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The Journal of Neuroscience, August 15, 1998, 18(16):6207-6217
Survival of Cultured Neurons from Amyloid Precursor Protein
Knock-Out Mice against Alzheimer's Amyloid- Toxicity and Oxidative
Stress
Anthony R.
White1,
Hui
Zheng2,
Denise
Galatis1,
Fran
Maher1,
Lars
Hesse3,
Gerd
Multhaup3,
Konrad
Beyreuther3,
Colin L.
Masters1, and
Roberto
Cappai1
1 Department of Pathology, The University of Melbourne
and The Mental Health Research Institute, Parkville, Victoria,
Australia, 3052, 2 Department of Genetics and Molecular
Biology, Merck Research Laboratories, Rahway, New Jersey 07065, and
3 Center of Molecular Biology, The University of
Heidelberg, 69120 Heidelberg, Germany
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ABSTRACT |
Studies on the amyloid precursor protein (APP) have suggested that
it may be neuroprotective against amyloid- (A) toxicity and
oxidative stress. However, these findings have been obtained from
either transfection of cell lines and mice that overexpress human APP
isoforms or pretreatment of APP-expressing primary neurons with
exogenous soluble APP. The neuroprotective role of endogenously expressed APP in neurons exposed to A or oxidative stress has not
been determined. This was investigated using primary cortical and
cerebellar neuronal cultures established from APP knock-out (APP /) and wild-type (APP+/+)
mice. Differences in susceptibility to A toxicity or oxidative stress were not found between APP/ and
APP+/+ neurons. This observation may reflect the
expression of the amyloid precursor-like proteins 1 and 2 (APLP1 and
APLP2) molecules and supports the theory that APP and the APLPs may
have similar functional activities. Increased expression of
cell-associated APLP2, but not APLP1, was detected in A-treated
APP/ and APP+/+ cultures but
not in H2O2-treated cultures. This suggests
that the A toxicity pathway differs from other general forms of
oxidative stress. These findings show that A toxicity does not
require an interaction of the A peptide with the parental molecule
(APP) and is therefore distinct from prion protein neurotoxicity that is dependent on the expression of the parental cellular prion protein.
Key words:
A25-35; cortical neurons; neurotoxicity; APP; Alzheimer's disease; knock-out mice
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INTRODUCTION |
An Alzheimer's disease (AD) brain
contains numerous plaques composed of the amyloid- (A) amyloid
peptide (A4), which is derived from the proteolytic
processing of the amyloid precursor protein (APP) (Glenner and Wong,
1984 ; Masters et al., 1985; Kang et al., 1987). APP is a transmembrane
glycoprotein that undergoes extensive alternative splicing (Sandbrink
et al., 1994). The APP751 and -770 isoforms contain a Kunitz-type
protease inhibitor domain (Tanzi et al., 1988), whereas APP695, which
lacks this domain, is expressed at high levels in CNS neurons
(Koo et al., 1990). APP is processed by endoproteases called
secretases. Constitutive cleavage within the A domain by
 -secretase results in the generation of secreted APP (sAPP).
Alternatively, APP can be cleaved by - and  -secretases to
generate A (for review, see Mattson, 1997). APP is one of a
multigene family that contains at least two other homologs known as
amyloid precursor-like proteins 1 and 2 (APLP1 and APLP2) (Wasco et
al., 1992; Sprecher et al., 1993; Slunt et al., 1994). The APLPs
contain most of the domains and motifs of APP, including a hydrophobic
membrane-spanning domain, N-glycosylation sites, copper and
zinc binding domains, and the KPI domain (only APLP2). Neither APLP1
nor APLP2 contains the A region and cannot directly contribute to
A deposition in Alzheimer's disease. The similarities between APP
and APLP (particularly APLP2) suggest that the APLPs could share and
compensate for the function of APP.
A number of activities for neuronal APP have been identified. In
vitro studies suggest that membrane-associated and secreted APP
have an important role in promoting cell-substratum adhesion, neurite
extension and development, and synaptic function in neurons (Schubert
et al., 1989; Milward et al., 1992; Salvietti et al., 1996). In
addition to a neuritogenic role, APP may also have a neuroprotective
effect. Neurotrophic factors and neuronal injury upregulate APP
expression and induce secretion of sAPP (Nakamura et al., 1992; Mattson
et al., 1993b; Ohyagi and Tabira, 1993; Schubert and Behl, 1993). The
addition of sAPP to culture medium protects cortical and hippocampal
neurons from neurotoxic insults induced by hypoglycemia and excitotoxic
amino acids. It is believed that sAPP acts by stabilizing intracellular
Ca2+ levels and reducing oxidative stress (Mattson
et al., 1993b; Goodman and Mattson, 1994; Barger et al., 1995).
Transfecting human cDNA into cell lines and transgenic mice can result
in protection against oxidative stress and increased resistance to
excitotoxicity (Schubert and Behl, 1993; Mucke et al., 1996). However,
increased ischemic brain damage has been reported in transgenic mice
that overexpress APP (Zhang et al., 1997). The pathways involved in these effects are yet to be determined.
The neuroprotective role of sAPP may also extend to A toxicity. The
A peptides (A1-40 and A1-42) can be toxic in
vitro to a wide variety of neuronal cell types through disruption
of Ca2+ homeostasis and increased oxidative stress
(Yankner et al., 1989, 1990; Roher et al., 1991; Mattson et al., 1993a;
Pike et al., 1993). In addition, A can potentiate excitotoxic,
hypoglycemic, and oxidative damage to neurons (Koh et al., 1990;
Lockhart et al., 1994). Treatment of rat hippocampal neurons with sAPP
results in a protective effect against A toxicity (Goodman and
Mattson, 1994) by reducing Ca2+ influx and levels of
reactive oxygen species. Similarly, it was shown that when the B103
neuronal cell line was transfected with human APP695 or APP751 it was
significantly more resistant to A toxicity compared with controls
(Schubert and Behl, 1993). The increased survival of APP-expressing
cells may reflect a heightened resistance to oxidative stress.
These studies indicate a role for APP in antioxidant responses. In AD,
the aberrant processing of APP may not only result in increased
deposition of toxic A but could also reduce the normal protective
function of sAPP. At present, there are no published studies
investigating the role of endogenous APP expression with respect to
toxicity in neuronal cells. To determine whether endogenous APP
expression alters the response to oxidative stress in neurons, we have
established primary neuronal cultures from APP knock-out (APP/) and wild-type (APP+/+)
mice and exposed them to toxic A peptide and different oxidative stresses. In contrast to previous experiments with exogenous sAPP or
transfected cell lines, our study did not identify differences in cell
survival in APP/ compared with
APP+/+ neurons when both were exposed to various
oxidative insults. This result may reflect expression of the APLP
molecules that were detected in the neuronal cultures. Because of their
close homology, the APLPs may have a function similar to that of APP and hence possess the ability to compensate for the absence of APP.
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MATERIALS AND METHODS |
Materials. Poly-L-lysine, 3,[4,5
dimethylthiazol-2-yl]-2,5 diphenyltetrazolium bromide (MTT), cytosine
arabinofuranoside (Ara C), basic FGF (bFGF), xanthine oxidase,
phenylmethylsulfonyl fluoride (PMSF), and protease inhibitors were
purchased from Sigma (St. Louis, MO). Glutamine, glutamate, glucose,
and gentamycin sulfate were obtained from Life Technologies
(Gaithersburg, MD). A25-35 was obtained from Auspep Pty.
Ltd. A1-42 was a gift from Dr. Ashley Bush (Massachusetts General
Hospital, Boston). Fetal calf serum (FCS) and horse serum (HS) were
from the Commonwealth Serum Laboratories. Xanthine was obtained from
Boehringer Ingelheim. For immunoblotting, 22C11 (anti-APP/APLP2) was
obtained from Boehringer Ingelheim. 25104 (anti-APLP1) is a rabbit
polyclonal antiserum raised to an unconjugated peptide corresponding to
APLP1 amino acids 499-557 (Paliga et al., 1997); 95/11 (anti-APLP2) is
a rabbit polyclonal antiserum raised to recombinant APLP2 amino acids
28-693 (Wasco et al., 1993) expressed in Pichia pastoris
(see below).
Primary neuronal cultures. The generation of the
APP/ mice has been described previously (Zheng
et al., 1995). Control mice (C57BL6J × 129/Sv) correspond to
genetically matched mice from which the APP/
mice were derived. Primary neuronal cultures of cerebral cortex and
cerebellum were established from APP/ and
APP+/+ mice. Cortices from embryonic day 14 (E14)
and cerebella from postnatal day 5-6 (P5-6) mice were removed,
dissected free of meninges, and dissociated in 0.025% trypsin.
Cortical cells were plated onto poly-L-lysine (5 µg/ml)-coated 24-well plates (Greiner) at a density of 450,000 cells/cm2 (high density) or 250,000 cells/cm2 (low density) in MEM (Life Technologies)
supplemented with 10% FCS, 10% HS, 2 mM glutamine, 25 mM KCl, and 5 gm/l glucose. Cerebellar granule neurons
(CGNs) were plated on identical plates at 350,000 cells/cm2 in BME (Life Technologies)
supplemented with 10% FCS, 2 mM glutamine, and 25 mM KCl. Gentamycin sulfate (100 µg/ml) was added to all plating media, and cultures were maintained at 37°C in 5%
CO2. For lactate dehydrogenase (LDH) assays and some MTT
assays, cortical cultures were placed in MEM with N2 supplements (Life
Technologies) at day 3 in vitro. Ara C (10 µM)
was added at day 1 (CGN cultures) or day 6 (cortical cultures). Where
stated, bFGF (10 ng/ml) was added to cultures at day 1 in
vitro. Neuronal purity of cultures was ~90-95% for cortical
cultures and 96-98% for the cerebellar granule cell cultures.
Measurement of neuronal cell viability and cell death. Cell
viability or redox potential was determined using the MTT assay. Culture medium was replaced with 0.6 mg/ml MTT in control salt solution
(Locke's buffer containing 154 mM NaCl, 5.6 mM
KCl, 2.3 mM CaCl2, 1.0 mM
MgCl2, 3.6 mM NaHCO3,
5 mM HEPES, and 5.6 mM glucose, pH 7.4) for 2 hr. The MTT was removed and cells were solubilized with dimethyl
sulfoxide. Aliquots (100 µl) were measured with a spectrophotometer
at 570 nm. Cell death was determined from culture supernatants free of
serum and cell debris using an LDH Cytotoxicity Detection Kit
(Boehringer Ingelheim) according to manufacturer's instructions. All
cell survival assays were performed with the MTT assay unless stated
otherwise.
Induction of oxidative stress. A25-35 was prepared as a
1 mM stock solution in dH2O and aged for 2-3 d
at 37°C. A1-42 was prepared by dissolving the peptide in
dH2O, sonicating, and centrifuging for 5 min in a
microfuge. The supernatant was adjusted to 200 µM and
added to cultures. After 3 d in culture, neuronal cells were
exposed to A25-35 or A1-42. Neuronal redox activity and cell
death were determined after 4 d of exposure to the peptide using
the MTT and LDH assays, respectively.
Intracellular peroxide-associated oxidative stress was induced by
addition of glutamate to immature cortical cultures (day 3 in
vitro) for 24 hr. Extracellular peroxide toxicity was induced in
cortical cultures by adding H2O2 to culture
media for 24 hr at day 4 in vitro. Superoxide anion
production was generated in cell culture medium at days 4 and 6 in vitro by adding 50 µM xanthine and
increasing concentrations of xanthine oxidase to culture wells for 24 hr. Hypoglycemia was induced by washing cultures twice with
glucose-free Locke's buffer followed by incubation in Locke's without
glucose (5.6 mM, pH 7.4). Data represent the SEM of
experiments performed in three to four cultures measured in triplicate.
Recombinant APP, APLP1, and APLP2. Recombinant secreted
APP751, APLP2, and APLP1 were produced in the methylotrophic yeast Pichia pastoris. APP751 has been described previously (Henry
et al., 1997). APLP2 was amplified by the PCR as a 1998 base pair DNA
fragment corresponding to residues 28-693 of human APLP2. Human APLP2 cDNA (Wasco et al., 1993) was used as the PCR
template (gift of Dr. Wilma Wasco, Massachusetts Institute of
Technology, Cambridge, MA). The oligonucleotides used were CCG AAT TCT
TGG CGC TGG CCG GCT ACA and CCC CTC TAG AAC TGC TAC TCA GAC TGA AGT C. The PCR fragment was digested with EcoRI and XbaI
and ligated into EcoRI/XbaI-digested pIC9
(Invitrogen, San Diego, CA). Human APLP1, corresponding to
amino acids 34-650, was amplified by PCR with the primers AAG CTT ACG
TAC AGC CCG CCA TCG GGA GCC TG and GGC AGC GGA AGG GCA CAA C. The PCR
fragment was digested with SnaB I and ScaI and
cloned into pIC9. The protein was expressed in the GS115 strain as
described previously (Henry et al., 1997).
Quantitative immunoblotting of APP, APLP1, and APLP2.
Cortical cultures (day 2 or 3 in vitro) were exposed to
A25-35 (10 µM) or H2O2 (25 µM) for 24 hr and then homogenized in TES buffer (20 mM Tris, pH 7.4, 1 mM EDTA, 0.25 M
sucrose) and the protease inhibitors pepstatin, aprotinin, and
leupeptin at 10 µg/ml and 0.1 mM PMSF. Protein
determination was performed using a BCA assay kit (Pierce, Rockford,
IL), and normalized protein samples were separated on a 12% SDS-PAGE
gel (25 µg/lane) at 30 mA/gel. Separated proteins were transferred to
PVDF membrane overnight, blocked overnight in 0.5% hydrolyzed casein,
and probed for 1 hr at room temperature (RT) with 22C11 (1:2000), 25104 (1:1000), or 95/11 (1:1000) in Tris-buffered saline (TBST) (10 mM Tris, 0.9% NaCl, and 0.1% Tween-20, pH 7.5). A
secondary antibody [1:2000, rabbit anti-mouse immunoglobulins
(Amersham, Arlington Heights, IL)] was applied to the 22C11-probed
membrane for 1 hr at RT. Blots were then probed with
125I-protein-A (Amersham) for 1 hr at RT (0.1 µCi/ml in
0.5% casein). Blots were washed three times (10 min each) between each
probing step with TBST, pH 8.5. Blots were analyzed and quantitated on a Fujix BAS 1000 Phosphorimager using MacBas1 software.
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RESULTS |
APP/ neuronal cell cultures
To determine whether there were differences in basal cell survival
in neurons from APP/ and
APP+/+ mice, we established primary cortical and
cerebellar neuronal cultures from mice at E14 and P5, respectively. As
shown in Table 1, growth of
APP/ cortical neurons in serum-containing media
(up to 7 d) or in serum-free N2-supplemented media (up to 14 d) showed no significant difference in cell survival compared with
APP+/+ neurons. The growth of cerebellar neurons in
serum-containing media also revealed no differences between
APP/ and wild-type mice. We conclude that growth
and survival of primary neuronal cultures at high or low density, with
and without serum (after day 3 in vitro) does not differ
significantly between APP/ and
APP+/+ neurons under basal conditions.
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Table 1.
APP/ cortical and cerebellar neurons reveal
no difference in survival at basal growth conditions compared with
APP+/+ neurons using the MTT assay
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A25-35 and A1-42 toxicity in APP/
and APP+/+ cortical neuronal cultures
To determine whether the APP molecule is directly involved in
neuronal responses to A peptide-mediated toxicity, we added A25-35 or A1-42 peptide to 3-d-old cultures of
APP/ and APP+/+ cortical
neurons. The A25-35 peptide was aged at 37°C for 2-3 d in
dH2O and showed considerable fibril formation (data not
shown). The A1-42 peptide also produced considerable fibril
formation when prepared as described in Materials and Methods. Cultures were assayed for neuronal redox activity using the MTT assay after exposure for 4 d to A peptides at different concentrations. The intracellular reduction of MTT to insoluble product is rapidly and
significantly inhibited in many types of neurons treated with A
peptides (Abe and Kimura, 1996). With this treatment regimen, 0.5 µM A25-35 induced a significant decline in MTT
reduction compared with non-A-treated control cultures (Fig.
1A). Increasing concentrations of A resulted in a further loss of MTT reduction up
to 25 µM A, the highest concentration used. A
significant difference was not observed in MTT reduction between
APP/ and APP+/+ cortical
cultures (Fig. 1A). Because APP expression and
resistance to oxidative stress could be affected by cell density,
low-density (250,000 cells/cm2) primary cortical
neuronal cultures were also established and exposed to A25-35.
Measurement of cell viability after exposure for 4 d revealed no
difference between APP/ and
APP+/+ cultures (Fig. 1B). Similar
results were obtained when cultures were exposed to A1-42. No
significant difference was observed in MTT readings between
APP/ and APP+/+ neurons after
4 d exposure to 5 or 20 µM A1-42 (Fig.
1C).
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Figure 1.
APP/ and
APP+/+ cortical neurons do not have differences in
susceptibility to A25-35 or A1-42 inhibition of MTT reduction.
Primary cortical neurons were grown at (A) high
density (450,000 cells/cm2) or
(B) low density (250,000 cells/cm2) for 3 d and exposed to A25-35
for an additional 4 d. No differences between MTT reduction were
observed between APP/ and
APP+/+ cortical neurons exposed to A25-35 at
either density. Treatment of cultures with 10 ng/ml bFGF (applied
concomitantly with A) resulted in a significant increase in cell
viability as compared with non-bFGF-treated cultures when measured
4 d after exposure to A25-35. *p < 0.05, **p < 0.01: differences in MTT reduction between
bFGF and non-bFGF-treated cultures were determined using ANOVA and
Newman-Keuls tests. C, APP/ and
APP+/+ cortical neurons reveal no differences in MTT
reduction when treated with A1-42. D,
APP/ and APP+/+ cortical
neurons have no differences in susceptibility to A25-35-induced
cell death as determined using the LDH assay. E,
APP/ and APP+/+ cortical
neurons reveal no differences in survival when exposed to
A1-42-induced cell death as determined using the LDH assay.
F, APP/ and
APP+/+ cerebellar granule neurons reveal no
differences in susceptibility to A25-35 inhibition of MTT
reduction. Primary cerebellar neurons were grown for 1 d and
exposed to A25-35 for an additional 6 d.
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Because there is still doubt concerning the direct relationship between
A inhibition of MTT levels and A-induced neuronal death (Shearman
et al., 1995; Abe and Kimura, 1996; Liu and Schubert, 1997), the LDH
assay was also used to determine actual cell death. Release of the
stable constitutive enzyme LDH occurs after cell lysis and therefore
gives an accurate estimation of the terminal response of neurons to
toxic A. Cortical neuronal cultures that had been treated with
A25-35 or A1-42 for 4 d were assayed for release of LDH. A
clear correlation between A concentration and the level of LDH
release into the culture medium was found (Fig. 1D,E). The level of cytotoxicity was lower for the
LDH assay compared with the MTT assay. This result was expected because
of the characteristic ability of A to rapidly lower the level of MTT
reduction in treated cultures without a direct increase in cell death
and the loss of membrane integrity required for LDH release (Shearman
et al., 1995; Abe and Kimura, 1996; Liu and Schubert, 1997). The LDH
assay also did not show a significant difference in A-mediated cell death between APP/ and APP+/+
neurons (Fig. 1D,E). These results reveal that a lack
of normal endogenous APP expression in mouse cortical neurons does not
affect the ability of A to lower neuronal redox activity or induce
cell death.
Effect of bFGF on A toxicity in APP/ and
APP+/+ cortical neurons
bFGF has been shown to reduce A toxicity in primary hippocampal
cultures (Mattson et al., 1993a) as well as increase APP expression in
neurons in vitro (Ohyagi and Tabira, 1993). If these effects
were linked, then bFGF treatment of neuronal cultures could result in
differential survival of APP/ and control
APP+/+ cultures after exposure to A. Treatment of
high- and low-density cultures with 10 ng/ml bFGF resulted in a
significant increase in cell viability after exposure to A25-35 for
4 d [Fig. 1A,B, *p < 0.05, **p < 0.01 (ANOVA and Newman-Keuls test)].
Interestingly, a difference was not observed between
APP/ and APP+/+ cultures at
low or high density, indicating that APP expression is not required for
the protective effect of bFGF on A toxicity in neurons.
A25-35 toxicity in APP/ and
APP+/+ cerebellar granule neuron cultures
To determine how other neuronal cell types are affected by a lack
of APP expression, we tested APP/ and
APP+/+ CGN cultures. A25-35 was added to CGN
cultures at day 1 in vitro (24 hr after plating) at 5 and 25 µM. Because CGN cultures do not reveal an immediate drop
in MTT reduction, characteristic for A-treated cortical neurons
(our unpublished observations), the MTT assay was used as a
measure of cell survival rather than cell redox activity alone. Cell
viability was determined after exposure for 6 d instead of 4 d because of increased resistance to A-induced toxicity in these
cultures. As with cortical neuronal cultures, differences were not
observed between APP/ and
APP+/+ CGN (Fig. 1F). bFGF
treatment did not alter the level of A toxicity in
APP/ and APP+/+ CGN cultures
(data not shown).
Effects of peroxide-associated oxidative stress on cell survival in
APP/ and APP+/+ neurons
in vitro
Intracellular peroxide generation can be induced in immature
neuronal cultures by exposure to high concentrations
(millimolar) of glutamate. This leads to glutathione (GSH)
depletion caused by competitive inhibition of cysteine uptake, which is
necessary for reduced GSH synthesis (Ratan et al., 1994), resulting in
a subsequent increase in intracellular peroxide levels. The role of GSH
depletion in this form of glutamate toxicity has been confirmed in our
laboratory by preventing toxicity with exogenous GSH (our unpublished
observations). This type of oxidative stress is reduced in a B103 cell
line transfected with human APP cDNA (Schubert and Behl,
1993), whereas secreted human APP [(hu) sAPP] applied to primary
cultures of human and animal cortical neurons results in reduced
peroxide generation (Mattson et al., 1993b).
To determine whether APP/ neurons are more
susceptible to oxidative stress than wild-type neurons, high
concentrations of glutamate were applied to immature cortical cultures
(3 d in vitro), and cell viability was determined after 24 hr. A significant decrease in cell survival was obtained with at least
5 mM glutamate (Fig. 2A). Significant
differences in toxicity were not observed between APP/ and APP+/+ cultures.
Similarly, exposure to exogenous hydrogen peroxide for 24 hr did not
affect cell viability of APP/ compared with
APP+/+ cultures (Fig. 2B). These
results indicate that endogenous APP expression does not significantly
reduce peroxide-associated oxidative stress in primary cortical
neurons.
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Figure 2.
APP/ and
APP+/+ neurons do not have differences in
susceptibility to intracellular- or extracellular-generated oxidative
stress. A, Primary cortical neurons were grown for
2 d and exposed to glutamate for 24 hr. B, Primary
cortical cultures were grown for 4 d and then exposed to
H2O2 for 24 hr. C, Primary
cerebellar granule neurons were grown for 7 d and then exposed to
glutamate for 30 min. D, Primary cortical cultures were
grown for 14 d and then exposed to glutamate for 30 min. Cell
viability was determined 24 hr later. E, Primary
cortical neurons were exposed to increasing concentrations of xanthine
oxidase and 50 µM xanthine for 24 hr at either 4 or
6 d in vitro. F, Primary cortical
neurons were grown for 14 d before incubation in glucose-free
Locke's media. Cell viability was determined after the given
incubation period.
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Effects of excitotoxicity-mediated oxidative stress on cell
survival in APP/ and APP+/+
neurons in vitro
Previous studies have demonstrated that (hu) sAPP applied to
primary cultures of human and animal neurons results in increased resistance to glutamate excitotoxicity (Mattson et al., 1993b). To
determine whether APP/ neurons have decreased
resistance to excitotoxicity, micromolar concentrations of glutamate
were added to APP/ and APP+/+
cultures of CGN and cortical neurons. The CGN neurons were used because
of their highly homogeneous nature (96%) and well characterized response to excitotoxins (Lindholm et al., 1993). Cortical neurons were
used because this cell type has previously been shown to be protected
from excitotoxic glutamate by (hu) sAPP (Mattson et al., 1993b). A 30 min exposure to glutamate in 7-d-old cultures of CGN or 14-d-old
cultures of cortical neurons resulted in cell death in both cultures as
measured by MTT reduction 24 hr after exposure (Fig. 2, C
and D, respectively). APP/ and
APP+/+ cultures did not differ significantly at any
concentration of glutamate used. The findings show that cortical
neurons and CGN that do not express APP do not differ in their ability
to survive excitotoxic insult.
Effects of superoxide anion-mediated oxidative stress on cell
survival in APP/ and APP+/+
neurons in vitro
An important form of oxidative stress in neurons is mediated by
the superoxide anion free radical (Sagara et al., 1996). To determine
whether the antioxidant pathway for superoxide removal (superoxide
dismutase pathway) involves APP expression, primary cortical neurons
were exposed to increasing concentrations of xanthine oxidase (XO) in
the presence of 50 µM xanthine, the combination of which
results in the generation of superoxide anions (Brown et al., 1996b).
Increasing concentrations of XO up to 400 mU/ml in the medium resulted
in increasing cell death in 4- and 6-d-old cultures (Fig.
2E) as measured 24 hr after addition of the enzyme. Little difference in the level of toxicity was observed between cultures at 4 and 6 d when exposed to 25-100 mU/ml XO. At
concentrations of 200 and 400 mU/ml XO, greater toxicity was seen in
4-d-old compared with 6-d-old cultures. Significant differences were
not observed in superoxide anion toxicity in
APP/ and APP+/+ cultures at
either 4 or 6 d in vitro (Fig. 2E).
These results reveal that the level of oxidative stress involving
O2 is not significantly affected by
expression of APP in cortical neuronal cultures.
Effects of hypoglycemia-mediated oxidative stress on cell survival
in APP/ and APP+/+ neurons
in vitro
Hypoglycemia can cause increased oxidative stress in neurons
through increased generation of reactive oxygen species. Mattson et al.
(1993b) have demonstrated a significant protective effect against
hypoglycemic-related cell death in neurons exposed to exogenous (hu)
sAPP. The role of endogenous APP expression in protection against
hypoglycemia was examined by exposing 14-d-old cortical neurons from
APP/ and APP+/+ to
glucose-free Locke's solution for 10, 18, and 24 hr. This resulted in
a significant and continuous reduction in cell survival over the 24 hr
period; again, differences between APP/ and
APP+/+ neurons were not observed (Fig.
2F). These results indicate that neurons capable of
expressing endogenous APP have no survival advantage under hypoglycemic
conditions.
Effect of A25-35 and H2O2 on APP and
APLP expression in primary cortical cultures
As APP is part of a multi-gene family, the similar survival
properties of APP/ and APP+/+
neurons in response to A or oxidative stress may be compensated for
by either of the APLPs. To test this hypothesis we exposed APP/ and APP+/+ neuronal
cultures to A25-35 (10 µM) or
H2O2 (25 µM) for 24 hr and
measured APP/APLP expression by quantitative Western blot analysis. The
specificities of the APLP2 and APLP1 antibodies are shown in Figure
3. The anti-APLP1 (25104) and anti-APLP2
(95/11) antibodies reacted only with their respective recombinant
proteins.
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Figure 3.
Characterization of the specificity of the APLP2
and APLP1 antibodies. Western blots of recombinant sAPP751 (lane
1), sAPLP2 (lane 2), and sAPLP1 (lane
3) probed with 22C11 (anti-APP/APLP2, 1:2000), 95/11
(anti-APLP2, 1:1000), or 25104 (anti-APLP1, 1:1000). The lower
bands correspond to breakdown products as described previously
(Henry et al., 1997). The position of the molecular weight markers is
indicated on the left.
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As expected, immunoblot analyses of untreated
APP/ cortical neurons did not reveal expression
of APP (Fig. 4A). In
addition, no increase in basal level expression of APLP1 or APLP2 was
observed in APP/ neurons when compared with
APP+/+ neurons (Fig. 4A). This is
consistent with previous data showing no increase in APLP2 expression
in total brain homogenates (Zheng et al., 1995). After a 20 hr exposure
to A25-35 (10 µM), APP+/+ neurons
showed a significant increase in cell-associated APP and APLP2
expression as determined by Western blotting with 22C11 and 95/11,
respectively (Fig. 4B,C; Table
2). Although 22C11 cross-reacts with
APLP2, differences in molecular weight between APP and APLP2 and a
comparison with APP/ lysates allowed us to
demonstrate specific increases in both APLP2 (110 kDa) and APP (95-105
kDa) proteins in APP+/+ cultures. Further
confirmation of protein specificity was obtained using the APP-specific
antibody Ab1-25 (data not shown). Blotting with 95/11 also revealed a
significant increase in the 110 kDa APLP2 band in
APP/ cultures exposed to A25-35 (Fig.
4C, Table 2). These results indicate that primary cortical
neurons respond to A toxicity by upregulating expression of both APP
and APLP2. Immunoblotting analysis with anti-APLP1 antibody did not
reveal any significant increase in this protein in
APP+/+ or APP/ cultures
exposed to A25-35 (Fig. 4D, Table 2). In contrast to the effect of A25-35, the treatment of APP+/+
and APP/ cortical cultures with 25 µM H2O2 for 24 hr resulted in no
significant change in the expression levels of APP or APLP (Fig.
4E-G). A similar result was also obtained when
3-d-old cultures were exposed to glutamate (15 mM) for 24 hr (data not shown). This indicates that the basal levels of APLP in
the APP/ cultures provide a similar level of
protection to APP and APLP in APP+/+ cultures.
View larger version (47K):
[in this window]
[in a new window]
|
Figure 4.
Quantitative immunoblotting of cell-associated
APP, APLP1, and APLP2 in neurons exposed to A25-35 and
H2O2. Primary cortical neurons were grown for
2 d and then exposed to either 10 µM
A25-35(A) or 25 µM
H2O2, or were untreated [control
(C)] for 24 hr. The antibodies are
anti-APP/APLP2 (22C11, 1:2000), anti-APLP2 (95/11, 1:1000), or
anti-APLP1 (25104, 1:1000). The brackets correspond to the proteins
described in Results and their molecular weights are as follows:
anti-APP (95-105), anti-APLP1 (87 and 126), and anti-APLP2 (110 kDa).
The position of the molecular weight markers is indicated on the
right-hand side. A, Analysis of APLP2,
APLP1, and APP expression under basal conditions. B,
Analysis of APP expression detected in A25-35-treated
APP+/+ cultures. C, Analysis of APLP2
expression in A25-35-treated APP/ and
APP+/+ neurons shows a significant increase in APLP2
expression in both APP/ and
APP+/+ neurons exposed to A. D,
Analysis of APLP1 expression in A25-35-treated
APP/ or APP+/+ neurons.
E, Analysis of APP expression in
APP+/+ neurons in response to
H2O2. F, Analysis of APLP2
expression in H2O2-treated
APP/ and APP+/+ neurons.
G, Analysis of APLP1 expression in
H2O2-treated APP/ and
APP+/+ neurons.
|
|
View this table:
[in this window]
[in a new window]
|
Table 2.
Quantitation of the immunoblot data on cell-associated APP,
APLP1, and APLP2 expression after A25-35 treatment as shown in
Figure 4B-D
|
|
| |
DISCUSSION |
The aim of this work was to investigate the neuroprotective role
of endogenously expressed APP in the CNS. It has been shown that
treatment of primary neurons with secreted forms of APP (Mattson et
al., 1993b; Goodman and Mattson, 1994) or transfection of cell lines
and mice with (hu)APP cDNA (Schubert and Behl, 1993;
Mucke et al., 1996) results in protection against oxidative toxicity induced by the A peptide, glutamate excitotoxicity, and
hypoglycemia. This increased survival may reflect a heightened
resistance to oxidative stress. Although these findings support a
neuroprotective activity for APP, the role of endogenous neuronal APP
in neuroprotection has not been assessed. We used APP knock-out primary
neurons to test the role of endogenous APP because they avoid the
problems associated with antisense procedures.
This study clearly demonstrates that endogenous APP expression does not
alter the survival of primary cortical or cerebellar neurons in
response to A-mediated toxicity or oxidative stress in
vitro. We found that the absence of APP expression had no effect on neuronal viability under basal conditions (Table 1), suggesting that
the neurotrophic activity of APP is not essential for cortical or
cerebellar neuronal growth in vitro. This differs slightly from a report on survival of hippocampal neurons cultured from APP/ mice. Perez et al. (1997) observed a small
but significant reduction in viability in APP/
as compared with APP+/+ hippocampal neurons in
primary culture. The results from our study may simply reflect
differences between cell types (cortical vs hippocampal), media, cell
densities, or the effect of astrocyte-conditioned media. There was no
observable increase in the basal levels of APLP1 and APLP2 expression
in APP/ neurons in our study, indicating that
under normal growth conditions in vitro, the absence of APP
does not require the compensatory increased expression of an APLP. We
evaluated the survival of APP/ and
APP+/+ neurons under neurotoxic conditions by
exposing them to a range of endogenously and exogenously generated
oxidative stresses. In all cases, differences in cell survival were not
observed between the APP/ and
APP+/+ neurons. However, changes in APP and APLP
expression were found after exposure to A but not to
H2O2. A treatment caused increased expression of APLP2 and APP in APP+/+ neurons and
APLP2 in APP/ neurons.
Our data demonstrating a similarity between the
APP/ and APP+/+ neurons do
not support a neuroprotective function for endogenous APP in
vitro. It is possible that the level of endogenous APP expression
and secretion in primary neuronal cultures is too low to influence the
level of neuroprotection against oxidative stress. If so, the absence
of APP in APP/ neurons would not be expected to
have any effect. The level of APP expression in transgenic mice is an
important factor in determining the degree of neuroprotection against
excitotoxic insult (Mucke et al., 1996). Although the specific level of
sAPP in primary neuronal cultures has not been reported, our
immunoblotting data and the study by Hung et al. (1992) show that
primary neuronal cultures express readily detectable levels of APP
(Fig. 4B). It is therefore unlikely that the level of
APP expression is too low to induce a measurable neuroprotective
effect.
The similarity between the APP/ and
APP+/+ neurons may reflect species-dependent
sequence variations between human and mouse APP. The effect of these
sequence differences on neuroprotection is unknown because all previous
studies have used human APP. The high homology between the coding
regions (97%) and the 5' regulatory regions of human and mouse APP (De
Strooper et al., 1991; Chernak, 1993) suggests a conserved function
between species and is therefore also an unlikely explanation. However,
it has been proposed that the A1-16 sequence is important for
neuroprotection, possibly through heparin binding. Interestingly, the
three differences between the mouse and human A sequences are all
contained within A1-16, including the histidine to arginine
substitution at position 13 that may be required for heparin binding
(Brunden et al., 1993).
We speculate that APP/ and
APP+/+ neurons do not display any differences
because APLP expression is able to compensate for the absence of APP.
This model would support the existing studies describing the
neuroprotective activity of APP but indicates that this function could
also be shared by the APLP molecules. There is considerable sequence
homology and conservation of putative functional motifs and domains
between APP and the APLP (Paliga et al., 1997). These similarities
suggest the APLPs could share and/or compensate for the function of
APP, thus reflecting a redundancy in the APP gene family. This is
supported by studies in which APP, APLP1, and APLP2 single knock-out
mice have been shown to survive with minor neurological dysfunction
(Zheng et al., 1995; Muller et al., 1997; von Koch et al., 1997).
However, APP/APLP2 double knock-outs are a lethal combination resulting
in death during embryonic to early postnatal development (von Koch et
al., 1997). This suggests that although early cerebral development may
not be severely impaired by a loss of either APP or APLP expression, cells cannot compensate for a simultaneous loss of both. The APLP could
be compensating for APP by binding to and stimulating the APP receptor.
Alternatively, APLP could be acting as a receptor to bind the APP
ligand and transmit the appropriate signal. However, the putative
compensatory role of APLP remains to be tested.
The finding that cell-associated APP and APLP2 expression is increased
after exposure to A is consistent with previous reports (Cribbs et
al., 1995; Saporito-Irwin et al., 1997; Schmitt et al., 1997). The
increased level of cell -associated protein may be attributable, at
least in part, to decreased secretion because 40% less sAPP was
detected in the media of A-treated cultures (data not shown). This
is supported by Schmitt et al. (1997) who reported that increases in
cellular APP expression induced by A25-35 were caused by decreases
in APP secretion. APP and APLP2 have similar expression and secretory
pathways (Nitsch et al., 1992; Webster et al., 1995). Interestingly, no
change was seen in sAPLP2 levels after A treatment (data not shown).
This would indicate that A can also induce increased expression of
APLP2, and possibly APP, and that A may have differential effects on some aspects of APP and APLP2 expression and processing. No change in
APLP1 expression was detected after exposure to A, and this may
reflect the lower homology of APLP1 with APP and APLP2. This data would
indicate that APLP2 expression, rather than APLP1, may be responsible
for compensatory activity in APP/ cells. To
clarify this issue, the neuroprotective activity of APLP2 and APLP1
needs to be determined. In addition, the susceptibility of neurons from
APLP1 and APLP2 single knock-outs and APP/APLP1, APP/APLP2, and
APLP1/APLP2 double knock-outs to oxidative stress also needs to be
investigated.
Our finding that APP and APLP expression levels remain unaltered in
H2O2 and glutamate-treated cultures is
important because it suggests that A-induced toxicity is distinct
from general oxidative stress and that A increases APP/APLP2
expression through a nonoxidative stress-related pathway. This could be
because A operates via an A-receptor (Yan et al., 1996, 1997),
which results in either localized oxidative stress or activation of a
specific cell-signaling pathway or both. In contrast,
H2O2 and glutamate produce pan-cellular
oxidative stress, which does not increase APP/APLP expression. It is
unlikely that the level of H2O2 or glutamate
exposure was too low to induce APP/APLP expression because the level of
toxicity obtained was similar to that induced by A.
It has been suggested that toxic, amyloidogenic peptides may share a
common toxic mechanism (Ridley and Baker, 1993). Our data would suggest
that the mechanism of A-toxicity is distinct from the amyloidogenic
prion protein-derived peptide PrP106-126 (Brown et al., 1996a). The
PrP106-126 peptide requires expression of normal cellular prion
protein (the parent molecule) to induce toxicity in neurons in
vitro (Brown et al., 1996a). In contrast, our results show that
A does not require the presence of the normal parent molecule (APP)
to induce neuronal toxicity (other than as a source of A in
vivo). This suggests that although A may induce a positive
feedback effect on APP processing leading to increased A formation
and aberrant APP metabolism (Cribbs et al., 1995), these factors do not
contribute to the short-term neuronal loss seen in vitro.
The effect of increased APP expression may result, however, in changes
to neuronal survival in the longer term. An alternative, and exciting,
explanation is that if the APLP molecules represent functional homologs
of APP then the A peptide could act through an APLP, and in
particular APLP2. This would suggest that APP/APLP double knock-out
neurons would be refractory to A toxicity and hence mimic the
prion-PrPc model.
If APP is involved in neuroprotection against A and oxidative stress
in vivo, then any perturbations to APP metabolism such as
those occurring in AD may not only result in increased A deposition but may also reduce neuroprotection (Mattson et al., 1993b). If the
APLP molecules, in particular APLP2, have a neuroprotective function
reflecting that seen with sAPP, then changes to APLP protein metabolism
may also result in decreased neuronal resistance to oxidative insults
or A. A neuroprotective role for APLP may have important
implications for Alzheimer's disease because therapeutic treatments
specifically aimed at increasing APLP expression may provide a means of
increasing neuroprotection without directly contributing to A
deposition. This may have the added benefit of replacing some of the
functions that are lost because of aberrant APP metabolism, such as
dendritic growth.
| |
FOOTNOTES |
Received Jan. 30, 1998; revised May 29, 1998; accepted June 8, 1998.
This work was supported in part by grants from the National Health and
Medical Research Council of Australia to C.L.M. K.B. is supported
by the Deutshe Forschungsgemeinschaft and the Bundesministerium fur
Forschung und Technologie.
Correspondence should be addressed to Dr. Roberto Cappai, Department of
Pathology, The University of Melbourne, Parkville Victoria 3052, Australia.
| |
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Copyright © 1998 Society for Neuroscience 0270-6474/98/18166207-11$05.00/0
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Top
Abstract
Introduction
