Inhibition of Lysosomal Degradative Functions in RPE
Cells by a Retinoid Component of Lipofuscin
Frank G. Holz,1 Florian Schiitt,1 Jilrgen Kopitz,2 Graig E. Eldred,3 Friedrich E. Kruse,1
Hans E. Volcker,1 and Michael Cantz1
PURPOSE. TO investigate the effect of the lipofuscin component iV-retinylidene-yV-retinylethanolamine (A2-E) on degradative functions of lysosomes in human retinal pigment epithelial (RPE) cells
and to evaluate its mechanism of action.
METHODS. A2-E was coupled to low-density lipoprotein (LDL). Human RPE cell cultures were loaded
with the A2-E/LDL complex, and controls were run with medium containing LDL alone. To
determine whether A2-E accumulated in lysosomes, cells were fractionated in a Percoll gradient,
and protein degradation was determined by metabolic labeling and measurement of the release of
low-molecular-weight radioactivity. Lysosomal degradation was distinguished from nonlysosomal
degradation by inclusion of NH4C1 in the medium. The metabolism of sulfated glycosaminoglycans
was studied by radiosulfate incorporation in pulse-chase experiments. Intralysosomal pH was
determined using a fluorescent lysosomotropic pH indicator.
RESULTS. A2-E accumulated almost exclusively in the lysosomal compartment. Lysosomal protein
degradation was reduced in a dose-dependent fashion in A2-E-treated cells. The selectivity of A2-E
on lysosomal function was demonstrated by its lack of effect on degradation of extralysosomal
protein. Lysosomal glycosaminoglycan catabolism of RPE cells was also strongly inhibited by A2-E.
Lysosomal pH was increased by A2-E.
CONCLUSIONS. The findings indicate that accumulation of A2-E in RPE cells interferes with lysosomal
functions as exemplified by its inhibitory effect on protein and glycosaminoglycan catabolic
pathways. The quaternary amine character of the A2-E apparently causes a perturbation of the
acidic intralysosomal milieu, resulting in diminished hydrolase action and consequent accumulation
of undegraded material. Such mechanism could be operative in retinal diseases associated with
excessive lipofuscin accumulation including age-related macular degeneration. (Invest Ophthalmol
Vis Set. 1999;40:737-743)
ccumulation of lipofuscin in the lysosomal compartment is a cytologic hallmark of aging in metabolically
active postmitotic cells, including neurons, cardiac
muscle cells, and the retinal pigment epithelium (RPE).' In the
latter, progressive lipofuscin accumulation is mainly a by-product of the constant phagocytosis of shed photoreceptor outer
segment discs. 2 3 The pathophysiological role of lipofuscin
accumulation in degenerative and hereditary retinal diseases,
including age-related macular degeneration (ARMD), a leading
cause of blindness in the Western world, and Best's disease and
Stargardt's disease, 4 " 7 has been debated without resolution. 8 " 16 Recently, a Schiff base reaction product, iV-retinylidene-/V-retinylethanolamine (A2-E), has been identified as a
major fluorophore in the chloroform-soluble fraction of lipo-
A
From the 'Department of Ophthalmology, Im Neuenheimer Feld
400, and institute of Pathochemistry and Neurochemistry, University
of Heidelberg, Heidelberg, Germany.
3
No current affiliation.
Supported by Deutsche Forschungsgemeinschaft, Bonn, Germany,
Grant VO 437/3-1.
Submitted for publication August 17, 1998; accepted September
30, 1998.
Proprietary interest category: N.
Reprint requests: Frank G. Holz, Department of Ophthalmology,
University of Heidelberg, Im Neuenheimer Feld 400, D-69120 Heidelberg, Germany.
fuscin in human RPE cells, 1 7 1 8 whereby A2-E represents only a
small proportion of the lipofuscin granule. The hypothesis was
raised that it might disturb lysosomal function because of its
detergent and base character. 17
To evaluate this hypothesis, we set up a cell culture model
suitable for investigating the effects of A2-E on lysosomal catabolic
processes in RPE cells. To enable efficient and specific transport
of the water-insoluble A2-E to the lysosomal compartment, A2-E
was complexed with low-density lipoprotein (LDL) particles,
which are transported to die lysosomes via receptor-mediated
endocytosis. The presence of LDL receptors in RPE plasma membrane was shown previously.19 The effects of A2-E loading on
catabolic padiways of lysosomes in human RPE cells were tested
using metabolic labeling in pulse-chase experiments. When challenged with A2-E, bodi lysosomal protein and glycosaminoglycan
catabolism were strongly inhibited. Because A2-E-treated RPE
cells showed an increase in lysosomal pH, A2-E seems to exert its
inhibitory effects at least partly via a decrease in proton concentration, thereby deranging the acidic environment required for
the action of lysosomal hydrolases.
METHODS
A2-E Loading of RPE Cells
Primary human RPE cell cultures were obtained and maintained as previously described. 20 A2-E was synthesized by cou-
Investigative Ophthalmology & Visual Science, March 1999, Vol. 40, No. 3
Copyright © Association for Research in Vision and Ophthalmology
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737
738
Holz et al.
pling all-trans retinaldehyde to ethanolamine (ratio 2:1).17
A2-E was complexed with LDL by adding 7 nmol A2-E to 5 mg
LDL particles (Sigma, Munich, Germany) in 1 ml culture medium (500 ml culture medium contained 250 ml medium
M-199, 250 ml Ham's F-12, 844 mg NaHC3, 3.5 mg glucose, 1
mg insulin, and 5 ml pyruvate 100 mM) and incubated at 37°C
for 2 hours. When the A2-E/LDL complex was analyzed by
ultracentrifugation,21 approximately 90% of the A2-E-associated fluorescence was recovered in the lipoprotein fraction.
RPE cell cultures were loaded with the A2-E/LDL complex (10
fxg per ml of culture medium) for 4 weeks, with fresh A2-E/LDL
medium exchanged every 3 to 4 days. Controls were run with
medium containing LDL without A2-E.
Subcellular Fractionation
RPE cells from 1 flask (75 cm2) treated for 4 weeks with
A2-E/LDL (10 /xg/ml medium) were harvested by trypsinization, suspended in 5 ml 5 mM HEPES, pH 7.6/0.25 M sucrose/
0.2 mM EDTA, and disrupted by nitrogen cavitation (10 minutes, 20 bar). The resulting homogenate was centrifuged
(lOOOg for 10 minutes). The postnuclear supernatant (2 ml)
was fractionated on 35 ml of a self-generating gradient of 30%
(vol/vol) Percoll (Pharmacia, Freiburg, Germany) in 5 mM
HEPES, pH 7.6/0.25 M sucrose/0.2 mM EDTA in a Sorvall rotor
T-865 (34,000 rpm, for 40 minutes) at 4°C.22 Fractions (0.8 ml)
were collected from the top of the gradient. Forty-six fractions
were obtained and analyzed by measuring marker enzyme
activities including lactate dehydrogenase (cytosol), phosphodiesterase (plasma membrane), galactosyltransferase (endoplasmic reticulum), j3-hexosaminidase (lysosomes), and succinate dehydrogenase (mitochondria).22'23 For determination of
the content of A2-E in the fractions, 500-/xl aliquots were
lyophilized, extracted with 300 jul methylene chloride/methanol (2:1; vol/vol), and dried again in a stream of nitrogen; then
the remainder was reextracted with methylene chloride, and
the fluorescence of the resulting extract was read against a
standard in a Kontron fluorometer (excitation 420 nm; emission 605 nm).
IOVS, March 1999, Vol. 40, No. 3
Catabolism of Sulfated Glycosaminoglycans
To analyze the catabolism of sulfated glycosaminoglycans, we
determined radiosulfate incorporation in pulse chase experiments by a modification of a previously published method.27
Briefly, cells were grown in 12-well tissue culture plates (approximately 2 X 105 cells per well) and pulse-labeled by
inclusion of 30 fxCi [35S]sulfate (Amersham) in 1 ml culture
medium for 3 days, followed by a 24-hour chase in nonradioactive medium. For determination of accumulation of radioactive sulfated glycosaminoglycans during the pulse phase and
their degradation during the chase, cells were harvested by
trypsinization, collected by centrifugation (1000 rpm), and
washed three times with 1 ml 0.9% NaCl. Then the cells were
solubilized in 400 /xl IN NaOH and counted for radioactivity in
a liquid scintillation counter. It had been shown in control
experiments that more than 90% of the intracellular radioactivity was glycosaminoglycan-associated. The experiments
were repeated with primary RPE cultures that originated from
donors 33, 74, and 84 years of age, to see whether the age of
the RPE cells is of importance.
Lysosomal pH
The intralysosomal pH was determined using a fluorescent
lysosomotropic compound, LysoSensor yellow/blue (Molecular Probes, Eugene, OR), according to the instructions of the
manufacturer.28 RPE cells with and without A2-E treatment
were cultured to a semiconfluent density on glass slides covered with regular medium. A third group of RPE cells without
A2-E treatment was incubated 20 minutes before pH measurements with 10 mM NH4C1 in the medium. After removal of
the medium, the cells were incubated with 25 n>\ LysoSensor
yellow/blue for 1 hour. The fluorescence was documented
with a fluorescence microscope (excitation 280 nm, emission
450 nm).
RESULTS
Protein Catabolism
Intracellular Localization of A2-E
To determine the effect of A2-E on lysosomal protein degradation, we performed pulse-chase experiments adapted from a
procedure developed for fibroblasts.24'25 Briefly, human RPE
cells were grown in 24-well tissue culture plates and treated
with A2-E/LDL (10 jag/ml culture medium) for 4 weeks. The
endogenous protein was then labeled by including 500 kBq/ml
[3H]-leucine (Amersham, Braunschweig, Germany) in the culture medium for 72 hours (pulse phase). After removal of the
radioactive medium, the cell layers were washed and chased
with nonradioactive medium (chase phase). Protein degradation was determined by measurement of the low-molecularweight radioactivity released into the medium and of the protein-bound radioactivity. Lysosomal (ammonia-sensitive)
protein degradation was blocked by adding 10 mM NH4C1 with
the chase medium, and lysosomal protein degradation was
calculated from the difference in the protein degradation rates
in the absence and presence of NH4C1.26 The lysosomal protein
degradation rates were also investigated in the presence of
various concentrations of A2-E. The experiments were repeated with primary RPE cultures that originated from donors
23, 58, and 84 years of age, to see whether age of the RPE cells
was important.
To examine the subcellular localization of the retinoid, A2-Eloaded RPE cells were fractionated in a Percoll gradient, and
the relative fluorescence of A2-E and the activities of marker
enzymes were determined in the fractions (Fig. 1). A2-E accumulated almost exclusively in the lysosomal compartment as
indicated by the identical peaks of the marker enzyme /3-hexosaminidase and the relative fluorescence of A2-E. Only a small
amount of A2-E appeared to associate with the cell membrane
as shown by a minor peak of A2-E corresponding to the distribution of phosphodiesterase activity. The results indicate that
the feeding of A2-E/LDL complexes to cultured RPE cells
proved to be highly effective in specific loading of the lysosomal compartment, providing a suitable cell culture model for
the investigation of A2-E effects on lysosomal function in RPE
cells.
Cell Morphology
Comparison of appearance of control cells and A2-E/LDL-fed
cells as a polygonal monolayer using a phase contrast microscope (Zeiss Axiovert 25; Jena, Germany) did not show any
morphologic differences. There was no obvious difference
between the control RPE cells and the A2-E/LDL-fed RPE cells
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IOVS, March 1999, Vol. 40, No. 3
Inhibition of Lysosomal Functions by A2-E
739
1 0 0 H,
10
20
30
Fractions
FIGURE 1. Subcellular distribution of cell-associated A2-E after A2-E/LDL loading of RPE cells. Isotonic
homogenates of RPE cells were fractionated in a Percoll gradient and the marker enzymes lactate
dehydrogenase ( • , cytosol), phosphodiesterase (A, plasma membrane), galactosyltransferase ( • , endoplasmic reticulum), j3-hexosaminidase (•, lysosomes), succinate dehydrogenase ( • , mitochondria), and
A2-E fluorescence (O) in the fractions determined as described in the Methods section.
regarding the occurrence of cell death (i.e., toxicity of A2-E
was not observed).
Protein Catabolism. The effect of A2-E on the degradation of intracellular proteins was determined by a method that
discriminated between lysosomal (ammonia-sensitive) and
nonlysosomal catabolism.24'26 As shown in Figure 2A, overall
endogenous protein catabolic rates were markedly reduced in
A2-E-treated cells. This reduction was exclusively due to the
ammonia-sensitive portion of protein degradation and, therefore, demonstrated the selective impact of A2-E on lysosomal
function. The lysosomal protein degradation rates were reduced by approximately 80% at 120 hours of chase in the cells
treated with A2-E (Fig. 2B). The effect of A2-E/LDL on lysosomal protein degradation was dose-dependent (Table 1); the
maximal effect with the minimum dose was achieved using a
concentration of 50 jag A2-E/LDL (containing 70 pmoles A2-E)
per milliliter of medium. When the inhibitory effect was compared in three primary RPE cell cultures originating from donors 23, 58, and 84 years of age, no difference in magnitude of
the effect was observed.
Sulfated Glycosaminoglycan Catabolism. As another
major lysosomal pathway, the metabolism of radiosulfate-labeled glycosaminoglycans was studied in pulse-chase experiments by a method devised to detect the abnormally increased
lysosomal accumulation of glycosaminoglycans in cell cultures
derived from patients with genetic mucopolysaccharidoses.25'27 The glycosaminoglycan-associated radioactivity of
control cells during the pulse phase reached a constant level
after approximately 24 hours, whereas the A2-E-treated cells
showed a greatly increased and continuing glycosaminoglycan
accumulation (Fig. 3A). During the chase, the rate of disappearance of prelabeled glycosaminoglycans was drastically reduced
in the A2-E-loaded cells (Fig. 3B). Apparently, A2-E-treated
cells are not able to catabolize endogenous glycosaminoglycans to the same extent as the controls with consequent
lysosomal storage. When the inhibitory effect was compared in
three primary RPE cell cultures originating from donors 23, 58,
and 84 years of age, no difference in magnitude of the effect
was observed.
Lysosomal pH. To find out whether the accumulated
A2-E would indeed derange the normally acidic lysosomal milieu, the intralysosomal pH was determined using a fluorescent
lysosomotropic compound, LysoSensor yellow/blue, that
would not interfere with A2-E fluorescence. As shown in Figure 4, there was yellow punctate fluorescence indicative of
acidic lysosomes in the control cells, whereas A2-E-treated
cells exhibited blue fluorescent lysosomes pointing to a pH
near neutrality. The same test was applied to RPE cells that had
been pretreated with NH4C1 and showed the expected blue
fluorescence of lysosomes (Fig. 4).
DISCUSSION
Lysosomes digest intra- and extracellular materials after autophagy, phagocytosis, and endocytosis with the help of some 40
lytic enzymes. The major material for phagocytosis in RPE cells
represents constantly shed outer segment discs of apposing
photoreceptor cells. Formation of cross-linked complexes that
are not degradable by lysosomal enzymes occurs particularly in
long-lived postmitotic cells. Lipofuscin, which accumulates
with age in the lysosomes of RPE cells, is a complex consisting
of at least 10 different fluorophores and various biomolecules,
including proteins and lipids.10 The major orange-emitting flu-
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IOVS, March 1999, Vol. 40, No. 3
LU
20
40
60
80
80
Time [hours]
100
120
Time [hours]
FIGURE 2. Degradation of endogenous protein in RPE cells. Protein degradation was determined in chase experiments after pulse-labeling with
[3H]-leucine in A2-E/LDL-treated (A, T) and control (A, V) cells, as described in the Methods section. (A) Endogenous protein degraded in absence
(A, A) and presence (•, V) of 10 mM NH4C1. (B) Ammonia-sensitive (lysosomal) protein degradation (•, A2-E-treated cells; O, controls). Results
are mean ± SD of four determinations.
orophore has recently been identified as A^-retinylidene-A^-retinylethanolamine (A2-E), which arises as a Schiff base reaction
product of ethanolamine and retinaldehyde.17'18 Both compounds are present in abundance in the outer retina, with
ethanolamine as a component of the membrane lipid phosphatidylethanolamine and retinaldehyde as the oxidized form
of vitamin A.
Although several retinal diseases including Stargardt's disease, Best's disease, and ARMD are associated with lipofuscin
1. Dose Dependency of Inhibition of
Endogenous Protein Degradation in Lysosomes after
Treatment with A2-E/LDL
TABLE
A2-E
(pmol/ml medium)
Endogenous Protein Degraded in
Lysosomes (%)*
0
7
35
53
70
175
•Values are means ± SD.
34.4 ±
31.8 ±
24.28 ±
16.15 ±
9.8 ±
9.98 ±
0.17
4.38
5.2
0.59
0.59
1.87
accumulation in the RPE,4'8'13 the question of whether or not
lipofuscin is detrimental to metabolic processes in the RPE cell
has been controversial. Although some have held the view that
the pigment is merely an accumulation of metabolically inert
residues, others have assumed that lipofuscin accumulation
interferes with cellular metabolism.1'29 The in vitro findings
reported herein demonstrate that a major component of lipofuscin indeed does affect the lysosomal metabolism in human
RPE cells. Accumulation of the retinoid A2-E potently impairs
lysosomal function as exemplified by its inhibitory effects on
protein and sulfated glycosaminoglycan catabolic pathways.
Because the intracellular degradation of proteins not only
occurs in the lysosomes but also in other cellular compartments, it is fitting that the inhibitory effect of A2-E was solely
confined to the lysosomal pathway. With regard to sulfated
glycosaminoglycans, it was known from studies in cultured
fibroblasts that they are exclusively degraded in lysosomes,25
so any major disturbance of lysosomal function should manifest
itself in a reduced degradation rate with a buildup of undigested material, as was indeed observed in the cells that had
been exposed to A2-E. The finding of a fluorescence shift of a
pH-sensitive lysosomotropic indicator dye shows that the accumulation of A2-E leads to an increase in lysosomal pH.
Evidently, the quaternary amine character of A2-E causes a
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Inhibition of Lysosomal Functions by A2-E
IOVS, March 1999, Vol. 40, No. 3
uu
741
«
90 ;
80 -
70 •
60 •
c
g
50
(D
Q
VK
V
^
40
30
20 -
10 0
Time [hours]
B
I
I
I
12
18
24
Time [hours]
FIGURE 3. Metabolism of [35S]-sulfate-labeled glycosaminoglycans in RPE cells. Intracellular radioactive glycosaminoglycans during the pulse (A)
and chase (B) were determined as described in the Methods section (A2-E-treated RPE cells from three different donors • , • , •; controls A, O,
• ) . Results are mean ± SD of four determinations.
perturbation of the acidic intralysosomal milieu that in turn
may lead to greatly diminished hydrolase action and accumulation of undegraded material in the lysosomal compartment.
Because A2-E also has detergent character,17 it was possible
that it might have disturbed lysosomal function by dissolving
the lysosomal membrane. If so, however, one would expect
the lysosomal marker enzyme /3-hexosaminidase to leak into
the cytosol, which was clearly not seen in- the subcellular
fractionation experiment shown in Figure 1, where both /3-hexosaminidase and A2-E codistributed in a sharp peak with a
density typical of lysosomes. A direct inhibitory interaction of
A2-E with lysosomal enzymes may also be considered; however, that was not investigated here. The few reports on agerelated changes in RPE lysosomal enzyme activities are thus far
conflicting and cannot clarify the issue.30 The importance of
the lysosomal apparatus for maintaining retinal integrity is
underscored by the existence of genetic lysosomal enzyme
deficiency diseases such as the mucopolysaccharidoses, in
which the intralysosomal accumulation of sulfated glycosaminoglycans may lead, among other signs, to retinal degeneration. 3132
Besides an inhibitory effect on lysosomal function, lipofuscin might act as a sensitizer for generation of reactive oxygen species and, thus, damage biological tissue. Generation of
free radicals on light irradiation has been demonstrated using
both isolated lipofuscin granules and synthetic A2-E.33'34 Further investigations are needed to evaluate whether this mechanism contributes to RPE cell dysfunction in association with
lipofuscin/A2-E accumulation in the lysosomal compartment.
The present results strengthen the view that lipofuscin
accumulation with age is of pathophysiological importance in
retinal disease. It has been postulated that excessive levels of
lipofuscin in the RPE contribute to the pathogenesis of
ARMD.9'35"37 Genetically determined macular degeneration including Stargardt's disease and Best's macular dystrophy4"7 has
been associated with a faster accumulation of lipofuscin in the
RPE. In Stargardt's disease the RPE contains up to seven times
more lipofuscin than normal, and this is associated with retinal
degeneration.38 Histopathologic investigations have demonstrated an association of abnormal accumulation of lipofuscin
with degeneration of RPE cells and adjacent photoreceptors in
an inherited retinal dystrophy of dogs.39 In humans, photoreceptor density was found to correlate with the lipofuscin
concentration of the apposing RPE cells.40 In vivo investigations using scanning laser ophthalmoscopy have demonstrated excessive lipofuscin accumulation in association with
various manifestations of ARMD.16'41 In areas of increased
lipofuscin-mediated fundus autofluorescence, the development of geographic atrophy has been demonstrated in a longitudinal observation in eyes with ARMD (authors' unpub-
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742
Holz et al.
fOVS, March 1999, Vol. 40, No. 3
CONCLUSIONS
The present results suggest that the retinoid A2-E that accumulates as a major lipofuscin component in aging RPE cells interferes with normal lysosomal function. Thus, the feeding of A2-E
to RPE cells causes a perturbation of the acidic intralysosomal
milieu that leads to diminished hydrolase action and accumulation of undegraded material. Loading of the lysosomal compartment of human RPE cells with A2-E after complexing to
LDL can therefore serve as an in vitro model for RPE aging and
for further studies on retinoid-related cellular effects. Our findings may contribute to the understanding of the pathogenesis
of degenerative diseases of the outer retina associated with
lipofuscin accumulation such as ARMD.
Acknowledgments
The authors thank Volker Ehemann for advice and Cornelia Lehmann
for skillful technical assistance (Institute of Pathochemistry and Neurochemistry, Heidelberg University).
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FIGURE 4. lntralysosomal pH of RPE cells. Control and A2-E/LDLtreated cells were probed with the fluorescent lysosomotropic indicator LysoSensor yellow/blue and observed with a fluorescence microscope. (A) Untreated control cells, yellow fluorescence indicates acidic
milieu. (B) A2-E-treated cells, blue fluorescence indicates milieu
around neutrality. (C) Control cells treated with NH4C1, blue fluorescence indicates neutrality. Original magnification, X1000.
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