? The?Journal?of?Clinical?Investigation http://www.jci.org
Cholinergic dysfunction in a mouse model
of Alzheimer disease is reversed
by an anti-Aβ antibody
Kelly R. Bales,1,2 Eleni T. Tzavara,1 Su Wu,1 Mark R. Wade,1 Frank P. Bymaster,1
Steven M. Paul,1 and George G. Nomikos1
1Neuroscience Discovery Research, Eli Lilly and Company, Indianapolis, Indiana, USA. 2Program of Medical Neurobiology,
Indiana University School of Medicine, Indianapolis, Indiana, USA.
The cholinergic neurotransmitter system in brain is critical for the
processing of information related to cognitive function (1). The
nearly complete destruction of cholinergic neurons located within
the nucleus basalis of Meynert in Alzheimer disease (AD), has led
many investigators to postulate that cholinergic dysfunction is a
primary cause of the memory decline associated with AD (2–4).
An alternative but not mutually exclusive hypothesis of AD patho-
genesis, the “amyloid cascade hypothesis,” postulates that memory
deficits are caused by increased brain levels of both soluble and
insoluble amyloid β (Aβ) peptide(s), which are derived from the
larger amyloid precursor protein (APP) by sequential proteolytic
processing (5). Although no direct clinical evidence in support
of this hypothesis is yet available, ample genetic evidence derived
from mutations within the APP gene associated with familial
early-onset forms of AD supports an important role for the Aβ
peptide(s) in AD pathogenesis (6). In addition to the abundant
deposits of Aβ in brain parenchyma of AD patients, there are also
neuritic plaques and neurofibrillary tangles within the basal fore-
brain and neocortical cholinergic pathways (3, 4). Although defi-
cits in several neurotransmitter systems have been observed in AD
brain, basal forebrain cholinergic neurons appear to be exquisitely
sensitive and susceptible to the disease process, and the majority
of currently available therapies, which do not alter disease progres-
sion, target the cholinergic synapse in an attempt to increase syn-
aptic levels of acetylcholine (ACh) in order to relieve the memory
deficits associated with disease progression.
Both soluble and insoluble forms of the Aβ peptide(s) have been
shown to disrupt synaptic transmission and inhibit long-term
potentiation in vivo as well as to cause memory impairment in
transgenic mouse models of AD, which overexpress mutations
associated with familial forms of AD (7, 8). Moreover several stud-
ies in humans have demonstrated significant correlations between
cognitive impairment and the level of soluble (9–11) and certain
deposited forms of Aβ (12). Additionally, we have recently demon-
strated that administration of the anti-Aβ antibody m266, which
binds with very high affinity to the mid-domain region of the sol-
uble forms of Aβ, is able to rapidly reverse memory impairment in
PDAPP mice following acute or subchronic administration with-
out any measurable change in brain Aβ burden (13).
To investigate whether the Aβ peptide(s) may directly affect cho-
linergic function in the absence of overt neurodegeneration, we
measured hippocampal ACh release using in vivo microdialysis in
awake, freely moving transgenic mice that overexpress a mutation
associated with familial AD (PDAPP mice). PDAPP mice represent
a well-characterized animal model of AD-like plaque pathology
with Aβ and amyloid deposition occurring in an age- and brain
region–dependent fashion (14). Although these mice have behav-
ioral deficits, they do not develop neurodegeneration or frank loss
of cholinergic neurons even as they age (15–17). Here we report an
Aβ-dependent disruption of hippocampal ACh release in PDAPP
mice that was associated with impaired habituation learning.
Kinetic analysis of high-affinity choline uptake into synaptosomes
prepared from the hippocampus of PDAPP mice demonstrated a
significant increase in Vmax without any measurable effect on Km.
We also observed a marked effect of Aβ42 on high-affinity choline
uptake in rat hippocampal synaptosomes or membranes prepared
from a stable cell line expressing the human choline transporter
(ChT-1). Additionally, we utilized coimmunoprecipitation fol-
Nonstandard?abbreviations?used: Aβ, amyloid β; ACh, acetylcholine; AD, Alzheimer
disease; APP, amyloid precursor protein; ChT-1, choline transporter.
Conflict?of?interest: All authors are or have been employed by Eli Lilly and Company.
Citation?for?this?article: J. Clin. Invest. doi:10.1172/JCI27120.
?? The?Journal?of?Clinical?Investigation http://www.jci.org
lowed by Western blot analysis to determine that Aβ could directly
associate with ChT-1 and potentially interfere with its normal
physiological role. Finally, acute treatment of PDAPP mice with
the anti-Aβ antibody m266 is able to fully restore cholinergic tone
and impaired habituation learning, suggesting that if the same sol-
uble “cholinotoxic” species of Aβ are involved with memory defi-
cits associated with early AD or mild cognitive impairment, then
passive administration with anti-Aβ antibodies may have similar
beneficial effects in the clinic.
Basal release and tissue levels of hippocampal ACh are reduced in PDAPP
mice. We first directly assessed ACh release in the hippocampus of
young (pre-plaque) PDAPP and WT mice under steady-state con-
ditions using in vivo microdialysis and found that basal levels of
hippocampal ACh release were markedly reduced in PDAPP mice
compared with age- and background-matched WT controls (Fig-
ure 1A; P < 0.05). Since in vivo microdialysis measures extracellular
concentrations of ACh, we also determined the tissue levels of ACh
in hippocampal and cortical homogenates from these mice. Similar
concentrations of ACh were measured in young mice (2 months of
age), but tissue levels of ACh were significantly reduced in PDAPP
mice at older ages (>4 months), confirming that the reduced level
of ACh release from the hippocampus as measured with in vivo
microdialysis mirrored those that were measured directly in tissue
homogenates (Figure 1, B and C; P < 0.05).
Evoked release of ACh is dysregulated in PDAPP mice. We next deter-
mined the functional responsiveness of hippocampal cholinergic
neurons in PDAPP and WT mice following a pharmacological
(systemic administration of scopolamine) or physiological (expo-
sure to a novel environment) challenge. Both conditions have been
shown to increase hippocampal ACh efflux and to constitute a reli-
able index of functional responsiveness of the cholinergic system
(18). As previously reported, when WT mice are placed into a novel
environment, a situation expected to evoke a physiological release
of ACh, hippocampal ACh efflux increases modestly (~50%) and
then returns to baseline levels within 60 minutes (18). By contrast,
when PDAPP mice were exposed to the same novel environment,
the level of hippocampal?ACh release was markedly elevated and
prolonged, increasing by more than 2.5-fold for nearly 90 minutes,
Hippocampal ACh release and tissue levels are reduced in PDAPP
transgenic mice. (A) Basal levels of hippocampal ACh release mea-
sured by in vivo microdialysis from WT and PDAPP transgenic mice
(n = 7–10 mice per group, 4–6 months of age). (B and C) Tissue levels
of ACh from the hippocampus (B) and cortex (C) of PDAPP and WT
mice (n = 6 mice per group). *P < 0.05 versus WT.
Hippocampal ACh release in PDAPP mice is restored to WT levels
after anti-Aβ treatment. (A) Hippocampal ACh release in WT and
PDAPP transgenic mice following exposure to a novel environment
(Novelty) and after treatment of PDAPP mice with the anti-Aβ antibody
m266 (PDAPP + m266; 500 μg i.p.). The arrows indicate the time at
which mice were placed into a novel environment (novelty) and back
into their home cage. (B) Release of ACh from the hippocampus of WT
and PDAPP mice following administration of scopolamine (0.3 mg/kg
i.p.) and after administration of m266. n = 7–10 mice per group, 4–6
months of age. (C) Extracellular levels of choline in brain measured by
in vivo microdialysis from WT and PDAPP mice. n = 5 mice per group,
4–6 months of age. (D) Basal levels of hippocampal ACh release in
WT, PDAPP, and PDAPP mice administered m266. n = 7–10 mice per
group, 4–6 months of age. *P < 0.05 versus WT; #P < 0.05, PDAPP
versus PDAPP + m266.
? The?Journal?of?Clinical?Investigation http://www.jci.org
compared with the levels of hippocampal ACh release observed
in WT mice measured under identical conditions (Figure 2A;
P < 0.05). Following administration of scopolamine (0.3 mg/kg
i.p.), a pan-muscarinic receptor antagonist, the level of ACh release
(resulting from blockade of inhibitory muscarinic receptors) from
the hippocampus of PDAPP mice was significantly reduced com-
pared with similarly treated WT mice (Figure 2B; P < 0.05). Under
all parameters measured (i.e., basal conditions), and following
physiological (exposure to novelty) or pharmacological (following
administration of scopolamine) stimuli, the efflux of ACh from
the hippocampus of PDAPP mice was significantly different from
that of age- and background-matched WT mice (Figure 2).
Extracellular levels of choline are increased in the hippocampus of PDAPP
mice. Could the reduced extracellular and tissue levels of ACh be due
to reduced availability of the ACh precursor choline? To address this
possibility, we determined the level of extracellular choline using in
vivo microdialysis. To our surprise, extracellular choline was sig-
nificantly increased in PDAPP mice compared with age-matched
WT controls (Figure 2C; P < 0.05), thus confirming that reduced
extracellular levels of the ACh precursor could not account for the
reduced level of ACh measured in either tissue homogenates or the
extracellular fluid with in vivo microdialysis (see Discussion).
Aβ directly interacts with ChT-1. One possible explanation for the
significant increase in extracellular levels of choline is that the
Aβ peptide interacts directly with ChT-1, effectively
blocking efficient transport and ACh biosynthesis. To
investigate this possibility, we prepared synaptosomes
from rat hippocampi and determined high-affin-
ity choline transport following exposure to human
Aβ42 (Figure 3A). There was a significant increase
in choline uptake when rat synaptosomes where
exposed to Aβ42 (100 nM; P < 0.05). Since nerve termi-
nal preparations contain elements of synaptic vesicles
and Aβ has been observed to have effects on multiple
intracellular systems including synaptic vesicles, we
next determined whether high-affinity choline uptake
would be affected when cells expressing human ChT-1
were exposed to human Aβ42 (Figure 3B). Similar to
the results obtained when rat synaptosomes were
exposed to Aβ42, there was a significant increase in
choline uptake even when cells expressing human
ChT-1 were exposed to very low concentrations of Aβ42
(100 nM; P < 0.05) for a relatively short (15-minute)
period of time. The reverse peptide (Aβ42–1) was with-
out effect while the Aβ-dependent increase in high-
affinity choline uptake occurred following exposure
to either freshly prepared or aged Aβ42 peptide (data
not shown). We next used coimmunoprecipitation to
determine which if any species of Aβ is associated with
ChT-1. When hippocampal homogenates prepared
from PDAPP mice were coimmunoprecipitated with
an anti-Aβ antibody, 4G8, an approximately 70-kDa
band representing ChT-1 was apparent (Figure 3C).
Conversely, an approximately 4-kDa band repre-
senting Aβ was clearly evident when hippocampal
homogenates from PDAPP mice were coimmunopre-
cipitated with an anti–ChT-1 antibody followed by
immunoblotting with the C-terminal–specific anti-
Aβ antibodies 21F12 and 2G3 (Figure 3D). No Aβ was
detected when hippocampal homogenates from WT
mice were coimmunoprecipitated with an anti–ChT-1 antibody
and probed with anti-Aβ antibodies (data not shown). ChT-1 was,
however, coimmunoprecipitated from hippocampal homogenates
prepared from WT mice by the anti-Aβ antibody 4G8 (Figure 3C).
Since our initial coimmunoprecipitation experiments utilized an
anti-Aβ antibody that recognizes the mid-domain region of the
Aβ peptide and would thus coimmunoprecipitate both Aβ40 and
Aβ42, we next sought to determine whether either or both species
of Aβ could interact with ChT-1. Again we employed coimmuno-
precipitation of Aβ utilizing anti-Aβ antibodies selective for Aβ
peptides ending in either 40 (2G3) or 42 (21F12) amino acids.
Similar to the results obtained following coimmunoprecipita-
tion with the anti-Aβ antibody 4G8, ChT-1 was detected when
hippocampal homogenates from PDAPP mice were coimmuno-
precipitated with Aβ42-selective antibody 21F12, but not with
Aβ40-selective antibody 2G3, followed by immunoblotting with an
anti–ChT-1 antibody (Figure 3E). ChT-1 was not detected when
hippocampal homogenates from WT mice were coimmunopre-
cipitated with either C-terminal–selective antibody (Figure 3E).
Since the highly hydrophobic nature of Aβ could result in a non-
specific association with ChT-1, we performed coimmunoprecipi-
tation experiments with an antibody directed against the gluta-
mate-1 transporter as well as with an irrelevant IgG. We did not
detect any Aβ when hippocampal homogenates from PDAPP mice
Aβ interacts with ChT-1. (A and B) High-affinity choline uptake into rat synap-
tosomes (A) and a cell line expressing the human ChT-1 (B) was significantly
increased after exposure to Aβ42. Data are the percent of choline uptake in untreat-
ed, control samples (mean ± SEM of triplicate values) from 1 representative experi-
ment, which was repeated 2–3 times with similar results. *P < 0.05. (C) ChT-1
was coimmunoprecipitated from hippocampal extracts by the anti-Aβ antibody 4G8
followed by Western blot analysis with an anti–ChT-1 antibody. (D) The Aβ peptide
was coimmunoprecipitated from hippocampal extracts prepared from PDAPP trans-
genic mice by an anti–ChT-1 antibody followed by Western blot analysis with bioti-
nylated anti-Aβ antibodies 21F12 and 2G3. (E) The anti-Aβ42 antibody 21F12, but
not the anti-Aβ40 antibody 2G3, coimmunoprecipitated ChT-1 from hippocampal
extracts prepared from PDAPP transgenic mice followed by Western blot analy-
sis with an anti–ChT-1 antibody. (F) Neither an irrelevant IgG nor an anti–gluta-
mate 1 transporter antibody (EAAT-1) coimmunoprecipitated Aβ from hippocampal
extracts prepared from PDAPP mice, whereas Aβ was readily detected following
immunoprecipitation with an antibody directed toward ChT-1 followed by Western
blot analysis with an anti-Aβ antibody.
were immunoprecipitated with an anti–glutamate-1 transporter
antibody or irrelevant IgG followed by probing for the presence
of Aβ (Figure 3F).
High-affinity choline uptake into hippocampal synaptosomes is increased
in PDAPP mice. Since the rate-limiting step in ACh biosynthesis is
the uptake of extracellular choline by the neuronal high-affinity
ChT-1, we next determined the level of ChT-1 mRNA in PDAPP
and WT mice, as a decrease in ChT-1 expression could explain
the reduced basal levels of ACh and higher levels of extracellular
choline in brain. There was no significant difference in the level of
hippocampal ChT-1 mRNA between PDAPP and WT controls as
determined by real-time quantitative PCR (Figure 4) at either 2 or
17 months of age. Interestingly, however, we found an age-depen-
dent increase in the level of ChT-1 mRNA in both WT and PDAPP
mice (Figure 4). To further explore the functional consequences
of Aβ overexpression on high-affinity choline transport, we mea-
sured high-affinity choline uptake by ChT-1 in hippocampal syn-
aptosomes from WT and PDAPP mice. The hippocampus was
chosen because of the well-characterized cholinergic innervation
to this brain region as well as the presence of high levels of the
Aβ peptide in PDAPP mice (14, 19–22). A kinetic analysis of high-
affinity choline uptake in PDAPP or WT hippocampal synapto-
somes revealed a significant increase (50.9%; P < 0.05) in the Vmax
of choline uptake in PDAPP versus WT control mice, with no mea-
surable change in apparent Km (Table 1).
Anti-Aβ antibody treatment restores the hippocampal ACh release
profile in PDAPP mice. We next investigated whether acute treat-
ment with the high-affinity anti-Aβ antibody m266 could restore
hippocampal ACh homeostasis, since we have previously demon-
strated that acute treatment of PDAPP mice with m266 was able
to reverse memory deficits without any effects on brain Aβ bur-
den and that m266 binds with high affinity to soluble forms of
Aβ (13, 23). PDAPP mice were treated with a single dose of m266
(500 μg) 24 hours prior to the establishment of baseline ACh
levels. ACh release from the hippocampi following scopolamine
administration in PDAPP mice administered m266 under basal
conditions were not significantly different from untreated WT
mice but were significantly different from untreated PDAPP mice
(Figure 2, B and D; P < 0.05). Similarly, when PDAPP mice were
exposed to a novel environment following m266 administration,
the magnitude and duration of hippocampal ACh release were
not significantly different from that of WT mice but were again
significantly different from untreated PDAPP mice (Figure 2A;
P < 0.05). To determine the specificity of m266 treatment, we
utilized an irrelevant IgG (isotype matched to m266) antibody as
well as an additional anti-Aβ antibody (3D6) that recognizes the
N terminus of the human Aβ peptide (20). Neither the irrelevant
IgG nor 3D6 were able to reverse or normalize hippocampal ACh
release in response to a novel environment or following scopol-
amine administration (Figure 5, A and B). However, treatment
with 3D6, but not with the irrelevant IgG, was able to restore
Relative mRNA level of ChT-1 in the hippocampus of young (2 months
of age) and old (17 months of age) WT and PDAPP transgenic mice as
determined by real-time quantitative PCR amplification. n = 5 mice per
group. **P < 0.01, *P < 0.05 versus respective 17-month-old groups.
Hippocampal ACh release is unaltered after treatment with other IgGs.
(A) Hippocampal release of ACh in WT and PDAPP transgenic mice
following exposure to a novel environment and 24 hours after the
administration of an irrelevant IgG (PDAPP + IgG; 500 μg i.p.) or the
anti-Aβ antibody 3D6 (PDAPP + 3D6; 500 μg i.p.). (B) Release of ACh
from the hippocampus of PDAPP and WT mice following administra-
tion of scopolamine (0.3 mg/kg i.p.) 24 hours after the administration of
an irrelevant IgG or the anti-Aβ antibody 3D6. No significant difference
between treated PDAPP and untreated PDAPP mice were observed
in either A or B. (C) Basal levels of hippocampal ACh release from WT
and PDAPP mice 24 hours following administration of an irrelevant IgG
or the anti-Aβ antibody 3D6. *P < 0.05 versus WT and PDAPP + 3D6.
n = 3–10 mice per group, 4–6 months of age.
basal hippocampal ACh release following administration to
PDAPP mice (Figure 5C). We also determined high-affinity
choline uptake in hippocampal synaptosomes prepared from
PDAPP mice that had been treated with m266 prior to prepara-
tion of the crude synaptosome fraction (22). When PDAPP mice
were administered m266 24 hours prior to measuring choline
uptake, the Vmax values were not significantly different between
PDAPP and WT control mice (Table 1).
Habituation learning in PDAPP mice is restored following m266 treat-
ment. Since habituation of locomotor activity to a novel environ-
ment provides a measure of learning processes related to primal
memory formation and since ACh levels are known to increase
when rodents are placed into a novel environment, we measured
habituation to a novel environment after PDAPP mice were treat-
ed with m266?(18, 24). As predicted, young (pre-plaque) PDAPP
mice, which display cholinergic hyperresponsiveness to novelty,
failed to habituate to a novel environment (data not shown).
Following m266 treatment, however, PDAPP mice habituated to
their novel environment in a manner similar to their WT coun-
terparts (Figure 6). By contrast, when PDAPP mice were treated
with 3D6, they, like untreated PDAPP mice, failed to habituate to
a novel environment (Figure 6).
Recently, Aβ1–42 and the α7 nicotinic ACh receptor (α7) was
reported to form a high-affinity, stable complex in vitro as well as
colocalize to neuritic plaques in AD brain, suggesting that Aβ1–42
may influence normal cholinergic neurotransmission by directly
modulating the α7 (25). To our knowledge, however, ours is the
first demonstration that the Aβ peptide(s) directly associate with
ChT-1 and perturb its physiological function, thereby impairing
cholinergic neurotransmission in vivo. Using a well-characterized
transgenic mouse model of AD — the PDAPP mouse — combined
with in vivo microdialysis, we quantified ACh release from the
hippocampi of awake and freely moving animals and observed a
significant reduction in ACh release in PDAPP mice under basal
conditions and a pronounced dysregulation of cholinergic neuro-
transmission in response to either pharmacological or physiologi-
cal stimuli, which assess and are associated with normal cogni-
tive function. The reduced hippocampal release and basal tissue
levels of ACh were not due to reduced mRNA levels of ChT-1
or extracellular levels of the ACh precursor choline. In contrast,
there was a significant increase in the rate of high-affinity choline
uptake when synaptosomes were prepared from PDAPP mice
that most likely represents a compensatory change in response
to a deficient cholinergic synapse. Furthermore we determined by
coimmunoprecipitation experiments that Aβ could directly associ-
ate with ChT-1 and thereby interfere with its intracellular traffick-
ing, localization, and normal physiological functions.
Administration of the anti-Aβ antibody m266 to PDAPP mice
restored hippocampal ACh release to near WT levels when mea-
sured under either basal conditions or after mice were exposed to a
novel environment. More dramatic, however, was the nearly com-
plete restoration of hippocampal ACh release when PDAPP mice
were administered m266 prior to scopolamine administration.
Additionally, the rate of choline uptake into hippocampal synap-
tosomes prepared from PDAPP mice following administration of
m266 was nearly completely restored to that observed in WT mice.
Taken together, our results suggest that the anti-Aβ antibody m266
can bind and neutralize cholinotoxic species of the Aβ peptide since
neither an irrelevant IgG nor another anti-Aβ antibody, 3D6, affect-
ed hippocampal ACh release when PDAPP mice were placed in a
novel environment or administered scopolamine.
Since habituation of locomotor activity in a novel environment
provides a measure of primal learning and memory processes and
is dependent on an integral cholinergic circuit, we used this readily
quantifiable behavior to examine the effect of acute treatment with
m266 (24).?As previously reported, PDAPP mice fail to habituate
to a novel environment to the same extent as WT mice (17). How-
ever, following acute treatment with the anti-Aβ antibody m266,
this behavioral deficit was reversed. Given previous reports dem-
onstrating transient elevations in brain ACh levels when rodents
are placed in a novel environment, our data suggest that “normal-
izing” hippocampal ACh release by acute treatment with m266
is most likely causally related to restored habituation to a novel
environment (24). Interestingly, the profile of hippocampal ACh
release we observed in PDAPP mice was similar to that of mice
lacking M2 muscarinic receptors, suggesting that Aβ may also
influence muscarinic inhibitory autoreceptors leading to ineffi-
cient regulation of synaptically released ACh (18).
Since we used young PDAPP mice, which have little to no immu-
noreactive Aβ deposits or frank amyloid (thioflavine-S–positive)
deposition, our results strongly implicate soluble forms of the Aβ
Kinetics of choline uptake into hippocampal synaptosomes
PDAPP + m266
26.61 ± 5.45
40.16 ± 7.87A
19.97 ± 6.62C
0.85 ± 0.14
1.23 ± 0.33B
0.50 ± 0.28B
Values are mean ± SEM of 2–5 experiments. AIncrease of 50.9% com-
pared with WT controls (P < 0.05). BNo significant difference compared
with WT mice. CDecrease of 50.2% compared with untreated PDAPP
Habituation learning in a novel open field in PDAPP mice
is normalized after treatment with m266. (A) PDAPP trans-
genic mice exhibited increased locomotor activity, which
was reduced following treatment with the anti-Aβ antibody
m266 (500 μg i.p.) but not following treatment with the anti-
Aβ antibody 3D6 (500 μg i.p.). (B) Accumulated locomotor
activity per 60-minute session. ***P < 0.001. n = 20 mice per
group, 4–6 months of age.
peptide(s) in disrupting basal and on-demand hippocampal ACh
release, both of which are likely required for normal memory func-
tion (1). Indeed there is now ample in vitro evidence that?suggests
that soluble forms of Aβ inhibit long-term potentiation as well
as dampen neuronal activity following depolarization (7, 26, 27).
Recently, oligomeric soluble forms of human Aβ were reported to
disrupt complex learning behavior in rats following intracerebro-
ventricular administration (28). Moreover, in vivo evidence from
various APP transgenic mouse models suggests that Aβ-directed
immunotherapy (either passive or active immunization) can effec-
tively reverse memory deficits without any measurable effect on
deposited (insoluble) Aβ (13, 29).
Our results do not eliminate the possibility that other frag-
ments of APP may modulate hippocampal ACh efflux. Since
certain forms of secreted APP have memory-enhancing effects, it
may be possible that other fragments of APP derived from pro-
teolytic cleavage would have differential effects on cholinergic
neurotransmission; this possibility will also need further investi-
gation (30). However, the fact that both the dysregulation in ACh
release and impaired habituation learning were rapidly reversed
by the anti-Aβ antibody m266 strongly argues for a pivotal role
of the Aβ peptide as a cholinotoxic substance. One intriguing
possibility is the potential for soluble forms of the Aβ peptide(s)
to interact and/or directly modulate ChT-1 since the activity of
the ACh biosynthetic enzyme choline acetyltransferase was not
decreased in brain samples from PDAPP mice (K.R. Bales and S.M.
Paul, unpublished observations) and patients with mild AD (31).
Moreover, recent reports suggest that Aβ can regulate the cell sur-
face expression of glutamate receptors, thereby affecting normal
glutamatergic neurotransmission (32).
High-affinity uptake of choline at cholinergic nerve terminals
is widely believed to be the rate-limiting step in the biosynthe-
sis of ACh, and a significant increase in high-affinity choline
transport was reported to occur in synaptosomes prepared from
rapid autopsy AD brain tissue; this most likely reflects a com-
pensatory response initiated by remaining cholinergic neurons
due to severe cholinergic deafferentation (33). Additionally,
deletion of 1 choline acetyltransferase allele in mice does not
result in a corresponding decrease in ACh biosynthesis since
enhanced recruitment and expression of ChT-1 results in nor-
mal ACh biosynthesis (34). Our finding of a significant increase
(~50%) in the rate of choline uptake into synaptosomes pre-
pared from PDAPP mice appears inconsistent with our observa-
tion of reduced basal/tissue levels of ACh. We hypothesize that
enhanced enrichment of ChT-1 to a synaptic vesicular pool in
PDAPP mice is the result of a compensatory response driven by
the inability of the cholinergic neuron to effectively transport
choline to the intracellular space for subsequent ACh biosyn-
thesis. Additionally, the significant increase in extracellular
choline levels we observed in PDAPP mice suggests that Aβ may
in some way interfere with efficient ChT-1–mediated trans-
port of choline and/or block normal cell surface redistribu-
tion of ChT-1, thereby resulting in impaired ACh biosynthesis
and reduced tissue levels (35). Further studies will be required
to fully delineate the exact interaction between ChT-1 and Aβ;
however, our results that Aβ directly associates with ChT-1,
potentially impeding ACh biosynthesis, bolster the hypothesis
of selective cholinergic membrane “autocannibalization” under
sustained ACh demand, which may explain the increased vul-
nerability of cholinergic neurons in AD brain (36). Additionally
our data do not eliminate the possibility that certain species of
Aβ (especially Aβ40) may subserve a physiologically relevant role,
while other species (especially Aβ42) may exert a more pathologi-
cal role on cholinergic neurotransmission. Interestingly, recent
reports have demonstrated that α-synuclein, another brain pro-
tein prone to aggregate in Parkinson disease, interacts with the
dopamine transporter, suggesting that abnormal protein aggre-
gation in general may interrupt neurotransmission by interfer-
ing with normal transporter function (37). Finally, it is also fea-
sible that up- and/or downregulation of pre- and post-synaptic
muscarinic receptors could influence, in an Aβ-dependent fash-
ion, the efflux of ACh from the hippocampus.
Although the cholinergic neurotransmitter system is neither
the only nor the first neurotransmitter system affected in AD, it is
widely believed that cholinergic deafferentation in AD brain con-
tributes to the memory decline associated with disease progression.
Further work will be required to address the exact mechanism(s)
by which Aβ influences cholinergic neurotransmission and neu-
rodegeneration; however, our data clearly demonstrate a direct
cholinotoxic effect of the Aβ peptide(s) on hippocampal ACh
release in PDAPP mice that was rapidly reversed by treatment with
the anti-Aβ antibody m266. Taken together, our findings suggest
that Aβ-mediated disruption of cholinergic neurotransmission
and memory can occur in the absence of overt neurodegeneration
and that administration of certain anti-Aβ antibodies may reverse
early memory impairment especially in patients with mild cogni-
tive impairment or early AD.
Transgenic mice. The PDAPP transgenic mice used in this study were
homozygous mice derived from a heterogeneous background compris-
ing the strains C57BL/6J, DBA/2J, and Swiss-Webster (14). Young (4–6
months of age) female PDAPP mice as well as age- and background-
matched WT controls were used for all in vivo microdialysis studies. For
determining open field activity in a novel environment, young PDAPP
males (4–6 months of age) as well as background-, age- and gender-
matched WT controls were assessed. All experiments utilizing animals
were reviewed and approved by the Internal Animal Care and Use Com-
mittee of Lilly Research Laboratories.
In vivo microdialysis. Mice were deeply anesthetized with a mixture of
chloral hydrate and pentobarbital, and a CMA/7 (2 mm) dialysis probe
(CMA Microdialysis) was implanted unilaterally into the hippocampus
(AP, –3.3; ML, +3.1; DV, –4.2). Mice were allowed to recover in their home
cages for more than 24 hours and on the morning of the experiments were
transferred to a Plexiglas cylinder (15 cm × 25 cm) with clean bedding.
The dialysis probe was connected to a liquid swivel (TCS-2; SciPro Inc.)
microdialysis instrument. Perfusate was delivered at a rate of 1.5 μl/min,
and samples were analyzed in-line with HPLC coupled to an electrochemi-
cal detection (EC) system as previously described (18). The perfusate was a
modified artificial Ringer’s solution supplemented with 0.33 μM neostig-
mine to prevent enzymatic degradation. The mice were allowed to accli-
mate to this setting for 4 hours, during which time dialysate samples were
collected every 15 minutes. Little variation (<10%) occurred in the ACh sig-
nal during this period, and baseline levels of ACh efflux were established as
the mean of 5 15-minute samples prior to any subsequent manipulation.
When mice were exposed to a novel environment, they were transferred
into a new cage that was approximately the same size but markedly dif-
ferent in color (dark red versus clear) and texture (thick rubber without
bedding versus Plexiglas with a bedding-covered floor). The mice remained
in this novel environment for 1 hour, during which time dialysate samples
were collected and analyzed as described above. After 1 hour, the mice were
transferred back into their original testing cage (Plexiglas cylinder), and
samples were collected for an additional 1 hour, during which time ACh
levels returned to baseline. For pharmacological manipulation, mice were
injected with scopolamine (0.3 mg/kg i.p.), and samples were collected for
an additional 3 hours. To assess the effects of various IgGs (anti-Aβ anti-
bodies m266 and 3D6 and irrelevant IgG, 500 μg i.p.), the same experimen-
tal paradigm described above for collecting and analyzing ACh efflux was
followed, except that the mice were administered IgG 24 hours prior to the
initial baseline collection and 24 hours after the probes were implanted.
Extracellular choline concentrations were assessed by in vivo microdialysis
essentially as described above. A modified Ringer’s solution devoid of neo-
stigmine was perfused, and 5-minute samples were collected and analyzed
online with HPLC-EC (38).
Tissue ACh levels. ACh levels in tissue homogenates were determined
essentially as described previously (39). Briefly, mice were quickly decapi-
tated, and brain regions were dissected and frozen on dry ice within 30
seconds. The tissues were homogenized in 10 volumes of 0.1 M trichlo-
roacetic acid containing 10 μM acetylthiocholine as an internal standard.
After centrifugation at 10,000 g, 20-μl aliquots of the supernatant were
analyzed for ACh with HPLC-EC (39).
Choline uptake. Choline uptake into hippocampal synaptosomes was mea-
sured as previously described (22). Briefly, hippocampi from 4- to 6-month-
old PDAPP mice and WT controls were dissected and pooled (n = 10–12
per experiment). Crude synaptosomes were prepared by homogenizing the
tissue in ice-cold buffer (0.32 M sucrose, 10 strokes Dounce homogenizer
AA068), and total protein content was determined (Pierce Biotechnology)
from P2 pellets after centrifugation (12,000 g for 20 minutes). Choline
uptake was determined in triplicate by incubating 0.1–0.2 mg of the crude
synaptosomal protein in choline uptake buffer (130 mM NaCl, 1.3 mM
KCl, 1.2 mM MgSO4, 1.2 mM KH2PO4, 10 mM glucose, 10 mM HEPES,
and 2.2 mM CaCl, pH 7.4) and [3H] choline (86 Ci/mmol; 10 nM; Perkin-
Elmer), with final choline concentrations ranging 0.03–5 μM, for 5 minutes
at 37°C. Uptake was terminated by returning samples to ice for 5 minutes
followed by aspiration onto polyethyleneimine-coated glass filters (Perkin-
Elmer). The specificity of uptake was determined by subtracting choline
uptake in the presence of hemicholinium (10 μM). Km and Vmax values were
determined by nonlinear regression analysis with GraphPad Prism soft-
ware (version 4.03; GraphPad Software Inc.). For determining the effects of
m266 on high-affinity choline uptake, PDAPP mice were administered 500
μg m266 i.p. 24 hours prior to the preparation of synaptosomes. For the
determination of high-affinity choline uptake into rat hippocampal nerve
termini, the above protocol was followed except that hippocampi from 2–3
rats were used. A stable cell line expressing the human ChT-1 was gener-
ated following selection in hygromycin after transfection into 293 cells. For
high-affinity choline uptake determinations, cells were plated onto poly-
l-lysine–coated Cytostar T?plates (60,000 cells/100 μl; Amersham Biosci-
ences) and incubated at 37°C for 24 hours prior to determining choline
uptake. For determination of high-affinity uptake, media was removed and
replaced with choline uptake?buffer with 14C-choline (0.2 μCi/ml; 3.75 μM
final concentration) and incubated for 70 minutes at room temperature.
The plates were counted in a MicroBeta counter (PerkinElmer) for 1 minute.
Specific uptake was determined by subtracting uptake in the presence of
hemicholinium (10 μM). To determine choline uptake following exposure
to human Aβ1–42 (AnaSpec), ChT-1 cells or rat hippocampal synaptosomes
were exposed to Aβ (0.01–1 μM), which was diluted from a stock solution
(1 mM), and incubated for 15 minutes at 37°C. Choline uptake was then
determined as described above
Coimmunoprecipitation. Hippocampi from PDAPP transgenic mice were
homogenized in buffer (1% Nonidet P-40, 0.1 % SDS, 50 mM Tris pH 8.0,
50 mM NaCl, 0.05% deoxycholate, and protease inhibitor), and protein
concentrations were determined (Pierce Biotechnology). Hippocampal
extracts (250 μg) were diluted in 250 μl PBS/0.2% BSA with antibodies
(2.5 μg; anti-Aβ, 2G3, and 21F12, all from Senetek; 4G8 from Signet Lab
Inc.; anti–ChT-1, anti–glutamate-1 transporter from Chemicon Interna-
tional; irrelevant IgG from?Harlan) for 8 hours at 4°C on a rocking plat-
form. Samples were incubated for an additional 16 hours after the addi-
tion of BSA (1%) blocked Protein G beads (100 μl; Pierce Biotechnology).
The beads were washed 3 times in PBS (500 μl),?and proteins were eluted
with sample loading buffer (30 μl; Novex, Invitrogen Corp.) prior to size
fractionation (4-12 % Bis Tris gel; Bio-Rad). Proteins were then transferred
onto PVDF membranes (Bio-Rad) and probed with an anti–ChT-1 anti-
body (Chemicon International) or anti-Aβ antibodies (21F12, 2G3) which
were biotinylated. The signal was visualized with enhanced chemilumines-
cence (Pierce Biotechnology).
Habituation learning. Locomotor activity as a measure of habituation to
a novel environment was assessed in photocell activity cages (San Diego
Instruments), which consisted of a standard plastic rodent cage (24 cm ×
45.5 cm × 15.5 cm) surrounded by a stainless steel frame. Locomotor activ-
ity frames consisted of 7 infrared photocell beams located across the long
axis of the frame, raised 2 cm above the floor and placed 5.5 cm apart.
The number of cage crossings, defined as consecutive interruptions of one
beam followed immediately by interruption of an adjacent beam, were
recorded by a computer system during consecutive 5-minute intervals for
60 minutes and used as a measure of spontaneous locomotion. Locomotor
activity was evaluated in animals that were naive to the test apparatus but
were habituated to the test room for approximately 1 hour prior to activity
measurements. PDAPP or WT mice were injected with various IgGs (500 μg
i.p.) or vehicle and assessed for locomotor activity 24 hours later.
mRNA analysis.?Total RNA was isolated from the hippocampus of 2- and
17-month-old PDAPP mice (n = 6) following saline perfusion. Briefly,
tissue was rapidly removed and stored on dry ice until homogenization.
Total RNA was isolated using the RNeasy Mini Kit (QIAGEN) according
to the manufacturer’s guidelines. cDNA was generated using the Super-
script (Invitrogen Corp.) preamplification system for first-strand synthe-
sis according to the manufacturer’s protocol and subsequently used (at
1:100 dilution) as the template for real-time quantitative PCR amplifica-
tion. Primers specific for ChT-1 (forward, 5′-CCAGCTTTTGGGTGCCTG;
reverse, 5′-TGTGGAAGCTCCAATAGCTCC; probe, 6FAM-TGATGGCTC-
TACCCGCCATATGC-TAMRA; 20-pmol and 5-pmol probe) were used.
Amplification conditions were as follows: 10 minutes at 95°C followed by
40 cycles at 95°C for 15 seconds and 60°C for 1 minute in a PerkinElmer
Applied Biosystems SDS7700 sequence detection system. Each sample was
run in triplicate with non–reverse transcribed template serving as control.
The relative level of ChT-1 mRNA was computed by the standard curve
method utilizing GAPDH amplification for normalization.
Statistics. The basal values of ACh and choline in tissue and microdialy-
sates in PDAPP and WT mice in response to antibody treatments were
evaluated by 2-tailed Student’s t test (Figure 1A and Figure 2C), 2-way
(genotype × age) ANOVA with repeated measures (Figure 1, B and C), or
1-way (treatment) ANOVA (Figure 2D and Figure 5C). The microdialysis
data were also expressed and presented as the percent change from base-
line (based on pretreatment values) and analyzed with a 2-way (treatment
× time) ANOVA (Figure 2, A and B, and Figure 5, A and B). In all cases,
significant interactions were evaluated using the Newman-Keuls test for
multiple comparisons. For high-affinity choline uptake experiments,
the data were analyzed with a 1-way (treatment) or 2-way (treatment ×
genotype) ANOVA and evaluated by the Newman-Keuls test for multiple
comparison. For habituation learning, the mean ambulations during a
60-minute session were used for a 2-way (genotype × treatment) com-
research article Download full-text
parison followed by the Tukey-Kramer test for multiple comparisons.
P < 0.05 was considered statistically significant.
The authors thank K. Svensson, P. May, C. Felder, and Y. Du for
comments on the manuscript as well as D. Koger and C. Salhoff
for technical assistance.
Received for publication October 12, 2005, and accepted in revised
form January 3, 2006.
Address correspondence to: K.R. Bales or S.M. Paul, DC0533 Eli
Lilly & Co., Neuroscience Discovery Research, 355 E. Merrill Street,
Indianapolis, Indiana 46285, USA. Phone: (317) 277-3061; Fax:
(317) 277-6146; E-mail: KRBales@Lilly.com (K.R. Bales). Phone:
(317) 277-8799; Fax: (317) 277-1125; E-mail: Paul_Steven_M@
Lilly.com (S.M. Paul).
K.R. Bales and E.T. Tzavara contributed equally to this work.
E.T. Tzavara’s present address is: INSERM U-513, Créteil, France.
1. Perry, E., Walker, M., Grace, J., and Perry, R. 1999.
Acetylcholine in mind: a neurotransmitter corre-
late of consciousness? Trends Neurosci. 22:273–280.
2. Bartus, R., Dean, R.L., Beer, B., and Lippa, A.S.1982.
The cholinergic hypothesis of geriatric memory
dysfunction. Science. 217:408–411.
3. Coyle, J.T., Price, D.L., and DeLong, M.R. 1983.
Alzheimer’s disease: a disorder of cortical cholin-
ergic innervation. Science. 219:1184–1190.
4. Whitehouse, P.J., et al. 1981. Alzheimer disease:
evidence for selective loss of cholinergic neurons in
the nucleus basalis. Ann. Neurol. 10:122–126.
5. Hardy, J., and Selkoe, D.J. 2002. The amyloid
hypothesis of Alzheimer’s disease: progress and
problems on the road to therapeutics. Science.
6. Selkoe, D.J. 2001. Alzheimer’s disease: genes, pro-
teins, and therapy. Physiol. Rev. 81:741–766.
7. Walsh, D.M., et al. 2002. Naturally secreted
oligomers of amyloid β protein potently inhibit
hippocampal long-term potentiation in vivo.
8. Westerman, M.A., et al. 2002. The relation-
ship between Aβ and memory in the Tg2576
mouse model of Alzheimer’s disease. J. Neurosci.
9. McLean, C.A., et al. 1999. Soluble pool of Aβ amyloid
as a determinant of severity of neurodegeneration in
Alzheimer’s disease. Ann. Neurol. 46:860–866.
10. Lue, L.F., et al. 1999. Soluble amyloid β peptide
concentration as a predictor of synaptic change in
Alzheimer’s disease. Am. J. Pathol. 155:853–862.
11. Wang, J., Dickson, D.W., Trojanowski, J.Q., and
Lee, V.M. 1999. The levels of soluble versus insol-
uble brain Aβ distinguish Alzheimer’s disease
from normal and pathologic aging. Exp. Neurol.
12. Parvathy, S., et al. 2001. Correlation between
Aβx-40-, Aβx-42-, and Aβx-43-containing amy-
loid plaques and cognitive decline. Arch. Neurol.
13. Dodart, J.C., et al. 2002. Immunization reverses
memory deficits without reducing brain Aβ bur-
den in Alzheimer’s disease model. Nat. Neurosci.
14. Games, D., et al. 1995. Alzheimer-type pathology
in transgenic mice overexpressing V717F beta-amy-
loid precursor protein. Nature. 373:523–527.
15. Irizarry, M., et al. 1997. Abeta deposition is asso-
ciated with neuropil changes, but not with overt
neuronal loss in the human amyloid precursor pro-
tein V717F (PDAPP) transgenic mouse. J. Neurosci.
16. German, D.G., et al. 2003. Cholinergic neuropa-
thology in a mouse model of Alzheimer’s disease.
J. Comp. Neurol. 462:371–381.
17. Dodart, J.C., Mathis, C., Bales, K.R., and Paul, S.M.
1999. Behavioral disturbances in transgenic mice
overexpressing the V717F β-amyloid precursor pro-
tein. Behav. Neurosci. 5:982–990.
18. Tzavara, E.T., et al. 2003. Dysregulated hippocampal
acetylcholine neurotransmission and impaired cog-
nition in M2, M4 and M2/M4 muscarinic receptor
knockout mice. Mol. Psychiatry. 8:673–679.
19. Yamamura, H., and Synder, S.H. 1972. Choline:
high affinity uptake by rat brain synaptosomes.
20. Johnson-Wood, K., et al.1997. Amyloid precursor
protein processing and Abeta 42 deposition in a
transgenic mouse model of Alzheimer disease. Proc.
Natl. Acad. Sci. U. S. A. 94:1550–1555.
21. Mesulam, M.M., and Mufson, E.J. 1984. Neural
inputs into the nucleus basalis of the substan-
tia inominata (Ch4) in the rhesus monkey. Brain.
22. Simon, J., Atweh, S., and Kuhar, M. 1976. Sodium-
dependent high affinity choline uptake: a regula-
tory step in the synthesis of acetylcholine J. Neuro-
23. DeMattos, R.B., et al. 2001. Peripheral anti-Aβ
antibody alters CNS and plasma Aβ clearance and
decreases brain Aβ burden in a mouse model of
AD. Proc. Natl. Acad. Sci. U. S. A. 15:8850–8855.
24. Acquas, E., Wilson, C., and Fibiger, H.C. 1996. Con-
ditioned and unconditioned stimuli increase fron-
tal cortical and hippocampal acetylcholine release:
effects of novelty, habituation, and fear. J. Neurosci.
25. Wang, H.-Y., et al. 2000. β-Amyloid 1-42 binds to α7
nicotinic acetylcholine receptor with high affinity.
J. Biol. Chem. 25:5626–5632.
26. Kamenetx, F., et al. 2003. APP processing and syn-
aptic function. Neuron. 37:925–937.
27. Selkoe, D.J. 2002. Alzheimer’s disease is a synaptic
failure. Science. 298:789–791.
28. Cleary, J.P., et al. 2005. Natural oligomers of the
amyloid-β protein specifically disrupt cognitive
function. Nat. Neurosci. 8:78–84.
29. Kotilinek, L.A., et al. 2002. Reversible memory loss
in a mouse transgenic model of Alzheimer’s dis-
ease. J. Neurosci. 22:6331–6335.
30. Meziane, H., et al. 1998. Memory-enhancing effects
of secreted forms of the β-amyloid precursor pro-
tein in normal and amnestic mice. Proc. Natl. Acad.
Sci. U. S. A. 95:12683–12688.
31. DeKosky, S.T., et al. 2002. Upregulation of choline
acetyltransferase activity in hippocampus and
frontal cortex of elderly subjects with mild cogni-
tive impairment. Ann. Neurol. 51:145–155.
32. Snyder, E.M., et al. 2005. Regulation of NMDA
receptor trafficking by amyloid-β Nat. Neurosci.
33. Slotkin, T.A., et al. 1990. Regulatory changes in
presynaptic cholinergic function assessed in rapid
autopsy material from patients with Alzheimer
disease: implications for etiology and therapy. Proc.
Natl. Acad. Sci. U. S. A. 87:2452–2455.
34. Brandon, E.P., et al. 2004. Choline transporter 1 main-
tains cholinergic function in choline acetyltransferase
haploinsufficiency. J. Neurosci. 24:5459–5466.
35. Gates, J., Ferguson, S.M., Blakely, R.D., and Appar-
sundaram, S. 2004. Regulation of choline trans-
porter surface expression and phosphorylation by
protein kinase C and protein phosphatase 1/2A.
J. Pharmacol. Exp. Therap. 310:536–545.
36. Wurtman, R.J. 1992. Choline metabolism as a basis
for the selective vulnerability of cholinergic neu-
rons. Trends Neurosci. 15:117–122.
37. Xu, J., et al. 2002. Dopamine-dependent neuro-
toxicity of α-synuclein: a mechanism for selective
neurodegeneration in Parkinson disease. Nat. Med.
38. Nomikos, G.G., Arborelius, L., and Svensson,
T.H. 1992. The novel 5-HT1A receptor antagonist
(S)-UH-301 prevents (R) OH-DPAT-induced decrease
in interstitial concentrations of serotonin in the rat
hippocampus. Eur. J. Pharmacol. 216:373–378.
39. Bymaster, F.P., Perry, K.W., and Wong, D.T. 1985.
Measurement of acetylcholine and choline in brain
by HPLC with electrochemical detection. Life Sci.