Fluorescent pigments of the retinal pigment epithelium and age-related macular degeneration

Article abstract of DOI:10.1016/S0960-894X(01)00314-6

The major hydrophobic fluorophore of the retinal pigment epithelium (RPE) is A2E, a pyridinium bis-retinoid derived from all-trans-retinal and phosphatidyl-ethanolamine. The accumulation of fluorophores such as A2E is implicated in the pathogenesis of age-related macular degeneration (AMD), a disease associated with the deterioration of central vision and a leading cause of blindness in the elderly. Recent chemical and biological studies have provided insight into the synthesis and biosynthesis of A2E, the spectroscopic properties of this pigment, and the role of A2E and RPE cell death.

Full text of DOI:10.1016/S0960-894X(01)00314-6

Bioorganic & Medicinal Chemistry Letters 11 (2001) 1533–1540
Fluorescent Pigments of the Retinal Pigment Epithelium and
Age-Related Macular Degeneration
Shimon Ben-Shabat,a Craig A. Parish,a Masaru Hashimoto,a,y Jianghua Liu,a
Koji Nakanishia,* and Janet R. Sparrowb,*
aDepartment of Chemistry, Columbia University, New York, NY 10027, USA
bDepartment of Ophthalmology, Columbia University, New York, NY 10032, USA
Received 14 December 2000; accepted 3 May 2001
Abstract—The major hydrophobic fluorophore of the retinal pigment epithelium (RPE) is A2E, a pyridinium bis-retinoid derived
from all-trans-retinal and phosphatidyl-ethanolamine. The accumulation of fluorophores such as A2E is implicated in the patho-
genesis of age-related macular degeneration (AMD), a disease associated with the deterioration of central vision and a leading cause
of blindness in the elderly. Recent chemical and biological studies have provided insight into the synthesis and biosynthesis of A2E,
the spectroscopic properties of this pigment, and the role of A2E and RPE cell death. # 2001 Published by Elsevier Science Ltd.
Age-related macular degeneration (AMD) results in a
loss of vision due to deterioration of the central region
of the retina (macula). This disease, the leading cause of
blindness in persons over the age of 60, manifests itself
in two ways—nonneovascular (atrophic, dry)1 and neo-
vascular (exudative, wet)2 forms. Nonneovascular
AMD is characterized by the deposition of material
(drusen) under the retinal pigment epithelium (RPE), by
changes in the distribution of melanin pigment, and by
the gradual degeneration of RPE cells. Progression to
the neovascular form of AMD occurs with the pro-
liferation of new blood vessels under the RPE and can
be accompanied by subretinal hemorrhage, RPE
detachment, and the ingrowth of scar tissue. The end
result of AMD is the loss of high acuity, central vision
due to RPE and photoreceptor cell death.
Schiff base linkage to the chromophore 11-cis-retinal. It
is the isomerization of the retinal chromophore from the
11-cis to the all-trans form by a photon of light that initi-
ates phototransduction.3 Photoreceptor cells, in turn, are
dependent on cooperative interactions with the underlying
retinal pigment epithelium (Fig. 1). It is these cells that
contain the machinery to regenerate 11-cis-retinal from all-
trans-retinal,4 the latter being released from opsin after
photoisomerization. RPE cells also phagocytose and
degrade outer segment membrane that is discarded daily
by the photoreceptor cell to balance the continual produc-
tion of new disks at the base of the photoreceptor outer
The cells intimately involved in visual transduction are
the rod and cone photoreceptor cells (Fig. 1). Rods and
cones are specialized for responding to dim light and
color vision, respectively. The outer segments of the
photoreceptor cells are replete with light-capturing pro-
teins consisting of rod or cone opsin bound through a
*Corresponding author. Fax: +1-212-932-8273; e-mail: kn5@columbia.
edu (K. Nakanishi). Fax: +1-212-305-9638; e-mail: jrs88@columbia.
edu (J. R. Sparrow).
yCurrent address: Faculty of Agriculture and Life Science, Hirosaki
University, Aomori 036, Japan.
Figure 1. Diagram of the eye. The macula is the central portion of the
retina, which contains photoreceptor cells (rods and cones) adjacent to
a monolayer of RPE. In this cartoon, the outer segment to RPE ratio
is simplified for clarity. RPE, retinal pigment epithelium.
0960-894X/01/$ - see front matter # 2001 Published by Elsevier Science Ltd.
PII: S0960-894X(01)00314-6

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S. Ben-Shabat et al. / Bioorg. Med. Chem. Lett. 11 (2001) 1533–1540
segments.5,6 Since RPE cells are essential to normal
visual function and to the health of neural retina, the
demise of these cells in some retinal disorders such as
AMD is a critical component of the disease process.
Working with the pooled lipofuscin from aged human
eyes, Eldred and Katz27 isolated fluorescent compounds
by chloroform–methanol extraction and an extensive
series of chromatographic separations. While the polar
extract contained only a weak blue fluorescence that
was not consistent with the orange colored emission of
lipofuscin, the hydrophobic extract could be separated
into ten fractions, a number of which contained mate-
rial that did correlate well with the expected orange
emission (ꢁ600 nm). Spectroscopic measurements indi-
cated that the first two of these fractions contained
retinol (vitamin A) and retinyl palmitate. The remaining
eight fractions, which emitted yellow to orange light
upon excitation, appeared to be responsible for the
characteristic fluorescence of lipofuscin. Isolation of the
most prominent of these compounds from more than
250 eyes of human donors, 40 years of age or older,
provided 100 micrograms of the major orange fluor-
ophore.28 Structural elucidation was attempted with this
material. The original compound as well as the perhy-
drogenated fluorophore and its acetate were submitted
to FAB tandem mass spectrometry with collisionally
activated decomposition analysis. These data, along
with NMRand UV analyses, led to the proposal of the
zwitterionic structure, N-retinylidene-N-retinyl-ethanol-
amine (Fig. 2).28 This structure was thought to be con-
firmed by the preparation of an identical substance by
the reaction of two equivalents of all-trans-retinal and
one equivalent of ethanolamine. However, several
aspects of this structural assignment could not be
reconciled: the mass spectrum of the proposed structure
would differ by two mass units from the measured
value, the hydroxyl group would not be ionized at bio-
logical pH, the iminium function would undergo
hydrolysis by silica gel during chromatographic separa-
tion, and the electronic spectrum of the fluorophore
showed none of the fine structure characteristic of the
retro retinoid moiety. After retraction of the originally
proposed structure,31 Eldred brought it to our attention
for further elucidation and we initiated a collaborative
effort.
RPE Lipofuscin
A major factor contributing to the deterioration and
death of RPE cells, particularly in the macula, may be
the accumulation of lipofuscin, autofluorescent granules
that localize in the RPE lysosomes and are comprised of
fluorescent pigments as well as an uncharacterized mix-
ture of lipids and proteins.7 The amassing of lipofuscin
(Fig. 1) is not a feature of aging RPE alone since excessive
amounts of these fluorophores are also present in the
RPE in some inherited retinal disorders such as Star-
gardt’s disease, Best’s macular dystrophy, and cone–rod
dystrophy.7ꢀ12 The deposition of lipofuscin occurs lar-
gely as a consequence of the phagocytotic burden placed
on the RPE cell, with the presumption being that the
components of the ingested outer segments that cannot
be degraded by the cohort of lysosomal enzymes present
in RPE accumulate as lipofuscin.13ꢀ17
Even at age 20 years, the content of lipofuscin is nota-
ble.9 By age 80, when it is estimated that approximately
90 million disk membranes will have been internalized,6
20% of the area of an RPE cell in the central retina is
occupied by lipofuscin.9 Moreover, histological analyses
of human donor eyes,18ꢀ20 in addition to in vivo (non-
invasive) fundus spectrophotometry21 and confocal
laser scanning ophthalmoscopy,22,23 have shown that
RPE cells overlying the macula, with the exception of
RPE in the macula’s cone-rich center (fovea), exhibit
the most pronounced accumulation of fluorescent
material. Lipofuscin levels in RPE cells are also topo-
graphically correlated with histopathological indicators
of AMD8,9 and with the loss of light absorbing photo-
receptor cells in aged eyes.8 While the accumulation of
fluorescent granules in RPE can be simulated by feeding
photoreceptor outer segments to RPE,24,25 it is not
known whether the fluorophores are comparable to
those present in native lipofuscin.
The ability to prepare the major fluorophore of RPE
lipofuscin by the reaction of retinal and ethanolamine28
provided significant quantities of this compound for
Chemical Composition of RPE Lipofuscin
In order to effectively investigate the impact of lipo-
fuscin in RPE cell function, the factors that contribute
to fluorescent pigment formation must be understood,
the molecular components of lipofuscin must be char-
acterized, and the specific effects of these pigments on
RPE cells must be defined. While an early hypothesis
attributed the accumulation of fluorescent pigments to
the oxidation of RPE lipids, controlled studies of lipid
oxidation did not provide material that contained the
major fluorophore present in aging RPE cells.26 The
most direct approach for identifying the fluorescent
constituents of lipofuscin is the isolation of the various
components followed by structural elucidation. In this
way, the prominent pigments of RPE lipofuscin have
been characterized.27ꢀ30
Figure 2. Structure of A2E. The original N-retinyl-N-retinylidene-
ethanolamine structure28 was later revised. Further structural elucida-
tion29 and synthesis32 has confirmed the pyridinium bis-retinoid
structure, A2E. The counterion of the pyridinium salt is presumably
chloride in vivo and is trifluoroacetate for synthetic samples.

S. Ben-Shabat et al. / Bioorg. Med. Chem. Lett. 11 (2001) 1533–1540
1535
further structural studies. The biomimetic protocol was
repeated on a larger scale, reacting one gram of all-
trans-retinal with ethanolamine in the presence of acetic
acid.29 This procedure yielded 5 mg of a compound that
was identical to that isolated from eye tissue. The
amount was sufficient for detailed NMRand additional
spectroscopic studies, and led to the elucidation of an
unprecedented pyridinium bis-retinoid structure, a rigid
amphiphilic wedge with a pyridinium polar head group
and two hydrophobic retinoid tails. This revised struc-
ture was given the name A2E (1) (Fig. 2) since it is
derived from two molecules of vitamin A aldehyde and
one molecule of ethanolamine.29 In the case of synthetic
A2E, the counterion of the pyridinium moiety is tri-
fluoroacetate, since trifluoroacetic acid is used during
A2E purification. However, in vivo, the anionic coun-
terion would most likely be chloride.
An alternative and efficient one-pot procedure for the
synthesis of key intermediate bis-aldehyde 4 was exe-
cuted starting with (E)-ethoxycarbonyl-2,4,6-trienal 6
(Fig. 4).33 Thus treatment of trienal 6 with two equiva-
lents of lithium bis-trimethylsilyl-amide in THF at room
temperature cleanly led via a Peterson reaction to the
corresponding N-TMS-1,2-dihydro derivative, which
underwent an aza-6p-electrocyclization of the resulting
azatriene. The reaction mixture containing the unstable
1,2-dihydropyridine was oxidized with DDQ, affording
the desired ethoxycarbonyl pyridine derivative 7. This
intermediate was converted to bis-aldehyde 4, the over-
all yield from trienal 6 being ca. 77%.
The most efficient method for the preparation of A2E is
an optimized one-step biomimetic synthesis (Fig. 5),
which provides A2E in ca. 50% yield.30 This result was
a significant enhancement over the original one-step
yield of 0.5%.29 The mechanism of A2E formation is
presented in Figure 5. Reaction of all-trans-retinal (8)
and ethanolamine (9) leads to Schiff base 10, which
tautomerizes to enamine 11. After a second molecule of
all-trans-retinal reacts with 11 to form iminium salt 12,
A2E (1) is generated after electrocyclization (to dihy-
dropyridine 13) and auto-oxidation. In the improved
biomimetic synthesis, a mixture of all-trans-retinal
(100 mg, 352 mmol) and ethanolamine (9.5 mg, 155 mmol)
was stirred in ethanol (3 mL) in the presence of one
equivalent of acetic acid (9.3 mL, 155 mmol). Optimization
of A2E formation under a variety of conditions indi-
cated that this process is not favored under strongly
acidic or basic milieu, consistent with the fact that A2E
is generated under physiological conditions. After stir-
ring the reaction for 2 days in the dark at room tem-
perature, the mixture was concentrated and purified by
silica gel chromatography (elution with methanol/
CH2Cl2/TFA) to provide 53.8 mg (76.3 mmol, 49%) of
A2E as the TFA salt. This sample contained 5% of an
additional component that was similar, but not iden-
tical, to A2E as characterized by NMRand UV. The
two fractions had identical masses as determined by
FAB mass spectrometry, but eluted separately by C18
reverse phase HPLC. Extensive NMRstudies (ROESY,
COSY) of this material showed that it was a double
bond isomer in which one double bond had undergone
an E to Z isomerization. This structure is referred to as
iso-A2E (14). Modeling studies have indicated that iso-
A2E forms a more streamlined conformation than A2E
Stepwise and Biomimetic Synthesis of A2E and
iso-A2E
An independent confirmation of the A2E structure was
attained by total stepwise synthesis of 1 through a con-
vergent double Wittig olefination of bis-aldehyde 4 with
Wittig reagent 5 (Fig. 3). Phosphonium salt 5 contains
the triene moiety common to both side arms of A2E.
The final step of the synthesis was the alkylation with
iodoethanol at the pyridine nitrogen, providing A2E
(1).32 To prepare bis-aldehyde 4, 2-hydroxy-4-methyl-
pyridine (2) was oxidized with SeO2 to give 2-hydroxy-
4-formyl-pyridine. The methyl group of 2 was activated
due to the coordination of SeO2 to the pyridine nitrogen
with concomitant H-bond formation with the ortho
hydroxyl group. Reaction of 2-hydroxy-4-formyl-pyri-
dine with Tf2O/pyridine and subsequent coupling with
vinyl stannane in the presence of a catalytic amount of
copper(I) iodide according to the procedure of Stille
provided vinyl pyridine 3. Subsequent oxidation with
SeO2 yielded the desired bis-aldehyde 4.
Figure 3. Total synthesis of A2E.32 Reagents and conditions: (a) SeO2,
dioxane (52%); (b) Tf2O, pyridine, CH2Cl2 (77%); (c)
Bu3SnCHC(CH3)CH3, Pd(PPh3)4, LiCl, CuI, dioxane (78%); (d) SeO2,
dioxane (83%); (e) 2.2 equiv 5, NaOEt, EtOH (76%); (f) ICH2CH2OH,
CH3NO2 (56%).
Figure 4. Alternate synthesis of bis-aldehyde 4.33 Reagents and con-
ditions: (a) 2 equiv LiN(TMS)2, THF; (b) 2 equiv DDQ; (c) LiAlH4,
diethyl ether (77%, three steps); (d) TBAF, THF; (e) MnO2, CH2Cl2
(quant, two steps).

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S. Ben-Shabat et al. / Bioorg. Med. Chem. Lett. 11 (2001) 1533–1540
Figure 5. Biomimetic synthesis of A2E from all-trans-retinal and ethanolamine. Two molecules of retinal and one molecule of ethanolamine react to
provide A2E. A2E equilibrates with iso-A2E, the 13-Z isomer, in the presence of light, providing a 4:1 A2E/iso-A2E mixture.
and hence may penetrate cell membranes even more
readily.
ison with the peaks observed in methanol (600 nm) and
PBS (610 nm).35 The emission maxima of intracellular
A2E (565–570 nm) corresponds most closely to that
observed in n-BuCl, indicating that A2E most likely
exists in a hydrophobic environment. These spectra are
significant in terms of the efforts to monitor lipofuscin
fluorescence by fundus spectrophotometry21,36,37 and
confocal laser scanning ophthalmoscopy.22,23
A2E: Spectroscopy, Photochemistry, and Analysis
A detailed description of the physical properties of A2E
is necessary both to understand its reactivity, photo-
chemistry, and spectroscopy as well as to contribute to
our understanding of A2E in a cellular environment.
Measurement of the absorbance, emission, and excita-
tion spectra of A2E provided data that was comparable
to that observed with the pigment isolated from lipo-
fuscin.27 The absorbance spectrum (Fig. 6) of synthetic
A2E in methanol has two peaks with lmax 439 nm
(e=36,900) and 336 nm (e=25,600).30 The excitation
spectrum, monitored at 600 nm emission, is similar in
shape with the maximum at 418 nm.34 Measurements of
the emission spectra of A2E in n-butyl chloride, metha-
nol and aqueous buffer have shown that the emission
maximum of A2E is dependent on the nature of the
solvent. In the more hydrophobic solvent, n-butyl chlo-
ride, the maximum is blue-shifted (585 nm) in compar-
When illuminated with room light in methanol, A2E
and iso-A2E undergo photoequilibration leading to a
4:1 A2E/iso-A2E mixture (Fig. 5).30 This isomerization
was also observed with monochromatic blue light
(430 nm), indicating that visible light that reaches the
retina is capable of accomplishing the same transfor-
mation. It is interesting to note that EPRstudies on
lipofuscin crude extracts and synthetic pigment mixtures
indicate that irradiation leads to the formation of radi-
cal species,38ꢀ40 which may be related to the A2E/iso-
A2E photoconversion.
While several protocols for HPLC detection of A2E
have been described,40,41 an optimized procedure30 with
excellent sensitivity employed C18 reversed-phase
HPLC (Fig. 7) to identify as little as 5 ng of A2E and
iso-A2E in aged human eyes. The quantity of A2E
detected in RPE-choroid isolates from individual
human eyes, as determined from integrated peak inten-
sities, was found to be in the 200–800 ng range. The
sensitivity of the HPLC procedure allowed the analysis
to be performed on only 5% of the sample from a single
human eye. When performed under dim light to prevent
photochemical degradation of the fluorophores, tissue
isolation and extraction with chloroform and methanol
provided a crude fluorophore mixture. This extract,
when characterized directly by HPLC (Fig. 7), not only
identified A2E and iso-A2E as the major hydrophobic
components,30 but also indicated that the mixture was
substantially simpler than the complex combination of
fluorophores originally observed by Eldred.27 Since
Eldred’s purification was more laborious, it may be
speculated that some fluorophore degradation may have
led to the formation of additional components that were
not observed with a more abbreviated protocol. It is
likely that one of Eldred’s components corresponded to
Figure 6. Absorbance (UV–vis), emission, and excitation spectra of
synthetic A2E in methanol. The excitation spectrum was measured
while detecting at 600 nm emission and the emission was measured
with an excitation wavelength of 400 nm.

S. Ben-Shabat et al. / Bioorg. Med. Chem. Lett. 11 (2001) 1533–1540
1537
iso-A2E. We have also observed what we believe to be
additional A2E double bond isomers in small quantities
(Fig. 7), and these too may have been isolated in some
amount without characterization by Eldred. Additional
peaks in the HPLC profile were identified as all-trans-
retinol (vitamin A) and 13-cis-retinol (Fig. 7).30 How-
ever, no all-trans-retinal was observed; this is not sur-
prising since free retinal is reduced to retinol by retinol
dehydrogenase in photoreceptor outer segments. Ret-
inal that escapes reduction is available for further reac-
tion, with A2E being one of the products.42 In contrast
to adult eyes, the extracts of choroid/RPE isolated from
two fetal eyes (18 and 20 weeks of gestation) did not
contain A2E.30
(Fig. 9A). Collision-induced dissociation analysis (FAB-
CID MS/MS) revealed the product ions m/z 551.4 and
408.2 for the synthetic NRPE (Fig. 9B) while only one
product ion was observed from A2-PE at m/z 672.8,
representing the phosphoryl-A2E fragment (Fig. 9C).42
FAB mass spectrometric analysis has clearly indicated
that A2-PE was generated when retinal was incubated
with ROS membranes. In the absence of retinal, no
peaks corresponding to A2-PE were observed.42
The observation that A2E is generated when A2-PE is
incubated with phospholipase D42 confirms that A2-PE
is the precursor to A2E. Several lines of evidence indi-
cate that A2-PE can form within the photoreceptor
outer segment membrane before phagocytosis by the
RPE cell. Thus, the absence of all-trans-retinal in RPE
cell extracts30 and the observation that A2-PE forma-
tion from all-trans-retinal and PE is optimal at neutral
pH42 are incompatible with the formation of A2E in the
acidic environment of RPE cell lysosomes.46 In addi-
tion, the abundant lipofuscin that is observed in RPE
cells of antioxidant-deficient rats47 and in the eyes of
patients with Stargardt’s disease and retinitis pigmen-
tosa20,48,49 coexists with deposits of autofluorescent
pigment in photoreceptor cells. Furthermore, mass
spectrometric analysis42 confirmed that A2-PE is the
orange-colored fluorophore17,28,50,51 present in degen-
erating photoreceptor outer segment debris of RCS rats,
an animal model of retinal degeneration. Significant
accumulations of A2-PE have also been observed in the
photoreceptor outer segments of knockout mice lacking
the abcr gene,44 which encodes a putative all-trans-ret-
inal transporter.52,53 And lastly the formation of [14C2]-
A2-PE has been measured in excised whole retinas fol-
lowing [14C2]-ethanolamine incorporation and illumina-
tion of the retinas to release endogenous all-trans-retinal
(unpublished observation). The observation that [14C2]-
A2-PE was not formed when retinas were incubated in
the dark (unpublished observation), together with the
finding that A2-PE and A2E formation in abcr null
Biosynthesis of A2E
Recent evidence indicates that the biogenesis of A2E is
initiated when all-trans-retinal (8), which is released
from photoactivated rod and cone visual pigment,
reacts with the membrane phospholipid phosphatidyl-
ethanolamine (PE, 15) to form a PE-all-trans-retinal
Schiff base conjugate (N-retinylidene-phosphatidyl-
ethanolamine; NRPE) 16 (Fig. 8).42ꢀ45 This adduct
undergoes a [1,6]-proton tautomerization generating the
phosphatidyl analogue of enamine 11, which reacts with
a second molecule of all-trans-retinal (cf. Fig. 5).30 After
aza-6p-electrocyclization and auto-oxidation, the fluor-
escent phosphatidyl-pyridinium bis-retinoid A2-PE (17)
is formed. Finally, hydrolysis of the phosphate ester of
A2-PE yields A2E (1).30,42 This biogenic scheme is
summarized in Figure 8.
Mass spectrometry was utilized to confirm the forma-
tion of A2-PE and to identify this pigment in complex
mixtures. In unpurified reaction samples prepared from
synthetic dipalmitoyl-PE, NRPE and A2-PE were iden-
tified by electrospray ionization (ESI) mass spectro-
metry. Protonated molecular ions of NRPE and A2-PE
provided peaks at m/z 958.6 and 1222.9, respectively
Figure 8. Biosynthesis of A2-PE and A2E in the retina. all-trans-Ret-
inal, which is released from opsin after photoisomerization of ground
state 11-cis-retinal, reacts with phosphatidyl-ethanolamine in the disk
membrane to produce the N-retinyl-phosphatidyl-ethanolamine Schiff
base (NRPE). If additional all-trans-retinal is present in the photo-
receptor outer segment, NRPE will react further to generate A2-PE.
Hydrolysis of the phosphate ester, potentially by phospholipase D in
the RPE, yields A2E.
Figure 7. HPLC of eye extracts. The chromatograms are the analysis
of an aged (80-year-old) human eye and fetal eye tissue by C18
reverse-phase HPLC, as described in Parish et al.30 Only 5% of the
aged eye (upper trace) was injected, while analysis of the fetal eye
(lower trace) indicated no A2E or iso-A2E.

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S. Ben-Shabat et al. / Bioorg. Med. Chem. Lett. 11 (2001) 1533–1540
mutant mice are dependent on light exposure44 also
confirms that the light-dependent release of all-trans-
retinal is a prerequisite of A2-PE formation.
neutral, unprotonated form of the amine can cross the
hydrophobic phospholipid membrane, but cannot traverse
back across the membrane after protonation. Accumula-
tion of lysosomotropic tertiary amines leads to an increase
in lysosomal pH. However, in the case of A2E, amine
protonation cannot be the explanation for lysosomal
compartmentalization and decreased acidity since A2E is a
quaternary pyridinium salt that clearly cannot be proto-
nated and deprotonated. An alternative explanation for
an A2E-associated change in lysosomal pH is that it
occurs as a result of detergent mediated perturbations of
the membrane-bound adenosine triphosphatase that
actively pumps protons into the lysosome.56ꢀ58 Indeed,
the ability of A2E to exert detergent-like activity on
membranes, leading to a loss of membrane integrity, has
not only been demonstrated,35 but it is also consistent
with the amphiphilic nature of A2E.
The cleavage of A2-PE by phospholipase D to generate
A2E indicates that, as opposed to being acid-mediated,
the in vivo hydrolysis of A2-PE may occur enzymati-
cally. Additional support for this notion is provided by
the fact that A2E is not generated when A2-PE is incu-
bated in an acidic environment that mimics the lysosome
(pH 5.0) (unpublished observations). Nevertheless, the
site of hydrolysis of A2-PE has not been clarified.
A2E in a Cellular Environment
When A2E is delivered to RPE cells in culture, it accu-
mulates intracellularly (Fig. 10) and demonstrates an
affinity for acidic organelles,35 a behavior which repli-
cates the lysosomal compartmentalization of A2E in
vivo. The mechanism by which A2E is amassed within
lysosomes is not understood, nor is the means known by
which A2E causes an elevation in lysosomal pH.23 In
general, lysosomotropic tertiary amines become locked
insides lysosomes as a result of being protonated within
the acidic environment of these organelles.54,55 The
While it is clear that there are levels of A2E that are
tolerated by RPE cells, critical intracellular concentra-
tions can be reached, above which A2E is damaging to
cells.35 Thus, not only can A2E mediate detergent-like
effects on cell membranes,35 but when accumulated by
cultured RPE cells, A2E also bestows a sensitivity to
blue light damage34,59 that is proportional to the A2E
content of the cells and that is not manifested by cells
devoid of A2E.34 While the blue region of the spectrum
has a marked ability to induce the death of A2E-con-
taining RPE, longer wavelength green light is con-
siderably less effective.34 This wavelength dependence is
consistent with the absorbance and excitation spectra of
A2E. Based on several criteria, the loss of A2E-loaded
RPE that are exposed to blue light occurs by means of
apoptotic cell death,34 and involves the activation of
cysteine-dependent proteases (caspases) to execute the
cell death program.60
Figure 9. Mass spectrometric analysis of NRPE and A2-PE. (A)
Dipalmitoyl-PE (DP-PE) was reacted with all-trans-retinal to provide
a mixture of PE, NRPE and A2-PE, which was analyzed by electro-
spray ionization MS. Peaks corresponding to the masses of DP-PE
(starting material), dipalmitoyl-NRPE (DP-NRPE), and dipalmitoyl-
A2-PE (DP-A2-PE) were observed. (B) Collision-induced dissociation
analysis of DP-NRPE provided peaks that correspond to the frag-
mentation shown in the figure. (C) Collision-induced dissociation
analysis of DP-A2-PE provided one fragment peak, which corre-
sponded to phosphoryl-A2E.
Figure 10. Fluorescence scanning confocal microscopic imaging of
RPE cells that have accumulated A2E in culture. Internalized A2E
presents as autofluorescent granules having a perinuclear distribution.
Nuclei (red) are stained with propidium iodide. Cell borders are out-
lined by immunolabeling with an antibody to ZO-1, a tight junction-
associated protein (arrows). In this image, A2E appears green because
of the use of FITC appropriate filters. Scale bar, 20 mM.

S. Ben-Shabat et al. / Bioorg. Med. Chem. Lett. 11 (2001) 1533–1540
1539
Figure 11. Model of the relationship between the ABCRtransporter and A2E biogenesis. The putative ligand exported by the ABCRtransporter is
NRPE, the Schiff base of all-trans-retinal with PE. It is proposed that NRPE is transported to the cytoplasmic face of the outer segment disk where
all-trans-retinal is reduced to all-trans-retinol. A retinol binding protein then carries retinol to the RPE where it is isomerized and oxidized back to
11-cis-retinal. The chromophore can then be reincorporated into opsin to form the ground state pigment, which is then ready to receive a photon of
light. When ABCR activity is reduced or absent, NRPE accumulates in the disk membrane. Reaction with an additional molecule of all-trans-retinal
generates A2-PE. Phagocytosis of shed outer segment membrane and phosphate hydrolysis of A2-PE, perhaps by phospholipase D (PLD), leads to
the deposition of A2E in the RPE cell. When accumulated to critical concentrations, A2E can lead to RPE cell death. This schematic was derived by
compiling existing models and published data.30,34,35,42,44,52,53,64,65 ABCR, ATP-binding cassette transporter in the retina; PE, phosphatidyl-etha-
nolamine; NRPE, retinal-PE Schiff base.
Clinical Significance of A2E Accumulation
Acknowledgements
Experiments performed in the authors’ laboratories
were supported by National Institutes of Health grants
GM-36564 (K.N.) and EY-12951 (J.R.S.), and unrest-
ricted funds from Research to Prevent Blindness (J.R.S.).
Studies demonstrating that A2E, when present in suffi-
cient intracellular concentrations, can be damaging to
RPE cells23,34,35,59 are particularly relevant to the non-
neovascular, atrophic form of AMD that accounts for
up to 21% of the blindness caused by AMD1 and that is
characterized by a massive accumulation of lipofuscin
preceding RPE cell death.23 While multiple factors con-
tribute to the onset of AMD, nonexudative AMD has
been linked, in some cases, to mutations in the gene
encoding for ABCR, the photoreceptor-specific ATP-
binding cassette transporter.61ꢀ63 Interestingly, the sub-
strate transported by ABCRin cone and rod outer seg-
ments64 is suggested to be NRPE (16), the Schiff base
adduct formed in the first step of A2E biogenesis (Fig.
10).52,53,65 It has been proposed that the function of
ABCRis to transport all- trans-retinal across the outer
segment disk membrane thereby aiding its reduction to
all-trans-retinol by retinol dehydrogenase (Fig.
11).52,53,65 Moreover, it is clear that reduced ABCR
activity results in NRPE accumulation and, with the
availability of free all-trans-retinal, production of A2-
PE in the disk membrane (Fig. 11). Subsequent phago-
cytosis of A2-PE-containing disk membranes together
with A2-PE hydrolysis leads to the deposition of A2E in
the RPE. Support for this model is provided by analyses
demonstrating that A2-PE and A2E levels are sig-
nificantly accentuated in photoreceptor outer seg-
ments44 and RPE cells,53 respectively, when ABCR
function is eliminated in mice. Moreover, the means by
which abcr mutations predispose the RPE to cell death
in nonneovascular AMD and some other forms of ret-
inal disease62,66,67 is understandable when considered
within the framework of A2E biogenesis and the harm-
ful effects of its accumulation in the cell.
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