The Journal of Neuroscience, July 2, 2003, 23(13):5531-5535
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BRIEF COMMUNICATION
The Production of Amyloid 
Peptide Is a Critical Requirement for the Viability of Central Neurons
Leigh D. Plant,1
John P. Boyle,2
Ian F. Smith,2
Chris Peers,2 and
Hugh A. Pearson1
1Schools of Biomedical Sciences and
2Institute for Cardiovascular Research, University of
Leeds, Leeds LS2 9JT, United Kingdom
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Abstract
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The amyloid 
peptide (A) is a product of the sequential
- and -secretase cleavage of amyloid precursor protein.
Inhibitors of secretase enzymes have been proposed as a potential therapeutic
strategy in the treatment of Alzheimer's disease. Here, we investigate the
effect of inhibiting these key enzymes on the viability of a range of cell
types.
Treatment of rat cortical neurons for 24 hr with secretase inhibitors or an
antibody that binds A resulted in a marked reduction in cell viability,
as measured by MTT reduction. Incubation with secretase inhibitors caused
similar effects on other neuronal cell types (rat cerebellar granule neurons
and the human SH-SY5Y cell line). Interestingly, rat astrocytes and a number
of non-neuronal cell lines investigated (HEK293, DDT1-FM2, and human
teratorhabdoid tumor cells) were unaffected by incubation with secretase
inhibitors.
The coincubation of A1–40 prevented the toxicity of
secretase inhibitors in neuronal cells. A1–40 was
protective in a concentration-dependent manner, and its effects were
significant at concentrations as low at 10 pM. Importantly, the
protective effects of A were A size-form specific, with the
A1–42 size form affording limited protection and the
A25–35 size form having very little protective
effect.
The present study demonstrates that inhibition of -or
-secretase activity induces death in neuronal cells. Importantly, this
toxicity, which our data suggest is a consequence of a decline in neuronal
A levels, was absent in non-neuronal cells. This study further supports
a key physiological role for the enigmatic A peptide.
Key words: Alzheimer's disease; amyloid; secretase; inhibitor; nerve; cell death; physiology
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Introduction
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Alzheimer's disease (AD) is characterized pathologically by progressive
neurodegeneration and the deposition of aggregated amyloid peptide
(A) as senile plaques. Although the mechanism underlying
neurodegeneration remains unclear, a growing body of evidence implicates
A in a pivotal role (Selkoe,
2001
). A is derived from the highly regulated and sequential
cleavage of amyloid precursor protein (APP) by proteases designated
-secretase and -secretase. The A peptide is readily
detected in human CSF as a range of isoforms between 38 and 43 amino acids in
length, the predominant isoforms being A1–40 (90%) and
the fibrilogenic A1–42 isoform (10%). In patients with
Alzheimer's disease, the relative proportions of 1–40 and 1–42
change to 
50% each (Mehta et al.,
2001). Furthermore, the total A burden in brain is increased
dramatically (Cummings and Cotman,
1995). Many drug development strategies for potential therapies in
AD therefore focus on reducing this A load. One such approach identifies
the - and -secretases as drug targets. Targeting these secretases
may, however, have unforeseen effects because a physiological role for A
has been proposed (Ramsden et al.,
2001). With this in mind, we studied the effects of - and
-secretase inhibition on the viability of central neurons. We report
evidence suggesting that loss of endogenous A by the pharmacological
inhibition of amyloidogenesis results in a severe reduction in the viability
of central neurons.
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Materials and Methods
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Inhibitors of - and -secretase were purchased from Calbiochem
(Nottingham, UK) and dissolved in dimethylsulfoxide and H2O,
respectively. A1–40 was a gift from GlaxoSmithKline
Research (Stevenage, UK). A1–42 was purchased from
Bachem (St. Helens, UK), and A25–35 was purchased from Sigma
(Poole, UK). 3D6 antibody was the kind gift from Elan Pharmaceuticals (San
Francisco, CA). Peptides were solubilized in dimethylsulfoxide and dissolved
in deoxygenated, deionized H2O at 100 µM, aliquoted
to 10 µl, and stored at –20°C.
Cell culture. Primary cultures of cortical neurons were obtained
from 16- to 18-d-old fetal Wistar rat neocortex by enzymatic and mechanical
dissociation (MacManus et al.,
2000). Cells were grown in 24-well plates in a humidified
atmosphere containing 5% CO2 and 95% air at 37°C. Culture
medium comprised minimal essential medium (MEM) supplemented with 10% fetal
bovine serum (FBS), 19 mM KCl, 2 mM L-glutamine, 26
mM glucose, 50 U/ml penicillin, and 50 µg/ml streptomycin. After
48 hr, 80 mM fluorodeoxyuridine was included in the culture medium
to prevent proliferation of non-neuronal cells. The culture medium was
exchanged every 3 d, and cells were used in experiments between days 5 and 8
in vitro. Primary cultures of rat cerebellar granule neurons (CGNs)
and primary cultures of rat astrocytes, obtained from 6- to 8-d-old Wistar
rats, were prepared and grown in the same manner as detailed by Plant et al.
(2002a). Cells were used in
experiments between days 7 and 12 in vitro.
Cell lines (DDT1-FM2, HEK293, and teratorhabdoid tumor cells) were cultured
using established methods (Shukla et al.,
2001; Plant et al.,
2002b) as continuous monolayer cultures in 5%
CO2–95% air at 37°C. Medium consisted of 50% MEM with
Earle's salts and L-glutamine and 50% F-12 neuronal supplement.
Media were supplemented with 10% FBS, 1% non-essential amino acids, penicillin
(50 U/ml), and streptomycin and gentamicin (both at 50 µg/ml).
3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
assay. Cell viability was assessed using the
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction
assay (Mosmann, 1983) as
described previously (Ramsden et al.,
2001; Shukla et al.,
2001). Absorbency was measured using a spectrophotometer at a test
wavelength of 570 nm and reference wavelength of 630 nm. Student's t
test (unpaired) was used to determine the significance of differences between
means, with p values <0.05 being considered significant.
Immunocytochemistry. For immunocytochemical experiments, cells
were washed with PBS before being fixed with paraformaldehyde (4% in PBS) for
20 min. After a second wash step, cells were permeabilized using PBS
containing 0.2% Triton X-100 and 10% normal goat serum (NGS). Cells were then
washed with PBS containing 1% NGS before being incubated overnight with a
monoclonal antibody (1:1000) raised against the first five N-terminal amino
acids of A (3D6), prepared in PBS containing 1% NGS. The secondary
antibody was added in PBS containing 1% NGS for 1 hr after a series of PBS
washes. Secondary antibody was a donkey anti-rabbit conjugated with a Cy3
fluorescent label (1:1000; Jackson ImmunoResearch, West Grove, PA). Coverslips
were mounted onto microscope slides using 50% glycerol in PBS and sealed using
standard nail lacquer. Slides were stored at 4°C in the dark until used.
Cells were viewed using a Zeiss (Oberkochen, Germany) Axioscop epifluorescence
microscope fitted with a rhodamine filter set. Images were captured using a
CCD camera and AcQuis image acquisition software (Synchroscopy, Cambridge,
UK). All images were acquired using identical exposures and settings.
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Results
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Inhibition of -secretase induces morphological changes in
cortical neurons
-Secretase activity is mediated by a multi-enzyme complex containing
presenilin and nicastrin and represents the rate-limiting step in
amyloidogenisis (Kaether et al.,
2002). The enzymatic activity of this complex is sensitive to the
peptide aldehyde 2-naphthoyl-VF-CHO (-IV). -IV is cell permeable
and has been shown to reversibly inhibit both A1–40 and
A1–42 production with ED50 values of 2.6 and
2.7 µM, respectively (Sinha
and Lieberburg, 1999). We treated primary cultures of rat
neocortical neurons with 10 µM -IV for 24 hr and noted
marked changes in the appearance of these cells. Application of -IV
induced cell shrinkage, granularization, and an apparent reduction in neuronal
cell number (Fig. 1).
Because the turnover of A is reported to be as rapid as 2 hr in
central neurons (Savage et al.,
1998), we hypothesized that preventing de novo
amyloidogenesis and allowing endogenous A levels to decline could
underlie the apparent toxicity of chronic -IV treatment. To test this
hypothesis, we coincubated neocortical neurons with -IV and 1
nM A1–40 for 24 hr. This strategy precluded
the apparent toxicity of -IV treatment and suggests that a decrease in
A levels underlies this effect (Fig.
1C).
Secretase inhibition is associated with decreased cell viability
The MTT assay was used to quantify the relative viability of neocortical
pyramidal neurons after chronic -IV treatment. This colorimetric assay
corroborated our morphological study. Thus, a 24 hr treatment with -IV
decreased relative viability of neocortical neurons to 43 ± 5%
(n = 9; p < 0.05) compared with untreated cells
(Fig. 2A). A second
inhibitor of -secretase,
N-[N-(3,5-difluorophenacetyl-L-alanyl)]-S-phenylglycine
t-butyl ester, designated -secretase inhibitor IX (200
nM) was similarly effective in reducing cortical cell survival
(Fig. 2A).
-Secretase activity is implicated in a number of neuronal pathways,
including Notch processing (Selkoe,
2001). It was crucial to determine the relative importance of
amyloidogenisis in contributing to the toxicity of -secretase
inhibition and exclude the possibility that other pathways might mediate this
effect. To resolve this issue, we sought to prevent A production by a
second method. H-KTEEISEVN-stat-VAEF-OH (SI) is a potent inhibitor of
-secretase activity (IC50 of 30 nM)
(Sinha et al., 1999),
preventing the initial cleavage of APP and therefore, subsequent A
production. A 24 hr treatment with SI (100 nM) mimicked the
decrease in relative cell viability measured in these neurons after treatment
with -secretase inhibitor (p < 0.05)
(Fig. 2A). A second
inhibitor of -secretase,
N-bennzyloxy-carbonyl-valine-leucine-leucinal (SI-II) (10
µM) (Abbenante et al.,
2000), was also highly effective in compromising cell survival
(Fig. 2A).
The ability of -IV to kill cells was time dependent.
Figure 2B shows the
effect of incubating cortical cultures with 10 µM -IV for
periods of 2–48 hr. Significant inhibition (n = 4; p
< 0.05) of viability occurred within 4 hr of the initial application of
-IV, and additional increases in toxicity were observed over the full
time course examined.
Secretase inhibitors are toxic to neurons but not to non-neuronal
cells
To assess the impact of secretase inhibition on other cell types, we
corroborated our investigation with primary cultures of rat CGNs and the human
neuroblastoma cell line SH-SY5Y. The MTT assay revealed the marked toxic
effects of both -IV and SI in both cell types. After 24 hr
(Fig. 2C) and 48 hr
(data not shown) treatment, cell viabilities were reduced to a similar extent
to that seen in the cortical neurons. Interestingly, primary cultured rat
astrocytes taken from the cerebral cortices of the same animals used to
produce cerebellar granule cultures were primarily unaffected by secretase
inhibition (Fig. 2C)
(see also background astrocytes in Fig.
1). This suggests that the toxic effects of secretase inhibitors
are neuron specific. To test this further, we applied secretase inhibitors to
a variety of non-neuronal cell types. These were the hamster smooth muscle
cell line DDT1-FM2, the human embryonic kidney cell line HEK293, and a primary
culture of cells isolated from a human teratorhabdoid tumor. No clear
induction of cell death was observed in any of these non-neuronal cell types
in response to incubation with either -IV or SI for up to 48 hr
(Fig. 2D).
We used an immunocytochemical approach to show that the secretase
inhibitors were effective in reducing A production under the conditions
used in our experiments. Figure
3 shows the effect of -IV and SI on endogenous
A immunostaining in cerebellar granule neurons. Under control
conditions, a dense, punctate staining could be observed in the cytoplasm and
membrane of cells (Fig.
3A). This staining was greatly reduced in cells that had
been treated with either -IV or SI for 24 hr
(Fig. 3B,C). Thus, a
clear and substantial reduction in endogenous A levels was effected by
the application of these inhibitors.
Cell death induced by secretase inhibitors is dependent on A
production
The independent inhibition of two distinct enzymes in distinct neuronal
populations provides evidence that amyloidogenesis plays an integral role in
determining the viability of central neurons grown in culture. To prove the
importance of A to this system, we attempted to recover the deficit in
relative cell viability associated with secretase inhibition by the addition
of exogenous A. In previous studies, we demonstrated that the incubation
of primary cultures of rat neurons with the physiologically prevalent
A1–40 isoform, at concentrations as high as 1
µM, is not associated with a reduction in relative cell
viability (Ramsden et al.,
2001). We coincubated cells with either-IV orSI and a
number of different A size forms at a range of concentrations for 24 hr.
Concentrations of A in the range of 10 pM to 1 nM
were effective in reversing the toxic effect of -secretase inhibition
(Fig. 4A). Indeed, 10
pM A afforded significant rescue of -IV-induced cell
death in both cortical and cerebellar granule cell cultures (n = 9 in
each case; p < 0.05). Such concentrations are within the range
reported from measurements of A concentrations in human CSF
(Mehta et al., 2001),
suggesting that this is a physiological effect. A was less effective in
reversing the toxicity of secretase inhibitors in SH-SY5Y. However, at higher
concentrations, the effectiveness of A became evident. For instance,
when cells were coincubated with 10 nM A and 10
µM -IV, cell viability was 69 ± 3% (n =
6) compared with 58 ± 7% (n = 8) in the presence of -IV
alone. Increasing the A concentration further to 100 nM or 1
µM increased viability to 80 ± 6% (n = 4) and 83
± 9% (n = 4), respectively.
Importantly, the ability of A to recover cell death in cortical
neurons treated with - or -secretase inhibitors was dependent on
the species of A used. A1–42 represents 10%
of total amyloid detected in human CSF. Coincubation of -IV or SI
with 1 nM A1–42 resulted in partial rescue
of the cell death associated with secretase inhibitors in cortical neurons and
granule cells (Fig.
4B). This isoform of A had no ability to recover
cell death in SH-SY5Y cells at 1 nM
(Fig. 4B) or at
concentrations up to 1 µM (data not shown). In addition, the
synthetic toxic portion of A, A25–35 (1
nM) (Kowall et al.,
1992), failed to rescue the deficit in cortical or granule neuron
viability associated with either secretase inhibitor
(Fig. 4B).
Additional evidence showing that endogenous A production is essential
for the survival of neurons is provided by experiments in which A was
bound by the application of the A-binding antibody 3D6. Incubation of
cortical neuron cultures with 3D6 (1 µg/ml) for 24 hr resulted in a
significant reduction in cell viability
(Fig. 4C). This was an
effect specific to the A-binding properties of the antibody because
coincubation with A1–40, but not
A25–35, was effective in reversing the toxic effect of
3D6.
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Discussion
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The data presented here indicate that inhibition of - or
-secretase in neurons can compromise cell viability. In three different
neuronal phenotypes, the pharmacological knock-down of amyloidogenic secretase
activity resulted in cell death.
Is the toxicity of these compounds attributable to inhibition of A
production? There are several lines of evidence that suggest this to be the
case. First, these compounds have been shown to produce a profound inhibition
of A production at the concentrations used in this study. Greater than
99% inhibition of de novo A production would be expected at
-IV concentrations used here (Beher
et al., 2001), and our own immunocytochemical data would appear to
confirm this. Second, the production of A has been prevented in two ways
via the pharmacological inhibition of two distinct and separate enzymes.
Importantly, all compounds used were structurally different, suggesting that
toxicity was not the nonspecific result of a particular chemical moiety.
Third, in experiments using the 3D6 antibody to bind and neutralize A,
we observed a toxic effect. This approach has been used previously to prevent
some of the effects mediated by A during hypoxia
(Green and Peers, 2001).
Significantly, we were again able to prevent the toxic effect of the antibody
by coincubation with exogenously added A. All of these data suggest that
the toxicity induced by secretase inhibitors in cells with a neuronal
phenotype is not simply a chemical toxicity but is related to an interaction
with neuron-specific pathways, such as A production. Finally, the lack
of toxicity in non-neuronal cell types argues strongly against a nonspecific
toxicity for these compounds.
The notion that toxicity is a result of inhibition of physiological A
production is given additional weight by evidence that the replacement of
endogenous A with picomolar concentrations of exogenous A recovers
cell viability. Importantly, the rescue of cell death by A is dependent
on the species of this peptide used. This effect is most pronounced when the
physiologically prevalent A species 1–40 is used.
A1–40 demonstrated rescue of toxicity in a
concentration-dependent manner and is effective in recovering total cell
viability to control levels at concentrations between 10 pM and 1
nM. This concentration range corresponds with that reported from
human CSF (Mehta et al.,
2001). Notably, A1–42, which is present at
much lower concentrations in human CSF, is less efficient in rescuing neuronal
cell viability. This may represent a lesser physiological requirement for
A1–42, a distinct protein that may be unable to fulfil
A1–40-specific roles. An additional possibility is that
A1–42 levels are not diminished as rapidly as
A1–40 levels after secretase inhibition. This is
supported by recent studies in which A1–42 production
was demonstrated in cells deficient for both presenilin 1 (PS1) and PS2, the
likely locus of -secretase inhibitor activity
(Wilson et al., 2002).
However, the effects of -secretase inhibitors in our study argue against
this possibility. In marked contrast to the protective effect of
A1–40, the 25–35 amino acid sequence of A
afforded no rescue to neuronal cells treated with inhibitors of either -
or -secretase. This synthetic peptide does not exist naturally in
either healthy individuals or Alzheimer's pathology. It is therefore not
surprising that A25–35 appears to be unable to
substitute for endogenous A in preventing secretase inhibitor
toxicity.
Pharmacological inhibitors of - or -secretase activities are
being developed at a prodigious rate. The question arises therefore of why
this overt neurotoxicity has not been reported previously. Many studies
explore the pharmacology of these compounds via cell-free processing often in
terms of the enzymatic activity of - and -secretases
(Sinha et al., 1999). In
vitro studies predominantly use a range of non-neuronal cell lines that
have been transformed to overexpress amyloid precursor protein
(Sinha and Lieberburg, 1999).
Because the data presented here suggests that these cells do not rely on
A production for viability, the toxicity of the secretase inhibitors
would not have been observed. Studies using neuronal cells have only examined
the efficacies of secretase inhibitors over short incubation times
(Beher et al., 2001). Allusions
have been made to the toxicity of secretase inhibitors, restricting the
concentrations of, for example, -IV that are suitable to explore
-secretase-dependent behavior in embryonic stem cells, although this
toxicity was not further investigated
(Wilson et al., 2002).
The effects of reduced secretase activity have also been investigated using
knock-out mice. -Secretase activity is mediated by BACE, or -site
APP cleavage enzyme (Vassar and Citron,
2000). BACE-1 knock-out mice have been used to probe the
functional importance of this enzyme and were found to have a normal phenotype
for up to 1 year with a reduced A production
(Roberds et al., 2001). This
might be taken to suggest that BACE-1 does not participate in the generation
of physiologically important proteins (Cai
et al., 2001; Luo et al.,
2001). Constitutive knock-down of the activity of a single enzyme
often leads to compensatory mechanisms in genetically manipulated organisms.
Compensation for this deficit by BACE-2 and as yet unidentified enzymes is a
possible explanation for this anomaly. Additional inhibition of BACE-2
activity would aid our understanding of the physiological relevance of
-secretase activity.
In contrast to BACE, a double-genetic knock-out of presenilin 1 and 2
activity proved to be fatal in utero
(Herreman et al., 1999). This
toxicity is primarily a result of inhibition of Notch signaling
(De Strooper et al., 1999), and
inhibition of Notch was suggested at the time to be a possible source of
-secretase inhibitor toxicity. Knock-out of PS1 alone does not have a
toxic effect, although A production is reduced by 80% in cells cultured
from such animals (De Strooper et al.,
1998; Annaert et al.,
2001). Thus, the physiological role of A remains unconfirmed
using such models.
An alternative to secretase knock-out is the knock-out of APP. This appears
to cause mild neurological and behavioral deficits in homozygous animals
(Zheng et al., 1995). Neurons
from such animals can be readily grown in culture
(Harper et al., 1998;
White et al., 1998),
suggesting that APP and its cleavage products are not required for cell
survival. However, recent studies have revealed that cleavage products of the
closely related APP-like proteins (APPLP1 and APPLP2) have a role as
transcriptional regulators (Scheinfeld et
al., 2002) and can substitute for APP itself. A combined APP/APPLP
knock-out displays growth retardation and early postnatal lethality
attributable to an unknown cause, supporting the hypothesis that the APP/APPLP
family and their products have important physiological functions
(Heber et al., 2000).
In conclusion, we showed that, in rodent and human neuronal cell types,
in vitro inhibition of - or -secretase production of
A and binding and neutralization of A with an antibody leads to
toxicity. Recovery of cell viability by the addition of physiologically
relevant concentrations of A suggest that this peptide may have a role
to play in the normal function of neuronal cells as suggested previously
(Ramsden et al., 2001).
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Footnotes
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Received Sep. 9, 2002;
revised Apr. 30, 2003;
accepted Mar. 7, 2003.
We gratefully acknowledge the assistance of the Medical Research Council,
the Wellcome Trust, and Pfizer Central Research in supporting this work.
Correspondence should be addressed to Dr. H. A. Pearson, School of
Biomedical Sciences, University of Leeds, Leeds LS2 9JT, UK. E-mail:
h.a.pearson{at}leeds.ac.uk.
Copyright © 2003 Society for Neuroscience
0270-6474/03/235531-05$15.00/0
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Top
Abstract
Introduction
