Proc. Natl. Acad. Sci.
USA
Vol. 93, pp. 8068-8071, July 1996
Pharmacology
Amyloid .3-protein reduces acetylcholine synthesis in a cell line
derived from cholinergic neurons of the basal forebrain
(acetylcholinesterase/Alzheimer disease/choline acetyltransferase/retinoic acid)
WARD A. PEDERSEN, MAREK A. KLOCZEWIAK, AND JAN KRZYSZTOF BLUSZTAJN*
Department of Pathology and Laboratory Medicine and Division of Psychiatry, Boston University School of Medicine, 85 East Newton Street, Boston, MA 02118
Communicated by Susan E. Leeman, Boston University School of Medicine, Boston, MA, March 25, 1996 (received for review September 29, 1995)
ABSTRACT The characteristic features of a brain with
Alzheimer disease (AD) include the presence of neuritic
plaques composed of amyloid 3-protein (A,B) and reductions
in the levels of cholinergic markers. Neurotoxic responses to
A,B have been reported in vivo and in vitro, suggesting that the
cholinergic deficit in AD brain may be secondary to the
degeneration of cholinergic neurons caused by Aj8. However,
it remains to be determined if A,B contributes to the cholin-
ergic deficit in AD brain by nontoxic effects. We examined the
effects of synthetic A,B peptides on the cholinergic properties
of a mouse cell line, SN56, derived from basal forebrain
cholinergic neurons. Aj8 1-42 and Aj3 1-28 reduced the ace-
tylcholine (AcCho) content of the cells in a concentration-
dependent fashion, whereas Af3 1-16 was inactive. Maximal
reductions of 43% and 33% were observed after a 48-h
treatment with 100 nM of A18 1-42 and 50 pM of A18 1-28,
respectively. Neither A,B 1-28 nor A,B 1-42 at a concentration
of 100 nM and a treatment period of 2 weeks was toxic to the
cells. Treatment of the cells with A,B 25-28 (48 h; 100 nM)
significantly decreased AcCho levels, suggesting that the
sequence GSNK (aa 25-28) is responsible for the AcCho-
reducing effect of Af3. The reductions in AcCho levels caused
by A,B 1-42 and Af3 1-28 were accompanied by proportional
decreases in choline acetyltransferase activity. In contrast,
acetylcholinesterase activity was unaltered, indicating that A18
specifically reduces the synthesis of AcCho in SN56 cells. The
reductions in AcCho content caused by AfJ 1-42 could be
prevented by a cotreatment with all-trans-retinoic acid (10
nM), a compound previously shown to increase choline acetyl-
transferase mRNA expression in SN56 cells. These results
demonstrate a nontoxic, suppressive effect of A,B on AcCho
synthesis, an action that may contribute to the cholinergic
deficit in AD brain.
An invariant pathological feature of a brain with Alzheimer
disease (AD) is the formation of the neuritic plaque, a
compacted extracellular deposit of amyloid filaments sur-
rounded by dystrophic neurites (1). This amyloid is composed
of the --4-kDa amyloid 13-protein (A13), which is proteolytically
derived from a 110-130 kDa transmembrane glycoprotein
known as the ,B-amyloid precursor protein (,BAPP) (1). Het-
erogeneous processing of 1APP results in the formation of A,B
peptides ranging from 39 to 43 amino acids in length (1).
Several missense mutations in the j3APP gene have been linked
to familial AD (2-4), and some of these have been shown to
result either in excess generation of AP3 (5-7) or in a shift to
the production of longer, more amyloidogenic forms (8).
Overexpression of the PAPP gene, which is located on the long
arm of chromosome 21, occurs in Down syndrome (trisomy 21)
patients, essentially all of whom exhibit AD brain pathology by
the age of 40 (9). Furthermore, transgenic mice overexpressing
a mutated human ,BAPP gene display many of the pathological
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8068
features of AD brain, including the neuritic plaque (10). These
observations suggest a primary role for f3APP/Af3 in the
pathogenesis of AD. However, the mechanisms by which AP3
causes the cognitive impairment characteristic of AD remain
obscure.
Among the numerous neurochemical abnormalities de-
scribed for the brains of AD patients, the decrease in the
activity of the acetylcholine (AcCho)-synthesizing enzyme,
choline acetyltransferase (ChAT), is the most prominent and
provides an excellent biochemical correlate of the severity of
dementia in this disorder (11-14). These findings support the
contention that cholinergic neurons are particularly vulnerable
in AD brain (15) and are consistent with the importance of
AcCho in memory and learning processes (16, 17). Peptides
derived from the human AP sequence have been shown to
cause toxic effects in cell lines and primary cultures of neuronal
origin (18-23) as well as in vivo (24, 25). Moreover, injections
of A13 1-40 or A,B 1-42 into the nucleus basalis magnocellularis
or the medial septum of the rat brain cause neurodegeneration
and reductions in the levels of cholinergic markers (26-28).
Therefore, the neurotoxic effect of AP3 peptides suggests that
the loss of cholinergic markers in AD brain is secondary to the
degeneration of cholinergic neurons. However, other studies
have shown that injection of A,B peptides into the medial
septum of the rat causes a reduction in AcCho release from the
hippocampus in the absence of toxicity (29). Thus, we sought
to determine if A,B affects the synthesis of AcCho in a basal
forebrain cholinergic cell line without causing toxicity.
MATERIALS AND METHODS
Aj3 Peptides. The AP3 peptides 1-16, 1-28, 1-42, and 25-35
were purchased from Bachem and the A(3 25-28 (GSNK)
peptide was synthesized by a standard fluorenylmethoxycar-
bonyl (Fmoc) solid-phase method (30) using the automated
peptide synthesizer PS-3 from Rainin Instruments. After
synthesis, the resin was washed with dimethylformamide and
methanol, and then dried under a vacuum overnight. The
peptide was cleaved from the resin with a mixture of 88%
trifluoroacetic acid/5% phenol/3% water/3% ethane di-
thiol/1% triisopropylsilane at room temperature for 2 h,
passed through a glass filter, precipitated with cold ethyl ether,
washed twice more with cold ethyl ether, and collected by
centrifugation. The pellet was dissolved in 10% acetic acid and
freeze-dried. The GSNK peptide was purified by reverse-phase
HPLC on a C18 preparative column using water-acetonitrile
mobile phase containing 0.05% trifluoroacetic acid, and re-
purified in a semipreparative C18 column. The purity of the
peptide was determined by analytical reverse-phase HPLC.
Abbreviations: AcCho, acetylcholine; AP3, amyloid (3-protein; AD,
Alzheimer disease; I3APP, 13-amyloid precursor protein, ChAT, cho-
line acetyltransferase; t-RA, all-trans-retinoic acid.
*To whom reprint requests should be addressed. e-mail:
jbluszta@bu.edu.
 Pharmacology: Pedersen et aL
Stock solutions of AP3 peptides were prepared in dimethyl
sulfoxide to a concentration of 1 mM and stored at -20°C.
Cell Culture and Treatment Protocol. The SN56 cell line
was generated by the fusion of N18TG2 neuroblastoma cells
(which do not express cholinergic markers) with septal neurons
obtained from postnatal day 21 mice (31, 32), and it therefore
provides an in vitro model for the study of basal forebrain
cholinergic neurons. The SN56 cells (clone SN56.B5.G4) were
maintained at 37°C in an atmosphere of 95% air/5% CO2 in
Dulbecco's modified Eagle's medium containing 10% fetal
bovine serum and 50 p,g/ml gentamicin. Media in stock flasks
were changed every 2-3 days, and the cells were subcultured
by mechanically removing them from the substratum with
squirts of fresh media. Cells of up to passage 25 were used. The
stock solutions of AP3 peptides were diluted in growth medium
immediately before application to the cells. (The highest
concentration of the peptides used was 1 AM, corresponding
to a final concentration of dimethyl sulfoxide of 0.1%, which
has no effect on the AcCho content of SN56 cells.) During
treatment periods, fresh control medium or one containing the
desired peptide was applied to the cells every 24 h. After
treatment, AcCho was extracted as described below.
Extraction and Measurement of AcCho and ChAT Activity.
Cells were grown to subconfluence in 35-mm culture dishes.
After the desired treatment, the medium was removed and the
cells were incubated for 1 h at 37°C in physiological salt
solution (pH 7.4) supplemented with 5 ,uM choline and 15 ,uM
neostigmine and consisting of the following: 135 mM NaCl, 5
mM KCl, 1 mM CaCl2, 0.75 mM MgCl2, 10 mM glucose, 10
mM Hepes. The cells were washed once with ice-cold, choline-
free physiological salt solution supplemented with 15 ,uM
neostigmine, methanol was added, the cells were scraped into
the methanol, and the suspension was transferred to a polypro-
pylene tube. A solution of 15% 1.0 M formic acid and 85%
acetone was added, the tubes were vortex mixed, and the
extracts centrifuged to pellet the protein. Both the protein
pellets and the supernatant fluids containing AcCho were
dried under a vacuum. The dry, AcCho-containing pellet was
resuspended in HPLC mobile phase [28 mM phosphate (pH
8.5) supplemented with Kathon CG Reagent], sonicated, and
filtered (0.2 ,um Nylon-66, Rainin Instrument). The content of
AcCho was determined by HPLC with an enzymatic reactor
containing acetylcholinesterase and choline oxidase and an
electrochemical detector using a commercial kit (Bioanalytical
Systems, West Lafayette, IN) based on the method of Potter
et al. (49). ChAT activity was measured in cell homogenates by
the radioenzymatic assay of Fonnum (33), and acetylcholines-
terase activity was measured by a modification of this method.
Protein content was determined using the bicinchoninic acid
assay with bovine serum albumin as the standard (34).
a3
350
0)
-.
300-
E
0 250-
E 200-
a-
0.
C 150-
0
_c-0 100-
50-
C.)
'.. 11
0 1 10 100
AB 1-42, nM
FIG. 1. The levels of AcCho in SN56 cells are reduced by AO3 1-42 and A,B 1-28 in a concentration-dependent manner. The cells were grown
for 2 days in the presence of either A,B 1-28 or Af3 1-42 at the concentrations indicated, and the levels of AcCho measured as described. The data,
from representative experiments, are reported as means ± SD of triplicate cultures.
Proc. Natl. Acad. Sci. USA 93 (1996) 8069
RESULTS AND DISCUSSION
We determined the effects of synthetic A,B 1-42, which rep-
resents the entire amyloid-forming sequence, and synthetic A,B
1-28, which is derived from a putative extramembraneous
region of ,BAPP and is considered nonamyloidogenic (18, 35,
36), on the intracellular levels of AcCho in SN56 cells. After
a 48-h treatment, both peptides reduced the intracellular levels
of AcCho in SN56 cells in a concentration-dependent and
saturable manner. A maximum reduction of 43% was observed
in cells treated with A,B 1-42 at a concentration of 100 nM (Fig.
1). A,B 1-28 was less effective but more potent than A13 1-42,
causing a maximal decrease in AcCho content of 33% at a
concentration of 50 pM (Fig. 1). Importantly, neither peptide
was cytotoxic, caused any morphological changes to the cells,
or reduced the rate of cell proliferation, even upon a prolonged
treatment of 2 weeks (data not shown).
The primary structural determinant for the formation of
stable aggregate structures by A,B are the hydrophobic residues
in its C terminus, amino acids 29-42 (36, 37). Because AP3 1-28
lacks this hydrophobic domain, it is far less effective than AP3
1-42 at forming stable aggregates and in eliciting neurotoxic
responses. In fact, concentrations of A,3 1-28 well into the
micromolar range were required to cause any degree of toxicity
in primary hippocampal cultures (18), whereas AP3 1-42 has
been reported to cause toxicity to rat cholinergic PC12 cells at
a concentration of 20 nm (38). Thus, our data demonstrating
that picomolar concentrations of AP3 1-28 reduced AcCho
levels in SN56 cells, along with the fact that the AP3 peptides
were not toxic to the cells under our treatment conditions,
suggest that this effect is aggregation-independent and is
mediated by a mechanism distinct from that responsible for the
neurotoxic action of AP3.
To determine which region of the A,B sequence is respon-
sible for the reductions in AcCho levels in SN56 cells, we
compared the effects of several peptides (all at 100 nM for
48 h) representing different regions of full-length A,B. AP3 1-16,
which is inactive in other systems (18), did not alter the AcCho
content of SN56 cells (Fig. 2). In contrast, AP3 25-35 decreased
the levels of AcCho in SN56 cells to the same extent as AP3 1-28,
although both peptides were less effective than A,B 1-42 (Fig.
2). The A,3 peptides that caused a reduction in AcCho levels
are characterized by a common sequence consisting of amino
acids 25-28 (GSNK). Treatment of SN56 cells with the peptide
GSNK reduced the cellular AcCho content by -25% (Fig. 2).
These results suggest that the region consisting of amino acids
25-28 is responsible for mediating the AcCho-reducing effect
of AP3. It is not clear why AP3 25-28, A,B 1-28, and AP3 25-35 are
less effective than AP3 1-42 at decreasing the levels of AcCho
in SN56 cells, but these results indicate that the maximal
AcCho-reductive effect requires the entire A,B sequence. A
ci) 3QQ A
a
300 A
._
250-
E
200-
0
ci_
d 150-
E 100-
0 50-
''
v,\ [r I. 1.1 I.
I. 1. I...
1000 0 1 10 100
AB 1-28, pM
8070 Pharmacology: Pedersen et al.
AG 1-42
AB 1-28
AB 25-35
AG 25-28
AB 1 --1_
0 20 40 60 80 100
Acetylcholine, %control
FIG. 2. The levels of AcCho in SN56 cells are reduced by AP3 25-28,
A,B 25-35, AP 1-28, and AP3 1-42, but not AP3 1-16. The cells were grown
for 48 h in the presence of the peptides indicated (100 nM each), with
the medium changed daily, and the levels of AcCho measured as
described. The results are reported as means ± SD of triplicate
cultures. With the exception of the difference between the control and
the A,B 1-16 treated group, the differences between the control and
each of the other groups were statistically significant (Dunnett's test;
P < 0.01). Moreover, a significant difference was found between the
AP3 1-42 treated group and each of the other groups (Tukey test; P <
0.01).
computer search of protein databases (using the BLAST pro-
gram) revealed that the sequence GSNKGA (A13 25-30) can be
found as a conserved motif only in 13APP and in a family of
proteins that includes calponin, a major component of the
smooth muscle thin filament (39), and a protein designated
NP25, which has been shown to be differentially expressed in
neuronal subpopulations of the rat central nervous system
(40). The functional significance of this conserved motif is as
yet undefined. It will be interesting to determine if the
anticholinergic actions of AP3 peptides described here are
related to their homology to calponin or other members of this
family of proteins.
We sought to determine if the AP3-induced reductions in the
intracellular levels of AcCho in SN56 cells are due to decreases
in ChAT activity. In cells treated with AP3 1-28 or AP3 1-42 at
a concentration of 100 nM for 48 h, ChAT activity was
decreased by 28% and 41%, respectively (Fig. 3), an effect in
excellent agreement with the observed reductions in AcCho
levels (Figs. 1 and 2). In contrast, acetylcholinesterase activity
was unaltered (Fig. 3), indicating that A,B specifically reduces
the synthesis of AcCho in SN56 cells and that the effect is not
due to generalized toxicity. Postmortem analysis of cortical
samples of AD brain revealed that ChAT activity is reduced by
Control
00 El AB 1-28
CD
E E HAB 1-42
0- 0 t-RAIA3 1-42, 10 nM _
Ca) Cf)
E E
a)
0
_ AB 1-42, lO nM
E E
O= 0 0-
Ui
FIG. 3. AP3 1-28 and AP3 1-42 reduce the activity of ChAT but do
not alter the activity of acetylcholinesterase (AChE) in SN56 cells. The
cells were grown for 2 days in the presence of 100 nM of either AP3 1-28
or A,B 1-42 and ChAT and AChE activities in cell homogenates were
determined by the method of Fonnum (33). The data, from a
representative experiment, are reported as means ± SD of triplicate
cultures. The differences in ChAT activity between control and AP3
1-28, and between control and A,B 1-42 treated groups were statisti-
cally significant (P < 0.01; Tukey test). AChE activity was not
statistically different among the groups.
Proc. Natl. Acad. Sci. USA 93 (1996)
30-80% relative to age-matched controls (11-14). Animal
studies have shown that significant impairments of memory
functions are observed in rats after bilateral ibotenic acid
lesions of the nucleus basalis magnocellularis (17), which
provides the major cholinergic innervation to the neocortex.
Those lesions reduced ChAT activity by 10% and 30% in the
parietal and frontal cortices, respectively (17). Thus, the
magnitude of the AP3-evoked reductions in ChAT activity
observed in our experimental system correlates well with that
observed in AD and with that necessary to cause memory
dysfunction in experimental animals.
In agreement with other reports of phenotypic actions of AP3
peptides at low nanomolar concentrations (41, 42), our results
indicate that some of the effects of AP3 are due to interactions
with high-affinity binding sites. Characterization of the puta-
tive high-affinity binding sites of A,B in basal forebrain cho-
linergic neurons could have therapeutic implications for AD in
that it might permit the development of antagonists to these
binding sites that would prevent the suppression of AcCho
synthesis in these cells. An alternative strategy for AD therapy
would be to use compounds that do not interact with the
binding sites of AP3, but would overcome the actions of the
peptides by enhancing the expression of cholinergic markers
through other mechanisms. We have previously shown that
all-trans-retinoic acid (t-RA) increases intracellular AcCho
content, ChAT activity, and ChAT mRNA levels in SN56 cells
(43, 44). Here we show that t-RA, at physiological concentra-
tions, can overcome the reductions in AcCho levels caused by
A,B in SN56 cells (Fig. 4). These results suggest that the AcCho
content of septal cells may be the result of a balance between
trophic factors and A,B, and that a shift in this balance could
either enhance or suppress the synthesis of this neurotrans-
mitter. This idea is consistent with the observations that
down-regulation of the cholinergic phenotype can occur in vivo
when cholinergic neurons are deprived of their targets and/or
trophic factors (45-48). Thus, we propose that a therapeutic
strategy for AD may include the use of compounds (e.g.,
retinoids) and growth factors that prevent the suppression of
AcCho synthesis caused by AP3.
We propose that the reduction in ChAT activity in cortical
and basal forebrain AD tissue is a consequence of both the
direct, nontoxic anticholinergic effects of A,B described here
and the toxic actions of the peptide described in other reports.
Evidence suggests that A,B has selective toxic effects on
different types of neurons. For instance, injection of A,B 1-42
into the medial septum of the rat brain caused degeneration of
cholinergic but not GABAergic neurons (27). Thus, although
speculative at present, it is possible that basal forebrain
t-RANAB 1-42, 1 nM
t-RA, lO nM
AB 1-42, 1 nM
Control
0 200 400 600 800
Acetylcholine, pmol/mg protein
FIG. 4. t-RA prevents the AP 1-42-evoked decreases in the content
of AcCho in SN56 cells. The cells were grown for 48 h in the presence
of A,B 1-42 (1 nM or 10 nM), t-RA (10 nM), or combinations of AP
1-42 and t-RA as indicated, and the levels of AcCho measured as
described. The results are reported as means ± SD of triplicate
cultures. The differences between the control and each of the other
groups were statistically significant (P < 0.01; Dunnett's test), but no
differences among the t-RA-treated groups were significant.
 Pharmacology: Pedersen et al.
cholinergic neurons are also particularly sensitive to the non-
toxic effects of AP3. A suppressive effect of A,B on the synthesis
of AcCho in basal forebrain cholinergic neurons would prob-
ably be most relevant in the early stages of AD, before
significant plaque formation and the apparent toxicity that
accompanies A,B deposition.
We are grateful to Dr. Bruce Wainer for the gift of SN56 cells. This
work was supported by the National Institute on Aging Grant AG-
09525.
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