(Received for publication, December 26, 1995; and in revised form, February 7, 1996)
From the
Many amyloid diseases are characterized by protein aggregations
linked to oxidative stress. Such diseases including those of the brain,
muscle, and blood vessels exhibit plaques containing -amyloid
(A
). Here we demonstrate that Alzheimer's precursor protein
(
APP) and A
are present at low levels in normal lenses and
increase in intact cultured monkey lenses treated with
H
O
or UV radiation (known cataractogenic
agents), and with phorbol 12-myristate 13-acetate. AP-1 factor binding,
shown by others to up-regulate
APP expression, increased in the
monkey lenses treated with H
O
, UV radiation, or
phorbol 12-myristate 13-acetate and paralleled the increase in
APP
expression. Rat lenses exposed to oxidative stress showed increased
APP in the anterior epithelium and cortex. Incubation of cultured
rabbit lens N/N1003A epithelial cells with A
induced inclusions
and vacuoles and was cytotoxic. A
cross-reacting protein was
readily detected in the cortex of a cataractous human lens. Our data
show that
APP and A
increase in mammalian lenses as part of a
response to H
O
or UV radiation and suggest that
they may contribute to the mechanism by which oxidative damage leads to
lens opacification.
Cataract impairs vision by opacification of the ocular
lens(1) . A large percentage of cataracts are of the
age-related (senile) type suggesting an environmental component to this
degenerative disease (1, 2, 3) . Both protein
aggregation (4, 5, 6, 7, 8) and vacuole
formation(9, 10, 11, 12, 13) are associated with several types of cataracts.
Alzheimer's protein is associated with amyloid diseases of the
muscle(14, 15) , brain(16, 17) , and
blood vessels(18) , which are also characterized by protein
aggregates. A pathological hallmark of Alzheimer's disease (AD) ()is the presence of amyloid plaques, which contain
-amyloid protein (A
) in brain, a proteolytic cleavage product
of the Alzheimer's precursor protein
(
APP)(15, 18) . In muscle fiber degeneration seen
in neuromuscular diseases, including inclusion body myositis,
A
-containing plaques are found(14, 15) . A
plaques have also been identified in the kidneys and lungs of patients
with Alzheimer's disease(19) . In the human eye, A
and
APP have been detected in the retina and are associated with
aging and retinal degeneration(20) .
The APP gene is
found on chromosome 21 (21) and almost all patients with
trisomy 21 (Down's syndrome) manifest AD, other A
plaque
formation diseases, and
cataracts(22, 23, 24) . A 4-5-fold
increase in
APP expression over normal levels has been documented
in cells from Down's syndrome patients(25) .
Interestingly, a 2- and 2.5-fold increase in
APP and A
,
respectively, has been demonstrated in fibroblasts from non-
APP
gene duplication familial AD associated with chromosome
14(26) .
In addition to A, neuritic plaques found in AD
brain tissue also contain
B-crystallin, a major ocular lens
protein(27) .
B-Crystallin has been associated with a host
of other neurodegenerative diseases (see (28) and (29) for reviews).
B-Crystallin is a small heat shock
protein(30) , possesses a chaperone-like anti-aggregative
function(31) , and mediates intermediate filament assembly in vitro(32) . However, the possible role of
B-crystallin in either promoting or inhibiting amyloid filament
formation is not known.
A belongs to a family of amyloidogenic
proteins(16) , which share a strong predisposition to form
insoluble
-sheet structures leading to fibrillar
aggregation(33) . A
and other amyloid proteins contain a
peptide consensus repeat motif: GXXX (where X is one
of 11 possible residues), that has been implicated in promoting
nucleation of amyloid fibrils(34, 35) . The seeding of
protein aggregate fibrillar structures by amyloid proteins like A
has been proposed as part of the pathogenic mechanism in amyloid
disease (35) .
A has also been demonstrated to be
cytotoxic; A
added exogenously to established neuronal or
cerebrovascular smooth muscle cell cultures induced vacuoles and
increased cell
death(15, 36, 37, 38, 39, 40) .
However, when added very early in the establishment of primary neuronal
cell cultures, A
can produce a neurotrophic effect(36) .
APP and specific cleavage products are normal constituents of many
cell types and appear to be involved with facilitating
membrane-associated functions(37, 38) . For example,
in fibroblasts,
APP is released from cells into the medium and has
an autocrine function in growth regulation(43) .
Oxidative
stress resulting from endogenous production of reactive oxygen species
is strongly linked with amyloid disease (44, 45) and
cataract(1, 2, 3) . A toxicity was
recently shown to be mediated by H
O
in PC12
cells(45) . The potential of antioxidants in inhibiting both
cataract and AD has been indicated. For example, populations with long
term consumption of vitamin C (
60 mg/day) from foods and/or
supplements have reduced risks of cataract (42, 46) .
Experimentally, vitamin E was demonstrated to protect nerve cells from
A
toxicity(40) .
Cellular responses to oxidative stress
from UV radiation and HO
include herpes and
human immunodeficiency virus induction and increases in specific gene
expression (47, 48, 49, 50) via
activation of signaling pathways which involve the Ras (50) and
Src (51) proteins, and include the transcription factors AP-1 (50) and NF-
B(49) . A direct role for AP-1 factors
in increased
APP expression by phorbol ester (phorbol 12-myristate
13-acetate, PMA) treatment in human glial cells and HeLa cells has been
described(52) . PMA stimulation leads to increased AP-1 binding
via a protein kinase C-mediated signaling pathway and has been used as
a positive control in experiments to test AP-1 activation during
oxidative stress(48, 50) . We have studied the effects
of oxidative stress using rat and rhesus monkey lenses in organ culture
as our model system. In the present report, we demonstrate that
APP and A
normally present in mammalian lenses are increased
by oxidative stress, that AP-1 binding is increased in both cultured
lenses and lens epithelial cells, and that A
is cytotoxic to
cultured lens epithelial cells.
Human eyes were obtained from the National Disease Research
Interchange. Human lenses were then frozen intact in embedding compound
for sectioning. Staining was carried out with a monoclonal directed
against A (DAKO) and developed using a Vectastain ABC and DAB kits
(Vector).
To begin our investigation of the lenticular expression of
Alzheimer's proteins following exposure to oxidative stress, we
assayed the levels of APP and A
proteins in clear intact
monkey lenses. Levels of
APP and A
increased in the monkey
lenses following treatment with either UV
radiation or
H
O
or PMA. Lenses were removed, incubated
overnight to acclimate in organ culture, and treated the following
morning with 1 mM H
O
, UV
/8 min, or 100 ng/ml PMA and incubated for an additional 6 h
unless otherwise indicated. Three cross-reacting bands (
92 kDa
(
APP),
32 kDa, and
4 kDa (A
)) increased following
treatment with 1 mM H
O
(6 or 24 h),
UV, or PMA (Fig. 1). PMA has been shown elsewhere to increase
APP expression in HeLa and glial cells through the activation of
AP-1(52) . Detectable amounts of Alzheimer's proteins in
unstressed control lenses (Fig. 1, control) may reflect
normal expression of this gene in the monkey lens. By contrast, an
example of gene expression that is not increased by oxidative stress is
the glyceraldehyde-3-phosphate dehydrogenase gene(58) .
Figure 1:
Western
blot of proteins from rhesus monkey lenses in organ culture treated as
follows: 100 ng/ml PMA for 6 h, UV for 8 min and
incubated for another 6 h at 37 °C under CO
, or 1
mM H
O
for 6 h or 24 h (see
``Materials and Methods''). The blot was probed with
antiserum directed against A
.
Evidence supporting expression of APP in untreated mammalian
lenses comes from RT-dependent PCR using RNA isolated immediately after
lens removal from 8-week-old FVB/N mice (Fig. 2). The presence
of
APP mRNA is indicated by the RT-dependent detection of a
473-base pair amplified product using oligonucleotide primers contained
within separate exons of the mouse
APP gene. These data indicate
that
APP is present at the RNA and protein levels in monkey and
mouse lenses.
Figure 2:
Agarose gel showing products of a RT-PCR
analysis of mouse APP RNA. For each reaction 1 µg of freshly
isolated total RNA from mouse lens was primer extended using Primer A
and subsequently amplified by PCR with Primers A and B. Left
lane, ethidium bromide-stained
X174 DNA molecular weight
markers (New England Biolabs); middle lane, no RT added; right lane, RT added for primer extension synthesis of
cDNA.
Activation of the Ras and Src pathways and increases
in AP-1 binding are known to occur during oxidative
stress(48, 50, 51) , and AP-1 was shown to
increase APP expression(52) . In this regard, we have
found a significant increase in AP-1 binding to oligonucleotides
containing its cognate binding site using standard electrophoretic
mobility shift assays after treating monkey lenses or cultured N/N1003A
rabbit lens epithelial cells with 100 ng/ml PMA, 1.0 mM H
O
or UV
radiation (Fig. 3). The increase in AP-1 binding paralleled the increase
in
APP which was demonstrated in the same monkey lens extracts in Fig. 1. Increased AP-1 binding was prevented in N/N1003A cells
by pretreatment with tyrophostin, an inhibitor of tyrosine
phosphorylation (49) (Fig. 3). These results are
consistent with the effects of H
O
and tyrosine
phosphorylation inhibitors on c-fos and c-jun expression in cultured rat lenses (58) . As c-fos and c-jun expression have been shown to increase with
oxidative stress in lens cells(58) , our experiments do not
necessarily distinguish between increased AP-1 activation and increased
AP-1 factor synthesis.
Figure 3:
Electrophoretic mobility shift assays. A, rhesus monkey lens in organ culture treated with 1.0 mM HO
, 100 ng/ml PMA, or UV
radiation for 5 or 10 min. After treatment lenses were incubated
for 6 or 24 h as indicated. B, N/N1003A cells were stimulated
with 100 ng/ml PMA or 50, 125, or 250 µM H
O
for 6 h. Lens or cell extracts bound to
AP1 cognate site-containing oligonucleotides (Santa Cruz) were resolved
on 5% acrylamide gels. Filled arrow indicates specific complex
formation, and open arrow indicates a nonspecific complex.
Tyrosine phosphorylation inhibitor tyrophostin (30 µM) was
added 1 h prior to treatment of the N/N1003A
cells.
We next localized the expression of APP
in adult rat lenses treated in organ culture for 24 h with 0, 125
µM, or 250 µM H
O
(Fig. 4A). The immunostaining of
APP in
H
O
-treated lenses was predominately in the
anterior epithelial layer and in cortical fiber cells. Low amounts of
APP were also detected in untreated rat lenses and may reflect
APP present in normal lenses and/or base-line oxidative stress
incurred during lens removal and culturing. A control SV40 T-antigen
antibody showed no staining in rat lenses either with or without
H
O
treatment (data not shown). Staining of a
cataractous human lens with a different monoclonal antibody directed at
A
is shown in Fig. 4B. We detected A
in the
cortical fiber cells below the epithelial cell surface layer (blue/black color) with this antibody in human lenses. These
results differ from H
O
treated rat lenses,
where
APP was detected in both the epithelial and cortical regions (Fig. 4A).
Figure 4:
A,
immunohistochemical detection of APP (BMB) in rat lenses treated
for 24 h. Rat lenses were placed in organ culture and
H
O
was added after 16 h culture and incubated
for an additional 24 h at 37 °C/5%CO
. Lenses were
subsequently frozen and sectioned for antibody staining (M. A.
Crawford, National Institutes of Health, Bethesda, MD). B, for
comparison, staining of a cataractous human lens with a monoclonal
antibody directed against A
(DAKO) or against SV40 large T antigen
(Oncogene).
To assess the effect of A on
cultured lens cells, we cultured N/N1003A cells in the presence of
A
-(1-40) as described elsewhere (38) . Extensive
inclusions and vacuoles were observed with exposure to 20 µg/ml
A
-(1-40) for 24 h (Fig. 5A). In addition,
<50% of the epithelial cells remained attached to the surface of the
culture dish after 5 days of culture in the presence of 50 µg/ml
A
-(1-40) (Fig. 5B). A
-(25-35)
peptide (15, 38) (20-50 µg/ml) was
cytotoxic for N/N1003A cells in culture (data not shown), producing
vacuoles and decreasing cell attachment. These cytotoxic effects were
produced by A
at similar concentrations in N/N1003A cells as for
neuronal cells(36) . N/N1003A cells cultured in the presence of
diluent alone, cytochrome c (20 µg/ml), or broad range
molecular weight standards (myosin,
-galactosidase, phosphorylase b, serum albumin, ovalbumin, carbonic anhydrase, trypsin
inhibitor, lysozyme, and aprotinin at 20 µg/ml each protein;
Bio-Rad) all appeared as in the control (Fig. 5A).
Experiments using cells of neuronal origen have indicated that
A
-(25-35) peptide, where the amino acid order has been
scrambled(40) , and A
-(1-40) peptide, where the
amino acid order has been scrambled had no effect on A
inducing
its own production in cultured muscle cells(15) .
Figure 5:
A, rabbit lens epithelial
N/N1003A cells in culture treated for 24 h or 42 h with 20 µg/ml
A (Bachem). Control cells were cultured in
solvent vehicle, cytochrome c (20 µg/ml), or broad range
protein mix (Bio-Rad) at 20 µg/ml and appeared as in the control. B, N/N1003A cells cultured in the presence of 50 µg/ml
A
-(1-40) for 5 days. Cell viability was performed by
counting >500 cells/unit surface area.
We have presented data showing that oxidative stress can
activate AP-1 factors in monkey lenses and rabbit lens epithelial cell
cultures and induce APP and A
in monkey and rat lenses in
organ culture. We have localized the increase in
APP in rat lenses
to the epithelium and cortex. Moreover, A
produces vacuoles and is
toxic to cultured rabbit lens epithelial cells. We have also detected
A
cross-reacting protein in the cortex of cataractous human lenses
in their opaque cortical regions. Together, these observations are
consistent with the possibility that
-amyloids may contribute to
the process of cataract formation.
Many cataracts, including
age-related
opacities(4, 5, 6, 7, 8) ,
the Nakano (59) and Emory ((60) ; see (61) for
review) mouse hereditary mouse opacities, and x-ray induced
opacities(62) , are associated with the accumulation of
insoluble protein aggregates. Since the ability of A to nucleate
protein aggregation in amyloid disease is well established (33, 34, 43) , it would seem from the present
data that a search for the involvement of
-amyloid proteins in
nucleation events associated with cataract is warranted. The formation
of heavy molecular weight protein fractions, believed to be
intermediates in protein insolubilization (see (7) for
review), and phase separation phenomena, which are reported to occur
during the early stages of
cataractogenesis(63, 64, 65, 66) ,
are processes that might be affected by A
. The present
immunohistochemical data in both rat and human lenses suggest that
-amyloids could contribute more to cortical than central nuclear
cataracts, as both A
and
APP were found predominately in the
epithelial and cortical regions rather than the central nuclear regions
of the lens. It is not known if the potential deleterious effects of
APP proteolytic products on lens cell development or homeostasis
require identical higher order A
structures as have been
implicated in AD.
APP and their cleavage products are normal
constituents of many cell types. However, the production of
A
-containing proteins capable of fibril formation and/or cytotoxic
effects appears to be a salient feature of amyloidogenic diseases where
A
plays a role. The up-regulation of A
is most dramatic in
trisomy 21 individuals (4-5-fold), where gene dosage plays a
role, however, both A
and
APP are also increased more than
2-fold in some familial AD involving chromosome 14, suggesting a
physiological cell signaling mechanism for A
up-regulation as
well.
The intracellular vacuoles associated with the cytotoxicity of
A in cultured N/N1003A lens epithelial cells in the present study
are also consistent with
-amyloid proteins contributing to
cataract. Intracellular vacuoles appear as a pathological hallmark in
diabetes-related sugar cataracts where both osmotic (67) and
oxidative (68) stresses are involved. Intracellular vacuoles
appear in lens epithelial cells cultured in the presence of low
concentrations of glucose and galactose (69) and appear in the
central epithelium as the first detectable abnormalities during
galactose cataract formation in rats(70) . Similar
vacuolization occurs in L-buthionine sulfoximine-induced
cataract in mice(71) . Moreover, recent experiments on cultured
rat lenses have indicated that oxidative stress caused by photochemical
insult leads to vacuole formation and irreversible damage to the
epithelial cells preceding and accompanying
opacification(72, 73) .
Since oxidative stress is
considered a major cause of
cataract(3, 67, 73) , our finding that
HO
and UV
radiation increase
APP and A
in cultured intact lenses supports the idea that
these
-amyloid proteins play a role in cataract formation. While
it has been shown that A
can induce H
O
in
mediating toxicity, and that anti-oxidants protect cells from A
toxicity in primary cultures and clonal cell lines derived from the
central nervous system(44) , to the best of our knowledge the
present study is the first report of oxidative stress increasing
Alzheimer's proteins and is consistent with the role of AP-1 in
APP gene expression. An induction of H
O
by
A
could provide a feedback loop mechanism in lenses allowing
A
to increase its own expression, as has been observed in some
muscle degeneration diseases(15) . The ability of oxidative
stress to induce A
suggests that the manifestation of amyloid
diseases could vary in different tissues depending on the amount of
exposure to oxidative stress. Moreover, the proliferative-like response
to oxidative stress involving such factors as AP-1 suggests that the
state of growth and/or differentiation of a given cell type could also
be a factor. Since our experiments suggest that both oxidative stress
and phorbol ester-mediated activation of AP-1 can increase
Alzheimer's disease proteins in lens, one must consider that
varied pathways and a multiplicity of signaling routes can potentially
increase the ectopic expression of deleterious proteins like A
.
The mechanism of neurotoxicity attributed to the A peptide
involves the generation of reactive oxygen species and destabilization
of cellular calcium homeostasis(74) . Indeed, recent
experiments indicate that different amyloidogenic peptides, such as
amylin, and
-microglobulin share this mechanism of
affecting neurotoxicity(74) . Thus it appears possible that in
addition to a multiplicity of signaling pathways, several amyloidogenic
peptides exist that potentially could give rise to toxic effects in
tissues including the lens.
The mammalian lens is composed of
anterior epithelial cells, which begin to elongate at the equatorial
margin. These cells withdraw from the cell cycle and produce large
amounts of crystallins. Terminal fiber cell differentiation is
associated with the degradation of cellular organelles, has some
characteristics of apoptosis(75) , and involves p53 (76) and retinoblastoma (77, 78, 79) proteins. As cells in the lens
cortex normally enter growth arrest, leading ultimately to nuclear
breakdown and DNA fragmentation in the central region of the
lens(75) , we infer that an ectopic proliferative-like response
to oxidative stress, which includes A up-regulation, may be of
greater consequence than a growth arrest DNA damage response (for
review see (80) ) to the lens. This is consistent with earlier
reports showing that x-irradiation-induced cataract in amphibian lenses
is greatly inhibited by preventing cell proliferation and/or
differentiation through hormonal
manipulation(81, 82, 83) . Similarly,
x-irradiation-induced cataracts do not develop in ground squirrels
during hibernation when there is no lens cell
proliferation(82, 83) .
Oxidative stress at the levels employed in our experiments exceed that which would be experienced under normal in vivo conditions where such stress is chronic, producing effects over decades. However, low level oxidation-induced perturbations in cell signaling processes including the Ras/Src-mediated pathway for AP-1 activation (47, 48, 49, 50, 51) may help explain the observed link between environmental oxidative stress and cataract and possibly also AD. Studies that have shown the activation of herpes viral proliferation and skin cancers by sunlight (84) support this idea.
In summary, cataract is a disease
that can involve protein aggregation and vacuole formation and thus
shares similarities with AD and related A pathologies. Oxidative
stress is an important etiological factor in these diseases and
up-regulates A
and
APP in lenses and lens cell cultures. In
addition, the vacuole formation and cytotoxicity induced in cultured
lens cells by A
are similar to neuronal cell cytotoxicity
demonstrated by others. Whether A
can elicit a trophic response in
primary lens cells as it does in neuronal cells remains to be
addressed. Indeed, the normal role for these proteins in neuronal cells
as well as lens cells is not known.
APP and A
proteins
expressed in mammalian lenses induced by oxidative stress parallel the
activation of AP-1 factor binding consistent with a role for
stress-induced cell signaling in cataract formation.
Taken together,
our data raise the possibility that oxidative stress, a known pathway
for cataract formation, stimulates APP formation or aberrant
APP protein cleavage in the ocular lens and imposes a strain on
protein organization and cell integrity, contributing to lens
opacification.