Alzheimer’s disease (AD) is characterized by accumulation of amyloid-? peptide (A?) in the brain regions that subserve memory and
investigated whether the amylin receptor antagonist, AC253, neutralizes the depressant effects of A?1–42and human amylin on hip-
recorded from the stratum radiatum layer of the CA1 area (cornu ammonis 1 region of the hippocampus) in response to electrical
where robust LTP is observed, 6- to 12-month-old TgCRND8 mice show blunted LTP that is significantly enhanced by application of
Alzheimer’s disease (AD) represents one of the most serious
proven treatment beyond symptomatic therapies. The accumu-
lation and deposition of amyloid-? peptide (A?), a 39–43 aa
peptide, in brain areas critical for cognition and memory is an
insoluble deposits of A? in plaque form coexist with small, dif-
fusible soluble oligomers of the peptide in cortical and limbic
trimers, tetramers and higher order assemblies, consistently in-
hibit hippocampal long-term potentiation (LTP), a surrogate in
Furthermore, naturally secreted A? oligomers potently inhibit hip-
pocampal LTP in vivo, which is reversible by passive infusions of
either A? antibodies or administration of an active A? vaccine
yloid precursor protein (APP), when treated with a ?-secretase in-
hibitor to reduce A? levels, demonstrate a profound restoration of
impaired LTP (Townsend et al., 2010). Thus, understanding the
mechanisms whereby soluble A? oligomers can disrupt synaptic
function is of great interest in preventing AD progression. Multiple
plicated in mediating A? disruption of synaptic processes and are
potential targets for developing anti-A? therapies (Huang and
target for the neuronal actions of A? (Jhamandas et al., 2011).
Human amylin (h-Amylin (islet amyloid polypeptide) is a 37-
residue amyloidogenic peptide first isolated from pancreatic is-
lets of Langerhans in patients with type 2 diabetes (Cooper et al.,
1987). Human amylin acts via a G-protein-coupled receptor
(amylin receptor) composed of the calcitonin receptor (CTR)
and a receptor-associated membrane protein (RAMP) and dem-
of A? (Jhamandas et al., 2011). Thus, blockade of the amylin
receptor with an antagonist (AC253) attenuates electrophysio-
logical and neurotoxic actions of A? on human and rat fetal
blunts A? toxicity Further, we have shown it is the amylin-3
(CTR ? RAMP3 ? AMY3), but not AMY1 or AMY2, subtype of
the amylin receptor that is most relevant for the activation of
ment of Medicine (Neurology), University of Alberta, Edmonton, AB, Canada T6G 2S2. E-mail: jack.
TheJournalofNeuroscience,November28,2012 • 32(48):17401–17406 • 17401
intracellular cAMP-dependent pathways and expression of A?
and h-Amylin toxicity (Fu et al., 2012). Collectively, these obser-
tor is also a target for the expression of deleterious effects of A?
on synaptic plasticity and learning.
of LTP is also observed for h-Amylin, and if so, are such effects
expressed via the amylin receptor. Furthermore, we investigated
the basis for depressed LTP observed in APP overexpressing
TgCRND8 mice with the notion that LTP in such mice could be
restored (to levels observed in age-matched wild-type mice) by
pharmacologically blocking the amylin receptor.
h-Amylin on LTP, we used 2- to 3-month-old male or female C57BL/6
mice. For experiments investigating the role of the amylin receptor in
LTP in mice overexpressing APP, we used transgenic (Tg) CRND8 mice
(human APP695 transgene array incorporating Swedish K670M/N671L
and Indiana V717F mutations superimposed upon a C57BL6/C3H ge-
netic background that have been described previously to exhibit A?
plaques and cognitive defects by 3 months of age (Chishti et al., 2001).
Specifically we used 6- to 12-month-old TgCRND8 and age-matched,
wild-type littermate (C57BL/6 background) male or female mice. All
experiments were performed in compliance with the relevant laws and
guidelines set by the Canadian Council for Animal Care and with the
approval of the Human Research Ethics Board and Animal Care Use
Committee (Health Sciences) at the University of Alberta.
Slice preparation and electrophysiology. Brains were quickly removed
pus. The aCSF contained (in mM) 124 NaCl, 3 KCl, 2.4 CaCl2, 2 MgCl2,
95% O2and 5% CO2. Hippocampal slices (400 ?m thick) were main-
tained in an aCSF-filled holding chamber at room temperature and
transferred to the submerged recording chamber constantly perfused
with aCSF (2 ml/min) at 30°C. Field EPSP (fEPSP) was recorded with a
fer collateral afferents were stimulated with 100 ?s test pulses via a bipo-
lar cluster electrode (FHC, Inc.) (Kimura and Ohno, 2009). To evaluate
basal synaptic transmission, we applied different stimulation strengths
(125–300 ?A in steps of 25 ?A) and plotted the amplitudes of presynap-
tic fiber volleys versus the corresponding fEPSP slopes to compare the
slope of input–output (I/O) curves of fEPSP. For LTP experiments, the
stimulus strength was set to elicit 40–50% of the maximum fEPSP am-
plitude and test pulses delivered to Schaffer collaterals once every 30 s.
LTP was induced by a 3 theta burst stimulation (3-TBS) protocol (each
burst consisted of 4 pulses at 100 Hz with a 200 ms interburst interval).
Before 3-TBS or drug application, the responses were monitored for at
least 10 min to ensure a stable baseline of fEPSP. To determine whether
the magnitude of LTP differed significantly between groups, average re-
were compared. To test paired-pulse facilitation, we measured the per-
ent interpulse intervals (20–500 ms).
Drugs and their application. Soluble oligomeric A?1–42, the reverse
nonfunctional sequence peptide ??42–1, and h-Amylin were used in the
present study and prepared as per previously published protocols (Stine
et al., 2003; Jhamandas et al., 2011). A?1–42and A?42–1were purchased
than a minute and a half.
Immunohistochemistry. Following electrophysiological recordings,
hippocampal brain slices from TgCRND8 and control wild-type mice
were fixed with 4% paraformaldehyde for immunohistochemical analy-
sis of A? plaque deposition (for details, see Jhamandas et al., 2011).
hippocampal LTP. A, A 50 nM concentration of A?1–42, but not A?42–1, impaired LTP in
hippocampal slices. LTP was induced by 3? TBS protocols at Schaffer collateral-CA1
before 50 nM A?1–42application and LTP was induced by 3? TBS protocols in the pres-
of application. All data are presented as mean ? SEM. Traces are the average of fEPSPs
recorded during baseline (black) and 40–60 min after 3? TBS (gray). Calibration: 0.2
mV, 20 ms (n ? 4–6 mice per group). C, The summary bar graphs show significant
*p ? 0.01 for control vs A?1–42and AC253 vs A?42–1, and **p ? 0.05 for A?1–42vs
Amylin receptor antagonist AC253 reverses A?1–42-induced depression of
17402 • J.Neurosci.,November28,2012 • 32(48):17401–17406Kimuraetal.•A?DepressionofLTPIsAmylinReceptorMediated
Briefly, 25-?m-thick hippocampal brain slices were sectioned, and free-
monoclonal antibody (Signet Laboratories) and visualized with Alexa
Fluor 546 anti-mouse antibody (Invitrogen).
Statistics. Results were expressed as means ? SE. Statistical analysis
was performed using one-way ANOVA followed by post hoc Tukey’s
honestly significant difference (HSD) test (for multiple comparisons) or
unpaired t test (for pairwise comparisons).
Application of soluble oligomeric A?1–42(50 nM) depressed LTP
induced by a weak tetanization protocol at the CA1 region of
wild-type mouse hippocampal slices. In contrast, using the same
protocol of drug application (for 5 min before and following
0.01 for AC253 vs h-Amylin, and **p ? 0.05 for h-Amylin vs h-Amylin with AC253; ANOVA
amyloid plaques are visualized within the brain parenchyma, and in 4, amyloid deposits are
fEPSP at various stimulus intensities (125–300 ?A in 25 ?A increments). Traces are fEPSP
Kimuraetal.•A?DepressionofLTPIsAmylinReceptorMediatedJ.Neurosci.,November28,2012 • 32(48):17401–17406 • 17403
impaired with 50 nM A?1–42(p ? 0.01;
unpaired t test) but not A?42–1(p ?
0.3148; unpaired t test) during the 40–60
min period after induction (Fig. 1A,C).
Application of A?1–42in the period after
LTP induction had no effect (data not
shown). To determine whether A?1–42-
induced reduction in LTP is expressed
SQELHRLQTYPRTNTGSNTY), a poly-
peptide that is a potent amylin receptor
antagonist. We applied 250 nM AC253
the hippocampal slices to 50 nM A?1–42
and for 5 min after LTP induction. The
based on prior studies that examined
the ability of this antagonist to block
h-Amylin and A? in rat and human fetal
neurons and HEK293 cells transfected
with subtypes of amylin receptors (Jha-
et al., 2011; Fu et al., 2012). AC253, ap-
plied alone did not affect control LTP
(Fig. 1B,C; p ? 0.9189, Tukey’s HSD
test), However, application of AC253
resulted in a significant restoration of
hippocampal LTP observed following ex-
posure of the brain slices A?1–42(Fig.
1B,C; p ? 0.05, Tukey’s HSD test).
We further investigated whether applica-
tion of h-Amylin also causes the basal de-
pression of hippocampal LTP induced by
3-TBS protocol in a manner similar to
that induced by A?1–42. Human amylin
(50 nM) application also resulted in im-
paired hippocampal LTP (Fig. 2A; p ?
0.01, unpaired t test), which could be reversed with preapplica-
tion of AC253 (250 nM; Fig. 2A,B; p ? 0.05, Tukey’s HSD test).
To identify a potential role for the amylin receptor in an animal
model where cognitive and memory deficits are attributable to
presence of significant brain levels of A?, we examined hip-
pocampal LTP in TgCRND8 mice that overexpress mutant
human APP695. TgCRND8 mice at 6 months and older dem-
onstrate marked amyloid deposition in the hippocampus and
cortex than age-matched wild-type littermate controls (Fig. 3A).
In hippocampal slices from 6- to 12-month-old TgCRND8, I/O
curves of fEPSPs in response to different stimulation strengths
average slope of I/O curves was reduced in TgCRND8 mice (p ?
evoke LTP at hippocampal Schaffer collateral-CA1 synapses of
in Figure 4B. LTP induced by 3-TBS was significantly depressed
with wild-type age-matched controls (p ? 0.05; unpaired t test).
relationships) and LTP at hippocampal CA1 are reduced in
First, we tested the effects of AC253 on paired-pulse facilitation
which represents a short-term form of synaptic plasticity reflect-
ing presynaptic transmitter release probability. Paired-pulse fa-
cilitation was indistinguishable between TgCRND mice and
controls at ?6 months of age (Fig. 4A). We next examined
TgCRND8 mice is dependent on the amylin receptor. Preappli-
and after 3-TBS, did not affect LTP during 40–60 min after in-
duction in wild-type mice (p ? 0.9999, Tukey’s HSD test, Fig.
4B). However, basal depression of LTP in TgCRND8 is restored
by application of 250 nM AC253 (p ? ? 0.05, Tukey’s HSD test)
to comparable levels that are observed in age-matched wild-type
effect on fEPSPs (data not shown).
17404 • J.Neurosci.,November28,2012 • 32(48):17401–17406Kimuraetal.•A?DepressionofLTPIsAmylinReceptorMediated
like soluble oligomeric A?, inhibits hippocampal LTP and both
the amylin receptor antagonist, AC253. Further, in the presence
of AC253, depression of LTP in transgenic mice that overexpress
APP (Tg CRND8) reported by others (Bellucci et al., 2006;
Arrieta-Cruz et al., 2010), and confirmed by us, is reversed to
levels seen in wild-type, age-matched control littermate mice.
The notion that h-Amylin is capable of disrupting synaptic plas-
ticity seems at first inconsistent with the multiple basic homeo-
static regulatory functions ascribed to this endogenous peptide.
However, available data indicate that amylin effects may be con-
centration dependent. The usual plasma concentration for
h-Amylin is in the low picomolar range, even following stimuli
that promote a release of the peptide in response to glucose load
ing and energy metabolism, the peptide seems nontoxic, and it is
only at higher concentrations (nanomolar to micromolar range)
that h-Amylin seems to exert its toxic effects (Fu et al., 2012).
Multiple receptors have been postulated as candidates for the
that recapitulate, in part, features of AD pathology (Patel and
Jhamandas, 2012). Of these, we have focused on the amylin re-
ceptor, which comprises of a combination of CTR and RAMP,
serves as a target receptor for human amylin, and has a wide-
hippocampus (Sexton et al., 1994; Paxinos et al., 2004). More-
over, discrete peptide sequences of A? and amylin have been
via the amylin receptor, and furthermore, data from HEK293
RAMP2) receptor that is the specific target for A? (Jhamandas and
ent data suggest that in an in vitro paradigm of synaptic plasticity
TgCRND8 mice encode a double mutant form of APP695
(KM670/671NL?V717F) under the control of the PrP gene pro-
moter and recapitulate many of the essential features of AD pa-
thology in an accelerated fashion (Chishti et al., 2001).
Histological deposition of A? in plaque form is evident in 100%
ropathological changes correlate with cognitive and behavioral
impediment that are observed as early as 3 months of age. Our
study demonstrates that electrophysiological markers of hip-
basal synaptic transmission (I/O curves) and LTP, are present in
all TgCRND8 mice by 6 months of age compared with age-
was identified. Short-lived presynaptic plasticity as measured by
paired pulse facilitation was normal in TgCRND8 and wild-type
mice as reported for other mouse models (Chapman et al., 1999;
with AC253 resulted in restoration of LTP recorded from
TgCRND8 mice to levels that are observed in wild-type controls.
However, in TgCRND8 mice, AC253 had no effect on fEPSPs
tion or in the absence of TBS. We and others have shown that
total hippocampal A? levels (in soluble oligomeric and plaque
form) in TgCRND8 mice ?6 months of age are markedly ele-
vated compared with wild-type littermates (Chishti et al., 2001;
Jhamandas et al., 2011). We suggest that the increase in ambient
that is observed in these animals as they get older and that block-
ade of the amylin receptor, which we have shown to serve as a
putative target receptor for A?, causes an improvement in LTP.
Interestingly, we have also observed that the age-dependent and
region-specific (cortex, hippocampus and septum) increases in
the amylin receptor (CTR and RAMP3) and A? in TgCRND8
mice are also apparent in cortex and hippocampus in postmor-
tem brain tissue from AD patients compared with age-matched
controls (our unpublished observations).
The nature and mechanisms of the soluble oligomeric A?
interaction with the amylin receptor, specifically the AMY3 moiety
We have previously shown that the locus of such a ligand-
receptor interplay is at the level of the postsynaptic membrane
where A?, through the amylin receptor, modulates activity of a
suite of ionic conductances, mostly potassium channels, that re-
calcium levels and activation of cAMP along with other down-
stream signal transduction mediators (Jhamandas and MacTavish,
rat brain slices, possibly via metabotropic glutamate receptors
scale (milliseconds to minutes) but have the potential to disrupt
intracellular homeostasis and synaptic plasticity. Longer expo-
sures to soluble oligomeric A? has been shown in many in vitro
mechanisms (Selkoe, 2008; Jhamandas et al., 2011). Thus, appli-
cation of amylin receptor antagonists or siRNA downregulation
utable to the soluble oligomeric A? species. The present study
provides first preclinical evidence for restoration of A?-induced
impairment of synaptic plasticity using blockade of the amylin
receptor and suggest that antagonists for this receptor, originally
ising therapeutic option for AD.
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