taken
together, the data suggest that atRAL dimer induces the G2/M
phase arrest via the ATM/ATR-Chk2-p53 pathway. Given that
p53 and Chk2 will be activated by the ATM/ATR signaling
pathway when DNA damage takes place within RPE cells,
atRAL dimer–induced direct DNA strand breaks was tested by
means of single-cell gel electrophoresis (comet assay). Comet
assay was carried out under alkaline conditions that permit
damaged DNA to be drawn out into a comet tail when
subjected to an electrical field. Indeed, the presence of DNA
strand breaks was visualized by the emergence of comet tails
from the nuclei of atRAL dimer–laden cells (Fig. 5A). By
contrast, the nuclei of control cells remained spherical. Tail
moment, a parameter whose magnitude reflects the frequency
of DNA strand breaks per nucleus,28–30 was remarkably
elevated in atRAL dimer–fed cells, indicative of DNA damage
response; the increased magnitude in tail moment was found
to be dependent on exposure concentrations of atRAL dimer
(Fig. 5B). The average comet tail moments of RPE cells exposed
to 20-, 40-, and 80-lM atRAL dimer, were measured as 34.14,
82.01, and 113.30 lm, respectively.
was performed. A total of Annexin Vþ/PI– and Annexin Vþ/PIþ
cells indicate cell death through both early- and late-stage
apoptosis. After treatment with atRAL dimer for 3 days, the
percentage of apoptotic cells increased concentration-depen-
dently (Fig. 6A), and was significant at the concentration of 80
lM versus the control (Fig. 6B). Disruption of DWm is an early
event in the process of apoptosis in mammalian cells,31
reflecting the mitochondrial membrane permeability transi-
tion. Results of staining with the DWm sensing dye JC-1
demonstrated that the untreated cells exhibited an intact
DWm, but atRAL dimer exposure significantly induced DWm
loss in RPE cells at the concentration of 80 lM, as indicated by
a shift in JC-1 fluorescence from aggregate (red) to monomer
(green) (Figs. 6E, 6F). To further corroborate this finding, we
evaluated the fluorescence of JC-1 with flow cytometry and
found that, compared with control cells (4.4%), the number of
cells with reduction of DWm was not significantly increased
after 20 lM (5.6%) and 40 lM (5.9%) atRAL dimer exposures
for 3 days, but was apparently raised following 80 lM (36.3%)
atRAL dimer treatment (Fig. 6C), consistent with quantitative
data on the amount of low DWm cells from three independent
experiments (Fig. 6D). On examination by confocal laser
scanning microscopy, treatment of RPE cells with atRAL dimer
clearly inflicted the release of cytochrome c from mitochondria
to the cytoplasm versus control cells that retained cytochrome
c in the mitochondria (Fig. 7A). In addition, we observed a
clear upregulation of the cleaved fragment of caspase-3 after 80
lM atRAL dimer exposure, which was accompanied by a
All-Trans-Retinal Dimer Causes Apoptosis, and
Activates the Mitochondria-Dependent Pathway in
RPE Cells
To assess whether atRAL dimer exhibited a proapoptotic effect
on RPE cells, flow cytometry analysis via Annexin V/PI staining
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FIGURE 7. All-trans-retinal dimer induced cytochrome c release from mitochondria and expression changes of typical apoptosis regulatory proteins
within RPE cells. (A) Retinal pigment epithelial cells, 3 days after exposing to 0, 20, 40, and 80 lM atRAL dimer, were examined by
immunofluorescence for cytochrome c localization. The distribution of cytochrome c (green) and MitoTracker (red) by confocal fluorescence
microscopy exhibited the release of cytochrome c into the cytosol. (B) Protein expression of Bcl-2, Bax, and Caspase-3, 3 days after cells were
treated with atRAL dimer (0, 20, 40, and 80 lM), was determined using Western blot, and quantified by Image Studio Lite Version 4.0 software. The
full-length blots are presented in Supplementary Figure S6. (C) The protein expression ratio of Bcl-2 to Bax was presented as fold change in treated
cells compared with control cells. Note that the normalization for band intensity of Bcl-2 and Bax against that of a-tubulin was individually made
before calculating the Bcl-2/Bax ratio. Each value represents mean 6 SEM from three independent experiments. *P < 0.05. (D) The protein
expression level of caspase-3 in atRAL dimer–treated cells versus control cells, including pro-caspase-3 and cleaved caspase-3. The data are shown as
mean 6 SEM; *P < 0.05, ***P < 0.001.
reduction in protein expression of pro-caspase-3 (Figs. 7B, 7D).
To further identify apoptosis onset in RPE cells via activation of
the mitochondrial pathway, Western blot analysis also was used
to detect antiapoptotic mitochondrial protein Bcl-2 and
proapoptotic mitochondrial protein Bax, and revealed that
atRAL dimer concentration-dependently caused downregula-
tion of Bcl-2 and upregulation of Bax (Fig. 7B). By comparison
with control cells, the ratio of Bcl-2/Bax decreased with
increasing atRAL dimer exposure concentrations, and was
significant at the concentration of 80 lM (Fig. 7C).
the RPE dissected from age-matched WT mice (Fig. 8A), thus
giving rise to a 93.94% decrease in the ratio of Bcl-2/Bax (Fig.
8C). Furthermore, protein expression of cleaved caspase-3 in
the RPE of Abca4ꢀ/ꢀRdh8ꢀ/ꢀ DKO mice was visibly raised,
along with a reduction of the pro-caspase-3 protein expression
(Fig. 8A). On the other hand, in vivo expression of Cyclin B1
and Cdc2 required for the G2/M phase transition of the cell
cycle was clearly altered in the RPE of Abca4ꢀ/ꢀRdh8ꢀ/ꢀ DKO
mice. We observed an obvious upregulation of Cyclin B1, as
well as an apparent downregulation of Cdc2 in the RPE of 4-
month-old Abca4ꢀ/ꢀRdh8ꢀ/ꢀ DKO mice versus that of age-
matched WT mice (Fig. 8D). Quantitative immunoblot analysis
demonstrated that there was an approximately 60% decline in
the Cdc2 protein expression level in the RPE of Abca4ꢀ/ꢀ
Rdh8ꢀ/ꢀ DKO mice at 4 months of age compared with age-
matched WT mice (Fig. 8E), whereas expression level of Cyclin
B1 protein showed an approximately 2-fold increase in the RPE
of Abca4ꢀ/ꢀRdh8ꢀ/ꢀ DKO mice (Fig. 8F).
p53-Dependent Apoptosis and G2/M Phase Cell-
Cycle Arrest Are Detected in the RPE of
Abca4ꢀ/ꢀRdh8ꢀ/ꢀ DKO Mice
Considering that an increased amount of atRAL dimer was
detected in eyes of Abca4ꢀ/ꢀRdh8ꢀ/ꢀ DKO mice versus WT
mice (Fig. 1D), in vivo expression of several proteins correlated
with apoptosis and G2/M phase arrest of the cell cycle was
examined (Figs. 8A, 8D). As expected, quantitative immuno-
blot analysis showed that p53 protein exhibited a 1.7-fold
increase in the RPE of 4-month-old Abca4ꢀ/ꢀRdh8ꢀ/ꢀ DKO
mice compared with age-matched WT mice (Fig. 8B).
Immunoblotting analysis of the RPE from Abca4ꢀ/ꢀRdh8ꢀ/ꢀ
DKO mice at age 4 months demonstrated the upregulation of
Bax protein and downregulation of Bcl-2 protein versus that of
DISCUSSION
There is a growing body of evidence to substantiate that A2E is
a representative and rich aging pigment in the RPE lipofus-
cin.11,15,32 A2E has been considered to cause damage to RPE
cells, including autophagic response,33 lysosomal alkaliniza-
tion,34 pro-apoptotic protein detachment from mitochondria,35
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FIGURE 8. Activation of p53-dependpent apoptosis and abnormal expression of key cell-cycle proteins Cdc2 and Cyclin B1 in the RPE of Abca4ꢀ/ꢀ
Rdh8ꢀ/ꢀ DKO mice. (A) Representative immunoblots for p53, Bcl-2, Bax, Caspase-3, and GAPDH protein from RPE homogenates (60 lg per lane) of
WT and Abca4ꢀ/ꢀRdh8ꢀ/ꢀ DKO mice at 4 months of age. The full-length blots are shown in Supplementary Figure S7. (B, C) Expression levels of
p53, Bcl-2, and Bax protein in the RPE of WT and Abca4ꢀ/ꢀRdh8ꢀ/ꢀ DKO mice were quantified using the Image Studio Lite software, and normalized
to that of GAPDH protein serving as the internal control. n ¼6, *P < 0.05 and **P < 0.01 versus WT mice. (D) Typical bands of Cdc2, Cyclin B1, and
GAPDH protein from RPE homogenates (60 lg per lane) of WT and Abca4ꢀ/ꢀRdh8ꢀ/ꢀ DKO mice at 4 months of age. The full-length blots are
presented in Supplementary Figure S7. (E, F) Expression levels of Cdc2 and Cyclin B1 protein in the RPE of WT and Abca4ꢀ/ꢀRdh8ꢀ/ꢀ DKO mice
were measured using the Image Studio Lite software and normalized to that of GAPDH protein. n ¼ 6, **P < 0.01 and ***P < 0.001 versus WT mice.
and inflammatory chemokine and cytokine production33,36 as
well as a detergent-like effect on the cytomembrane.37 By
contrast, atRAL dimer is barely detected in eyes of WT mice
compared with A2E.19 However, the amount of atRAL dimer
dramatically increased in eyes of Abca4ꢀ/ꢀRdh8ꢀ/ꢀ DKO mice
versus WT mice (Fig. 1D),19 and was even much greater than
that of A2E in eyes of Abca4ꢀ/ꢀRdh8ꢀ/ꢀ DKO mice (Fig. 1E).
Abca4ꢀ/ꢀRdh8ꢀ/ꢀ DKO mice are established by cross-breeding
Abca4ꢀ/ꢀ mice, a model of recessive Stargardt disease,38 with
Rdh8ꢀ/ꢀ mice. In the DKO mouse RPE cells, the deposition of
A2E and atRAL dimer was already apparent by 2 months of age,
and although it continued to increase until approximately 6
months of age (atRAL dimer, 93.34 6 4.828 pmol/eye; A2E,
21.18 6 1.129 pmol/eye), thereafter it declined (Fig. 1E). This
trend of change in the quantity of endogenous atRAL dimer and
A2E is possibly because the attenuation in the rate of the visual
cycle after the age of 6 months slows their accumulation in
Abca4ꢀ/ꢀRdh8ꢀ/ꢀ DKO mice, whereas continuous light-in-
duced oxidation and cleavage leads to the decrease in levels of
atRAL dimer and A2E to a certain degree.39–41 Another possible
explanation for the phenomenon is due to exacerbated loss of
photoreceptors and removal of atrophic RPE cells in Abca4ꢀ/ꢀ
Rdh8ꢀ/ꢀ DKO mice aged older than 6 months. The finding is
supported by early studies regarding changes in the amount of
RPE lipofuscin in humans with age.42–45
coworkers37 demonstrated that an estimated 5% to 10% of
A2E in the medium is internalized by the RPE cells, and
compared the level of A2E internalized by cultured RPE cells
with an estimated amount of endogenous A2E in RPE cells
isolated from healthy human donor eyes at ages ranging from
58 to 79, thus concluding that 10- to 25-lM concentrations of
A2E in the medium are required to achieve intracellular levels
that approximate those amounts of A2E measured in human
RPE. In the present study, HPLC quantification of atRAL dimer
and A2E in eyes of Abca4ꢀ/ꢀRdh8ꢀ/ꢀ DKO mice revealed that
the amount of atRAL dimer is more than four times greater than
that of A2E (Fig. 1E). Accordingly, it may be physiologically
relevant for the concentrations at which atRAL dimer is
presented in the cell-based experiments of this work.
Although A2E and atRAL dimer are both derived from atRAL
or 11-cis-retinal,9,14,46 there is a distinct difference between
them on the chemical structure. A2E contains a pyridinium
ring on which a positive charge on the nitrogen atom linked to
a saturated alcohol group (Fig. 1A), whereas atRAL dimer is a
nonpyridinium bisretinoid with a simpler cyclohexadiene ring
that is attached by an aldehyde moiety (Fig. 1B). Moreover,
atRAL dimer is much more photosensitive than A2E.41 Previous
evidence demonstrated the inability of A2E to damage DNA of
RPE cells at a 20-lM concentration in the medium.47
Conversely, comet assay revealed that atRAL dimer significantly
induced DNA damage on RPE cells at concentrations starting
from 20 lM (Fig. 5).
Using HPLC to quantify atRAL dimer within cultured RPE
cells, we found that the amount of atRAL dimer internalized by
the cells varied with the concentration of atRAL dimer in the
medium and with the incubation period, and tentatively
estimated that 6% to 13% of atRAL dimer in the medium was
internalized by the RPE cells. By contrast, Sparrow and
Although it is clear that there are levels of atRAL dimer that
are tolerated by RPE cells, intracellular concentrations can be
reached above which atRAL dimer is damaging to RPE cells. As
depicted in Figure 9, we did verify that atRAL dimer inhibited
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FIGURE 9. Mechanism for atRAL dimer induced proliferation inhibition and apoptosis of RPE cells. When endogenously produced to a critical level,
atRAL dimer, an indigestible lipofuscin pigment in the RPE, triggers a DNA damage response and activates the ATM/ATR-Chk2-p53 pathway. Chk2
can phosphorylate Cdc25C and thereby inhibit the Cdc25C-mediated activation of Cyclin B1-Cdc2 complexes, which is essential for the G2/M phase
transition of the cell cycle. Phosphorylation of Cdc25C by Chk2 creates a binding site for the 14-3-3 family of phosphoserine binding proteins, and
14-3-3 proteins can regulate Cdc25C by sequestering it from the nucleus to the cytoplasm. Furthermore, upregulation of p53 also activates p21, an
inhibitor of activation of the Cyclin B1-Cdc2 complexes. Taken together, atRAL dimer–triggered suppression of cell proliferation is likely an
important cause of RPE degeneration. On the other land, DNA damage–induced activation of p53 by atRAL dimer in the RPE results in aberrant
expression of the apoptosis-related proteins Bax and Bcl-2, the release of cytochrome c from mitochondria, and Caspase-3 activation, indicating that
atRAL dimer–provoked RPE degeneration is also involved with the mitochondrial apoptosis pathway.
RPE cell proliferation (Figs. 3D, 3E) and induced cell-cycle
arrest at the G2/M phase via activation of the ATM/ATR-Chk2-
p53 signaling pathway (Fig. 4), as well as caused DNA damage
(Fig. 5) and mitochondrion-associated apoptosis in RPE cells
(Figs. 6, 7). In addition, although atRAL dimer could have
clearly inflicted membrane damage (Figs. 3A, 3B), we observed
that LDH levels following exposure of RPE cells to atRAL dimer
for 5 days, first increased in a concentration-dependent
manner, but gave a trend to decrease at 80 lM (Fig. 3B).
Moreover, in contrast to the control group, DAPI-stained cells
showed a significant decrease in the count and density with
increasing atRAL dimer exposure concentrations (Figs. 3C,
3D). These phenomena are likely caused by the occurrence of
proliferation inhibition in RPE cells induced by atRAL dimer,
which leads to a rapid reduction in the number of cells.
Evidence from immunoblot studies on the RPE of Abca4ꢀ/ꢀ
Rdh8ꢀ/ꢀ DKO mice manifested that excess buildup of atRAL
dimer, at least in part, contributes to the degeneration of the
RPE in vivo (Fig. 8).
The rd8 mutation of the Crb1 gene is unexpectedly
detected in vendor strains of C57BL/6N mice.22 Thus, it is
suspected that Crb1rd8 mutation might be responsible for the
retinal degeneration in several mutant mouse models exhibit-
ing AMD-like lesions.48,49 Here we found that Crb1rd8 mutation
was present in Abca4ꢀ/ꢀRdh8ꢀ/ꢀ DKO mice (Supplementary
Figs. S8A, S8B). However, Crb1rd8 mice usually exhibit retinal
folds and pseudorosettes at 2 months of age,50 which are not
observed in Abca4ꢀ/ꢀRdh8ꢀ/ꢀ DKO mice. In contrast,
Abca4ꢀ/ꢀRdh8ꢀ/ꢀ DKO mice used in this study develop
lipofuscin, drusen, basal laminar deposits, Bruch’s membrane
thickening, and choroidal neovascularization,19 which are
cardinal to AMD pathology but not shown in Crb1rd8 mutant
mice. In Crb1rd8 mutant mice, the shortening of photoreceptor
inner and outer segments is observed within 2 weeks after
birth, suggestive of a developmental defect.50 Distinctively,
retinal structure is normal in Abca4ꢀ/ꢀRdh8ꢀ/ꢀ DKO mice by 3
weeks of age, and the phenotypes are aggravated with age.
Even more importantly, retinal lesions are exacerbated by light
exposure, and rescued by the administration of retinylamine, a
visual cycle inhibitor, implying that AMD-like lesions in
Abca4ꢀ/ꢀRdh8ꢀ/ꢀ DKO mice are more likely due to the
abnormal accumulation of atRAL and its derived products.19
Therefore, we concluded that the phenotypes observed in the
RPE of Abca4ꢀ/ꢀRdh8ꢀ/ꢀ DKO mice were largely due to
retinoid dysregulation. On the other hand, deletion of CRB1
could alter cell-cycle distribution of retinal progenitor cells and
thereby more cells reside in the G2/M phases,51 suggesting that
Crb1 mutation may affect the expression levels of cell-cycle–
related proteins. In this study, we have shown that the
expression of Cyclin B1 and Cdc2, two key cell-cycle proteins,
is significantly altered in atRAL dimer–laden RPE cells (Fig. 4C)
and the RPE of Abca4ꢀ/ꢀRdh8ꢀ/ꢀ DKO mice (Figs. 8D–F). The
expression of Cyclin B1 was decreased in atRAL dimer–treated
RPE cells (Figs. 4C, 4E), but increased in the RPE of Abca4ꢀ/ꢀ
Rdh8ꢀ/ꢀ DKO mice (Figs. 8D, 8F). To exclude the possibility
that Crb1rd8 might contribute to the difference of Cyclin B1
expression in vitro and in vivo, its protein level in the RPE of
C57BL/6J, C57BL/6N and Abca4ꢀ/ꢀRdh8ꢀ/ꢀ DKO mice was
measured by Western blot (Supplementary Fig. S8C), which
indicated that the increase of Cyclin B1 in the RPE of Abca4ꢀ/ꢀ
Rdh8ꢀ/ꢀ DKO mice was not due to the mutation in the Crb1
gene. Nevertheless, whether rd8 mutation partially accounts
for the retinal degeneration observed in Abca4ꢀ/ꢀRdh8ꢀ/ꢀ
DKO mice is worth further investigation. Furthermore, the
expression of Cdc2 was downregulated both in vitro (Figs. 4C,
4E) and in vivo (Figs. 8D, 8E), suggesting that the formation of
Cyclin B1-Cdc2 complexes was positively suffocated. Reduced
formation of Cyclin B1-Cdc2 complexes would suppress
progression of the cell cycle from the G2 phase to the M
phase.52
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13. Li J, Yao K, Yu X, et al. Identification of a novel lipofuscin
pigment (iisoA2E) in retina and its effects in the retinal
pigment epithelial cells. J Biol Chem. 2013;288:35671–
35682.
Delayed clearance of atRAL in the photoreceptors would
result in excess accumulation of atRAL dimer in the RPE.19 The
finding that atRAL dimer is much more abundant than A2E in
eyes of Abca4ꢀ/ꢀRdh8ꢀ/ꢀ DKO mice reveals potential impor-
tance of atRAL dimer in the RPE degeneration. Most recently,
we reported that initially, the conversion of atRAL to atRAL
dimer in the retina is likely a protective pathway that detoxifies
free atRAL with a highly active aldehyde moiety53; however,
when atRAL dimer accumulated beyond a critical level, it
surely provoked adverse effects on the RPE. This work expands
the understanding of different biological properties of atRAL
dimer, and may make it as a potential target of gene-based and
drug therapies that aim to alleviate age-dependent RPE
degeneration.
14. Fishkin NE, Sparrow JR, Allikmets R, Nakanishi K. Isolation
and characterization of a retinal pigment epithelial cell
fluorophore: an all-trans-retinal dimer conjugate. Proc Natl
Acad Sci U S A. 2005;102:7091–7096.
15. Wu Y, Fishkin NE, Pande A, Pande J, Sparrow JR. Novel
lipofuscin bisretinoids prominent in human retina and in a
model of recessive Stargardt disease. J Biol Chem. 2009;284:
20155–20166.
16. Wu Y, Jin Q, Yao K, et al. Retinal metabolism in humans
induces the formation of an unprecedented lipofuscin
fluorophore ‘pdA2E.’ Biochem J. 2014;460:343–352.
17. Zhao J, Yao K, Jin Q, et al. Preparative and biosynthetic
insights into pdA2E and isopdA2E, retinal-derived fluoro-
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Ophthalmol Vis Sci. 2014;55:8241–8250.
Acknowledgments
Supported in part by China National Natural Science Foundation
Grants 81570857 (YW) and 81271018 (YW); the Fundamental
Research Funds for the Central Universities grant (YW); and
Natural Science Foundation of Fujian Province Grant 2016J01412
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Disclosure: J. Zhao, None; Y. Liao, None; J. Chen, None; X.
Dong, None; Z. Gao, None; H. Zhang, None; X. Wu, None; Z.
Liu, None; Y. Wu, None
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